Combination therapy for the treatment and prevention of biofilms

Abstract

To provide a combination therapy for the treatment and prevention of biofilms.SOLUTION: Provided herein are compositions and combinations for therapeutic and diagnostic use in treating and preventing biofilms and related disorders using high mobility group box protein (HMGB) polypeptides, mutants and / or fragments thereof, and anti-DNABII antibodies, fragments or variants thereof. The polypeptide and antibody may be administered as the same or separate compositions. Provided herein are combinations of HMG-box polypeptides (non-limiting examples of which include HMGB1 polypeptides) and anti-DNABII antibodies or antigen-binding fragments thereof (e.g., antibodies that specifically recognize and bind to the apical or chimeric apical domain of DNABII), or equivalents of each thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 049,065, filed July 7, 2020, and U.S. Provisional Application No. 63 / 175,487, filed April 15, 2021, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Federal Government Support Statement This invention was made with government support under Grant No. DC011818 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy was created on July 2, 2021, is named 106887-7960_ST25.txt, and is 233,163 bytes in size. [Background technology]

[0004] background Proteins of the DNABII family are naturally found on the outside of bacterial cells and contribute to biofilm formation. At least one protein from the DNABII family is found in all known eubacteria. Although these proteins elicit strong innate and adaptive immune responses, host subjects are unable to naturally produce immunoprotective antibodies against family members as a result of infection. A major problem with bacterial biofilms is the inaccessibility of the host immune system and / or antibiotics and other antimicrobial agents to the bacteria protected within the biofilm.

[0005] Bacteria within the human host favor community architectures, also known as biofilms. Biofilms form when free-living (planktonic) bacteria attach to each other (aggregate biofilms) or to surfaces (attached biofilms), initiating a developmental program that includes changes in gene expression, cell-to-cell communication, and, importantly, the production of a self-produced extracellular matrix (extracellular polymeric substances or EPS). Resident bacteria within each of these community architectures are refractory to the host immune system and antimicrobial agents (Slinger et al., Diagn Microbiol Infect Dis, 2006. 56(3): pp. 247-53; and Starner et al., Antimicrob Agents Chemother, 2008. 52(1): pp. 137-45), allowing bacteria to persist and serving as a reservoir for maintaining chronic and recurrent infections. Therefore, there is a critical need to develop targeted strategies for dissolving bacterial biofilms.

[0006] Biofilms are also present in industrial environments. For example, they are implicated in a wide range of petroleum processing problems, from production sites to gas station storage tanks. At the sites, sulfate-reducing biofilm bacteria produce hydrogen sulfide (soured oil). In process pipelines, biofilm activity generates slime that clogs filters and openings. Biofilms and biofilm organisms also cause corrosion in pipelines and petroleum processing equipment. These problems can be manifested throughout oil or gas production facilities to the extent that fouling and corrosive biofilm organisms are even found on the surfaces of end-product storage tanks.

[0007] Biofilms are relevant to a wide range of water treatment applications, both domestic and industrial. They grow on the surfaces of treatment equipment and can impair equipment performance (e.g., degraded heat transfer or blockage of filters and membranes). Biofilms growing on cooling tower fill can add enough weight to cause the fill to collapse. Biofilms even cause corrosion of highly specialized stainless steel. Biofilms in water treatment, for example, biofilm contamination in paper processing or even the attachment of single cells to silicon chips, can degrade the value of the end product. Biofilms growing in drinking water delivery systems can harbor potential pathogenic, corrosive, or bacterial organisms that degrade the aesthetic quality of the water. In the home, biofilms are found in or on any surface that supports microbial growth, such as drains, cooking surfaces, toilets, and swimming pools and spas. Thus, there is a need to penetrate the protective biofilm barrier in order to treat or kill associated bacterial infections and remove them from surfaces and water systems. The present disclosure fulfills this need and also provides related advantages. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Slinger et al., Diagn Microbiol Infect Dis, 2006. 56(3): p. 247-53 [Non-patent document 2] Starner et al., Antimicrob Agents Chemother, 2008. 52(1): p. 137-45 Summary of the Invention [Means for solving the problem]

[0009] overview The present specification provides a combination of an HMG box polypeptide (non-limiting examples of which include HMGB1 polypeptide) with an anti-DNABII antibody or its antigen-binding fragment (for example, an antibody that specifically recognizes and binds to the tip or chimeric tip domain of DNABII), or each of their equivalents. Furthermore, polynucleotides, vectors, and host cells that express the elements of the combination alone or in combination with each other are provided. Furthermore, compositions, combinations, and kits that comprise, consist essentially of, or even consist of the combination, or the polynucleotide or vector or host cell that expresses the combination, or both, are provided, along with methods for producing or using them. Without wishing to be bound by theory, the disclosed combinations exhibit synergistic effects in preventing biofilm formation or destroying biofilms.

[0010] In one aspect, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide, further optionally comprising one or more mutations (i.e., a mutant HMGB1 (mHMGB1) polypeptide), or a fragment thereof, optionally comprising, consisting essentially of, or consisting of an HMGB, optionally the B box or the A box, or both (e.g., the AB box) of an HMGB1 polypeptide, and optionally the HMGB polypeptide or HMGB1 polypeptide or fragment thereof is isolated or engineered, or both; and (b) (i) a heavy chain (HC) immunoglobulin variable domain comprising, alternatively consisting essentially of, or alternatively consisting of amino acids (aa) 25 to aa 144 of any one of SEQ ID NOs: 13, 24, or 26, or their respective equivalents; and / or (ii) a light chain (LC) immunoglobulin variable domain comprising, alternatively consisting essentially of, or alternatively consisting of aa21 to aa132 of SEQ ID NO: 14 or 25, or aa21 to aa126 of SEQ ID NO: 27, or their respective equivalents. An anti-DNABII antibody or antigen-binding fragment thereof comprising, or alternatively consisting essentially of, or alternatively consisting of There is provided a composition or combination comprising, consisting essentially of, or alternatively consisting of:

[0011] In a further aspect, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide, further optionally comprising one or more mutations (i.e., a mutant HMGB1 (mHMGB1) polypeptide), or a fragment thereof, optionally comprising, consisting essentially of, or consisting of an HMGB, optionally the B box or the A box, or both (e.g., the AB box) of an HMGB1 polypeptide, and optionally the HMGB polypeptide or HMGB1 polypeptide or fragment thereof is isolated or engineered, or both; and (b) (i) a heavy chain (HC) comprising, alternatively consisting essentially of, or alternatively consisting of, any one or more of SEQ ID NOs: 13, 24, or 26, or their respective equivalents; and / or (ii) a light chain (LC) comprising, alternatively consisting essentially of, or alternatively consisting of, any one or more of SEQ ID NOs: 14, 25, or 27, or their respective equivalents. An anti-DNABII antibody or antigen-binding fragment thereof comprising, or alternatively consisting essentially of, or alternatively consisting of There is provided a composition or combination comprising, consisting essentially of, or alternatively consisting of:

[0012] In still a further aspect, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide, optionally comprising one or more mutations (i.e., a mutant HMGB1 (mHMGB1) polypeptide (or corresponding mutations in HMGB2, HMGB3, or HMGB4), or a fragment thereof, optionally comprising, consisting essentially of, or consisting of an HMGB, optionally the B box or the A box, or both (e.g., the AB box) of an HMGB1 polypeptide, and optionally the HMGB polypeptide or HMGB1 polypeptide or fragment thereof is isolated, engineered, or both; and (b) (i) to (vi) of the following: (i): a heavy chain complementarity determining region 1 (CDRH1) comprising, alternatively consisting essentially of, or alternatively consisting of any one or more of GFTFXXY (amino acids (aa) 50 to aa 56 of SEQ ID NO: 13), GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 1 or 2 or 3 or 24), or GFTFSRY (aa 50 to aa 56 of SEQ ID NO: 4 or 5 or 6 or 26), where X is any amino acid or amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 1-6; (ii): a heavy chain complementarity determining region 2 (CDRH2) comprising, alternatively consisting essentially of, or alternatively consisting of any one or more of XSXXXX (amino acids (aa) 76 to aa 81 of SEQ ID NO: 13), GSDRRH (aa 76 to aa 81 of SEQ ID NO: 1 or 2 or 3 or 24), or SSGGSY (aa 76 to aa 81 of SEQ ID NO: 4 or 5 or 6 or 26), where X is any amino acid or amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 1-6; (iii): a heavy chain complementarity determining region 3 (CDRH3) comprising, alternatively consisting essentially of, or alternatively consisting of any one or more of XXXXXXXYXXFDX (amino acids (aa) 121 to aa 133 of SEQ ID NO: 13), VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 1, or 2, or 3, or 24), or ERHGGDGYWYFDV (aa 121 to aa 133 of SEQ ID NO: 4, or 5, or 6, or 26), where X is any amino acid or amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 1-6; (iv): a light chain complementarity determining region 1 (CDRL1) comprising, alternatively consisting essentially of, or alternatively consisting of any one or more of QXXXXXXXXXX (aa47 to aa57 of SEQ ID NO: 14), QXXXXX (aa47 to aa52 of SEQ ID NO: 14), QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 7 or 8 or 9 or 25), or QDISNY (aa47 to aa52 of SEQ ID NO: 10 or 11 or 12 or 27), where X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 7 to 12; (v): a light chain complementarity determining region 2 (CDRL2) comprising, alternatively consisting essentially of, or alternatively consisting of any one or more of XXS (aa75 to aa77 of SEQ ID NO: 14), LVS (aa75 to aa77 of SEQ ID NO: 7 or 8 or 9 or 25), or YTS (aa70 to aa72 of SEQ ID NO: 10 or 11 or 12 or 27), where X is any amino acid or amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 7 to 12; and (vi) A light chain complementarity determining region 3 (CDRL3) comprising, alternatively consisting essentially of, or alternatively consisting of, any one or more of the following: XQGXXXXXT (aa114 to aa122 of SEQ ID NO: 14), WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 7 or 8 or 9 or 25), or QQGNPLRT (aa109 to aa116 of SEQ ID NO: 10 or 11 or 12 or 27), where X is any amino acid or amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 7 to 12. An anti-DNABII antibody or antigen-binding fragment thereof comprising, or consisting essentially of, or further consisting of one, two, three, four, five, or all six of: There is provided a composition or combination comprising, consisting essentially of, or alternatively consisting of:

[0013] In one aspect, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide, optionally comprising one or more mutations (i.e., a mutant HMGB1 (mHMGB1) polypeptide or corresponding mutations in HMGB2, HMGB3, or HMGB4), or a fragment thereof, optionally comprising, consisting essentially of, or consisting of an HMGB, optionally the B box or the A box, or both (e.g., the AB box) of an HMGB, optionally an HMGB1 polypeptide, wherein optionally the HMGB polypeptide or HMGB1 polypeptide or fragment thereof is isolated, engineered, or both; and (b) (i) to (vi) of the following: (i) a heavy chain complementarity-determining region 1 (CDRH1) comprising, alternatively consisting essentially of, or alternatively consisting of GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 1, 2, 3, or 24); (ii) a heavy chain complementarity-determining region 2 (CDRH2) comprising, alternatively consisting essentially of, or alternatively consisting of GSDRRH (aa 76 to aa 81 of SEQ ID NO: 1, 2, 3, or 24); (iii) a heavy chain complementarity-determining region 3 (CDRH3) comprising, alternatively consisting essentially of, or alternatively consisting of VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 1, 2, 3, or 24); (iv) a light chain complementarity-determining region 1 (CDRL1) comprising, alternatively consisting essentially of, or alternatively consisting of QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7, 8, 9, or 25); (v) a light chain complementarity-determining region 2 (CDRL2) comprising, or alternatively consisting essentially of, or alternatively consisting of LVS (aa75 to aa77 of SEQ ID NO: 7, 8, 9, or 25); and (vi) a light chain complementarity-determining region 3 (CDRL3) comprising, or consisting essentially of, or alternatively consisting of WQGTHFP (aa114 to aa120 of SEQ ID NO: 7, 8, 9, or 25); An anti-DNABII antibody or antigen-binding fragment thereof comprising, or consisting essentially of, or further consisting of one, two, three, four, five, or all six of: There is provided a composition or combination comprising, consisting essentially of, or alternatively consisting of:

[0014] In another aspect, (a) a high mobility group box protein HMGB polypeptide, optionally an HMGB1 polypeptide, or a fragment thereof comprising, consisting essentially of, or consisting of its B box, A box, or AB box; and (b) (i) a heavy chain complementarity-determining region 1 (CDRH1) comprising, alternatively consisting essentially of, or alternatively consisting of GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 1, 2, 3, or 24); (ii) a heavy chain complementarity-determining region 2 (CDRH2) comprising, alternatively consisting essentially of, or alternatively consisting of GSDRRH (aa 76 to aa 81 of SEQ ID NO: 1, 2, 3, or 24); (iii) a heavy chain complementarity-determining region 3 (CDRH3) comprising, alternatively consisting essentially of, or alternatively consisting of VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 1, 2, 3, or 24); (iv) a light chain complementarity-determining region 1 (CDRL1) comprising, alternatively consisting essentially of, or alternatively consisting of QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7, 8, 9, or 25); (v) a light chain complementarity-determining region 2 (CDRL2) comprising, or alternatively consisting essentially of, or alternatively consisting of LVS (aa75 to aa77 of SEQ ID NO: 7, 8, 9, or 25); and (vi) a light chain complementarity-determining region 3 (CDRL3) comprising, or consisting essentially of, or alternatively consisting of WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 7, 8, 9, or 25); An anti-DNABII antibody or antigen-binding fragment thereof comprising, consisting essentially of, or further consisting of There is provided a composition or combination comprising, consisting essentially of, or alternatively consisting of:

[0015] In yet another aspect, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide or a fragment thereof comprising, consisting essentially of, or consisting of a B box, an A box, or an AB box; and (b) an antibody or antigen-binding fragment thereof that specifically recognizes and binds to the tip domain of the DNABII protein; A composition or combination comprising, consisting essentially of, or even consisting of however, (i) the composition or combination does not comprise SEQ ID NO: 52, or (ii) the antigen-binding fragment does not include the Fab, optionally the Fab of a polyclonal antibody, or the antibody does not include a polyclonal antibody; or Both (i) and (ii) The present invention provides a composition or combination, wherein

[0016] In one aspect, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide, optionally comprising one or more mutations (i.e., a mutant HMGB1 (mHMGB1) polypeptide or corresponding mutations in HMGB2, HMGB3, or HMGB4, or a fragment thereof, optionally comprising, consisting essentially of, or consisting of an HMGB, optionally the B box or the A box, or both (e.g., the AB box) of an HMGB, optionally an HMGB1 polypeptide, wherein optionally the HMGB polypeptide or HMGB1 polypeptide or fragment thereof is isolated, engineered, or both; and (b) (i) CDR1-3 of any one of SEQ ID NOs: 1-6, 13, 24 or 26, or their respective equivalents; and / or (ii) CDR1 to CDR3 of any one of SEQ ID NOs: 7 to 12, 14, 25, or 27, or their respective equivalents. An anti-DNABII antibody or antigen-binding fragment thereof comprising, or alternatively consisting essentially of, or alternatively consisting of There is provided a composition or combination comprising, alternatively consisting essentially of, or alternatively consisting of:

[0017] In a further aspect, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide, optionally comprising one or more mutations (i.e., a mutant HMGB1 (mHMGB1) polypeptide), or a fragment thereof, optionally comprising, consisting essentially of, or consisting of an HMGB, optionally the B box or the A box or both (e.g., the AB box) of an HMGB, optionally an HMGB1 polypeptide, wherein optionally the HMGB polypeptide or HMGB1 polypeptide or fragment thereof is isolated or engineered, or both; and (b) an anti-DNABII antibody or antigen-binding fragment thereof that competes for epitope binding with an antibody or antigen-binding fragment thereof disclosed herein; There is provided a composition or combination comprising, alternatively consisting essentially of, or alternatively consisting of:

[0018] In still a further aspect, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide, optionally comprising one or more mutations (i.e., a mutant HMGB1 (mHMGB1) polypeptide or corresponding mutations in HMGB2, HMGB3, or HMGB4), or a fragment thereof, optionally comprising, consisting essentially of, or consisting of an HMGB, optionally the B box or the A box, or both (e.g., the AB box) of an HMGB, optionally an HMGB1 polypeptide, wherein optionally the HMGB polypeptide or HMGB1 polypeptide or fragment thereof is isolated, engineered, or both; and (b) a polypeptide comprising a complementarity-determining region (CDR) disclosed herein In some embodiments, the CDRs comprise, consist essentially of, or further consist of any one or more of heavy chain (HC) CDR1 (CDRH1), HCCDR2 (CDRH2), HCCDR3 (CDRH3), light chain (LC) CDR1 (CDRL1), LCCDR2 (CDRL2), or LCCDR3 (CDRL3).

[0019] In one aspect, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide, further optionally comprising one or more mutations (i.e., a mutant HMGB1 (mHMGB1) polypeptide or corresponding mutations in HMGB2, HMGB3, or HMGB4), or a fragment thereof, optionally comprising, consisting essentially of, or consisting of an HMGB, optionally the B box or the A box, or both (e.g., the AB box) of an HMGB, optionally an HMGB1 polypeptide, wherein optionally the HMGB polypeptide or HMGB1 polypeptide or fragment thereof is isolated, engineered, or both; and (b) Below: (i) A heavy chain complementarity determining region 1 (CDRH1) comprising, alternatively consisting essentially of, or alternatively consisting of, any one of the following: GFTFXXY (amino acids (aa) 50 to 56 of SEQ ID NO: 13, where X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 1 to 6), GFTFRTY (aa 50 to 56 of SEQ ID NO: 1, 2, 3, or 24), GFTFSRY (aa 50 to 56 of SEQ ID NO: 4, 5, 6, or 26), GFTFRTYA (aa 50 to 56 of SEQ ID NO: 1, 2, 3, or 24), or 7), aASGFTFRTYAMS (aa47 to aa59 of SEQ ID NO:24: where lowercase a is A, i.e., aa47 to aa59 of SEQ ID NO:1 or 2, or where lowercase a is K, i.e., aa47 to aa59 of SEQ ID NO:3), GFTFSRYG (aa50 to aa57 of SEQ ID NO:4 or 5 or 6 or 26), or aASGFTFSRYGMS (aa47 to aa59 of SEQ ID NO:26: where lowercase a is A, i.e., aa47 to aa59 of SEQ ID NO:4 or 5, or where lowercase a is T, i.e., aa47 to aa59 of SEQ ID NO:6); (ii) Heavy chain complementarity determining region 2 (CDRH2) comprising, alternatively consisting essentially of, or alternatively consisting of, any one of the following: XSXXXX (amino acids (aa) 76 to aa81 of SEQ ID NO: 13, where X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 1 to 6), GSDRRH (aa 76 to aa81 of SEQ ID NO: 1 or 2 or 3 or 24), SSGGSY (aa 76 to aa81 of SEQ ID NO: 4 or 5 or 6 or 26), IGSDRRHT ... sequence number 1 or 2 or 3 or 24), IGSDRRHTY (aa75 to aa83 of SEQ ID NO: 1 or 2 or 3 or 24), TIGSDRRHTY (aa74 to aa83 of SEQ ID NO: 1 or 2 or 3 or 24), WVATIGSDRRHTYYP (aa71 to aa85 of SEQ ID NO: 1 or 2 or 3 or 24), ISSGGSYT (aa75 to aa82 of SEQ ID NO: 4 or 5 or 6 or 26), or TISSGGSYTY (aa74 to aa83 of SEQ ID NO: 4 or 5 or 6 or 26); (iii) a heavy chain complementarity determining region 3 (CDRH3) comprising, alternatively consisting essentially of, or alternatively consisting of, any one of the following: XXXXXXXYXXFDX (amino acids (aa) 121 to aa 133 of SEQ ID NO: 13, where X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 1 to 6), VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 1 or 2 or 3 or 24), ERHGGDGYWYFDV (aa 121 to aa 133 of SEQ ID NO: 4 or 5 or 6 or 26), VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 1 or 2 or 3 or 24), or ER (aa 121 to aa 122 of SEQ ID NO: 4 or 5 or 6 or 26); (iv) Light chain complementarity determining region 1 (CDRL1) comprising, alternatively consisting essentially of, or alternatively consisting of, any one of the following: QXXXXXXXXXX (aa47 to aa57 of SEQ ID NO: 14, where X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 7 to 12), QXXXXX (aa47 to aa52 of SEQ ID NO: 14, where X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 7 to 12), QSL LDSDGKTF (aa47 to aa57 of SEQ ID NO: 7 or 8 or 9 or 25), QDISNY (aa47 to aa52 of SEQ ID NO: 10 or 11 or 12 or 27), rSSQSLLDSDGKTFLN (aa44 to aa59 of SEQ ID NO: 25, where lowercase r is R, i.e., aa44 to aa59 of SEQ ID NO: 7 or 8, or where lowercase r is K, i.e., aa44 to aa59 of SEQ ID NO: 9, or RASQDISNYLN (aa44 to aa54 of SEQ ID NO: 10 or 11 or 12 or 27); (v) A light chain complementarity-determining region 2 (CDRL2) comprising, alternatively consisting essentially of, or alternatively consisting of, any one of the following: XXS (aa 75 to aa 77 of SEQ ID NO: 14, where X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 7 to 12), LVS (aa 75 to aa 77 of SEQ ID NO: 7, or 8, or 9, or 25), YTS (aa 70 to aa 77 of SEQ ID NO: 10, or 11, or 12, or 27), aa72), LVSKlDS (aa75 to aa81 of SEQ ID NO: 25: where the lowercase l is L, i.e., aa75 to aa81 of SEQ ID NO: 7 or 9, or where the lowercase l is R, i.e., aa75 to aa81 of SEQ ID NO: 8), YLVSKlDS (aa74 to aa81 of SEQ ID NO: 25: where the lowercase l is L, i.e., aa74 to aa81 of SEQ ID NO: 7 or 9, or where the lowercase l is R, i.e., aa74 to aa81 of SEQ ID NO: 8 aa81), LVSKlDSG (aa75 to aa82 of SEQ ID NO: 25: where the lowercase l is L, i.e., aa75 to aa82 of SEQ ID NO: 7 or 9, or where the lowercase l is R, i.e., aa75 to aa82 of SEQ ID NO: 8), YLVSKlDSGV (aa74 to aa83 of SEQ ID NO: 25: where the lowercase l is L, i.e., aa74 to aa83 of SEQ ID NO: 7 or 9, or where the lowercase l is R, i.e., aa 74 to 83), RLIYLVSKlDSGVPD (aa71 to aa85 of SEQ ID NO:25: where the lowercase l is L, i.e., aa71 to aa85 of SEQ ID NO:7 or 9, or where the lowercase l is R, i.e., aa71 to aa85 of SEQ ID NO:8), YTSRLHS (aa70 to aa76 of SEQ ID NO:10 or 11 or 12 or 27); or YYTSRLHS (aa69 to aa76 of SEQ ID NO:10 or 11 or 12 or 27); and (vi) A light chain complementarity-determining region 3 (CDRL3) comprising, alternatively consisting essentially of, or alternatively consisting of, any one of the following: XQGXXXXXT (aa114 to aa122 of SEQ ID NO: 14, where X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 7 to 12), WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 7, 8, 9, or 25), QQGNPLRT (aa109 to aa112 of SEQ ID NO: 10, 11, 12, or 27), 116), WQGTHFP (aa114 to aa120 of SEQ ID NO: 7, or 8, or 9, or 25), WQGTHFPY (aa114 to aa121 of SEQ ID NO: 7, or 8, or 9, or 25), WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 7, or 8, or 9, or 25), or QQ (aa109 to aa110 of SEQ ID NO: 10, or 11, or 12, or 27). There is provided a composition or combination comprising, alternatively consisting essentially of, or alternatively consisting of:

[0020] In a further aspect, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide, further optionally comprising one or more mutations (i.e., a mutant HMGB1 (mHMGB1) polypeptide or corresponding mutations in HMGB2, HMGB3, or HMGB4), or a fragment thereof, optionally comprising, consisting essentially of, or consisting of an HMGB, optionally the B box or the A box, or both (e.g., the AB box) of an HMGB, optionally an HMGB1 polypeptide, wherein optionally the HMGB polypeptide or HMGB1 polypeptide or fragment thereof is isolated, engineered, or both; and (b) an isolated polypeptide comprising, consisting essentially of, or even consisting of any one or more of SEQ ID NOs: 1-14 or 24-27, or their respective equivalents. There is provided a composition or combination comprising, alternatively consisting essentially of, or alternatively consisting of:

[0021] In yet a further aspect, (a) an isolated polynucleotide encoding an HMGB polypeptide disclosed herein, optionally an HMGB1 polypeptide, or a fragment thereof, or an equivalent of each thereof, and optionally operably linked to a regulatory sequence that directs its expression, such as a promoter or enhancer, or both; and (b) an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof disclosed herein, or their respective equivalents, optionally operably linked to regulatory sequences that direct its expression, e.g., a promoter or enhancer, or both; There is provided a composition or combination comprising, alternatively consisting essentially of, or alternatively consisting of:

[0022] In one aspect, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide, or a fragment thereof comprising, consisting essentially of, or consisting of the B box, A box, or AB box thereof; and (b) an anti-DNABII antibody or antigen-binding fragment thereof disclosed herein, e.g., (i) a heavy chain complementarity-determining region 1 (CDRH1) comprising, alternatively consisting essentially of, or alternatively consisting of GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 1, 2, 3, or 24); (ii) a heavy chain complementarity-determining region 2 (CDRH2) comprising, alternatively consisting essentially of, or alternatively consisting of GSDRRH (aa 76 to aa 81 of SEQ ID NO: 1, 2, 3, or 24); (iii) a heavy chain complementarity-determining region 3 (CDRH3) comprising, alternatively consisting essentially of, or alternatively consisting of VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 1, 2, 3, or 24); (iv) a light chain complementarity-determining region 1 (CDRL1) comprising, alternatively consisting essentially of, or alternatively consisting of QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7, 8, 9, or 25); (v) a light chain complementarity-determining region 2 (CDRL2) comprising, or alternatively consisting essentially of, or alternatively consisting of LVS (aa75 to aa77 of SEQ ID NO: 7, 8, 9, or 25); and (vi) a light chain complementarity-determining region 3 (CDRL3) comprising, or consisting essentially of, or alternatively consisting of WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 7, 8, 9, or 25); An antibody or antigen-binding fragment thereof comprising, consisting essentially of, or alternatively consisting of In accordance with the present invention, there is provided a polypeptide comprising, consisting essentially of, or alternatively consisting of:

[0023] In a further aspect, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide, or a fragment thereof comprising, consisting essentially of, or consisting of the B box, A box, or AB box thereof; and (b) an antibody or antigen-binding fragment thereof that specifically recognizes and binds to the tip domain of the DNABII protein; A polypeptide comprising, consisting essentially of, or alternatively consisting of, however, (i) the polypeptide does not comprise SEQ ID NO: 52, or (ii) the antigen-binding fragment does not include the Fab, optionally the Fab of a polyclonal antibody, or the antibody does not include a polyclonal antibody; or Both (i) and (ii) A polypeptide is provided, wherein:

[0024] In one aspect, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide, or a fragment thereof comprising, consisting essentially of, or consisting of the B box, A box, or AB box thereof; and (b) an anti-DNABII antibody or antigen-binding fragment thereof disclosed herein, e.g., (i) a heavy chain complementarity-determining region 1 (CDRH1) comprising, alternatively consisting essentially of, or alternatively consisting of GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 1, 2, 3, or 24); (ii) a heavy chain complementarity-determining region 2 (CDRH2) comprising, alternatively consisting essentially of, or alternatively consisting of GSDRRH (aa 76 to aa 81 of SEQ ID NO: 1, 2, 3, or 24); (iii) a heavy chain complementarity-determining region 3 (CDRH3) comprising, alternatively consisting essentially of, or alternatively consisting of VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 1, 2, 3, or 24); (iv) a light chain complementarity-determining region 1 (CDRL1) comprising, alternatively consisting essentially of, or alternatively consisting of QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7, 8, 9, or 25); (v) a light chain complementarity-determining region 2 (CDRL2) comprising, or alternatively consisting essentially of, or alternatively consisting of LVS (aa75 to aa77 of SEQ ID NO: 7, 8, 9, or 25); and (vi) a light chain complementarity-determining region 3 (CDRL3) comprising, or consisting essentially of, or alternatively consisting of WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 7, 8, 9, or 25); An antibody or antigen-binding fragment thereof comprising, consisting essentially of, or alternatively consisting of a polynucleotide encoding Alternatively, a polynucleotide complementary thereto is provided.

[0025] In a further aspect, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide, or a fragment thereof comprising, consisting essentially of, or consisting of the B box, A box, or AB box thereof; and (b) an antibody or antigen-binding fragment thereof that specifically recognizes and binds to the tip domain of the DNABII protein; or a polynucleotide complementary thereto, however, (i) the polynucleotide does not encode SEQ ID NO: 52, or (ii) the antigen-binding fragment does not include the Fab, optionally the Fab of a polyclonal antibody, or the antibody does not include a polyclonal antibody; or Both (i) and (ii) A polynucleotide is provided, wherein:

[0026] In one aspect, a vector is provided that comprises, or alternatively, essentially consists of, or even consists of the polynucleotide disclosed herein. In some embodiments, the vector is a non-viral vector (e.g., a plasmid) or a viral vector. In further embodiments, the viral vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector. Additionally or alternatively, the vector further comprises a regulatory sequence that directs the expression of the polynucleotide.

[0027] Provided herein are host cells comprising one or more of the compositions or combinations described herein, the polypeptides disclosed herein, the polynucleotides disclosed herein, or the vectors disclosed herein.

[0028] In one aspect, a method is provided for inhibiting or competing with the binding of a DNABII polypeptide or protein to microbial DNA, the method comprising, alternatively consisting essentially of, or alternatively consisting of, contacting a DNABII polypeptide or protein with a combination or composition disclosed herein.

[0029] In another aspect, a method of disrupting a biofilm is provided, the method comprising, alternatively consisting essentially of, or alternatively consisting of, contacting the biofilm with a composition or combination disclosed herein.

[0030] In yet another aspect, a method is provided for preventing the formation of or disrupting a biofilm on a surface, the method comprising, or alternatively consisting essentially of, or even consisting of contacting the biofilm with a combination or composition disclosed herein, or treating a surface susceptible to or containing a biofilm with a combination or composition disclosed herein.

[0031] In a further aspect, there is provided a method for preventing the formation of or disrupting a biofilm in a subject, the method comprising, alternatively consisting essentially of, or alternatively consisting of, administering to the subject a composition or combination disclosed herein.

[0032] In yet a further aspect, there is provided a method for inhibiting, preventing or treating a biofilm-producing microbial infection in a subject, the method comprising, alternatively consisting essentially of, or even consisting of, administering to the subject a combination or composition disclosed herein.

[0033] In one aspect, a method is provided for treating a condition characterized by biofilm formation in a subject, the method comprising, alternatively consisting essentially of, or alternatively consisting of, administering to the subject a composition or combination disclosed herein.

[0034] In one aspect, a method is provided for one or more of: (A) preventing biofilm formation or disrupting biofilms in vitro or ex vivo, (B) preventing biofilm formation or disrupting biofilms in a subject, (C) inhibiting, preventing, or treating a biofilm-producing microbial infection in a subject, or (D) treating a condition characterized by biofilm formation in a subject. The method comprises administering to a subject: (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide or a fragment thereof comprising, consisting essentially of, or alternatively consisting of, a B box, an A box, or an AB box; and (b) an anti-DNABII antibody or antigen-binding fragment thereof disclosed herein; The method may comprise, alternatively consist essentially of, or alternatively further consist of, administering

[0035] In one aspect, a method is provided for inducing or increasing the formation of neutrophil extracellular traps (NETs) immediately adjacent to a biofilm in a subject, and for disrupting the biofilm, optionally without inducing a pro-inflammatory response. The method comprises administering to a subject: (a) a high mobility group box 1 protein (HMGB1) polypeptide comprising the amino acid sequence of SEQ ID NO: 52, or a fragment thereof comprising, consisting essentially of, or alternatively consisting of the B box, A box, or AB box thereof; and (b) an anti-DNABII antibody or antigen-binding fragment thereof disclosed herein; The method may comprise, alternatively consist essentially of, or alternatively further consist of, administering

[0036] In one aspect, a method is provided for one or more of: (A) preventing biofilm formation or disrupting biofilms in vitro or ex vivo, (B) preventing biofilm formation or disrupting biofilms in a subject, (C) inhibiting, preventing, or treating a biofilm-producing microbial infection in a subject, or (D) treating a condition characterized by biofilm formation in a subject, the method comprising, consisting essentially of, or even further consisting of administering to the subject one or more of a composition or combination disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, or a host cell disclosed herein.

[0037] In one aspect, a method is provided for inducing or increasing the formation of neutrophil extracellular traps (NETs) immediately adjacent to a biofilm in a subject, and for disrupting the biofilm, optionally without inducing a pro-inflammatory response. The method comprises, or consists essentially of, or further consists of administering to the subject one or more of the compositions or combinations disclosed herein, the polypeptides disclosed herein, the polynucleotides disclosed herein, the vectors disclosed herein, or the host cells disclosed herein, with the proviso that the HMGB1 polypeptide comprises, consists essentially of, or further consists of SEQ ID NO:52.

[0038] Additionally, kits for use in the methods disclosed herein are provided, which comprise, consist essentially of, or even consist of instructions for use and one or more of a composition or combination disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, or a host cell disclosed herein. In an embodiment of the present invention, for example, the following items are provided: (Item 1) (a) a high mobility group box protein (HMGB) polypeptide, or a fragment thereof containing the B box, A box, or AB box; and (b) (i) heavy chain complementarity-determining region 1 (CDRH1) containing GFTFRTY (aa50 to aa56 of SEQ ID NO: 1, 2, 3, or 24); (ii) heavy chain complementarity-determining region 2 (CDRH2) containing GSDRRH (aa76 to aa81 of SEQ ID NO: 1 or 2 or 3 or 24); (iii) heavy chain complementarity-determining region 3 (CDRH3) containing VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 1, 2, 3, or 24); (iv) light chain complementarity-determining region 1 (CDRL1) comprising QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 7 or 8 or 9 or 25); (v) light chain complementarity-determining region 2 (CDRL2) containing LVS (aa75 to aa77 of SEQ ID NO: 7, 8, 9, or 25); and (vi) Light chain complementarity-determining region 3 (CDRL3) containing WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 7, 8, 9, or 25) an anti-DNABII antibody or an antigen-binding fragment thereof comprising A composition or combination comprising: (Item 2) 2. The composition or combination of item 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain (HC) immunoglobulin variable domain comprising amino acids 25 to 144 of any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. (Item 3) 3. The composition or combination according to item 2, wherein the antibody or antigen-binding fragment thereof comprises a light chain (LC) immunoglobulin variable domain comprising amino acids 21 to 132 of any one of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. (Item 4) 4. The composition or combination according to item 3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain (HC) immunoglobulin variable domain comprising amino acids 25 to 144 of any one of SEQ ID NO: 1, and the LC immunoglobulin variable domain comprises amino acids 21 to 132 of SEQ ID NO: 7. (Item 5) 4. The composition or combination according to item 3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain (HC) immunoglobulin variable domain comprising amino acids 25 to 144 of any one of SEQ ID NO: 1, and the LC immunoglobulin variable domain comprises amino acids 21 to 132 of SEQ ID NO: 8. (Item 6) 4. The composition or combination according to item 3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain (HC) immunoglobulin variable domain comprising amino acids 25 to 144 of any one of SEQ ID NO: 1, and the LC immunoglobulin variable domain comprises amino acids 21 to 132 of SEQ ID NO: 9. (Item 7) 7. The composition or combination according to any one of items 1 to 6, wherein the antibody comprises a constant region selected from an IgA constant region, an IgD constant region, an IgE constant region, an IgG constant region, or an IgM constant region. (Item 8) 8. The composition or combination according to any one of items 1 to 7, wherein the antigen-binding fragment thereof comprises Fab, F(ab')2, Fab', scFv, or Fv. (Item 9) 9. The composition or combination according to any one of items 1 to 8, wherein the antibody or antigen-binding fragment thereof is modified. (Item 10) 10. The composition or combination according to item 9, wherein the antibody or antigen-binding fragment thereof is modified by a process selected from pegylation, polysialylation, HESylation, or glycosylation. (Item 11) 11. The composition or combination according to any one of items 1 to 10, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody or an antigen-binding fragment of the monoclonal antibody. (Item 12) 12. The composition or combination according to any one of items 1 to 11, wherein the antibody or antigen-binding fragment thereof comprises a humanized or human framework. (Item 13) 13. The composition or combination according to any one of items 1 to 12, wherein the HMGB polypeptide is an HMGB1 polypeptide, optionally further comprising one or more mutations selected from the group consisting of mutations at K12, C23, C45, C106, or K114, or an HMGB polypeptide selected from the group consisting of HMGB2, HMGB3, or HMGB4 polypeptides having a mutation corresponding to the HMGB1 polypeptide comprising one or more mutations selected from the group consisting of mutations at K12, C23, C45, C106, or K114. (Item 14) 14. The composition or combination according to any one of items 1 to 13, wherein the HMGB polypeptide, optionally the A box of the HMGB11 polypeptide, further comprises one or more mutations selected from the group consisting of mutations at K12, C23, or C45, or, if the HMGB polypeptide is selected from the group consisting of HMGB2, HMGB3, or HMGB4 polypeptides having a mutation corresponding to the mutated HMGB1 polypeptide, a corresponding mutation. (Item 15) 15. The composition or combination according to any one of items 1 to 14, wherein the HMGB, optionally the B box of the HMGB1 polypeptide, further comprises one or both mutations at C106 or K114, or, if the HMGB polypeptide is selected from the group consisting of HMGB2, HMGB3, or HMGB4 polypeptides, a mutation corresponding to the mutated HMGB1 polypeptide. (Item 16) 16. The composition or combination according to any one of items 13 to 15, wherein the one or more mutations are mutations to serine, glycine, alanine, valine, isoleucine, or threonine. (Item 17) 17. The composition or combination according to any one of items 1 to 16, wherein the HMGB polypeptide, optionally the HMGB1 polypeptide, further comprises one or more mutations selected from C23S, C45S, and C106S, or the HMGB polypeptide is selected from the group of HMGB2, HMGB3, or HMGB4 polypeptides having one or more mutations corresponding to the mutated HMGB1 polypeptide. (Item 18) 18. The composition or combination according to any one of items 1 to 17, wherein the HMGB polypeptide, optionally the HMGB1 polypeptide, further comprises an HMGB polypeptide selected from the group consisting of HMGB2, HMGB3, or HMGB4 polypeptides having a C45S mutation or a mutation corresponding to the mutated HMGB1 polypeptide. (Item 19) (a) the HMGB polypeptide, and optionally the HMGB1 polypeptide, further comprising a C45S mutation; and (b) the anti-DNABII antibody or antigen-binding fragment thereof, comprising the heavy chain (HC) immunoglobulin variable domain comprising amino acids 25 to 144 of SEQ ID NO: 1 and the light chain (LC) immunoglobulin variable domain comprising amino acids 21 to 132 of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. 19. The composition or combination according to any one of items 1 to 18, comprising: (Item 20) (a) a high mobility group box 1 protein (HMGB1) polypeptide, or a fragment thereof containing the B box, A box, or AB box; and (b) an antibody or antigen-binding fragment thereof that specifically recognizes and binds to the tip domain of the DNABII protein; A composition or combination comprising: With the proviso that (i) the composition or combination does not comprise SEQ ID NO: 52, or (ii) the antigen-binding fragment does not comprise a Fab, optionally a Fab of a polyclonal antibody, or the antibody does not comprise a polyclonal antibody, or both (i) and (ii). (Item 21) The tip domain is NFELRDKSSRPGRNPKTGDVV (SEQ ID NO: 31); SLHHRQPRLGRNPKTGDSVNL (SEQ ID NO: 32); [ka] (wherein "X" is an optional amino acid linker sequence and "X1" is any amino acid) (SEQ ID NO: 38); [ka] (wherein "X1" is any amino acid) (SEQ ID NO: 39); or [ka] 21. The composition or combination according to item 20, comprising one or more amino acid sequences selected from: (Item 22) (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide or a fragment thereof containing the B box, A box, or AB box; and (b) (i) heavy chain complementarity-determining region 1 (CDRH1) containing GFTFRTY (aa50 to aa56 of SEQ ID NO: 1, 2, 3, or 24); (ii) heavy chain complementarity-determining region 2 (CDRH2) containing GSDRRH (aa76 to aa81 of SEQ ID NO: 1 or 2 or 3 or 24); (iii) heavy chain complementarity-determining region 3 (CDRH3) containing VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 1, 2, 3, or 24); (iv) light chain complementarity-determining region 1 (CDRL1) comprising QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 7 or 8 or 9 or 25); (v) light chain complementarity-determining region 2 (CDRL2) containing LVS (aa75 to aa77 of SEQ ID NO: 7, 8, 9, or 25); and (vi) Light chain complementarity-determining region 3 (CDRL3) containing WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 7, 8, 9, or 25) A polypeptide comprising an anti-DNABII antibody or an antigen-binding fragment thereof comprising: (Item 23) (a) a high mobility group box protein (HMGB), optionally an HMGB1 polypeptide, or a fragment thereof comprising the B box, A box, or AB box; and (b) an antibody or antigen-binding fragment thereof that specifically recognizes and binds to the tip domain of the DNABII protein; A polypeptide comprising: With the proviso that (i) the polypeptide does not comprise SEQ ID NO: 52, or (ii) the antigen-binding fragment does not comprise a Fab, optionally a Fab of a polyclonal antibody, or the antibody does not comprise a polyclonal antibody, or both (i) and (ii). (Item 24) The tip domain is NFELRDKSSRPGRNPKTGDVV (SEQ ID NO: 31); SLHHRQPRLGRNPKTGDSVNL (SEQ ID NO: 32); [ka] (wherein "X" is an optional amino acid linker sequence and "X1" is any amino acid) (SEQ ID NO: 38); [ka] (wherein "X1" is any amino acid) (SEQ ID NO: 39); or [ka] 24. The polypeptide according to item 23, comprising one or more amino acid sequences selected from: (Item 25) 25. The polypeptide according to any one of items 22 to 24, further comprising a cleavable peptide located between (a) and (b). (Item 26) (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide, or a fragment thereof containing the B box, A box, or AB box; and (b) (i) heavy chain complementarity-determining region 1 (CDRH1) containing GFTFRTY (aa50 to aa56 of SEQ ID NO: 1, 2, 3, or 24); (ii) heavy chain complementarity-determining region 2 (CDRH2) containing GSDRRH (aa76 to aa81 of SEQ ID NO: 1 or 2 or 3 or 24); (iii) heavy chain complementarity-determining region 3 (CDRH3) containing VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 1, 2, 3, or 24); (iv) light chain complementarity-determining region 1 (CDRL1) comprising QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 7 or 8 or 9 or 25); (v) light chain complementarity-determining region 2 (CDRL2) containing LVS (aa75 to aa77 of SEQ ID NO: 7, 8, 9, or 25); and (vi) Light chain complementarity-determining region 3 (CDRL3) containing WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 7, 8, 9, or 25) a polynucleotide encoding an anti-DNABII antibody or an antigen-binding fragment thereof comprising: Or a polynucleotide complementary thereto. (Item 27) (a) a high mobility group box protein (HMGB), optionally an HMGB1 polypeptide, or a fragment thereof comprising the B box, A box, or AB box; and (b) an antibody or antigen-binding fragment thereof that specifically recognizes and binds to the tip domain of the DNABII protein; or a polynucleotide complementary thereto, provided that (i) the polynucleotide does not encode SEQ ID NO: 52, or (ii) the antigen-binding fragment does not comprise a Fab, optionally a Fab of a polyclonal antibody, or the antibody does not comprise a polyclonal antibody, or both (i) and (ii). (Item 28) The tip domain is NFELRDKSSRPGRNPKTGDVV (SEQ ID NO: 31); SLHHRQPRLGRNPKTGDSVNL (SEQ ID NO: 32); [ka] (wherein "X" is an optional amino acid linker sequence and "X1" is any amino acid) (SEQ ID NO: 38); [ka] (wherein "X1" is any amino acid) (SEQ ID NO: 39); or [ka] 28. The polynucleotide of item 27, comprising one or more amino acid sequences selected from: (Item 29) A vector comprising the polynucleotide according to any one of Items 26 to 28. (Item 30) A host cell comprising one or more of the composition or combination according to any one of items 1 to 21, the polypeptide according to any one of items 22 to 25, the polynucleotide according to any one of items 26 to 28, or the vector according to item 29. (Item 31) 31. The host cell according to item 30, which secretes the HMGB polypeptide, optionally the HMGB1 polypeptide or the fragment thereof; and the anti-DNABII antibody or antigen-binding fragment thereof. (Item 32) (A) preventing the formation of or disrupting biofilms in vitro or ex vivo; (B) preventing the formation of or disrupting a biofilm in a subject; (C) inhibiting, preventing, or treating a biofilm-producing microbial infection in a subject; or (D) Treating a condition characterized by biofilm formation in a subject. A method for one or more of To the subject, (a) a high mobility group box protein (HMGB) polypeptide, optionally an HMGB1 polypeptide or a fragment thereof comprising the B box, A box, or AB box; and (b) (i) heavy chain complementarity-determining region 1 (CDRH1) containing GFTFRTY (aa50 to aa56 of SEQ ID NO: 1, 2, 3, or 24); (ii) heavy chain complementarity-determining region 2 (CDRH2) containing GSDRRH (aa76 to aa81 of SEQ ID NO: 1 or 2 or 3 or 24); (iii) heavy chain complementarity-determining region 3 (CDRH3) containing VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 1, 2, 3, or 24); (iv) light chain complementarity-determining region 1 (CDRL1) comprising QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 7 or 8 or 9 or 25); (v) light chain complementarity-determining region 2 (CDRL2) containing LVS (aa75 to aa77 of SEQ ID NO: 7, 8, 9, or 25); and (vi) Light chain complementarity-determining region 3 (CDRL3) containing WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 7, 8, 9, or 25) an anti-DNABII antibody or an antigen-binding fragment thereof comprising The method of claim 1, further comprising administering (Item 33) 33. The method of claim 32, wherein (i) the HMGB1 polypeptide or fragment thereof does not comprise SEQ ID NO: 52, or (ii) the antigen-binding fragment does not comprise a Fab, optionally a Fab of a polyclonal antibody, or the antibody does not comprise a polyclonal antibody, or both (i) and (ii). (Item 34) 1. A method for inducing or increasing the formation of neutrophil extracellular traps (NETs) immediately adjacent to a biofilm in a subject, and optionally for disrupting said biofilm without inducing a pro-inflammatory response, comprising: To the subject, (a) a high mobility group box 1 protein (HMGB1) polypeptide comprising the amino acid sequence of SEQ ID NO: 52, or a fragment thereof comprising, consisting essentially of, or alternatively consisting of, the B box, A box, or AB box thereof; and (b) (i) heavy chain complementarity-determining region 1 (CDRH1) containing GFTFRTY (aa50 to aa56 of SEQ ID NO: 1, 2, 3, or 24); (ii) heavy chain complementarity-determining region 2 (CDRH2) containing GSDRRH (aa76 to aa81 of SEQ ID NO: 1 or 2 or 3 or 24); (iii) heavy chain complementarity-determining region 3 (CDRH3) containing VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 1, 2, 3, or 24); (iv) light chain complementarity-determining region 1 (CDRL1) comprising QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 7 or 8 or 9 or 25); (v) light chain complementarity-determining region 2 (CDRL2) containing LVS (aa75 to aa77 of SEQ ID NO: 7, 8, 9, or 25); and (vi) Light chain complementarity-determining region 3 (CDRL3) containing WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 7, 8, 9, or 25) an anti-DNABII antibody or an antigen-binding fragment thereof comprising The method of claim 1, further comprising administering (Item 35) 35. The method according to any one of items 32 to 34, wherein the administration of (a) and the administration of (b) are carried out simultaneously or sequentially. (Item 36) 36. The method of any one of items 32 to 35, wherein the administration of (a) and (b) is repeated at least once, at least twice, at least three times, or more times. (Item 37) (A) preventing the formation of or disrupting biofilms in vitro or ex vivo; (B) preventing the formation of or disrupting a biofilm in a subject; (C) inhibiting, preventing, or treating a biofilm-producing microbial infection in a subject; or (D) Treating a condition characterized by biofilm formation in a subject. A method for one or more of 29. A method comprising administering to the subject one or more of the composition or combination according to any one of items 1 to 21, the polypeptide according to any one of items 22 to 25, the polynucleotide according to any one of items 26 to 28, the vector according to item 29, or the host cell according to item 30 or 31. (Item 38) A method for inducing or increasing the formation of neutrophil extracellular traps (NETs) immediately adjacent to a biofilm in a subject, and optionally disrupting the biofilm without inducing a pro-inflammatory response, the method comprising the step of administering to the subject one or more of the composition or combination described in any one of Items 1 to 21, the polypeptide described in any one of Items 22 to 25, the polynucleotide described in any one of Items 26 to 28, the vector described in Item 29, or the host cell described in Item 30 or 31, wherein the HMGB1 polypeptide comprises the amino acid sequence of SEQ ID NO: 52, or a fragment thereof comprising, consisting essentially of, or further consisting of the B box, A box, or AB box thereof. (Item 39) 39. The method of any one of paragraphs 32 to 38, further comprising administering to the subject one or more of a DNase enzyme, an antibiotic, an antimicrobial, an anti-infective, an anti-fungal, an anti-parasitic, an anti-viral, or an antibody or antigen-binding fragment thereof that specifically recognizes and binds to OMP P5, rsPilA, OMP 26, OMP P2, or type IV Pilin. (Item 40) 40. The method of any one of items 32, 33, 35-37, and 39, wherein the condition characterized by biofilm formation comprises one or more of chronic non-healing wounds, lung infections caused by Burkholderia sp., venous ulcers, diabetic foot ulcers, ear infections, sinus infections, urinary tract infections, gastrointestinal tract diseases, hospital-acquired pneumonia, ventilator-associated pneumonia, surgical implant-associated infections, lung infections, airway infections, cystic fibrosis, chronic obstructive pulmonary disease, catheter-associated infections, indwelling device-associated infections, infections associated with implanted prostheses, osteomyelitis, cellulitis, abscesses, or periodontal disease. (Item 41) A kit for use in the method according to any one of Items 32 to 40, comprising instructions for use and one or more of the composition or combination according to any one of Items 1 to 21, the polypeptide according to any one of Items 22 to 25, the polynucleotide according to any one of Items 26 to 28, the vector according to Item 29, or the host cell according to Item 30 or 31. [Brief explanation of the drawings]

[0039] [Figure 1] Figure 1 provides a model of HMGB1-mediated regulation of the eDNA-dependent bacterial biofilm matrix.

[0040] [Figure 2]Figures 2A–2E provide composite photographs showing the zone between the biofilm and abundant PMNs induced to the site of infection. Figure 2A is a representative low-magnification light micrograph of an H&E-stained cryosection of a 17-day-old biofilm produced by nontypeable Haemophilus influenzae (NTHI) in the middle ear of a chinchilla during experimental otitis media. The area of ​​dense PMN infiltration is located primarily in the region in the upper right corner, while the NTHI-induced biofilm occupies the lower left region of the image. The zone where these two regions meet is demarcated by a dashed line. Bar = 100 μm. Figure 2B is a representative image of a serial section of the in situ biofilm shown in Figure 2A, in which the PMN-rich region immunolabeled with an antibody against elastase to demarcate PMNs intersects with the NTHI biofilm immunolabeled with an antibody against an NTHI outer membrane protein to demarcate the NTHI-induced biofilm; the intersecting region appears intertwined with both fluorescent dyes. Bar = 100 µm. High-magnification immunolabeling confocal images of 11-day-old NTHI biofilms recovered from chinchilla middle ears: Figure 2C shows an NTHI biofilm labeled almost exclusively with an antibody against the DNABII protein HU, where HU labeling is detected on strands of bacterial eDNA; Figure 2D provides the region where the NTHI-induced biofilm intersects with a PMN-rich region, where anti-DNABII (HU) labeling is evident, as well as labeling with anti-HMGB1; d1 and d2 are sequential 1 µm Z-plane images of the inset, demonstrating the absence of physical overlap between DNABII and HMGB1 labeling. Figure 2E provides a PMN-rich region where labeling is exclusively for anti-HMGB1. Bars in panels C-E = 5 µm.

[0041] [Figure 3]Figures 3A-3B show that HMGB1 variants disrupted biofilms formed by diverse high-priority human pathogens. Figure 3A shows that the indicated isoforms of HMGB1 (200 nM unless otherwise indicated) were added to 24-hour biofilms in vitro for 16 hours. For each pathogen tested, five bars are shown, representing, from left to right, data obtained from the control, α-IHFEc, rHMGB1, mHMGB1, and nHMGB1. Exceptions were as follows: 800 nM rHMGB1 or 200 nM mHMGB1 for S. aureus (designated S in ESKAPE); 800 nM rHMGB1, 800 nM mHMGB1, 3.3 mM α-IHFEc IgG for E. faecium (designated E in ESKAPE), for only 1 hour to avoid potential degradation by proteases produced by E. faecium. Biofilms were stained with LIVE / DEAD® stain, visualized via confocal laser scanning microscopy (CLSM), and analyzed by COMSTAT to calculate the average thickness. The percent change in biofilm thickness compared to the control was plotted. Bars indicate SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 via unpaired t-test. Figure 3B provides representative images of UPEC biofilms incubated with the indicated concentrations of rHMGB1. Together, the data show that rHMGB1, nHMGB1, and mHMGB1 significantly disrupted bacterial biofilms formed by diverse human pathogens, and further demonstrate that rHMGB1 induced dose-dependent disruption of UPEC biofilms.

[0042] [Figure 4]Figures 4A-4B show that HMGB1 disrupted biofilms and released biofilm-resident bacteria into a planktonic state. 24-h UPEC biofilms were incubated with rHMGB1 (200 nM) for 16 hours, and then planktonic bacteria (conditioned medium) versus biofilm-resident bacteria (adherent bacteria) were counted. Total CFU (planktonic + biofilm) are plotted in Figure 4A. The relative percentage of the total number of planktonic versus biofilm-resident bacteria is plotted in Figure 4B. Bars indicate SEM. *P<0.05 via paired t-test. Note that rHMGB1 had no bactericidal effect but induced the partitioning of bacteria from biofilm-resident to planktonic states.

[0043] [Figure 5] Figures 5A-5B show that rHMGB1 synergized with antibiotics in eradicating planktonic and biofilm-resident bacteria in vitro. Twenty-four-hour NTHI biofilms were incubated with rHMGB1 (200 nM) alone or in combination with ampicillin (32 μg / ml) or amoxicillin-clavulanate (1 μg / ml) for 16 hours, and then the planktonic bacteria (conditioned medium) versus the biofilm-resident bacteria (adherent bacteria) were counted. Biofilm CFU (Figure 5A) and planktonic CFU (Figure 5B) were plotted. Bars indicate SEM. *P<0.05, **P<0.01 via unpaired t-test. Note that rHMGB1 had no bactericidal effect, but when delivered in combination with antibiotics, rHMGB1 promoted the killing of both planktonic and biofilm-resident bacteria.

[0044] [Figure 6-1]Figures 6A-6C show that oxidation of rHMGB1 negatively affected its antibiofilm activity (Figure 6A), whereas acetylation and phosphorylation of rHMGB1 did not affect its antibiofilm activity (Figures 6B and 6C). In Figure 6A, 24-hour biofilms formed by UPEC in vitro were incubated with ox-rHMGB1 (200 nM) for 16 hours. Biofilms were stained with LIVE / DEAD® stain, visualized via confocal laser scanning microscopy (CLSM), and analyzed by COMSTAT to calculate the average thickness. Bars indicate SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, as assessed by unpaired t-test. Note that the antibiofilm function of HMGB1 was significantly reduced upon oxidation of rHMGB1. In Figure 6B, the acetylated or phosphorylated forms were confirmed by Triton acetate urea gel (TAU gel; top) and Western blot using α-acetyl-lysine (α-Ac-Lys; bottom). In Figure 6C, 24-hour biofilms formed in vitro by (Bc - B. cenocepacia; E - Enterobacter spp.; or K - K. pneumoniae) were significantly disrupted by both Ac-rHMGB1 and PrHMGB1 compared with controls. Bars indicate SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, as assessed by unpaired t-test. Note that the antibiofilm function of HMGB1 remained unchanged regardless of either acetylation or phosphorylation. [Figure 6-2] Same as above.

[0045] [Figure 7]Figure 7 shows that the engineered single amino acid variant mHMGB1 retained its ability to bind to HJ DNA. 5'-end-labeled 6-carboxyfluorescein HJ DNA (20 nM) was incubated with increasing concentrations (50-500 nM) of IHF, rHMGB1, or mHMGB1 and then separated by non-denaturing PAGE. The arrow indicates the HJ DNA-protein complex. Note that mHMGB1 retained its ability to bind to HJ DNA.

[0046] [Figure 8] Figures 8A–8C show that HMGB1 bound to but failed to stabilize HJ DNA. 5′-end-labeled 32P HJ DNA was incubated with increasing concentrations (25–500 nM) of IHF (Figure 8A), rHMGB1 (Figure 8B), or RuvA (Figure 8C) for 10 min at either room temperature (RT) or 55°C and then separated on a 6% non-denaturing PAGE. Asterisks indicate melted oligos, and arrows indicate DNA-protein complexes. The DNA-HMGB1 complex was stable at RT but unstable at 55°C, resulting in an increase in the abundance of melted component oligos. This result contrasts with that observed with both IHF and the prototypical HJ DNA-binding protein, RuvA.

[0047] [Figure 9]Figure 9 shows that HMGB1 isoforms disrupted the lattice-like eDNA meshwork within K. pneumoniae biofilms in vitro. 24-hour K. pneumoniae biofilms were incubated with the indicated proteins (200 nM) for 16 hours. Unfixed biofilms were incubated with an α-dsDNA monoclonal antibody followed by goat α-mouse IgG conjugated to AlexaFluor 488 (lower panel). K. pneumoniae were stained with FilmTracer FM 4-64 (upper panel). Biofilms were visualized by CLSM. Note the intertwined spider web structure in the control and the disruption of the spider web structure by rHMGB1 and mHMGB1. Scale bar indicates 10 μm.

[0048] [Figure 10] Figures 10A-10B show that HMGB1 disrupted biofilms through its ability to bind to HJ-like structures within the biofilm extracellular matrix. Figure 10A shows 24-hour biofilms formed in vitro by UPEC incubated with mHMGB1 (200 nM) for 16 hours in the presence or absence of each of the indicated proteins. Figure 10B shows 24-hour biofilms formed in vitro by UPEC incubated with rHMGB1 (200 nM) or NEM-rHMGB1 (200 nM) for 16 hours. Biofilms were stained with LIVE / DEAD® stain, visualized via confocal laser scanning microscopy (CLSM), and analyzed by COMSTAT to calculate the mean thickness. Bars indicate SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 as assessed by unpaired t-test. Note that the antibiofilm function of HMGB1 was lost in the presence of competitors (HU, RuvA) that directly bind to eDNA and therefore compete with HMGB1 for binding to eDNA, or upon modifications of rHMGB1 that directly affect its ability to bind HJ DNA.

[0049] [Figure 11] Figures 11A-11B show that NEM-rHMGB1 does not bind to HJ DNA. Figure 11A provides evidence that NEM-modified rHMGB1 was confirmed by triton acetate urea gel (TAU gel). As shown in Figure 11B, 5'-end-labeled 6-carboxyfluorescein HJ DNA (20 nM) was incubated with increasing concentrations (250-500 nM) of IHF, rHMGB1, or NEM-rHMGB1 and then separated by non-denaturing PAGE. The arrow indicates the HJ DNA-protein complex. Note that NEM-rHMGB1 lost its ability to bind to HJ DNA.

[0050] [Figure 12] Figure 12 shows that HMGB1 isoforms induced varying degrees of neutrophil-mediated NET formation in vitro. Neutrophils were incubated with the indicated proteins (200 nM) for 3.5 hours. Neutrophils were fixed and then incubated with α-dsDNA monoclonal antibody and α-neutrophil elastase antibody, followed by incubation with goat α-mouse IgG conjugated to AlexaFluor 488 and goat α-rabbit IgG conjugated to AlexaFluor 594. Neutrophils were stained with wheat germ agglutinin (WGA) conjugated to AlexaFluor 350. NETs were visualized by CLSM. Note that intertwined NETs formed specifically, albeit to varying degrees, upon incubation of neutrophils with rHMGB1, mHMGB1, and NEM-rHMGB1. Scale bars indicate 10 μm.

[0051] [Figure 13-1]Figures 13A-13F show that HMGB1 promotes clearance of B. cenocepacia aggregates from mouse lungs. Figure 13A provides representative IF images of lung sections harvested from mice infected with B. cenocepacia. C57BL / 6 mice were exposed to 10 CFU it and received 0.2 nmol of the indicated HMGB1 variant either simultaneously (prevention) or 24 hours later (treatment). Bronchoalveolar lavage (BAL) samples were collected at 18 hpi (prevention, Figure 13B) or 72 hpi (treatment, Figure 13C) and then analyzed for CFU. Figure 13D provides representative images stained with H&E (10x and 40x magnification). As shown in Figure 13E, cells in the BAL were stained with α-CD45, CD11b, and Ly-6G and analyzed by flow cytometry to measure relative neutrophil influx (Figure 13F). Bars indicate SD. *P<0.05, **P<0.01 as assessed by Mann-Whitney test. HMGB1 treatment significantly reduced CFU of B. cenocepacia in mouse lungs, and treatment with the engineered C45S mutation in mHMGB1 abolished the pro-inflammatory activity. [Figure 13-2] Same as above.

[0052] [Figure 14-1]Figures 14A-14C show that mHMGB1, despite reduced phagocytosis, cleared B. cenocepacia from mouse lungs and attenuated neutrophil recruitment into the peritoneal cavity. Figure 14A shows C57BL / 6 mice exposed to 10 CFU of B. cenocepacia and treated with 0.2 nmol of rHMGB1 or mHMGB1 24 hours later. Forty-eight hours after treatment, mice were sacrificed, and lung sections were labeled with E. coli α-EF-Tu monoclonal antibody to label B. cenocepacia and DAPI to detect neutrophils. Figure 14B shows macrophages treated with 5 μg / ml rHMGB1, 5 μg / ml mHMGB1, or 10 μM cytochalasin D for 2 hours, followed by the addition of pHrodo Red E. coli bioparticles and incubation for 2 hours. Any excess bioparticles were removed by washing the cells with HBSS, and the phagocytosed bioparticles were measured using a plate reader (560 / 585 nm). Figure 14C shows that C57BL / 6 mice were intraperitoneally injected with the indicated HMGB1 isoforms, and 24 hours later, the influx of neutrophils into the peritoneal cavity was determined by flow cytometry using α-CD45, CD11b, and Ly-6G antibodies. n = 3. Bars indicate SD. *P < 0.05. Treatment with rHMGB1 induced significant migration of neutrophils into the peritoneal cavity, whereas treatment with mHMGB1 significantly attenuated this pro-inflammatory response despite a modest reduction in phagocytosis. [Figure 14-2] Same as above.

[0053] [Figure 15]Figure 15 shows that rHMGB1 used to treat biofilms in vivo did not induce a dysregulated host response to infection associated with septic shock in mice. Mice were injected i.p. with either 0.2 nmol of endotoxin-free HMGB1, 5 mg / kg LPS, or both and then monitored for signs of septic shock for 24 hours. Serum TNF-α was measured by ELISA 24 hours later. Bars indicate SD. LoD: limit of detection. Note that the same concentration of rHMGB1 used to treat in vivo biofilms did not induce septic shock, as indicated by an increase in TNF-α.

[0054] [Figure 16-1]Figures 16A-16J show that mHMGB1 mediated the clearance of biofilm-resident NTHI, eradication of established mucosal biofilms, and resolution of experimental disease, an outcome that was enhanced upon co-delivery with an antibody fragment (Tip-Chimer Fab) against the immunoprotective domain of the DNABII protein. Figure 16A provides a study timeline for evaluating the relative ability of rHMGB1 or mHMGB1 to dissolve established NTHI biofilms in chinchilla middle ears. Figure 16B provides the relative amounts of NTHI resident within the mucosal biofilm and NTHI adherent to the middle ear mucosa one day after completion of treatment. Figure 16C shows annotations used to qualitatively assess the amount of middle ear mucosal biofilm remaining one day after completion of treatment. Figure 16D provides the relative amount of mucosal biofilm in each middle ear per cohort. Figure 16E shows annotations used to qualitatively assess the amount of middle ear mucosal inflammation one day after completion of treatment. Figure 16F provides the relative amount of mucosal inflammation within each middle ear per cohort. Figure 16G provides representative images of middle ears from each cohort to demonstrate the relative presence / clearance of mucosal biofilms and the inflammatory / non-inflammatory state. Figure 16H is a study timeline to evaluate the additive potential of mHMGB1 co-delivered with tip-chimer Fab to dissolve established NTHI biofilms in chinchilla middle ears. Figure 16I provides the relative amounts of NTHI resident within mucosal biofilms and NTHI adherent to the middle ear mucosa 24 hours after one or two treatment doses. Figure 16J provides the relative amounts of mucosal biofilm within each middle ear per cohort 24 hours after one or two treatment doses. Both rHMGB1 and mHMGB1 induced rapid clearance of biofilm-resident NTHI and eradication of established mucosal biofilms, whereas only mHMGB1 induced limited mucosal inflammation. Furthermore, co-delivery of mHMGB1 with tip-chimer Fab fragments was highly effective in eradicating NTHI and associated biofilms from the middle ear. [Figure 16-2] Same as above.

[0055] [Figure 17] Figure 17 shows that the pro-inflammatory cytokines IL-1β and IL-17A were significantly more abundant in middle ear fluid collected from chinchillas treated with rHMGB1, whereas anti-inflammatory cytokines predominated in the mHMGB1-treated cohort. Six days after NTHI challenge (one day after completion of treatment), middle ear fluid was collected and screened by cytometric bead array for relative amounts of a panel of pro- and anti-inflammatory cytokines. Each data point represents an individual middle ear fluid and the average for each cohort indicated. Note the significantly elevated concentrations of the pro-inflammatory cytokines IL-1β and IL-17A in the rHMGB1-treated chinchillas (p<0.05), whereas significantly elevated concentrations of the anti-inflammatory cytokine IL-10 were observed in the mHMGB1-treated cohort (p<0.01). DETAILED DESCRIPTION OF THE INVENTION

[0056] Detailed Description definition It should be understood that section or subsection headings, if used herein, are for organizational purposes only and should not be construed as limiting and / or separating the subject matter described.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All nucleotide sequences provided herein are shown in the 5' to 3' direction. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, certain non-limiting exemplary methods, devices, and materials are described herein. All technical and patent publications cited herein are incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior invention.

[0058] 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, which are within the skill of the art. See, e.g., Sambrook and Russell eds., (2001) Molecular Cloning: A Laboratory Manual, 3 rd edition; Series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; Series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5 th edition; 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;Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986));Perbal (1984) A Practical Guide to Molecular Cloning;Miller and Calos eds, (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory);Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds. (1996) Weir's Handbook of Experimental Immunology.

[0059] All numerical designations, including ranges, e.g., pH, temperature, time, concentration, and molecular weight, are approximations which are varied (+) or (-) by increments of 1.0 or 0.1, as appropriate, or by a variation of + / - 15%, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is to be understood, although not always expressly indicated, that all numerical designations are preceded by the term "about." It is also to be understood, although not always expressly indicated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0060] The term "about," as used herein, when referring to a measurable value, such as an amount or concentration, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0061] 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 "a polypeptide" includes multiple polypeptides, including mixtures thereof.

[0062] As used herein, the term "comprising" is intended to 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, means excluding any other elements that are essential to the combination for its 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-buffered saline, preservatives, and the like. "Consisting of" means excluding more than trace elements of other ingredients 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.

[0063] "Optionally" or "as needed" means that the subsequently described condition may or may not occur, and thus the description includes cases where the condition occurs and cases where the condition does not occur.

[0064] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives ("or").

[0065] "Substantially" or "essentially" means almost entirely or completely, e.g., 95% or more of a given amount. In some embodiments, "substantially" or "essentially" means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0066] The terms "acceptable," "effective," or "sufficient," when used to describe the selection of any components, ranges, levels, rates, dosage forms, etc. disclosed herein, are intended to mean that those components, ranges, levels, rates, dosage forms, etc. are suitable for the purpose disclosed.

[0067] As used herein, relative terms, such as higher, lower, increase, decrease, reduction, or any grammatical variation thereof, can refer to a certain variation from the reference. In some embodiments, such a variation can refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1-fold, or about 2-fold, or about 3-fold, or about 4-fold, or about 5-fold, or about 6-fold, or about 7-fold, or about 8-fold, or about 9-fold, or about 10-fold, or about 20-fold, or about 30-fold, or about 40-fold, or about 50-fold, or about 60-fold, or about 70-fold, or about 80-fold, or about 90-fold, or about 100-fold or more. In some embodiments, such a variation may refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 0%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the reference.

[0068] The term "biofilm" refers to an organized microbial community in which microorganisms may attach to structural surfaces, which may be organic or inorganic, along with macromolecules such as DNA that are secreted, released, and / or become available in the extracellular environment due to bacterial lysis. Biofilms are highly resistant to microbiotics and antimicrobial agents. They survive on gum tissue, teeth, and restorations, causing dental caries and periodontal disease, also known as periodontal plaque disease. They also cause chronic middle ear infections. Biofilms can also form on the surfaces of dental implants, stents, catheter systems, and contact lenses. They grow on pacemakers, heart valve replacements, artificial joints, and other surgical implants. The Centers for Disease Control estimates that more than 65% of nosocomial (hospital-acquired) infections are caused by biofilms. They cause chronic vaginal infections and life-threatening systemic infections in people with compromised immune systems. Biofilms are also involved in many diseases. For example, cystic fibrosis patients often have Pseudomonas infections, which result in antibiotic-resistant biofilms. In one embodiment, the biofilm comprises a DNABII polypeptide or protein. In a further embodiment, the biofilm comprises IHF and / or HU. In yet a further embodiment, the biofilm comprises IHFA and / or IHFB.

[0069] The term "neutrophil" refers to a type of granulocyte, a type of white blood cell, and a type of phagocyte in mammals, which are first responders to inflammation. Neutrophils are granule-containing polymorphonuclear leukocytes that arise from myeloid precursors in the bone marrow. They play a key role in the innate immune response, either intracellularly by destroying foreign particles in phagosomes or extracellularly by releasing neutrophil extracellular traps (NETs) and promoting acute inflammation. In humans, neutrophils are the most abundant circulating leukocyte, accounting for 50-70% of white blood cells, whereas 10-25% of circulating mouse leukocytes are neutrophils. Neutrophils can be identified visually based on their nuclear shape and cytoplasmic granularity, but can also be identified based on the expression of certain markers. Murine neutrophils are commonly identified based on cell surface expression of Ly-6G and CD11b / integrin alpha M. Because mouse granulocytic myeloid-derived suppressor cells can also express these markers, neutrophils are often distinguished from these cells in mice based on the lack of expression of M-CSF R / CD115 and CD244 / SLAMF4, along with the absence of immunosuppressive properties. In humans, neutrophils are distinguished from eosinophils and monocytes based on the expression of both CD15 and CD16 / Fc gamma RIII on human neutrophils, along with the lack of expression of CD14. In addition, CD66b / CEACAM-8, CD11b / integrin alpha M, CD33, and the cytoplasmic marker myeloperoxidase are other common markers used to identify human neutrophils.

[0070] As used herein, neutrophil extracellular traps (NETs) refer to a meshwork of extracellular fibers composed primarily of neutrophil-derived DNA that binds pathogens. NETs enable neutrophils to kill extracellular pathogens while minimizing damage to host cells. High-resolution scanning electron microscopy has shown that NETs consist of stretches of DNA and globular protein domains with diameters of 15–17 nm and 25 nm, respectively. These aggregate into larger threads with diameters of 50 nm. However, under flow conditions, NETs can form much larger structures, reaching lengths and widths of hundreds of nanometers. Immunofluorescence analysis has confirmed that NETs contain azurophil granules (neutrophil elastase, cathepsin G, and myeloperoxidase), specific granules (lactoferrin), tertiary granules (gelatinase), and proteins from the cytoplasm; however, CD63, actin, tubulin, and various other cytoplasmic proteins are absent in NETs. In some embodiments, NETs can be measured by assessing one or more of their components (including, but not limited to, neutrophil elastase), for example, by immunofluorescence. NET activation and release is referred to herein as NETosis, a dynamic process that can occur in two forms: suicidal NETosis and vital NETosis.

[0071] As used herein, the phrase "directly adjacent" refers to a location that is directly connected to a reference location or structure, without any gap or space between them.

[0072] In some embodiments, the term "disrupt" refers to a reduction in the formation of the DNA / protein matrix, which is a component of a microbial biofilm. Additionally or alternatively, the term "disrupt" refers to a reduction in the formed biofilm, e.g., partial or complete dissipation of the biofilm. Such a reduction can be indicated by various parameters. For example, the biofilm biomass or bacterial load, or both, can be assessed before and after treatment, and the reduction after treatment can be used to indicate the effectiveness of the treatment. Another example of a parameter is the relative mucosal biofilm score or biomass score given by a blinded assessor. Other suitable parameters are shown in the examples disclosed herein. In some embodiments, the treatment reduces the biofilm to at least about 90% (including but not limited to, at least about 85%, or at least about 80%, or at least about 75%, or at least about 70%, or at least about 65%, or at least about 60%, or at least about 55%, or at least about 50%, or at least about 45%, or at least about 40%, or at least about 35%, or at least about 30%, or at least about 25%, or at least about 20%, or at least about 15%, or at least about 10%, or at least about 9%, or at least about 8%, or at least about 7%, or at least about 6%, or at least about 5%, or at least about 4%, or at least about 3%, or at least about 2%, or at least about 1%, or less than 1%, or about 0%) of the biofilm before the treatment. In certain embodiments, disrupting a biofilm refers to dissipating the biofilm (completely or partially), releasing the microorganisms from the DNA / protein matrix of the biofilm, and, if necessary, allowing the microorganisms to be killed by host immune effectors and / or antibiotics.

[0073] "DNABII polypeptide or protein" refers to a DNA-binding protein or polypeptide that consists of a DNA-binding domain and therefore has specific or general affinity for microbial DNA. In one embodiment, they bind to the minor groove of DNA. Non-limiting examples of DNABII proteins are integration host factor (IHF) protein and histone-like protein from E. coli strain U93 (HU). Other DNA-binding proteins that may be associated with biofilms include 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).

[0074] "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 IHF protein subunits in E. coli are the himA (GenBank accession number POA6X7.1) and himD (POA6Y1.1) genes. Homologs of these genes have been found in other organisms. In certain embodiments, the term "IHF" refers to one or both of the two IHF subunits: integration host factor subunit alpha (IHFA or IhfA) and integration host factor subunit beta (IHFB or IhfB).

[0075] "HU" or "histone-like protein from E. coli strain U93" refers to a class of heterodimeric proteins typically 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. Antibodies against the HU protein are commercially available from Abeam. The genes encoding the HU protein subunits in E. coli are hupA and hupB, which correspond to SEQ ID NOs: 29 and 30, respectively. Homologs of these genes have been found in other organisms, and peptides corresponding to these genes from other organisms can be found in Table 10 of WO2011 / 123396.

[0076] The term "surface antigen" or "surface protein" refers to a protein or peptide on the surface of a cell, for example, a bacterial cell. Examples of surface antigens include OMP P5 (GenBank accession number YP_004139079.1), OMP P2 (GenBank accession number ZZX87199.1), and OMP P26 (GenBank accession number YP_665091.1), and examples of outer membrane proteins include rsPilA or recombinant soluble PilA (GenBank accession number EFU96734.1) and type IV Pilin (GenBank accession number Yp_003864351.1).

[0077] 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 and contain IhfA, ihfB, and hupA genes or proteins. Some non-limiting examples of different strains of Haemophilus influenzae include Rd KW20, 86-028NP, R2866, PittGG, PittEE, R2846, and 2019.

[0078] By "microbial DNA" is intended single-stranded or double-stranded DNA from microorganisms that are incorporated into a biofilm.

[0079] By "inhibiting, preventing or disrupting" a biofilm is intended a prophylactic or therapeutic reduction in biofilm structure.

[0080] By "bent polynucleotide" is intended a double-stranded polynucleotide containing a small loop in one strand that does not pair with the other strand. In some embodiments, the loop is 1 to about 20 bases in length, or alternatively 2 to about 15 bases in length, or alternatively about 3 to about 12 bases in length, or alternatively about 4 to about 10 bases in length, or alternatively has about 4, 5, 6, 7, 8, 9, or 10 bases.

[0081] "A polypeptide that competes with DNABII binding, e.g., IHF in binding to DNA" refers to a protein or peptide that competes with DNABII (e.g., IHF) in binding to bent or distorted DNA structures, but does not form a biofilm 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.

[0082] A "subject" of diagnosis or treatment is a cell or an animal, e.g., a mammal or a human. Non-human animals for diagnosis or treatment include non-human animals or animal models subject to infection, such as primates, murines, e.g., rats, mice, chinchillas, canines, e.g., dogs, lagomorphs, e.g., rabbits, livestock, sport animals, and pets. The terms "subject," "host," "individual," and "patient," when used interchangeably herein, refer to an animal, typically a mammal. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), livestock animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal may be of any age or at any stage of development (e.g., an adult, adolescent, child, juvenile, or in utero mammal). The mammal may be male or female. In some embodiments, the subject is a human.

[0083] The terms "protein," "peptide," and "polypeptide" are used interchangeably and in their broadest sense to 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 esters, ethers, etc. A protein or peptide must contain at least two amino acids, and no limit is placed on the maximum number of amino acids that may make up a protein's or peptide's sequence. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acids, amino acid analogs, and peptidomimetics, including glycine and both D and L optical isomers.

[0084] As used herein, "complementary" sequences refer to two nucleotide sequences that contain multiple individual nucleotide bases that pair with each other when aligned antiparallel to each other. The pairing of nucleotide bases forms hydrogen bonds, thus stabilizing the double-stranded structure formed by complementary sequences. For sequences to be considered "complementary," it is not necessary for all nucleotide bases in two sequences to pair with each other. For example, sequences can be considered complementary if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in the two sequences pair with each other. In some embodiments, the term "complementary" refers to 100% of the nucleotide bases in the two sequences pair with each other. In addition, sequences can still be considered "complementary" if the total lengths of the two sequences are significantly different from each other. For example, when a plurality of individual nucleotide bases of the primer are paired with the nucleotide bases in the longer polynucleotide, and the primer is aligned in antiparallel with a specific region of the longer polynucleotide, a 15-nucleotide primer can be considered "complementary" to a longer polynucleotide containing several hundred nucleotides.Nucleotide base pairing is known in the art; for example, in DNA, purine adenine (A) is paired with pyrimidine thymine (T), and pyrimidine cytosine (C) is always paired with purine guanine (G), while in RNA, adenine (A) is paired with uracil (U), and guanine (G) is paired with cytosine (C).Furthermore, the nucleotide bases that are aligned in antiparallel with each other but are not paired in two complementary sequences are referred to herein as mismatches.

[0085] By "C-terminal polypeptide" is intended at least 10, or alternatively at least 15, or alternatively at least 20, or at least 25 C-terminal amino acids, or alternatively half of the polypeptide. In another embodiment, for a polypeptide containing 90 amino acids, the C-terminal polypeptide would include amino acids 46 to 90. In one embodiment, the term contemplates the carboxy-terminal to C-terminal 20 amino acids.

[0086] The "tip fragment" of a DNABII polypeptide refers to a DNABII polypeptide that forms the two arms of the protein, using IHF alpha and IHF beta as examples. Non-limiting examples include the following: IhfA, A tip fragment: NFELRDKSSRPGRNPKTGDVV, SEQ ID NO: 31, and IhfB, B tip fragment: SLHHRQPRLGRNPKTGDSVNL, SEQ ID NO: 32, as illustrated below, and the amino acid sequence of a DNABII polypeptide from another species (e.g., another bacterium) aligned with SEQ ID NO: 31 or 32, and a peptide containing two tip domains or modified domains to provide the necessary confirmation, such as mIhFB4 and IhfA tip domains.

[0087] By "tail fragment" of a DNABII polypeptide is intended the region of the protein that is exposed to the bulk medium and is not occluded by DNA or other polypeptides.

[0088] As used herein, ESKAPE pathogens include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. These pathogens are the leading cause of hospital-acquired infections worldwide.

[0089] "HMG domain," "high mobility group (HMG) box domain," or "HMGB" refers to an amino acid sequence involved in DNA binding (Stros et al., Cell Mol Life Sci. 64(19-20):2590-606(2007)). In one embodiment, the structure of an HMG box domain consists of three helices in a disordered arrangement. In another embodiment, the HMG box domain enables the protein to bind to non-B-form DNA conformations (twisted or unwound) with high affinity. HMG box domains are found in high mobility group proteins involved in regulating DNA-dependent processes, such as transcription, replication, and DNA repair, all of which require changes in chromatin conformation (Thomas (2001) Biochem. Soc. Trans. 29(Pt 4):395-401).

[0090] HMG box proteins are found in various eukaryotes and can be roughly divided into two groups based on sequence-dependent and sequence-independent DNA recognition; the former usually contain one HMG box motif, while the latter may contain multiple HMG box motifs. Non-limiting examples of polypeptides containing HMG box domains include HMG1 (HMGB1), HMG2 (HMGB2), HMGB3, and HMGB4 non-histone components of chromatin; SRY (sex-determining region Y protein), which is involved in differential gonad formation; SOX family transcription factors (Harley et al. (2003) Endocr. Rev. 24(4):466-87); and the sequence-specific LEF1 (lymphoid enhancer-binding factor 1) and TCF-1 (T-cell factor 1), which are involved in regulating organogenesis and thymocyte differentiation (Labbe et al. (2000) Proc. Natl. Acad. Sci. USA 97(15):8358-63); structure-specific recognition protein SSRP involved in transcription and replication; MTF1 mitochondrial transcription factor; nucleolar transcription factor UBF 1 / 2 (upstream binding factor) involved in transcription by RNA polymerase I; Abf2 yeast ARS-binding factor (Cho et al. (2001) Biochim. Biophys. Acta. 1522(3):175-86); yeast transcription factors lxr1, Rox1, Nhp6b, and Spp41; mating type protein (MAT) involved in sexual reproduction in fungi (Barve et al. (2003) Fungal Genet. Biol. 39(2):151-67); and YABBY plant-specific transcription factor.

[0091] "HMGB1" refers to the high mobility group box (HMGB) 1 protein, which is reported to bind to and distort the minor groove of DNA. Recombinant or isolated proteins and polypeptides are commercially available from Atgenglobal, ProSpecBio, Protein1, and Abnova. HMGB1 polypeptide is used herein to refer to an HMGB1 protein or its equivalent. In some embodiments, the HMGB1 protein comprises, consists essentially of, or even consists of SEQ ID NO: 51. In some embodiments, the HMGB1 equivalent comprises, consists essentially of, or even consists of an HMG box protein, such as those disclosed herein. In some embodiments, the HMGB1 equivalent comprises, consists essentially of, or even consists of an HMG box domain, such as those disclosed herein. In further embodiments, the HMGB1 equivalent comprises, consists essentially of, or even consists of one or more of HMGB2, HMGB3, or HMGB4. Additionally or alternatively, the HMGB1 equivalent comprises, consists essentially of, or further consists of one or more mutations disclosed herein, hi some embodiments, the HMGB1 equivalent comprises, consists essentially of, or further consists of any one or more of SEQ ID NOs: 52-58, 68-74, 84-90, or 100-114, or their respective equivalents.

[0092] Exemplary sequences of polypeptides containing an HMG box domain include NP_002119 (human HMGB1), NP_001124160 (human HMGB2), NP_005333 (human HMGB3), and NP_660206 (human HMGB4). Approximately 9 to 76 amino acid residues of human HMGB1, for example, form an HMG box domain, and approximately 90 to 138 amino acid residues form another HMG box domain. For example, an HMGB1 fragment containing either of these two HMG box domains also constitutes a polypeptide containing an HMG box domain within the meaning of the present disclosure. In the examples described herein, recombinant HMGB1 (derived from a human and recombinantly expressed and purified in E. coli) was used as a comparison to mHMGB1(C45S), and has the sequence: [ka] It has.

[0093] As used herein, amino acid mutations can be identified by two letters separated by a number. The first letter refers to the original amino acid residue, the number indicates the position of the mutation in the reference sequence, and the second letter refers to the mutated amino acid residue. For example, an HMGB1 polypeptide containing a C45S mutation indicates that the 45th amino acid residue of HMGB1, cysteine ​​(C), has been mutated to serine (S). In further embodiments, the full-length sequence, e.g., SEQ ID NO: 51, can be used as a reference, for example, when identifying an amino acid position in a fragment. In one example, a B-box fragment of an HMGB1 polypeptide can contain a C106S mutation, where the number 106 refers to the amino acid residue position in the full-length HMGB1 polypeptide instead of the B-box fragment. In some embodiments, the reference is SEQ ID NO: 51. In some embodiments, the second letter can be omitted when referring to a position.

[0094] As used herein, amino acid positions in a sequence are identified by a letter followed by a number. The letter refers to the amino acid residue at that position, while the number indicates the position of the mutation in the reference sequence. For example, C45 refers to the 45th amino acid residue of HMGB1, which is cysteine ​​(C). In further embodiments, the full-length sequence, e.g., SEQ ID NO: 51, can be used as a reference, for example, when identifying an amino acid position in a fragment. In one example, a B-box fragment of an HMGB1 polypeptide can contain a C106S mutation, where the number 106 refers to the amino acid residue position in the full-length HMGB1 polypeptide instead of the B-box fragment. In some embodiments, the reference is SEQ ID NO: 51.

[0095] The terms "modified high mobility group box 1 domain" and mHMGB1, as used herein, refer to HMGB1 that has been mutated, for example, through substitution of cysteine ​​residues at positions 23, 45, and / or 106 based on the consensus sequence polypeptide of HMGB1 derived from humans: SEQ ID NO: 51.

[0096] Non-limiting exemplary sequences of modified high mobility group box 1 domains (i.e., mHMGB1) include, but are not limited to: mHMGB1(C23S), which is SEQ ID NO: 51, further comprising one mutation at C23S, i.e., SEQ ID NO: 53; mHMGB1(C45S), which is SEQ ID NO: 51, further comprising one mutation at C45S, i.e., [ka] mHMGB1 (C106S) which is SEQ ID NO: 51 and further contains one mutation at C106S, i.e., SEQ ID NO: 54; mHMGB1 (C23S, C45S double mutant) which is SEQ ID NO: 51 and further contains two mutations at C23S and C45S, i.e., SEQ ID NO: 55; mHMGB1 (C23S, C106S double mutant) which is SEQ ID NO: 51 and further contains two mutations at C23S and C106S, i.e., SEQ ID NO: 56; mHMGB1 (C45S, C106S double mutant) which is SEQ ID NO: 51 and further contains two mutations at C45S and C106S, i.e., SEQ ID NO: 57; and mHMGB1 (C23S, C45S, C106S triple mutant) which is SEQ ID NO: 51 and further contains three mutations at C23S, C45S, and C106S, i.e., SEQ ID NO: 58.

[0097] An exemplary nucleic acid sequence encoding human HMGB1 is provided below: [ka]

[0098] Nucleic acid sequences encoding these modified high mobility group box 1 domains (i.e., mHMGB1) are also provided: mHMGB1(C23S), which is SEQ ID NO:59 further comprising AGT encoding C23S, i.e., SEQ ID NO:59 further comprising one T to A mutation at nucleotide residue 67 of SEQ ID NO:59, see SEQ ID NO:60; mHMGB1(C45S), which is SEQ ID NO:59 further comprising AGT encoding C45S, i.e., SEQ ID NO:59 further comprising one T to A mutation at nucleotide residue 133 of SEQ ID NO:59 and one C to T mutation at nucleotide residue 135 of SEQ ID NO:59, see SEQ ID NO:61. See SEQ ID NO: 59, mHMGB1(C106S), which is SEQ ID NO: 59 further comprising an AGC encoding C106S, i.e., one mutation from T to A at nucleotide residue 316 of SEQ ID NO: 59; see SEQ ID NO: 62, mHMGB1(C23S,C45S), which is SEQ ID NO: 59 further comprising an AGT encoding C23S and an AGT encoding C45S, i.e., one mutation from T to A at nucleotide residue 67 of SEQ ID NO: 59, one mutation from T to A at nucleotide residue 133 of SEQ ID NO: 59, and one mutation from C to T at nucleotide residue 135 of SEQ ID NO: 59. mHMGB1(C23S, C106S - double mutant), see SEQ ID NO: 63; mHMGB1(C23S, C106S - double mutant), see SEQ ID NO: 64, which is SEQ ID NO: 59 further comprising AGT encoding C23S and AGC encoding C106S, i.e., further comprising one T to A mutation at nucleotide residue 67 of SEQ ID NO: 59 and one T to A mutation at nucleotide residue 316 of SEQ ID NO: 59; mHMGB1(C45S, C106S - double mutant), see SEQ ID NO: 65, which is SEQ ID NO: 59 further comprising AGT encoding C45S and AGC encoding C106S, i.e., further comprising one T to A mutation at nucleotide residue 133 of SEQ ID NO: 59, one C to T mutation at nucleotide residue 135 of SEQ ID NO: 59, and one T to A mutation at nucleotide residue 316 of SEQ ID NO: 59;and mHMGB1 (C23S, C45S, C106S - triple mutant), SEQ ID NO: 66, which is SEQ ID NO: 59 further comprising AGT encoding C23S, AGT encoding C45S, and AGC encoding C106S, i.e., one T to A mutation at nucleotide residue 67 of SEQ ID NO: 59, one T to A mutation at nucleotide residue 133 of SEQ ID NO: 59, one C to T mutation at nucleotide residue 135 of SEQ ID NO: 59, and one T to A mutation at nucleotide residue 316 of SEQ ID NO: 59;

[0099] The term "modified high mobility group box 1 domain" is understood to further encompass equivalents that have at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% identity to the HMGB1 consensus sequence or any one of SEQ ID NOs: 51-58 and contain the same substitutions at corresponding positions in the equivalent sequence based on alignment to the HMGB1 consensus sequence or any one of SEQ ID NOs: 51-58.

[0100] It is understood that among the equivalents to the modified high mobility group box 1 domain are modified high mobility group box 2, modified high mobility group box 3, and modified high mobility group box 4, which contain cysteine ​​to serine substitutions at one or more amino acid positions corresponding to C23S, C45S, and C106S of the modified high mobility group box 1 or its equivalent. As will be understood by those skilled in the art, mutations in more than one HMG box protein (e.g., HMGB1, HMGB2, HMGB3, or HMGB4) are considered equivalent if the mutated positions in more than one HMG box protein are aligned with each other in a sequence alignment of more than one HMG box protein. Additionally and optionally, the original amino acid residue to be mutated is the same in an equivalent mutation. Furthermore and optionally, the amino acid residue to which the mutation is mutated is the same in an equivalent mutation. Therefore, an equivalent of an HMGB1 polypeptide can be a wild-type HMGB1 mutated at one or more positions disclosed herein, such as C23S, C45S, C106S, or any combination thereof. Furthermore, an equivalent of an HMGB1 polypeptide can be an HMG box protein other than HMGB1 that contains an equivalent mutation, such as HMGB2, HMGB3, or HMGB4. One non-limiting example is the HMGB1 C45S mutant, which is equivalent to the HMGB2 C45S mutant, as well as HMGB3 C45S or HMGB4 C45S. Alternatively, without being bound by theory, the applicant believes that the modified HMGB1 fragments disclosed herein function similarly, and therefore the C to S mutations specified herein can be considered equivalent to each other, regardless of their position.

[0101] The corresponding amino acid substitutions to generate a modified high mobility group box 2 are found in the consensus sequence polypeptide of HMGB2 from humans: [ka] Based on this, the cysteine ​​residues at positions 23, 45, and / or 106 may be used.

[0102] Non-limiting exemplary sequences of modified high mobility group box 2 domains include, but are not limited to, mHMGB2(C23S), which is SEQ ID NO: 67, further comprising one mutation at C23S, i.e., SEQ ID NO: 68; mHMGB2(C45S), which is SEQ ID NO: 67, further comprising one mutation at C45S, i.e., SEQ ID NO: 69; mHMGB2(C106S), which is SEQ ID NO: 67, further comprising one mutation at C106S, i.e., SEQ ID NO: 70; mHMGB2(C23S,C45S - double mutant), which is SEQ ID NO: 67, further comprising two mutations at C23S and C45S, i.e., SEQ ID NO: 71; mHMGB2(C23S,C106S -), which is SEQ ID NO: 67, further comprising two mutations at C23S and C106S, i.e., SEQ ID NO: 72; mHMGB2 (C45S, C106S - double mutant), i.e., SEQ ID NO: 72; mHMGB2 (C45S, C106S - double mutant), which is SEQ ID NO: 67 and further contains two mutations, C45S and C106S, i.e., SEQ ID NO: 73; and mHMGB2 (C23S, C45S, C106S - triple mutant), which is SEQ ID NO: 67 and further contains three mutations, C23S, C45S, and C106S, i.e., SEQ ID NO: 74.

[0103] An exemplary nucleic acid sequence encoding human HMGB2 is provided below: [ka]

[0104] Nucleic acid sequences encoding these modified high mobility group box 2 domains are also provided: mHMGB2(C23S), which is SEQ ID NO:75 further comprising AGT encoding C23S, i.e., SEQ ID NO:75 further comprising one T to A mutation at nucleotide residue 67 of SEQ ID NO:75 and one C to T mutation at nucleotide residue 69 of SEQ ID NO:75, see SEQ ID NO:76; mHMGB2(C45S), which is SEQ ID NO:75 further comprising AGT encoding C45S, i.e., SEQ ID NO:75 further comprising one T to A mutation at nucleotide residue 133 of SEQ ID NO:75, see SEQ ID NO:77; mHMGB2(C106S), which is SEQ ID NO:75 further comprising AGC encoding C106S, i.e., SEQ ID NO:75 further comprising one T to A mutation at nucleotide residue 316 of SEQ ID NO:75, see SEQ ID NO:78. see mHMGB2(C23S,C45S-double mutant), SEQ ID NO:79, which is SEQ ID NO:75 further comprising AGT encoding C23S and AGT encoding C45S, i.e., further comprising one T to A mutation at nucleotide residue 67 of SEQ ID NO:75, one C to T mutation at nucleotide residue 69 of SEQ ID NO:75, and one T to A mutation at nucleotide residue 133 of SEQ ID NO:75; see mHMGB2(C23S,C45S-double mutant), SEQ ID NO:75 further comprising AGT encoding C23S and AGC encoding C106S, i.e., one T to A mutation at nucleotide residue 67 of SEQ ID NO:75, one C to T mutation at nucleotide residue 69 of SEQ ID NO:75, and one T to A mutation at nucleotide residue 316 of SEQ ID NO:75. mHMGB2 (C45S, C106S - double mutant), see SEQ ID NO: 80; mHMGB2 (C45S, C106S - double mutant), see SEQ ID NO: 81, which is SEQ ID NO: 75 further comprising AGT encoding C45S and AGC encoding C106S, i.e., one T to A mutation at nucleotide residue 133 of SEQ ID NO: 75 and one T to A mutation at nucleotide residue 316 of SEQ ID NO: 75;and mHMGB2 (C23S, C45S, C106S - triple mutant), SEQ ID NO: 82, which is SEQ ID NO: 75 further comprising AGT encoding C23S, AGT encoding C45S, and AGC encoding C106S, i.e., one T to A mutation at nucleotide residue 67 of SEQ ID NO: 75, one C to T mutation at nucleotide residue 69 of SEQ ID NO: 75, one T to A mutation at nucleotide residue 133 of SEQ ID NO: 75, and one T to A mutation at nucleotide residue 316 of SEQ ID NO: 75;

[0105] The corresponding amino acid substitutions to generate modified high mobility group box 3 (mHMGB3) are the consensus sequence polypeptide of HMGB3 from humans: [ka] Based on this, substitutions can be made at the cysteine ​​residues at positions 23, 45, and / or 104. As will be understood by those skilled in the art, mHMGB3(C104S) is the equivalent of mHMGB1(C106S) or mHMGB2(C106S).

[0106] Non-limiting exemplary sequences of modified high mobility group box 3 domains include, but are not limited to, mHMGB3(C23S), which is SEQ ID NO: 83, further comprising one mutation at C23S, i.e., SEQ ID NO: 84; mHMGB3(C45S), which is SEQ ID NO: 83, further comprising one mutation at C45S, i.e., SEQ ID NO: 85; mHMGB3(C104S), which is SEQ ID NO: 83, further comprising one mutation at C104S, i.e., SEQ ID NO: 86; mHMGB3(C23S,C45S - double mutant), which is SEQ ID NO: 83, further comprising two mutations at C23S and C45S, i.e., SEQ ID NO: 87; mHMGB3(C23S,C104S -), which is SEQ ID NO: 83, further comprising two mutations at C23S and C104S, i.e., SEQ ID NO: 88 mHMGB3 (C45S, C104S - double mutant), i.e., SEQ ID NO: 88; mHMGB3 (C45S, C104S - double mutant), which is SEQ ID NO: 83 and further contains two mutations, C45S and C104S, i.e., SEQ ID NO: 89; and mHMGB3 (C23S, C45S, C104S - triple mutant), which is SEQ ID NO: 83 and further contains three mutations, C23S, C45S, and C104S, i.e., SEQ ID NO: 90.

[0107] An exemplary nucleic acid sequence encoding human HMGB3 is provided below: [ka]

[0108] Nucleic acid sequences encoding these modified high mobility group box 3 domains are also provided herein below: mHMGB3(C23S), which is SEQ ID NO:91 further comprising AGT encoding C23S, i.e., SEQ ID NO:91 further comprising one T to A mutation at nucleotide residue 67 of SEQ ID NO:91 and one C to T mutation at nucleotide residue 69 of SEQ ID NO:91, see SEQ ID NO:92; mHMGB3(C45S), which is SEQ ID NO:91 further comprising AGT encoding C45S, i.e., SEQ ID NO:91 further comprising one T to A mutation at nucleotide residue 133 of SEQ ID NO:91 and one C to T mutation at nucleotide residue 135 of SEQ ID NO:91, see SEQ ID NO:93; A encoding C104S, See SEQ ID NO:94, mHMGB3(C104S), which is SEQ ID NO:91 further comprising GC, i.e., one T to A mutation at nucleotide residue 310 of SEQ ID NO:91 and one T to C mutation at nucleotide residue 312 of SEQ ID NO:91; mHMGB3(C23S,C45S), which is SEQ ID NO:91 further comprising AGT encoding C23S and AGT encoding C45S, i.e., one T to A mutation at nucleotide residue 67 of SEQ ID NO:91, one C to T mutation at nucleotide residue 69 of SEQ ID NO:91, one T to A mutation at nucleotide residue 133 of SEQ ID NO:91, and one C to T mutation at nucleotide residue 135 of SEQ ID NO:91. mHMGB3 (C23S, C104S double mutant), see SEQ ID NO: 95; mHMGB3 (C23S, C104S double mutant), see SEQ ID NO: 91, further comprising AGT encoding C23S and AGC encoding C104S, i.e., SEQ ID NO: 91, further comprising one T to A mutation at nucleotide residue 67 of SEQ ID NO: 91, one C to T mutation at nucleotide residue 69 of SEQ ID NO: 91, one T to A mutation at nucleotide residue 310 of SEQ ID NO: 91, and one T to C mutation at nucleotide residue 312 of SEQ ID NO: 91; mHMGB3 (C45S, C104S - double mutant), see SEQ ID NO: 96; mHMGB3 (C45S, C104S - double mutant), see SEQ ID NO: 91, which further comprises AGT encoding C45S and AGC encoding C104S, i.e., one T to A mutation at nucleotide residue 133 of SEQ ID NO: 91, one C to T mutation at nucleotide residue 135 of SEQ ID NO: 91, one T to A mutation at nucleotide residue 310 of SEQ ID NO: 91, and one T to C mutation at nucleotide residue 312 of SEQ ID NO: 91; mHMGB3 (C23S, C45S, C104S - triple mutant), see SEQ ID NO:98, which is SEQ ID NO:91 further comprising AGT encoding C23S, AGT encoding C45S, and AGC encoding C104S, i.e., one T to A mutation at nucleotide residue 67 of SEQ ID NO:91, one C to T mutation at nucleotide residue 69 of SEQ ID NO:91, one T to A mutation at nucleotide residue 133 of SEQ ID NO:91, one C to T mutation at nucleotide residue 135 of SEQ ID NO:91, one T to A mutation at nucleotide residue 310 of SEQ ID NO:91, and one T to C mutation at nucleotide residue 312 of SEQ ID NO:91.

[0109] The corresponding amino acid substitutions to generate modified high mobility group box 4 (mHMGB4) are the consensus sequence polypeptide of HMGB4 from humans: [ka] Based on the above, the cysteine ​​residues at positions 45, 104, 164, and / or 178 may be used.

[0110] Non-limiting exemplary sequences of modified high mobility group box 4 domains include, but are not limited to, mHMGB4(C45S) which is SEQ ID NO: 99 and further contains one mutation at C45S, i.e., SEQ ID NO: 100; mHMGB4(C104S) which is SEQ ID NO: 99 and further contains one mutation at C104S, i.e., SEQ ID NO: 101; mHMGB4(C164S) which is SEQ ID NO: 99 and further contains one mutation at C164S, i.e., SEQ ID NO: 102; mHMGB4(C178S) which is SEQ ID NO: 99 and further contains one mutation at C178S, i.e., SEQ ID NO: 103; mHMGB4(C45S,C104S -) which is SEQ ID NO: 99 and further contains two mutations at C45S and C104S. mHMGB4 (C45S, C164S double mutant) which is SEQ ID NO: 104 and further contains two mutations, C45S and C164S, and is SEQ ID NO: 99; mHMGB4 (C45S, C164S double mutant) which is SEQ ID NO: 105 and further contains two mutations, C45S and C178S, and is SEQ ID NO: 99; mHMGB4 (C45S, C178S double mutant) which is SEQ ID NO: 106 and further contains two mutations, C104S and C164S, and is SEQ ID NO: 99; mHMGB4 (C104S, C178S double mutant) which is SEQ ID NO: 107; mHMGB4 (C104S, C178S double mutant) which is SEQ ID NO: 99 and further contains two mutations, C104S and C178S, i.e., SEQ ID NO: 108; mHMGB4 (C164S, C178S double mutant) which is SEQ ID NO: 99 and further contains two mutations, C164S and C178S, i.e., SEQ ID NO: 109; mHMGB4 (C45S, C104S, C164S triple mutant) which is SEQ ID NO: 110; mHMGB4 (C45S, C104S, C178S triple mutant) which is SEQ ID NO: 99 and further contains three mutations, C45S, C104S, and C178S mHMGB4 (C45S, C164S, C178S triple mutant) which is SEQ ID NO: 99 and further contains three mutations, C45S, C164S, and C178S, i.e., SEQ ID NO: 112; mHMGB4 (C104S, C164S, C178S triple mutant) which is SEQ ID NO: 99 and further contains three mutations, C104S, C164S, and C178S, i.e., SEQ ID NO: 113; and mHMGB4 (C45S, C104S, C164S, C178S quadruple mutant) which is SEQ ID NO: 99 and further contains four mutations, C45S, C104S, C164S, and C178S, i.e., SEQ ID NO: 114.

[0111] An exemplary nucleic acid sequence encoding human HMGB4 is provided below: [ka]

[0112] Nucleic acid sequences encoding these modified high mobility group box 4 domains are also provided herein below: mHMGB4(C45S), which is SEQ ID NO:115 further comprising AGT encoding C45S, i.e., SEQ ID NO:115 further comprising one mutation from T to A at nucleotide residue 133 of SEQ ID NO:115, see SEQ ID NO:116; mHMGB4(C45S), which is SEQ ID NO:115 further comprising AGT encoding C104S, i.e., SEQ ID NO:115 further comprising one mutation from T to A at nucleotide residue 310 of SEQ ID NO:115 and SEQ ID NO:115 mHMGB4(C104S), which is SEQ ID NO: 115, further comprising one C to T mutation at 312, see SEQ ID NO: 117; mHMGB4(C164S), which is SEQ ID NO: 115, further comprising AGT encoding C164S, see SEQ ID NO: 118; mHMGB4(C178S), which is SEQ ID NO: 115, further comprising AGC encoding C178S, see SEQ ID NO: 119; mHMGB4(C45S,C104S), which is SEQ ID NO: 115, further comprising AGT encoding C45S and AGT encoding C104S mHMGB4 (C45S,C164S - double mutant), see SEQ ID NO: 120; mHMGB4 (C45S,C164S - double mutant), which is SEQ ID NO: 115 further comprising an AGT encoding C45S and an AGT encoding C164S, see SEQ ID NO: 121; mHMGB4 (C45S,C178S - double mutant), which is SEQ ID NO: 115 further comprising an AGT encoding C45S and an AGC encoding C178S, see SEQ ID NO: 122; mHMGB4 (C104S,C164S - double mutant), which is SEQ ID NO: 115 further comprising an AGT encoding C104S and an AGT encoding C164S, see SEQ ID NO: 123; mHMGB4 (C104S,C178S - double mutant), which is SEQ ID NO: 115 further comprising an AGT encoding C104S and an AGC encoding C178S. mHMGB4 (C164S, C178S - double mutant), see SEQ ID NO: 124; mHMGB4 (C164S, C178S - double mutant), see SEQ ID NO: 115, which further comprises AGT encoding C164S and AGC encoding C178S;mHMGB4 (C45S, C104S, C164S - triple mutant) which is SEQ ID NO: 115 further comprising AGT encoding C45S, AGT encoding C104S, and AGT encoding C164S, see SEQ ID NO: 126; mHMGB4 (C45S, C104S, C178S - triple mutant) which is SEQ ID NO: 115 further comprising AGT encoding C45S, AGT encoding C104S, and AGC encoding C178S, see SEQ ID NO: 127; mHMGB4 (C45S, C164S, C178S - triple mutant) which is SEQ ID NO: 115 further comprising AGT encoding C45S, AGT encoding C164S, and AGC encoding C178S, see SEQ ID NO: 128; mHMGB4 (C104S, C164S, C178S - triple mutant), see SEQ ID NO: 128; mHMGB4 (C104S, C164S, C178S - triple mutant), see SEQ ID NO: 115, further comprising AGT encoding C104S, AGT encoding C164S, and AGC encoding C178S, see SEQ ID NO: 129; mHMGB4 (C45S, C104S, C164S, C178S - quadruple mutant), see SEQ ID NO: 115, further comprising AGT encoding C45S, AGT encoding C104S, AGT encoding C164S, and AGC encoding C178S, see SEQ ID NO: 130;

[0113] Additional exemplary nucleic acid sequences encoding human HMGB4 are provided below: [ka]

[0114] Nucleic acid sequences encoding these modified high mobility group box 4 domains are also provided herein below: mHMGB4(C45S), which is SEQ ID NO: 135 further comprising AGT encoding C45S, i.e., SEQ ID NO: 135 further comprising one T to A mutation at nucleotide residue 133 of SEQ ID NO: 135, see SEQ ID NO: 136; mHMGB4(C104S), which is SEQ ID NO: 135 further comprising AGT encoding C104S, i.e., SEQ ID NO: 135 further comprising one T to A mutation at nucleotide residue 310 of SEQ ID NO: 135 and optionally one C to T mutation at nucleotide residue 312 of SEQ ID NO: 135, see SEQ ID NO: 137; mHMGB4(C104S), which is SEQ ID NO: 135 further comprising AGT encoding C164S, i.e., SEQ ID NO: 135 See SEQ ID NO: 138, mHMGB4(C164S), which is SEQ ID NO: 135 further comprising one T to A mutation at nucleotide residue 532 of SEQ ID NO: 135; see SEQ ID NO: 139, mHMGB4(C178S), which is SEQ ID NO: 135 further comprising AGC encoding C178S, i.e., one T to A mutation at nucleotide residue 532 of SEQ ID NO: 135; see SEQ ID NO: 135 further comprising AGT encoding C45S and AGT encoding C104S, i.e., one T to A mutation at nucleotide residue 133 of SEQ ID NO: 135, one T to A mutation at nucleotide residue 310 of SEQ ID NO: 135, and optionally one C to T mutation at nucleotide residue 312 of SEQ ID NO: 135. mHMGB4 (C45S, C164S - double mutant), see SEQ ID NO: 140; mHMGB4 (C45S, C164S - double mutant), see SEQ ID NO: 141, which is SEQ ID NO: 135 further comprising AGT encoding C45S and AGT encoding C164S, i.e., one T to A mutation at nucleotide residue 133 of SEQ ID NO: 135 and one T to A mutation at nucleotide residue 490 of SEQ ID NO: 135;mHMGB4 (C45S, C178S; mHMGB4(C104S,C164S - double mutant), see SEQ ID NO: 142; mHMGB4(C104S,C164S - double mutant), see SEQ ID NO: 143, which is SEQ ID NO: 135 further comprising AGT encoding C104S and AGT encoding C164S, i.e., SEQ ID NO: 135 further comprising one T to A mutation at nucleotide residue 310 of SEQ ID NO: 135, optionally one C to T mutation at nucleotide residue 312 of SEQ ID NO: 135, and one T to A mutation at nucleotide residue 490 of SEQ ID NO: 135; mHMGB4(C104S,C178S - double mutant), see SEQ ID NO: 135 further comprising AGT encoding C104S and AGC encoding C178S, i.e., SEQ ID NO: 135 further comprising one T to A mutation at nucleotide residue 310 of SEQ ID NO: 135, optionally one C to T mutation at nucleotide residue 312 of SEQ ID NO: 135, and one T to A mutation at nucleotide residue 532 of SEQ ID NO: 135. mHMGB4 (C164S, C178S - double mutant), see SEQ ID NO: 144; mHMGB4 (C164S, C178S - double mutant), see SEQ ID NO: 135, which further comprises AGT encoding C164S and AGC encoding C178S, i.e., SEQ ID NO: 135, which further comprises one T to A mutation at nucleotide residue 490 of SEQ ID NO: 135 and one T to A mutation at nucleotide residue 532 of SEQ ID NO: 135; mHMGB4 (C45S, C104S, C164S - triple mutant), see SEQ ID NO: 146, which is SEQ ID NO: 135 further comprising AGT encoding C45S, AGT encoding C104S, and AGT encoding C164S, i.e., one T to A mutation at nucleotide residue 133 of SEQ ID NO: 135, one T to A mutation at nucleotide residue 310 of SEQ ID NO: 135, optionally one C to T mutation at nucleotide residue 312 of SEQ ID NO: 135, and one T to A mutation at nucleotide residue 490 of SEQ ID NO: 135;mHMGB4(C45S, C104S, C178S-), which is SEQ ID NO: 135, further comprising AGT encoding C45S, AGT encoding C104S, and AGC encoding C178S, i.e., SEQ ID NO: 135, further comprising one T to A mutation at nucleotide residue 133 of SEQ ID NO: 135, one T to A mutation at nucleotide residue 310 of SEQ ID NO: 135, optionally one C to T mutation at nucleotide residue 312 of SEQ ID NO: 135, and one T to A mutation at nucleotide residue 532 of SEQ ID NO: 135; mHMGB4 (C45S, C164S, C178S - triple mutant), see SEQ ID NO: 147; mHMGB4 (C45S, C164S, C178S - triple mutant), see SEQ ID NO: 135, which further comprises AGT encoding C45S, AGT encoding C164S, and AGC encoding C178S, i.e., SEQ ID NO: 135, which further comprises one T to A mutation at nucleotide residue 133 of SEQ ID NO: 135, one T to A mutation at nucleotide residue 490 of SEQ ID NO: 135, and one T to A mutation at nucleotide residue 532 of SEQ ID NO: 135; mHMGB4 (C104S, C164S, C178S - triple mutant), see SEQ ID NO: 148; mHMGB4 (C104S, C164S, C178S - triple mutant), see SEQ ID NO: 149, which is SEQ ID NO: 135 further comprising AGT encoding C104S, AGT encoding C164S, and AGC encoding C178S, i.e., one T to A mutation at nucleotide residue 310 of SEQ ID NO: 135, optionally one C to T mutation at nucleotide residue 312 of SEQ ID NO: 135, one T to A mutation at nucleotide residue 490 of SEQ ID NO: 135, and one T to A mutation at nucleotide residue 532 of SEQ ID NO: 135;See SEQ ID NO: 135, which further comprises AGT encoding C45S, AGT encoding C104S, AGT encoding C164S, and AGC encoding C178S, e.g., mHMGB4 (C45S, C104S, C164S, C178S - quadruple mutant), SEQ ID NO: 150, which is SEQ ID NO: 135 further comprising one T to A mutation at nucleotide residue 133 of SEQ ID NO: 135, one T to A mutation at nucleotide residue 310 of SEQ ID NO: 135, optionally one C to T mutation at nucleotide residue 312 of SEQ ID NO: 135, one T to A mutation at nucleotide residue 490 of SEQ ID NO: 135, and one T to A mutation at nucleotide residue 532 of SEQ ID NO: 135;

[0115] It is understood that among equivalents to the modified high mobility group box 1 domain are modified high mobility group box 2, modified high mobility group box 3, and modified high mobility group box 4, which contain cysteine ​​to serine substitutions at one or more amino acid positions corresponding to C23S, C45S, and C106S in modified high mobility group box 1. For modified high mobility group box 2, the corresponding cysteine ​​residues are found at positions 23, 45, and 106; for modified high mobility group box 3, the corresponding cysteine ​​residues are found at positions 23, 45, and 104; and for modified high mobility group box 4, the corresponding cysteine ​​residues are found at positions 45, 104, 164, and 178. Accordingly, Applicants believe that references to a modified high mobility group box 1 containing one or more substitutions selected from the group of C23S, C45S, and C106S are equally applicable to the above-mentioned high mobility group box species having one or more cysteine ​​to serine substitutions at the recited positions, e.g., a modified high mobility group box 2 containing one or more substitutions selected from the group of C23S, C45S, and C106S; a modified high mobility group box 3 containing one or more substitutions selected from the group of C23S, C45S, and C104S; and a modified high mobility group box 4 containing one or more substitutions selected from the group of C45S, C104S, C164S, and C178S. In one aspect, equivalents of such polypeptides or proteins include those having a percent identity of at least 70% or higher (as described herein), provided that certain substituted amino acids, e.g., C23S, C45S, and / or C106S, are maintained.

[0116] An "A box" polypeptide refers to a polypeptide comprising the A box domain of an HMGB1 protein. The A box polypeptide may be mutated or contain additional sequences, such as a linker sequence, a signal sequence, or a secretory sequence. One or more point mutations may be introduced at amino acids K12, C23, and C45. In some embodiments, the A box polypeptide comprises, consists essentially of, or even consists of a fragment of an HMGB1 polypeptide disclosed herein aligned with aa9-aa79 of SEQ ID NO: 51 or an equivalent thereof, e.g., a fragment of an HMGB1 polypeptide disclosed herein aligned with aa9-aa79 of SEQ ID NO: 51. In some embodiments, the A box polypeptide comprises, consists essentially of, or even consists of a fragment of an HMGB1 polypeptide disclosed herein aligned with aa1-aa79 of SEQ ID NO: 51 or an equivalent thereof, e.g., a fragment of an HMGB1 polypeptide disclosed herein aligned with aa1-aa79 of SEQ ID NO: 51. In some embodiments, the A box polypeptide comprises, consists essentially of, or even consists of a fragment of an HMGB1 polypeptide disclosed herein aligned with aa1-aa70 of SEQ ID NO: 51 or an equivalent thereof, e.g., a fragment of an HMGB1 polypeptide disclosed herein aligned with aa1-aa70 of SEQ ID NO: 51. Examples of A box polypeptides include: [ka] Comprising, consisting essentially of, or also consisting of.

[0117] A "B box" polypeptide refers to a polypeptide comprising the B box domain of the HMGB1 protein. The B box polypeptide may be mutated or contain additional sequences, such as a linker sequence, a signal sequence, or a secretory sequence. Point mutations at amino acids K114 or C106 may be introduced to confer DNA binding, inflammatory properties, and anti-biofilm activity. In some embodiments, the B box polypeptide comprises, consists essentially of, or even consists of a fragment of the HMGB1 polypeptide disclosed herein aligned with aa95 to aa163 of SEQ ID NO:51 or an equivalent thereof, e.g., a fragment of the HMGB1 polypeptide disclosed herein aligned with aa95 to aa163 of SEQ ID NO:51. In some embodiments, the B box polypeptide comprises, consists essentially of, or even consists of a fragment of the HMGB1 polypeptide disclosed herein aligned with aa88 to aa164 of SEQ ID NO:51 or an equivalent thereof, e.g., a fragment of the HMGB1 polypeptide disclosed herein aligned with aa88 to aa164 of SEQ ID NO:51. In some embodiments, the B box polypeptide comprises, consists essentially of, or even further consists of a fragment of an HMGB1 polypeptide disclosed herein aligned with aa80 to aa164 of SEQ ID NO: 51 or an equivalent thereof, for example, a fragment of an HMGB1 polypeptide disclosed herein aligned with aa80 to aa164 of SEQ ID NO: 51. In some embodiments, the B box polypeptide comprises, consists essentially of, or even further consists of a fragment of an HMGB1 polypeptide disclosed herein aligned with aa80 to aa176 of SEQ ID NO: 51 or an equivalent thereof, for example, a fragment of an HMGB1 polypeptide disclosed herein aligned with aa80 to aa176 of SEQ ID NO: 51. In some embodiments, the B box polypeptide comprises, consists essentially of, or even further consists of a fragment of an HMGB1 polypeptide disclosed herein aligned with aa90 to aa176 of SEQ ID NO: 51 or an equivalent thereof, for example, a fragment of an HMGB1 polypeptide disclosed herein aligned with aa90 to aa176 of SEQ ID NO: 51. In some embodiments, the B box polypeptide comprises, consists essentially of, or even consists of a fragment of an HMGB1 polypeptide disclosed herein aligned with aa89 to aa162 of SEQ ID NO: 51, or an equivalent thereof, e.g., a fragment of an HMGB1 polypeptide disclosed herein aligned with aa89 to aa162 of SEQ ID NO: 51. Examples of B box polypeptides include: [ka] Comprising, consisting essentially of, or also consisting of.

[0118] By "C-box" polypeptide is intended a polypeptide comprising the C-tail domain of an HMGB1 protein. The C-tail polypeptide may be mutated or may contain additional sequences, such as a linker sequence, a signal sequence, or a secretory sequence. In some embodiments, the C-box polypeptide comprises, consists essentially of, or even consists of aa 186 to aa 215 of SEQ ID NO: 51, or an equivalent thereof, e.g., a fragment of an HMGB1 polypeptide disclosed herein aligned with aa 186 to aa 215 of SEQ ID NO: 51. In some embodiments, the C-box polypeptide comprises, consists essentially of, or even consists of EEEEDEEDEEEEEEEDEEDEEEDDDDE (SEQ ID NO: 133).

[0119] An "AB box" polypeptide refers to a polypeptide comprising the A and B box domains of an HMGB1 protein fused together, but without the corresponding amino acids of the full-length wild-type protein. The AB box polypeptide may be mutated or contain additional sequences, such as a linker sequence, a signal sequence, or a secretory sequence. One or more point mutations at amino acids described herein (e.g., at amino acids K12, C23, C45, C106, and / or K114) may be introduced to confer DNA binding, inflammatory properties, and anti-biofilm activity. In some embodiments, the AB box polypeptide comprises, consists essentially of, or even consists of a fragment of an HMGB1 polypeptide disclosed herein aligned with aa1-aa176 of SEQ ID NO: 51 or its equivalent, e.g., aa1-aa176 of SEQ ID NO: 51. In some embodiments, the AB box polypeptide comprises, consists essentially of, or even further consists of a fragment of an HMGB1 polypeptide disclosed herein aligned with aa1-aa162 of SEQ ID NO: 51 or an equivalent thereof, e.g., a fragment of an HMGB1 polypeptide disclosed herein aligned with aa1-aa162 of SEQ ID NO: 51. In some embodiments, the AB box polypeptide comprises, consists essentially of, or even further consists of a fragment of an HMGB1 polypeptide disclosed herein aligned with aa1-aa164 of SEQ ID NO: 51 or an equivalent thereof, e.g., a fragment of an HMGB1 polypeptide disclosed herein aligned with aa1-aa164 of SEQ ID NO: 51.

[0120] In some embodiments, equivalents to the HMGB1 polypeptides described herein may comprise, consist essentially of, or even consist of proteins or protein fragments containing one or more of the HMG box domain truncations and / or variants described herein, or the HMG box domains, truncations, variants, or equivalents of proteins or fragments having the disclosed amino acid substitutions. In some embodiments, fragments of HMGB1 polypeptides comprise, consist essentially of, or even consist of one or more of the A box polypeptides disclosed herein, the B box polypeptides disclosed herein, or the AB box polypeptides disclosed herein.

[0121] In some embodiments, the A box polypeptide can further comprise, or alternatively consist essentially of, or even consist of, one or more amino acid mutations selected from K12, C23, and C45 (e.g., a mutation of the native K or C modified to an amino acid from the group selected from serine, glycine, alanine, valine, isoleucine, or threonine), or equivalents thereof containing one or more amino acid mutations selected from K12, C23, and C45 (e.g., a mutation of the native K or C modified to an amino acid from the group selected from serine, glycine, alanine, valine, isoleucine, or threonine). In one aspect, the mutation is a C45S mutation. The A box polypeptide can further comprise a linker or peptide sequence located at one or both termini. An example of a peptide linker is PPKGETKKKF (SEQ ID NO: 131).

[0122] When recombinantly produced, A box polypeptides can be prepared using methods known in the art, e.g., Olia AS et al., (2015) ACS chemical biology. 10(9): 2034-47. doi: 10.1021 / acschembio.5b00342, PubMed PMID: 26083674; PubMed Central PMCID: PMC4610810; Ugrinova I et al., (2102) Molecular Biology Reports, 2012; 39(11): 9947-53. Epub 2012 / 06 / 29. doi: 10.1007 / s11033-012-1863-x. PubMed PMID: 22740141; and Ito The A box polypeptide may be partially or fully acetylated, oxidized, or phosphorylated using methods known from T et al. (2007) JTH, 5(1):109-16. doi:10.1111 / j.1538-7836.2006.02255.x. PubMed PMID:17239166. In one embodiment, the A box polypeptide comprises, consists essentially of, or even consists of amino acids 1-70 of a wild-type HMGB1 polypeptide having the aforementioned mutations. Examples of A box polypeptides include:

[0123] In some embodiments, the B-box polypeptide can comprise, or alternatively consist essentially of, or even consist of, a mutation at amino acid C106 or K114, or both (e.g., mutation of the native cysteine ​​to an amino acid from the group selected from serine, glycine, alanine, valine, isoleucine, or threonine), or an equivalent thereof comprising a mutation at amino acid C106 or K114, or both (e.g., mutation of the native cysteine ​​to an amino acid from the group selected from serine, glycine, alanine, valine, isoleucine, or threonine). In one aspect, the B-box polypeptide comprises, consists essentially of, or even consists of about 80 to about 176, or about 88 to about 164, or about 89 to about 162, or even about 80 to about 164 of a wtHMGB1 polypeptide having the aforementioned mutations.

[0124] The B box polypeptide may further comprise a linker or peptide sequence located at one or both termini. An example of a peptide linker is PPKGETKKKF (SEQ ID NO: 131). When recombinantly produced, the disclosed B-box polypeptides can be synthesized using methods known in the art, e.g., Olia AS et al. (2015) ACS chemical biology. 10(9):2034-47. doi:10.1021 / acschembio.5b00342, PubMed PMID:26083674; PubMed Central PMCID:PMC4610810; Ugrinova I et al. (2102) Molecular Biology Reports, 2012;39(11):9947-53. Epub 2012 / 06 / 29. doi:10.1007 / s11033-012-1863-x. PubMed PMID:22740141; and Ito It may be partially or fully acetylated, oxidized, or phosphorylated using methods known in T et al. (2007) JTH, 5(1):109-16. doi:10.1111 / j.1538-7836.2006.02255.x. PubMed PMID: 17239166.

[0125] In some embodiments, the AB box polypeptide may comprise, alternatively consist essentially of, or additionally consist of one or more amino acid mutations selected from K12, C23, C45, C106, or K114 (e.g., a native K or C modified to an amino acid from the group selected from serine, glycine, alanine, valine, isoleucine, or threonine), or an equivalent thereof comprising one or more amino acid mutations selected from K12, C23, C45, C106, or K114 (e.g., a native K or C modified to an amino acid from the group selected from serine, glycine, alanine, valine, isoleucine, or threonine). In one aspect, the mutation is a C45S mutation. In another embodiment, the polypeptide comprises a mutation at amino acid C106 (e.g., mutation of the native cysteine ​​to an amino acid from the group selected from serine, glycine, alanine, valine, isoleucine, or threonine), or an equivalent thereof comprising one or more amino acid mutations selected from K12, C23, C45 and a mutation at amino acid C106 (e.g., mutation of the native cysteine ​​to an amino acid from the group selected from serine, glycine, alanine, valine, isoleucine, or threonine). In one embodiment, the AB box polypeptide comprises the C45S and C106S mutations, and equivalents retain these mutations. In one embodiment, the AB box polypeptide comprises, consists essentially of, or even consists of amino acids 1-176, or 1-162, or even 1-164 of a wild-type HMGB1 polypeptide having the aforementioned mutations.

[0126] In some embodiments, the AB box polypeptide further comprises a linker polypeptide located between the A box polypeptide and the B box polypeptide, and in one aspect, a second linker linking the B box and C box polypeptide. When recombinantly produced, the AB or A, B, and C box polypeptides can be partially or fully acetylated, oxidized, or phosphorylated. An example of a peptide linker is PPKGETKKKF (SEQ ID NO: 131). In one aspect, methods for linking AB or A, B, and C box polypeptides can be described in detail in the art, e.g., Olia AS et al., (2015) ACS chemical biology. 10(9):2034-47. doi:10.1021 / acschembio.5b00342, PubMed PMID:26083674; PubMed Central PMCID:PMC4610810; Ugrinova I et al., (2102) Molecular Biology Reports, 2012;39(11):9947-53. Epub 2012 / 06 / 29. doi:10.1007 / s11033-012-1863-x. PubMed PMID:22740141; and Ito T et al., (2007) JTH,5(1):109-16. doi:10.1111 / j.1538-7836.2006.02255.x. PubMed PMID:17239166.

[0127] Examples of AB box polypeptides have the aforementioned mutations: [ka] Comprise, consist essentially of, or even consist of.

[0128] In a further embodiment, the AB box polypeptide further comprises a linker polypeptide positioned between the A box polypeptide and the B box polypeptide.

[0129] In some embodiments, the HMGB1 polypeptide or fragment thereof comprises, consists essentially of, or further consists of the A, B, and C domains, and the polypeptide comprises, consists essentially of, or further consists of one or more amino acid mutations selected from K12, C23, C45, C106, or K114, or equivalents thereof, or equivalents thereof containing one or more amino acid mutations selected from K12, C23, C45, C106, or K114.

[0130] As used herein, a polypeptide equivalent refers to a sequence that is at least about 70%, or alternatively at least about 75%, or alternatively at least about 80%, or alternatively at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively at least about 98%, or alternatively at least about 99% identical to a reference polypeptide, and in one embodiment, retains a variant amino acid(s). In some embodiments, a polypeptide equivalent retains the desired function and / or structural characteristics of a polypeptide, for example, containing an HMG box domain, but optionally without inducing a pro-inflammatory response. In one embodiment, an equivalent polypeptide includes a domain that is at least about 70%, or alternatively at least about 80%, or alternatively at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively at least about 98%, or alternatively at least about 99% identical to an HMG box domain, and in one embodiment, retains a variant amino acid(s). In some embodiments, such an equivalent domain retains the function and / or structural characteristics of the HMB box domain, for example, binding to an HMB box binding target, but optionally without inducing a pro-inflammatory response. In one embodiment, the equivalent polypeptide is encoded by a polynucleotide that is capable of hybridizing under stringent conditions to a polynucleotide encoding an HMB box domain polypeptide.

[0131] In some embodiments, the HMGB1 box polypeptide further comprises a linker polypeptide located between the A box polypeptide and the B box polypeptide, and a second linker polypeptide connecting the B box polypeptide and the C box polypeptide. An example of a peptide linker is PPKGETKKKF (SEQ ID NO: 131).

[0132] An immunodominant antigen is intended to be a region of a protein that is recognized by an antibody and binds to it with high affinity.

[0133] An immunoprotective antigen refers to a region of a protein that is recognized by an antibody, binds to it with high affinity, and interferes with the function of the protein; antibodies generated against an immunoprotective antigen are characterized by an enhanced or optimal effect on the target indication, in this case, an improved ability to remove biofilm, as a result of interfering with the function of the protein.

[0134] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. 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, ESTs, 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 can 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 may 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 the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0135] A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, if the polynucleotide is RNA, uracil (U) instead of thymine. Thus, the term "polynucleotide sequence" is an alphabetic representation of a polynucleotide molecule. This alphabetic representation can be input into a database on a computer with a central processing unit and used in bioinformatics applications, such as functional genomics and homology searching.

[0136] The terms "isolated" or "recombinant," as used herein with respect to nucleic acids, e.g., DNA or RNA, refer to molecules separated from other DNAs or RNAs and polypeptides, respectively, present in the natural source of the polymer. The term "isolated or recombinant nucleic acid" is intended to include nucleic acid fragments not naturally occurring as fragments, not found in the natural state. The term "isolated" is also used herein to refer to polynucleotides, polypeptides, and proteins 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 being separated from cellular components and other cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragment(s) thereof, with which it is otherwise normally associated in nature. For example, an isolated cell is one that is separated from tissues or cells of a dissimilar phenotype or genotype. An isolated polynucleotide is separated from the 3' and 5' flanking nucleotides with which it is normally associated in its native or natural environment, e.g., on a 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 distinguish it from its naturally occurring counterpart.

[0137] The term "isolated," as used herein, refers to a molecule, biologic, cellular material, cell, or biological sample that is substantially free of other materials. In one aspect, the term "isolated" refers to a nucleic acid, e.g., a DNA or RNA, or a protein or polypeptide (e.g., an antibody or derivative thereof), or a cell or cell organelle, or a tissue or organ, that has been separated from other DNA or RNA, or proteins or polypeptides, or cells or cell organelles, or tissues or organs, respectively, that are present in the natural source.

[0138] In some embodiments, the term "engineered" refers to the inclusion of at least one modification not normally found in its naturally occurring counterpart, wild-type or parent. In some embodiments, the term "engineered" is used interchangeably with "recombinant" to refer to synthesized by humans.

[0139] Where the present disclosure relates to a polypeptide, protein, polynucleotide, or antibody, it should be assumed that equivalents or biological equivalents of such are intended within the scope of the present disclosure, without explicit recitation and unless otherwise intended. As used herein, the term "biological equivalent thereof" when referring to a reference protein, antibody, fragment, polypeptide, or nucleic acid is intended to be synonymous with "equivalent thereof," and is intended to be synonymous with "equivalent thereof," and to have minimal homology while still maintaining the desired structure or functionality. Unless otherwise recited herein, any polynucleotide, polypeptide, or protein listed herein is also intended to include its equivalent. In one aspect, an equivalent polynucleotide is a polynucleotide that hybridizes under stringent conditions to 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 is intended to be an antibody or antigen-binding polypeptide that binds to a reference antibody or antigen-binding fragment with at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% affinity or higher. In another embodiment, the equivalent competes with the antibody or antigen-binding fragment's binding to its antigen in a competitive ELISA assay. In another embodiment, an equivalent is intended to be at least about 80% homologous or identical, and alternatively at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively 98% percent homologous or identical, and exhibits substantially equivalent biological activity to the reference protein, polypeptide, or nucleic acid. Examples of biologically equivalent polypeptides are provided in Table 9 of WO2011 / 123396, which identifies conservative amino acid substitutions for the disclosed amino acid sequences.

[0140] In some embodiments, equivalents to an amino acid sequence include polypeptides having at least 80% amino acid identity to the amino acid sequence, and / or equivalents to an amino acid sequence include polypeptides encoded by polynucleotides that hybridize under high stringency conditions to the complement of a polynucleotide encoding the amino acid sequence. In further embodiments, equivalents to an amino acid sequence include polypeptides having at least 90% amino acid identity to the amino acid sequence, and / or equivalents to an amino acid sequence include polypeptides encoded by polynucleotides that hybridize under high stringency conditions to the complement of a polynucleotide encoding the amino acid sequence. In still further embodiments, equivalents to an amino acid sequence include polypeptides having at least 95% amino acid identity to the amino acid sequence, and / or equivalents to an amino acid sequence include polypeptides encoded by polynucleotides that hybridize under high stringency conditions to the complement of a polynucleotide encoding the amino acid sequence. In one embodiment, equivalents to an amino acid sequence include polypeptides having at least 96% amino acid identity to the amino acid sequence and / or equivalents to an amino acid sequence include polypeptides encoded by polynucleotides that hybridize under high stringency conditions to the complement of a polynucleotide encoding the amino acid sequence. In a further embodiment, equivalents to an amino acid sequence include polypeptides having at least 97% amino acid identity to the amino acid sequence and / or equivalents to an amino acid sequence include polypeptides encoded by polynucleotides that hybridize under high stringency conditions to the complement of a polynucleotide encoding the amino acid sequence.In still further embodiments, equivalents to an amino acid sequence include polypeptides having at least 98% amino acid identity to the amino acid sequence, and / or equivalents to an amino acid sequence include polypeptides encoded by polynucleotides that hybridize under high stringency conditions to the complement of a polynucleotide encoding the amino acid sequence. In one embodiment, equivalents to an amino acid sequence include polypeptides having at least 99% amino acid identity to the amino acid sequence, and / or equivalents to an amino acid sequence include polypeptides encoded by polynucleotides that hybridize under high stringency conditions to the complement of a polynucleotide encoding the amino acid sequence.

[0141] 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 comparing the two sequences. Alignment and percent homology or sequence identity can be determined using software programs known in the art, such as the software programs described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. In certain embodiments, default parameters are used for the alignment. A non-limiting exemplary alignment program is BLAST, using default parameters. In particular, exemplary programs include BLASTN and BLASTP, which use the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; prediction = 10; matrix = BLOSUM62; description = 50 sequences; classification = high score; database = non-redundant GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percent identity were determined by incorporating them into clustalW (available at the web address align.genome.jp (last accessed March 7, 2011)). In some embodiments, Clustal Omega, accessible at www.ebi.ac.uk / Tools / msa / clustalo / , is used in sequence alignment or in determining percent identity. In further embodiments, default settings are applied.

[0142] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that can be aligned for comparison purposes. 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 shared by the sequences. 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.

[0143] "Homology" or "identity" or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions.

[0144] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can occur through Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific manner. The complex can include two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction can constitute a step in a more extensive process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0145] 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 about 1, 2, or 15 minutes. SSC is a 0.15M NaCl and 15mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be used.

[0146] 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 then 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.

[0147] The term "encoding," when applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide when, in its native state, or when manipulated by methods well known to those of skill in the art, it is capable of being transcribed and / or translated to produce mRNA and the polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0148] As used herein, the terms "treat," "treatment," and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect can be preventative, in that it is a complete or partial prevention of a disorder or its signs or symptoms, and / or therapeutic, in that it is a partial or complete cure of the disorder and / or adverse effects resulting from the disorder. As used herein, "treating" or "treatment" of a disease in a subject can also refer to (1) preventing a symptom or disease from occurring in a subject who is susceptible to the disease or who does not yet exhibit symptoms of the disease; (2) inhibiting or arresting the development of the disease; or (3) ameliorating or causing regression of the disease or symptoms of the disease. As understood in the art, "treatment" is an approach to obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results may include, but are not limited to, one or more of: alleviation or amelioration of one or more symptoms; attenuation of the severity of a condition (including a disease); stabilization (i.e., non-worsening) of a condition (including a disease); delay or slowing of a condition (including a disease); progression, improvement or alleviation of a condition (including a disease), condition and remission (whether partial or total), whether detectable or undetectable. In one embodiment, treatment excludes prevention.

[0149] By prevent is intended preventing a disorder or effect in vitro or in vivo in a system or subject susceptible to the disorder or effect, one example of which is preventing biofilm formation in a system infected with a microorganism known to produce biofilms.

[0150] A "composition" is intended to mean a combination of an active agent with another compound or composition, inert (e.g., a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc., and includes a pharmaceutically acceptable carrier. Carriers also include pharmaceutical excipients and additives, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-oligosaccharides, and oligosaccharides; derivatized sugars, e.g., alditols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, including 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin, e.g., human serum albumin (HSA), and the like. These include recombinant human albumin (rHA), gelatin, casein, etc. Representative amino acids / antibody components that may also function in buffering capacity include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. Carbohydrate excipients are also contemplated within the scope of this technology, examples of which include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and myo-inositol.

[0151] A "pharmaceutical composition" is intended to include a combination of an active agent with an inert or active carrier that makes the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0152] "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 phosphate, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference textbook in this field. They may be selected with regard to the intended dosage form, ie, oral tablet, capsule, elixir, syrup, etc., and consistent with conventional pharmaceutical practice.

[0153] Compositions used in accordance with the present disclosure may be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of composition calculated to produce the desired response in association with its administration, i.e., an appropriate route and regimen. The amount to be administered, both by number of treatments and unit dose, depends on the desired outcome and / or protection. The precise amount of the composition also depends on the judgment of the practitioner and is peculiar to each individual. Factors affecting dosage include the subject's physical and clinical condition, the route of administration, the intended purpose of treatment (relief of symptoms versus cure), and the efficacy, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the administration formulation and in an amount that is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, for example, the types of injectable solutions described herein.

[0154] Combination, as used herein, contemplates that the individual active ingredients of the composition may be formulated separately for combined use and packaged separately, with or without specific dosages. The active ingredients of the combination may be administered simultaneously or sequentially.

[0155] By "bioactive agent" or active agent as disclosed herein is intended one or more of an isolated or recombinant polypeptide, an isolated or recombinant polynucleotide, a vector, an isolated host cell, or an antibody, and compositions comprising one or more of these.

[0156] "Administration" can be achieved in one dose, continuously, or intermittently throughout the course of treatment. Methods for determining the most effective means and dosage of administration are known to those skilled in the art and vary depending on the composition used for treatment, the purpose of treatment, the target cells being treated, and the subject being treated. Single or multiple administrations can be performed at dose levels and patterns selected by the treating physician. Suitable administration formulations and methods for administering the active substance are known in the art. In addition, 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 for treatment, the purpose of treatment, the health condition or disease stage of the subject being treated, and the target cells or tissues being treated. Non-limiting examples of administration routes include oral administration, nasal administration, injection, and topical application.

[0157] The agents of the present disclosure may be administered for therapy by any suitable route of administration, it being understood that the most suitable route will vary with the condition and age of the recipient and the disease being treated.

[0158] The term "contacting" refers to a direct or indirect binding or interaction between two or more entities. A specific example of a direct interaction is binding. A specific example of an indirect interaction is when one entity acts on an intermediate molecule, which in turn acts on a second reference entity. Contacting, as used herein, includes contacting in solution, in solid phase, in vitro, ex vivo, in a cell, and in vivo. In vivo contacting can be referred to as administering or administration.

[0159] The term "effective amount" refers to an amount sufficient to achieve a desired effect. For therapeutic or prophylactic applications, the effective amount depends on the type and severity of the condition in question, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. For immunogenic compositions, in some embodiments, the effective amount is an amount sufficient to produce a protective response against a pathogen. In other embodiments, the effective amount of an immunogenic composition is an amount sufficient to produce antibody production against an antigen. In some embodiments, the effective amount is the amount necessary to confer passive immunity to a subject in need thereof. For 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 will be able to determine the appropriate amount depending on these and other factors.

[0160] In the case of intravital 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 intravital target and the method of use. Those skilled in the art can determine the effective amount based on these and other considerations. The effective amount may include one or more administrations of the composition depending on the embodiment.

[0161] The term "contacting" refers to a direct or indirect binding or interaction between two or more entities. A specific example of a direct interaction is binding. A specific example of an indirect interaction is when one entity acts on an intermediate molecule, which in turn acts on a second reference entity. Contacting, as used herein, includes contacting in solution, in solid phase, in vitro, ex vivo, in a cell, and in vivo. In vivo contacting can be referred to as administering or administration.

[0162] 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 the residue of the chimeric polypeptide or can form a covalent bond with a linker that then forms a covalent bond with the residue of the chimeric polypeptide.

[0163] A "peptide conjugate" refers to the association of one or more polypeptides with another chemical or biological compound, either covalently or non-covalently. In a non-limiting example, the "conjugation" of a polypeptide with a chemical compound results in improved stability or efficacy of the polypeptide for its intended purpose. In one embodiment, the peptide is conjugated to a carrier, and the carrier is a liposome, a micelle, or a pharmaceutically acceptable polymer.

[0164] "Liposomes" are microscopic vesicles composed of concentric lipid bilayers. Structurally, liposomes range in size and shape from elongated tubes to spheres, with dimensions ranging from hundreds of angstroms to fractions of a millimeter. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity in the final complex, providing 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 and either saturated or containing one or more double C=C bonds. Examples of lipids that can form stable liposomes, alone or in combination with other lipid components, are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC). soy phosphatidylcholine), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC) These are palmitoyloteoylphosphatidylcholine, palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimido-triethyl)cyclohexane-1-carboxylate (DOPE-mal). Additional non-phosphorus-containing lipids that can be incorporated into 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 listed 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 dicetyl phosphate, which can form vesicles. Typically, liposomes can be divided into three categories based on their overall size and the nature of their lamellar structure. The three classifications developed by the December 1977 New York Academy of Sciences conference, "Liposomes and Their Use in Biology and Medicine," 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.

[0165] "Micelles" are aggregates of surfactant molecules dispersed in a liquid colloid. Typical micelles in aqueous solution form aggregates with their hydrophilic "head" regions in contact with the surrounding solvent and their hydrophobic tail regions sequestered in the center of the micelle. This type of micelle is known as a normal-phase micelle (oil-in-water micelle). Reverse micelles have head groups in the center and tails pointing outward (water-in-oil micelle). Micelles can be used to bind polynucleotides, polypeptides, antibodies, or compositions described herein to facilitate efficient delivery to target cells or tissues.

[0166] The phrase "pharmaceutically acceptable polymer" refers to a group of compounds that can be conjugated to one or more polypeptides described herein. It is contemplated that conjugation of a polymer to a polypeptide can 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. Bioactive agents can be conjugated to pharmaceutically acceptable polymers for administration according to the methods described herein.

[0167] " Gene delivery vehicle " is defined as any molecule that can carry inserted polynucleotide into host cell. Examples of gene delivery vehicles include liposomes, micelles, biocompatible polymers including natural and synthetic polymers, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial virus envelopes, metal particles, and bacteria or viruses, such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors, and other recombinant vehicles typically used in the art, which have been described for expression in various eukaryotic and prokaryotic hosts and can be used for gene therapy and simple protein expression.

[0168] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vehicles. 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 transfer. 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) and 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 typically requires that the introduced polynucleotide either 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, several vectors are known to be capable of mediating the transfer of genes into mammalian cells.

[0169] The term "regulatory sequence," "regulatory element," "expression control element," or "promoter," as used herein, refers to a polynucleotide that is operably linked to a polynucleotide to be transcribed and / or replicated and promotes the expression and / or replication of the polynucleotide. Non-limiting examples of regulatory sequences include promoters, enhancers, or polyadenylation sequences.

[0170] The term "promoter," as used herein, refers to any sequence that regulates the expression of a coding sequence, such as a gene. A promoter can be, for example, constitutive, inducible, repressible, or tissue-specific. A "promoter" is a regulatory sequence, which is a region of a polynucleotide sequence at which the initiation and rate of transcription are controlled. A "promoter" can contain genetic elements at which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. Non-limiting examples of promoters include the cytomegalovirus CMV promoter or retroviral long terminal repeat (LTR) promoter. See, for example, Weber et al. Hum Gene Ther. 2007 Sep;18(9):849-60.

[0171] An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter / enhancer" is a polynucleotide containing a sequence that can provide both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. Enhancers / promoters can be "endogenous," "exogenous," or "heterologous." An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that is placed in juxtaposition to a gene by genetic engineering (i.e., molecular biological techniques) so that the linked enhancer / promoter directs transcription of that gene.

[0172] As used herein, the term "eDNA" refers to extracellular DNA found as a component of pathogenic biofilms.

[0173] A "plasmid" is an extrachromosomal DNA molecule that is separate from chromosomal DNA and can replicate independently of it. It is often circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and typically provide a selective advantage under given environmental conditions. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively, the proteins produced may act as toxins under similar conditions.

[0174] "Plasmids" used in genetic engineering are called "plasmid vectors." Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of the plasmid, which also contains a gene that makes the cell resistant to a particular antibiotic and a multiple cloning site (MCS, or polylinker), a short region containing several commonly used restriction sites and allowing easy insertion of DNA fragments at this location. Another major use of plasmids is to produce large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces the protein that confers its antibiotic resistance, it can also be induced to produce large amounts of the protein from the inserted gene. This is an inexpensive and easy way to mass-produce a gene or the protein it encodes.

[0175] "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 required for replication and storage in yeast cells. Once assembled using initial circular plasmids, they can be linearized using restriction enzymes, and then DNA ligase can be used to add sequences or genes of interest into the linear molecule using the cohesive ends. Yeast expression vectors, such as YACs, YIp (yeast integrating plasmid), and YEp (yeast episomal plasmid), are very useful because yeast is itself a eukaryotic cell, allowing the production of eukaryotic protein products with post-translational modifications. However, YACs are known to be more unstable than BACs and to produce chimeric effects.

[0176] A "viral vector" is defined as a recombinant virus or virus particle containing a polynucleotide to be delivered to a host cell, 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 are sometimes used to manufacture 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 Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also 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, vector construct refers to a polynucleotide comprising the retroviral genome or portion thereof and a therapeutic gene.

[0177] As used herein, "retroviral-mediated gene transfer" or "retroviral transduction" have the same meaning and refer to the process by which a gene or nucleic acid sequence is stably transferred into a host cell by a virus that enters the cell and integrates the viral genome into the host cell genome. The virus may enter the host cell through its normal infection mechanism, or the virus may be modified to bind to a different host cell surface receptor or ligand and 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.

[0178] 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 that integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0179] In embodiments in which gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or an adeno-associated virus (AAV), the term vector construct refers to a polynucleotide comprising the viral genome or a portion thereof and a transgene. Adenoviruses (Ad) are a relatively well-characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., PCT International Application Publication No. WO95 / 27071. Ad does not require integration into the host cell genome. Recombinant Ad-derived vectors have also been constructed, particularly those with reduced potential for recombination and wild-type virus generation. See, e.g., PCT International Application Publication Nos. WO95 / 00655 and WO95 / 11984. Wild-type AAV has high infectivity and specificity for integration into the host cell genome. See Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0180] Vectors containing both a promoter and a cloning site to which a polynucleotide can be operably linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from suppliers 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 extra, 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 improve expression.

[0181] Gene delivery vehicles also include DNA / liposome complexes, micelles, and targeted viral protein-DNA complexes. Liposomes containing targeting antibodies or antigen-binding fragments thereof can also be used in the methods disclosed herein. In addition to delivery of polynucleotides to cells or cell populations, direct introduction of the proteins described herein into cells or cell populations can be achieved by, but is not limited to, protein transfection techniques, or culture conditions that allow for enhanced expression and / or activity of the proteins disclosed herein, among other techniques.

[0182] As used herein, the terms "antibody," "antibodies," and "immunoglobulin" include whole antibodies and any antigen-binding fragments or single chains thereof. Thus, the term "antibody" includes any protein- or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule. The terms "antibody," "antibodies," and "immunoglobulin" also include 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 kappabodies; antibody fragments and multispecific antibody fragments formed from one or more isolated fragments. Examples of such antibodies include, but are not limited to, the complementarity-determining regions (CDRs) of a heavy or light chain or its ligand-binding portion, heavy or light chain variable regions (also referred to herein as variable domains), heavy or light chain constant regions (also referred to herein as constant domains), framework (FR) regions, or any portion thereof, at least a portion of a binding protein, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with antigens. The constant regions of antibodies (Abs) can mediate the binding of the immunoglobulin to host tissues. The term "anti-" when used before a protein name, e.g., anti-DNABII, anti-IHF, anti-HU, anti-OMP P5, refers to a monoclonal or polyclonal antibody that exhibits binding to and / or affinity for a specific protein. For example, "anti-IHF" refers to an antibody that binds to the IHF protein. A specific antibody has affinity for or is capable of binding to a protein other than the protein against which the antibody was raised.For example, an anti-IHF may be raised specifically against the IHF protein, but may also bind to other proteins related through sequence homology or through structural homology.

[0183] Complementarity-determining regions (CDRs) are portions of the variable regions of antibodies or T cell receptors produced by B cells and T cells, respectively, where these molecules bind to their specific antigen (also called epitope). In certain embodiments, the terms "variable region" and "variable domain" are used interchangeably to refer to polypeptides in the light or heavy chain of an antibody that vary greatly in sequence of amino acid residues from one antibody to another and determine the conformation of the binding site that confers the antibody's specificity for a particular antigen. In further embodiments, the variable region is about 90 to about 200 amino acids in length, including, but not limited to, about 100 amino acids in length, or alternatively about 110 amino acids in length, or alternatively about 120 amino acids in length, or alternatively about 130 amino acids in length, or alternatively about 140 amino acids in length, or alternatively about 150 amino acids in length, or alternatively about 160 amino acids in length, or alternatively about 170 amino acids in length, or alternatively about 180 amino acids in length, or alternatively about 190 amino acids in length. In certain embodiments, a variable region of an amino acid sequence, as used herein, refers to the first about 100 amino acids, or alternatively about 110 amino acids, or alternatively about 120 amino acids, or alternatively about 130 amino acids, or alternatively about 140 amino acids, or alternatively about 150 amino acids (including or excluding the signal peptide, if applicable) of an amino acid sequence being the variable region.

[0184] A set of CDRs constitutes a paratope, also called an antigen-binding site, which is the part of an antibody that recognizes and binds to an antigen. Three CDRs (CDR1, CDR2, and CDR3) are present in the amino acid sequence of the variable region of an antigen receptor, such as a heavy or light chain, non-contiguously arranged, optionally from the amino terminus to the carboxy terminus. As used herein, CDRn refers to a CDRn in an immunoglobulin chain or a CDRn derived from an immunoglobulin chain, where the number n is selected from 1 to 3. In one embodiment, CDRLn refers to a CDRn in a light chain or a CDRn derived from a light chain, where the number n is selected from 1 to 3, while CDRHn refers to a CDRn in a heavy chain or a CDRn derived from a heavy chain, where the number n is selected from 1 to 3. In certain embodiments, framework region (FR) refers to a portion of the variable region that is not a CDR. In certain embodiments, FRn refers to a FR in a heavy or light chain or a FR derived from a heavy or light chain, where the number n is selected from 1 to 4. In certain embodiments, the variable region comprises, consists essentially of, or even consists of the following (optionally in the order provided, and optionally from amino terminus to carboxy terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0185] The variable regions and / or CDRs of an antibody or fragment thereof can be determined by one of skill in the art using, for example, publicly available or commercially available tools. Non-limiting examples of such tools include IgBlast (accessible at www.ncbi.nlm.nih.gov / igblast / ), Scaligner (available from drugdesigntech at www.scaligner.com / ), IMGT rules and / or tools (see, e.g., www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html and accessible at www.imgt.org / ), Chothia Canonical Assignment (accessible at www.bioinf.org.uk / abs / chothia.html), Antigen Receptor Numbering And Receptor Calibration (ANARCI, accessible at opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / anarci / ), Kabat numbering method / scheme (e.g., Kabat, EA, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), or the Paratome web server (accessible at www.ofranlab.org / paratome / ; see Vered Kunik, et al, Nucleic Acids Research, Volume 40, Issue W1, 1 July 2012, Pages W521-W524).

[0186] Antibodies can be polyclonal, monoclonal, multispecific (e.g., bispecific) and antibody fragments, so long as they exhibit the desired biological activity. Antibodies can be isolated from any suitable biological source, for example, murines, rats, sheep and canines.

[0187] The terms "polyclonal antibody" or "polyclonal antibody composition," as used herein, refer to a preparation of antibodies derived from different B-cell lines. They are a mixture of immunoglobulin molecules secreted against a particular antigen, each recognizing a different epitope.

[0188] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. Because each monoclonal antibody is directed against a single determinant on the antigen, monoclonal antibodies are highly specific. Antibodies can be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, etc. Antibodies can be further conjugated to other moieties, such as members of specific binding pairs, for example, biotin (a member of the biotin-avidin specific binding pair), etc. Antibodies can also be bound to solid supports, including, but not limited to, polystyrene plates or beads.

[0189] Monoclonal antibodies can be produced using hybridoma technology or recombinant DNA methods known in the art. Hybridomas are cells produced in a laboratory from the fusion of antibody-producing lymphocytes with non-antibody-producing cancer cells, usually myelomas or lymphomas. The hybridomas grow and produce continuous samples of specific monoclonal antibodies. Alternative techniques for producing or selecting antibodies include in vitro exposure of lymphocytes to the antigen of interest and screening antibody display libraries in cell, phage, or similar systems.

[0190] 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, e.g., a mouse, have been grafted onto human framework sequences. Thus, as used herein, the term "human antibody" refers to an antibody in which substantially any portion of the protein (e.g., CDRs, framework, C L , C H Domain (e.g., CH1 , C H2 , C H3 ), hinge, (VL, VH)) also refer to antibodies that are substantially non-immunogenic in humans and involve only minor sequence changes or variations. Similarly, primate (monkey, baboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guinea pig, hamster, etc.), and other mammalian designations of antibodies refer to such species-, subgenus-, genus-, subfamily-, and family-specific antibodies. Furthermore, chimeric antibodies include any combination of the above. Such changes or variations optionally retain or reduce immunogenicity in humans or other species compared to unmodified antibodies. Thus, human antibodies are distinct 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, it may contain a linker peptide not found in native human antibodies. For example, an Fv may include a linker peptide, e.g., two to about eight glycine or other amino acid residues, which connects the heavy chain variable region and the light chain variable region, and such a linker peptide may be of human origin.

[0191] As used herein, a human antibody is "derived" from a particular germline sequence if the antibody is obtained from a system that uses human immunoglobulin sequences, for example, by immunizing a transgenic mouse harboring human immunoglobulin genes or by screening a human immunoglobulin gene library. A human antibody "derived" from a human germline immunoglobulin sequence can itself be identified by comparing the amino acid sequence of the human antibody to the amino acid sequence of a human germline immunoglobulin. Typically, the selected human antibody is at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as a human antibody when compared to the germline immunoglobulin amino acid sequence of another species (e.g., a murine germline sequence). In certain cases, the human antibody may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence displays no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

[0192] As used herein, the term "humanized antibody" or "humanized immunoglobulin" refers to a human / non-human chimeric antibody containing minimal sequence derived from a non-human immunoglobulin. For the most part, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a variable region or fragment thereof (e.g., one, two, three, four, five, or all six CDRs) of the recipient are replaced by residues from a variable region or fragment thereof (e.g., one, two, three, four, five, or all six CDRs) of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity. A humanized antibody may contain residues that are not found in the recipient antibody or in the donor antibody. A humanized antibody may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin; a non-human antibody containing one or more amino acids in the framework region, constant region, or CDR replaced with correspondingly positioned amino acids from a human antibody. Without wishing to be bound by theory, humanized antibodies result in a reduced immune response in a human host compared to a non-humanized version of the same antibody. Humanized antibodies may have conservative amino acid substitutions that have no substantial effect on antigen binding or other antibody functions. Conservative substitution classes include glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serine-threonine, and asparagine-glutamine. Specifically, the humanized antibodies disclosed herein contain, within certain ranges, the following: EC 50 , K. on , K. off , K. A and / or K DIn one or more of the above, the humanized antibody specifically binds to the DNABII polypeptide or a fragment thereof (such as the tip chimeric peptide or the tail chimeric peptide) and inhibits or releases a specific cytokine when treating a subject. In a further embodiment, a humanized antibody that specifically binds to the tip region of the DNABII polypeptide (such as the tip chimeric peptide) destroys biofilms both in vivo and in vitro. In addition, although the humanization process is a rational design process, it may produce unexpected changes (positive or negative) in binding affinity, antigen specificity, or physical properties such as solubility or aggregation, and therefore the properties of the humanized antibody cannot be essentially predicted from the properties of the starting non-human antibody.

[0193] In some embodiments, the antibody used herein may be a recombinant antibody. The term "recombinant human antibody" as used herein includes all antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies isolated from transgenic or transchromosomal animals (e.g., mice) for 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 antibody libraries; and antibodies prepared, expressed, generated, or isolated by any other means involving splicing of immunoglobulin (Ig) gene sequences into other DNA sequences. However, in certain embodiments, such recombinant antibodies may be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, if an animal transgenic for Ig sequences is used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that may not naturally exist in the antibody germline repertoire in vivo. Methods for producing these antibodies are described herein.

[0194] In some embodiments, an antibody as used herein may be a chimeric antibody. As used herein, a chimeric antibody is an antibody whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species.

[0195] As used herein, the term "antibody derivative" includes a full-length antibody or a fragment of an antibody in which one or more of the amino acids have been chemically modified, for example, by alkylation, pegylation, acylation, ester formation, or amide formation, to link the antibody to a second molecule, including, but not limited to, pegylated antibodies, cysteine-pegylated antibodies, and variants thereof.

[0196] 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 rotatory 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 also contemplates a polynucleotide or protein, such as an antibody. The term also encompasses sequences conjugated to a polynucleotide that provide a signal upon expression of the inserted sequence, such as green fluorescent protein (GFP). Labels may be detectable by themselves (e.g., radioisotope labels or fluorescent labels) or, in the case of enzymatic labels, may catalyze a chemical change in a detectable substrate compound or composition. Labels may be suitable for small-scale detection or may be more suitable for high-throughput screening. Suitable labels thus include, but are not limited to, magnetically rotatory isotopes, non-radioactive isotopes, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. Labels may be simply detected, and labels may be quantified. A simply detected response generally includes a response whose presence is simply confirmed, while a quantified response generally includes a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other property. In luminescent or fluorescent assays, the detectable response can be generated directly using a luminophore or fluorophore associated with the assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent labels that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. The detectable luminescent response generally involves a change in, or the generation of, a luminescent signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art, see, for example, Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0197] As used herein, the term "immunoconjugate" includes an antibody or antibody derivative associated with or linked to a second agent, e.g., 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.

[0198] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, 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 (6 th ed.).

[0199] In another embodiment, the fluorescent label is functionalized to facilitate covalent binding to cellular components present in cells or tissues, or on the cell or tissue surface, such as cell surface markers.Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which can be used to bind the fluorescent label to a second molecule.The choice of functional group of the fluorescent label depends on the binding site to either a linker, an agent, a marker, or a second labeling agent.

[0200] "Eukaryotic cells" include all kingdoms of life except the kingdom Monera. They can be easily distinguished by a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes, or organisms in which the cell is organized into a complex structure by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless otherwise recited, the term "host" includes eukaryotic hosts, including, for example, yeast, higher plants, insects, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include primates, bovines, porcines, murines, rats, birds, reptiles, and humans.

[0201] "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.

[0202] A "native" or "natural" antigen is one that has been isolated from a natural biological source and that is capable of binding to an antigen receptor, particularly a T cell antigen receptor (TCR), in a subject. A polypeptide, protein, or fragment containing an epitope capable of specifically binding to a target polypeptide (e.g., a polypeptide-binding protein or fragment containing an epitope) is a polypeptide, protein, or fragment containing an epitope capable of specifically binding to a target polypeptide (e.g., a polypeptide-binding protein or fragment containing an epitope) or ...

[0203] The terms "antigen" and "antigenic" refer to a molecule capable of being recognized by an antibody or otherwise acting as a member of an antibody-ligand pair. "Specific binding" or "binding" refers to the interaction of an antigen with the variable regions of immunoglobulin heavy and light chains. 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 act as antigens. Those skilled in the art will further understand that nucleic acids encoding proteins with the potential to act as antibody ligands necessarily encode antigens. Those skilled in the art will further understand that antigens are not limited to full-length molecules but can also include partial molecules. The term "antigenic" is an adjective that refers to a molecule having the properties of an antigen. This term encompasses substances that are immunogenic, i.e., immunogens, and substances that induce immunological unresponsiveness or anergy, i.e., anergens.

[0204] An "altered antigen" is an antigen having a primary sequence different from that of the corresponding wild-type antigen. Altered antigens can be produced by synthetic or recombinant methods and include, but are not limited to, antigenic peptides that are differentially modified during or after translation, for example, by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, or linkage to antibody molecules, membrane molecules, or other ligands. (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320) The synthetic or altered antigens disclosed herein are intended to bind to the same TCR as the natural epitope.

[0205] An "autoantigen," also referred to herein as a native or wild-type antigen, is an antigenic peptide that elicits little or no immune response in a subject due to self-tolerance to that antigen. An example of an autoantigen is the melanoma-specific antigen gp100.

[0206] "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 inducing an immune response. All immunogens are antigens, however, not all antigens are immunogenic. The immune responses disclosed herein can be humoral (via antibody activity) or cell-mediated (via T cell activation). The response can occur in vivo or in vitro. Those skilled in the art will understand that various macromolecules, including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides, and polysaccharides, have the potential to be immunogenic. Those skilled in the art will further understand that a nucleic acid encoding a molecule capable of inducing 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.

[0207] 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 when maternal antibodies are transferred to a fetus. Passive immunity can also occur artificially when an antibody composition is administered to a non-immune subject. Antibody donors and recipients can be human or non-human subjects. Depending on the embodiment, antibodies can be polyclonal or monoclonal, generated in vitro or in vivo, and purified, partially purified, or unpurified. In some embodiments described herein, passive immunity is provided to a subject in need thereof through the administration of an antibody or antigen-binding fragment that specifically recognizes or binds to a particular antigen. In some embodiments, passive immunity is provided through the administration of an isolated or recombinant polynucleotide encoding an antibody or antigen-binding fragment that specifically recognizes or binds to a particular antigen.

[0208] In the context of the present disclosure, a "ligand" is a polypeptide. In one aspect, the term "ligand" as used herein refers to any molecule that binds to a specific site on another molecule. In other words, the ligand confers specificity to the protein in its reaction with immune effector cells or antibodies to the protein or DNA to the protein. In one aspect, it is the ligand site within the protein that directly combines with the complementary binding site on the immune effector cell.

[0209] As used herein, the terms "solid phase support" or "solid support," which are used interchangeably, are not limited to a particular type of support. Rather, numerous supports are available and known to those skilled in the art. Solid phase supports include silica gel, resins, derivatized plastic films, glass beads, cotton, plastic beads, and alumina gel. As used herein, "solid support" also includes synthetic antigen-presenting matrices, cells, and liposomes. A suitable solid phase support can be selected based on the desired end use and suitability for various protocols. For example, for 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), polystyrene resin grafted with polyethylene glycol (TentaGel®, Rapp Polymere, Tübingen, Germany) or polydimethylacrylamide resin (available from Milligen / Biosearch, Calif.).

[0210] Examples of solid supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. The nature of the support can be either soluble to some extent or insoluble. The support material can have virtually any possible structural configuration, so long as the attached molecule can bind to a polynucleotide, polypeptide, or antibody. Thus, the support configuration can be spherical, as in a bead, or cylindrical, as in the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface can be flat, such as a sheet, test strip, or polystyrene beads. Those skilled in the art will know many other suitable supports for binding antibodies or antigens, or can ascertain such by use of routine experimentation.

[0211] As used herein, a biological sample, or sample, can be obtained from a subject, a cell line, or a cultured cell or tissue. Exemplary samples include, but are not limited to, cell samples, tissue samples, liquid samples such as blood and other liquid samples of biological origin (including, but not limited to, ocular fluid (aqueous humor and vitreous humor), peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menstrual fluid, pus, sebum, vomit, vaginal secretions / washings, synovial fluid, mucosal secretions, stool water, pancreatic juice, nasal lavage fluid, bronchopulmonary aspirate, blastocyst cavity fluid, blastocyst fluid, cy ... In one embodiment, the biological sample is suspected of having a biofilm. In another embodiment, the biological sample comprises a biofilm.

[0212] As used herein, the term "signal peptide" or "signal polypeptide" refers to an amino acid sequence that is usually present at the N-terminus of a newly synthesized secreted or membrane polypeptide or protein. It acts to direct the polypeptide to a specific cellular location, for example, across the cell membrane, into the cell membrane, or into the nucleus. In some embodiments, the signal peptide is removed from the following locations: Examples of signal peptides are well known in the art; non-limiting examples are those described in U.S. Patent Nos. 8,853,381, 5,958,736, and 8,795,965.

[0213] As used herein, a cleavable peptide, also referred to as a cleavable linker, refers to a peptide that can be cleaved, for example, by an enzyme. A single translated polypeptide containing such a cleavable peptide can produce two end products, thus enabling the expression of more than one polypeptide from a single open reading frame. One example of a cleavable peptide is a self-cleaving peptide, such as the 2A self-cleaving peptide. 2A self-cleaving peptides are a class of 18-22 aa-long peptides that can induce cleavage of recombinant proteins in cells. In some embodiments, the 2A self-cleaving peptide is selected from P2A, T2A, E2A, F2A, and BmCPV2A. See, for example, Wang Y, et al. 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori. Sci Rep. 2015;5:16273. Published 2015 Nov 5.

[0214] As used herein, the terms "T2A" and "2A peptide" are used interchangeably to refer to any 2A peptide or fragment thereof, any 2A-like peptide or fragment thereof, or an artificial peptide containing the required amino acids in a relatively short peptide sequence (on the order of 20 amino acids in length, depending on the virus of origin) containing the consensus polypeptide motif DV / IEXNPGP, where X refers to any amino acid that is generally considered to be self-cleaving (SEQ ID NO: 134).

[0215] As used herein, the term "chimera" or "chimeric peptide" refers to a recombinant polypeptide comprising, or alternatively consisting essentially of, or further consisting of, two or more fragments or domains of a DNABII polypeptide that are directly or indirectly (e.g., via a linker) conjugated to each other. In one embodiment, the domain is a conformational tip domain and / or a conformational tail domain. Additionally or alternatively, the two or more fragments or domains are derived from the same or different DNABII polypeptides. In one embodiment, the chimeric peptide comprises, or alternatively consists essentially of, or further consisting of, the tip domain of IhfA and the tip domain of IhfB that are directly or indirectly (e.g., via a linker) conjugated to each other. In another embodiment, the chimeric peptide comprises, or alternatively consists essentially of, or further consisting of, the tail domain of IhfA and the tail domain of IhfB that are directly or indirectly (e.g., via a linker) conjugated to each other. A "conformational tip domain" of a polypeptide refers to a polypeptide that comprises a primary amino acid sequence having an antiparallel beta ribbon structure with a sharp turn, typically mediated by a proline residue. The "tip" of an IHF polypeptide is shown in Figure 1 of WO2018 / 129078.

[0216] As used herein, the phrase "derived from" means isolated from, purified from, or manipulated from, or any combination thereof.

[0217] In certain embodiments, the antibody [ka] (wherein "X" is an optional amino acid linker sequence comprising, consisting essentially of, or alternatively consisting of, 1 to 20 amino acids, as appropriate, and where "X1" is any amino acid, or alternatively, "X1" is selected from the amino acids Q, R, K, S, or T). NTHI In a further aspect, "X1" is K or Q. In a further embodiment, the tip-chimeric peptide IhfA5-mIhfB4 NTHI teeth, [ka] (where "X" is optionally an optional amino acid linker sequence comprising, consisting essentially of, or alternatively consisting of 1-20 amino acids). In yet a further embodiment, the tip-chimeric peptide IhfA5-mIhfB4 NTHI teeth, [ka] The polypeptide sequence of the present invention may comprise, consist essentially of, or even consist of the polypeptide sequence of

[0218] In certain embodiments, the antibody comprises a tail chimeric peptide IhfA3-IhfB2 comprising, consisting essentially of, or alternatively consisting of, a polypeptide sequence of FLEEIRLSLESGQDVKLSGF-X-TLSAKEIENMVKDILEFISQ (SEQ ID NO: 41), where "X" is optionally an amino acid linker sequence comprising, consisting essentially of, or alternatively consisting of, 1-20 amino acids. NTHI In certain embodiments, the linker is selected from any one or more of SEQ ID NOs: 42 to 49. In one embodiment, the tail chimeric peptide IhfA3-IhfB2 NTHI comprises, consists essentially of, or alternatively further consists of FLEEIRLSLESGQDVKLSGFGPSLTLSAKEIENMVKDILEFISQ (SEQ ID NO: 50).

[0219] As used herein, "EC 50 " refers to the concentration of an antibody or antigen-binding fragment thereof that induces a response (e.g., binding between an antibody or antigen-binding fragment thereof and its target) that is intermediate between baseline and maximum after a specified exposure time.

[0220] Several parameters are used herein to describe the binding and non-binding reactions of receptor (R, e.g., an antibody or antigen-binding fragment thereof) and ligand (L, e.g., the target of the antibody or antigen-binding fragment thereof) molecules, and are formalized as R + L ⇔ RL. This reaction is expressed as M -1 s -1 and s -1 The on-rate constant k has units of on and the off-rate constant k off At equilibrium, the forward bonding transition R+L → RL must be balanced by the backward non-bonding transition RL → R+L. That is, k on [R][L]=k off[RL], where [R], [L], and [RL] represent the concentration of unbound free receptor, the concentration of unbound free ligand, and the concentration of the receptor-ligand complex. Furthermore, the equilibrium dissociation constant, K D " is [R] × [L] / [RL] k off / k on whereas the equilibrium binding constant "K A " is [RL] / ([R] × [L]) k on / k off It can be calculated as:

[0221] As used herein, the term "cytokine" refers, without limitation, to small proteins (approximately 5-20 kDa) important in cell signaling, including, but not limited to, chemokines, interferons, interleukins (ILs), lymphokines, and tumor necrosis factors, generally non-hormonal. Cytokines are peptides that cannot cross the lipid bilayer of cells and enter the cytoplasm. Inflammatory or proinflammatory cytokines are a type of signaling molecule (cytokine) secreted by immune cells such as helper T cells (Th) and macrophages, as well as certain other cell types, that promote inflammation. These include (but are not limited to) interleukin-1 (IL-1), IL-12, and IL-18, tumor necrosis factor alpha (TNF-α), interferon gamma (IFNγ), and granulocyte-macrophage colony-stimulating factor (GM-CSF), and play an important role in mediating the innate immune response. Proinflammatory cytokines are preferentially produced by and are involved in the upregulation of inflammatory responses. The term "anti-inflammatory cytokine" includes immunoregulatory 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 pro-inflammatory cytokine inhibitors. Methods for measuring cytokine levels, including anti-inflammatory and pro-inflammatory cytokines, are well known in the art. For example, serum cytokine levels can be measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits.

[0222] As used herein, the terms "pro-inflammatory response" and "inflammatory response" are used interchangeably to refer to a subject's biological response to a pathogen, e.g., a bacterium, virus, or other microorganism that can cause disease. In some embodiments, a pro-inflammatory response or inflammatory response refers to an immune response involving both specific and non-specific defense systems. A specific defense system response is a specific immune system response to an antigen. Examples of specific defense system responses include antibody responses. A non-specific defense system response is an inflammatory response mediated by leukocytes, e.g., macrophages, eosinophils, and neutrophils, which are generally incapable of immunological memory. In some embodiments, the immune response comprises, consists essentially of, or even further consists of the secretion of pro-inflammatory cytokines, resulting in elevated pro-inflammatory cytokine levels. Additionally or alternatively, the immune response comprises, consists essentially of, or even further consists of a reduction in the level of anti-inflammatory cytokines. Thus, a pro-inflammatory response can comprise, consist essentially of, or even further consist of the secretion of pro-inflammatory cytokines, or the reduction of anti-inflammatory cytokines, or both.

[0223] As used herein, the term "anti-infective agent" refers to an agent capable of inhibiting the spread of or completely killing infectious organisms. This term includes, but is not limited to, antibiotics, antifungals, anthelmintics, antimalarials, antiprotozoal agents, antituberculosis agents, and antivirals. Antifungal agents are also called antimycotic agents. They kill or inactivate fungi and are used to treat fungal infections (including yeast infections). One non-limiting example, polyene antifungals, are used to treat fungal infections of the gastrointestinal tract, such as thrush, because they are not absorbed when administered orally. Other non-limiting examples include azole antifungals, which are synthetic fungistatic agents with broad-spectrum activity; echinocandins, which are lipopeptide molecules that non-competitively inhibit the (1,3) beta-d-glucan synthase enzyme and target fungal cell walls; Fulvicin U / F (i.e., griseofulvin); Grifulvin V(Pro) (i.e., griseofulvin); Lamisil(Pro) (i.e., terbinafine); Gris-PEG(Pro) (i.e., griseofulvin); Ancobon(Pro) (i.e., flucytosine); Fulvicin P / G (i.e., griseofulvin); and Terbinex(Pro) (i.e., terbinafine). Additional anti-infective agents can be found, for example, at www.drugbank.ca / categories / DBCAT000065. The terms "anti-viral" or "antiviral" refer to a class of drugs used to treat viral infections. Most antiviral drugs target specific viruses, while broad-spectrum antivirals are effective against a wide range of viruses. Unlike most antibiotics, antiviral drugs do not destroy their target pathogens, but instead inhibit their development.Some of the ways in which they can act include inhibiting viral DNA polymerase; binding to specific cell surface receptors to inhibit viral penetration or uncoating; inhibiting viral protein synthesis; or preventing viral replication by blocking late stages of viral assembly. Non-limiting examples of antiviral agents can be found, for example, at www.drugbank.ca / categories / DBCAT000066.

[0224] As used herein, the term "antiparasitic drug" refers to a class of drugs indicated for the treatment of parasitic diseases, such as diseases caused by helminths, amoebae, ectoparasites, parasitic fungi, and protozoa, among others. Non-limiting examples of antiparasitic drugs can be found, for example, at www.drugbank.ca / categories / DBCAT000522. Modes for Carrying Out This Disclosure

[0225] Although the component molecules of extracellular polymeric substances (EPS) vary among bacterial species, extracellular DNA (eDNA) serves as a common underlying structural component across diverse bacterial biofilms (Flemming et al., Nat Rev Microbiol, 2010. 8(9): pp. 623-33). The eDNA structure has been further characterized, and it has been determined that the eDNA lattice is composed of Holliday junction (HJ)-like structures that are essential for the stability of eDNA-dependent bacterial biofilm EPS (Devaraj et al., Proc Natl Acad Sci USA, 2019 Dec 10;116(50):25068-25077). DNA-binding proteins of the bacterial DNABII family, including integration host factors (IHFs) and histone-like proteins (HUs), bind to these HJ-like structures within the eDNA lattice and act as linchpins maintaining the structural integrity of the eDNA-dependent EPS (Devaraj et al., 2019). al.). Sequestration of free DNABII protein (via exposure to a specific antibody against the DNA-binding domain of DNABII protein (α-DNABII)) shifts the equilibrium from the DNA-bound to the unbound state, which in turn causes the breakdown of the bacterial biofilm (Devaraj et al.; Goodman et al., Mucosal Immunol, 2011. 4(6): p. 625-37; Gustave et al., J Cyst Fibros, 2013. 12(4): p. 384-9; Novotny et al., PLoS One, 2013. 8(6): p. e67629; Brockson et al., Mol Microbiol, 2014. 93(6): p. 1246-58; Brandstetter et al., The Laryngoscope, 2013. 123(11): p. 2626-2632;Rocco et al., Mol Oral Microbiol, 2016;Novotny et al., EBioMedicine, 2016. 10: p. 33-44;Devaraj et al. al., Microbiologyopen, 2017;Freire et al., Mol Oral Microbiol, 2017. 32(1): p. 74-88; and Novotny et al., NPJ Vaccines, 2019. 4: p. 43.).

[0226] DNABII proteins are absent in vertebrates, but eukaryotes possess a partially functional orthologue, HMGB1, that binds to similar curved DNA structures, e.g., HJ DNA (Bianchi et al., Science, 1989. 243(4894 Pt 1): p. 1056-9). HMGB1 is a ubiquitous protein in eukaryotes and is a native part of chromatin (Bianchi and Beltrame, Am J Hum Genet, 1998. 63(6): p. 1573-7; and Agresti et al., Mol Cell, 2005. 18(1): pp. 109-21). They function as monomers, consist of two tandem DNA-binding domains and an acidic C-terminal tail (Bianchi et al., EMBO J, 1992. 11(3): pp. 1055-63), and often have post-translational modifications (PTMs) that define their location (nuclear, cytoplasmic, or extracellular) and activity (reviewed in Kang et al., Mol Aspects Med, 2014. 40: pp. 1-116). HMGB1 acts as an accessory protein in multiple DNA-protein transactions, including recombination, DNA repair, and transcription, through its ability to bind and bend DNA in a sequence-independent manner (Little et al., Nucleic Acids Res, 2013. 41(5): pp. 3289-301; Sutrias-Grau et al., J Biol Chem, 1999. 274(3): pp. 1628-34; and Yuan et al., J Biol Chem, 2004. 279(20): pp. 20935-40). Upon its release from eukaryotic cells into the extracellular environment, HMGB1 also functions as a damage-associated molecular pattern (DAMP) molecule that induces a pro-inflammatory cascade via the NF-κB pathway by binding to TLR2, TLR4, TLR9, and RAGE (Klune et al., Mol Med, 2008. 14(7-8): p. 476-84; Park et al., Am J Physiol Cell Physiol, 2003. 284(4): p. C870-9; Silva et al., Intensive Care Med, 2007. 33(10): p. 1829-39; and He et al., Asian Pac J Cancer Prev, 2012. 13(4): p. 1365-70), thus acting as an alarmin with the potential to cause sepsis with devastating consequences for the host (Qin et al., J Exp Med, 2006. 203(7): p. 1637-42; and Diener et al., Immunol Cell Biol, 2013. 91(7): p. Extracellularly, HMGB1 also has a wide range of functions, including tissue regeneration and wound healing, aging, and, at very high concentrations (1.75 μM to 12 μM), bacterial killing (Ranzato et al., Mol Cell Biochem, 2009. 332(1-2): pp. 199-205; Gong et al., J Biomed Sci, 2009. 16: pp. 83; Davalos et al., J Cell Biol, 2013. 201(4): pp. 613-29; and Zetterstrom et al., Pediatr Res, 2002. 52(2): pp. 148-54). Perhaps most importantly, extracellular HMGB1 is an integral part of the eDNA of neutrophil extracellular traps (NETs), the host's first resort for sequestering bacteria for further clearance (Tadie et al., Am J Physiol Lung Cell Mol Physiol, 2013. 304(5): p. L342-9; Remijsen, et al., Cell Death Differ, 2011. 18(4): p. 581-8; Brinkmann et al., Science, 2004. 303(5663): p. 1532-5; and Peng et al., Sci Rep, 2017. 7(1): p. 16628), and, as proposed herein, can also serve as a strategy for preventing bacterial biofilm growth.

[0227] The eukaryotic host innate immune effector HMGB1 and bacterial DNABII proteins play similar roles as accessory proteins in nucleoprotein transduction in the host and bacteria, respectively (reviewed in Kang et al. and Browning et al., Curr Opin Microbiol, 2010. 13(6): pp. 773-80). Although HMGB1 and DNABII proteins share no discernible sequence identity or secondary structure, they can nevertheless functionally substitute for each other in in vitro transduction. Thus, HMGB1 was originally considered a functional ortholog of DNABII proteins (Paull et al., Genes Dev, 1993. 7(8): pp. 1521-34; and Segall et al., EMBO J, 1994. 13(19): pp. 4536-48). Both proteins bind and bend DNA, but they do so via distinct mechanisms. Both HMGB1 (as a monomer) and DNABII proteins (as dimers) bind to DNA through their minor grooves, but HMGB1 stabilizes DNA bending from its convex surface, and DNABII proteins stabilize DNA bending from its concave surface (Sanchez-Giraldo et al., Acta Crystallogr D Biol Crystallogr, 2015. 71(Pt 7): p. 1423-32; and Rice et al., Cell, 1996. 87(7): p. Given their remarkable functional similarities within cells, and because HMGB1 and DNABII proteins are also found extracellularly, the interaction of HMGB1 within the eDNA-dependent EPS of bacterial biofilms is further disclosed herein.

[0228] A previously unknown extracellular function of vertebrate high-mobility group box 1 protein (HMGB1) in bacterial biofilm growth is described and exemplified herein. Within host cells, HMGB1 functions as a DNA architectural protein similar to the ubiquitous DNABII family of bacterial proteins, despite the lack of amino acid sequence identity between these proteins. Extracellularly, HMGB1 induces a pro-inflammatory immune response, while DNABII proteins stabilize the extracellular DNA-dependent matrix that maintains bacterial biofilms. Without wishing to be bound by theory, when both proteins target eDNA within bacterial biofilms, HMGB1, unlike DNABII proteins, disrupted biofilms both in vitro (including high-priority ESKAPEE pathogens) and in vivo in two separate animal models, despite inducing a strong inflammatory response (which was attenuated by a single engineered amino acid change). A model is proposed herein in which extracellular HMGB1 balances the degree of induced inflammation with biofilm containment without excessive release of biofilm-resident bacteria.

[0229] While interfering with the binding between DNABII protein and extracellular DNA (for example, by administering anti-DNABII antibody) and providing HMGB1 polypeptide, a surprising beneficial effect, namely, complete biofilm eradication, has been demonstrated and observed. See Example 1 and Figures 16I and 16J. It has further been noted that repeated administration of HMGB1 polypeptide alone or anti-DNABII antibody alone does not reach the biofilm reduction level achieved by a single administration of the combination of HMGB1 polypeptide and anti-DNABII antibody, suggesting a more-than-additive (i.e., synergistic) effect. Such observation is consistent with the mechanism proposed herein. Without being bound by theory, such synergistic effects are predicted for the combination of other HMGB polypeptides and anti-DNA antibodies, for example, antibodies or fragments thereof that bind to tip polypeptides or tip chimer polypeptides. ***

[0230] In one aspect, a composition or combination is provided that comprises, alternatively consists essentially of, or further consists of: (a) a high mobility group box 1 protein (HMGB1) polypeptide, or a fragment thereof comprising, or consisting essentially of, or alternatively consisting of, the B box, A box, or AB box thereof; and (b) an anti-DNABII antibody, or an antigen-binding fragment thereof, disclosed herein.

[0231] In another aspect, the composition or combination comprises, consists essentially of, or even further consists of (a) an HMGB1 polypeptide further comprising a C45S mutation; and (b) an anti-DNABII antibody or antigen-binding fragment thereof disclosed herein.

[0232] In one aspect, there is provided a composition or combination comprising, consisting essentially of, or further consisting of (a) a high mobility group box 1 protein (HMGB1) polypeptide, or a fragment thereof; and (b) an antibody or antigen-binding fragment thereof that specifically recognizes and binds to the tip domain of a DNABII protein, with the proviso that (i) the composition or combination does not comprise one or more of SEQ ID NOs: 51-58, for example SEQ ID NO: 52, or (ii) the antigen-binding fragment does not comprise a Fab, optionally a Fab of a polyclonal antibody, or the antibody does not comprise a polyclonal antibody, or both (i) and (ii).

[0233] In one aspect, a polypeptide is provided that comprises, consists essentially of, or even consists of: (a) a high mobility group box 1 protein (HMGB1) polypeptide, or a fragment thereof; and (b) an anti-DNABII antibody, or an antigen-binding fragment thereof.

[0234] In one aspect, a polypeptide is provided comprising, consisting essentially of, or alternatively consisting of: (a) a high mobility group box 1 protein (HMGB1) polypeptide, or a fragment thereof; and (b) an antibody or antigen-binding fragment thereof that specifically recognizes and binds to the apical domain of a DNABII protein, with the proviso that (i) the polypeptide does not comprise one or more of SEQ ID NOs: 51-58, e.g., SEQ ID NO: 52, or (ii) the antigen-binding fragment does not comprise a Fab, optionally a Fab of a polyclonal antibody, or the antibody does not comprise a polyclonal antibody, or both (i) and (ii).

[0235] In some embodiments, the polypeptide further comprises a cleavable peptide located between (a) and (b).

[0236] In a further aspect, there is provided a polynucleotide encoding a polypeptide disclosed herein, or a polynucleotide complementary thereto.

[0237] In one aspect, there is provided a polynucleotide encoding, or complementary to, (a) a high mobility group box 1 protein (HMGB1) polypeptide, or a fragment thereof; and (b) an anti-DNABII antibody, or an antigen-binding fragment thereof.

[0238] In one aspect, there is provided a polynucleotide encoding (a) a high mobility group box 1 protein (HMGB1) polypeptide, or a fragment thereof; and (b) an antibody or antigen-binding fragment thereof that specifically recognizes and binds to the apical domain of a DNABII protein, or a polynucleotide complementary thereto, with the proviso that (i) the polynucleotide does not encode one or more of SEQ ID NOs: 51-58, for example, SEQ ID NO: 52, or (ii) the antigen-binding fragment does not include a Fab, optionally a Fab of a polyclonal antibody, or the antibody does not include a polyclonal antibody, or both (i) and (ii).

[0239] In some embodiments, (a) and (b) are encoded by a single contiguous polynucleotide, e.g., under the direction of the same regulatory sequence(s). In further embodiments, (a) and (b) are encoded by a single contiguous polynucleotide, under the direction of different regulatory sequences, i.e., the polynucleotide is bicistronic. In other embodiments, (a) and (b) are encoded by two polynucleotides.

[0240] In one aspect, a vector is provided that comprises, consists essentially of, or even consists of a polynucleotide disclosed herein.

[0241] In one aspect, a host cell is provided that comprises one or more of the compositions or combinations disclosed herein, the polypeptides disclosed herein, the polynucleotides disclosed herein, or the vectors disclosed herein.In some embodiments, the host cell secretes an HMGB1 polypeptide or a fragment thereof; and an anti-DNABII antibody or an antigen-binding fragment thereof.In some embodiments, the host cell is used to produce the compositions or combinations disclosed herein, the polypeptides disclosed herein, the polynucleotides disclosed herein, or the vectors disclosed herein, for example, by culturing the host cell and collecting the compositions or combinations, polypeptides, polynucleotides, or vectors.

[0242] In one aspect, a method for producing a composition or combination disclosed herein is provided. The method comprises, or consists essentially of, or further consists of culturing a host cell containing a polynucleotide disclosed herein and isolating an HMBG1 polypeptide or a fragment thereof and an anti-DNABII antibody or an antigen-binding fragment thereof from the cell culture. In another aspect, a method for producing a polypeptide disclosed herein is provided. The method comprises, or consists essentially of, or further consists of culturing a host cell containing a polynucleotide disclosed herein and isolating the polypeptide from the cell culture. In some embodiments, the method further comprises introducing the polynucleotide or a vector containing the polynucleotide into a host cell.

[0243] In one aspect, a method is provided for one or more of: (A) preventing biofilm formation or disrupting biofilms in vitro or ex vivo, (B) preventing biofilm formation or disrupting biofilms in a subject, (C) inhibiting, preventing, or treating a biofilm-producing microbial infection in a subject, or (D) treating a condition characterized by biofilm formation in a subject. The method comprises administering to a subject: (a) a high mobility group box 1 protein (HMGB1) polypeptide, or a fragment thereof comprising, consisting essentially of, or alternatively consisting of, the B box, A box, or AB box thereof; and (b) an anti-DNABII antibody or antigen-binding fragment thereof disclosed herein; The method may comprise, alternatively consist essentially of, or alternatively further consist of, administering

[0244] In one aspect, a method is provided for inducing or increasing the formation of neutrophil extracellular traps (NETs) immediately adjacent to a biofilm in a subject, and for disrupting the biofilm, optionally without inducing a pro-inflammatory response. The method comprises administering to a subject: (a) a high mobility group box 1 protein (HMGB1) polypeptide comprising any one or more amino acid sequences of SEQ ID NOs: 51-58, or a fragment thereof comprising, consisting essentially of, or alternatively consisting of, the B box, A box, or AB box thereof; and (b) an anti-DNABII antibody or antigen-binding fragment thereof disclosed herein; The method may comprise, alternatively consist essentially of, or alternatively further consist of, administering

[0245] In one aspect, a method is provided for one or more of: (A) preventing biofilm formation or disrupting biofilms in vitro or ex vivo, (B) preventing biofilm formation or disrupting biofilms in a subject, (C) inhibiting, preventing, or treating a biofilm-producing microbial infection in a subject, or (D) treating a condition characterized by biofilm formation in a subject, the method comprising, consisting essentially of, or even further consisting of administering to the subject one or more of a composition or combination disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, or a host cell disclosed herein.

[0246] In one aspect, a method is provided for inducing or increasing the formation of neutrophil extracellular traps (NETs) immediately adjacent to a biofilm in a subject, and for disrupting the biofilm, optionally without inducing a pro-inflammatory response, comprising, consisting essentially of, or further consisting of, administering to the subject one or more of a composition or combination disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, or a host cell disclosed herein, wherein the HMGB1 polypeptide comprises, consists essentially of, or further consists of any one or more of SEQ ID NOs: 51-58.

[0247] In some embodiments, the method further includes administering to the subject one or more of a DNase enzyme, an antibiotic, an antimicrobial, an anti-infective, an anti-fungal, an anti-parasitic, an anti-viral, or an antibody or antigen-binding fragment thereof that specifically recognizes and binds to OMP P5, rsPilA, OMP 26, OMP P2, or type IV Pilin.

[0248] In some embodiments, the condition characterized by biofilm formation comprises, consists essentially of, or even consists of one or more of chronic non-healing wounds, Burkholderia infections, lung infections caused by Burkholderia sp., venous ulcers, diabetic foot ulcers, ear infections, sinus infections, urinary tract infections, gastrointestinal tract diseases, hospital-acquired pneumonia, ventilator-associated pneumonia, surgical implant-associated infections, lung infections, airway infections, cystic fibrosis, chronic obstructive pulmonary disease, catheter-associated infections, indwelling device-associated infections, infections associated with implanted prostheses, osteomyelitis, cellulitis, abscesses, or periodontal disease.

[0249] In some embodiments, the administration of (a) and the administration of (b) occur simultaneously or sequentially. Additionally or alternatively, the administration of (a) and (b) is repeated at least once, at least twice, at least three times, or more times.

[0250] In some embodiments of any method(s) disclosed herein, optionally including an administration step, the antibody or its antigen-binding fragment reduces one or more pro-inflammatory cytokines and increases one or more anti-inflammatory cytokines in a subject.With respect to the methods disclosed herein, applicant has unexpectedly discovered that the HMGB1 fragments disclosed herein are not post-translationally modified, but still retain DNA binding activity.Without being bound by theory, applicant believes that modified HMGB1 fragments function similarly.

[0251] In some aspects of any aspect(s), any embodiment(s) and / or method(s) disclosed herein, the HMGB1 polypeptide or fragment thereof disclosed herein and the anti-DNABII antibody or antigen-binding fragment thereof disclosed herein are combined in or contained within the same composition (e.g., in the same pharmaceutical composition optionally further comprising a pharmaceutically acceptable carrier, and / or by contacting or administering simultaneously as one composition in a method disclosed herein).

[0252] In another aspect of any aspect(s), any embodiment(s) and / or method(s) disclosed herein, the HMGB1 polypeptide or fragment thereof disclosed herein is provided as a first composition (herein referred to as the HMGB1 composition, which may optionally further comprise a pharmaceutically acceptable carrier), and the anti-DNABII antibody or antigen-binding fragment thereof is provided as a second or separate individual composition (herein referred to as the anti-DNABII antibody composition, which may be a separate pharmaceutical composition). In a further aspect, the mixture of the HMGB1 composition and the anti-DNABII antibody composition comprises, consists essentially of, or consists of both the HMGB1 polypeptide or fragment thereof and the anti-DNABII antibody or antigen-binding fragment thereof disclosed herein used in the methods disclosed herein. In one aspect, the HMGB1 composition is contacted or administered separately from the anti-DNABII antibody composition (i.e., in the absence of the other) in the methods disclosed herein. Additionally or alternatively, the HMGB1 composition is contacted or administered together with the anti-DNABII antibody composition (e.g., within 0.5 hours) in the methods disclosed herein. In yet another embodiment, the HMGB1 composition is contacted or administered sequentially with the anti-DNABII antibody composition in the methods disclosed herein, with or without a gap between them. Such a gap can be from a few minutes to a few weeks, as needed, selected from, but not limited to, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1.5 weeks, 2 weeks, or longer. Additionally or alternatively, the HMGB1 composition and the anti-DNABII antibody composition can be administered in the methods disclosed herein by the same route of administration, e.g., intravenously, or by different routes of administration. In a further aspect, the composition is suitable for the intended route(s) of administration, e.g., has a suitable pH.Without wishing to be bound by theory, in one embodiment, such composition(s), pharmaceutical composition(s), and / or pharmaceutically acceptable carrier(s) stabilize the HMGB1 polypeptide or fragment thereof and / or anti-DNABII antibody or antigen-binding fragment thereof and / or prevent degradation of the HMGB1 polypeptide or fragment thereof and / or anti-DNABII antibody or antigen-binding fragment thereof.

[0253] In some embodiments of the methods disclosed herein, the method further comprises contacting a sample suspected of containing a biofilm with an antibody or an antigen-binding fragment thereof that binds to a DNABII polypeptide, and detecting the biofilm by detecting the binding of the antibody or its fragment to the biofilm.In one embodiment, the detection antibody or its antigen-binding fragment binds to the tip region of the DNABII polypeptide.In another embodiment, the detection antibody or its antigen-binding fragment binds to the tail region of the DNABII polypeptide.

[0254] Also provided is a method for screening a subject for use of the compositions or methods described herein. This screening method comprises, or alternatively consists essentially of, or alternatively consists of, contacting an anti-DNABII antibody, anti-DNABII polypeptide, or antigen-binding fragment thereof disclosed herein with a biological sample isolated from a subject, the biological sample containing a biofilm, and detecting binding of the antibody or antigen-binding fragment thereof to any biofilm in the sample. In one embodiment, the antigen-binding fragment of the antibody is selected from the group of Fab, F(ab')2, Fab', scFv, or Fv. Additionally or alternatively, the antibody or antigen-binding fragment thereof specifically binds to the tip region of the DNABII peptide. In one embodiment, the DNABII peptide is an IHF peptide. In one embodiment, the screening antibody or antigen-binding fragment binds to the tip region of the DNABII polypeptide. In another embodiment, the screening antibody or antigen-binding fragment binds to the tail region of the DNABII polypeptide.

[0255] In one aspect, kits for use in the methods disclosed herein are provided, which comprise, consist essentially of, or even consist of instructions for use and one or more of a composition or combination disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein (e.g., a polynucleotide disclosed herein), a vector disclosed herein, or a host cell disclosed herein.

[0256] HMGB composition In some embodiments, the HMGB is human HMGB1 or mouse HMGB1. In some embodiments, an HMGB polypeptide, as used herein, comprises, consists essentially of, or further consists of an HMG box domain (e.g., HMGB1, HMGB2, HMGB3, or HMGB4) or a mutated modified high mobility group box domain (e.g., mHMGB1, mHMGB2, mHMGB3, or mHMGB4) disclosed herein. In some embodiments, an HMGB polypeptide comprises, consists essentially of, or further consists of any one or more of SEQ ID NOs: 51-58, 68-74, 84-90, or 100-114.

[0257] In some embodiments, HMGB1 is replaced by HMGB2, or HMGB3, or HMGB4, or another equivalent disclosed herein.

[0258] In some embodiments, the HMGB1 polypeptide further comprises one or more mutations selected from the group consisting of mutations at K12, C23, C45, C106, or K114. In some embodiments, the A box of the HMGB1 polypeptide further comprises one or more mutations selected from the group consisting of mutations at K12, C23, or C45. In some embodiments, the B box of the HMGB1 polypeptide further comprises one or both mutations at C106 or K114. In some embodiments, the one or more mutations are mutations to serine, glycine, alanine, valine, isoleucine, or threonine. In some embodiments, the HMGB1 polypeptide further comprises one or more mutations selected from C23S, C45S, and C106S. In some embodiments, the HMGB1 polypeptide further comprises a C45S mutation. In a further embodiment, the HMGB polypeptide is a modified version of HMGB2, HMGB3, or HMGB4, or a modified version having a mutation to serine, glycine, alanine, valine, isoleucine, or threonine at positions corresponding to modified HMGB1 at K12, C23, or C45. Additionally or alternatively, mutant HMGBs (mHMGBs) contain one or more mutations at positions corresponding to C23S, C45S, and C106S. In a further aspect, the polypeptide further contains a mutation at the corresponding C45S.

[0259] In some embodiments, one or more of the HMGB1 mutations are selected from the group of mutations at K12, C23, C45, C106, or K114. In a further aspect, one or more of the HMGB1 mutations are selected from the group of mutations at K12, C23, C45, C106, or K114 to serine, glycine, alanine, valine, isoleucine, or threonine. Additionally or alternatively, the mutant HMGB1 (mHMGB1) comprises one or more mutations selected from C23S, C45S, and C106S. In a further embodiment, the HMGB polypeptide is HMGB2, HMGB3, or HMGB4, or a modified version having a mutation at K12, C23, C45, C106, or K114 corresponding to the modified HMGB1 to serine, glycine, alanine, valine, isoleucine, or threonine. Additionally or alternatively, mutant HMGB (mHMGB) contains one or more mutations at positions corresponding to C23S, C45S, and C106S.

[0260] In some embodiments, a fragment of an HMGB1 polypeptide, e.g., an HMGB1 polypeptide, comprises, consists essentially of, or even consists of the B box, A box, or AB box thereof. In some embodiments, a fragment of an HMGB1 polypeptide or an HMGB1 polypeptide comprises a B box polypeptide disclosed herein, an A box polypeptide disclosed herein, or an A box polypeptide disclosed herein. It comprises, consists essentially of, or alternatively further consists of a B box polypeptide.

[0261] antibody composition In some embodiments, an antibody or antigen-binding fragment thereof, as used herein, comprises, consists essentially of, or even consists of 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 the DNABII protein. In certain embodiments, the antibody or antigen-binding fragment thereof binds to a DNABII peptide (e.g., the tip region or tip chimer of IHF or HU, the tip region of IHFA or IHFB, and / or the tip-chimeric peptide IhfA5-mIhfB4). NTHI and / or the tail region of IHF or HU, the tail region of IHFA or IHFB, and / or the tail-chimeric peptide IhfA3-IhfB2. NTHI In one embodiment, the antibody or antigen-binding fragment thereof binds to the tip-chimeric peptide IhfA5-mIhfB4 NTHI In another embodiment, the antibody or antigen-binding fragment thereof binds to the tail-chimeric peptide IhfA3-IhfB2 NTHI Combine with.

[0262] In some embodiments, the antibody or antigen-binding fragment thereof binds to the tip region (i.e., tip domain) or tip chimer of the DNABII peptide. In one aspect, the DNABII peptide is an IHF peptide. In some embodiments, the tip domain is NFELRDKSSRPGRNPKTGDVV (SEQ ID NO: 31); SLHHRQPRLGRNPKTGDSVNL (SEQ ID NO: 32); [ka] (wherein "X" is an optional amino acid linker sequence and "X1" is any amino acid) (SEQ ID NO: 38); [ka] (wherein "X1" is any amino acid) (SEQ ID NO: 39); or [ka] The amino acid sequence of the present invention may comprise, consist essentially of, or alternatively consist of, one or more amino acid sequences selected from:

[0263] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain complementarity determining region 1 (CDRH1) comprising, consisting essentially of, or consisting of GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 1, 2, 3, or 24); (ii) a heavy chain complementarity determining region 2 (CDRH2) comprising, consisting essentially of, or consisting of GSDRRH (aa 76 to aa 81 of SEQ ID NO: 1, 2, 3, or 24); (iii) a heavy chain complementarity determining region 3 (CDRH3) comprising, consisting essentially of, or consisting of VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 1, 2, 3, or 24); (iv) (v) light chain complementarity determining region 2 (CDRL2) comprising, consisting essentially of, or consisting of LVS (aa75 to aa77 of SEQ ID NO:7, or 8, or 9, or 25); and (vi) light chain complementarity determining region 3 (CDRL3) comprising, consisting essentially of, or consisting of WQGTHFPYT (aa114 to aa122 of SEQ ID NO:7, or 8, or 9, or 25).

[0264] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain. In further embodiments, the HC immunoglobulin variable domain comprises, consists essentially of, or alternatively consists of amino acids 25-144 of any one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a light chain (LC) immunoglobulin variable domain. In further embodiments, the LC immunoglobulin variable domain comprises, consists essentially of, or alternatively consists of amino acids 21-132 of any one of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain and a LC immunoglobulin variable domain. In further embodiments, the HC immunoglobulin variable domain comprises, consists essentially of, or even consists of amino acids 25-144 of any one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In still further embodiments, the LC immunoglobulin variable domain comprises, consists essentially of, or even consists of amino acids 21-132 of any one of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, the HC immunoglobulin variable domain comprises, consists essentially of, or even consists of amino acids 25-144 of any one of SEQ ID NO:1, and the LC immunoglobulin variable domain comprises, consists essentially of, or even consists of amino acids 21-132 of SEQ ID NO:7. In some embodiments, the HC immunoglobulin variable domain comprises, consists essentially of, or even consists of amino acids 25-144 of any one of SEQ ID NO:1, and the LC immunoglobulin variable domain comprises, consists essentially of, or even consists of amino acids 21-132 of SEQ ID NO:8.In some embodiments, the HC immunoglobulin variable domain comprises, consists essentially of, or even further consists of amino acids 25-144 of any one of SEQ ID NO: 1, and the LC immunoglobulin variable domain comprises, consists essentially of, or even further consists of amino acids 21-132 of SEQ ID NO: 9.

[0265] In some embodiments, the antibody comprises a constant region optionally selected from an IgA constant region, an IgD constant region, an IgE constant region, an IgG constant region, or an IgM constant region.

[0266] In some embodiments, the antigen-binding fragment comprises a Fab, F(ab')2, Fab', scFv, or Fv.

[0267] In some embodiments, the antibody or antigen-binding fragment thereof is modified. In further embodiments, the antibody or antigen-binding fragment thereof is modified by a process selected from pegylation, polysialylation, HESylation, or glycosylation.

[0268] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, or an antigen-binding fragment of a monoclonal antibody.

[0269] In some embodiments, the antibody or antigen-binding fragment thereof comprises a humanized or human framework.

[0270] In some embodiments, the antibody or antigen-binding fragment is derived from a mammal. In further embodiments, the antibody or antigen-binding fragment is derived from a non-human mammal, such as a mouse, rat, pig, cow, rabbit, goat, chicken, horse, dog, cat, or llama. In other embodiments, the antibody or antigen-binding fragment is derived from a human. In yet other embodiments, the antibody or antigen-binding fragment is humanized. Non-limiting examples of antibodies or antigen-binding fragments can be found in U.S. Patent Nos. 8,999,291, 9,745,366, 10,940,204; U.S. Patent Application Publication Nos. 2016-0175440, 2018-0303900, 2019-0338018, 2019-0337996, 2020-0190170, and 2021-0139551; and PCT Publication No. WO2021 / 007260.

[0271] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of a sequence selected from the group of amino acids (aa) 25 to aa 144 of SEQ ID NO: 1-6, 13, 24 or 26, or an equivalent of each thereof; and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of a sequence selected from the group of aa 21 to aa 132 of SEQ ID NO: 7-9, 14 or 25, aa 21 to aa 126 of SEQ ID NO: 10-12 or 27, or an equivalent of each thereof.

[0272] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain (HC) that comprises, consists essentially of, or consists of a sequence selected from the group of aa25 to aa473 of SEQ ID NO: 1-6, 13, 24 or 26, or an equivalent of each thereof; and / or a light chain (LC) that comprises, consists essentially of, or consists of a sequence selected from the group of aa21 to aa239 of SEQ ID NO: 7-9, 14 or 25, aa21 to aa233 of SEQ ID NO: 10-12 or 27, or an equivalent of each thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, or alternatively consists essentially of, or alternatively consists of a heavy chain (HC) comprising, or alternatively consisting essentially of, or alternatively consisting of a sequence selected from the group of amino acids (aa) 25 to aa 473 of SEQ ID NO: 13, 24 or 26, or an equivalent of each thereof; and / or a light chain (LC) comprising, or alternatively consisting essentially of, or alternatively consisting of a sequence selected from the group of aa 21 to aa 239 of SEQ ID NO: 14 or 25, aa 21 to aa 233 of SEQ ID NO: 27, or an equivalent of each thereof.

[0273] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consisting essentially of, or consisting of a sequence selected from the group of SEQ ID NOs: 1-6, 13, 24, or 26, or an equivalent of each thereof; and / or a light chain (LC) comprising, consisting essentially of, or consisting of a sequence selected from the group of SEQ ID NOs: 7-12, 14, 25, or 27, or an equivalent of each thereof.

[0274] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of, any one or any two or all three CDRs of a sequence selected from the group of SEQ ID NOs: 1-6, 13, 24 or 26, or their respective equivalents; and / or any one or any two or all three CDRs of a sequence selected from the group of SEQ ID NOs: 7-12, 14, 25 or 27, or their respective equivalents.

[0275] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or also consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of a sequence selected from the group of aa25 to aa144 of SEQ ID NO: 13, 24 or 26, or an equivalent thereof; and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of a sequence selected from the group of aa21 to aa132 of SEQ ID NO: 14 or 25, aa21 to aa126 of SEQ ID NO: 27, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of a sequence selected from the group of aa25 to aa144 of SEQ ID NO: 1-6, 13, 24 or 26, or an equivalent of each thereof; and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of a sequence selected from the group of aa21 to aa132 of SEQ ID NO: 7-9, 14 or 25, aa21 to aa126 of SEQ ID NO: 10-12 or 27, or an equivalent of each thereof.

[0276] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or also consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 1, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa21 to aa132 of any one of SEQ ID NOs: 7-9, 14 or 25, aa21 to aa126 of SEQ ID NOs: 10-12 or 27, or an equivalent of each thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 2, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa132 of any one of SEQ ID NOs: 7-9, 14 or 25, aa21 to aa126 of SEQ ID NOs: 10-12 or 27, or an equivalent of each thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or also consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 3, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa21 to aa132 of any one of SEQ ID NOs: 7-9, 14 or 25, aa21 to aa126 of SEQ ID NOs: 10-12 or 27, or an equivalent of each thereof.

[0277] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or also consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 4, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of any one of aa21 to aa132 of SEQ ID NO: 7-9, 14 or 25, aa21 to aa126 of SEQ ID NO: 10-12 or 27, or an equivalent of each thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or also consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 5, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of any one of aa21 to aa132 of SEQ ID NO: 7-9, 14 or 25, aa21 to aa126 of SEQ ID NO: 10-12 or 27, or an equivalent of each thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or also consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 6, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of any one of aa21 to aa132 of SEQ ID NO: 7-9, 14 or 25, aa21 to aa126 of SEQ ID NO: 10-12 or 27, or an equivalent of each thereof.

[0278] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or also consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa25 to aa144 of any one of SEQ ID NOs: 1-6, 13, 24 or 26, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 7, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or also consists of a heavy chain (HC) immunoglobulin variable domain sequence, and a light chain (LC) immunoglobulin variable domain sequence, comprising, consisting essentially of, or consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 8, or an equivalent thereof, which comprises, consists essentially of, or consists of the amino acid sequence from aa25 to aa144 of any one of SEQ ID NOs: 1-6, 13, 24 or 26, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or also consists of a heavy chain (HC) immunoglobulin variable domain sequence, and a light chain (LC) immunoglobulin variable domain sequence, comprising, consisting essentially of, or consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 9, or an equivalent thereof, which comprises, consists essentially of, or consists of the amino acid sequence from aa25 to aa144 of any one of SEQ ID NOs: 1-6, 13, 24 or 26, or an equivalent thereof.

[0279] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of any one of SEQ ID NOs: 1-6, 13, 24 or 26, or an equivalent of each thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 10, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of any one of SEQ ID NOs: 1-6, 13, 24 or 26, or an equivalent of each thereof, and a light chain (LC) immunoglobulin variable domain comprising, consisting essentially of, or consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 11, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or further consists of a light chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence from aa 25 to aa 144 of any one of SEQ ID NOs: 1-6, 13, 24 or 26, or an equivalent of each thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising the amino acid sequence from aa 21 to aa 126 of SEQ ID NO: 12, or an equivalent thereof.

[0280] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or further consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 1, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 7, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or further consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 1, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 8, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or also consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 1, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 9, or an equivalent thereof.

[0281] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, or alternatively consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 2, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, or alternatively consisting essentially of, or consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 7, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa25 to aa144 of SEQ ID NO: 2, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, or alternatively consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 8, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or also consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO:2, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO:9, or an equivalent thereof.

[0282] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 3, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising or consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 7, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 3, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 8, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 3, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising the amino acid sequence from aa21 to aa132 of SEQ ID NO: 9, or an equivalent thereof.

[0283] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 4, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 10, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 4, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 11, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or also consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 4, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 12, or an equivalent thereof.

[0284] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 5, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 10, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 5, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 11, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or also consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 5, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or also consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 12, or an equivalent thereof.

[0285] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 6, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 10, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 6, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 11, or an equivalent thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or also consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 6, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 12, or an equivalent thereof.

[0286] In one aspect, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 24, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 25, or an equivalent thereof. In a further embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of the tip region of a DNABII peptide (including, but not limited to, the tip region of IHF or HU, the tip region of IHFA or IHFB, and / or the tip-chimeric peptide IhfA5-mIhfB4 NTHI In one embodiment, the antibody or antigen-binding fragment thereof binds to the tip-chimeric peptide IhfA5-mIhfB4 NTHI In yet a further embodiment, the fragment is an antigen-binding fragment.

[0287] In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 1, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 7, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 1, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 8, or an equivalent thereof. In yet another embodiment, the antibody or antigen-binding fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 1, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 9, or an equivalent thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consisting essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 2, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 7, or an equivalent thereof.In another embodiment, the antibody or fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 2, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 8, or an equivalent thereof. In yet another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 2, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 9, or an equivalent thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 3, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 7, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consisting essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 3, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa132 of SEQ ID NO: 8, or an equivalent thereof.In yet another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or further consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or further consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 3, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or further consisting of the amino acid sequence from aa21 to aa132 of SEQ ID NO: 9, or an equivalent thereof.

[0288] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 26, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 27, or an equivalent thereof. In further embodiments, the antibody or antigen-binding fragment thereof comprises a tail region of a DNABII peptide (including, but not limited to, the tail region of IHF or HU, the tail region of IHFA or IHFB, and / or the tail chimeric peptide IhfA3-IhfB2 NTHI In one embodiment, the antibody or antigen-binding fragment thereof binds to the tail chimeric peptide IhfA3-IhfB2 NTHIIn yet a further embodiment, the fragment is an antigen-binding fragment. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa 25 to aa 144 of SEQ ID NO: 4, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa 21 to aa 126 of SEQ ID NO: 10, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 4, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 11, or an equivalent thereof. In yet another embodiment, the antibody or antigen-binding fragment thereof comprises, consisting essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 4, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 12, or an equivalent thereof.In one embodiment, the antibody or antigen-binding fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 5, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 10, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consisting essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 5, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 11, or an equivalent thereof. In yet another embodiment, the antibody or antigen-binding fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 5, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 12, or an equivalent thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consisting essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa25 to aa144 of SEQ ID NO: 6, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence from aa21 to aa126 of SEQ ID NO: 10, or an equivalent thereof.In another embodiment, the antibody or fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 6, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 11, or an equivalent thereof. In yet another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa25 to aa144 of SEQ ID NO: 6, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of aa21 to aa126 of SEQ ID NO: 12, or an equivalent thereof.

[0289] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consisting essentially of, or further consisting of the amino acid sequence of SEQ ID NO: 24 or an equivalent thereof, and / or a light chain (LC) comprising, consisting essentially of, or further consisting of the amino acid sequence of SEQ ID NO: 25 or an equivalent thereof. In further embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or further consists of the distal region of a DNABII peptide (the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal-chimeric peptide IhfA5-mIhfB4). NTHI In one embodiment, the antibody or antigen-binding fragment thereof binds to the tip-chimeric peptide IhfA5-mIhfB4 NTHIIn yet a further embodiment, the fragment is an antigen-binding fragment. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 1, or an equivalent thereof, and / or a light chain (LC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 7, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 1, or an equivalent thereof, and / or a light chain (LC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 8, or an equivalent thereof. In yet another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 1, or an equivalent thereof, and / or a light chain (LC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 9, or an equivalent thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 2, or an equivalent thereof, and / or a light chain (LC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 7, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or further consists of a heavy chain (HC) comprising, consisting essentially of, or further consisting of the amino acid sequence of SEQ ID NO: 2 or an equivalent thereof, and / or a light chain (LC) comprising, consisting essentially of, or further consisting of the amino acid sequence of SEQ ID NO: 8 or an equivalent thereof.In yet another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 2, or an equivalent thereof, and / or a light chain (LC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 9, or an equivalent thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 3, or an equivalent thereof, and / or a light chain (LC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 7, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 3, or an equivalent thereof, and / or a light chain (LC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 8, or an equivalent thereof. In yet another embodiment, the antibody or antigen-binding fragment thereof comprises, consisting essentially of, or alternatively consists of a heavy chain (HC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 3, or an equivalent thereof, and / or a light chain (LC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 9, or an equivalent thereof.

[0290] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 26 or an equivalent thereof, and a light chain (LC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 27 or an equivalent thereof. In further embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of, the tail region of a DNABII peptide (the tail region of IHF or HU, the tail region of IHFA or IHFB, and / or the tail-chimeric peptide IhfA3-IhfB2). NTHI In one embodiment, the antibody or antigen-binding fragment thereof binds to the tail-chimeric peptide IhfA3-IhfB2 NTHIIn yet a further embodiment, the fragment is an antigen-binding fragment. In one embodiment, the antibody or antigen-binding fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) comprising, essentially consists of, or alternatively consists of the amino acid sequence of SEQ ID NO: 4, or an equivalent thereof, and / or a light chain (LC) comprising, essentially consists of, or alternatively consists of the amino acid sequence of SEQ ID NO: 10, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, essentially consists of, or alternatively consists of a heavy chain (HC) comprising, essentially consists of, or alternatively consists of the amino acid sequence of SEQ ID NO: 4, or an equivalent thereof, and / or a light chain (LC) comprising, essentially consists of, or alternatively consists of the amino acid sequence of SEQ ID NO: 11, or an equivalent thereof. In yet another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 4, or an equivalent thereof, and / or a light chain (LC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 12, or an equivalent thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 5, or an equivalent thereof, and / or a light chain (LC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 10, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or further consists of a heavy chain (HC) comprising, consisting essentially of, or further consisting of the amino acid sequence of SEQ ID NO: 5 or an equivalent thereof, and / or a light chain (LC) comprising, consisting essentially of, or further consisting of the amino acid sequence of SEQ ID NO: 11 or an equivalent thereof.In yet another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 5 or its equivalent, and / or a light chain (LC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 12 or its equivalent. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or alternatively consists of a heavy chain (HC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: 6 or its equivalent, and / or a light chain (LC) comprising, consisting essentially of, or alternatively consisting of the amino acid sequence of SEQ ID NO: ...

Claims

[Claim 1] The invention described in the specification.

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