Antibody fragments for treatment of biofilm-related disorders

Abstract

To provide antibody fragments for treatment of biofilm-related disorders.SOLUTION: This disclosure provides antibody fragments and isolated polypeptides comprising the antibody fragments that are useful as a therapeutic for those with an existing infection characterized by formation of biofilms. The antibody fragments and isolated polypeptides comprising the antibody fragments can be administered to detect, treat or prevent infection and / or remedy biofilms. Bacteria that cannot form functional biofilms are more readily cleared by the remainder of the host's immune system and / or traditional antibiotics.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under U.S.C. §119(e) of U.S. Provisional Application No. 62 / 453,921, filed February 2, 2017, and U.S. Provisional Application No. 62 / 442,307, filed January 4, 2017, the contents of each of which are incorporated herein by reference in their entirety.

[0002] STATEMENT OF GOVERNMENT RIGHTS This disclosure was made in part with government support under Grant No. NIH R01 DC11818 awarded by the National Institutes of Health. The government has certain rights in this disclosure.

[0003] Field The present disclosure generally relates to methods and compositions for reducing and / or eliminating bacterial biofilms and treating biofilm-associated diseases or disorders using novel antibody fragments and compositions containing these fragments. [Background technology]

[0004] background At least one protein from the DNABII family of proteins is found in all known eubacteria and is naturally found on the outside of bacterial cells. This family elicits a strong innate immune response, but host subjects are unable to naturally produce specific protective antibodies against family members as a result of infection. DNABII proteins and extracellular DNA (eDNA) contribute to the lattice structure of a "biofilm." A major problem with bacterial biofilms is the inability of the host immune system and / or antibiotics and other antimicrobial agents to access the bacteria protected within the biofilm.

[0005] Biofilms also exist in industrial settings. For example, biofilms are associated with a wide range of petroleum processing problems, from production sites to gas station storage tanks. In the field, sulfate-reducing biofilm bacteria produce hydrogen sulfide (soured oil). In processing pipelines, biofilm activity creates slime that clogs filters and openings. Biofilms and biofilm organisms also cause corrosion in pipelines and petroleum processing equipment. These problems can manifest throughout oil or gas production facilities, to the point where fouling and corrosive biofilm organisms are found on the surface of the end product in storage tanks.

[0006] In homes, biofilms are found in or on any surface that supports microbial growth, such as in drains, on countertops, in toilets, and in swimming pools and spas. Biofilms are implicated in a wide range of water processing applications, both domestic and industrial. Biofilms can grow on the surfaces of processing equipment and impair its performance, such as reducing heat transfer efficiency or clogging filters and membranes. Biofilms growing on cooling tower fill can add enough weight to cause the fill to collapse. Biofilms can corrode even highly specialized stainless steel. Biofilms in water processing, such as biofilm contamination in paper processing or the attachment of even single cells to silicon chips, can degrade the value of the final product. Biofilms growing in drinking water distribution systems can harbor potential pathogenic, corrosive, or bacterial organisms that degrade the aesthetic quality of the water. partial sequence table SEQ ID NO: 1: Full length wild type (wt) 86-028NP Haemophilus influenzae IhfA; Genbank accession number: AAX88425.1 (last accessed March 21, 2011): MATITKLDIIEYLSDKYHLSKQDTKNVVENFLEEIRLSLESGQDVKLSGFGNFELRDKSSRPGRNPKTGDVVPVSARRVVITKPGQKLRARVEKIK. SEQ ID NO: 2: Full length wild-type (wt) 86-028NP Haemophilus influenzae IhfB; Genbank accession number: AAX88699.1 (last accessed May 13, 2015): MTKSELMEKLSAKQPTLSAKEIENMVKDILEFISQSLENGDRVEVRGFGSFSLHHRQPRLGRNPKTGDSVNLSAKSVPYFKAGKELKARV DVQA. SEQ ID NO: 3: Full length wt86-028NP Haemophilus influenzae HU; Genbank accession number: YP_248142.1 (last accessed March 21, 2011): MRFVTIFINHAFNSSQVRLSFAQFLRQIRKDTFKESNFLFNRRYKFMNKTDLIDAIANAAELNKKQAKAALEATLDAITASLKEGEPVQLIGFGTFKVNERAARTGRNPQTGAEIQIAASKVPAFVSGKALKDAIK. SEQ ID NO: 4: Full length wt R2846 Haemophilus influenzae IhfA; Genbank accession number: ADO96375 (last accessed March 21, 2011): MATITKLDIIEYLSDKYHLSKQDTKNVVENFLEEIRLSLESGQDVKLSGFGNFELRDKSSRPGRNPKTGDVVPVSARRVVTFKPGQKLRARVEKTK. SEQ ID NO: 5: Full length wt Rd Haemophilus influenzae IhfA; Genbank accession number: AAC22959.1 (last accessed March 21, 2011): MATITKLDIIEYLSDKYHLSKQDTKNVVENFLEEIRLSLESGQDVKLSGFGNFELRDKSSRPGRNPKTGDVVPVSARRVVTFKPGQKLRARVEKTK. SEQ ID NO: 6: Full length wt E. coli K12 IhfA; Genbank accession number: AAC74782.1 (last accessed March 21, 2011): MALTKAEMSEYLFDKLGLSKRDAKELVELFFEEIRRALENGEQVKLSGFGNFDLRDKNQRPGRNPKTGEDIPITARRVVTFRPGQKLKSRVENASPKDE; DNA Genbank No. NC_000913. SEQ ID NO: 7: Full length wt E. coli K12 IhfB; Genbank accession number: BAA35656 (last accessed May 19, 2015): MTKSELIERLATQQSHIPAKTVEDAVKEMLEHMASTLAQGERIEIRGFGSFSLHYRAPRTGRNPKTGDKVELEGKYVPHFKPGKELRDRANIYG. SEQ ID NO: 8: E. coli hupA; Genbank accession number: AP_003818 (last accessed March 21, 2011): MNKTQLIDVIAEKAELSKTQAKAALESTLAAITESLKEGDAVQLVGFGTFKVNHRAERTGRNPQTGKEIKIAAANVPAFVSGKALKDAVK. SEQ ID NO: 9: E. coli hupB; Genbank accession number: AP_001090.1 (last accessed March 21, 2011): MNKSQLIDKIAAGADISKAAAGRALDAIIASVTESLKEGDDVALVGFGTFAVKERAARTGRNPQTGKEIAAAKVPSFRAGKALKDAVN. SEQ ID NO: 10: Full length wt P. aeruginosa PA 01 IhfA; Genbank accession number: AAG06126.1 (last accessed March 21, 2011): MGALTKAEIAERLYEELGLNKREAKELVELFFEEIRQALEHNEQVKLSGFGNFDLRDKRQRPGRNPKTGEEIPITARRVVTFRPGQKLKARVEAYAGTKS. SEQ ID NO: 11: Full length wt P. aeruginosa PA 01 IhfB; Genbank accession number: AAF72950.1 (last accessed May 19, 2015): MTKSELIERIVTHQGQLSAKDVELAIKTMLEQMSQALATGDRIEIRGFGSFSLHYRAPRVGRNPKTGESVRLDGKFVPHFKPGKELRDRVNEPE. SEQ ID NO: 12: Haemophilus influenzae IhfA, A-3 fragment: FLEEIRLSLESGQDVKLSGF. SEQ ID NO: 13: Haemophilus influenzae IhfA, A5 fragment: RPGRNPKTGDVVPVSARRVV. SEQ ID NO: 14: Haemophilus influenzae HU, A5 fragment: RTGRNPQTGAEIQIAASKVP. SEQ ID NO: 15: Haemophilus influenzae IhfB, B2 fragment: TLSAKEIENMVKDILEFISQ. SEQ ID NO: 16: Haemophilus influenzae IhfB, B4 fragment: RGFGSFSLHHRQPRLGRNPK. SEQ ID NO: 17: Haemophilus influenzae IhfB, modified B4 (mB4) fragment: FSLHHRQPRLGRNPKTGDSV. SEQ ID NO: 18: Haemophilus influenzae IhfA, A-1 fragment: MATITKLDIIEYLSDKYHLS. SEQ ID NO: 19: Haemophilus influenzae IhfA, A2 fragment: KYHLSKQDTKNVVENFLEEI. SEQ ID NO: 20: Haemophilus influenzae IhfA, A4 fragment KLSGFGNFELRDKSSRPGRN. SEQ ID NO: 21: Haemophilus influenzae IhfA, A6 fragment: ARRVVTFKPGQKLRARVEKTK. SEQ ID NO: 22: Haemophilus influenzae IhfB, B1 fragment: MTKSELMEKLSAKQPTLSAK. SEQ ID NO: 23: Haemophilus influenzae IhfB, B3 fragment: EFISQSLENGDRVEVRGFGS. SEQ ID NO: 24: Haemophilus influenzae IhfB, B5 fragment: GRNPKTGDSVNLSAKSVPYF. SEQ ID NO: 25: Haemophilus influenzae IhfB, B6 fragment: SVPYFKAGKELKARVDVQA. SEQ ID NO: 26: Haemophilus influenzae IhfA, A tip fragment: NFELRDKSSRPGRNPKTGDVV. SEQ ID NO: 27: Haemophilus influenzae IhfB, B tip fragment: SLHHRQPRLGRNPKTGDSVNL. SEQ ID NO: 28: Haemophilus influenzae HU fragment: MNKTDLIDAIANAAELNKKQAK. SEQ ID NO: 29: Haemophilus influenzae HU fragment: KKQAKAALEATLDAITASLKEG. SEQ ID NO: 30: Haemophilus influenzae HU fragment: SLKEGEPVQLIGFGTFKVNERA. SEQ ID NO: 31: Haemophilus influenzae HU fragment: VNERAARTGRNPQTGAEIQIAA. SEQ ID NO: 32: Haemophilus influenzae HU fragment: IQIAASKVPAFVSGKALKDAIK. SEQ ID NO: 33: Haemophilus influenzae HU fragment, A3 fragment: KKQAKAALEATLDAITASLKEG. SEQ ID NO: 34: Human IgD constant region, Uniprot: P01880: APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFV VGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK. SEQ ID NO: 35: Human IgG1 constant region, Uniprot: P01857: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. SEQ ID NO: 36: Human IgG2 constant region, Uniprot: P01859: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. SEQ ID NO: 37: Human IgG3 constant region, Uniprot: P01860: ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK. SEQ ID NO: 38: Human IgM constant region, Uniprot: P01871: GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKV SVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFA IPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPARE QLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY. SEQ ID NO: 39: Human IgG4 constant region, Uniprot: P01861: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. SEQ ID NO: 40: Human IgA1 constant region, Uniprot: P01876: ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPER DLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY. SEQ ID NO: 41: Human IgA2 constant region, Uniprot: P01877: ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCY SVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY. SEQ ID NO: 42: Human Ig kappa constant region, Uniprot: P01834: TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. SEQ ID NO: 43: Non-limiting exemplary linker: GPSLKL. SEQ ID NO: 44: Non-limiting exemplary linker: GGG. SEQ ID NO: 45: Non-limiting exemplary linker: GPSL. SEQ ID NO: 46: Non-limiting exemplary linker: GPS. SEQ ID NO: 47: Non-limiting exemplary linker: PSLK. SEQ ID NO: 48: Non-limiting exemplary linker: GPSLK. SEQ ID NO: 49: Non-limiting exemplary linker: SLKL. SEQ ID NO: 50: Non-limiting exemplary heavy chain variable region nucleotide sequence, IhfA5 fragment: GAGGTGCAGCTGCAGGAGTCTGGACCTGGCCTGGTGACGCCCTCACAGAGCCTGTCCATGACTTGCACTGTCTCTGGGTTTTCATTAACCAGCTATAGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGAGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAG CACAAATTATAATTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGGACAGTCTGCAAACTGATGACACAGCCATATACTACTGTGCCAGAGAGGACTCCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA. SEQ ID NO: 51: Non-limiting exemplary heavy chain variable region amino acid sequence, IhfA5 fragment: EVQLQESGPGLVTPSQSLSMTCTVSGFSLTSYSVHWVRQPPGKSLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMDSLQTDDTAIYYCAREDSWGQGTSVTVSS. SEQ ID NO: 52: Non-limiting exemplary heavy chain variable region nucleotide sequence, IhfmB4 fragment:GAGGTGCAGCTGCAGGAGTCTGGGGCAGAGCTTGTGAGGTCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACTACTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAAAATGATGATACTGAATATGTCCCGAAGTTCCAGGGCAAGGCCAGTATGACTGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTACAGAGCTCGGAGCTTACTGGGGCCAGGGGACTCTGGTC. SEQ ID NO: 53: Non-limiting exemplary heavy chain variable region amino acid sequence, IhfmB4 fragment: EVQLQESGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQGLEWIGWIDPENDDTEYVPKFQGKASMTADTSSNTAYLQLSSLTSEDTAVYYCTELGAYWGQGTLV. SEQ ID NO: 54: Non-limiting exemplary light chain variable region nucleotide sequence, IhfA5 fragment: GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTAC CGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCCACGTTCGGAGGGGGGACCAAGTTGGAAATAAAA. SEQ ID NO: 55: Non-limiting exemplary light chain variable region amino acid sequence, IhfA5 fragment: DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPTFGGGTKLEIK. SEQ ID NO: 56: Non-limiting exemplary light chain variable region nucleotide sequence, IhfmB4 fragment: GATGTTGTGATGACCCAGATTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTAATGGAAAGACATATTTGAATTGGTTGTTTCAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTTGAGGCTGAGGATTTGGGAATTTATTATTGCTGGCAAAGTACACATTTTCCTCACACGTTCGGAGGGGGGACCAAGTTGGAAATCAAA. SEQ ID NO: 57: Non-limiting exemplary light chain variable region amino acid sequence, IhfmB4 fragment: DVVMTQIPLTLSVTIGQPASISCKSSQSLLDSNGKTYLNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQSTHFPHTFGGGTKLEIK. SEQ ID NO: 58: Non-limiting exemplary partial CDRH1 sequence, IhfA5 fragment: FSLTSYS. SEQ ID NO: 59: Non-limiting exemplary partial CDRH1 sequence, IhfA5 fragment: FSLTSYSV. SEQ ID NO: 60: Non-limiting exemplary partial CDRH1 sequence, IhfA5 fragment: FSLTSYSVH. SEQ ID NO: 61: Non-limiting exemplary partial CDRH1 sequence, IhfA5 fragment: GFSLTSYS. SEQ ID NO: 62: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: IWAGGST. SEQ ID NO: 63: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: VIWAGGST. SEQ ID NO: 64: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: GVIWAGGST. SEQ ID NO: 65: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: LGVIWAGGST. SEQ ID NO: 66: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: WLGVIWAGGST. SEQ ID NO: 67: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: IWAGGSTN. SEQ ID NO: 68: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: VIWAGGSTN. SEQ ID NO: 69: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: GVIWAGGSTN. SEQ ID NO: 70: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: LGVIWAGGSTN. SEQ ID NO: 71: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: WLGVIWAGGSTN. SEQ ID NO: 72: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: IWAGGSTNY. SEQ ID NO: 73: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: VIWAGGSTNY. SEQ ID NO: 74: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: GVIWAGGSTNY. SEQ ID NO: 75: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: LGVIWAGGSTNY. SEQ ID NO: 76: Non-limiting exemplary partial CDRH2 sequence, IhfA5 fragment: WLGVIWAGGSTNY. SEQ ID NO: 77: Non-limiting exemplary partial CDRH3 sequence, IhfA5 fragment: REDS. SEQ ID NO: 78: Non-limiting exemplary partial CDRH3 sequence, IhfA5 fragment: AREDS. SEQ ID NO: 79: Non-limiting exemplary partial CDRL1 sequence, IhfA5 fragment: QNVGTN. SEQ ID NO: 80: Non-limiting exemplary partial CDRL1 sequence, IhfA5 fragment: QNVGTNV. SEQ ID NO: 81: Non-limiting exemplary partial CDRL1 sequence, IhfA5 fragment: QNVGTNVA. SEQ ID NO: 82: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: SAS. SEQ ID NO: 83: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: YSAS. SEQ ID NO: 84: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: IYSAS SEQ ID NO: 85: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: LIYSAS. SEQ ID NO: 86: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: ALIYSAS. SEQ ID NO: 87: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: SASY. SEQ ID NO: 88: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: YSASY. SEQ ID NO: 89: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: IYSASY. SEQ ID NO: 90: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: LIYSASY. SEQ ID NO: 91: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: ALIYSASY. SEQ ID NO: 92: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: SASYR. SEQ ID NO: 93: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: YSASYR. SEQ ID NO: 94: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: IYSASYR. SEQ ID NO: 95: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: LIYSASYR. SEQ ID NO: 96: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: ALIYSASYR. SEQ ID NO: 97: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: SASYRY. SEQ ID NO: 98: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: YSASYRY. SEQ ID NO: 99: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: IYSASYRY. SEQ ID NO: 100: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: LIYSASYRY. SEQ ID NO: 101: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: ALIYSASYRY. SEQ ID NO: 102: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: SASYRYS. SEQ ID NO: 103: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: YSASYRYS. SEQ ID NO: 104: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: IYSASYRYS. SEQ ID NO: 105: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: LIYSASYRYS. SEQ ID NO: 106: Non-limiting exemplary partial CDRL2 sequence, IhfA5 fragment: ALIYSASYRYS. SEQ ID NO: 107: Non-limiting exemplary linker: GGSGGS. SEQ ID NO: 108: Non-limiting exemplary partial CDRL3 sequence, IhfA5 fragment: QQYNSYP. SEQ ID NO: 109: Non-limiting exemplary partial CDRL3 sequence, IhfA5 fragment: QQYNSYPT. SEQ ID NO: 110: Non-limiting exemplary partial CDRH1 sequence, IhfmB4 fragment: FNIKDYY. SEQ ID NO: 111: Non-limiting exemplary partial CDRH1 sequence, IhfmB4 fragment: FNIKDYYM. SEQ ID NO: 112: Non-limiting exemplary partial CDRH1 sequence, IhfmB4 fragment: FNIKDYYMH. SEQ ID NO: 113: Non-limiting exemplary partial CDRH1 sequence, IhfmB4 fragment: GFNIKDYY. SEQ ID NO: 114: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment: IDPENDDT. SEQ ID NO: 115: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment: WIDPENDDT. SEQ ID NO: 116: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment: GWIDPENDDT. SEQ ID NO: 117: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment: IGWIDPENDDT. SEQ ID NO: 118: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment: WIGWIDPENDDT. SEQ ID NO: 119: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment: IDPENDDTE. SEQ ID NO: 120: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment:WIDPENDDTE. SEQ ID NO: 121: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment: GWIDPENDDTE. SEQ ID NO: 122: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment: IGWIDPENDDTE. SEQ ID NO: 123: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment: WIGWIDPENDDTE. SEQ ID NO: 124: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment: IDPENDDTEY. SEQ ID NO: 125: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment:WIDPENDDTEY. SEQ ID NO: 126: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment: GWIDPENDDTEY. SEQ ID NO: 127: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment: IGWIDPENDDTEY. SEQ ID NO: 128: Non-limiting exemplary partial CDRH2 sequence, IhfmB4 fragment: WIGWIDPENDDTEY. SEQ ID NO: 129: Non-limiting exemplary partial CDRH3 sequence, IhfmB4 fragment: TELGAY. SEQ ID NO: 130: Non-limiting exemplary partial CDRL1 sequence, IhfmB4 fragment: QSLLDSNGKTY. SEQ ID NO: 131: Non-limiting exemplary partial CDRL1 sequence, IhfmB4 fragment: QSLLDSNGKTYL. SEQ ID NO: 132: Non-limiting exemplary partial CDRL1 sequence, IhfmB4 fragment: QSLLDSNGKTYLN. SEQ ID NO: 133: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: LVS. SEQ ID NO: 134: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: YLVS. SEQ ID NO: 135: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: IYLVS. SEQ ID NO: 136: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: LIYLVS. SEQ ID NO: 137: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: RLIYLVS. SEQ ID NO: 138: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment:LVSK. SEQ ID NO: 139: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: YLVSK. SEQ ID NO: 140: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: IYLVSK. SEQ ID NO: 141: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: LIYLVSK. SEQ ID NO: 142: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: RLIYLVSK. SEQ ID NO: 143: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment:LVSKL. SEQ ID NO: 144: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: YLVSKL. SEQ ID NO: 145: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: IYLVSKL. SEQ ID NO: 146: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: LIYLVSKL. SEQ ID NO: 147: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: RLIYLVSKL. SEQ ID NO: 148: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment:LVSKLD. SEQ ID NO: 149: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: YLVSKLD. SEQ ID NO: 150: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: IYLVSKLD. SEQ ID NO: 151: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: LIYLVSKLD. SEQ ID NO: 152: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: RLIYLVSKLD. SEQ ID NO: 153: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: LVSKLDS SEQ ID NO: 154: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: YLVSKLDS. SEQ ID NO: 155: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: IYLVSKLDS. SEQ ID NO: 156: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: LIYLVSKLDS. SEQ ID NO: 157: Non-limiting exemplary partial CDRL2 sequence, IhfmB4 fragment: RLIYLVSKLDS. SEQ ID NO: 158: Non-limiting exemplary partial CDRL3 sequence, IhfmB4 fragment: WQSTHFPH. SEQ ID NO: 159: Non-limiting exemplary partial CDRL3 sequence, IhfmB4 fragment: WQSTHFPHT.

[0007] Table Description Table 1 lists non-limiting exemplary antibody-producing hybridomas.

[0008] Table 2 is a non-limiting list of exemplary antibodies and their corresponding CDRs.

[0009] Table 3 is a non-limiting list of exemplary antibodies and their corresponding heavy and light chain variable regions.

[0010] Table 4 outlines the scoring method used in the otitis media model of Example 3 to generate a mucosal biomass score.

[0011] Table 5 summarizes the potency of IgG Fab fragments against intact rabbit polyclonal IgG and mouse monoclonal IgG. Summary of the Invention [Problem to be solved by the invention]

[0012] Within bacterial cells, DNABII proteins are DNA-binding proteins that, upon binding, naturally bend the DNA substrate. Similarly, DNA already in a bent conformation is an exemplary substrate, as it eliminates the energy required for bending.

[0013] The DNABII family is a member of a class of proteins called nuclear inclusion-associated proteins (NAPs), bacterial proteins that partially form bacterial nuclear inclusions within cells (Browning et al. (2010) Curr. Opin. Microbiol. 13:773-780). In addition, this family is ubiquitous, expressed by virtually all eubacteria. All family members characterized to date function as either homodimers or heterodimers of subunits. The family is divided into two classes: HU (histone-like proteins) and IHF (integration host factors). The major distinction between these family members is that HU binds to DNA in a sequence-independent manner, whereas IHF binds to a consensus sequence (WATCAANNNNTTR [where W is A or T, R is purine, and N is any base] (SEQ ID NO: 160)) that is conserved across the genus (Swinger et al. (2004) Curr. Opin. Struct. Biol., 14:28-35). All DNABII proteins bind to and bend DNA significantly; for example, E. coli IHF can bend DNA into a virtual U-shaped turn (Rice et al. (1996), Cell, 87:1295-1306). In addition, all family members are likely to adopt pre-bent or curved DNA structures, such as Holliday junctions, cruciform-like structures that are central to DNA recombination. Indeed, DNABII proteins function as accessory factors that facilitate all DNA functions within the cell, including gene expression, recombination, repair, and replication (Swinger et al. (2004), Curr. Opin. Struct. Biol., 14:28-35). page). [Means for solving the problem]

[0014] In one aspect of the present disclosure, applicants provide an antibody or antigen-binding fragment of the antibody that is specific to DNABII polypeptide. When the antibody or antigen-binding fragment is administered to a host, it reduces or eradicates existing or pre-formed biofilms. Thus, in one aspect, the present disclosure relates to one or more antibodies or antigen-binding fragments that specifically bind to DNABII polypeptides and their use in degrading, remediating or destroying biofilms in vitro or in vivo.

[0015] In another embodiment, the antibody or antigen-binding fragment is described as an isolated polypeptide comprising, alternatively consisting essentially of, or even consisting of, a Fab fragment of an antibody that specifically recognizes and specifically binds to a DNABII-binding polypeptide produced by any suitable organism, such as a Haemophilus influenzae IHF alpha, IHF beta, or HU polypeptide. Non-limiting examples of such are selected from the group of engineered conformational apex regions, A5 or mB4 peptide fragments, or their respective equivalents. In one embodiment, the fragment is selected from the group of engineered conformational apex regions, such as mIhfB4. NHTI In one embodiment, the fragment is derived from the IHF A5 antibody or an equivalent of that antibody.

[0016] In certain aspects, the present disclosure provides mIhfB4 NTHIThe present disclosure relates to an isolated polypeptide comprising, or alternatively consisting essentially of, an Fab fragment of an IHF fragment B4, mIhfB4, or A5 antibody, or an equivalent of each of these polypeptides. In another aspect, the present disclosure relates to an isolated polypeptide consisting essentially of an Fab fragment of an IHF fragment B4, mIhfB4, or A5 antibody, or an equivalent of each of these polypeptides. In some embodiments, the antibody fragment or antigen-binding fragment is derived from polyclonal serum. In other aspects, the antibody fragment or antigen-binding fragment is not derived from a polyclonal antibody. In some embodiments, the antibody fragment or antigen-binding fragment is derived from a monoclonal antibody. Table 1 lists non-limiting exemplary monoclonal antibodies from which the fragments and polypeptides containing the fragments of the present disclosure can be derived, as well as the hybridomas that produce them. For example, hybridoma cell lines producing monoclonal antibodies that specifically recognize and bind to Haemophilus influenzae IhfA fragment A5 (SEQ ID NO: 13) or IhfB fragment B4 (SEQ ID NO: 16) and IhfB fragment mB4 (SEQ ID NO: 17) were deposited with the American Type Culture Collection (ATCC) on July 30, 2015, in accordance with the provisions of the Budapest Treaty, under the accession numbers listed in Table 1.Further non-limiting exemplary antibodies include those that specifically recognize and specifically bind to Haemophilus influenzae IhfA fragment A3 (SEQ ID NO: 12) or IhfB fragment B2 (SEQ ID NO: 15) produced by hybridoma cell lines IhfA3 NTHI 9B10.F2.H3, IhfB2 NTHI 7A4.E4.G4, and IhfB2 NTHI 7A4.E4.G11 (deposited with the American Type Culture Collection (ATCC) under the provisions of the Budapest Treaty on August 1, 2016 under the accession numbers listed in Table 1); and antibodies that specifically recognize or specifically bind to a polypeptide comprising a polypeptide selected from the group of SEQ ID NO: 31, SEQ ID NO: 33, a DNA BII polypeptide comprising the amino acid sequence NPXT (wherein X is any amino acid or X is selected from the amino acids Q, R, K, S, or T), or their respective equivalents.

[0017] The antibodies and antibody-derived fragments of the present disclosure may be derived from mammalian polyclonal antibodies. In another embodiment, the mammalian polyclonal antibody is selected from the group of rabbit polyclonal antibodies, mouse polyclonal antibodies, sheep polyclonal antibodies, horse polyclonal antibodies, dog polyclonal antibodies, or human polyclonal antibodies. In another embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a monoclonal antibody, a humanized antibody, or a human antibody. In another embodiment, the mammalian monoclonal antibody is selected from the group of rabbit monoclonal antibodies, mouse monoclonal antibodies, sheep monoclonal antibodies, horse monoclonal antibodies, or dog monoclonal antibodies. In another embodiment, the antibody is a monoclonal antibody produced by a hybridoma listed in Table 1. The antibody may be of any isotype, for example, IgM or IgG.

[0018] In another embodiment, the isolated antibody fragment comprises a detectable label and / or a purification label.

[0019] In a further aspect, compositions comprising, consisting essentially of, or even consisting of a polypeptide described herein and a carrier, non-limiting examples of which include a preservative or stabilizer, are also provided. In one aspect, the composition is processed for ease of transportation, e.g., lyophilized.

[0020] In another aspect, an isolated polynucleotide encoding an antibody fragment or polypeptide described herein is also provided. In another aspect, the isolated polynucleotide further comprises a detectable label and / or a purification label. In another aspect, the detectable label is not a conjugated naturally occurring fluorescent polynucleotide. In embodiments, the isolated polynucleotide is operably linked to one or more regulatory elements for replication and / or expression, as appropriate, contained in an expression vector or host cell.

[0021] In another aspect, a composition is provided comprising, consisting essentially of, or even consisting of an isolated polynucleotide as described herein and a carrier, hi another aspect, the carrier comprises a preservative or stabilizer.

[0022] In another aspect, vectors and / or host cells containing the isolated polynucleotides, including, as appropriate, expression or replication vectors described herein, are also provided. In one aspect, the host cell is a prokaryotic or eukaryotic cell, non-limiting examples of which are disclosed herein.

[0023] In another aspect, there is provided a method for producing a polypeptide described herein, comprising, alternatively consisting essentially of, or even consisting of, culturing an isolated host cell disclosed herein under conditions that allow expression of a polynucleotide into a polypeptide. In another aspect, the method further comprises isolating the expressed polypeptide from the host cell or cell culture medium. The host cell can be a eukaryotic or prokaryotic cell.

[0024] In another aspect, a method is provided for preventing the formation of or disrupting a biofilm associated with an industrial process, the method comprising treating a surface susceptible to or containing a biofilm with an antibody fragment, antigen-binding fragment, or polypeptide described herein.

[0025] In another aspect, a method is provided for identifying a binding moiety, e.g., an antibody, antigen-binding fragment, small molecule, or other agent, that disrupts a biofilm, comprising contacting the candidate binding moiety with a biofilm component and at least one polypeptide described herein, and assaying the binding of the binding moiety to DNABII in the biofilm in comparison with the binding of the polypeptide described herein to DNABII in the biofilm, wherein a binding moiety that has substantially the same or stronger binding compared to the binding of the polypeptide is a binding moiety that disrupts a biofilm.

[0026] In another aspect, a method is provided for identifying an agent, e.g., an antibody, antigen-binding agent, small molecule, polynucleotide, or other therapeutic or diagnostic agent, that reverses drug resistance in multiple bacterial species, comprising evaluating the agent for activity in disrupting biofilms produced by the multiple species, wherein an agent that disrupts biofilms and has substantially equivalent activity to the antibody fragment, antigen-binding fragment, or polypeptide described herein (and optionally, an agent that binds to multiple of said proteins) is identified as an agent that reverses drug resistance. In one aspect, the agent is effective against biofilms derived from at least two bacterial species, including a Gram-positive species and a Gram-negative species; non-limiting examples of such bacterial species include S. aureus, P. aeruginosa, and B. cenocepacia.

[0027] In another aspect, a method for treating a condition characterized by biofilm formation in a subject and / or detecting biofilm formation in a subject is provided, comprising, alternatively consisting essentially of, or even consisting of administering to, or treating a subject in need thereof, an antibody fragment, antigen-binding fragment, or polypeptide described herein. The amount to be administered must be effective to disrupt or detect biofilm. In one aspect, if biofilm is to be detected, the method further comprises imaging and / or monitoring biofilm formation and / or disruption in the subject before, during, and after administration of a composition containing an antibody fragment, antigen-binding fragment, or polypeptide described herein. In one aspect, if biofilm is to be detected, the antibody fragment, antigen-binding fragment, or polypeptide is chemically modified. In one aspect, if biofilm is to be detected, the antibody fragment, antigen-binding fragment, or polypeptide is conjugated to a diagnostic or detectable label or agent. In one aspect, if a biofilm is being detected, the method further comprises observing complex formation of the antibody fragment, antigen-binding fragment, or polypeptide described herein with any biofilm present. In one aspect, the condition to be treated is selected from the group of chronic non-healing wounds, including venous ulcers and diabetic foot ulcers, ear infections, sinus infections, urinary tract infections, lung infections, cystic fibrosis, chronic obstructive pulmonary disease, catheter-related infections, infections associated with implanted prostheses, or periodontal disease.

[0028] In another aspect, a method of monitoring the development or progression of a condition characterized by biofilm formation in a subject is provided, comprising treating the subject or administering to the subject an antibody fragment, antigen-binding fragment, or polypeptide described herein. In one aspect, the method further comprises observing complex formation of the antibody fragment, antigen-binding fragment, or polypeptide described herein with any present biofilm. In one aspect, the polypeptide is chemically modified. In one aspect, the polypeptide is conjugated to a diagnostic or detectable label or agent.

[0029] In another aspect, a method for monitoring treatment of a condition characterized by biofilm formation in a subject is provided, comprising, alternatively consisting essentially of, or even consisting of administering to the subject an antibody fragment, antigen-binding fragment, or polypeptide described herein. In one aspect, the method further comprises imaging and / or monitoring biofilm formation and / or destruction in the subject. In one aspect, the polypeptide is chemically modified. In one aspect, the polypeptide is conjugated to a detectable label or diagnostic agent.

[0030] In another aspect, there is provided a method of preventing biofilm formation or disrupting biofilms in a subject in need thereof, comprising, alternatively consisting essentially of, or even consisting of administering to the subject an effective amount of an antibody fragment, antigen-binding fragment, or polypeptide described herein.

[0031] In another aspect, there is provided a non-physiological surface coated with an antibody fragment, antigen-binding fragment or polypeptide described herein, optionally wherein the surface is in an industrial environment, such as in a pipe.

[0032] In one aspect, provided herein is one or more methods for treating a subject infected with a biofilm-producing organism, for disrupting a biofilm in a subject, and / or for treating a subject suffering from a condition associated with the presence of a biofilm, etc., comprising, alternatively consisting essentially of, or even consisting of, administering to the subject an effective amount of an antibody fragment, antigen-binding fragment, or polypeptide described herein, wherein the antibody fragment, antigen-binding fragment, or polypeptide further induces the production of anti-inflammatory cytokines in the subject. In another aspect, the anti-inflammatory cytokines include one or more of IL-4, IL-13, or IL-10. In another aspect, the subject is a mammal. In another aspect, the mammal is a human. In yet another aspect, the subject is a pediatric patient.

[0033] In another aspect, the above method is combined with the administration of an effective amount of an antibiotic. In another aspect, a method is provided for increasing the efficacy of an antibiotic by administering an effective amount of an antibody fragment, antigen-binding fragment, or polypeptide described herein.

[0034] Kits are also provided that include the isolated antibody fragments, antigen-binding fragments, or polypeptides described herein and instructions for use. In one embodiment, the kits may be used for diagnostic or therapeutic use and thus include the isolated antibody fragments, antigen-binding fragments, or polypeptides described herein and instructions for use. In another embodiment, the kits may be used to monitor biofilm formation and / or destruction. In another embodiment, the kits further include an effective amount of a detectable or diagnostic label and / or an antibiotic. [Brief explanation of the drawings]

[0035] [Figure 1]Figure 1 depicts the generation of Fab fragments by digestion of monoclonal antibodies. Fab fragments were generated by digestion of mouse monoclonal antibodies with ficin in the presence of cysteine ​​(see Mariani et al., "A new enzymatic method to obtain high-yield F(ab)2 suitable for clinical use from mouse IgG1," Molec. Immunol. (1991) January-February, 28(1-2):69-77, PMID: 2011130). Fab fragments were purified, and the purity of each preparation was assessed using 10% Bis-Tris PAGE (BioRad) and Coomassie Fluor Orange.

[0036] [Figure 2] Figure 2 depicts the disruption of biofilms formed by Haemophilus influenzae (NTHI) 86-028NP by incubation with mAb mIhfB4NTHI Fab (also referred to as IHF mB4 or alternatively as mB4 in this disclosure). NTHI biofilms were incubated with medium (control), mAb IgG1 Fab (control), mAb IhfB2NTHI Fab, and mAb mIhfB4NTHI Fab.

[0037] [Figure 3]Figure 3 depicts the disruption of biofilms formed by Haemophilus influenzae (NTHI) 86-028NP, S. aureus 29213, P. aeruginosa 27853, and B. cenocepacia K56-2 by incubation with mAb mIhfB4NTHI Fab. Biofilms formed by Haemophilus influenzae (NTHI) 86-028NP, S. aureus 29213, P. aeruginosa 27853, and B. cenocepacia K56-2 were incubated with mouse IgG1, mouse IgG1 Fab, mAb IhfB2NTHI, mAb IhfB2NTHI Fab, mAb mIhfB4NTHI, and mAb mIhfB4NTHI Fab.

[0038] [Figure 4] Figure 4 depicts how the disruption of biofilms formed by Haemophilus influenzae (NTHI) 86-028NP in the middle ear of adult chinchillas was analyzed. On day 0 (day 0), 1,000 colony-forming units (CFU) of Haemophilus influenzae (NTHI) 86-028NP were inoculated into the middle ear of adult chinchillas. As shown, by day 4, 83-100% of the ears developed mucosal biofilms. On days 4 and 5, Fab fragments were administered (342 nM) by direct delivery into the middle ear. On day 6, biofilms were assessed using otoscopy, imaging of mucosal biofilms, collection of middle ear fluid, middle ear mucosa, and attached bacterial biofilms, enumeration of bacterial load, and assessment of cytokine levels in the middle ear fluid. The experiment included cohorts using mouse mAb IgG1 Fab fragment (4 chinchillas), mouse mAb IhfB2NTHI Fab fragment (4 chinchillas), and mouse mAb mIhfB4NTHI Fab fragment (4 chinchillas).

[0039] [Figure 5A]Figure 5(A) depicts the mean otoscopic scores on days 4, 5, and 6 after Haemophilus influenzae (NTHI) 86-028NP challenge for chinchillas administered either IgG1 Fab, IhfB2NTHI Fab, or murine mAb mIhfB4NTHI Fab. Using a WelchAllyn MacroView otoscope, the tympanic membrane of each animal was visualized and blindly ranked on a 0-4+ scale, with a score of ≥2.0 indicating the presence of fluid in the middle ear cavity, i.e., the presence of otitis media (Novotny et al., (2006) Vaccine 24:4804). Administration of mIhfB4NTHI Fab demonstrated lower mean otoscopic scores compared with the control IgG1 Fab and IhfB2NTHI Fab. P values ​​are indicated: *P ≤ 0.05 and **P ≤ 0.01. Figure 5(B) shows representative otoscopic images of chinchillas administered either IgG1 Fab, IhfB2NTHI Fab, or mouse mAb mIhfB4NTHI Fab on days 0, 4, and 6 after Haemophilus influenzae (NTHI) 86-028NP challenge. On day 4, middle ear fluid is seen behind the tympanic membrane. On day 6, middle ear fluid is seen in the chinchilla administered IgG1 Fab (control), and thickening of the tympanic membrane is sometimes seen in the chinchilla administered IhfB2NTHI Fab, indicating the presence of otitis media in these animals. On day 6, a non-inflamed tympanic membrane is seen in the chinchilla administered mAb mIhfB4NTHI Fab. [Figure 5B]Figure 5(A) depicts the mean otoscopic scores on days 4, 5, and 6 after Haemophilus influenzae (NTHI) 86-028NP challenge for chinchillas administered either IgG1 Fab, IhfB2NTHI Fab, or murine mAb mIhfB4NTHI Fab. Using a WelchAllyn MacroView otoscope, the tympanic membrane of each animal was visualized and blindly ranked on a 0-4+ scale, with a score of ≥2.0 indicating the presence of fluid in the middle ear cavity, i.e., the presence of otitis media (Novotny et al., (2006) Vaccine 24:4804). Administration of mIhfB4NTHI Fab demonstrated lower mean otoscopic scores compared with the control IgG1 Fab and IhfB2NTHI Fab. P values ​​are indicated: *P ≤ 0.05 and **P ≤ 0.01. Figure 5(B) shows representative otoscopic images of chinchillas administered either IgG1 Fab, IhfB2NTHI Fab, or mouse mAb mIhfB4NTHI Fab on days 0, 4, and 6 after Haemophilus influenzae (NTHI) 86-028NP challenge. On day 4, middle ear fluid is seen behind the tympanic membrane. On day 6, middle ear fluid is seen in the chinchilla administered IgG1 Fab (control), and thickening of the tympanic membrane is sometimes seen in the chinchilla administered IhfB2NTHI Fab, indicating the presence of otitis media in these animals. On day 6, a non-inflamed tympanic membrane is seen in the chinchilla administered mAb mIhfB4NTHI Fab.

[0040] [Figure 6A]Figure 6(A) shows quantification of colony-forming units (CFU) Haemophilus influenzae (NTHI) 86-028NP per milligram (mg) of mucosal biofilm in chinchillas administered either IgG1 Fab, IhfB2NTHI Fab, or murine mAb mIhfB4NTHI Fab after Haemophilus influenzae (NTHI) 86-028NP challenge and biofilm formation. Quantification was performed on day 6 after Haemophilus influenzae (NTHI) 86-028NP challenge (treatment was performed on days 4 and 5).

[0041] [Figure 6B] Figure 6(B) shows quantification of colony-forming units (CFU) Haemophilus influenzae (NTHI) 86-028NP per milligram (mg) of middle ear fluid in chinchillas administered either IgG1 Fab, IhfB2NTHI Fab, or mouse mAb mIhfB4NTHI Fab after Haemophilus influenzae (NTHI) 86-028NP challenge and biofilm formation.

[0042] [Figure 7A]Figure 7(A) shows quantification of the mass (grams, g) of middle ear mucosa and mucosal biofilm collected from chinchillas administered either IgG1 Fab, IhfB2NTHI Fab, or murine mAb mIhfB4NTHI Fab after Haemophilus influenzae (NTHI) 86-028NP challenge and biofilm formation. Quantification was performed on day 6 after Haemophilus influenzae (NTHI) 86-028NP challenge (treatment was administered on days 4 and 5). Figure 7(B) shows quantification of the volume (microliters, mL) of middle ear fluid (MEF) collected from chinchillas administered either IgG1 Fab, IhfB2NTHI Fab, or murine mAb mIhfB4NTHI Fab after Haemophilus influenzae (NTHI) 86-028NP challenge and biofilm formation. Quantification was performed on day 6 after Haemophilus influenzae (NTHI) 86-028NP challenge (treatment was administered on days 4 and 5). [Figure 7B] Figure 7(A) shows quantification of the mass (grams, g) of middle ear mucosa and mucosal biofilm collected from chinchillas administered either IgG1 Fab, IhfB2NTHI Fab, or murine mAb mIhfB4NTHI Fab after Haemophilus influenzae (NTHI) 86-028NP challenge and biofilm formation. Quantification was performed on day 6 after Haemophilus influenzae (NTHI) 86-028NP challenge (treatment was administered on days 4 and 5). Figure 7(B) shows quantification of the volume (microliters, mL) of middle ear fluid (MEF) collected from chinchillas administered either IgG1 Fab, IhfB2NTHI Fab, or murine mAb mIhfB4NTHI Fab after Haemophilus influenzae (NTHI) 86-028NP challenge and biofilm formation. Quantification was performed on day 6 after Haemophilus influenzae (NTHI) 86-028NP challenge (treatment was administered on days 4 and 5).

[0043] [Figure 8A] Figure 8(A) shows the mucosal biomass scores, as blindly examined by seven individuals, for chinchillas administered either IgG1 Fab, IhfB2 NTHI Fab, or mIhfB4 NTHI Fab after Haemophilus influenzae (NTHI) 86-028NP challenge and biofilm formation. A mucosal biofilm score scale was used to rank the remaining biofilm in the middle ear cavity. Using established criteria, a score of 0 to 4 was assigned to each image as follows: zero (0): no visible biofilm; 1: biofilm filled >0 to ≤25% of the middle ear cavity; 2: biofilm filled >25% to ≤50% of the middle ear cavity; 3: biofilm filled >50% to ≤75% of the middle ear cavity; and 4: biofilm filled >75% to ≤100% of the middle ear cavity. Significantly lower mucosal biomass scores were observed in chinchillas treated with mIhfB4NTHI Fab compared to chinchillas administered the IgG1 Fab control or IhfB2NTHI Fab control. P values ​​are indicated: *P≦0.05 and ***P≦0.001. Figure 8(B) depicts representative middle ear images obtained by otoscopic examination 1 day after receiving the final dose of IgG1 Fab, IhfB2NTHI Fab, or murine mAb mIhfB4NTHI Fab following Haemophilus influenzae (NTHI) 86-028NP challenge and biofilm formation. [Figure 8B]Figure 8(A) shows the mucosal biomass scores, as blindly examined by seven individuals, for chinchillas administered either IgG1 Fab, IhfB2 NTHI Fab, or mIhfB4 NTHI Fab after Haemophilus influenzae (NTHI) 86-028NP challenge and biofilm formation. A mucosal biofilm score scale was used to rank the remaining biofilm in the middle ear cavity. Using established criteria, a score of 0 to 4 was assigned to each image as follows: zero (0): no visible biofilm; 1: biofilm filled >0 to ≤25% of the middle ear cavity; 2: biofilm filled >25% to ≤50% of the middle ear cavity; 3: biofilm filled >50% to ≤75% of the middle ear cavity; and 4: biofilm filled >75% to ≤100% of the middle ear cavity. Significantly lower mucosal biomass scores were observed in chinchillas treated with mIhfB4NTHI Fab compared to chinchillas administered the IgG1 Fab control or IhfB2NTHI Fab control. P values ​​are indicated: *P≦0.05 and ***P≦0.001. Figure 8(B) depicts representative middle ear images obtained by otoscopic examination 1 day after receiving the final dose of IgG1 Fab, IhfB2NTHI Fab, or murine mAb mIhfB4NTHI Fab following Haemophilus influenzae (NTHI) 86-028NP challenge and biofilm formation.

[0044] [Figure 9] Figure 9 shows the relative amounts of a panel of pro- and anti-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-10, IL-17A, IL-12p70, and TNF) in clarified middle ear fluid from chinchillas administered either IgG1 Fab, IhfB2NTHI Fab, or mIhfB4NTHI Fab after Haemophilus influenzae (NTHI) 86-028NP challenge and biofilm formation. Quantitation was performed to provide picograms (pg) cytokines per milliliter (ml) MEF.

[0045] [Figure 10] Figure 10 shows histological analysis of the middle ear in chinchillas. The image on the left shows a middle ear from an animal administered the mouse IhfB2NTHI Fab control. The image on the right shows a middle ear from an animal administered the mouse mIhfB4NTHI Fab. Staining was performed using hematoxylin and eosin staining. Images were obtained using a 10X objective. The scale bar corresponds to 100 μm.

[0046] [Figure 11] Figure 11 shows how the disruption of biofilms formed by Haemophilus influenzae (NTHI) 86-028NP was analyzed in the middle ear of adult chinchillas using polyclonal rabbit anti-IhfB2NTHI, anti-mIhfB4NTHI, and Fab fragments generated from naive rabbit serum. The experiment included cohorts using naive rabbit serum IgG Fab fragments, rabbit anti-IhfB2NTHI Fab fragments, and rabbit anti-mIhfB4NTHI Fab fragments. On day zero (0), chinchillas were inoculated into the middle ear with Haemophilus influenzae (NTHI) 86-028NP. Subsequently, on days 4 and 5 (two-dose experiment) or days 4, 5, and 6 (three-dose experiment), Fab fragments were administered via direct delivery to the middle ear (342 nM). For those administered on days 4 and 5, three chinchillas per cohort were sacrificed on either day 6 or day 12. For those dosed on days 4, 5, and 6, three (3) chinchillas per cohort were sacrificed on day 13. After sacrifice, chinchillas were imaged, middle ear mucosa was collected, adherent biofilm was assessed, middle ear fluid was collected, bacterial quantification was performed, and middle ear fluid was evaluated using a cytokine multiplex assay. The purity of each Fab preparation is also shown (three separate preparations: naive, IhfB2, and mIhfB4), as confirmed by 10% Bis-Tris PAGE (BioRad) and SYPRO Orange protein gel staining (Invitrogen).

[0047] [Figure 12] Figure 12 shows quantification of colony-forming units (CFU) of Haemophilus influenzae (NTHI) 86-028NP per milligram (mg) of mucosal biofilm in chinchillas administered either rabbit IgG1 Fab, rabbit IhfB2 NTHI Fab, or rabbit mIhfB4 NTHI Fab after Haemophilus influenzae (NTHI) 86-028NP challenge and biofilm formation. Quantification was performed in three separate experiments: (1) two doses of each Fab were administered and sacrificed one day later; (2) two doses of each Fab were administered and sacrificed seven days later; and (3) three doses of each Fab were administered and sacrificed seven days later. The Fab doses were administered on days 4 and 5 (two doses) or days 4, 5, and 6 (three doses) after Haemophilus influenzae (NTHI) 86-028NP challenge. P values ​​are indicated: **P≦0.01.

[0048] [Figure 13] Figure 13 shows quantification of colony-forming units (CFU) of Haemophilus influenzae (NTHI) 86-028NP per milliliter (ml) of middle ear fluid in chinchillas administered either rabbit IgG1 Fab, IhfB2 NTHI Fab, or mIhfB4 NTHI Fab after Haemophilus influenzae (NTHI) 86-028NP challenge and biofilm formation. Quantification was performed in three separate experiments: (1) two doses of each Fab administered and sacrificed one day later; (2) two doses of each Fab administered and sacrificed seven days later; and (3) three doses of each Fab administered and sacrificed seven days later. The Fab doses were administered on days 4 and 5 (two doses) or days 4, 5, and 6 (three doses) after Haemophilus influenzae (NTHI) 86-028NP challenge. P values ​​are indicated: *P≦0.05 and **P≦0.01.

[0049] [Figure 14]Figure 14 shows the mean mucosal biofilm scores (seven blinded examiners) of chinchillas administered either rabbit IgG1 Fab, IhfB2 NTHI Fab, or mIhfB4 NTHI Fab after Haemophilus influenzae (NTHI) 86-028 NP challenge and biofilm formation. Quantification was performed in three separate experiments: (1) two doses of each Fab administered and sacrificed one day later; (2) two doses of each Fab administered and sacrificed seven days later; and (3) three doses of each Fab administered and sacrificed seven days later. The Fab doses were administered on days 4 and 5 (two doses) or days 4, 5, and 6 (three doses) after Haemophilus influenzae (NTHI) 86-028 NP challenge. A mucosal biofilm score scale was used to rank the biofilm remaining in the middle ear cavity. Using established criteria, a score from 0 to 4 was assigned to each image as follows: zero (0): no visible biofilm; 1: biofilm fills >0 to ≤25% of the middle ear cavity; 2: biofilm fills >25% to ≤50% of the middle ear cavity; 3: biofilm fills >50% to ≤75% of the middle ear cavity; and 4: biofilm fills >75% to 100% of the middle ear cavity. P values ​​are indicated: *P ≤ 0.05 and **P ≤ 0.01.

[0050] [Figure 15] Figure 15 shows the relative amounts of a panel of pro- and anti-inflammatory cytokines in clarified middle ear fluid from chinchillas administered either naive rabbit IgG Fab, rabbit anti-mIhfB2NTHI IgG Fab, or rabbit anti-mIhfB4NTHI IgG Fab after Haemophilus influenzae (NTHI) 86-028NP challenge and biofilm formation. Proinflammatory cytokines measured included IL-1β, IL-6, IL-8, IL-12p70, IL-17A, TNF, and IFNγ. Anti-inflammatory cytokines measured included IL-4, IL-10, and IL-13. DETAILED DESCRIPTION OF THE INVENTION

[0051] Detailed Description Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All nucleotide sequences provided herein are presented in a 5' to 3' orientation. Although any methods and materials similar or equivalent to those described herein can be used in practicing or testing the present disclosure, specific, non-limiting, exemplary methods, devices, and materials are described herein. All technical publications and patent disclosures cited herein are incorporated by reference in their entirety. Nothing herein shall be construed as an admission that the present disclosure is not entitled to antedate such disclosure by way of prior invention.

[0052] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, within the skill of the art. See, e.g., Green and Sambrook (eds.) (2012), Molecular Cloning: A Laboratory "PCR Manual," 4th edition; Ausubel et al. (eds.), (2015), "Current Protocols in Molecular Biology"; "Methods in Enzymology" series (Academic Press, Inc., NY); MacPherson et al. (2015), "PCR 1: A Practical Approach" (Oxford University Press, IRL Press); MacPherson et al. (1995), "PCR 2: A Practical Approach"; McPherson et al. (2006), "PCR: The Basics," (Garland Science); Harlow and Lane (eds.), (1999), "Antibodies, A Laboratory Manual"; Greenfield (ed.) (2014) "Antibodies, A Laboratory Manual"; Freshney (ed.) (2010) "Culture of Animal Cells: A Manual of Basic Technique", 6th ed.; Gait (ed.) (1984) "Oligonucleotide Synthesis"; U.S. Patent No. 4,683,195; Hames and Higgins (eds.) (1984) "Nucleic Acid Hybridization"; Anderson (1999) "Nucleic Acid Hybridization"; Herdewijn (eds.) (2005) "Oligonucleotide Synthesis: Methods and Applications"; Hames and Higgins (eds.) (1984) "Transcription and Translation"; Buzdin and Lukyanov (eds.) (2007) "Nucleic Acids Hybridization: Modern Applications"; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi (ed.) (2007) "In Vitro Transcription and Translation (2006), "Immobilization of Enzymes and Cells," Perbal (1988), "A Practical Guide to Molecular Cloning," 2nd ed.; Miller and Calos (1987), "Gene Transfer Vectors for Mammalian Cells" (Cold Spring Harbor Laboratory); Makrides (2003), "Gene Transfer and Expression in Mammalian Cells"; Mayer and Walker (1987), "Immunochemical Methods in Cell and Molecular Biology" (Academic Press, London); Lundblad and Macdonald (2010), "Handbook of Biochemistry and Molecular Biology," 4th ed.; and Herzenberg et al. (1996), "Weir's Handbook of Experimental Immunology," 5th ed.

[0053] All numerical designations, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximate and may vary (+) or (-) by 1.0 or 0.1 increments, as appropriate, or alternatively, by a variance of + / - 15%, or 10%, or 5%, or 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." It is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary, and that equivalents thereof are known in the art, although not always explicitly stated.

[0054] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "polypeptide" includes multiple polypeptides, including mixtures thereof.

[0055] As used herein, the term "comprising" shall mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, shall mean excluding any other elements of essential importance for the combination for the intended use. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from isolation and purification methods and pharmaceutically acceptable carriers, e.g., phosphate saline, preservatives (e.g., sodium benzoate, potassium sorbate, and methyl hydroxybenzoate), etc. "Consisting of" shall mean excluding more than trace elements of other components and substantial method steps for administering the compositions disclosed herein. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0056] The term "biofilm" refers to an organized community of microorganisms, sometimes attached to the surface of a structure, which may be organic or inorganic, along with polymers such as DNA secreted and / or released by the microorganisms. Biofilms are highly resistant to microbiotics and antimicrobial agents. Biofilms inhabit gum tissue, teeth, and restorations, causing dental caries and periodontal disease, also known as periodontal plaque disease. Biofilms also cause middle ear infections. Biofilms can also form on the surfaces of dental implants, stents, catheter lines, and contact lenses. Biofilms grow on pacemakers, heart valve replacements, artificial joints, and other surgical implants. The Centers for Disease Control estimate that more than 65% of nosocomial infections (hospital-acquired infections) are caused by biofilms. Biofilms cause vaginal infections and lead to life-threatening systemic infections in people with compromised immune systems. Biofilms also cause Aggregatibacter actinomycetemcomitans, Borrelia burgdorferi (e.g., B31), Bordetella pertussis (e.g., Tohama I), Burkholderia pseudomallei (e.g., 668), Burkholderia cenocepacia (e.g., HI2424), Escherichia coli (e.g., K12 MG1655), Enterococcus faecalis (e.g., V583), Haemophilus influenzae (e.g., Rd KW20), Helicobacter pylori (e.g., 26695), Klebsiella pneumoniae, Moraxella catarrhalis (e.g., RH4), Mycobacterium smegmatis (e.g., MC2), Mycobacterium tuberculosis (e.g., CDC1551), Neisseria gonorrhoeae (e.g., FA1090), Neisseria meningitidis (e.g., MC58), Pseudomonas aeruginosa, Porphyromonas gingivalis (e.g., W83), Prevotella intermedia (e.g., 17), Prevotella melaninogenica (e.g., ATCC (registered trademark) 25845), Staphylococcus aureus (e.g., MW2), Staphylococcus epidermidis (e.g., RP62A), Streptococcus agalactiae (e.g., 2603V / R), Streptococcus bovis, Streptococcus gallolyticus (e.g., UCN34), Streptococcus gordonii (e.g., NCTC 7868 (Challis)), Streptococcus mutans (e.g., UA159), Streptococcus pneumoniae (e.g., R6), Streptococcus pyogenes (e.g., MGAS10270), Streptococcus sobrinus (e.g., 6715), SalmonellaBiofilms are also implicated in numerous diseases, including, but not limited to, diseases caused by Enterococcus enterica (e.g., Typhi, CT18), Treponema denticola (e.g., ATCC® 35405), Treponema palladum (e.g., Nichols), and Vibrio cholera (e.g., El Tor, N16961). Additional organisms known to be associated with and / or form biofilms include, but are not limited to, Campylobacter species, Candida species, Legionella pneumophila, and Listeria monocytogenes. For example, cystic fibrosis patients have Pseudomonas infections, which often result in antibiotic-resistant biofilms. Other diseases associated with biofilms include lung infections, otitis media, and post-tympanostomy ear discharge in cystic fibrosis patients. These include, but are not limited to, bacterial infections caused by the organisms listed above (e.g., Listeria monocytogenes, urinary tract infection ... Foodborne pathogens, such as, but not limited to, some of the Salmonella species (S. monocytogenes, Escherichia coli, Salmonella enterica), can also form biofilms on the foods they contaminate. Biofilms that cause disease in animals (e.g., Escherichia coli, Salmonella species, and Shigella species) can also cause food contamination and / or disease in downstream human hosts. Furthermore, biofilms need not be a homogenous population of microorganisms but may incorporate other pathogens, and even host cells. In addition to being associated with disease (both hospital-based and other) and food contamination, biofilms are often a source of industrial contamination, particularly associated with process water and surfaces that come into contact with it. Problems involving biofilm-forming organisms as industrial contaminants include, but are not limited to, biocorrosion, biofouling, and equipment damage as a result of biofilm formation. Non-limiting exemplary biofilm-associated organisms in industrial settings include those disclosed in Ferrera et al. (2015), Biofouling, 31(2):173-180, and Desulfovibrio species. Further details regarding biofilms can be found, for example, in Donlan (2002), Emerging Infectious Diseases, 8(9):881-890.

[0057] The terms "preventing the formation of" or "disrupting a biofilm" refer to the prevention, reduction or disruption of the formation of, or the prevention, reduction or disruption of the structure of, the DNA / protein matrix that is a component of a microbial biofilm.

[0058] As used herein, the term "nuclear inclusion-associated protein" or "NAP" refers to a class of proteins that influence the dynamic spatial organization of nucleic acids within the nuclear inclusions of prokaryotic cells. These proteins organize the genome through DNA bending, binding, and aggregation. Certain NAPs are DNA-binding proteins that may be associated with biofilms, including DNABII proteins, DPS (GenBank Accession No. CAA49169), H-NS (GenBank Accession No. CAA47740), Hfq (GenBank Accession No. ACE63256), CbpA (GenBank Accession No. BAA03950), and CbpB (GenBank Accession No. NP_418813). Among NAPs, DNABII proteins are distinct and generally share strong sequence identity with an alpha-helix dimerization domain, and may contain antiparallel beta ribbons, which contain a tip that binds to, inserts into, and bends the minor groove of DNA. [ka] [wherein X can be any amino acid or selected from the amino acids Q, R, K, S, or T]. A functional protomer is a dimer of identical or homologous subunits.

[0059] "DNABII polypeptide or DNABII protein" refers to a DNA-binding protein or polypeptide that is composed of a DNA-binding domain and therefore has a specific or general affinity for microbial DNA. In one embodiment, the DNABII polypeptide or DNABII protein binds to DNA in the minor groove. Non-limiting examples of DNABII proteins are integration host factor (IHF) protein and histone-like protein (HU).

[0060] "Integration host factor" or "IHF" proteins are bacterial proteins used by bacteriophages to integrate their DNA into host bacteria. They also bind to extracellular microbial DNA. The genes encoding the IHF protein subunits in E. coli are the himA gene (Genbank accession number: POA6X7.1) and the himD gene (POA6Y1.1). Homologs to these genes are found in other organisms, and peptides corresponding to these genes from other organisms can be found in Table 10 of U.S. Pat. No. 8,999,291, incorporated herein by reference.

[0061] "HMGB1" is the high mobility group box (HMGB) 1 protein, which is reported to bind to and distort the minor groove of DNA, and is an example of an agent. Recombinant or isolated proteins and polypeptides are commercially available from Atgenglobal, ProSpecBio, Protein1, and Abnova.

[0062] "HU" or "histone-like protein" refers to a class of heterodimeric proteins commonly associated with E. coli. HU proteins are known to bind to DNA junctions. Related proteins have been isolated from other microorganisms. The complete amino acid sequence of E. coli HU was reported by Laine et al. (1980) Eur. J. Biochem. 103(3):447-481. The genes encoding the HU protein subunits in E. coli are hupA and hupB, which correspond to SEQ ID NOs:8 and 9, respectively. The Haemophilus influenzae homologs from nontypeable Haemophilus influenzae (NTHI) correspond to SEQ ID NO:3. Homologs of these genes are found in other organisms, and peptides corresponding to these genes from other organisms can be found in Table 10 of U.S. Pat. No. 8,999,291, incorporated herein by reference.

[0063] The term "surface antigen" or "surface protein" refers to a protein or peptide on the surface of a cell, such as a bacterial cell, that is at least partially exposed to the extracellular environment. Examples of surface antigens include outer membrane proteins such as OMP P5 (Genbank accession number: YP_004139079.1), OMP P2 (Genbank accession number: ZZX87199.1), OMP P26 (Genbank accession number: YP_665091.1), rsPilA or recombinant soluble PilA (Genbank accession number: EFU96734.1), and type IV Pilin (Genbank accession number: Yp_003864351.1).

[0064] The term "Haemophilus influenzae" refers to a pathogenic bacterium that can cause many different infections, such as ear infections, eye infections, and sinusitis. Many different strains of Haemophilus influenzae have been isolated that contain the IhfA gene or protein. Some non-limiting examples of different strains of Haemophilus influenzae include Rd KW20, 86-028NP, R2866, PittGG, PittEE, R2846, and 2019.

[0065] "Microbial DNA" means single-stranded or double-stranded DNA derived from a biofilm-producing microorganism.

[0066] A "conformational apical domain" of a polypeptide refers to a polypeptide that comprises a primary amino acid sequence whose structure has an antiparallel beta ribbon with a tight turn, typically mediated by a proline residue.

[0067] "Inhibiting or preventing biofilms" means therapeutically reducing or preventing the formation of biofilms.

[0068] "Treating an infection" refers to the reduction of the number of microorganisms, such as bacteria, associated with the formation of a biofilm. Methods for determining whether the number of microorganisms has been reduced are known in the art, including in vivo and ex vivo assays, and the alleviation of clinical symptoms of the infection. Because bacteria are protected by biofilms, they become resistant to the use of antibacterial agents. By destroying biofilms, bacterial resistance to antibacterial agents and other agents can be reduced or inhibited, and bacterial infections can be treated.

[0069] "Bent polynucleotide" refers to a double-stranded polynucleotide that contains a small loop on one strand that does not pair with the other strand. In some embodiments, the loop is from 1 to about 20 bases in length, or from 2 to about 15 bases in length, or from about 3 to about 12 bases in length, or from about 4 to about 10 bases in length, or about 4, 5, or 6, or 7, or 8, or 9, or 10 bases in length.

[0070] "Polypeptides that compete with DNABII proteins for DNA binding" refers to proteins or peptides that compete with IHF or HU for binding to bent or distorted DNA structures but do not form biofilms with DNA. Examples include, but are not limited to, fragments of IHF containing one or more DNA-binding domains of IHF, or biological equivalents thereof.

[0071] As used herein, the term "specifically recognize or specifically bind to" intends that binding of an agent, e.g., a Fab (fragment antigen binding) or monoclonal antibody, to its intended target or binding partner is more likely than non-binding.

[0072] A "subject" of diagnosis or treatment is a cell or an animal, such as a mammal or a human. Non-human animals are animals or animal models that are subject to diagnosis or treatment and are susceptible to infection, such as monkeys, rats, mice, murines such as chinchillas, canines such as dogs, leporids such as rabbits, farm animals, sport animals, and pets.

[0073] The terms "protein," "peptide," and "polypeptide" are used interchangeably and, in their broadest sense, refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as ester bonds, ether bonds, etc. A protein or peptide must contain at least two amino acids, there is no limit on the maximum number of amino acids, and may include a protein sequence or a peptide sequence. As used herein, the term "amino acid" refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs, and peptidomimetics.

[0074] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST tags, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment disclosed herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms that are known or predicted to be in double-stranded form.

[0075] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, if the polynucleotide is RNA, uracil (U) is substituted for thymine. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database in a computer having a central processing unit and used for bioinformatics applications, such as functional genomics and homology searching.

[0076] The terms "isolated" or "recombinant," when used herein with reference to nucleic acids, such as DNA or RNA, refer to molecules separated from other DNA or RNA, respectively, present in the macromolecule and the natural source of the polypeptide. The term "isolated or recombinant nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in nature. The term "isolated" is also used herein to refer to polynucleotides, polypeptides, and proteins that have been isolated from other cellular proteins and is intended to encompass both purified and recombinant polypeptides. In other embodiments, the term "isolated or recombinant" refers to a cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof, separated from components, cells, and other items with which it is normally associated in nature. For example, an isolated cell is one that is separated from tissues or cells of a different phenotype or genotype. An isolated polynucleotide is separated from its native or natural environment, e.g., the 3' and 5' contiguous nucleotides with which it is normally associated on the chromosome. As will be apparent to one of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof does not require "isolation" to be distinguished from its naturally occurring counterpart.

[0077] Without explicit recitation or unless otherwise intended, when the present disclosure relates to a polypeptide, protein, polynucleotide, or antibody, it should be assumed that its equivalent or biological equivalent is intended within the scope of the present disclosure. As used herein, "biological equivalent thereof" is intended to be synonymous with the term "equivalent thereof" when referring to a reference protein, antibody, fragment, polypeptide, or nucleic acid, and means one that has minimal homology while still maintaining the desired structure or functionality. Unless specifically recited herein, any polynucleotide, polypeptide, or protein referred to herein is also considered to encompass its equivalent. In one aspect, an equivalent polynucleotide is a polynucleotide that hybridizes under stringent conditions with the polynucleotide or complement of the polynucleotide described herein for use in the described method. In another embodiment, an equivalent antibody or antigen-binding polypeptide refers to an antibody or antigen-binding polypeptide that binds to a reference antibody or reference antigen-binding fragment or Fab (antigen-binding fragment) with at least 70%, alternatively at least 75%, alternatively at least 80%, alternatively at least 85%, alternatively at least 90%, alternatively at least 95% affinity or greater. In another embodiment, the equivalent competes with the antibody or antigen-binding fragment for binding to its antigen in a competitive ELISA assay. In another embodiment, equivalent refers to a percentage of homology or identity of at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95%, alternatively 98%, and exhibits substantially the same biological activity as the reference protein, polypeptide, or nucleic acid. Examples of biologically equivalent polypeptides are provided in Table 9 of U.S. Pat. No. 8,999,291, incorporated herein by reference, which identifies conservative amino acid substitutions for the disclosed amino acid sequences.

[0078] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in the two sequences being compared. Alignment and percentages of homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Appendix 30, section 7.7.18, table 7.7.1. In certain embodiments, default parameters are used for alignment. A non-limiting exemplary alignment program is BLAST using default parameters. Specifically, exemplary programs include BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identities and percentage identities were determined by importing them into clustalW (available at the web address: genome.align.jp (last accessed March 7, 2011)).

[0079] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions within each sequence, which can be aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions the sequences share. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[0080] "Homology" or "identity" or "similarity" may also refer to two nucleic acid molecules that hybridize under stringent conditions.

[0081] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized by hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogsteen binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a larger process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0082] Examples of stringent hybridization conditions include an incubation temperature of about 25°C to about 37°C, a hybridization buffer concentration of about 6xSSC to about 10xSSC, a formamide concentration of about 0% to about 25%, and a wash solution of about 4xSSC to about 8xSSC. Examples of moderate hybridization conditions include an incubation temperature of about 40°C to about 50°C, a buffer concentration of about 9xSSC to about 2xSSC, a formamide concentration of about 30% to about 50%, and a wash solution of about 5xSSC to about 2xSSC. Examples of high stringency conditions include an incubation temperature of about 55°C to about 68°C, a buffer concentration of about 1xSSC to about 0.1xSSC, a formamide concentration of about 55% to about 75%, and a wash solution of about 1xSSC, 0.1xSSC, or deionized water. Generally, hybridization incubation times range from 5 minutes to 24 hours, with one, two, or more wash steps, with wash incubation times of approximately 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalent SSCs using other buffer systems can be used.

[0083] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0084] The term "encoding," when applied to a polynucleotide, refers to a polynucleotide that, in its native state or when manipulated by methods well known to those of skill in the art, is considered to "encode" a polypeptide that can be transcribed and / or translated to produce mRNA for the polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

[0085] As used herein, the terms "treating," "treatment," and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be preventative, in that a disorder or its signs or symptoms are completely or partially prevented, and / or may be therapeutic, in that a disorder and / or adverse effects resulting from the disorder are partially or completely cured. Methods for determining whether treatment has occurred are known in the art and are outlined herein.

[0086] By prevent is meant preventing a disorder or effect in vitro or in vivo in a system or subject predisposed to the disorder or effect, such as preventing biofilm formation in a system infected with a microorganism known to produce biofilms.

[0087] A "composition" is intended to mean a combination of an active agent and another compound or composition, either inert (e.g., a detectable agent or label) or active, such as an adjuvant.

[0088] A "pharmaceutical composition" is intended to include a combination of an active agent with a carrier, inert or active, forming a composition suitable for use in diagnosis or treatment in vitro, in vivo or ex vivo.

[0089] "Pharmaceutically acceptable carrier" refers to any diluent, excipient, or carrier that can be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, salts or electrolytes such as sodium chloride and zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, microspheres, microparticles, or nanoparticles (including, for example, biodegradable polymers such as Poly(lactic acid-co-glycolic acid)), and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. Suitable pharmaceutical carriers can be selected with respect to the intended form of administration, ie, oral tablet, capsule, elixir, syrup, etc., and consistent with conventional pharmaceutical practice.

[0090] As disclosed herein, a "biologically active agent" or active agent refers to one or more of an isolated or recombinant polypeptide, an isolated or recombinant polynucleotide, a vector, an isolated host cell, or an antibody, as well as compositions containing one or more of them.

[0091] "Administration" or "delivery" of a therapeutic or other agent can be carried out in a single dose, continuously, or intermittently throughout the course of treatment. Methods for determining the most effective means and dosages are known to those skilled in the art and vary depending on the composition used in the therapy, the purpose of the therapy, the target cells being treated, and the subject being treated. Single or multiple administrations can be carried out, with the dosage level and pattern selected by the treating physician. Appropriate dosage formulations and methods for administering agents are known in the art. The route of administration can be determined, and methods for determining the most effective route of administration are known to those skilled in the art and vary depending on the composition used in the therapy, the purpose of the therapy, the health condition or stage of disease of the subject being treated, and the target cells or tissues. Non-limiting examples of routes of administration include oral administration, nasal administration, inhalation, injection, and topical application. Administration can be for use in industrial and therapeutic applications.

[0092] The agents of the present disclosure (antibodies or fragments thereof, polypeptides or polynucleotides, or cells) can be administered for treatment by any suitable route. It should also be understood that the optimal route will vary depending on the condition and age of the recipient and the disease being treated. The agents may also be used in industrial settings and for the treatment of animals. When used in industrial settings, biofilms are contacted with the agent, for example, a Fab (fragment antigen binding) or antibody.

[0093] The term "effective amount" refers to an amount sufficient to achieve a desired effect. In the context of therapeutic or prophylactic use, the effective amount depends on the type and severity of the condition in question and individual subject characteristics, such as general health, age, sex, weight, species, and tolerance to the pharmaceutical composition. In the context of the present disclosure, in some embodiments, an effective amount is an amount sufficient to cause a reduction in biofilm mass or biofilm degradation. In other aspects, the amount is effective to treat a biofilm-associated bacterial infection in a subject. In other embodiments, in the context of a Fab (fragment antigen binding) or antibody, an effective amount is an amount sufficient to degrade, reduce, or destroy a biofilm. In other embodiments, an effective amount of an agent or immunogenic composition is an amount sufficient to cause antibody production against an antigen. In some embodiments, an effective amount is the amount necessary to confer passive immunity to a subject in need thereof. With respect to compositions, in some embodiments, the effective amount depends, in addition to the factors described above, on the intended use and the health / responsiveness of the subject's immune system. Those skilled in the art will be able to determine the appropriate amount depending on these and other factors.

[0094] In the case of in vitro applications, in some embodiments, the effective amount depends on the size and nature of the application in question. The effective amount also depends on the nature and sensitivity of the target in vitro and the method used. Those skilled in the art can determine the effective amount based on these and other considerations. Depending on the embodiment, the effective amount may include one or more administrations of the composition.

[0095] The term "potency," as it relates to the efficacy of drugs such as antibiotics, refers to a measure of drug activity expressed in terms of the amount required to produce an effect of a given strength. Highly potent drugs elicit a given response at low concentrations, while less potent drugs elicit the same response only at higher concentrations. Potency depends on both affinity and efficacy.

[0096] The term "conjugated moiety" refers to a moiety that can be added to an isolated chimeric polypeptide by forming a covalent bond with a residue of the chimeric polypeptide. The moiety can be directly attached to a residue of the chimeric polypeptide or can form a covalent bond with a linker, which in turn forms a covalent bond between the linker and a residue of the chimeric polypeptide. A "peptide conjugate" refers to the covalent or noncovalent association of one or more polypeptides with another chemical or biological compound. In a non-limiting example, the "conjugation" of a polypeptide with a chemical compound improves the stability or effectiveness of the polypeptide for its intended purpose. In one embodiment, a polypeptide of the present disclosure is conjugated to a carrier, where the carrier is a liposome, a micelle, or a pharmaceutically acceptable polymer.

[0097] "Liposomes" are microscopic vesicles composed of concentric lipid bilayers. Liposomes are an example of a carrier, e.g., a pharmaceutically acceptable carrier. Structurally, liposomes range in size and shape from long tubes with dimensions ranging from a few hundred angstroms to a fraction of a millimeter to spheres. Vesicle-forming lipids are selected to achieve a specific degree of fluidity or rigidity in the final complex, resulting in the lipid composition of the outer layer. They can be neutral (cholesterol) or bipolar, and include phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM), as well as other types of bipolar lipids, including, but not limited to, dioleoylphosphatidylethanolamine (DOPE), with hydrocarbon chain lengths ranging from 14 to 22, saturated or containing one or more C=C double bonds. Examples of lipids that can be used alone or in combination with other lipid components to create stable liposomes include phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, distearoylphosphatidylethan-olamine (DSPE), dioleoyl These include dipalmitoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloteoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimido-triethyl)cyclohexane-1-carboxylate (DOPE-mal).Additional non-phosphorus-containing lipids that can be incorporated into the liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine lauryl sulfate, alkyl-aryl sulfates, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the cationic lipids described above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol). Negatively charged lipids include phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioteoylphosphatidylglycerol (DOPG), and dicetylphosphate, which can form vesicles. Generally, liposomes can be divided into three categories based on their overall size and lamellar structure. The three classifications developed by the New York Academy Sciences Meeting, "Liposomes and Their Use in Biology and Medicine," December 1977, are multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), and large unilamellar vesicles (LUVs). Bioactive agents can be encapsulated in such vesicles for administration according to the methods described herein.

[0098] "Micelles" are aggregates of surfactant molecules dispersed in a liquid colloid. Typical micelles in aqueous solution have hydrophilic "head" regions in contact with the surrounding solvent, forming aggregates with sequestered hydrophobic tail regions in the center of the micelle. This type of micelle is known as a normal-phase micelle (oil-in-water micelle). Reverse micelles have a central head group with protruding tails (water-in-oil micelle). Micelles can be used to attach polynucleotides, polypeptides, antibodies, or compositions described herein to facilitate efficient delivery to target cells or tissues.

[0099] The phrase "pharmaceutically acceptable polymer" refers to a group of compounds that can be conjugated to one or more polypeptides or antibodies described herein. Conjugating a polymer to a polypeptide or antibody may extend the half-life of the polypeptide in vivo and in vitro. Non-limiting examples include polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, cellulose derivatives, polyacrylates, polymethacrylates, sugars, polyols, and mixtures thereof. Biologically active agents can be conjugated to pharmaceutically acceptable polymers for administration according to the methods described herein.

[0100] " Gene delivery vehicle " is defined as any molecule that can carry inserted polynucleotide into host cell.Examples of gene delivery vehicle include biocompatible polymers, including liposomes, micelles, natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial virus envelopes; metal particles; and bacteria or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors, and other recombinant vehicles commonly used in the art, which are described for expression in various eukaryotic and prokaryotic hosts, and can be used for gene therapy and for the expression of simple proteins.

[0101] The polynucleotides disclosed herein can be delivered to cells or tissues using a gene delivery vehicle. As used herein, "gene delivery," "gene transfer," "transduction," and the like are terms that refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for the introduction. Such methods include various well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes), as well as techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques used to introduce polynucleotides). The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance generally requires that the introduced polynucleotide contain a replication origin compatible with the host cell or be integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. As known in the art and described herein, a number of vectors are known to be capable of mediating the transfer of genes into mammalian cells.

[0102] As used herein, the term "eDNA" refers to extracellular DNA found as a component to pathogenic biofilms.

[0103] A "plasmid" is an extrachromosomal DNA molecule that is separate from chromosomal DNA and can replicate independently of it. Plasmids are often circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and generally confer a selective advantage under given environmental conditions. Plasmids may carry genes that confer resistance to naturally occurring antibiotics in a competitive environmental niche, or the proteins produced may function as toxins under similar circumstances.

[0104] "Plasmids" used in genetic engineering are called "plasmid vectors." Many plasmids for such use are commercially available. The gene to be replicated is inserted into a copy of the plasmid, which contains a multiple cloning site (MCS, or polylinker), a short region containing several commonly used restriction sites that allow genes that make cells resistant to a particular antibiotic and DNA fragments to be easily inserted into this location. Another major use of plasmids is to produce large amounts of proteins. In this case, researchers grow bacteria containing a plasmid with the gene of interest. Just as bacteria produce proteins to confer antibiotic resistance, they can also be induced to produce large amounts of proteins from the inserted gene. This is an inexpensive and easy way to mass-produce a gene, or in turn, the protein it encodes.

[0105] "Yeast artificial chromosome" or "YAC" refers to a vector used to clone large DNA fragments (greater than 100 kb, up to 3000 kb). It is an artificially constructed chromosome that contains telomere, centromere, and origin of replication sequences necessary for replication and storage in yeast cells. It is constructed using an initial circular plasmid, which is linearized using restriction enzymes. The target sequence or gene can then be added to the linear molecule using DNA ligase and overhanging ends. Yeast expression vectors, such as YACs, YIps (yeast integrating plasmids), and YEps (yeast episomal plasmids), are very useful because yeast is itself a eukaryotic cell, allowing for post-translationally modified eukaryotic protein products to be obtained. However, YACs are more unstable than BACs and have been found to produce chimeric effects.

[0106] A "viral vector" is an in vivo vector containing a polynucleotide to be delivered to a host cell. Viral vectors are defined as viruses or viral particles produced by recombinant means, either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and alphavirus vectors. Infectious tobacco mosaic virus (TMV)-based vectors can be used to produce proteins and have been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15):6099-6104). Alphavirus vectors, such as Semulki Forest virus-based vectors and Sindbis virus-based vectors, have been developed for use in gene therapy and immunotherapy. See Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. In embodiments where gene transfer is mediated by a retroviral vector, the vector construct refers to a polynucleotide comprising the retroviral genome or a portion thereof and a therapeutic gene. Further details about current vector methods for use in gene transfer can be found, for example, in Kotterman et al. (2015), "Viral Vectors for Gene Therapy: Translational and Clinical Outlook Annual Review of Biomedical Engineering," p. 17.

[0107] As used herein, "retroviral-mediated gene transfer" or "retroviral transduction" have the same meaning and refer to the process by which a virus enters a cell and integrates its genome into the host cell genome, thereby stably transferring a gene or nucleic acid sequence into the host cell. The virus can enter the host cell through its normal infection mechanism or can be modified to bind to a different host cell surface receptor or ligand to enter the cell. As used herein, a retroviral vector refers to a viral particle that can introduce exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.

[0108] Retroviruses carry their genetic information in the form of RNA; however, when the virus infects a cell, the RNA is reverse transcribed into a DNA form and integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0109] In embodiments in which gene transfer is mediated by a DNA viral vector, such as adenovirus (Ad) or adeno-associated virus (AAV), the vector construct refers to the polynucleotide comprising the viral genome or a portion thereof and the transgene. Adenoviruses (Ad) are a relatively well-characterized, historically homogenous group of viruses, including over 50 serotypes. See, e.g., PCT International Patent Application Publication No. WO 95 / 27071. Ad does not require integration into the host cell genome. Recombinant Ad-derived vectors have also been constructed, particularly those that reduce the potential for recombination and generation of wild-type virus. See, PCT International Patent Application Publication Nos. WO 95 / 00655 and WO 95 / 11984. Wild-type AAV has high infectivity and specificity for integration into the host cell genome. Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA Vol. 81 :6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:398 See pages 8-3996.

[0110] Vectors containing both a promoter and a cloning site into which a polynucleotide can be operably linked are well known in the art. Such vectors can transcribe RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to eliminate additional, potentially inappropriate, alternative translation initiation codons or other sequences that may interfere with or reduce expression at either the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' of the initiation codon to enhance expression.

[0111] Gene delivery vehicles also include DNA / liposome complexes, micelles, and targeted viral protein-DNA complexes. Liposomes containing targeting antibodies or fragments thereof can be used in the methods disclosed herein. In addition to delivering polynucleotides to cells or cell populations by non-limiting techniques of protein transfection, the proteins described herein can be directly introduced into cells or cell populations, or culture conditions that enhance the expression and / or activity of the proteins disclosed herein are other non-limiting techniques.

[0112] As used herein, the terms "antibody," "antibody," and "immunoglobulin" include whole antibodies and any antigen-binding fragments or single chains thereof. Thus, the term "antibody" encompasses any protein or peptide containing molecule comprising at least a portion of an immunoglobulin molecule. The terms "antibody," "antibody," and "immunoglobulin" also encompass immunoglobulins of any isotype, fragments of antibodies that retain specific binding to an antigen, including, but not limited to, Fab, Fab', F(ab)2, Fv, scFv, dsFv, Fd fragments, dAb, VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies; multispecific antibody fragments formed from antibody fragments, and isolated fragments of one or more antibodies. Examples of such antibodies include, but are not limited to, a heavy or light chain complementarity-determining region (CDR) or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, at least a portion of a binding protein, a chimeric antibody, a humanized antibody, a species-specific antibody, a single-chain antibody, and fusion proteins and non-antibody proteins comprising the antigen-binding portion of an antibody. The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with antigens. The constant region of an antibody (Ab) can mediate the binding of the immunoglobulin to host tissues. When the term "anti-" is used before the name of a protein, e.g., anti-IHF, anti-HU, anti-OMP P5, refers to a monoclonal or polyclonal antibody that binds to and / or has affinity for a specific protein. For example, "anti-IHF" refers to an antibody that binds to the IHF protein. A specific antibody may have affinity for or bind to proteins other than the protein against which it was raised. For example, anti-IHF may be raised specifically against the IHF protein but may also bind to other proteins related by sequence or structural homology.

[0113] Antibodies can be polyclonal, monoclonal, multispecific (e.g., bispecific), diabodies, and antibody fragments, so long as they exhibit the desired biological activity. Antibodies can be isolated from any suitable biological source, for example, human, mouse, rat, sheep, and dog.

[0114] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. Monoclonal antibodies are highly specific because each is directed against a single determinant on an antigen. Antibodies can be detectably labeled, for example, with radioisotopes, enzymes that generate detectable products, fluorescent proteins, etc. Antibodies can also be conjugated to other moieties, such as members of specific binding pairs, for example, biotin (a member of the biotin-avidin specific binding pair). Antibodies can also be bound to solid supports, including, but not limited to, polystyrene plates or beads.

[0115] Monoclonal antibodies can be produced using hybridoma or recombinant DNA methods known in the art. Hybridomas are cells produced in laboratories by fusing antibody-producing lymphocytes with non-antibody-producing cancer cells, usually myeloma or lymphoma. Hybridomas grow and produce continuous samples of specific monoclonal antibodies. Alternative techniques for producing or selecting antibodies include exposing lymphocytes to the antigen of interest in vitro and screening antibody display libraries in cell, phage, or similar systems.

[0116] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Thus, as used herein, the term "human antibody" refers to an antibody in which substantially all portions of the protein (e.g., CDRs, framework, C L Domain, C H Domain (e.g., C H1 , C H2 , C H3 (VL, VH) refers to antibodies in which the VL, VH) are substantially non-immunogenic in humans and contain only minor sequence changes or mutations. Similarly, antibodies designated for primates (monkeys, baboons, chimpanzees, etc.), rodents (mouse, rats, rabbits, guinea pigs, hamsters, etc.), and other mammals refer to antibodies specific for such species, subgenus, genus, subfamily, or family. Furthermore, chimeric antibodies include any combination of the above. Such changes or mutations may retain or be less immunogenic in humans or other species than the unmodified antibody, as the case may be. Thus, human antibodies differ from chimeric or humanized antibodies. It is noted that human antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. Furthermore, when a human antibody is a single-chain antibody, the human antibody may contain a linker peptide not found in native human antibodies. For example, an Fv may include a linker peptide, such as two to about eight glycine or other amino acid residues, connecting the heavy chain variable region and the light chain variable region, and such a linker peptide is considered to be of human origin.

[0117] As used herein, a human antibody is "derived from" a particular germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, for example, by immunizing a transgenic mouse carrying human immunoglobulin genes or by screening a human immunoglobulin gene library. A human antibody "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequence of a human germline immunoglobulin. The selected human antibody generally has at least 90% amino acid sequence identity to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as human compared to germline immunoglobulin amino acid sequences of other species (e.g., mouse germline sequences). In some cases, the human antibody has at least 95%, or even at least 96%, 97%, 98%, or even 99% amino acid sequence identity to the amino acid sequence encoded by the germline immunoglobulin gene. Generally, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In some cases, a human antibody may display no more than 5, or even no more than 4, no more than 3, no more than 2, or no more than 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0118] "Human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. This term also refers to recombinant human antibodies. Methods for making these antibodies are described herein.

[0119] The term "recombinant human antibody," as used herein, encompasses all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed to express the antibody, e.g., from transfectomas, antibodies isolated from recombinant, combinatorial human antibody libraries, and antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) such that the amino acid sequences of the recombinant antibody VH and VL regions are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally occur in the human antibody germline repertoire in vivo. Methods for making these antibodies are described herein.

[0120] As used herein, a chimeric antibody is an antibody whose light and heavy chain genes have been constructed, generally by genetic engineering, from antibody variable and constant region genes belonging to different species.

[0121] As used herein, the term "humanized antibody" or "humanized immunoglobulin" refers to a human / non-human chimeric antibody that contains minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient's variable region are replaced with residues from the variable region of a non-human species (donor antibody) such as a mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. Humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. Humanized antibodies may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The non-human antibody contains one or more amino acids in the framework regions, constant region, or CDRs that are substituted for similarly positioned amino acids from a human antibody. In general, humanized antibodies are expected to generate a reduced immune response in a human host compared to non-humanized versions of the same antibody. Humanized antibodies may have conservative amino acid substitutions that have substantially no effect on antigen binding or other antibody functions. Conservative substitution groupings include glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serine-threonine, and asparagine-glutamine. The term "speciation" refers to antibodies that have been modified in the same or similar manner for non-human species.

[0122] The terms "polyclonal antibody" or "polyclonal antibody composition," as used herein, refer to a preparation of antibodies derived from different B-cell lines. These are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope. In some embodiments, the antibody or antigen-binding fragment is not a polyclonal antibody.

[0123] As used herein, the term "antibody derivative" includes a full-length antibody or an antibody fragment in which one or more of the amino acids have been chemically modified, such as by alkylation, pegylation, acylation, ester formation, or amide formation, for example, to link the antibody to a second molecule. Antibody derivatives include, but are not limited to, pegylated antibodies, cysteine-pegylated antibodies, and variants thereof. The present disclosure also provides antibody derivatives of antibody fragments, such as polypeptides that are conjugated to another molecule, such as PEG, or further modified by acylation.

[0124] As used herein, the term "immunoconjugate" includes an antibody, antibody fragment, or antibody derivative associated with or linked to a second agent, such as a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semi-synthetic antibody, or a multispecific antibody. The present disclosure provides immunoconjugates comprising, as one component, an antibody polypeptide as described herein.

[0125] As used herein, the term "label" refers to a directly or indirectly detectable compound or composition, e.g., an N-terminal histidine tag (N-His), a magnetically active isotope, e.g., a nucleotide sequence, which is directly or indirectly conjugated to a composition to be detected to produce a "labeled" composition. 115 Sn, 117 Sn and 119 Sn, non-radioactive isotopes, e.g. 13 C and 15The term "label" refers to a polynucleotide or protein, such as an antibody, such as N. The term also encompasses sequences conjugated to a polynucleotide that provide a signal when the inserted sequence is expressed, such as green fluorescent protein (GFP). The term also includes purification tags or labels that aid in the isolation of biological materials from a mixed population. While the term "label" generally contemplates a composition covalently attached to the composition to be detected, in one aspect, the term "label" specifically excludes naturally occurring nucleosides and amino acids that are known to fluoresce under certain conditions (e.g., temperature, pH, etc.) when placed in a polynucleotide or protein in their natural environment, and generally excludes any natural fluorescence that may be present in the composition to be detected. Labels may be detectable themselves (e.g., radioisotope labels or fluorescent labels) or, in the case of enzymatic labels, may catalyze chemical alteration of a substrate compound or composition that is detectable. Labels may be suitable for small-scale detection or may be more suitable for high-throughput screening. As such, suitable labels include, but are not limited to, magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. Labels can be simply detected or quantified. A simply detected reaction generally involves a reaction whose presence is simply confirmed, while a quantified reaction generally involves a reaction that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other properties. In luminescent or fluorescent assays, the detectable reaction can be generated directly using a luminophore or fluorophore attached to the assay component actually involved in the binding, or indirectly using a luminophore or fluorophore attached to another component (e.g., a reporter or indicator). Examples of luminescent labels that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent reaction generally involves a change or appearance of a luminescent signal.Suitable methods and luminophores for luminescently labeling assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th Edition). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0126] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, CASCADE BLUE™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

[0127] In another embodiment, the fluorescent label is functionalized to facilitate covalent attachment to cellular components present within or on the surface of cells or tissues, such as cell surface markers. Suitable functional groups, including but not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, can all be used to attach the fluorescent label to a second molecule. The choice of functional group on the fluorescent label depends on the site of attachment, either to a linker, agent, marker, or second labeling agent.

[0128] "Eukaryotic cells" include all kingdoms of life except Monera. Eukaryotes can be easily distinguished by their membrane-bound nuclei. Animals, plants, fungi, and protists are eukaryotic organisms or organisms whose cells are organized into complex structures by internal membranes and cytoskeleton. The most distinctive membrane-bound structure is the nucleus. Unless otherwise specified, the term "host" encompasses eukaryotic hosts, including, for example, yeast, higher plant, insect, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include monkeys, cows, pigs, mice, rats, birds, reptiles, and humans.

[0129] Prokaryotic cells typically lack a nucleus or any other membrane-bound organelles and are divided into two domains: bacteria and archaea. In addition to chromosomal DNA, these cells may also contain genetic information in circular loops called episomes. Bacterial cells are very small, roughly the size of animal mitochondria (approximately 1-2 μm in diameter and 10 μm in length). Prokaryotic cells are characterized by three main shapes: rod-shaped, spherical, and spiral. Instead of undergoing an elaborate replication process like eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, Bacillus, E. coli, and Salmonella.

[0130] A "native" or "natural" antigen is a polypeptide, protein, or fragment that contains an epitope, has been isolated from a natural biological source, and is capable of specifically binding to an antigen receptor, specifically a T cell antigen receptor (TCR), in a subject.

[0131] The terms "antigen" and "antigenic" refer to a molecule capable of being recognized by an antibody or otherwise serving as a member of an antibody-ligand pair. "Specific binding" refers to the interaction of an antigen with the variable regions of the heavy and light chains of an immunoglobulin. Antibody-antigen binding can occur in vivo or in vitro. Those skilled in the art will understand that macromolecules, including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides, and polysaccharides, have the potential to serve as antigens. Those skilled in the art will further understand that nucleic acids encoding proteins with the potential to serve as antibody ligands necessarily encode antigens. Those skilled in the art will further understand that antigens are not limited to full-length molecules but may also include partial molecules. The term "antigenic" is an adjective reference to a molecule having the properties of an antigen. This term encompasses substances that are immunogenic, i.e., immunogens, as well as substances that induce immunological unresponsiveness or anergy, i.e., anergens.

[0132] An "altered antigen" is an antigen having a primary sequence that differs from that of the corresponding wild-type antigen. The polypeptide mB4 (SEQ ID NO: 17) is an example of an altered antigen (SEQ ID NO: 16). Altered antigens can be produced by synthetic or recombinant methods and include, but are not limited to, antigenic peptides that have been differentially modified during or after translation by, for example, phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, or linkage to an antibody molecule, membrane molecule, or other ligand (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320). The synthetic or altered antigens disclosed herein will bind to the same TCR as the native epitope.

[0133] "Autoantigens," also referred to herein as native antigens or wild-type antigens, are antigenic peptides that induce little or no immune response in a subject due to self-tolerance to the antigen. An example of an autoantigen is the melanoma-specific antigen gp100.

[0134] "Immune response" broadly refers to an antigen-specific response of lymphocytes to a foreign substance. The terms "immunogen" and "immunogenic" refer to a molecule capable of eliciting an immune response. All immunogens are antigens, but not all antigens are immunogenic. The immune responses disclosed herein can be humoral (through antibody activity) or cell-mediated (through T cell activation). The response can occur in vivo or in vitro. Those skilled in the art will understand that a variety of macromolecules have the potential to be immunogenic, including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides, and polysaccharides. Those skilled in the art will further understand that a nucleic acid encoding a molecule capable of eliciting an immune response necessarily encodes an immunogen. Those skilled in the art will further understand that an immunogen is not limited to a full-length molecule but can include a partial molecule.

[0135] The term "passive immunity" refers to the transfer of immunity from one subject to another through the transfer of antibodies. Passive immunity can occur naturally, such as when a mother's antibodies are transferred to her fetus. Passive immunity can also occur artificially, such as when an antibody composition is administered to a non-immune subject. The donor and recipient of the antibody can be a human or non-human subject. The antibody can be polyclonal or monoclonal, can be generated in vitro or in vivo, and can be purified, partially purified, or unpurified, depending on the embodiment. In some embodiments described herein, passive immunity is conferred by administering to a subject in need thereof an antibody or antigen-binding fragment that specifically recognizes or binds to a particular antigen. In some embodiments, passive immunity is conferred by administering an isolated or recombinant polynucleotide encoding an antibody or antigen-binding fragment that specifically recognizes or binds to a particular antigen.

[0136] As used herein, the term "inducing an immune response in a subject" is a term well understood in the art and means being able to detect or measure an increase in the immune response to an antigen (or epitope) of at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold or more after introducing the antigen (or epitope) into a subject, compared to the immune response (if any) before introducing the antigen (or epitope) into the subject. An immune response to an antigen (or epitope) includes, but is not limited to, the production of antigen-specific (or epitope-specific) antibodies and the production of immune cells expressing on their surface a molecule that specifically binds to the antigen (or epitope). Methods for determining whether an immune response to a given antigen (or epitope) has been induced are well known in the art. For example, antigen-specific antibodies can be detected using any of a variety of immunoassays known in the art, including, but not limited to, ELISA, in which binding of antibodies in a sample to an immobilized antigen (or epitope) is detected using a detectably labeled second antibody (e.g., an enzyme-labeled mouse anti-human Ig antibody).

[0137] As used herein, the term "anti-inflammatory cytokine" includes immunomodulatory molecules that control proinflammatory cytokine responses. Cytokines act together with specific cytokine inhibitors and soluble cytokine receptors to regulate human immune responses. Major anti-inflammatory cytokines include interleukin (IL)-1 receptor antagonists, IL-4, IL-6, IL-10, IL-11, and IL-13. Specific cytokine receptors for IL-1, tumor necrosis factor alpha, and IL-18 also function as proinflammatory cytokine inhibitors. Methods for measuring cytokine levels, including anti-inflammatory cytokine levels, are well known in the art. For example, serum cytokine levels can be measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits.

[0138] The term "effective amount" refers to an amount sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount depends on the type and severity of the condition in question and the characteristics of the individual subject, such as overall health, age, sex, weight, and tolerance to the pharmaceutical composition. In some embodiments, in the context of an immunogenic composition, an effective amount is an amount sufficient to produce a protective response against a pathogen. In other embodiments, an effective amount of an immunogenic composition is an amount sufficient to produce antibody production against an antigen. In some embodiments, an effective amount is the amount necessary to confer passive immunity to a subject in need thereof. With respect to immunogenic compositions, in some embodiments, the effective amount depends on the intended use, the degree of immunogenicity of the particular antigenic compound, and the health / responsiveness of the subject's immune system, in addition to the factors described above. Those skilled in the art can determine the appropriate amount depending on these and other factors.

[0139] In the case of in vitro application, in some embodiments, the effective amount depends on the size and nature of the application in question. It also depends on the nature and sensitivity of the target in vitro and the method used. Those skilled in the art can determine the effective amount based on these and other considerations. Depending on the embodiment, the effective amount may include one or more administrations of the composition.

[0140] As used herein, the terms "solid support" and "solid support" are used interchangeably and are not limited to a particular type of support. Rather, numerous supports are available and known to those skilled in the art. Solid supports include silica gel, resins, derivatized plastic films, glass beads, cotton, plastic beads, and alumina gel. As used herein, "solid support" also encompasses synthetic antigen-presenting matrices, cells, and liposomes. An appropriate solid support can be selected based on the desired end use and suitability for various protocols. For example, with respect to peptide synthesis, a solid support may refer to a resin such as polystyrene (e.g., PAM-resin available from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE® resin (available from Aminotech, Canada), polyamide resin (available from Peninsula Laboratories), polyethylene glycol-grafted polystyrene resin (TentaGel®, Rapp Polymere, Tübingen, Germany) or polydimethylacrylamide resin (available from Milligen / Biosearch, Calif.).

[0141] Examples of solid supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural cellulose, modified cellulose, polyacrylamide, gabbro, and magnetite. The nature of the carrier can be soluble to some extent or insoluble. The support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a polynucleotide, polypeptide, or antibody. Thus, the configuration of the support can be spherical, such as a bead, or cylindrical, such as the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface can be flat, e.g., a sheet, test strip, or polystyrene beads. Those of skill in the art will know many other suitable carriers for binding antibodies or antigens, or will be able to ascertain such by use of routine experimentation.

[0142] MODE FOR CARRYING OUT THE INVENTION Antibody fragments and their derivatives The present disclosure provides antibody fragments (e.g., Fab fragments or antigen-binding fragments). The present disclosure also provides isolated polypeptides comprising, alternatively consisting essentially of, or even consisting of the amino acid sequence of a Fab fragment or antigen-binding fragment (e.g., Fab fragment), wherein the fragment or polypeptide binds to and / or specifically recognizes a DNABII polypeptide and / or a biofilm component comprising a DNABII protein or polypeptide.

[0143] The antibody fragment or isolated polypeptide comprising, essentially consisting of, or even consisting of an antibody fragment can be any Fab fragment derived from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, a veneered antibody, a diabody, a humanized antibody, an antibody derivative, or a recombinant humanized antibody.Antibodies can be derived from any suitable species, for example, mammalian species, for example, mouse, cat, dog, rabbit, or human.Methods for preparing Fab antibody fragments are known in the art and are briefly described herein.One exemplary method is outlined in Figure 1.

[0144] Antibody fragments are derived from or generated from antibodies that specifically recognize and specifically bind to the DNABII protein; non-limiting examples of such antibody fragments include IHF alpha, IHF beta, and HU. Non-limiting examples of such polypeptides and antibodies that bind to such polypeptides are provided in U.S. Patent No. 8,999,291, which is incorporated herein by reference. In one embodiment, the antibody specifically binds to the "tip domain" of the DNABII polypeptide, which contains a conformational epitope. In one embodiment, the DNABII polypeptide comprises, consists essentially of, or even consists of one of SEQ ID NOS: 1-33. Further non-limiting examples of exemplary DNABII polypeptides include conformational epitopes within the apical domain of, for example, IHF alpha, IHF beta, or HU, as provided in, for example, SEQ ID NOS: 13, 14, 17, 26-32. In one embodiment, SEQ ID NOS: 13, 14, 17, and 26-32 are contained within a larger polypeptide that is not a wild-type naturally occurring polypeptide, for example, SEQ ID NOS: 1-11. The larger polypeptide may contain additional amino acids at the amine and / or carboxy termini, for example, at least 3, 4, 5, 10, 15, or 20 or more amino acids at one or both termini.

[0145] Additionally, the antibody may be directed to the "tip" region of the DNABII protein, which in one embodiment is the antiparallel beta ribbon turn and / or sequence. [ka] [where "X" refers to any amino acid, or X is selected from the amino acids Q, R, K, S, or T]. Such antibodies can also be raised against "tip" regions containing, or altered to contain, between about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids on either or both sides of this sequence, as appropriate. [ka] A non-limiting example of such a fragment of the DNABII protein is the consensus amino acid sequence [ka] In another aspect, the antibody is directed to the tip region of the DNABII protein, having the consensus sequence: [ka] [wherein "X" is any amino acid, or alternatively, X is selected from the amino acids Q, R, K, S, or T]. Those skilled in the art will appreciate that in any one of these sequences, the residue at position X can be substituted with any amino acid, for example, Q, R, K, S, or T. Examples include: SEQ ID NO: 13: Haemophilus influenzae IhfA, A5 fragment: [ka] SEQ ID NO: 14: Haemophilus influenzae HU, A5 fragment: [ka] SEQ ID NO: 17: Haemophilus influenzae IhfB, modified B4 (mB4) fragment: [ka] SEQ ID NO: 26: Haemophilus influenzae IhfA, A tip fragment: [ka] SEQ ID NO: 27: Haemophilus influenzae IhfB, B tip fragment: [ka] SEQ ID NO: 31 Haemophilus influenzae HU, fragment: [ka] Includes:

[0146] In some embodiments, [ka] is at least about 20 amino acids in length, [ka] is centrally located within the sequence. Non-limiting examples of such sequences include SEQ ID NOs: 13, 14, and 31. Alternatively, the amino acid sequence may be modified by either substitution or deletion of one or more amino acids. [ka] To remove the motif, [ka] Polypeptides having motifs (e.g., those mentioned above) can also be modified and monoclonal antibodies can be raised against these polypeptides. [ka] It has been determined that Fab fragments of antibodies raised against DNABII polypeptides lacking the motif are useful in diagnostic methods for imaging and monitoring biofilm formation and / or disruption. [ka] a Fab fragment of an antibody raised against or binding to a DNABII having the motif; [ka] Also provided is a kit that includes the Fab fragment of an antibody that is raised against or binds to a DNABII (e.g., a modified polypeptide or a naturally occurring polypeptide) that lacks a motif.For example, the kit can include an antibody that recognizes and binds to polypeptide A5, B4, or mB4 (therapeutic), and can be combined in the kit with the Fab fragment of an antibody that recognizes and binds to polypeptide A3 or B2 (diagnostic).The kit is useful for diagnosis, treatment, and monitoring of biofilm treatment.

[0147] In one embodiment, the isolated polypeptide is mIhfB4 NTHI Fab fragment of antibody or mIhfB4 NTHI It may comprise, alternatively consist essentially of, or even consist of, equivalents of antibodies, examples of which are provided herein.

[0148] In another embodiment, Fab fragments are prepared from antibodies produced by hybridoma cell lines, eg, the hybridomas listed in Table 1. [Table 1]

[0149] As mentioned above, the present disclosure also provides antibodies disclosed in Table 1, e.g., IhfmB4 NTHI Also provided is an antibody fragment, antigen-binding fragment, or polypeptide comprising, consisting essentially of, or consisting of, a Fab fragment of the antibody or antibody equivalent. NTHI It comprises, alternatively consists essentially of, or even further consists of, an Fab fragment of an antibody or antibody equivalent.

[0150] In a further aspect, the present disclosure provides an isolated antibody disclosed in Table 1 or an antigen-binding fragment of an antibody disclosed in Table 1 that is at least 85% identical, alternatively at least 90%, or alternatively at least 95%, or alternatively at least 100% identical to an antibody fragment disclosed in Table 1 or derived from an antibody selected from the group consisting of: (i) an antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) an antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) an antibody produced by hybridoma cell line mIhfB4 NTHI 12E6.F8.D12.D5.

[0151] In one aspect, the present disclosure provides an isolated antibody fragment, antigen-binding fragment, or polypeptide comprising a CDR of (i) an antibody produced by a hybridoma cell line disclosed in Table 1, e.g., IhfA5 NTHI 14G8.F5.G6, (ii) an antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, or (iii) an antibody produced by hybridoma cell line mIhfB4 NTHI 12E6.F8.D12.D5. In one aspect, the disclosure provides an isolated antibody fragment, antigen-binding fragment, or polypeptide derived from an antibody having CDRs that are at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 100% identical to (i) an antibody produced by a hybridoma disclosed in Table 1, e.g., cell line IhfA5 NTHI 14G8.F5.G6, (ii) an antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, or (iii) an antibody produced by hybridoma cell line mIhfB4 NTHI 12E6.F8.D12.D5.

[0152] In some embodiments of the antibody fragments, antigen-binding fragments, or polypeptides provided herein, the HC variable domain sequence of the antibody providing such is selected from the group consisting of (i) an antibody produced by a hybridoma cell line disclosed in Table 1, e.g., IhfA5 NTHI 14G8.F5.G6, (ii) an antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, or (iii) an antibody produced by hybridoma cell line MIhfB4 The HC variable domain sequence includes the HC variable domain sequence of the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, or (iii) the LC variable domain sequence of the antibody produced by hybridoma cell line mIhfB4 NTHI 12E6.F8.D12.D5, and / or the LC variable domain sequence includes the LC variable domain sequence of an antibody disclosed in Table 1, for example, (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, or (iii) the antibody produced by hybridoma cell line mIhfB4 NTHI 12E6.F8.D12.D5.

[0153] In some embodiments of the antibody fragments, antigen-binding fragments, or polypeptides provided herein, the HC variable domain sequence of the antibody providing such a sequence comprises an HC variable domain sequence that is at least 85% identical, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 100% identical to the HC variable domain sequence of an antibody disclosed in Table 1, e.g., (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfB4 NTHI 12E6.F8.D12.D5; 14G8.F5.G6, (ii) an antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) an antibody produced by hybridoma cell line mIhfB4 NTHI 12E6.F8.D12.D5, comprising an LC variable domain sequence that is at least 85% identical, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 100% identical to the LC variable domain sequence of an antibody produced by hybridoma cell line mIhfB4 NTHI 12E6.F8.D12.D5.

[0154] In one aspect, the disclosure provides a Fab fragment comprising a heavy chain (HC) variable domain sequence and a light chain (LC) variable domain sequence, wherein the heavy and light chain immunoglobulin variable domain sequences form an antigen binding site that binds to an epitope of a DNABII protein.

[0155] In some embodiments, the heavy chain variable region is selected from the group consisting of antibodies disclosed in Table 1, such as (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfB4 NTHI The antibody may comprise, alternatively consist essentially of, or even further comprise, an amino acid sequence comprising the CDRH1 of any one of the antibodies produced by 12E6.F8.D12.D5 or their respective equivalents.

[0156] In some embodiments, the heavy chain variable region is selected from the group consisting of antibodies disclosed in Table 1, such as (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfB4 NTHI The antibody may comprise, alternatively consist essentially of, or even further comprise, an amino acid sequence comprising the CDRH2 of any one of the antibodies produced by 12E6.F8.D12.D5 or their respective equivalents.

[0157] In some embodiments, the heavy chain variable region is selected from the group consisting of antibodies disclosed in Table 1, such as (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfB4 NTHI The antibody may comprise, alternatively consist essentially of, or even further comprise, an amino acid sequence comprising the CDRH3 of any one of the antibodies produced by 12E6.F8.D12.D5 or their respective equivalents.

[0158] In some embodiments, the heavy chain variable region is selected from the group consisting of antibodies disclosed in Table 1, such as (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfB4 NTHI 12E6.F8.D12.D5 or their respective equivalents.

[0159] In some embodiments, the light chain variable region is selected from the group consisting of the antibodies disclosed in Table 1, such as (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfB4 NTHI The antibody may comprise, alternatively consist essentially of, or even further comprise, an amino acid sequence comprising the CDRL1 of any one of the antibodies produced by 12E6.F8.D12.D5 or their respective equivalents.

[0160] In some embodiments, the light chain variable region is selected from the group consisting of the antibodies disclosed in Table 1, such as (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfB4 NTHI The antibody may comprise, alternatively consist essentially of, or even further comprise, an amino acid sequence comprising the CDRL2 of any one of the antibodies produced by 12E6.F8.D12.D5 or their respective equivalents.

[0161] In some embodiments, the light chain variable region is selected from the group consisting of the antibodies disclosed in Table 1, such as (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfB4 NTHI The antibody may comprise, alternatively consist essentially of, or even further comprise, an amino acid sequence comprising the CDRL3 of any one of the antibodies produced by 12E6.F8.D12.D5 or their respective equivalents.

[0162] In some embodiments, the light chain variable region is selected from the group consisting of the antibodies disclosed in Table 1, such as (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfB4 NTHI It comprises, alternatively consists essentially of, or even further consists of, a polypeptide encoded by a polynucleotide sequence comprising the light chain variable region sequence of any one of the antibodies produced by 12E6.F8.D12.D5 or their respective equivalents.

[0163] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH1 sequence comprising, alternatively consisting essentially of, or even further consisting of an amino acid sequence comprising FSLTSYS (SEQ ID NO: 58), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of FSLTSYSV (SEQ ID NO: 59), FSLTSYSVH (SEQ ID NO: 60), GFSLTSYS (SEQ ID NO: 61), or their respective equivalents.

[0164] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH1 sequence comprising, alternatively consisting essentially of, or even consisting of an amino acid sequence comprising FNIKDYY (SEQ ID NO: 110), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of FNIKDYYM (SEQ ID NO: 111), FNIKDYYMH (SEQ ID NO: 112), GFNIKDYY (SEQ ID NO: 113), or their respective equivalents.

[0165] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH2 sequence comprising, alternatively consisting essentially of, or even consisting of an amino acid sequence comprising IWAGGST (SEQ ID NO:62), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of VIWAGGST (SEQ ID NO:63), GVIWAGGST (SEQ ID NO:64), LGVIWAGGST (SEQ ID NO:65), WLGVIWAGGST (SEQ ID NO:66), IWAGGSTN (SEQ ID NO:67), VIWAGGSTN (SEQ ID NO:68), GVIWAGGSTN (SEQ ID NO:69), LGVIWAGGSTN (SEQ ID NO:70), WLGVIWAGGSTN (SEQ ID NO:71), IWAGGSTNY (SEQ ID NO:72), VIWAGGSTNY (SEQ ID NO:73), GVIWAGGSTNY (SEQ ID NO:74), LGVIWAGGSTNY (SEQ ID NO:75), WLGVIWAGGSTNY (SEQ ID NO:76), or their respective equivalents.

[0166] In some embodiments, the heavy chain variable region of the antibody or fragment thereof has an amino acid sequence comprising IDPENDDT (SEQ ID NO: 114), and is selected from the group consisting of WIDPENDDT (SEQ ID NO: 115), GWIDPENDDT (SEQ ID NO: 116), IGWIDPENDDT (SEQ ID NO: 117), WIGWIDPENDDT (SEQ ID NO: 118), IDPENDDTE (SEQ ID NO: 119), WIDPENDDTE (SEQ ID NO: 120), GWIDPENDDTE (SEQ ID NO: 121), IGWIDPENDDTE (SEQ ID NO: 122), WIGWIDPENDDTE (SEQ ID NO: 123), IDPENDDTEY (SEQ ID NO: 124), WIDPENDDTEY (SEQ ID NO: 125). CDRH2 sequences including, alternatively consisting essentially of, or even consisting of, an amino acid sequence such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of GWIDPENDDTEY (SEQ ID NO: 126), IGWIDPENDDTEY (SEQ ID NO: 127), WIGWIDPENDDTEY (SEQ ID NO: 128), or their respective equivalents.

[0167] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH3 sequence that comprises, alternatively consists essentially of, or even consists of an amino acid sequence comprising AREDS (SEQ ID NO: 77), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of AREDS (SEQ ID NO: 78) or an equivalent.

[0168] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises a CDRH3 sequence comprising, alternatively consisting essentially of, or even consisting of, an amino acid sequence comprising TELGAY (SEQ ID NO: 129) or an equivalent thereof.

[0169] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises the polynucleotide sequence referred to below: or alternatively consisting essentially of, or even further consisting of, a polypeptide encoded by GAGGTGCAGCTGCAGGAGTCTGGACCTGGCCTGGTGACGCCCTCACAGAGCCTGTCCATGACTTGCACTGTCTCTGGGTTTTCATTAACCAGCTATAGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGAGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAGCACAAATTATAATTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGGACAGTCTGCAAACTGATGACACAGCCATATACTACTGTGCCAGAGAGGACTCCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 50) or an equivalent thereof.

[0170] In some embodiments, the heavy chain variable region of the antibody or fragment thereof has the amino acid sequence: EVQLQESGPGLVTPSQSLSMTCTVSGFSLTSYSVHWVRQPPGKSLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMDSLQTDDTAIYYCAREDSWGQGTSVTVSS (SEQ ID NO: 51) or an equivalent thereof.

[0171] In some embodiments, the heavy chain variable region of the antibody or fragment thereof comprises the polynucleotide sequence referred to below: or alternatively consisting essentially of, or even further consisting of, a polypeptide encoded by GAGGTGCAGCTGCAGGAGTCTGGGGCAGAGCTTGTGAGGTCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACTACTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAAAATGATGATACTGAATATGTCCCGAAGTTCCAGGGCAAGGCCAGTATGACTGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTACAGAGCTCGGAGCTTACTGGGGCCAGGGGACTCTGGTC (SEQ ID NO: 52) or an equivalent thereof.

[0172] In some embodiments, the heavy chain variable region of the antibody or fragment thereof has the amino acid sequence: EVQLQESGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQGLEWIGWIDPENDDTEYVPKFQGKASMTADTSSNTAYLQLSSLTSEDTAVYYCTELGAYWGQGTLV (SEQ ID NO: 53) or an equivalent thereof.

[0173] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises a CDRL1 sequence comprising, alternatively consisting essentially of, or even further consisting of an amino acid sequence comprising QNVGTN (SEQ ID NO: 79), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of QNVGTNV (SEQ ID NO: 80), QNVGTNVA (SEQ ID NO: 81), or their respective equivalents.

[0174] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises a CDRL1 sequence comprising, alternatively consisting essentially of, or even further consisting of an amino acid sequence comprising QSLLDSNGKTY (SEQ ID NO: 130), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of QSLLDSNGKTYL (SEQ ID NO: 131), QSLLDSNGKTYLN (SEQ ID NO: 132), or their respective equivalents.

[0175] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises an amino acid sequence comprising SAS (SEQ ID NO: 82), and is selected from the group consisting of YSAS (SEQ ID NO: 83), IYSAS (SEQ ID NO: 84), LIYSAS (SEQ ID NO: 85), ALIYSAS (SEQ ID NO: 86), SASY (SEQ ID NO: 87), YSASY (SEQ ID NO: 88), IYSASY (SEQ ID NO: 89), LIYSASY (SEQ ID NO: 90), ALIYSASY (SEQ ID NO: 91), SASYR (SEQ ID NO: 92), YSASYR (SEQ ID NO: 93), IYSASYR (SEQ ID NO: 94), LIYSASYR (SEQ ID NO: 95), ALIYSASYR (SEQ ID NO: 96), SASYRY (SEQ ID NO: 97), and CDRL2 sequences comprising, alternatively consisting essentially of, or even consisting of, an amino acid sequence such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of YSASYRY (SEQ ID NO: 98), IYSASYRY (SEQ ID NO: 99), LIYSASYRY (SEQ ID NO: 100), ALIYSASYRY (SEQ ID NO: 101), SASYRYS (SEQ ID NO: 102), YSASYRYS (SEQ ID NO: 103), IYSASYRYS (SEQ ID NO: 104), LIYSASYRYS (SEQ ID NO: 105), ALIYSASYRYS (SEQ ID NO: 106), or their respective equivalents.

[0176] In some embodiments, the light chain variable region of the antibody or fragment thereof has an amino acid sequence comprising LVS (SEQ ID NO: 133), and is selected from the group consisting of YLVS (SEQ ID NO: 134), IYLVS (SEQ ID NO: 135), LIYLVS (SEQ ID NO: 136), RLIYLVS (SEQ ID NO: 137), LVSK (SEQ ID NO: 138), YLVSK (SEQ ID NO: 139), IYLVSK (SEQ ID NO: 140), LIYLVSK (SEQ ID NO: 141), RLIYLVSK (SEQ ID NO: 142), LVSKL (SEQ ID NO: 143), YLVSKL (SEQ ID NO: 144), IYLVSKL (SEQ ID NO: 145), LIYLVSKL (SEQ ID NO: 146), RLIYLVSKL (SEQ ID NO: 147), LVSKLD (sequence and / or CDRL2 sequences that comprise, alternatively consist essentially of, or even consist of, an amino acid sequence such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of: YLVSKLD (SEQ ID NO: 148), YLVSKLD (SEQ ID NO: 149), IYLVSKLD (SEQ ID NO: 150), LIYLVSKLD (SEQ ID NO: 151), RLIYLVSKLD (SEQ ID NO: 152), LVSKLDS (SEQ ID NO: 153), YLVSKLDS (SEQ ID NO: 154), IYLVSKLDS (SEQ ID NO: 155), LIYLVSKLDS (SEQ ID NO: 156), RLIYLVSKLDS (SEQ ID NO: 157), or their respective equivalents.

[0177] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises a CDRL3 sequence comprising, alternatively consisting essentially of, or even consisting of an amino acid sequence comprising QQYNSYP (SEQ ID NO: 108), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of QQYNSYPT (SEQ ID NO: 109), or an equivalent thereof.

[0178] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises a CDRL3 sequence comprising, alternatively consisting essentially of, or even consisting of an amino acid sequence comprising WQSTHFPH (SEQ ID NO: 158), such as, but not limited to, an amino acid sequence beginning with, ending with, or consisting essentially of WQSTHFPHT (SEQ ID NO: 159) or an equivalent.

[0179] In some embodiments, the light chain variable region of the antibody or fragment thereof comprises, alternatively consists essentially of, or even consists of a polypeptide encoded by the polynucleotide sequence: GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCCACGTTCGGAGGGGGGACCAAGTTGGAAATAAAA (SEQ ID NO: 54) or an equivalent.

[0180] In some embodiments, the light chain variable region of the antibody or fragment thereof has the amino acid sequence: DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPTFGGGTKLEIK (SEQ ID NO: 55) or an equivalent thereof.

[0181] In some embodiments, the light chain variable region comprises the polynucleotide sequence: GATGTTGTGATGACCCAGATTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTAATGGAAAGACATATTTGAATTGGTTGTTTCAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTTGAGGCTGAGGATTTGGGAATTTATTATTGCTGGCAAAGTACACATTTTCCTCACACGTTCGGAGGGGGGACCAAGTTGGAAATCAAA (SEQ ID NO: 56) or an equivalent thereof.

[0182] In some embodiments, the light chain variable region has the amino acid sequence: DVVMTQIPLTLSVTIGQPASISCKSSQSLLDSNGKTYLNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQSTHFPHTFGGGTKLEIK (SEQ ID NO: 57) or an equivalent thereof.

[0183] Exemplary antibodies comprising the disclosed CDR sequences and heavy and light chain variable sequences from which antibody fragments, antigen-binding fragments, or polypeptides are derived are disclosed in Tables 2 and 3, respectively. Alternative CDR predictions can be made based on the heavy and / or light chain sequences (e.g., based on the Kabat definition, the Clothia definition, the AbM definition, or the contact definition of CDR specificity; details of these CDR prediction methods are known in the art (see, e.g., bioinf.org.uk / abs / #cdrid) and / or are commercially available). Those disclosed in Table 2 are the results of utilizing the CDR prediction algorithm provided by Ofran Lab (Paratome, available at ofranservices.biu.ac.il / site / services / paratome / index.html) and Green Mountain Antibodies' CDR prediction program. [Table 2] [Table 3]

[0184] In one aspect, the present disclosure provides an antibody fragment (e.g., a Fab fragment) of an isolated antibody that is at least 85%, or alternatively at least 90%, or alternatively at least 95% or 100% identical to an antibody disclosed in Table 1, e.g., an antibody selected from the group consisting of Ihf A5, Ihf mB4, or a biological equivalent of each thereof.

[0185] In one aspect, the present disclosure provides an isolated antibody fragment comprising the CDRs of Ihf A5. In one aspect, the present disclosure provides an isolated antibody fragment of an antibody that is at least 85%, or alternatively at least 90%, or alternatively at least 95%, or 100% identical to Ihf A5 or a biological equivalent thereof.

[0186] In one aspect, the present disclosure provides an isolated antibody fragment comprising the CDRs of Ihf mB4. In one aspect, the present disclosure provides an isolated antibody fragment of an isolated antibody that is at least 85%, or alternatively at least 90%, or alternatively at least 95%, or 100% identical to Ihf mB4 or a biological equivalent thereof.

[0187] In some embodiments of the antibody fragments provided herein, the HC variable domain sequence comprises the variable domain sequence of Ihf A5, and the LC variable domain sequence comprises the variable domain sequence of Ihf A5.

[0188] In some embodiments of the antibody fragments provided herein, the HC variable domain sequence comprises the variable domain sequence of Ihf mB4, and the LC variable domain sequence comprises the variable domain sequence of Ihf mB4.

[0189] In another aspect of the present technology, the antibody fragment has the following characteristics: (a) the light chain immunoglobulin variable domain sequence comprises one or more CDRs that are at least 85%, or alternatively at least 90%, or alternatively at least 95%, or 100% identical to the CDRs of the light chain variable domain of any of the disclosed light chain sequences; (b) the heavy chain immunoglobulin variable domain sequence comprises one or more CDRs at least 85%, or alternatively at least 90%, or alternatively at least 95%, or 100% identical to the CDRs of the heavy chain variable domain of any of the disclosed heavy chain sequences; (c) the light chain immunoglobulin variable domain sequence is at least 85%, or alternatively at least 90%, or alternatively at least 95%, or 100% identical to the light chain variable domain of any of the disclosed light chain sequences; (d) the HC immunoglobulin variable domain sequence is at least 85%, or alternatively at least 90%, or alternatively at least 95%, or 100% identical to the heavy chain variable domain of any of the disclosed light chain sequences; and (e) the antibody binds to an epitope that overlaps with the epitope bound by any of the disclosed sequences Contains one or more of the following:

[0190] In some embodiments of the antibodies provided herein, the antibody fragment, antigen-binding fragment, or polypeptide is a nucleotide sequence that binds to a DNABII protein. -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 It binds with a dissociation constant (KD) of less than M. In some embodiments of the antibody fragments or polypeptides provided herein, the antigen-binding site specifically binds to a DNABII protein. In another embodiment, the affinity of the antibody or antigen-binding fragment is less than or about 1000 picomolar (pM), 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, about 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, and 8 pM.

[0191] In some embodiments of the antibodies provided herein, the antibody fragment or antigen-binding fragment is a soluble Fab. In other embodiments, the polypeptide comprises, alternatively consists essentially of, or even consists of a soluble Fab or equivalent of a soluble Fab of an antibody disclosed herein.

[0192] In some embodiments of the antibodies provided herein, the antibody from which the antibody fragment, antigen-binding fragment, or polypeptide is derived is a rabbit antibody. In some embodiments of the antibodies provided herein, the antibody from which the antibody fragment, antigen-binding fragment, or polypeptide is derived is a human antibody or humanized antibody, or is non-immunogenic in humans.

[0193] In some embodiments of the antibodies provided herein, the antibody from which the antibody fragment, antigen-binding fragment, or polypeptide is derived comprises a framework region of a human antibody (e.g., a humanized antibody). In some embodiments of the antibodies from which the antibody fragment, antigen-binding fragment, or polypeptide is derived provided herein, the antibody comprises an antibody framework region derived from a non-human species, such as monkey, horse, cat, dog, cow, pig, goat, or sheep. In some embodiments of the antibodies from which the antibody fragment, antigen-binding fragment, or polypeptide is derived, the antibody is a chimeric antibody. In any one or more of these embodiments, the antibody may further comprise one or more of the CDR regions and / or heavy and / or light chains disclosed herein.

[0194] In other embodiments, one or more residues within the CDRs of the antibodies provided herein are substituted with another amino acid. The substitutions may be "conservative," meaning that they are substitutions within the same amino acid family. Naturally occurring amino acids can be divided into four families, and conservative substitutions will occur within these families: 1) Amino acids with basic side chains: lysine, arginine, histidine. 2) Amino acids with acidic side chains: aspartic acid, glutamic acid. 3) Amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, and tyrosine. 4) Amino acids with nonpolar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.

[0195] In another embodiment, one or more residues are added to or deleted from one or more CDRs of the antibody, either at the N-terminus or C-terminus of the CDR or at a position within the CDR.

[0196] By altering the amino acid sequence of the antibody CDR by adding, deleting, or substituting amino acids, a variety of effects can be achieved, such as increasing the binding affinity for the target antigen.

[0197] It will be appreciated that antibodies of the present disclosure containing such variations in the CDR sequences will still bind to DNABII proteins with a specificity and sensitivity profile similar to the disclosed antibodies, which can be determined by binding assays.

[0198] The constant region of the antibody can also be altered. For example, antibodies can be provided with the Fc region of any isotype: IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), or IgM. Furthermore, the constant region of the antibody can be adapted to a specific therapeutic indication, for example, following the teachings of Irani et al. (2015), Molecular Immunol. 67:171-182.

[0199] As mentioned in Irani et al. (2015), Fc engineering has been shown to increase potent and specific activity. This is important for developing therapeutic antibodies that are compatible with Fc engineering and therefore reduce both dosage and potential side effects. Understanding the differences across IgG subclasses has been used in Fc engineering, and studies have shown that introducing specific residues from one subclass into another can convert certain effector functions while retaining others (Armour et al. (1999), Eur. J. Immunol., 29:2613-2624; Armour et al. (2003), Mol. Immunol., 40:585-593; Hessell et al. (2007), Nature, 449:101-104; Redpath et al. (1998), Hum. Immunol., 59:720-727; Vafa et al. (2014), Methods (San Diego, CA), 65:114-126). These Fc engineering methods This is relevant because in the context of disease, the effector functions of specific antibodies are often crucial for the efficient elimination of pathogens.

[0200] The structural and functional properties of IgG subclasses vary, as do their response profiles to different infectious diseases, and these differences can be exploited in the development of effective therapeutic antibodies (Carter (2006), Nat. Rev. Immunol., 6:343-357; Jefferis (2012), Arch. Biochem. Biophys., 526:1 (pp. 59-166). Heavy chains share greater than 90% sequence identity across IgG subclasses (Rispens and Vidarsson (2014) in Nimmerjahn, MEA (ed.), Chapter 9, Human IgG Subclasses, Academic Press, Boston, pp. 159-177). However, there are differences in surface-exposed residues in the constant (CH1, CH2, and CH3) domains, as well as substantial variation within the hinge region. This confers many of the unique properties of each IgG subclass, such as stability, flexibility, and the distance over which the two Fabs and associated Fc span. The hinge structure contributes to the binding of mAbs (Liu and May (2012), mAbs 4:17-23; Roux et al. (1997), J. Immunol. (Baltimore, MD: 1950), 159:33 (pp. 72-3382; Tian et al. (2014) Pharm. Sci. 103:1701-1710). Importantly, some regions of the Fc and hinge that differ among IgG subclasses clearly overlap with residues known to be involved in binding to both activating and inhibitory Fcγ receptors (FcγRs), the neonatal receptor for IgG (FcRn), and the complement component C1q. The occurrence of key amino acid differences within the binding sites of these effector molecules helps explain the observed differences in the effector properties of IgG subclasses. This structural and molecular information is important when selecting subclass frameworks for therapeutic antibodies or when introducing key amino acid changes to tailor antibodies for specific purposes.

[0201] Non-limiting examples of constant region sequences include: Human IgD constant region, Uniprot:P01880 SEQ ID NO:34: APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFV VGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK. Human IgG1 constant region, Uniprot:P01857 SEQ ID NO:35: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. Human IgG2 constant region, Uniprot:P01859 SEQ ID NO:36: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. Human IgG3 constant region, Uniprot:P01860 SEQ ID NO:37: ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK. Human IgM constant region, Uniprot:P01871 SEQ ID NO:38: GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKV SVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFA IPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPARE QLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY. Human IgG4 constant region, Uniprot:P01861 SEQ ID NO:39: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. Human IgA1 constant region, Uniprot:P01876 SEQ ID NO:40: ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPER DLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY. Human IgA2 constant region, Uniprot:P01877 SEQ ID NO:41: ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCY SVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY. Human Ig kappa constant region, Uniprot:P01834 SEQ ID NO:42: TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0202] In some embodiments, the antibody fragment, antigen-binding fragment, or polypeptide is an IhfA fragment. The parent antibody is derived from an antibody specific for A5 (SEQ ID NO: 13) and comprises a constant region derived from IgM or IgG, such as, but not limited to, IgG2 (in certain embodiments, IgG2a). When the parent antibody is a monoclonal antibody, exemplary isotypes include, but are not limited to, IgG, such as IgG1, IgG2, IgG3, and IgG4; IgM; IgA, such as IgA1 and IgA2; IgD; and IgE, and may preferably include IgG and IgM. The isotype and subclass of a monoclonal antibody can be determined, for example, by Ouchterlony test, ELISA, or radioimmunoassay (hereinafter referred to as "RIA"). Commercially available kits for identification (e.g., Mouse Typer Kit; Bio-Rad Laboratories, Inc., and Rat Monoclonal Antibody Isotyping Test Kit: AbD Serotec) can be used.

[0203] In some embodiments, the antibody fragment, antigen-binding fragment or polypeptide is derived from an antibody or equivalent specific for the IhfB fragment mB4 (SEQ ID NO: 17) and comprises a constant region derived from an IgG, such as, but not limited to, IgG1.

[0204] In some embodiments, the antibody fragment, antigen-binding fragment, or polypeptide is derived from an antibody that comprises a heavy chain constant region at least 80% identical, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 100% identical to the heavy chain constant region sequence of any one of the following antibodies: (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfB4 NTHI 12E6.F8.D12.D5, or their respective equivalents.

[0205] In some embodiments, the antibody fragment, antigen-binding fragment, or polypeptide is derived from an antibody that comprises a light chain constant region at least 80% identical, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 100% identical to the light chain constant region sequence of any one of the following parent antibodies: (i) the antibody produced by hybridoma cell line IhfA5 NTHI 14G8.F5.G6, (ii) the antibody produced by hybridoma cell line IhfB4 NTHI 4E11.E5.G2, and (iii) the antibody produced by hybridoma cell line mIhfB4 NTHI 12E6.F8.D12.D5, or their respective equivalents.

[0206] In some embodiments of the antibody fragments, antigen-binding fragments or polypeptide-derived antibodies provided herein, the parent antibody or Fab (fragment antigen-binding) fragment binds to the epitope bound by the DNABII antibody.

[0207] In some embodiments of the parent antibodies provided herein, the antibody fragment contains structural modifications that facilitate rapid binding and cellular uptake and / or slow release.

[0208] Antibody fragments, antigen-binding fragments and polypeptides and their equivalents can be combined with a detectable or purification label and / or a carrier, e.g., a pharmaceutically acceptable carrier or other agent, to provide a formulation for use and / or storage. Methods for antibody production

[0209] The general structure of an antibody is known in the art and will only be briefly summarized here. An immunoglobulin monomer contains two heavy chains and two light chains connected by disulfide bonds. Each heavy chain pairs with one of the light chains to which it is directly linked via a disulfide bond. Each heavy chain contains a constant region (which varies depending on the antibody isotype) and a variable region. The variable region contains three hypervariable regions (or complementarity-determining regions), designated CDRH1, CDRH2, and CDRH3, supported within framework regions. Each light chain contains a constant region and a variable region, and the variable region, like the heavy chain variable region, contains three hypervariable regions (designated CDRL1, CDRL2, and CDRL3), supported by framework regions.

[0210] The hypervariable regions of each pair of heavy and light chains cooperate with each other to form an antigen-binding site capable of binding to a target antigen. The binding specificity of a pair of heavy and light chains is defined by the sequences of CDR1, CDR2, and CDR3 of the heavy and light chains. Therefore, once a set of CDR sequences (i.e., the sequences of CDR1, CDR2, and CDR3 of the heavy and light chains) that confer a particular binding specificity is determined, the set of CDR sequences can, in principle, be inserted into an appropriate position in any other antibody framework region linked to the constant region of any antibody to create a different antibody with the same antigen-binding specificity.

[0211] Antibodies for producing Fab fragments can be produced using conventional techniques known in the art and fully described in the literature. There are several methodologies for producing polyclonal antibodies. For example, polyclonal antibodies are usually produced by immunizing suitable mammals, such as, but not limited to, chickens, goats, sheep, guinea pigs, hamsters, horses, dogs, mice, rats, and rabbits. When an antigen is injected into a mammal, it induces B lymphocytes to produce immunoglobulins specific to the antigen. Immunoglobulins can be purified from the serum of the mammal. Antibodies specific to the IHFα subunit and / or IHFβ subunit can be produced by injecting polypeptides corresponding to different epitopes of IHFα and IHFβ. For example, antibodies can be produced using 20 amino acids from each subunit, such as SEQ ID NOs: 12 and 13 (fragments A3 and A5 of IHF, respectively) for IHFα, SEQ ID NO: 14 (fragment A5 of HU) for HU, and SEQ ID NOs: 15 to 17 (fragments B2, B4, and mB4 of IHF) for IHFβ.

[0212] Variations of this method include modifying the adjuvant, route and site of administration, injection volume per site, and number of sites per animal to optimize production and humanely handle animals. For example, adjuvants are typically used to improve or enhance the immune response to an antigen. Most adjuvants cause antigen retention at the injection site, allowing for stowed release of the antigen into the draining lymph nodes. Other adjuvants include surfactants, which promote the concentration of protein antigen molecules over a large surface area, and immunostimulatory molecules. Non-limiting examples of adjuvants for generating polyclonal antibodies include Freund's adjuvant, the Ribi adjuvant system, adjuvants derived from MPL, adjuvants derived from E. coli heat-labile enterotoxin (e.g., dmLT = double mutant unstable toxin), and Titermax. Polyclonal antibodies can be produced using methods known in the art, some of which are described in U.S. Patent Nos. 7,279,559; 7,119,179; 7,060,800; 6,709,659; 6,656,746; 6,322,788; 5,686,073; and 5,670,153.

[0213] Monoclonal antibodies can be produced using conventional hybridoma methods, which are known in the art and fully described in the literature. For example, hybridomas can be produced using a suitable immortal cell line (e.g., Sp2 / 0 cells, Sp2 / 0-AG14 cells, NSO cells, NS1 cells, NS2 cells, AE-1 cells, L.5 cells, P3X63Ag8,653 cells, Sp2 SA3 cells, Sp2 MAI cells, Sp2 SS1 cells, Sp2 SA5 cells, U397 cells, MIA 144 cells, ACT IV cells, MOLT4 cells, DA-1 cells, JURKAT cells, WEHI cells, K-562 cells, COS cells, RAJI cells, NIH 313 cells, HL-60 cells, MLA 144 cells, NAMAIWA cells, NEURO cells, or the like). Myeloma cell lines, such as, but not limited to, myeloma cell lines, such as, but not limited to, 2A cells, CHO cells, PerC.6 cells, YB2 / O cells, or heteromyelomas, fusion products thereof, or any cells or fusion cells derived therefrom, or any other suitable cell line known in the art (see cell lines at the following web addresses, e.g., atcc.org, lifetech.com, last accessed November 26, 2007), isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B-cell containing cells, or recombinant or endogenous viruses, bacteria, algae, prokaryotes, amphibians, insects, reptiles, fish, mammals, rodents, horses, sheep (o The antibodies are produced by fusing with antibody-producing cells such as, but not limited to, any other cell that expresses heavy chain constant sequences or heavy chain variable sequences or heavy chain framework sequences or heavy chain CDR sequences or light chain constant sequences or light chain variable sequences or light chain framework sequences or light chain CDR sequences as endogenous or heterologous nucleic acids as nucleic acids of eukaryotic genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single-stranded, double-stranded, or triple-stranded nucleic acids, hybridized nucleic acids, etc., or any combination thereof, of a mammalian animal.Antibody-producing cells can also be obtained from the peripheral blood, or in certain embodiments, the spleen or lymph nodes, of humans or other suitable animals immunized with the antigen of interest. Any other suitable host cells can also be used to express heterologous or endogenous nucleic acid encoding the antibodies, specified fragments, or variants thereof of the present disclosure. Fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other appropriate known methods and cloned via limiting dilution, cell sorting, or other known methods. Some embodiments disclosed herein relate to specific hybridomas producing monoclonal antibodies against IhfA5 NTHI 14G8.F5.G6 (ATCC No.: PTA-122334), IhfB4 NTHI 4E11.E5.G2 (ATCC No.: PTA-122336), mIhfB4 NTHI 12E6.F8.D12.D5 (ATCC No.: PTA-122335).

[0214] Other suitable methods for producing or isolating antibodies with the required specificity can be used, including, but not limited to, selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to, bacteriophage display libraries, ribosome display libraries, oligonucleotide display libraries, cDNA display libraries, etc.; for example, commercially available libraries from commercial sources such as MorphoSys (Martinsreid / Planegg, Del.), BioInvent (Lund, Sweden), and Affitech (Oslo, Norway) using methods known in the art). Methods known in the art are described in the patent literature, some of which include U.S. Patent Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; and 5,976,862. An alternative method relies on immunization of transgenic animals (e.g., SCID mice; Nguyen et al. (1977), Microbiol. Immunol. 41:901-907 (1997); Sandhu et al. (1996), Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998), Mumma 93:154-161) to produce a repertoire of human antibodies as known in the art and / or as described herein. Such techniques include ribosome display (e.g., Wanes et al. (1997), Proc. Natl. Acad. Sci. USA 94:4937-4942; Hanes et al. (1998), Proc. Natl. Acad. Sci. USA 95:14130-14135), single-cell antibody production methods (e.g., the selected lymphocyte antibody method ("SLAM") (U.S. Patent No. 5,627,052; Wen et al. (1987), J. Immunol. 17:887-892; Babcook et al. (1996), Proc. Natl. Acad. Sci. USA 93:7843-7848), gel microdroplets and flow cytometry (Powell et al. (1990), Biotechnol. 8:333-337; One Cell Systems (Cambridge, Mass); Gray et al. (1995), J. Imm. Meth., 182:155-163; and Kenny et al. (1995), Bio. Technol., 13:787-790), B cell selection (Steenbakkers et al. (1994), Molec. Biol. Reports, 19:125-134). Additional methods, see U.S. Pat. No. 7,939,344, rely on isolation of human antibodies from human serum using techniques known in the art.

[0215] Antibodies can also be produced by inducing in vivo production in lymphocyte populations, or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents (Orlandi et al., 1989). , PNAS, 86:3833-3837; Winter et al. (1991), Nature, 349:293-299).

[0216] Alternatively, techniques for producing single chain antibodies can be used. Single chain antibodies (scF v ) comprises a heavy chain variable region and a light chain variable region connected by a linker peptide (typically about 5 to 25 amino acids in length). vWithin an scF, the heavy and light chain variable regions may be derived from the same antibody or from different antibodies. v have used recombinant techniques to express scF in a host organism such as E. coli. v scF can be synthesized by expression of a vector encoding v The DNA encoding scF can be obtained by using, as a template, DNA selected from DNA encoding the heavy chain or the heavy chain variable region of the above-mentioned antibody and DNA encoding the light chain or the light chain variable region thereof, which DNA encodes the entire amino acid sequence or a partial DNA encoding a desired amino acid sequence, and performing PCR amplification using primer pairs that define both ends of the DNA, and further performing amplification using DNA encoding a polypeptide linker portion combined with primer pairs that define both ends of the DNA, and ligating both ends of the linker to the heavy chain and the light chain, respectively. v An expression vector containing DNA encoding the above and a host transformed with the expression vector can be obtained according to conventional methods known in the art.

[0217] Antigen-binding fragments, such as F(ab')2 fragments, can be generated by pepsin digestion of antibody molecules, and Fab fragments can be generated by reducing the disulfide bridges of F(ab')2 fragments. Fab fragments, particularly mouse Fab fragments generated from mouse monoclonal antibodies, can also be generated by digestion of antibodies with the thiol protease ficin in the presence of cysteine. Fab fragments, particularly rabbit Fab fragments generated from rabbit polyclonal antibodies, can also be generated by digestion of antibodies with the protease papain in the presence of cysteine-HCl. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al. (1989), Science 256:1). (pp. 275-1281) Once the fragments are generated, they can be isolated and sequenced using conventional techniques to determine the amino acid sequence.

[0218] Antibody derivatives of the present disclosure can also be prepared by delivering a polynucleotide encoding an antibody fragment, antigen-binding fragment, or polypeptide as disclosed herein to a suitable host to provide a transgenic animal or mammal, such as a goat, cow, horse, sheep, etc., that produces such antibodies in its milk. These methods are known in the art and are described, for example, in U.S. Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.

[0219] The term "antibody derivative" includes post-translational modifications to the linear polypeptide sequence of an antibody or fragment. For example, U.S. Patent No. 6,602,684 B1 describes methods for producing modified glycoforms of antibodies, including whole antibody molecules, antibody fragments, or fusion proteins, that contain a region equivalent to the Fc region of an immunoglobulin and have enhanced Fe-mediated cytotoxicity, and the glycoproteins thus produced.

[0220] The parent antibodies from which the antibody fragments, antigen-binding fragments, and polypeptides disclosed herein are derived also include derivatives modified by the covalent attachment of any type of molecule to the antibody, provided that the covalent attachment does not prevent the antibody from eliciting an anti-idiotypic response. Antibody derivatives include, but are not limited to, antibodies modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization via known protecting / blocking groups, proteolytic cleavage, conjugation to intracellular ligands or other proteins, and the like. Non-limiting examples of modified antibodies contemplated herein are unglycosylated whole antibody molecules, antibody fragments, or fusion proteins containing a region equivalent to the Fc region of an immunoglobulin. Such unglycosylated forms can be produced, for example, in host cells lacking the ability to modify proteins with N-linked glycans, or by mutating N-linked consensus sites on the antibody of interest. In addition, derivatives can contain one or more non-classical amino acids.

[0221] Antibody derivatives of parent antibodies can also be prepared by generating transgenic plants and cultured plant cells (e.g., but not limited to, tobacco, corn, and duckweed) that deliver the polynucleotides disclosed herein to produce such antibodies, specified portions, or variants in plant parts or cultured cells derived therefrom. For example, Cramer et al. (1999), Curr. Top. Microbol. Immunol., 240:95-118, and references cited therein, describe the production of transgenic tobacco leaves that express large amounts of recombinant proteins, e.g., using inducible promoters. Transgenic corn has been used to express mammalian proteins at commercial production levels with biological activity equivalent to mammalian proteins produced in other recombinant systems or purified from natural sources. See, e.g., Hood et al. (1999), Adv. Exp. Med. Biol., 464:127-147, and references cited therein. Antibody derivatives have also been produced in large quantities from transgenic plant seeds, including tobacco seeds and potato tubers, that contain antibody fragments such as single-chain antibodies (scFv). See, e.g., Conrad et al. (1998), Plant Mol. Biol., 38:101-109, and references cited therein. Thus, antibodies can also be produced using transgenic plants according to known methods.

[0222] Antibody derivatives of a parent antibody can also be produced, for example, by adding exogenous sequences to modify immunogenicity or to reduce, enhance, or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic. Generally, some or all of the non-human or human CDR sequences are maintained while substituting human or other amino acids for the non-human sequences in the variable and constant regions.

[0223] Humanization or engineering of antibodies can be carried out using any known method, such as, but not limited to, those described in U.S. Patent Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.

[0224] Further provided is an isolated polypeptide comprising, alternatively consisting essentially of, or even consisting of, a Fab fragment or antigen-binding fragment or polypeptide disclosed herein, further comprising a detectable label or purification label as described herein.

[0225] Chimeric, humanized, or primatized parent antibodies of the antibody fragments, antigen-binding fragments, or polypeptides of the present disclosure can be prepared based on the sequence of a reference monoclonal antibody prepared using standard molecular biology techniques. Such antibodies can be used to generate antibody fragments (e.g., Fab fragments or antigen-binding fragments) of the present disclosure.

[0226] DNA encoding heavy and light chain immunoglobulins can be obtained from the desired hybridoma and engineered to contain non-canonical (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create chimeric antibodies, mouse variable regions can be linked to human constant regions using methods known in the art (U.S. Patent No. 4,816,567). To create humanized antibodies, mouse CDR regions can be inserted into a human framework using methods known in the art (U.S. Patent No. 5,225,539, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370). Similarly, to create primatized antibodies, mouse CDR regions can be inserted into a primate framework using methods known in the art (PCT International Patent Application Publication Nos. WO93 / 02108 and WO99 / 55369).

[0227] Techniques for producing partially to fully human antibodies are known in the art, and any such technique can be used. According to one embodiment, fully human antibody sequences are produced in transgenic mice engineered to express human heavy and light chain antibody genes. Several such transgenic mouse strains have been generated that are capable of producing different classes of antibodies. B cells from transgenic mice producing the desired antibody can be fused to generate hybridoma cell lines for continuous production of the desired antibody (see, e.g., Russell et al. (2000), Infection and Immunity, April 2000:1820-1826; Gallo et al. (2000), European J. of Immun., 30:534-540; Green (1999), J. of Immun. Methods, 231:11-23; Yang et al. (1999A), J. of Leukocyte Biology, 66:401-410; Yang (1999B), Cancer Research, 59(6):1236-1243; Jakobovits (1998), Advanced Drug Reviews, 31:33-42; Green and Jakobovits (1998), J. Exp. Med., 188(3):483-495; Jakobovits (1998), Exp. Opin. Invest. Drugs, 7(4):607-614; Tsuda et al. (1997), Genomics, 42:413-421; Sherman-Gold (1997), Genetic Engineering News, 17(14); Mendez et al. (1997), Nature Genetics, 15:146-156; Jakobovits (1996), "Weir's Handbook of Experimental Immunology, The Integrated Immune System," IV, 194.1-194.7; Jakobovits (1995), Current Opinion in Biotechnology, 6:561-566; Mendez et al. (1995), Genomics, 26:294-307; Jakobovits (1994), Current Biology, 4(8):761-763; Arbones et al. (1994), Immunity, 1(4):247-260; Jakobovits (1993), Nature, 362(6417):255-258; Jakobovits et al. (1993), Proc. Natl. Acad. Sci. USA, 90(6):2551-2555; and U.S. Patent No. 6,075,181).

[0228] The antibodies disclosed herein can also be modified to create chimeric antibodies, which are antibodies in which the various domains of the antibody's heavy and light chains are encoded by DNA from multiple species. See, e.g., U.S. Patent No. 4,816,567.

[0229] Alternatively, the antibodies disclosed herein can also be modified to create veneered antibodies. A veneered antibody is an antibody in which the exterior amino acid residues of an antibody from one species have been carefully replaced, or "veneered," with exterior amino acid residues from a second species, so that the antibody from the first species is not immunogenic in the second species, thereby reducing the immunogenicity of the antibody. Because the immunogenicity of a protein depends primarily on the nature of its surface, substituting exposed residues that differ from those typically found in antibodies from another mammalian species may reduce the immunogenicity of the antibody. Such careful substitution of exterior residues should have little or no effect on internal domains or interdomain contacts. Therefore, changes limited to framework residues in the variable regions should not affect the binding properties of the ligand. This process is referred to as "veneering," because only the outer surface, or skin, of the antibody is altered, while supporting residues remain unperturbed.

[0230] The "veneering" procedure utilizes available sequence data for human antibody variable domains compiled by Kabat et al. (1987), "Sequences of Proteins of Immunological Interest," 4th ed., Bethesda, Md., National Institutes of Health, updates to this database, and other accessible databases (both nucleic acid and protein databases) in the United States and abroad. Non-limiting examples of methods used to produce veneered antibodies include EP 519596, U.S. Pat. No. 6,797,492, and are also described in Padlan et al. (1991), Mol. Immunol. 28(4-5):489-498.

[0231] Antibody fragments, antigen-binding fragments, and polypeptides as disclosed herein can be recovered and purified from recombinant cell cultures by known methods, including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

[0232] Antibodies for preparation of antibody fragments, antigen-binding fragments, or antibodies of the present disclosure include naturally occurring purified products, products of chemical synthetic procedures, and products produced via recombinant methods from eukaryotic hosts, including, for example, yeast, higher plants, insect, and mammalian cells, or alternatively, from prokaryotic hosts as described above. Birch and Radner (2006), Adv. Drug Delivery Rev., 58:671-685, describe many antibody production systems.

[0233] If the test antibody fragment binds to a protein or polypeptide, the test antibody fragment and the antibody fragment provided by the present disclosure are equivalent. It is also possible to determine, without undue experimentation, whether an antibody fragment has the same specificity as the antibody fragment disclosed herein by determining whether the test antibody prevents the antibody fragment from binding to the protein or polypeptide with which it is normally reactive. If the test antibody fragment competes with the antibody fragment disclosed herein, as indicated by reduced binding by the antibody fragment disclosed herein, the two antibody fragments are likely to bind to the same epitope or a closely related epitope. Alternatively, the antibody fragment disclosed herein can be preincubated with the protein with which it is normally reactive to determine whether the test antibody fragment's ability to bind to the antigen is inhibited. If the test antibody fragment is inhibited, then the antibody almost certainly has the same epitope specificity as the antibody fragment disclosed herein or a closely related epitope specificity.

[0234] The terms "antibody" and "antibody fragment" are also intended to encompass antibodies of all immunoglobulin isotypes and immunoglobulin subclasses. A particular isotype of monoclonal antibody can be prepared directly by selection from the initial fusion, or can be subsequently prepared from parent hybridomas secreting monoclonal antibodies of a different isotype by using sib selection methods to isolate class-switched variants using procedures described in Steplewski et al. (1985), Proc. Natl. Acad. Sci. USA 82:8653, or Spira et al. (1984), J. Immunol. Methods 74:307. Alternatively, recombinant DNA methods can be used.

[0235] Isolation of other monoclonal antibodies for use in generating antibody fragments, antigen-binding fragments, and polypeptides with the specificity of the monoclonal antibodies described herein can also be achieved by one skilled in the art through the generation of anti-idiotypic antibodies (Herlyn et al. (1986) Science 232:100). Anti-idiotypic antibodies are antibodies that recognize unique determinants present in the monoclonal antibody of interest.

[0236] In some embodiments disclosed herein, it is useful to detectably or therapeutically label antibody fragments, antigen-binding fragments, or polypeptides.Suitable labels are described herein.Methods for conjugating antibodies and polypeptides with these agents are known in the art.For illustrative purposes only, antibody fragments, antigen-binding fragments, or polypeptides can be labeled with detectable moieties, such as radioactive atoms, chromophores, fluorophores, etc.Such labeled antibodies can be used in diagnostic techniques either in vivo or in isolated test samples.

[0237] The sensitivity of antibodies, fragments, and polypeptides in assays can be increased by coupling them to low molecular weight haptens. The hapten can then be specifically detected by a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific anti-hapten antibodies. See Harlow and Lane (1988), supra.

[0238] The variable regions of the parent antibodies of the present disclosure can be modified by mutating amino acid residues within the VH CDR1 and / or VL CDR1 regions, VH CDR2 and / or VL CDR2 regions, and / or VH CDR3 and / or VL CDR3 regions to improve one or more binding characteristics (e.g., affinity) of the antibody. Mutations can be introduced via site-directed mutagenesis or PCR-mediated mutagenesis, and the effect on antibody binding or other functional properties of interest can be assessed in appropriate in vitro or in vivo assays. In certain embodiments, conservative modifications are introduced, typically altering no more than one, two, three, four, or five residues within the CDR regions. Mutations can be amino acid substitutions, additions, or deletions.

[0239] For example, antibodies can be modified with framework modifications to reduce immunogenicity by "backmutating" one or more framework residues to the corresponding germline sequence.

[0240] In addition, the antibody fragments, antigen-binding fragments, or polypeptides disclosed herein can be engineered to include modifications in the Fc region that alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Such modifications include, but are not limited to, altering the number of cysteine ​​residues in the hinge region to facilitate assembly of the light and heavy chains or to increase or decrease antibody stability (U.S. Patent No. 5,677,425), and mutating amino acids in the Fc hinge region that decrease the biological half-life of the antibody (U.S. Patent No. 6,165,745).

[0241] In addition, the antibody fragments disclosed herein can also be chemically modified. For example, the glycosylation of an antibody can be altered by modifying one or more glycosylation sites within the antibody sequence to increase the affinity of the antibody for an antigen (U.S. Patent Nos. 5,714,350 and 6,350,861). Alternatively, to increase antibody-dependent cell-mediated cytotoxicity, the antibody can be expressed in a host cell with an altered glycosylation machinery to produce a hypofucosylated antibody with reduced amounts of fucosyl residues or an antibody with increased bisecting GlcNac structures (Shields et al., (2002), J. Biol. Chem., 277:26733-26740; Umana et al., 1999, Nat. Biotech., 17:176-180).

[0242] The antibodies, antigen-binding fragments, or polypeptides disclosed herein can be PEGylated to extend their biological half-life by reacting them with polyethylene glycol (PEG), a reactive ester of PEG, or an aldehyde derivative of PEG under conditions that result in attachment of one or more PEG groups to the antibody or antibody fragment. PEGylation can be carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG used to derivatize other proteins, such as mono(C1-C10)alkoxypolyethyleneglycol or aryloxypolyethyleneglycol, or polyethyleneglycolmaleimide. The antibody to be PEGylated can be an aglycosylated antibody. Methods for PEGylation of proteins are known in the art and can be applied to the antibodies disclosed herein (EP 0154316 and EP 0401384).

[0243] Additionally, antibody-binding fragments, antigen-binding fragments, or polypeptides can be chemically modified by conjugating or fusing the antigen-binding region to serum proteins, such as human serum albumin, to increase the half-life of the resulting molecule. Such techniques are described, for example, in EP0322094 and EP0486525.

[0244] The antibody fragments, antigen-binding fragments, or polypeptides disclosed herein can be conjugated with diagnostic agents and used for diagnosis, e.g., to monitor the onset or progression of disease and determine the effectiveness of a given treatment regimen. Examples of diagnostic agents include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, various positron-emitting metals used in positron emission tomography, and non-radioactive paramagnetic metal ions. The detectable substance can be directly coupled or conjugated to the antibody or its fragment, or indirectly linked or conjugated to the antibody or its fragment via a linker using techniques known in the art. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include: 125 I, 131I, Indium 111, Lutetium 171, Bismuth 212, Bismuth 213, Astatine 211, Copper 62, Copper 64, Copper 67, Yttrium 90, Iodine 125, Iodine 131, Phosphorus 32, Phosphorus 33, Scandium 47, Silver 111, Gallium 67, Praseodymium 142, Samarium 153, Terbium 161, Dysprosium 166, Holmium 166, These include rhenium-186, rhenium-188, rhenium-189, lead-212, radium-223, actinium-225, iron-59, selenium-75, arsenic-77, strontium-89, molybdenum-99, rhodium-1105, palladium-109, praseodymium-143, promethium-149, erbium-169, iridium-194, gold-198, gold-199, and lead-211. Monoclonal antibodies can be indirectly conjugated to radioactive metal ions by using bifunctional chelating agents that are covalently bound to the antibodies. Chelating agents can be attached through amines (Meares et al., (1984) Anal. Biochem. 142:68-78), sulfhydral groups of amino acid residues (Koyama (1994) Chem. Abstr. 120:217-262t), and carbohydrate groups (Rodwell et al., (1986) PNAS USA 83:2632-2636; Quadri et al., (1993) Nucl. Med. Biol. 20:559-570).

[0245] Furthermore, the antibody fragments, antigen-binding fragments, or polypeptides of the present disclosure can also be conjugated to a therapeutic agent. Suitable therapeutic agents include antibiotics or, for example, antimicrobial agents, or host defense peptides (e.g., specifically targeted antimicrobial peptides (STAMPs)).

[0246] Additional suitable conjugated molecules include ribonucleases (RNases), DNases, antisense nucleic acids, inhibitory RNA molecules such as siRNA molecules, immunostimulatory nucleic acids, aptamers, ribozymes, triplex-forming molecules, and external guide sequences. Aptamers are small nucleic acids ranging from 15 to 50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets, and can bind to small molecules such as ATP (U.S. Pat. No. 5,631,146) and theophylline (U.S. Pat. No. 5,580,737), as well as large molecules such as reverse transcriptase (U.S. Pat. No. 5,786,462) and thrombin (U.S. Pat. No. 5,543,293). Ribozymes are nucleic acid molecules capable of catalyzing chemical reactions intramolecularly or intermolecularly. Ribozymes typically cleave nucleic acid substrates by recognizing and binding to the target substrate, which is then cleaved. Nucleic acid molecules with triplex-forming function can interact with double-stranded nucleic acids by forming triplexes, and can also interact with single-stranded nucleic acids, where the triplex of DNA relies on both Watson-Crick and Hoogsteen base pairing to form a complex.Triplex molecules can bind to target regions with high affinity and specificity.Suitable conjugate molecules can also include any protein that binds to DNA, provided that it does not create or stabilize biofilm architecture, and it is assumed that at least a subset of such proteins can promote the binding kinetics of the agents disclosed herein.

[0247] Functional nucleic acid molecules may act as effectors, inhibitors, regulators, and stimulators of specific activities possessed by target molecules, or may possess novel activities that are independent of other molecules.

[0248] Therapeutic agents can be linked directly or indirectly to the fragments or polypeptides using any of a number of available methods. For example, agents can be attached to the hinge region of a reduced antibody component via disulfide bond formation using a cross-linking agent such as N-succinyl-3-(2-pyridyldithio)propionate (SPDP), or via carbohydrate moieties in the Fc region of the antibody (Yu et al. (1994) Int. J. Cancer 56:244; Upeslacis et al., "Modification of Antibodies by Chemical Methods," in Monoclonal antibodies: principles and applications, Birch et al. (eds.), pp. 187-230 (Wiley-Liss, Inc., 1995); Price, "Modification of Antibodies by Chemical Methods," in Monoclonal antibodies: Production, Engineering and Clinical Application, Characterization of Synthetic Peptide-Derived Antibodies, Ritter et al. (eds.), pp. 60-84 (Cambridge University Press, 1995).

[0249] Techniques for conjugating therapeutic agents to antibodies and antibody fragments are well known (see, for example, Amon et al., "Monoclonal Antibodies For Immunotargeting of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc., 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Developments in Monoclonal Antibodies For Cancer Detection And Therapy," in Prospect of the Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy,” Baldwin et al. (eds.), pp. 303-16 (Academic Press, 1985); and Thorpe et al. (1982) Immunol. Rev. 62:119-58).

[0250] The fragments or polypeptides can be linked to another functional molecule, such as another antibody or a receptor ligand, to produce bispecific or multispecific molecules that bind to at least two or more different binding sites or target molecules. Linking an antibody to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, can be achieved, for example, via chemical coupling, genetic fusion, or noncovalent association. Multispecific molecules can further include a third binding specificity in addition to the first and second target epitopes.

[0251] Bispecific and multispecific molecules can be prepared using methods known in the art.For example, each binding unit of a bispecific molecule (hi-specific) can be produced separately, and then they can be conjugated with each other.When the binding molecule is a protein or peptide, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (sulfo-SMCC) (Karpovsky et al. (1984) J. Exp. Med., 160:1686; Liu et al. (1985) Proc. Natl. Acad. Sci. USA, 82:8648). When the binding molecule is an antibody, it can be conjugated by sulfhydryl bonding of the C-terminal hinge regions of the two heavy chains.

[0252] Antibody fragments, antigen-binding fragments, or polypeptides as disclosed herein can also be attached to solid supports, which are particularly useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0253] Antibody fragments, antigen-binding fragments, or polypeptides can also be bound to many different carriers. Thus, the present disclosure also provides compositions containing antigens or polypeptides and other active or inactive substances. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be soluble or insoluble, depending on the purpose disclosed herein. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such carriers using routine experimentation.

[0254] Further provided herein are compositions comprising, alternatively consisting essentially of, or even consisting of one or more of the above embodiments. Also provided are polynucleotides encoding the amino acid sequences of antibody fragments, antigen-binding fragments, or polypeptides, as well as methods for recombinantly producing or chemically synthesizing fragments and polypeptides. Antibody polypeptides and fragments can be produced in eukaryotic or prokaryotic cells, or by other methods known in the art and outlined herein.

[0255] The fragments and isolated polypeptides comprising the fragments disclosed herein can be selected so that they have a high level of epitope binding specificity and high binding affinity to biofilms.Generally, the greater the binding affinity of an antibody or antibody fragment or the isolated polypeptide comprising an antibody fragment, the more stringent the washing conditions can be implemented in immunoassays to remove non-specifically bound substances without removing the target.Therefore, the fragments and isolated polypeptides comprising the antibody fragments of the present technology useful in the disclosed method are usually at least 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , or 10 -12 In certain embodiments, the fragments, including antibody fragments, and isolated polypeptides have a kinetic on-rate sufficient to reach equilibrium under standard conditions in at least 12 hours, at least 5 hours, at least 1 hour, or at least 30 minutes. In another embodiment, the affinity of the antibody or antigen-binding fragment is less than or about 1000 picomolar (pM), less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, or about 10 pM, or about 9 pM, or about 8 pM, or alternatively, less than about 4 pM, or alternatively, less than about 2 pM.

[0256] In any of the above embodiments, a peptide linker can be added to the N-terminus or C-terminus of the antibody fragment, antigen-binding fragment, or polypeptide disclosed herein. A "linker" or "peptide linker" refers to a peptide sequence linked to either the N-terminus or C-terminus of a polypeptide sequence. In one aspect, the linker is from about 1 to about 20 amino acid residues in length, or alternatively from 2 to about 10 amino acid residues, or from about 3 to about 5 amino acid residues in length. An example of a peptide linker is Gly-Pro-Ser-Leu-Lys-Leu (SEQ ID NO: 43). Other examples include Gly-Gly-Gly (SEQ ID NO: 44); Gly-Pro-Ser-Leu (SEQ ID NO: 45); Gly-Pro-Ser (SEQ ID NO: 46); Pro-Ser-Leu-Lys (SEQ ID NO: 47); Gly-Pro-Ser-Leu-Lys (SEQ ID NO: 48), and Ser-Leu-Lys-Leu (SEQ ID NO: 49).

[0257] Polynucleotides encoding polypeptides Further provided herein are isolated polynucleotides encoding the above-mentioned antibody fragments, antigen-binding fragments, and polypeptides, as well as vectors and host cells containing them, and methods for recombinantly producing polypeptides using recombinant cell systems known in the art and described herein. The polynucleotides may be DNA or RNA. The polynucleotides may be operably linked to regulatory sequences, promoters, enhancers, etc., for recombinant reproduction of the polynucleotides and / or recombinant production of polypeptides. The polynucleotides may be inserted into vectors (e.g., plasmids or viruses) and inserted into appropriate host cells (eukaryotic or prokaryotic) for recombinant reproduction or expression. Thus, antibody fragments or polypeptides can be recombinantly produced by culturing host cells under conditions for expression of the polynucleotide. In one embodiment, the polypeptides are isolated from the cells or culture medium. The present application also provides the polynucleotides described herein conjugated to a detectable agent for use in diagnostic methods.

[0258] Further provided is a vector or antibody that comprises isolated or recombinant polynucleotide, examples of which are known in the art and are briefly described herein.In one embodiment, where more than one isolated or recombinant polynucleotide is to be expressed as a single unit, isolated or recombinant polynucleotide can be contained in a polycistronic vector.Polynucleotide can be DNA, RNA, mRNA, or interference RNA such as siRNA, miRNA or dsRNA.

[0259] The present disclosure also provides isolated or recombinant polynucleotides operably linked to a promoter of RNA transcription and other regulatory sequences for DNA or RNA replication and / or transient or stable expression. As used herein, the term "operably linked" means that the promoter is positioned to direct transcription of RNA from the DNA molecule. Examples of such promoters are SP6, T4, and T7. In certain embodiments, cell-specific promoters are used to direct cell-specific expression of an inserted polynucleotide. Vectors containing a promoter or promoter / enhancer, along with a termination codon and selectable marker sequence, and a cloning site into which a small piece of DNA can be operably linked to the promoter, are known in the art and commercially available. For general methods and cloning strategies, see "Gene Expression Technology" (Goeddel, ed., Academic Press, Inc. (1991)) and the references cited therein, as well as "Vectors: Essential Data Series" (Gacesa and Ramji, eds., John Wiley & Sons, NY (1994)), which contains references to maps, functional characteristics, commercial sources, and GenEMBL accession numbers for a variety of suitable vectors.

[0260] In one embodiment, polynucleotides derived from the polynucleotides disclosed herein encode polypeptides or proteins having diagnostic and therapeutic utility as described herein, as well as probes for identifying protein transcripts, which may or may not be present. These nucleic acid fragments can be prepared, for example, by restriction enzyme digestion of larger polynucleotides, and then labeled with a detectable marker. Alternatively, nick translation of molecules can be used to generate random fragments. For methods of preparing and labeling such fragments, see Sambrook et al. (1989), supra.

[0261] Expression vectors containing these nucleic acids are useful for creating host-vector systems for producing proteins and polypeptides. It has been shown that these expression vectors must be replicable in the host organism, either as episomes or as an integral part of the chromosomal DNA. Non-limiting examples of suitable expression vectors include plasmids, yeast vectors, viral vectors, and liposomes. Adenoviral vectors are particularly useful for introducing genes into tissues in vivo because they efficiently transform cells with high levels of expression both in vitro and in vivo. When the nucleic acid is inserted into a suitable host cell, such as a prokaryotic or eukaryotic cell, and the host cell replicates, the protein can be recombinantly produced. Suitable host cells depend on the vector and can include mammalian, animal, human, simian, insect, yeast, and bacterial cells constructed using known methods. See Sambrook et al. (1989), supra. In addition to using viral vectors to insert exogenous nucleic acids into cells, nucleic acids can be inserted into host cells by methods known in the art, such as transformation for bacterial cells; transfection using calcium phosphate precipitation or DEAE-dextran for mammalian cells; electroporation; or microinjection. For methods, see Sambrook et al. (1989), supra. Thus, the present disclosure also provides host cells, e.g., prokaryotic cells such as mammalian cells, animal cells (rat or mouse), human cells, or bacterial cells, containing a polynucleotide encoding a protein or polypeptide or antibody.

[0262] Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be added before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can also be further modified after polymerization, such as by conjugation with a labeled component. This term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment disclosed herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms that are known or predicted to be in a double-stranded form.

[0263] The present disclosure also provides genetically modified cells that contain and / or express the polynucleotides disclosed herein. Genetically modified cells can be produced by inserting upstream regulatory sequences, such as promoters or gene activators (see U.S. Patent No. 5,733,761).

[0264] Polynucleotides can be conjugated to detectable markers, such as enzyme labels or radioisotopes, for detecting nucleic acid and / or gene expression in cells.A wide variety of suitable detectable markers are known in the art, including fluorescent ligands, radioactive ligands, enzyme ligands, or other ligands such as avidin / biotin, which can produce detectable signals.In one embodiment, it may be desirable to use fluorescent labels or enzyme tags such as urease, alkaline phosphatase, or peroxidase instead of radioactive or other environmentally undesirable reagents.In the case of enzyme tags, calorimetric indicator substrates can be used to provide visual or spectrophotometrically detectable means for identifying specific hybridization with complementary nucleic acid-containing samples. Therefore, the present disclosure also provides a method for detecting a single-stranded polynucleotide or its complement by contacting the target single-stranded polynucleotide with a labeled single-stranded polynucleotide (probe) that is part of the polynucleotide disclosed herein under conditions that allow hybridization of complementary single-stranded polynucleotides (optionally, moderately stringent hybridization conditions), or optionally, under highly stringent hybridization conditions. Hybridized polynucleotide pairs are separated from unhybridized single-stranded polynucleotides. Hybridized polynucleotide pairs are detected using methods known to those skilled in the art, for example, the methods described in Sambrook et al. (1989), supra.

[0265] The polynucleotides embodied in the present disclosure can be obtained by chemical synthesis, recombinant cloning, PCR, or any combination thereof.Chemical polynucleotide synthesis methods are known in the art and do not need to be described in detail herein.Those skilled in the art can use the sequence data provided herein to obtain desired polynucleotides by using a DNA synthesizer or by requesting commercial services.

[0266] The polynucleotides disclosed herein can be isolated or replicated using PCR. PCR technology is the subject of U.S. Pat. Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202, and is described in "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., Birkhauser Press, Boston (1994)) or MacPherson et al. (1991) and (1995), supra, and the references cited therein. Alternatively, one skilled in the art can replicate DNA using the sequences provided herein and a commercially available DNA synthesizer. Thus, the present disclosure also provides a process for obtaining the polynucleotides disclosed herein by providing the linear sequence of the polynucleotide, nucleotides, appropriate primer molecules, chemicals such as enzymes, and instructions for replicating them, and chemically replicating or linking the nucleotides in the appropriate orientation to obtain a polynucleotide. In another embodiment, these polynucleotides are further isolated. Furthermore, those skilled in the art can insert the polynucleotide into an appropriate replicable vector and then insert the vector into an appropriate host cell (prokaryotic or eukaryotic cell) for replication and amplification. The DNA thus amplified can be isolated from the cell by methods known to those skilled in the art. Also provided herein is a process for obtaining a polynucleotide by this method, as well as the polynucleotide thus obtained.

[0267] RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell. The DNA can be delivered by any suitable method, for example, by using an appropriate gene delivery vehicle (e.g., liposome, plasmid, or vector), or by electroporation. After the cells have replicated and the DNA has been transcribed into RNA, the RNA can then be isolated using methods known to those skilled in the art, for example, the methods set forth in Sambrook et al. (1989), supra. For example, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. (1989), supra, or extracted with nucleic acid-binding resins according to the accompanying instructions provided by the manufacturer.

[0268] Polynucleotides exhibiting sequence complementarity or sequence homology to the polynucleotides disclosed herein are useful as hybridization probes or as equivalents of the specific polynucleotides identified herein. Because the complete coding sequence of the transcript is known, any portion of this sequence or a homologous sequence can be used in the methods disclosed herein.

[0269] It is known in the art that a "perfect match" probe is not required for specific hybridization. Minor changes in the probe sequence, achieved by substitution, deletion, or insertion of a small number of bases, do not affect the specificity of hybridization. Generally, a base pair mismatch of around 20% (when optimally aligned) can be tolerated. In some embodiments, probes useful for detecting the aforementioned mRNAs are at least about 80% identical to the homologous region. In some embodiments, the probe is 85% identical to the corresponding gene sequence after alignment with the homologous region, and in some embodiments, the probe exhibits 90% identity.

[0270] These probes can be used in radioassays (for example, Southern blot analysis and Northern blot analysis) to detect, prognose, diagnose, or monitor various cells or tissues containing these cells.Probes can also be attached to solid supports or arrays such as chips for use in high-throughput screening assays to detect the expression of genes corresponding to the polynucleotides disclosed herein.Therefore, the present disclosure also provides probes that comprise or correspond to the polynucleotides disclosed herein, or their equivalents, or their complements, or their fragments, attached to solid supports for use in high-throughput screening.

[0271] The overall size of the fragment, as well as the size of the complementary stretch, will depend on the intended use or application of the particular nucleic acid segment. Smaller fragments are generally used in hybridization embodiments, and the length of the complementary region can vary, such as between at least 5-10 nucleotides and about 100 nucleotides, or even full length, depending on the complementary sequence desired to be detected.

[0272] Nucleotide probes with complementary sequences spanning a stretch greater than 5 to 10 nucleotides in length are generally preferred to increase hybrid stability and selectivity, thereby improving the specificity of the resulting hybrid molecules. In certain embodiments, polynucleotides can be designed with gene-complementary stretches of 10 nucleotides or more, or even longer if desired. Such fragments can be readily prepared, for example, by direct synthesis of the fragments via chemical means, by applying nucleic acid reproduction techniques such as the PCR technique involving two priming oligonucleotides described in U.S. Pat. No. 4,603,102, or by introducing selected sequences into a recombinant vector for recombinant production. In one aspect, the probes are about 50 to 75 nucleotides or more in length, alternatively, 50 to 100 nucleotides in length.

[0273] The polynucleotides of the present disclosure can be used as primers for detecting genes or gene transcripts expressed in the cells described herein. In this context, amplification refers to any method that utilizes a primer-dependent polymerase that can replicate the target sequence with reasonable fidelity. Amplification can be performed with natural or recombinant DNA polymerases, such as T7 DNA polymerase, the Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. For illustrative purposes only, the primers are the same length as those identified for the probe.

[0274] One method for amplifying polynucleotides is PCR, and PCR amplification kits are commercially available. After amplification, the resulting DNA fragments can be detected by any suitable method known in the art, such as agarose gel electrophoresis, followed by visualization with ethidium bromide staining and ultraviolet illumination.

[0275] Methods for administering an effective amount of gene delivery vector or vehicle to cells have been developed and are known to those skilled in the art, and are described herein.Methods for detecting gene expression in cells are known in the art, and include techniques such as hybridization to DNA microarrays, in situ hybridization, PCR, RNase protection assay and Northern blot analysis.Such methods are useful for detecting and quantifying the expression of genes in cells.Alternatively, the expression of encoded polypeptides can also be detected by various methods.In particular, it is useful to prepare polyclonal or monoclonal antibodies that are specifically reactive with target polypeptides.Such antibodies are useful for visualizing cells that express polypeptides, for example, using techniques such as immunohistochemistry, ELISA and Western blotting.These techniques can be used to determine the expression level of expressed polynucleotides.

[0276] As noted above, antibodies, antibody fragments, proteins, and polypeptides can be obtained by a number of processes known to those skilled in the art, including purification, chemical synthesis, and recombinant methods. Polypeptides can be isolated from preparations such as host cell lines by standard techniques such as immunoprecipitation with antibodies, as well as gel filtration, ion exchange, reverse-phase, and affinity chromatography. For such methods, see, for example, Deutscher et al. (1999), "Guide to Protein Purification: Methods in Enzymology" (Vol. 182, Academic Press). Thus, the present disclosure also provides processes for obtaining these polypeptides, as well as products that can be obtained by these processes and the products obtained.

[0277] Polypeptides can also be obtained by chemical synthesis using a commercially available automated peptide synthesizer, such as an automated peptide synthesizer manufactured by Perkin / Elmer / Applied Biosystems, Inc., Model 430A or 431A, Foster City, Calif., USA. The synthesized polypeptides can be precipitated and further purified, for example, by high performance liquid chromatography (HPLC). Thus, the present disclosure also provides a process for chemically synthesizing the proteins disclosed herein by providing the protein sequences and reagents, such as amino acids and enzymes, and linking the amino acids together in the proper orientation and linear sequence.

[0278] composition Compositions are also provided. The compositions include a carrier and one or more of the antibody fragments or antigen-binding fragments or isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the isolated host cells disclosed herein, small molecules, antibodies, or antibody fragments (e.g., Fab (fragment antigen-binding) fragments) disclosed herein. The carrier may be one or more of a solid support or a pharmaceutically acceptable carrier. The composition may further include an adjuvant or other ingredients suitable for administration as a vaccine. In one embodiment, the composition is formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, and / or adjuvants. Additionally, embodiments of the compositions of the present disclosure include one or more of an isolated polypeptide disclosed herein, an isolated polynucleotide disclosed herein, a vector disclosed herein, a small molecule, an isolated host cell disclosed herein, an antibody or an antibody fragment (e.g., a Fab (fragment antigen-binding) fragment) of the present disclosure formulated with one or more pharmaceutically acceptable substances.

[0279] For oral preparations, any one or more of the isolated or recombinant polypeptides described herein, the isolated or recombinant polynucleotides described herein, the vectors described herein, the isolated host cells as described herein, the small molecules, or the antibodies described herein can be used alone or in a pharmaceutical formulation disclosed herein comprising or consisting essentially of the compound in combination with suitable excipients for making tablets, powders, granules, or capsules, e.g., conventional excipients such as lactose, mannitol, corn starch, or potato starch; binders such as microcrystalline cellulose, cellulose derivatives, acacia gum, corn starch, or gelatin; disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose; lubricants such as talc or magnesium stearate; and, if desired, diluents, buffers, humectants, preservatives, and flavoring agents. Pharmaceutically compatible binders and / or adjuvant materials can be incorporated as part of the composition.Tablets, pills, capsules, troches, etc. can contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0280] Pharmaceutical formulations and unit dosage forms suitable for oral administration are particularly useful in the treatment of chronic conditions, infectious diseases, and therapies where the patient self-administers the drug. In one embodiment, the formulation is specific for pediatric administration.

[0281] The present disclosure provides pharmaceutical formulations in which one or more of the antibody fragments, antigen-binding domains, isolated polypeptides, isolated polynucleotides disclosed herein, vectors disclosed herein, isolated host cells disclosed herein, or antibodies disclosed herein, each as disclosed herein, are formulated into a preparation for injection in accordance with the present disclosure by dissolving, suspending, or emulsifying in an aqueous solvent or a non-aqueous solvent such as a vegetable oil or other similar oil, a synthetic aliphatic acid glyceride, an ester of a higher aliphatic acid, or propylene glycol, and, if desired, with conventional additives such as solubilizing agents, isotonicity agents, suspending agents, emulsifying agents, stabilizers (e.g., gum arabic, alginic acid and sodium alginate), and preservatives, or other antimicrobial agents. Non-limiting examples of such include surface antigens, e.g., OMP P5, OMP 26, OMP P2, or vaccine components, e.g., type IV pilin proteins (see Jurcisek and Bakaletz (2007), J. of Bacteriology, 189(10):3868-3875; and Murphy, TF et al. (2009), The Pediatric Infectious Disease Journal, 28:S121-S126), and other antigens, such as antimicrobial agents. Another therapeutic agent is an antibody against the vaccine component. For intravenous administration, suitable carriers include physiological bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In either case, compositions for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists.

[0282] The aerosol formulations provided by the present disclosure can be administered via inhalation, and can be propellant-based or non-propellant-based.For example, the embodiments of the pharmaceutical formulations disclosed herein include the compounds disclosed herein that are formulated into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, etc. For inhalation administration, the compounds can be delivered in the form of aerosol spray from a pressurized container or dispenser containing a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer.A non-limiting example of a non-propellant is a pump spray that is propelled from a closed container by mechanical force (i.e., by pressing down the plunger with a finger, or by compressing the container through the compressive force applied to the wall of the container, or the elastic force exerted by the wall itself (for example, through an elastic bladder)).

[0283] The suppositories disclosed herein can be prepared by mixing the compounds disclosed herein with any of a variety of bases, such as emulsifying bases or water-soluble bases.The pharmaceutical formulations of the compounds disclosed herein can be administered rectally via suppositories.Suppositories can contain vehicles such as cocoa butter, carbowax, and polyethylene glycol, which melt at body temperature but solidify at room temperature.

[0284] It is possible to provide a unit dosage form for oral or rectal administration, such as syrup, elixir, and suspension, and each dosage unit, for example, a teaspoonful, a spoonful, a tablet, or a suppository, contains a predetermined amount of the composition containing one or more compounds disclosed herein.Similarly, a unit dosage form for injection or intravenous administration can contain the compound disclosed herein in a composition as a solution in sterile water, normal saline, or another pharmaceutically acceptable carrier.

[0285] The embodiments of the pharmaceutical formulations disclosed herein include pharmaceutical formulations in which one or more of the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the small molecules used in this disclosure, the isolated host cells disclosed herein, or the antibodies disclosed herein are formulated into an injectable composition.The injectable pharmaceutical formulations disclosed herein are prepared as solutions or suspensions, or as solid forms suitable for dissolving or suspending in a liquid vehicle before injection.The preparations can also be emulsified, or the active ingredient can be encapsulated in a liposome vehicle, according to other embodiments of the pharmaceutical formulations disclosed herein.

[0286] In certain embodiments, one or more of the isolated polypeptides disclosed herein, isolated polynucleotides disclosed herein, vectors disclosed herein, isolated host cells disclosed herein, antibodies or antibody fragments (e.g., Fab (fragment antigen-binding) fragments) disclosed herein are formulated for delivery via a sustained delivery system. The term "sustained delivery system" is used interchangeably herein with "controlled delivery system" and encompasses sustained (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, etc., a wide variety of which are known in the art.

[0287] Mechanical infusion pump or electromechanical infusion pump may also be suitable for use with the present disclosure.Examples of such devices include, for example, the devices described in U.S. Patent No. 4,692,147; U.S. Patent No. 4,360,019; U.S. Patent No. 4,487,603; U.S. Patent No. 4,360,019; U.S. Patent No. 4,725,852; U.S. Patent No. 5,820,589; U.S. Patent No. 5,643,207; U.S. Patent No. 6,198,966, etc. Generally, the delivery of the compounds disclosed herein can be achieved using any of a variety of rechargeable pump systems.The pump provides a constant controlled release over a period of time.In some embodiments, the compounds disclosed herein are in a liquid formulation in a drug-impermeable reservoir and are continuously delivered to individuals.

[0288] In one embodiment, the drug delivery system is at least partially an implantable device. The implantable device can be implanted at any suitable implantation site using methods and devices known in the art. An implantation site is a site within a subject's body where the drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to, subdermal, intramuscular, or other suitable sites within a subject's body. In some embodiments, a subcutaneous implantation site is used because it allows for convenient implantation and removal of the drug delivery device.

[0289] Drug release devices suitable for use in the present disclosure may be based on any of a variety of actuation modes. For example, the drug release device may be based on a diffusion system, a convection system, or an erosion system (e.g., an erosion-based system). For example, the drug release device may be an electrochemical pump, an osmotic pump, an electroosmotic pump, a vapor pressure pump, or an osmotic bursting matrix, e.g., a pump in which a drug is incorporated into a polymer that releases a drug formulation concurrently with the degradation of a drug-impregnated polymeric material (e.g., a biodegradable drug-impregnated polymeric material). In other embodiments, the drug release device is based on an electrodiffusion system, an electrolytic pump, a bubble pump, a piezoelectric pump, a hydrolysis system, or the like.

[0290] Drug release devices based on mechanical infusion pumps or electromechanical infusion pumps can also be suitable for use with the present disclosure.Examples of such devices include, for example, the devices described in United States Patent No. 4,692,147; United States Patent No. 4,360,019; United States Patent No. 4,487,603; United States Patent No. 4,360,019; United States Patent No. 4,725,852, etc. Generally, the treatment of the subject can be achieved using any of a variety of rechargeable non-replaceable pump systems.Pumps and other convective systems can be utilized because of their generally constant and highly controlled release over a period of time.In some embodiments, osmotic pumps are used because of the advantages of combining constant and highly controlled release with relatively small size (see, for example, PCT International Patent Application Publication No. WO 97 / 27840; and United States Patent No. 5,985,305 and 5,728,396). Exemplary osmotically driven devices suitable for use in the present disclosure include those described in U.S. Patent Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202; 4,111,203; 4,203,440; Such devices include, but are not necessarily limited to, those described in: 4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396, etc. A further exemplary device that may be adapted for use with the present disclosure is the Synchromed infusion pump (Medtronic).

[0291] In some embodiments, the drug delivery device is an implantable device. The drug delivery device can be implanted at any suitable implantation site using methods and devices known in the art. As referred to herein, an implantation site is a site within a subject's body where the drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to, subdermal (subcutaneous) sites, intramuscular sites, or other suitable sites within a subject's body.

[0292] Suitable excipient vehicles for the compounds disclosed herein are, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof.In addition, if desired, vehicle can also contain minor amounts of auxiliary substances, such as wetting agents, emulsifying agents, or pH buffering agents.In view of the present disclosure, those skilled in the art will know or will be able to understand how to prepare such dosage forms.See, for example, "Remington's Pharmaceutical Sciences", Mack Publishing Company, Easton, Pa., 17th Edition, 1985.In any case, the composition or formulation to be administered contains a sufficient amount of compound to achieve the desired state in the treated subject.

[0293] The compositions of the present disclosure include compositions comprising sustained-release or controlled-release matrices. Additionally, embodiments of the present disclosure can be used in conjunction with other treatments that utilize sustained-release formulations. As used herein, a sustained-release matrix is ​​a matrix made of a material, usually a polymer, that is degradable through enzymatic or acid-based hydrolysis or dissolution. Once inserted into the body, the matrix is ​​acted upon by enzymes and bodily fluids. The sustained-release matrix is ​​preferably selected from biocompatible materials such as liposomes, polylactide (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide-co-glycolide (copolymer of lactic and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, and isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicone. Exemplary biodegradable matrices include polylactide matrices, polyglycolide matrices, and polylactide-co-glycolide (copolymers of lactic and glycolic acid) matrices.

[0294] In another embodiment, the antibody fragment (as well as the combination composition) is delivered by a controlled release system. For example, the compounds disclosed herein can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump is used (Sefton (1987), CRC Crit. Ref. Biomed. Eng., 14:201; Buchwald et al. (1980), Surgery, 88:507; Saudek et al. (1989), N. Engl. J. Med., 321:574). In another embodiment, a polymeric material is used. In yet another embodiment, the controlled release system is placed near the therapeutic target, i.e., the liver, thereby requiring only a fraction of the systemic dose. In yet another embodiment, the controlled release system is placed near the therapeutic target, thereby requiring only a fraction of the systemic dose. Other controlled release systems are reviewed by Langer (1990) Science 249:1527-1533.

[0295] In another embodiment, the compositions of the present disclosure (as well as the individual or combined compositions) include compositions formed by impregnating the inhibitors described herein into absorbent materials such as sutures, bandages, and gauze, or compositions coated onto the surface of solid materials such as surgical staples, zippers, and catheters for delivery of the compositions. Other delivery systems of this type will be readily apparent to those skilled in the art with the present disclosure in mind.

[0296] The present disclosure provides methods and compositions for administering one or more antibody fragments to treat microbial infections. In various embodiments, the methods disclosed herein span almost any available method and route suitable for delivering a drug, including in vivo and ex vivo methods, as well as systemic and local administration routes.

[0297] In a specific aspect, the present disclosure provides a formulation or co-formulation comprising an antibody or antibody fragment (e.g., a Fab (fragment antigen-binding) fragment) that specifically recognizes or specifically binds to a DNABII protein or polypeptide or an isolated or recombinant polypeptide consisting essentially of an amino acid sequence selected from SEQ ID NOs: 12 to 17, SEQ ID NO: 33, or their respective equivalents. The antibodies or antibody fragments (e.g., Fab (fragment antigen-binding) fragments) disclosed herein can be selected to have a high level of epitope binding specificity and high binding affinity for biofilms. Generally, the greater the binding affinity of an antibody or antibody fragment (e.g., a Fab (fragment antigen-binding) fragment), the more stringent washing conditions can be performed in an immunoassay to remove non-specifically bound materials without removing the target. Thus, the antibodies or antibody fragments (e.g., Fab (fragment antigen-binding) fragments) of the present technology useful in the disclosed methods typically have a binding affinity of at least 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , or 10 -12 In certain embodiments, the antibody or antibody fragment (e.g., Fab (fragment antigen-binding) fragment) has a kinetic binding rate sufficient to reach equilibrium under standard conditions in at least 12 hours, at least 5 hours, at least 1 hour, or at least 30 minutes. In other embodiments, the affinity of the antibody or antigen-binding fragment is 1000 picomolar (pM), 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, about 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, M, or less than 4 pM or about 1000 picomolar (pM), 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, about 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, or 4 pM.

[0298] In some embodiments, the antibody is present in the formulation at a concentration of from about 0.1 mg / mL to about 200 mg / mL, or alternatively, from about 1 to about 150 mg / mL, or alternatively, from about 2 mg / mL to about 100 mg / mL, or alternatively, from about 3 mg / mL to about 80 mg / mL, or alternatively, from about 4 mg / mL to about 50 mg / mL, or alternatively, from about 5 mg / mL to about 20 mg / mL. In some embodiments, the antibody has a concentration of at least about 1 mg / mL, or alternatively, at least about 2 mg / mL, at least about 3 mg / mL, or alternatively, at least about 4 mg / mL, or alternatively, at least about 5 mg / mL, or alternatively, at least about 6 mg / mL, or alternatively, at least about 7 mg / mL, or alternatively, at least about 8 mg / mL, or alternatively, at least about 9 mg / mL, or alternatively, at least about 10 mg / mL, or alternatively, at least about 15 mg / mL, or alternatively, at least about 20 mg / mL, Or alternatively, it is present at a concentration of at least about 30 mg / mL, or alternatively at least about 40 mg / mL, or alternatively at least about 50 mg / mL, or alternatively at least about 60 mg / mL, or alternatively at least about 70 mg / mL, or alternatively at least about 80 mg / mL, or alternatively at least about 90 mg / mL, or alternatively at least about 100 mg / mL, or alternatively at least about 120 mg / mL, or alternatively at least about 150 mg / mL, or alternatively at least about 200 mg / mL.In some embodiments, at least one of the plurality of antibodies has a concentration of at least about 1 mg / mL, or alternatively, at least about 2 mg / mL, or alternatively, at least about 3 mg / mL, or alternatively, at least about 4 mg / mL, or alternatively, at least about 5 mg / mL, or alternatively, at least about 6 mg / mL, or alternatively, at least about 7 mg / mL, or alternatively, at least about 8 mg / mL, or alternatively, at least about 9 mg / mL, or alternatively, at least about 10 mg / mL, or alternatively, at least about 15 mg / mL, or alternatively, at least In some embodiments, the hydroxybenzoates are present at a concentration of at least about 20 mg / mL, or alternatively at least about 30 mg / mL, or alternatively at least about 40 mg / mL, or alternatively at least about 50 mg / mL, or alternatively at least about 60 mg / mL, or alternatively at least about 70 mg / mL, or alternatively at least about 80 mg / mL, or alternatively at least about 90 mg / mL, or alternatively at least about 100 mg / mL, or alternatively at least about 120 mg / mL, or alternatively at least about 150 mg / mL, or alternatively at least about 200 mg / mL.

[0299] In some embodiments in which multiple different antibodies are included in the antibody combination formulation, the different antibodies may be present at substantially equal concentrations. In another aspect of such embodiments, one or more of the different antibodies may be present at a substantially higher concentration than the other antibodies, for example, at a ratio of about 1.5:1, or alternatively about 1.5:1:1, or alternatively about 1.5:1:1:1, or alternatively about 2:1, or alternatively about 2:1:1, or alternatively about 2:1:1:1, or alternatively at least about 2.5:1, or alternatively at least about 2.5:1:1, or alternatively at least about 2.5:1:1. Methods for stably formulating antibody formulations and co-formulations can be performed according to techniques disclosed in the art (see, e.g., U.S. Patent Application No. 12 / 875,083 (published as US2011 / 0059079)).

[0300] Diagnostic and Therapeutic Methods Also provided are methods for inhibiting, competing with, or titrating the binding of a DNABII polypeptide or protein to microbial DNA by contacting the DNABII polypeptide or protein or microbial DNA with an antibody fragment, antigen-binding fragment, or composition described herein, thereby inhibiting, competing with, or titrating the binding of the DNABII protein or polypeptide to the microbial DNA. In a further embodiment, the DNABII polypeptide and the microbial DNA are detectably labeled, for example, with a luminescent molecule that emits a signal when they come into close contact with each other. The contacting may be in It can be carried out in vitro or in vivo.

[0301] In another aspect, a method is provided for inhibiting, preventing, or destroying microbial biofilms by contacting the biofilm with an antibody fragment, its antigen-binding fragment, or polypeptide described herein, thereby inhibiting, preventing, or destroying the microbial biofilm. In a further aspect, the DNABII polypeptide and the microbial DNA are detectably labeled, for example, with a luminescent molecule that generates a signal when they come into close contact with each other. The contacting can be performed in vitro or in vivo.

[0302] When carried out in vitro, the method is useful for screening or confirming antibody fragments and polypeptides that have the same, similar, or opposite abilities as the polypeptides, polynucleotides, antibodies, antibody fragments (e.g., Fab (fragment antigen-binding) fragments), host cells, small molecules, and compositions disclosed herein. Alternatively, they can be used to identify which antibody fragments or polypeptides are suitable for treating microbial infections. For example, by having two samples containing a DNABII polypeptide, microbial DNA, and a test agent, it is possible to screen, for example, new agents or combination therapies. The second sample contains a DNABII polypeptide, microbial DNA, and a known active agent, such as an anti-IHF antibody or a small molecule used as a positive control. In a further embodiment, several samples are provided, and the agent is added to the system at increasing dilutions to determine the optimal dose that is likely to be effective in treating subjects in a clinical setting. As will be apparent to those skilled in the art, a negative control containing a DNABII polypeptide and microbial DNA can be provided. In another embodiment, DNABII polypeptide and microbial DNA are detectably labeled with luminescent molecules, which generate signals when they are in close contact with each other.Sample is kept under the same conditions for the time that the agent is effective for inhibiting, competing with or gradually reducing the interaction between DNABII polypeptide and microbial DNA, and then the sample is assayed for the generation of signals from luminescent molecules.If sample generates signals, the agent is not effective for inhibiting binding.

[0303] In another embodiment, an in vitro method is performed in a miniaturized chamber slide system in which isolates of infectious disease-causing microorganisms (e.g., bacteria) can be isolated from humans / animals and then cultured and grown as biofilms in vitro; see, e.g., Experiment 1 below. An agent (such as an anti-IHF antibody) or potential agent biofilm is added to the culture with or without increasing dilutions of the potential agent or agent, such as anti-IHF (or other antibodies, small molecules, agents, etc.), alone or in combination with another agent, to find the optimal dose that, when delivered to a subject with an infection, is likely to be effective in treating the patient. As will be apparent to one skilled in the art, positive and negative controls can be performed simultaneously.

[0304] In a further embodiment, the method is carried out in a high-throughput platform with antibody fragments and / or potential agents (alone or in combination with another agent) in a flow cell. Anti-IHF antibodies or fragments thereof or potential agents are added to cultures with or without increasing dilutions of the potential agent or agent, such as anti-IHF (or other antibodies, small molecules, agents, etc.), alone or in combination with another agent, to find the optimal dose that is likely to be effective in treating the patient when delivered to a subject with an infection. Biofilm isolates are sonicated to separate biofilm bacteria from DNABII polypeptides such as IHF that are bound to microbial DNA. DNABII polypeptide-DNA complexes are isolated by anti-IHF antibodies on the platform. Microbial DNA is then released, for example, by salt washing, and used to identify the added biofilm bacteria. The released DNA is then identified, for example, by PCR sequencing. If DNA is not released, the agent has successfully functioned or bound to microbial DNA. If DNA is found in the sample, the agent did not interfere with the binding between the DNABII polypeptide and the microbial DNA. As will be apparent to those skilled in the art, positive and / or negative controls can be performed simultaneously.

[0305] In another aspect, one or more of the antibody fragments, antigen-binding fragments, polypeptides, or compositions disclosed herein are used in a method for detecting biofilms in vivo. In a further embodiment, the antibody fragments, polypeptides, or compositions are detectably labeled, for example, with a luminescent or fluorescent molecule. Further applications of the methods disclosed herein include the use of such antibody fragments, polypeptides, or compositions, for example, to image biofilms using a detectably labeled primary antibody fragment, polypeptide, or composition that produces a detectable signal when bound to a biofilm, or a detectably labeled secondary antibody fragment, polypeptide, or composition that binds to the primary antibody fragment, polypeptide, or composition when bound to a biofilm.

[0306] The above method can also be used as a diagnostic test, as a given bacterial species may respond better to one agent than another for reversal of its biofilm, and this rapid, high-throughput assay system would allow one skilled in the art to assay a panel of potential anti-IHF-like agents to identify the most effective group.

[0307] An advantage of these methods is that most clinical microbiology laboratories in hospitals are already equipped to perform these types of assays (i.e., determining MIC and MBC values) using bacteria grown in liquid culture (or in planktonic state). As will be apparent to those skilled in the art, bacteria generally do not persist in planktonic state when causing disease. Instead, they grow as stable biofilms, and these biofilms are significantly more resistant to treatment with antibiotics, antibodies, or other therapeutic agents. This resistance is the reason why most MIC / MBC values ​​do not accurately predict in vivo efficacy. Therefore, by determining which "dose" of an agent can reverse bacterial biofilms in vitro (as described above), Applicants' preclinical assays will become a more reliable predictor of clinical efficacy, even in personalized medicine applications.

[0308] In addition to clinical settings, this method can also be used to identify infectious disease-causing microorganisms and / or identify effective alternative agents in industrial settings, for example, in pipes.

[0309] In a further embodiment of the above method, an antibiotic or antimicrobial agent known to inhibit the progression of the underlying infection is added sequentially or simultaneously to determine whether the infection can be inhibited. To assay for biofilm inhibition, the antibody fragment, polypeptide or composition can be added to the microbial DNA or DNABII polypeptide before adding the complex.

[0310] When performed in vivo in non-human animals such as chinchillas, the methods provide preclinical screening to identify antibody fragments, polypeptides or compositions that can be used alone or in combination with other agents to degrade biofilms.

[0311] In another aspect, provided herein are methods of inhibiting, preventing, or disrupting a biofilm in a subject by administering to the subject an effective amount of an antibody fragment, polypeptide, or composition, thereby inhibiting, preventing, or disrupting the microbial biofilm.

[0312] Alternatively or additionally, methods for inhibiting, preventing, or disrupting biofilms can be performed in vitro and / or ex vivo and involve providing a biofilm sample (taken from a subject or generated in vitro) and administering an effective amount of an antibody fragment, polypeptide, or composition, thereby inhibiting, preventing, or disrupting the microbial biofilm. Similarly, the antibody fragments, polypeptides, or compositions disclosed herein can also be used in method embodiments for inhibiting, preventing, or disrupting microbial biofilms on surfaces colonized by biofilms, such as, but not limited to, hospital instruments, industrial equipment, and other materials not contained in living tissue.

[0313] In a further aspect, the method comprises, or alternatively consists essentially of, and / or further consists of, administering to the subject an effective amount of one or more of an antimicrobial agent, an antigenic peptide, or an adjuvant.

[0314] Non-limiting examples of antibacterial agents are antibodies directed against surface antigens, e.g., vaccine components such as OMP P5, rsPilA, OMP 26, OMP P2, or type IV Pilin protein (see Jurcisek and Bakaletz (2007), J. Bacteriology, 189(10):3868-3875; Murphy et al. (2009), The Pediatric Infectious Disease Journal, 28:S121-S126; Novotny et al. (2015), Mol Microbiol., 96(2):276-92).

[0315] The agents and compositions disclosed herein can be administered simultaneously or sequentially with other antibacterial agents and / or surface antigens. In a specific embodiment, administration is local administration to the site of infection, for example, by direct injection or inhalation. Other non-limiting examples of administration include administration by one or more methods, including transdermal, urethral, ​​sublingual, rectal, vaginal, ocular, subcutaneous, intramuscular, intraperitoneal, intranasal, by inhalation, or orally.

[0316] Microbial infections and diseases that can be treated by the methods disclosed herein include, but are not limited to, infections by a variety of organisms associated with biofilm formation, including, but not limited to, those disclosed in Examples 4-9. Non-limiting examples of responsible organisms (and their exemplary strains in parentheses) include Aggregatibacter actinomycetemcomitans, Borrelia burgdorferi (e.g., B31), Bordetella pertussis (e.g., Tohama I), Burkholderia pseudomallei (e.g., 668), Burkholderia cenocepacia (e.g., HI2424), Escherichia coli (e.g., K12 MG1655), Enterococcus faecalis (e.g., V583), Haemophilus influenzae (e.g., Rd KW20), Helicobacter pylori (e.g., 26695), Klebsiella pneumoniae, Moraxella catarrhalis (e.g., RH4), Mycobacterium smegmatis (e.g., MC2), Mycobacterium tuberculosis (e.g., CDC1551), Neisseria gonorrhoeae, and the like. gonorrhoeae (e.g., FA1090), Neisseria meningitidis (e.g., MC58), Pseudomonas aeruginosa, Porphyromonas gingivalis (e.g., W83), Prevotella intermedia (e.g., 17), Prevotella melaninogenica (e.g., ATCC (registered trademark) 25845), Staphylococcus aureus (e.g., MW2), Staphylococcus epidermidis (e.g., RP62A), Streptococcus agalactiae (e.g., 2603V / R), Streptococcus bovis, Streptococcus gallolyticus (e.g., UCN34), Streptococcus gordonii (e.g., NCTC 7868 (Challis)), Streptococcus mutans (e.g., UA159), Streptococcus pneumoniae (e.g., R6), Streptococcus pyogenes (e.g., MGAS10270), Streptococcus sobrinus (e.g., 6715), Salmonella enterica (e.g., Salmonella typhi, CT18), Treponema denticola (e.g., ATCC® 35405), Treponema palladum (e.g., Nichols), Vibrio cholera (e.g., El Tor, N16961). Additional organisms known to be associated with and / or form biofilms include, but are not limited to, Campylobacter species, Candida species, Legionella pneumophila, and Listeria monocytogenes. For example, cystic fibrosis patients often suffer from Pseudomonas infection, which often results in antibiotic-resistant biofilms.Exemplary diseases associated with biofilms include, but are not limited to, lung infections in cystic fibrosis patients, otitis media, native valve infective endocarditis, osteomyelitis, rhinosinusitis, prostatitis, recurrent urinary tract infections, wounds, dental caries, and periodontitis.Conditions such as infected prosthetic devices, joints, catheters, stents, or other surgical implants are also associated with the formation of biofilms.

[0317] These microbial infections can be present in the upper, middle, and lower respiratory tract (such as otitis, sinusitis, bronchitis, but also exacerbations of chronic obstructive pulmonary disease (COPD), chronic cough, complications and / or primary causes of cystic fibrosis (CF), and community-acquired pneumonia (CAP). Thus, by practicing the in vivo methods disclosed herein, these diseases and complications resulting from these infections can also be prevented or treated.

[0318] Infections can also occur in the oral cavity (dental caries, periodontitis) and are caused by Streptococcus mutans, Porphyromonas gingivalis, and Aggregatibacter actinomvctemcomitans. Infections can also be localized to the skin (abscesses, "staph" infections, impetigo, secondary infections of burns, Lyme disease) and are caused by Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Borrelia burdorferi. Infections of the urinary tract (UTI) are typically caused by Escherichia coli and can also be treated. Infections of the gastrointestinal (GI) tract (diarrhea, cholera, gallstones, stomach ulcers) are typically caused by Salmonella enterica serovar, Vibrio cholerae, and Helicobacter pylori. Infections of the genital tract are typically caused by Neisseria gonorrhoeae. The infection can be a bladder infection or an indwelling device infection caused by Enterococcus faecalis. Infections associated with implanted prosthetic devices, such as artificial hip or knee joints, or dental implants, or medical devices, such as pumps, catheters, stents, or monitoring systems, are typically caused by various bacteria and can be treated by the methods disclosed herein. These devices can also be coated with or conjugated to the agents described herein. Thus, the in By practicing in vivo methods, these diseases and complications resulting from these infections can also be prevented or treated.

[0319] Infections caused by Streptococcus agalactiae, which is a leading cause of bacterial sepsis in newborns, can also be treated with the methods disclosed herein. Infections caused by Neisseria meningitidis, which can also cause meningitis, can also be treated.

[0320] Thus, administration routes applicable to the methods disclosed herein include intranasal, intramuscular, urethral, ​​intratracheal, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral, inhalation, and other enteral and parenteral routes of administration. Administration routes can be combined if desired and tailored depending on the agent and / or desired effect. Active agents can be administered in a single dose or multiple doses. Suitable embodiments of these methods and routes of delivery include systemic or local routes. In general, administration routes suitable for the methods disclosed herein include, but are not limited to, direct injection, enteral, parenteral, or by inhalation.

[0321] Parenteral administration routes other than administration by inhalation include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the digestive tract. Parenteral administration can be carried out to result in systemic or local delivery of the inhibitor. When systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of the pharmaceutical preparation.

[0322] The antibody fragments, polypeptides, or compositions disclosed herein can also be delivered to a subject by enteral administration, including, but not limited to, oral and rectal delivery (e.g., using a suppository).

[0323] Methods of administering active substances through the skin or mucosa include, but are not limited to, topical application of appropriate pharmaceutical preparations, transcutaneous transmission, transdermal transmission, injection, and epidermal administration. For transdermal penetration, absorption enhancers or iontophoresis are suitable methods. Iontophoretic penetration can be achieved using commercially available "patches" that deliver their products continuously through intact skin via electrical pulses for periods of several days or longer.

[0324] In various embodiments of the methods disclosed herein, the antibody fragment, polypeptide, or composition is administered by inhalation, injection, or orally continuously, daily, at least once daily (QD), and in various embodiments twice daily (BID), three times daily (TID), or even four times daily. A daily therapeutically effective dose will typically be at least about 1 mg, or at least about 10 mg, or at least about 100 mg, or about 200 mg to about 500 mg, and in some cases as much as about 1 g to about 2.5 g, depending on the compound.

[0325] The present disclosure provides methods and compositions for inhibiting or preventing infection of a host or host cells by bacteria that export a DNABII protein, comprising administering to a tissue exposed to or infected with the bacteria an effective amount of a bacteria-relevant antibody fragment, antigen-binding fragment thereof, polypeptide or antibody fragments that specifically recognize and bind to the DNABII protein. The present invention provides methods and compositions comprising, alternatively consisting essentially of, or even consisting of administering a composition to inhibit or prevent infection of a host or host cells by bacteria. Multiple antibody fragments, polypeptides or compositions can be administered concomitantly or sequentially, along with supportive care as referred to herein.

[0326] The present disclosure provides a method for inhibiting or preventing infection of cells by bacteria that export DNABII protein. The method comprises, or alternatively consists essentially of, or even consists of, administering to the tissue infected with the bacteria an effective amount of an antibody fragment, polypeptide, or composition that specifically recognizes and specifically binds to DNABII protein, thereby inhibiting or preventing infection by the bacteria. Multiple antibody fragments, polypeptides, or compositions can be administered together or sequentially, along with the supportive care referred to herein.

[0327] The administration can be in vitro, in culture, or in vivo, to a patient infected with bacteria. When performed in vivo, the method can be used to treat a subject infected with bacteria by administering an effective amount of an antibody to the infected subject. In addition, when the subject is a non-human animal, the method can be used to investigate possible treatments or combination therapies before administration to humans. When performed in vitro, the method is useful for screening other therapeutic agents and combination therapies, such as small molecule drugs that inhibit or prevent bacterial infections in tissues.

[0328] Also provided is a method for treating bacterial infection in a subject in need thereof, the subject being infected with a bacterium containing a DNABII protein, comprising, alternatively consisting essentially of, or even consisting of, administering to the subject an effective amount of an antibody fragment, polypeptide, or composition that specifically recognizes and specifically binds to the DNABII protein, thereby inhibiting or preventing bacterial infection. The antibody fragment can be generated from a polyclonal antibody, a monoclonal antibody, or an antibody derivative that recognizes and binds to the bacteria-associated DNABII protein. The source of antibodies against the DNABII protein can be induced by active vaccination of the host with the DNABII protein, or by passive transfer of antisera or antibodies against the DNABII protein. Multiple antibody fragments, polypeptides, or compositions can be administered together or sequentially, along with the supportive therapy described herein.

[0329] Furthermore, there is also provided a method for treating a condition in a subject in need thereof, said condition being associated with infection with a bacterium expressing a DNABII protein, comprising, alternatively consisting essentially of, or even consisting of, administering to the subject an effective amount of an antibody fragment, polypeptide, or composition that specifically recognizes and specifically binds to the DNABII protein, thereby inhibiting or preventing infection by the bacterium. The source of the antibody that resulted in the antibody fragment, polypeptide, or composition against the DNABII protein can be induced by active vaccination of the host with the DNABII protein, or by passive transfer of antisera or antibodies against the DNABII protein. The antibody may be a polyclonal antibody, a monoclonal antibody, or an antibody derivative that recognizes and binds to the bacteria-associated DNABII protein. Multiple antibody fragments, polypeptides, or compositions can be administered together or sequentially, along with the supportive therapies mentioned herein.

[0330] Any of the above methods may further comprise, alternatively consist essentially of, or even consist of administering to the subject or to the in vitro tissue or cell culture an effective amount of one or more of an antimicrobial agent, an antigenic peptide, or an adjuvant. In some aspects, the subject is a non-human animal or a human patient.

[0331] The antibody fragment, polypeptide or composition is administered locally or systemically by any suitable method, for example, topically to the site of infection or biofilm, rectally, vaginally, ophthalmically, subcutaneously, intramuscularly, intraperitoneally, urethrally, intranasally, by inhalation, or orally.

[0332] In some aspects, the subject is a pediatric patient and the antibody is administered in a pediatric formulation.

[0333] Also disclosed are screens for identifying potential therapeutic agents that inhibit or prevent infection of cells by bacteria that export DNABII proteins and / or disrupt or prevent biofilm formation. The screening method comprises, alternatively, essentially consists of, and / or further consists of contacting an agent with a bacterially infected tissue in vitro, or administering an agent to a bacterially infected tissue in vivo, and determining whether the agent binds to the DNABII protein. Methods for determining binding are known in the art, and some non-limiting examples are described herein. In one aspect, if the agent binds to the protein, the agent is a potential therapeutic agent; if the agent does not bind to the protein, the agent is not a potential therapeutic agent. In another aspect, if infection or biofilm is inhibited, disrupted, or prevented in vivo, the agent is a potential therapeutic agent; if infection is not inhibited or prevented, the agent is not a potential therapeutic agent. Methods for determining whether an infection is inhibited or prevented are known in the art, some non-limiting examples of which are described herein; methods for determining whether a biofilm is disrupted or prevented are also known in the art, and are further disclosed herein. Non-limiting examples of potential therapeutic agents are selected from the group of antibodies, antibody derivatives, polypeptides, or small molecules. Multiple antibodies can be administered concurrently or sequentially, along with the supportive therapies referred to herein.

[0334] In a further embodiment, the agent binds to the protein and the binding is compared to the binding of anti-DNABII antisera to the antibody fragment or polypeptide.

[0335] It will be appreciated that any of the general properties envisioned for antibody fragments, polypeptides or compositions for inhibiting, reducing or competing with the binding of DNABII protein to microbial DNA also apply to the methods disclosed above for bacterial infections.

[0336] Dosing can be achieved using capsules, tablets, oral suspensions, intramuscular injection suspensions, intravenous injection suspensions, topical application gels or creams, or intra-articular injection suspensions according to the methods disclosed herein.

[0337] The dosage, toxicity, and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as determining the LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. In certain embodiments, the compositions exhibit a high therapeutic index. Compounds that exhibit toxic side effects may be used, but care should be taken to design a delivery system that targets such compounds to the affected tissue site to minimize potential damage to uninfected cells and thereby reduce side effects.

[0338] Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds lies (in certain embodiments) within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages may vary within this range depending on the dosage form and route of administration utilized. For any compound used in the methods, a therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound at which half-maximal inhibition of symptoms is achieved) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0339] In some embodiments, an effective amount of the composition sufficient to achieve a therapeutic or prophylactic effect ranges from about 0.000001 mg per kilogram of body weight per administration to about 10,000 mg per kilogram of body weight per administration. The dosage is suitably from about 0.0001 mg per kilogram of body weight per administration to about 100 mg per kilogram of body weight per administration. Administration can be as an initial dose followed by one or more "booster" doses. The booster doses can be administered 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months after the initial dose. In some embodiments, the booster dose is administered after assessing the subject's response to the previous dose.

[0340] Those skilled in the art will recognize that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or can include a series of treatments.

[0341] Functional analysis using antibodies The antibodies and antibody fragments (e.g., Fab (fragment antigen-binding) fragments) disclosed herein can be used to purify the polypeptides disclosed herein and to identify biologically equivalent polypeptides and / or polynucleotides. They can also be used to identify agents, such as aptamers, polynucleotides, and small molecules, that modify the function of the polypeptides disclosed herein. These antibodies include polyclonal antisera, monoclonal antibodies, and various reagents derived from these preparations that are well known to those of skill in the art and described above.

[0342] Antibodies and antibody fragments (e.g., Fab (fragment antigen-binding) fragments) that neutralize the activity of the proteins encoded by the identified genes can also be used to detect the activity of proteins in vivo and in vitro by adding such neutralizing antibodies to in vivo and in vitro test systems. They can also be used to demonstrate in vitro function. Such neutralizing antibodies are also useful as pharmaceutical agents that modulate the activity of the polypeptides disclosed herein.

[0343] Various antibody preparations and antibody fragments (e.g., Fab (fragment antigen-binding) fragments) can also be used in analytical methods such as ELISA assays or Western blots to confirm the expression of the proteins encoded by the identified genes by test cells in vitro or in vivo. Fragments of such proteins generated by metabolic protease degradation can also be identified by using appropriate polyclonal antisera with samples derived from experimental samples.

[0344] Furthermore, in some embodiments, the antibodies and antibody fragments (e.g., Fab (fragment antigen-binding) fragments) disclosed herein can be used to visualize and / or detect biofilms. In such embodiments, the antibodies can be detectably labeled, for example, with a radioisotope, an enzyme that produces a detectable product, a fluorescent protein, or can be conjugated to other moieties, such as, for example, a member of a specific binding pair, e.g., biotin (a member of the biotin-avidin specific binding pair). The detectably labeled antibody can then be introduced into a sample suspected of being colonized with a biofilm and visualized and / or detected by microscopy or other methods known to detect the associated label, e.g., spectroscopy, cytometry, or other common techniques. Conjugated or unlabeled antibodies can likewise be identified by known analytical methods directed to the conjugated moiety or antibody, respectively. For example, in some embodiments, detectably labeled secondary antibodies specific for isotypes of the antibodies and / or antibody fragments (e.g., Fab (fragment antigen-binding) fragments) disclosed herein can also be used in visualizing and / or detecting biofilms; such embodiments can involve administering an antibody specific for the "tail" of the DNABII protein to a surface suspected of being colonized by a biofilm, followed by administration of a detectably labeled secondary antibody and subsequent detection of the associated label.

[0345] The antibody fragments, antigen-binding domains, polypeptides or compositions disclosed herein can be used alone, in combination with peptide- or protein-based vaccines or dendritic cell-based vaccines, for vaccination, or to boost vaccination.

[0346] Screening assays The present disclosure provides methods for screening for equivalent agents, such as monoclonal antibodies and fragments thereof, equivalent to the exemplary antibodies described herein, as well as various agents that modulate the activity of the active agents and pharmaceutical compositions disclosed herein, or the function of the polypeptide or peptide products encoded by the polynucleotides disclosed herein. For purposes of this disclosure, "agent" is intended to include, but is not limited to, biological or chemical compounds, such as simple or complex organic or inorganic molecules, peptides, proteins (e.g., antibodies), polynucleotides (antisense), or ribozymes. A wide variety of compounds can be synthesized, including polymers such as polypeptides and polynucleotides, and synthetic organic compounds based on various core structures; these are also encompassed by the term "agent." Additionally, various natural sources, such as plant or animal extracts, can also provide compounds for screening. Although not always explicitly stated, it is understood that an agent may be used alone or in combination with another agent that has the same or different biological activity as the agent identified by the screens of the present invention.

[0347] For the purpose of this disclosure, "binding moiety" is intended to include, but is not limited to, biological or chemical compounds, such as simple or complex organic or inorganic molecules, peptides, proteins (e.g., antibodies), polynucleotides (antisense) or ribozymes, which have affinity for at least one DNABII protein.A wide variety of compounds can be synthesized, for example, polymers such as polypeptides and polynucleotides, and synthetic organic compounds based on various core structures, and these are also included in the term "binding moiety".In addition, various natural sources, such as plant extracts or animal extracts, can also provide compounds for screening.

[0348] To carry out in vitro screening method, first provide suitable cell culture or tissue that is infected with the microorganism to be treated.Cell is cultured for an appropriate length of time under the conditions (temperature, growth medium or culture medium, and gas (CO2)) to achieve exponential growth without density-dependent constraints.In addition, it is also desirable to maintain another separate cell culture that is not infected as a control.

[0349] As will be apparent to one of skill in the art, suitable cells can be cultured in microtiter plates and several agents can be assayed simultaneously by looking for genotypic changes, phenotypic changes, or reduction in microbial titer.

[0350] If the agent is a composition other than DNA or RNA, such as a small molecule as described above, the agent can be added directly to the cell culture or to the culture medium for supplementation. As will be apparent to those skilled in the art, an "effective" amount must be added, which can be determined empirically.

[0351] If the agent is an antibody or antigen-binding fragment, the agent can be contacted with or incubated with the target antigen and polyclonal antibody described herein under conditions for performing a competitive ELISA. Such methods are known to those skilled in the art.

[0352] The assay can also be performed in a subject. When the subject is an animal such as a rat, chinchilla, mouse, or monkey, the method provides a convenient animal model system that can be used before clinical testing of an agent in human patients. In this system, if the symptoms of disease or microbial infection are alleviated or eliminated compared to untreated animals with the same infection, the candidate agent is a potential drug. It can also be useful to have a separate negative control group of healthy, untreated cells or animals to provide a basis for comparison.

[0353] Additional screening assays specific for the antibodies contemplated herein include methods suitable for identifying antibodies or agents that specifically bind to one or more of the polypeptides disclosed herein, which involve identifying antibodies that bind to the site on the antigen to which an antibody of the invention binds.

[0354] The antibody binding site can be determined by methods known to those skilled in the art. For example, if an antibody binds to or recognizes a partial conformation of an antigen, the antibody binding site can be determined by identifying amino acid residues on the antigen adjacent to the antibody using X-ray structural analysis. For example, an antibody or a fragment thereof and an antigen or a fragment thereof can be bound to each other and crystallized, followed by structural analysis to identify each amino acid residue on the antigen that has an interaction distance with the antibody. The interaction distance is 8 angstroms or less, preferably 6 angstroms or less, and more preferably 4 angstroms or less. One or more such amino acid residues that have an interaction distance with the antibody can constitute the site on the antigen to which the antibody binds (epitope). Two or more such amino acid residues cannot be adjacent to each other in the primary sequence. Such structural analysis can reveal the three-dimensional structure of the protein and thus can provide both the sequence and conformation of the identified epitope.

[0355] A test antibody or test agent can be contacted with one or more of the polypeptides disclosed herein.The distance between the substance and the amino acid residues that make up the epitope to which any one of the antibodies disclosed herein binds is then measured.The test antibody or test agent can be determined to specifically bind if it is within an appropriate interaction distance from each of the residues.In some embodiments, the test antibody is not a polyclonal antibody.

[0356] In some embodiments, a test antibody or test agent is analyzed based on its interaction with a conformational epitope identified by structural analysis of the antibody-antigen interaction. A non-limiting example of such a conformational epitope is a β-hairpin, which may be contained within one or more of the polypeptides disclosed herein. A further non-limiting exemplary epitope is the consensus amino acid sequence [ka] [where "X" refers to any amino acid]. In some embodiments, X is selected from the amino acids Q, R, K, S, or T. Non-limiting examples of such are provided herein as consensus amino acid sequences. [ka] The skilled artisan will recognize that in any one of these sequences, the residue at position X (in the example below, [ka] It is understood that the amino acid residues (marked with a ) can be substituted with any amino acid (e.g., Q, R, K, S, or T). SEQ ID NO: 13: Haemophilus influenzae IhfA, A5 fragment: [ka] . SEQ ID NO: 14: Haemophilus influenzae HU, A5 fragment: [ka] . SEQ ID NO: 17: Haemophilus influenzae IhfB, modified B4 (mB4) fragment: [ka] . SEQ ID NO: 26: Haemophilus influenzae IhfA, A tip fragment: [ka] . SEQ ID NO: 27: Haemophilus influenzae IhfB, B tip fragment: [ka] . SEQ ID NO: 31 Haemophilus influenzae HU, fragment: [ka] .

[0357] In some embodiments, [ka] is at least about 20 amino acids in length, and NPXT is located approximately centrally within the sequence. Non-limiting examples of such sequences include SEQ ID NOs: 13, 14, and 31.

[0358] The agents and compositions can be used in the manufacture of medicaments and, as the active ingredient of a pharmaceutical composition, can be used to treat humans and other animals by administration in accordance with conventional procedures. Combination Therapy

[0359] The compositions and related methods of the present disclosure can be used in combination with the administration of other therapies, including, but not limited to, the administration of DNase enzymes, antibiotics, antimicrobial agents, vaccine adjuvants or other antibodies.

[0360] In some embodiments, the methods and compositions include a deoxyribonuclease (DNase) enzyme that acts synergistically with an anti-DNABII antibody. DNase is any enzyme that catalyzes the cleavage of phosphodiester bonds in the DNA backbone. Three non-limiting examples of DNase enzymes that are known to target not only cruciform structures but also various secondary structures of DNA include DNase I, T4 Endo VII, and T7 Endo I. In certain embodiments, when combined with DNase, the effective amount of anti-DNABII antibody required to destabilize biofilms is reduced. When administered in vitro, DNase can be added directly to the assay or in an appropriate buffer known to stabilize the enzyme. The effective unit dose of DNase and assay conditions can vary and can be optimized according to procedures known in the art.

[0361] In other embodiments, the methods and compositions can be combined with antibiotics and / or antimicrobial agents. Antimicrobial agents are substances that kill or inhibit the growth of microorganisms such as bacteria, fungi, or protozoa. Biofilms are generally resistant to the action of antibiotics, but the compositions and methods described herein can be used to sensitize biofilm-associated infections to conventional therapies for treating infections. In other embodiments, the use of antibiotics or antimicrobial agents in combination with the methods and compositions described herein allows for a reduction in the effective amount of the antimicrobial agent and / or biofilm-reducing agent. Some non-limiting examples of antimicrobial agents and antibiotics useful in combination with the methods of the present disclosure include amoxicillin, amoxicillin-clavulanic acid, cefdinir, azithromycin, and sulfamethoxazole-trimethoprim. Therapeutically effective amounts of antimicrobial agents and / or antibiotics to be combined with a biofilm-reducing agent can be readily determined by conventional methods. In some embodiments, the dose of the antimicrobial agent combined with the biofilm reducing agent is the average effective dose shown to be effective in other bacterial infections, for example, bacterial infections whose etiology does not involve biofilms. In other embodiments, the dose is 0.1, 0.15, 0.2, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.8, 0.85, 0.9, 0.95, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, or 5 times the average effective dose. The antibiotic or antimicrobial agent can be added before, simultaneously with, or after the addition of the anti-DNABII antibody.

[0362] In other embodiments, the methods and compositions can be combined with antibodies to treat bacterial infections. One example of an antibody useful in combination with the methods and compositions described herein is an antibody directed against an unrelated outer membrane protein (i.e., OMP P5). Treatment with this antibody alone does not reduce biofilms in vitro. Combination therapy with this antibody and a biofilm-reducing agent results in a greater effect than can be achieved by using either agent alone at the same concentration. Other antibodies that may provide a synergistic effect when used in combination with a biofilm-reducing agent or method for reducing biofilm include anti-rsPilA preparations, anti-OMP26 preparations, anti-OMP P2 preparations, and anti-total OMP preparations.

[0363] The compositions and methods described herein can be used to sensitize biofilm-associated bacterial infections to common therapeutic modalities that are effective in treating non-biofilm-associated bacterial infections but are otherwise ineffective in treating biofilm-associated bacterial infections. In other embodiments, the compositions and methods described herein can be used in combination with therapeutic modalities that are effective in treating or preventing biofilm-associated bacterial infections, where the combination of such additional therapy with a biofilm-reducing agent or method results in a synergistic effect that may reduce the effective amount of either the biofilm-reducing agent or the additional therapeutic agent. In other cases, the combination of such additional therapy with a biofilm-reducing agent or method results in a synergistic effect that enhances treatment. Enhanced treatment can be evidenced by a reduction in the length of time required to treat the infection.

[0364] The additional therapeutic treatment can be added before, in conjunction with, or after the method or composition used to reduce the biofilm, and can be included in the same formulation or as a separate formulation.

[0365] Kits containing the necessary agents and instructions for carrying out the in vitro and in vivo methods described herein are also claimed.Thus, the present disclosure provides kits for carrying out these methods, which may include instructions for carrying out the methods disclosed herein, such as collecting the antibody fragments or antigen-binding fragments thereof disclosed herein and tissues, and / or carrying out screening, and / or analyzing the results, and / or administering an effective amount of the antibody fragments or antigen-binding fragments thereof as defined herein.These can be used alone or in combination with other suitable antimicrobial agents.

[0366] For example, the kit may comprise, alternatively consist essentially of, or even consist of any one or more of the antibody fragments, polypeptides, or compositions of the present disclosure and instructions for use. The kit may further comprise one or more of an adjuvant, an antigenic peptide, or an antimicrobial agent. Examples of carriers include liquid carriers, pharmaceutically acceptable carriers, solid-phase carriers, pharmaceutically acceptable carriers, pharmaceutically acceptable polymers, liposomes, micelles, implants, stents, pastes, gels, dental implants, or medical implants.

[0367] The following examples are intended to illustrate, but not limit, the embodiments disclosed herein. [Example]

[0368] Example 1 Generation of IHF and HU antibodies and Fab fragments IHF and HU antibodies, proteins and polypeptides have been generated against IHF and HU in general and against specific fragments.

[0369] See, e.g., Granston and Nash (1993), J. Mol. Biol., 234:45-5. 9; Nash et al. (1987), Journal of Bacteriology, 169(9):412 Methods for their production are well known to those skilled in the art, as described in Rice et al. (1996), Cell 87:1295-1306; Briefly, to overproduce IHF-α (SEQ ID NO: 6), the himA gene was inserted into the PAD284 plasmid, a bacterial plasmid. L The desired plasmid was inserted downstream of the promoter. Transformants of strain K5607, a lambda lysogen of strain C600himA42, were identified by screening ampicillin-resistant transformants for their ability to grow bacteriophage mu. Standard DNA isolation methods were used to isolate hima + DNA was prepared from the transformants, and the orientation of the himA gene was determined by restriction enzyme digestion. L pP, a plasmid containing the himA gene in the correct orientation for promoter-driven expression L himA-1 was transformed into strain N5271, which contains a lambda cryptic prophage expressing the cI857 heat-inducible repressor, resulting in strain K5770.

[0370] To overproduce the IHF-β subunit (SEQ ID NO: 7), the P L The plasmid pKT23-hip323, which contains a fusion of the IHF-beta coding sequence with the promoter, was used. pKT23-hip323 was introduced into N5271 to generate strain E443. To facilitate selection of pKT23-hip323 in the presence of other pla...

Claims

[Claim 1] The invention as set forth in the drawings.

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