Combination therapies for the treatment and prevention of biofilms

Abstract

Provided herein are compositions and combinations for the treatment and diagnosis of biofilms and related diseases using high mobility group protein (HMGB) polypeptides, mutants and / or fragments thereof and anti-DNABII antibodies, fragments or variants thereof. The polypeptides and antibodies can be used with identical or separate compositions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Nos. 63 / 049,065 and 63 / 175,487, filed on July 7, 2020 and April 15, 2021, respectively, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Government Support Statement

[0004] 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.

[0005] Sequence Listing

[0006] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on July 2, 2021, is named 106887-7960_ST25.txt, and is 233,163 bytes in size. Background Art

[0007] The DNABII family of proteins naturally exists outside bacterial cells and contributes to biofilm formation. At least one protein from the DNABII family is found in all known eubacteria. While these proteins trigger strong innate and adaptive immune responses, the host is unable to naturally produce protective antibodies against family members in response to infection. A major problem with bacterial biofilms is the inability of the host immune system and / or antibiotics and other antimicrobial agents to reach the bacteria protected within the biofilm.

[0008] Bacteria in the human host prefer community structures, also known as biofilms. Biofilms form when free-living (planktonic) bacteria adhere to each other (aggregate biofilms) or to surfaces (attached biofilms) and initiate a developmental program that includes changes in gene expression, intercellular communication, and importantly, the production of a self-made extracellular matrix (extracellular polymeric substances or EPS). The resident bacteria within each of these community structures are resistant to the host immune system and antimicrobial agents (Slinger et al., Diagn Microbiol Infect Dis, 2006. 56(3): p. 247-53; and Starner et al., Antimicrob Agents Chemother, 2008. 52(1): p. 137-45), which allows the bacteria to persist and serve as a reservoir for chronic and recurrent infections. Therefore, there is an urgent need to develop targeted strategies to address bacterial biofilms.

[0009] Biofilms are also present in industrial environments. For example, biofilms are implicated in a wide range of oil process problems, from production sites to gas station storage tanks. In the field, sulfate-reducing biofilm bacteria produce hydrogen sulfide (acidifying the oil). In process piping, biofilm activity forms a sludge that blocks filters and orifices. Biofilms and biofilm organisms can also cause corrosion on piping and oil processing equipment. These problems can manifest throughout an oil or gas production facility, with fouling and corrosive biofilm organisms even being found on the surfaces of final product storage tanks.

[0010] Biofilms are involved in a wide range of water treatment processes, both domestic and industrial. They can grow on process equipment surfaces and hinder their performance, such as degrading heat transfer or clogging filters and membranes. Biofilms growing on cooling tower fill can add enough weight to cause the fill to collapse. Biofilms can even corrode highly specialized stainless steel. Biofilms in water treatment processes can reduce the value of the final product, such as biofilm fouling in paper processing or the attachment of single cells to silicon wafers. Biofilms growing in drinking water distribution systems may contain potentially pathogenic, corrosive organisms, or bacteria, reducing the aesthetic quality of the water. In the home, biofilms are present in or on any surface that supports microbial growth, such as in drains, on food processing surfaces, in toilets, and in swimming pools and baths.

[0011] Therefore, there is a need to penetrate the protective barrier of biofilms to treat or kill the associated bacterial infections and remove them from surfaces and water systems. The present disclosure satisfies this need and also provides related advantages.

[0012] Overview

[0013] Provided herein are combinations of HMG-box polypeptides (non-limiting examples of which include HMGB1 polypeptides) and anti-DNABII antibodies or their antigen-binding fragments (e.g., antibodies specifically recognizing and binding the head (tip) or chimeric head domains of DNABII) or their respective equivalents. In addition, polynucleotides, vectors, and host cells expressing the elements of the combination, either alone or in combination with one another, are also provided. Compositions, combinations, and test kits are also provided in addition, comprising the polynucleotides or vectors or host cells or both of the combination or expressing the combination, and methods for producing or using the combination, or consisting essentially of, or consisting of. Without wishing to be bound by theory, the disclosed combination demonstrates a synergistic effect in preventing biofilm formation or destroying biofilm.

[0014] In one aspect, provided herein is a composition or combination comprising, consisting essentially of, or consisting of:

[0015] (a) a high mobility group box protein (HMGB) polypeptide, which is optionally an HMGB1 polypeptide, further optionally comprising one or more mutations (i.e., a mutant HMGB1 (mHMGB1) polypeptide), or a fragment thereof, which fragment optionally comprises, consists essentially of, or consists of the B box or the A box or both (e.g., the AB box) of an HMGB (optionally HMGB1) polypeptide, optionally wherein the HMGB polypeptide or HMGB1 polypeptide or fragment thereof is isolated or engineered or both; and

[0016] (b) an anti-DNABII antibody or an antigen-binding fragment thereof comprising, consisting essentially of, or consisting of:

[0017] (i) a heavy chain (HC) immunoglobulin variable domain comprising, consisting essentially of, or consisting of amino acids (aa) 25 to aa 144 of any one of SEQ ID NO: 13, 24, or 26, or an equivalent of each thereof; and / or

[0018] (ii) a light chain (LC) immunoglobulin variable domain comprising, consisting essentially of, or consisting of aa 21 to aa 132 of SEQ ID NO: 14 or 25, aa 21 to aa 126 of SEQ ID NO: 27, or an equivalent of each thereof.

[0019] In another aspect, provided herein is a composition or combination comprising, consisting essentially of, or consisting of:

[0020] (a) a high mobility group protein (HMGB) polypeptide, which is optionally an HMGB1 polypeptide, further optionally comprising one or more mutations (e.g., a mutant HMGB1 (mHMGB1) polypeptide), or a fragment thereof, which fragment optionally comprises, consists essentially of, or consists of the Bbox or A box, or both (e.g., the AB box) of the HMGB polypeptide, optionally wherein the HMGB1 polypeptide or fragment thereof is isolated or engineered, or both; and

[0021] (b) an anti-DNABII antibody or an antigen-binding fragment thereof comprising, consisting essentially of, or consisting of:

[0022] (i) a heavy chain (HC) comprising, consisting essentially of, or consisting of any one or more of SEQ ID NO: 13, 24 or 26, or an equivalent of each thereof; and / or

[0023] (ii) a light chain (LC) comprising, consisting essentially of, or consisting of any one or more of SEQ ID NO: 14, 25 or 27, or an equivalent of each thereof.

[0024] In another aspect, provided herein is a composition or combination comprising, consisting essentially of, or consisting of:

[0025] (a) a high mobility group box protein (HMGB) polypeptide, which is 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, which fragment optionally comprises, consists essentially of, or consists of the B box or the A box, or both (e.g., the AB box) of the HMGB1 polypeptide, optionally wherein the HMGB1 polypeptide or fragment thereof is isolated or engineered, or both; and

[0026] (b) an anti-DNABII antibody or an antigen-binding fragment thereof, which comprises, consists essentially of, or consists of one, two, three, four, five, or all six of the following components (i) to (vi):

[0027] (i) a heavy chain complementarity determining region 1 (CDRH1) comprising, consisting essentially of, or consisting of: GFTFXXY (amino acids (aa) 50 to aa 56 of SEQ ID NO: 13), GFTFRTY (aa50 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), wherein X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 1-6;

[0028] (ii) a heavy chain complementarity determining region 2 (CDRH2) comprising, consisting essentially of, or consisting of the sequence: XSXXXX (amino acids (aa) 76 to aa 81 of SEQ ID NO: 13), GSDRRH (aa76 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), wherein X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 1-6;

[0029] (iii) a heavy chain complementarity determining region 3 (CDRH3) comprising, consisting essentially of, or consisting of the sequence: 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), wherein X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 1-6;

[0030] (iv) a light chain complementarity determining region 1 (CDRL1) comprising, consisting essentially of, or consisting of the sequence: QXXXXXXXXXX (aa 47 to aa 57 of SEQ ID NO: 14), QXXXXX (aa 47 to aa 52 of SEQ ID NO: 14), QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7 or 8 or 9 or 25), or QDISNY (aa 47 to aa 52 of SEQ ID NO: 10 or 11 or 12 or 27), wherein X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 7-12;

[0031] (v) a light chain complementarity determining region 2 (CDRL2) comprising, consisting essentially of, or consisting of XXS (aa 75 to aa 77 of SEQ ID NO: 14), LVS (aa 75 to aa 77 of SEQ ID NO: 7 or 8 or 9 or 25), or YTS (aa 70 to aa 72 of SEQ ID NO: 10 or 11 or 12 or 27), wherein X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 7-12; and

[0032] (vi) a light chain complementarity determining region 3 (CDRL3) comprising, consisting essentially of, or consisting of the sequence: XQGXXXXXT (aa 114 to aa 122 of SEQ ID NO: 14), WQGTHFPYT (aa 114 to aa 122 of SEQ ID NO: 7 or 8 or 9 or 25), or QQGNPLRT (aa 109 to aa 116 of SEQ ID NO: 10 or 11 or 12 or 27), wherein X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 7-12.

[0033] In one aspect, a composition or combination is provided comprising, consisting essentially of, or consisting of:

[0034] (a) a high mobility group box protein (HMGB) polypeptide, which is optionally an HMGB1 polypeptide, further optionally comprising one or more mutations (i.e., a mutant HMGB1 (mHMGB1) polypeptide or a corresponding mutation in HMGB2, HMGB3 or HMGB4)), or a fragment thereof, which fragment optionally comprises, consists essentially of, or consists of the B box or the A box or both (e.g., the AB box) of the HMGB1 polypeptide, optionally wherein the HMGB1 polypeptide or fragment thereof is isolated or engineered or both; and

[0035] (b) an anti-DNABII antibody or an antigen-binding fragment thereof, which comprises, consists essentially of, or consists of one, two, three, four, five, or all six of the following components (i) to (vi):

[0036] (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 or 2 or 3 or 24);

[0037] (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 or 2 or 3 or 24);

[0038] (iii) a heavy chain complementarity determining region 3 (CDRH3) comprising, consisting essentially of, or consisting of the following sequence: VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 1 or 2 or 3 or 24);

[0039] (iv) a light chain complementarity determining region 1 (CDRL1) comprising, consisting essentially of, or consisting of the following sequence: QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7 or 8 or 9 or 25);

[0040] (v) a light chain complementarity determining region 2 (CDRL2) comprising, consisting essentially of, or consisting of: LVS (aa 75 to aa 77 of SEQ ID NO: 7 or 8 or 9 or 25); and

[0041] (vi) a light chain complementarity determining region 3 (CDRL3) comprising, consisting essentially of, or consisting of the following sequence: WQGTHFP (aa 114 to aa 120 of SEQ ID NO: 7 or 8 or 9 or 25).

[0042] In another aspect, a composition or combination is provided comprising, consisting essentially of, or consisting of:

[0043] (a) a high mobility group protein HMGB polypeptide, which is optionally an HMGB1 polypeptide, or a fragment thereof, which fragment comprises, consists essentially of, or consists of a B box, an A box, or an AB box thereof; and

[0044] (b) an anti-DNABII antibody or an antigen-binding fragment thereof comprising, consisting essentially of, or consisting of:

[0045] (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 or 2 or 3 or 24);

[0046] (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 or 2 or 3 or 24);

[0047] (iii) a heavy chain complementarity determining region 3 (CDRH3) comprising, consisting essentially of, or consisting of the following sequence: VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 1 or 2 or 3 or 24);

[0048] (iv) a light chain complementarity determining region 1 (CDRL1) comprising, consisting essentially of, or consisting of the following sequence: QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7 or 8 or 9 or 25);

[0049] (v) a light chain complementarity determining region 2 (CDRL2) comprising, consisting essentially of, or consisting of: LVS (aa 75 to aa 77 of SEQ ID NO: 7 or 8 or 9 or 25); and

[0050] (vi) a light chain complementarity determining region 3 (CDRL3) comprising, consisting essentially of, or consisting of the following sequence: WQGTHFP (aa 114 to aa 120 of SEQ ID NO: 7 or 8 or 9 or 25).

[0051] In another aspect, a composition or combination is provided comprising, consisting essentially of, or consisting of:

[0052] (a) a high mobility group protein HMGB polypeptide, which is optionally an HMGB1 polypeptide, or a fragment thereof, which fragment comprises, consists essentially of, or consists of a B box, an A box, or an AB box thereof; and

[0053] (b) an antibody or an antigen-binding fragment thereof that specifically recognizes and binds to the head domain of the DNABII protein,

[0054] The condition is

[0055] (i) the composition or combination does not comprise SEQ ID NO: 52, or

[0056] (ii) the antigen-binding fragment does not comprise a Fab, optionally a polyclonal antibody or a Fab of an antibody that does not comprise a polyclonal antibody, or

[0057] Both (i) and (ii).

[0058] In one aspect, a composition or combination is provided comprising, consisting essentially of, or consisting of:

[0059] (a) a high mobility group box protein (HMGB) polypeptide, which is 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, which fragment optionally comprises, consists essentially of, or consists of the B box or the A box or both (e.g., the AB box) of the HMGB1 polypeptide, optionally wherein the HMGB1 polypeptide or fragment thereof is isolated or engineered or both; and

[0060] (b) an anti-DNABII antibody or an antigen-binding fragment thereof comprising, consisting essentially of, or consisting of:

[0061] (i) CDRs 1-3 of any one of SEQ ID NOs: 1-6, 13, 24 or 26, or an equivalent of each thereof; and / or

[0062] (ii) CDRs 1-3 of any one of SEQ ID NOs: 7-12, 14, 25 or 27, or an equivalent of each thereof.

[0063] In another aspect, a composition or combination is provided comprising, consisting essentially of, or consisting of:

[0064] (a) a high mobility group box protein (HMGB) polypeptide, which is optionally an HMGB1 polypeptide, optionally comprising one or more mutations (i.e., a mutant HMGB1 (mHMGB1) polypeptide), or a fragment thereof, which fragment optionally comprises, consists essentially of, or consists of a B box or an A box, or both (e.g., an AB box) of an HMGB1 polypeptide, optionally wherein the HMGB polypeptide (optionally an HMGB1 polypeptide) or fragment thereof is isolated or engineered, or both; and

[0065] (b) an anti-DNABII antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof disclosed herein for binding to the epitope.

[0066] In another aspect, a composition or combination is provided comprising, consisting essentially of, or consisting of:

[0067] (a) a high mobility group box protein (HMGB) polypeptide, which is optionally an HMGB1 polypeptide, optionally comprising one or more mutations (e.g., a mutant HMGB1 (mHMGB1) polypeptide or a corresponding mutation in an HMGB2, HMGB3, or HMGB4 polypeptide), or a fragment thereof, which fragment optionally comprises, consists essentially of, or consists of the B box or the A box, or both (e.g., the AB box) of the HMGB1 polypeptide, optionally wherein the HMGB1 polypeptide or fragment thereof is isolated or engineered, or both; and

[0068] (b) polypeptides comprising the complementarity determining regions (CDRs) disclosed herein. In some embodiments, the combination is formulated separately for combined administration. In some embodiments, the CDRs comprise, consist essentially of, or consist of any one or more of heavy chain (HC) CDR 1 (CDRH1), HC CDR 2 (CDRH2), HC CDR3 (CDRH3), light chain (LC) CDR1 (CDRL1), LC CDR 2 (CDR L2), or LC CDR 3 (CDRL3).

[0069] In one aspect, a composition or combination is provided comprising, consisting essentially of, or consisting of:

[0070] (a) a high mobility group box protein (HMGB) polypeptide, which is optionally an HMGB1 polypeptide, further optionally comprising one or more mutations (e.g., a mutant HMGB1 (mHMGB1) polypeptide or a corresponding mutation in an HMGB2, HMGB3, or HMGB4 polypeptide), or a fragment thereof, which fragment optionally comprises, consists essentially of, or consists of the B box or the A box, or both (e.g., the AB box) of the HMGB1 polypeptide, optionally wherein the HMGB1 polypeptide or fragment thereof is isolated or engineered, or both; and

[0071] (b) a complementarity determining region (CDR) comprising, consisting essentially of, or consisting of any one or more of the following sequences:

[0072] (i) a heavy chain complementarity determining region 1 (CDRH1) comprising, consisting essentially of, or consisting of any one of the following sequences: GFTFXXY (amino acids (aa) 50 to aa 56 of SEQ ID NO: 13, wherein X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 1-6), GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 1 or 2 or 3 or 24), GFTFSRY (aa 50 to aa 56 of SEQ ID NO: 4 or 5 or 6 or 26), GFTFRTYA (aa 50 to aa 57 of SEQ ID NO: 1 or 2 or 3 or 24), aASGFTFRTYAMS (aa 47 to aa 59 of SEQ ID NO: 24, wherein lowercase a is A (i.e., aa 47 to aa 59 of SEQ ID NO: 1 or 2), or wherein lowercase a is K (i.e., aa 58 to aa 59 of SEQ ID NO: 24). NO:3), GFTFSRYG (aa 50 to aa 57 of SEQ ID NO:4 or 5 or 6 or 26), or aASGFTFSRYGMS (aa 47 to aa 59 of SEQ ID NO:26, wherein lowercase a is A (i.e., aa 47 to aa 59 of SEQ ID NO:4 or 5) or wherein lowercase a is T (i.e., aa 47 to aa 59 of SEQ ID NO:6));

[0073] (ii) a heavy chain complementarity determining region 2 (CDRH2) comprising, consisting essentially of, or consisting of any one of the following sequences: XSXXXX (amino acids (aa) 76 to aa 81 of SEQ ID NO: 13, wherein X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 1-6), GSDRRH (aa76 to aa 81 of SEQ ID NO: 1 or 2 or 3 or 24), SSGGSY (aa 76 to aa 81 of SEQ ID NO: 4 or 5 or 6 or 26), IGSDRRHT (aa 75 to aa 82 of SEQ ID NO: 1 or 2 or 3 or 24), IGSDRRHTY (aa 75 to aa 83 of SEQ ID NO: 1 or 2 or 3 or 24), TIGSDRRHTY (aa 74 to aa 83 of SEQ ID NO: 1 or 2 or 3 or 24), WVATIGSDRRHTYYP (SEQ ID NO: 1 or 2 or 3 or 24), or NO: 1 or 2 or 3 or 24), ISSGGSYT (aa 75 to aa 82 of SEQ ID NO: 4 or 5 or 6 or 26), or TISSGGSYTY (aa 74 to aa 83 of SEQ ID NO: 4 or 5 or 6 or 26);

[0074] (iii) a heavy chain complementarity determining region 3 (CDRH3) comprising, consisting essentially of, or consisting of any one of the following sequences: XXXXXXXYXXFDX (amino acids (aa) 121 to aa 133 of SEQ ID NO: 13, wherein X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 1-6), 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);

[0075] (iv) a light chain complementary determining region 1 (CDRL1) comprising, consisting essentially of, or consisting of any one of the following sequences: QXXXXXXXXXX (aa 47 to aa 57 of SEQ ID NO: 14, wherein X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 7-12), QXXXXX (aa 47 to aa 52 of SEQ ID NO: 14, wherein X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 7-12), QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7 or 8 or 9 or 25), QDISNY (aa 47 to aa 52 of SEQ ID NO: 10 or 11 or 12 or 27), rSSQSLLDSDGKTFLN (aa 44 to aa 59 of SEQ ID NO: 25, wherein lowercase r is R (i.e., aa 47 to aa 52 of SEQ ID NO: 7 or 8), 44 to aa 59) or wherein lowercase r is K (i.e., aa 44 to aa 59 of SEQ ID NO: 9)), or RASQDISNYLN (aa 44 to aa 54 of SEQ ID NO: 10 or 11 or 12 or 27);

[0076] (v) a light chain complementary determining region 2 (CDRL2) comprising, consisting essentially of, or consisting of any one of the following sequences: XXS (aa 75 to aa 77 of SEQ ID NO: 14, wherein X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 7-12), LVS (aa 75 to aa 77 of SEQ ID NO: 7 or 8 or 9 or 25), YTS (aa 70 to aa 72 of SEQ ID NO: 10 or 11 or 12 or 27), LVSK1DS (aa 75 to aa 81 of SEQ ID NO: 25, wherein lowercase 1 is L (i.e., aa 75 to aa 81 of SEQ ID NO: 7 or 9) or wherein lowercase 1 is R (i.e., aa 75 to aa 81 of SEQ ID NO: 8)), YLVSK1DS (aa 74 to aa 85 of SEQ ID NO: 25), 81, wherein lowercase letter 1 is L (i.e., aa 74 to aa 81 of SEQ ID NO: 7 or 9) or wherein lowercase letter 1 is R (i.e., aa 74 to aa 81 of SEQ ID NO: 8), LVSK1DSG (aa 75 to aa 82 of SEQ ID NO: 25, wherein lowercase letter 1 is L (i.e., aa 75 to aa 82 of SEQ ID NO: 7 or 9) or wherein lowercase letter 1 is R (i.e., aa 75 to aa 82 of SEQ ID NO: 8)), YLVSK1DSGV (aa 74 to aa 83 of SEQ ID NO: 25, wherein lowercase letter 1 is L (i.e., aa 74 to aa 83 of SEQ ID NO: 7 or 9) or wherein lowercase letter 1 is R (i.e., aa 74 to aa 83 of SEQ ID NO: 8)), RLIYLVSK1DSGVPD (SEQ ID NO: 25, wherein lowercase letter 1 is L (i.e., aa 74 to aa 83 of SEQ ID NO: 7 or 9) or wherein lowercase letter 1 is R (i.e., aa 74 to aa 83 of SEQ ID NO: 8)), 7 to aa 85 of SEQ ID NO: 25, wherein lowercase l is L (i.e., aa 71 to aa 85 of SEQ ID NO: 7 or 9) or wherein lowercase l is R (i.e., aa 71 to aa 85 of SEQ ID NO: 8)), YTSRLHS (aa 70 to aa 76 of SEQ ID NO: 10 or 11 or 12 or 27), or YYTSRLHS (aa 69 to aa 76 of SEQ ID NO: 10 or 11 or 12 or 27); and

[0077] (vi) a light chain complementarity determining region 3 (CDRL3) comprising, consisting essentially of, or consisting of any one of the following sequences: XQGXXXXXT (aa 114 to aa 122 of SEQ ID NO: 14, wherein X is any amino acid or an amino acid at an aligned aa position of a sequence selected from SEQ ID NOs: 7-12), WQGTHFPYT (aa 114 to aa 122 of SEQ ID NO: 7 or 8 or 9 or 25), QQGNPLRT (aa 109 to aa 116 of SEQ ID NO: 10 or 11 or 12 or 27), WQGTHFP (aa 114 to aa 120 of SEQ ID NO: 7 or 8 or 9 or 25), WQGTHFPY (aa 114 to aa 121 of SEQ ID NO: 7 or 8 or 9 or 25), WQGTHFPYT (aa 114 to aa 122 of SEQ ID NO: 7 or 8 or 9 or 25), 114 to aa 122), or QQ (aa 109 to aa 110 of SEQ ID NO: 10 or 11 or 12 or 27).

[0078] In another aspect, a composition or combination is provided comprising, consisting essentially of, or consisting of:

[0079] (a) a high mobility group protein (HMGB) polypeptide, which is optionally an HMGB1 polypeptide, further optionally comprising one or more mutations (i.e., a mutant HMGB1 (mHMGB1) polypeptide or a corresponding mutation in an HMGB2, HMGB3 or HMGB4 polypeptide), or a fragment thereof, which fragment optionally comprises, consists essentially of, or consists of the B box or the A box or both (e.g., the AB box) of the HMGB1 polypeptide, optionally wherein the HMGB1 polypeptide or fragment thereof is isolated or engineered or both; and

[0080] (b) an isolated polypeptide comprising, consisting essentially of, or consisting of any one or more of SEQ ID NOs: 1-14 or 24-27, or an equivalent of each thereof.

[0081] In another aspect, a composition or combination is provided comprising, consisting essentially of, or consisting of:

[0082] (a) an isolated polynucleotide encoding an HMGB polypeptide (optionally an HMGB1 polypeptide) disclosed herein, or a fragment thereof, or an equivalent of each thereof, and optionally operably linked to a regulatory sequence (e.g., a promoter or enhancer or both) that directs its expression; and

[0083] (b) an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof disclosed herein, or an equivalent of each thereof, and optionally operably linked to a regulatory sequence (e.g., a promoter or enhancer or both) that directs its expression.

[0084] In one aspect, a polypeptide is provided comprising, consisting essentially of, or consisting of:

[0085] (a) a high mobility group protein (HMGB) polypeptide, which is optionally an HMGB1 polypeptide, or a fragment thereof, which fragment comprises, consists essentially of, or consists of a B box, A box, or AB box thereof; and

[0086] (b) The anti-DNABII antibody or antigen-binding fragment thereof disclosed herein, e.g., an antibody or antibody-binding fragment thereof, comprises, consists essentially of, or consists of the following components:

[0087] (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 or 2 or 3 or 24);

[0088] (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 or 2 or 3 or 24);

[0089] (iii) a heavy chain complementarity determining region 3 (CDRH3) comprising, consisting essentially of, or consisting of the following sequence: VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 1 or 2 or 3 or 24);

[0090] (iv) a light chain complementarity determining region 1 (CDRL1) comprising, consisting essentially of, or consisting of the following sequence: QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7 or 8 or 9 or 25);

[0091] (v) a light chain complementarity determining region 2 (CDRL2) comprising, consisting essentially of, or consisting of: LVS (aa 75 to aa 77 of SEQ ID NO: 7 or 8 or 9 or 25); and

[0092] (vi) a light chain complementarity determining region 3 (CDRL3) comprising, consisting essentially of, or consisting of the following sequence: WQGTHFP (aa 114 to aa 120 of SEQ ID NO: 7 or 8 or 9 or 25).

[0093] In another aspect, a polypeptide is provided comprising, consisting essentially of, or consisting of:

[0094] (a) a high mobility group protein (HMGB) polypeptide, which is optionally an HMGB1 polypeptide, or a fragment thereof, which fragment comprises, consists essentially of, or consists of a B box, A box, or AB box thereof; and

[0095] (b) an antibody or an antigen-binding fragment thereof that specifically recognizes and binds to the head domain of the DNABII protein,

[0096] The condition is

[0097] (i) the polypeptide does not comprise SEQ ID NO: 52, or

[0098] (ii) the antigen-binding fragment does not comprise a Fab, optionally a polyclonal antibody or a Fab of an antibody that does not comprise a polyclonal antibody, or

[0099] Both (i) and (ii).

[0100] In one aspect, a polynucleotide is provided that encodes:

[0101] (a) a high mobility group protein (HMGB) polypeptide, which is optionally an HMGB1 polypeptide, or a fragment thereof, which fragment comprises, consists essentially of, or consists of a B box, A box, or AB box thereof; and

[0102] (b) The anti-DNABII antibody or antigen-binding fragment thereof disclosed herein, e.g., an antibody or antibody-binding fragment thereof, comprises, consists essentially of, or consists of the following components:

[0103] (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 or 2 or 3 or 24);

[0104] (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 or 2 or 3 or 24);

[0105] (iii) a heavy chain complementarity determining region 3 (CDRH3) comprising, consisting essentially of, or consisting of the following sequence: VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 1 or 2 or 3 or 24);

[0106] (iv) a light chain complementarity determining region 1 (CDRL1) comprising, consisting essentially of, or consisting of the following sequence: QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7 or 8 or 9 or 25);

[0107] (v) a light chain complementarity determining region 2 (CDRL2) comprising, consisting essentially of, or consisting of: LVS (aa 75 to aa 77 of SEQ ID NO: 7 or 8 or 9 or 25); and

[0108] (vi) a light chain complementarity determining region 3 (CDRL3) comprising, consisting essentially of, or consisting of the following sequence: WQGTHFPYT (aa 114 to aa 122 of SEQ ID NO: 7 or 8 or 9 or 25),

[0109] or a polynucleotide complementary thereto.

[0110] In another aspect, a polynucleotide is provided that encodes:

[0111] (a) a high mobility group protein (HMGB) polypeptide, which is optionally an HMGB1 polypeptide, or a fragment thereof, which fragment comprises, consists essentially of, or consists of a B box, A box, or AB box thereof; and

[0112] (b) an antibody or an antigen-binding fragment thereof that specifically recognizes and binds to the head domain of the DNABII protein,

[0113] or a polynucleotide complementary thereto,

[0114] The condition is

[0115] (i) the polynucleotide does not encode SEQ ID NO: 52, or

[0116] (ii) the antigen-binding fragment does not comprise a Fab, optionally a polyclonal antibody or a Fab of an antibody that does not comprise a polyclonal antibody, or

[0117] Both (i) and (ii).

[0118] In one aspect, a vector is provided, comprising, or consisting essentially of, or consisting of a 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 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.

[0119] Provided herein is a host cell comprising one or more of the following: a composition or combination described herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, or a vector disclosed herein.

[0120] In one aspect, a method for inhibiting or competing for DNABII polypeptide or protein binding to microbial DNA is provided. The method comprises contacting a DNABII polypeptide or protein with a combination or composition disclosed herein, or consisting essentially of it, or consisting of it.

[0121] In another aspect, a method of disrupting a biofilm is provided, comprising contacting the biofilm with, consisting essentially of, or consisting of a composition or combination disclosed herein.

[0122] In another aspect, a method of preventing biofilm formation on a surface or disrupting a biofilm on a surface is provided. The method comprises contacting the biofilm with a combination or composition disclosed herein, or treating a surface susceptible to or containing a biofilm with a composition or composition disclosed herein, or consisting essentially of, or consisting of.

[0123] In another aspect, a method of preventing biofilm formation or disrupting a biofilm in a subject is provided. The method comprises administering to the subject, or consisting essentially of, or consisting of a composition or combination disclosed herein.

[0124] In another aspect, a method for inhibiting, preventing, or treating a biofilm-producing microbial infection in a subject is provided. The method comprises administering to the subject, or consisting essentially of, or consisting of a composition or combination disclosed herein.

[0125] In one aspect, a method of treating a condition characterized by biofilm formation in a subject is provided. The method comprises administering to the subject, or consisting essentially of, or consisting of a composition or combination disclosed herein.

[0126] In one aspect, a method is provided for one or more of: (A) preventing biofilm formation or disrupting a biofilm in vitro or ex vivo, (B) preventing biofilm formation 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. The method comprises, consists essentially of, or consists of administering to the subject:

[0127] (a) a high mobility group protein (HMGB) polypeptide, which is optionally an HMGB1 polypeptide, or a fragment thereof, which fragment comprises, consists essentially of, or consists of a B box, A box, or AB box thereof; and

[0128] (b) an anti-DNABII antibody or antigen-binding fragment thereof disclosed herein.

[0129] In one aspect, a method for inducing or increasing the formation of neutrophil extracellular traps (NETs) in close proximity to a biofilm in a subject and disrupting the biofilm, optionally without inducing a proinflammatory response, is provided. The method comprises, consists essentially of, or consists of administering to the subject:

[0130] (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 consisting of the B box, A box, or AB box thereof; and

[0131] (b) an anti-DNABII antibody or antigen-binding fragment thereof disclosed herein.

[0132] In one aspect, a method is provided for one or more of: (A) preventing biofilm formation or disruption in vitro or ex vivo, (B) preventing biofilm formation or disruption 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 the subject, or consisting essentially of, or consisting of, one or more of the following: a composition or combination disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, or a host cell disclosed herein.

[0133] In one aspect, a method for inducing or increasing the formation of neutrophil extracellular traps (NETs) in close proximity to a biofilm in a subject and disrupting the biofilm without inducing a proinflammatory response is provided. The method comprises administering to the subject one or more of, or consisting essentially of, or consisting 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, provided that the HMGB1 polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 52.

[0134] Also provided is a kit for use in the methods disclosed herein, comprising 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, or consisting essentially of the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1 Provides a model for HMGB1-mediated eDNA-dependent control of the bacterial biofilm matrix.

[0136] Figures 2A-2E A composite of images depicting the area between the biofilm and the abundant PMNs induced to the site of infection is provided. Figure 2A Representative low-magnification light micrographs of H&E-stained frozen sections of a 17-day biofilm produced by nontypeable Haemophilus influenzae (NTHI) in the middle ear of a chinchilla during experimental otitis media. The dense PMN infiltrate is primarily located in the upper right region, while the NTHI-induced biofilm occupies the lower left region of the image. The intersection of these two regions is indicated by a dashed line. Scale bar = 100 μm. Figure 2B yes Figure 2A Representative images of a series of cross-sections of the in situ biofilm shown, where a PMN-rich region intersects an NTHI biofilm, immunolabeled with antibodies against elastase to label PMNs and against an NTHI outer membrane protein to label the NTHI-induced biofilm, and where the intersection region is visible with a mixture of both fluorescent dyes. Scale bar = 100 μm. High-magnification confocal image of an 11-day-old immunolabeled NTHI biofilm recovered from the middle ear of a chinchilla: Figure 2C shows an NTHI biofilm almost completely labeled with the DNABII protein antibody HU, where the HU label was detected on the bacterial eDNA strand; Figure 2DA region is presented where the NTHI-induced biofilm intersects the PMN-rich region, where anti-DNABII (HU) labeling as well as anti-HMGB1 labeling are now evident; d1 and d2 are consecutive 1 μm Z-plane images of the inset, which demonstrate that there is no physical overlap of DNABII and HMGB1 labeling. Figure 2E PMN-rich regions are shown, where labeling was performed with anti-HMGB1 only. Scale bars in panels CE = 5 μm.

[0137] Figures 3A-3B HMGB1 variants were shown to disrupt biofilms formed by multiple high-priority human pathogens. Figure 3A Shown are the indicated isoforms of HMGB1 (200 nM unless otherwise stated) added to 24-hour biofilms for 16 hours in vitro. For each pathogen tested, five bars are shown, representing from left to right the changes in activity from control, α-IHF, Ec , rHMGB1, mHMGB1, and nHMGB1. Exceptions were: for S. aureus (S as indicated in ESKAPE), 800 nM rHMGB1 or 200 nM mHMGB1; for E. faecium (E as indicated in ESKAPE), 800 nM rHMGB1, 800 nM mHMGB1, and 3.3 mM α-IHF. Ec IgG, only 1 hour to avoid potential degradation by proteases produced by E. faecium. The biofilms were stained and visualized by confocal laser scanning microscopy (CLSM) and analyzed by COMSTAT to calculate the mean thickness. The percentage change in biofilm thickness compared to the control is plotted. Bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001 by unpaired t-test. Figure 3B Representative images of UPEC biofilms incubated with the indicated concentrations of rHMGB1 are provided. Together, the data demonstrate that rHMGB1, nHMGB1, and mHMGB1 significantly disrupt bacterial biofilms formed by multiple human pathogens and further indicate that rHMGB11 induces dose-dependent disruption of UPEC biofilms.

[0138] Figures 4A-4B HMGB1 was shown to disrupt biofilms and release biofilm-resident bacteria into the planktonic state. 24-hour UPEC biofilms were incubated with rHMGB1 (200 nM) for 16 hours, and then bacteria in the planktonic state (conditioned medium) were counted relative to those in the biofilm state (adherent bacteria). Total CFU (planktonic + biofilm) were calculated as Figure 4A shown. Figure 4BThe percentages of total bacteria in the planktonic state relative to those in the biofilm state are plotted. Bars represent mean squared error (SEM). *P < 0.05 by paired t-test. Note that rHMGB1 had no bactericidal effect but induced bacterial partitioning from the biofilm to the planktonic state.

[0139] Figures 5A-5B The synergistic effect of rHMGB1 and antibiotics in eradicating planktonic and biofilm-resident bacteria in vitro was shown. 24-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 bacteria in the planktonic state (conditioned medium) were counted relative to the biofilm state (adherent bacteria). Biofilm CFU ( Figure 5A ) and planktonic CFU (Table 5B). Bars represent SEM. *P < 0.05, **P < 0.01 by unpaired t-test. Note that rHMGB1 had no bactericidal effect, but when delivered in combination with antibiotics, rHMGB1 promoted the killing of planktonic and biofilm-resident bacteria.

[0140] Figures 6A-6C showed that oxidation of rHMGB1 negatively affected its antibiofilm activity ( Figure 6A ), while acetylation and phosphorylation of rHMGB11 did not affect the anti-biofilm activity of rHMGB1 ( Figure 6B and 6C ).exist Figure 6A In the experiment, 24-hour biofilms formed by UPEC in vitro were incubated with ox-rHMGB1 (200 nM) for 16 hours. The cells were stained and visualized by confocal laser scanning microscopy (CLSM) and analyzed by COMSTAT to calculate the mean thickness. Bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001, assessed by unpaired t-test. It was noted that the anti-biofilm function of HMGB1 was significantly reduced when rHMGB1 was oxidized. Figure 6B In the , acetylated or phosphorylated forms were confirmed by triton acetate urea gel (TAU gel; upper panel) and Western blotting with anti-acetyl lysine (α-Ac-Lys; lower panel). Figure 6CIn the study, Ac- and PrHMGB1 significantly disrupted 24-hour biofilms formed by (Bc – Burkholderia cenocepacia; E – Enterobacter spp.; or K – Klebsiella pneumoniae) compared with controls. Bars represent mean square error (SEM). *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001, assessed by unpaired t-test. Note that the antibiofilm function of HMGB1 remains unchanged regardless of whether it is acetylated or phosphorylated.

[0141] Figure 7 This study demonstrates that engineered single-amino acid variants of mHMGB1 retain their 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 resolved by native polyacrylamide gel electrophoresis (PAGE). Arrows indicate HJ DNA-protein complexes. Note that mHMGB1 retains its ability to bind to HJ DNA.

[0142] Figures 8A-8C It showed that HMGB1 binds to HJ DNA but cannot stabilize HJ DNA. 32 pHJ DNA was incubated with increasing concentrations (25–500 nM) of IHF ( Figure 8A )、rHMGB1( Figure 8B ) or RuvA( Figure 8C ) for 10 minutes and then resolved on 6% native PAGE. Asterisks indicate molten oligomers, and arrows indicate DNA-protein complexes. Although the DNA-HMGB1 complex is stable at room temperature, it is unstable at 55°C, resulting in an increased abundance of oligomers in the molten fraction. This result contrasts with that observed for IHF and the prototypic HJ DNA-binding protein RuvA.

[0143] Figure 9 HMGB1 isoforms were shown to disrupt the lattice-like eDNA network within Klebsiella pneumoniae biofilms in vitro. 24-hour Klebsiella pneumoniae biofilms were incubated with the indicated proteins (200 nM) for 16 hours. Unfixed biofilms were incubated with anti-dsDNA monoclonal antibodies and then with goat anti-mouse IgG conjugated to AlexaFluor 488 (bottom panel). Klebsiella pneumoniae were stained with FilmTracer FM 4-64 (top panel). Biofilms were visualized by CLSM. Note the interwoven reticular structure in the control and the disruption of the reticular structure by rHMGB1 and mHMGB1. Scale bar represents 10 μm.

[0144] Figures 10A-10B showed that HMGB1 disrupts biofilms through its ability to bind to HJ-like structures within the biofilm extracellular matrix. Figure 10A Shown are 24-hour biofilms formed by UPEC in vitro incubated with mHMGB1 (200 nM) for 16 hours in the presence or absence of each of the indicated proteins. Figure 10B Figure 2 shows 24-hour biofilms formed by UPEC in vitro and incubated with rHMGB1 (200 nM) or NEM-rHMGB11 (200 nM) for 16 hours. The stain was stained, visualized by confocal laser scanning microscopy (CLSM), and analyzed by COMSTAT to calculate the mean thickness. Bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001, assessed by unpaired t-test. It is noted that the anti-biofilm function of HMGB1 was lost in the presence of competitors (HU, RuvA) that directly bind to eDNA and thus compete with HMGB1 for binding to eDNA, or after modifications of rHMGB1 that directly affect its ability to bind to HJ DNA.

[0145] Figures 11A-11B showed that NEM-rHMGB1 did not bind to HJ DNA. Figure 11A The invention provides rHMGB1 modified with NEM, which is confirmed by triton acetate urea gel (TAU gel). Figure 11B As shown, 5'-end labeled 6-carboxyfluorescein HJ DNA (20 nM) was incubated with increasing concentrations (250-500 nM) of IHF, rHMGB1, or NEM-rHMGB11 and then resolved by native PAGE. Arrows indicate HJ DNA-protein complexes. Note that NEM-rHMGB1 lost its ability to bind to HJ DNA.

[0146] Figure 12 HMGB1 isoforms are shown to induce 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 anti-dsDNA monoclonal antibodies and anti-neutrophil elastase antibodies, followed by incubation with goat anti-mouse IgG conjugated to AlexaFluor 488 and goat anti-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 interwoven NETs were particularly formed when neutrophils were incubated with rHMGB1, mHMGB1, and NEM-rHMGB11, although to varying degrees. Scale bar represents 10 μm.

[0147] Figures 13A-13F showed that HMGB1 promotes the clearance of Burkholderia cepacia aggregates from the lungs of mice. Figure 13A Representative IF images of lung sections recovered from mice infected with Burkholderia cepacia are provided. 7 CFU challenge, and received 0.2 nmol of the indicated HMGB1 variants simultaneously (prevention) or 24 h later (treatment). Figure 13B ) or 72hpi (treatment, Figure 13C ) Bronchoalveolar lavage (BAL) was collected and then analyzed for CFU. Figure 13D Representative images (10x and 40x magnification) stained with H&E are provided. Figure 13E As indicated, cells in BAL were stained with anti-CD45, CD11b, and Ly-6G and analyzed by flow cytometry to measure relative neutrophil influx ( Figure 13F ). Bars represent SD. *P < 0.05, **P < 0.01, assessed by Mann-Whitney test. HMGB1 treatment significantly reduced the CFU of Burkholderia cepacia in the lungs of mice, and treatment with the engineered C45S mutation within mHMGB1 abolished its proinflammatory activity.

[0148] Figures 14A-14C showed that despite reduced phagocytosis, mHMGB1 cleared Burkholderia cepacia from the lungs of mice and exhibited attenuated neutrophil recruitment to the peritoneal cavity. Figure 14A It was shown that C57BL / 6 mice were treated with 10 7 The mice were challenged with CFU of Burkholderia cepacia and treated with 0.2 nmol rHMGB1 or mHMGB1 24 hours later. 48 hours after treatment, the mice were sacrificed and lung sections were labeled with Escherichia coli anti-EF-Tu monoclonal antibody to identify Burkholderia cepacia and DAPI to detect neutrophils. Figure 14B As shown, macrophages were treated for 2 hours with 5 μg / ml rHMGB1, 5 μg / ml mHMGB1, or 10 μM cytochalasin D, followed by the addition of pHrodo Red E. coli bioparticles and incubation for 2 hours. Cells were washed with HBSS to remove any excess bioparticles, and phagocytosed bioparticles were measured by a plate reader (560 / 585 nm). Figure 14CShown are C57BL / 6 mice intraperitoneally injected with the indicated HMGB1 isoforms. Neutrophil influx into the peritoneal cavity was measured 24 hours later by flow cytometry using antibodies against CD45, CD11b, and Ly-6G. n = 3. Bars represent SD. *P < 0.05. Although rHMGB1 treatment induced a significant influx of neutrophils into the peritoneal cavity, mHMGB1 treatment significantly attenuated this proinflammatory response, despite a slight decrease in phagocytosis.

[0149] Figure 15 This study demonstrates that rHMGB1, used to treat biofilms in vivo, does not induce a dysregulated host response associated with septic shock in mice. Mice were intraperitoneally injected with 0.2 nmol of endotoxin-free HMGB1, 5 mg / kg LPS, or both and then monitored for septic shock symptoms for 24 hours. Serum TNF-α was measured 24 hours later by ELISA. Bars represent mean ± standard deviation (SD). LoD: limit of detection. Note that the same concentration of rHMGB1 used to treat biofilms in vivo did not induce septic shock, as indicated by an increase in TNF-α.

[0150] Figures 16A-16J We show that mHMGB1 mediates clearance of biofilm-resident NTHI, elimination of established mucosal biofilms, and resolution of experimental disease, an outcome that is enhanced upon co-delivery with an antibody fragment (head chimera Fab) directed against the immunoprotective domain of the DNABII protein. Figure 16A A timeline for studies evaluating the relative ability of rHMGB1 or mHMGB1 to resolve established NTHI biofilms in the chinchilla middle ear is provided. Figure 16B The relative amount of NTHI residing within the mucosal biofilm and adhered to the middle ear mucosa is provided 1 day after completion of treatment. Figure 16C As shown, the scoring criteria were used to qualitatively assess the amount of middle ear mucosal biofilm remaining 1 day after treatment completion. Figure 16D The relative amount of biofilm in each middle ear mucosa within each cohort is provided. Figure 16E In the 24-hour follow-up, the scoring criteria were used to qualitatively assess the amount of middle ear mucosal inflammation 1 day after completion of treatment. Figure 16F The relative amount of inflammation in each middle ear mucosa within each cohort is provided. Figure 16G Representative images of the middle ear in each cohort are provided to demonstrate the relative presence / clearance of mucosal biofilm and inflammatory / non-inflammatory states. Figure 16H The timeline for the study is to evaluate the additive potential of mHMGB1 co-delivered with a head chimeric Fab to resolve established NTHI biofilms in the chinchilla middle ear. Figure 16I The relative amount of NTHI resident within the mucosal biofilm and adhered to the middle ear mucosa within 24 hours after 1 or 2 therapeutic doses is provided. Figure 16JThe relative amount of mucosal biofilm per middle ear in each cohort 24 hours after one or two treatment doses is provided. Although both rHMGB1 and mHMGB1 induced rapid clearance of biofilm-resident NTHI and clearance of established mucosal biofilms, only mHMGB11 induced limited mucosal inflammation. Furthermore, co-delivery of mHMGB1 with a head chimeric Fab fragment was highly effective in eradicating NTHI and associated biofilms from the middle ear.

[0151] Figure 17 The proinflammatory cytokines IL-1β and IL-17A were significantly more abundant in the middle ear fluid recovered from gray rats treated with rHMGB1, while anti-inflammatory cytokines predominated in the cohort treated with mHMGB1. Six days after NTHI challenge (one day after the end of treatment), the middle ear fluid was recovered and screened for the relative amounts of pro-inflammatory and anti-inflammatory cytokines by cytometric bead array. Each data point represents a single middle ear fluid, and the mean for each cohort is shown. It was noted that the concentrations of the proinflammatory cytokines IL-1β and IL-17A were significantly increased in the rHMGB1-treated gray rats (p<0.05), while a significantly increased concentration of the anti-inflammatory cytokine IL-10 was observed in the mHMGB1-treated cohort (p<0.01).

[0152] Detailed description

[0153] definition

[0154] It should be understood that the section or subsection headings used herein are for organizational purposes only and are not to be construed as limiting and / or separating the subject matter described.

[0155] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. All nucleotide sequences provided herein are arranged in a 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, specific, non-limiting exemplary methods, devices and materials are now described. All techniques and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein should be construed as an admission that this disclosure is not entitled to precede this disclosure by reason of prior invention.

[0156] 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. For example, see Sambrook and Russell eds, (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Ausubelet al. eds. (2007) Current Protocols in Molecular Biology series; Methods in Enzymology (Academic Press, Inc., NY) series; MacPherson et al. (1991) PCR 1: APractical 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 BasicTechnique, 5th 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) APractical Guide to Molecular Cloning; Miller and Calos eds, (1987) Gene TransferVectors 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. .

[0157] All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate values ​​with variations (+) or (-) in increments of 1.0 or 0.1, as appropriate, or with variations of + / - 15%, or 10%, or 5%, or 2%. It should be understood that all numerical designations are preceded by the term "about," although not always explicitly stated. It should also be understood that the reagents described herein are exemplary only, and that equivalents thereof are known in the art.

[0158] As used herein, the term "about" when referring to a measurable value (eg, an amount or concentration, etc.) is intended to encompass variations of 20%, 10%, 5%, 1%, 0.5% or even 0.1% of the particular value.

[0159] As used in the specification and claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a polypeptide" includes a plurality of polypeptides including mixtures thereof.

[0160] As used herein, the term "comprising / including" means that the compositions and methods include the elements mentioned, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of shall mean excluding other elements of any substantial significance to the combination of the intended use. Thus, a composition consisting essentially of the elements defined herein will not exclude trace contaminants and pharmaceutically acceptable carriers (e.g., phosphate buffered saline, preservatives, etc.) from separation and purification methods. "Consisting of" means excluding trace elements that exceed 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 the present disclosure.

[0161] "Optional" or "optionally" means that the subsequently described event may or may not occur, so that the description includes instances where the event occurs and instances where it does not.

[0162] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted as an alternative ("or").

[0163] "Substantially" or "substantially" refers to nearly all or completely, for example, 95% or more of a given amount. In some embodiments, "substantially" or "substantially" refers to 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%.

[0164] The terms "acceptable," "effective," or "sufficient" when used to describe the selection of any ingredient, range, level, rate, dosage form, etc. disclosed herein mean that the ingredient, range, level, rate, dosage form, etc. is suitable for the disclosed purpose.

[0165] As used in this article, comparative terms used herein, such as higher, lower, increase, decrease, lowering, or any grammatical variation thereof, can refer to certain changes relative to a reference. In some embodiments, such changes 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 relative to a reference. In some embodiments, such a change 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 a reference.

[0166] "Biofilm" refers to an organized community of microorganisms that sometimes adhere to the surface of a structure that may be organic or inorganic, along with polymers (such as DNA) that they secrete, release, and / or become available in the extracellular environment due to bacterial lysis. Biofilms are highly resistant to microbial and antimicrobial agents. They live on gum tissue, teeth, and restorations, causing caries and periodontal disease, also known as periodontal plaque. They can also lead to chronic middle ear infections. Biofilms can also form on the surfaces of dental implants, stents, catheter lines, and contact lenses. They grow on pacemakers, heart valve substitutes, artificial joints, and other surgical implants. The U.S. Centers for Disease Control estimates that more than 65% of nosocomial (hospital-acquired) infections are caused by biofilms. They cause chronic vaginal infections and lead to life-threatening systemic infections in people with compromised immune systems. Biofilms are also associated with many diseases. For example, Pseudomonas infections in cystic fibrosis patients often lead to antibiotic-resistant biofilms. In one embodiment, the biofilm comprises a DNABII polypeptide or protein. In another embodiment, the biofilm comprises IHF and / or HU. In another embodiment, the biofilm comprises IHFA and / or IHFB.

[0167] The term “neutrophil” refers to a type of granulocyte, a type of white blood cell, a phagocyte in mammals, and a first responder to inflammation. Neutrophils are granular, polymorphonuclear leukocytes that develop in the bone marrow from myeloid precursors. They play a central role in the innate immune response, destroying foreign particles and promoting acute inflammation by either intracellularly within phagosomes or by releasing neutrophil extracellular traps (NETs) extracellularly. In humans, neutrophils are the most abundant circulating leukocytes, comprising 50–70% of leukocytes, while 10–25% of circulating mouse leukocytes are neutrophils. Although neutrophils can be visually identified based on the shape of their nucleus and the granularity of their cytoplasm, they can also be identified based on their expression of several markers. Mouse neutrophils are typically identified based on cell surface expression of Ly-6G and CD11b / integrin αM. In mice, neutrophils are often distinguished from eosinophils and monocytes based on their lack of expression of M-CSF R / CD115 and CD244 / SLAMF4, as well as their lack of immunosuppressive properties, since mouse granulocyte myeloid-derived suppressor cells also express these markers. In humans, neutrophils are distinguished from eosinophils and monocytes based on their expression of CD15 and CD16 / FcγRIII and lack of CD14 expression on human neutrophils. In addition, CD66b / CEACAM-8, CD11b / integrin αM, CD33, and the cytoplasmic marker myeloperoxidase are other commonly used markers to identify human neutrophils.

[0168] As used herein, neutrophil extracellular traps (NETs) refer to an extracellular fibrous network composed mainly of DNA from neutrophils, which binds to pathogens. NETs allow neutrophils to kill extracellular pathogens while minimizing damage to host cells. High-resolution scanning electron microscopy shows that NETs are composed of elongated DNA and globular protein domains with diameters of 15-17nm and 25nm, respectively. These aggregate into larger wires with a diameter of 50nm. However, under flow conditions, NETs can form larger structures reaching hundreds of nanometers in length and width. Analysis by immunofluorescence confirmed that NETs contain proteins from azuroic granules (neutrophil elastase, cathepsin G, and myeloperoxidase), specific granules (lactoferrin), tertiary granules (gelatinase), and cytoplasm; however, CD63, actin, tubulin, and various other cytoplasmic proteins are not present 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. The activation and release of NETs, ​​referred to herein as NETosis, is a dynamic process that can take two forms: suicidal and survival NETosis.

[0169] As used herein, the phrase "immediately adjacent" refers to a position that is directly connected to a referenced position or structure without gaps or spaces.

[0170] In some embodiments, the term "disruption" refers to reducing the formation of the DNA / protein matrix that is a component of a microbial biofilm. Additionally or alternatively, the term "disruption" refers to reducing the formation of a biofilm, for example, partially or completely dispersing the biofilm. This reduction can be demonstrated in a variety of parameters. For example, the biofilm biomass or bacterial load, or both, can be assessed before and after treatment, and its reduction after treatment can be used to demonstrate the effectiveness of the treatment. Another example of such a parameter is a relative mucosal biofilm score or biomass score assigned by blinded assessors. Other suitable parameters are shown in the Examples disclosed herein. In some embodiments, the treatment reduces the biofilm to at least about 90% of the biofilm before treatment (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%). In certain embodiments, disrupting a biofilm refers to (completely or partially) breaking up the biofilm, releasing the microorganisms from the DNA / protein matrix of the biofilm, and optionally killing the microorganisms by host immune effectors and / or antibiotics.

[0171] " DNABII polypeptide or albumen " means the DNA binding protein or polypeptide being made up of DNA binding domain, and therefore has specific or general affinity to microbial DNA. In one aspect, they bind to the DNA in the minor groove. The non-limiting example of DNABII protein is integration host factor (IHF) albumen and the histone-like protein from Escherichia coli (E.coli) strain U93 (HU). Other DNA binding proteins that may be relevant to biofilm 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).

[0172] " integration host factor " or " IHF " albumen is the bacterial protein that phage is used to incorporate its DNA into host bacteria.They are also in conjunction with extracellular microbial DNA.The gene of encoding IHF protein subunit in intestinal bacteria is himA (GenbankAccession No.:POA6X7.1) and himD (POA6Y1.1) gene.The homologue of these genes is also found in other organisms.In certain embodiments, term " IHF " refers to one or two of two IHF subunits: integration host factor subunit α (IHFA or IhfA) and integration host factor subunit β (IHFB or IhfB).

[0173] "HU" or "histone-like protein from Escherichia coli strain U93" refers to a class of heteromeric proteins commonly associated with E. coli. HU proteins are known to bind to DNA junctions. Related proteins have been isolated from other microorganisms. The complete amino acid sequence of E. coli HU is reported in Laine et al. (1980) Eur. J. Biochem 103(3) 447-481. Antibodies to the HU protein can be purchased from Abeam. The genes encoding the HU protein subunits in E. coli are hupA and hupB corresponding to SEQ ID NOs: 29 and 30, respectively. Homologs of these genes have also been found in other organisms, and peptides corresponding to these genes from other organisms can be found in Table 10 of WO 2011 / 123396.

[0174] The term "surface antigen" or "surface protein" refers to a protein or peptide on the surface of a cell (e.g., a bacterial cell). Examples of outer membrane proteins include OMP P5 (Genbank Accession No.: YP-004139079.1), OMP P2 (Genbank Accession No.: ZZX87199.1), and OMP P26 (Genbank Accession No.: YP-665091.1), and surface antigens include rsPilA or recombinant soluble PilA (Genbank Accession No.: EFU96734.1) and type IV fimbriae protein (Pilin) ​​(Genbank Accession No.: Yp-003864351.1).

[0175] 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 possess the ihfA, ihfB, and hupA genes or proteins. Some non-limiting examples of different Haemophilus influenzae strains include RdKW20, 86-028NP, R2866, PittGG, PittEE, R2846, and 2019.

[0176] "Microbial DNA" refers to single-stranded or double-stranded DNA from microorganisms that are incorporated into a biofilm.

[0177] "Inhibiting, preventing or disrupting" a biofilm means prophylactically or therapeutically reducing the structure of the biofilm.

[0178] A "bent polynucleotide" refers to a double-stranded polynucleotide that contains a small loop on one strand that is not paired with the other strand. In some embodiments, the loop is from 1 base to about 20 bases long, or from 2 bases to about 15 bases long, or from about 3 bases to about 12 bases long, or from about 4 bases to about 10 bases long, or has about 4, 5, or 6, or 7, or 8, or 9, or 10 bases.

[0179] " polypeptide (for example DNA combines in the IHF) in conjunction with DNABII competition " means in conjunction with bending or twisted DNA structure and DNABII (for example IHF) competition but do not form biomembrane protein or peptide with DNA.Example comprises the fragment of the IHF of one or more DNA binding structural domains or its biological equivalent that comprise IHF, but is not limited to this.

[0180] The "subject" of diagnosis or treatment is a cell or animal, such as a mammal or a human. Non-human animal subjects receiving diagnosis or treatment are those infected or animal models, for example, apes, rodents (e.g., rats, mice, chinchillas), canines (e.g., dogs), lagomorphs (e.g., rabbits), livestock, sports animals, and pets. The terms "subject," "host," "individual," and "patient" are used interchangeably herein to refer to animals, typically mammals. 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), farm animals (e.g., horses, cattle, goats, sheep, pigs), and experimental animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal can be at any age or at any stage of development (e.g., an adult, adolescent, child, infant, or mammal in utero). The mammal can be male or female. In some embodiments, the subject is a human.

[0181] The terms "protein," "peptide," and "polypeptide" are used interchangeably and in their broadest sense refer to a compound of two or more subunits of amino acids, amino acid analogs, or peptidomimetics. These subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds (e.g., esters, ethers, etc.). A protein or polypeptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that may comprise a protein or polypeptide sequence. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including glycine and the D and L optical isomers, amino acid analogs, and peptidomimetics.

[0182] As used herein, "complementary" sequences refer to sequences that contain multiple individual nucleotide bases that pair with each other when two nucleotide sequences are arranged in antiparallel relation to each other. The pairing of nucleotide bases forms hydrogen bonds, thereby stabilizing the double-stranded structure formed by the complementary sequences. It is not necessary for every nucleotide base in the two sequences to pair with each other to be considered "complementary". For example, if the nucleotide bases in the two sequences are at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% paired with each other, the sequences can be considered to be complementary. In some embodiments, the term complementary refers to that 100% of the nucleotide bases in the two sequences pair with each other. In addition, when the total lengths of the two sequences are significantly different, the sequences can still be considered to be "complementary". For example, when a primer is arranged antiparallel to a specific region of a longer polynucleotide, a primer of 15 nucleotides can be considered to be "complementary" to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer are paired with the nucleotide bases of the longer polynucleotide. Nucleotide base pairing is well known in the art, for example, in DNA, adenine (A) pairs with thymine (T), and cytosine (C) always pairs with guanine (G); whereas in RNA, adenine (A) pairs with uracil (U), and guanine (G) pairs with cytosine (C). In addition, nucleotide bases that are arranged antiparallel to each other in two complementary sequences but are not a pair are referred to herein as mismatches.

[0183] "C-terminal polypeptide" refers to at least 10, or at least 15, or at least 20, or at least 25 C-terminal amino acids or half of a polypeptide. In another aspect, for a polypeptide containing 90 amino acids, the C-terminal polypeptide will include amino acids 46 to 90. In one aspect, the term refers to the C-terminal 20 amino acids starting from the carboxyl terminus.

[0184] " head fragment " of DNABII polypeptide means DNABII polypeptide, and taking IHF α and IHF β as example, it forms two arms of protein.This non-limiting example includes IhfA, A head fragment: NFELRDKSSRPGRNPKTGDVV, SEQ ID NO:31, and IhfB, B head fragment: SLHHRQPRLGRNPKTGDSVNL, SEQ ID NO:32, and with SEQ ID NO:31 or 32 align the amino acid sequence of the DNABII polypeptide of another species (such as another bacterium) and the peptide containing two head domains or modified domains, to provide necessary confirmation, such as hereinafter exemplified mIhFB4 and the IhfA head domain.

[0185] The "tail segment" of the DNABII polypeptide refers to the region of the protein that is both exposed to the bulk medium and not shielded by DNA or other polypeptides.

[0186] As used herein, ESKAPE pathogens include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. These pathogens are the leading causes of nasal infections worldwide.

[0187] "HMG domain," "high mobility group (HMG) box domain," or "HMGB" refers to an amino acid sequence involved in binding to DNA (Stros et al., Cell Mol Life Sci. 64(19-20):2590-606 (2007)). In one embodiment, the structure of the HMG-box domain consists of three irregularly arranged helices. In another embodiment, the HMG-box domain enables the protein to bind to non-B-form DNA conformations (kinked or unwound) with high affinity. HMG-box domains can be found in high mobility group proteins, which are 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).

[0188] HMG-box proteins are present in various eukaryotic organisms and can be broadly divided into two groups based on sequence-dependent and sequence-independent DNA recognition; the former usually contain one HMG-box motif, while the latter can 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), involved in the development of different gonads; the SOX family of transcription factors (Harley et al. (2003) Endocr. Rev. 24(4):466-87); sequence-specific LEF1 (lymphoid enhancer binding factor 1) and TCF-1 (T cell factor 1), involved in regulating organogenesis and thymocyte differentiation (Labbé 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 RNA polymerase I transcription; Abf2 yeast ARS binding factor (Cho et al. al. (2001) Biochim. Biophys. Acta. 1522(3):175-86); yeast transcription factors lxr1, Rox1, Nhp6b, and Spp41; mating type protein (MAT) involved in fungal sexual reproduction (Barve et al. (2003) Fungal Genet. Biol. 39(2):151-67); and the YABBY plant-specific transcription factor.

[0189] "HMGB1" is a high mobility group protein (HMGB) 1, which is reported to bind to and distort the minor groove of DNA. Recombinant or isolated proteins and polypeptides can be purchased from Atgenglobal, ProSpecBio, Protein1, and Abnova. As used herein, an HMGB1 polypeptide refers to an HMGB1 protein or an equivalent thereof. In some embodiments, the HMGB1 protein comprises, consists essentially of, or consists of SEQ ID NO: 51. In some embodiments, an HMGB1 equivalent comprises, consists essentially of, or consists of an HMG-box protein, such as those disclosed herein. In some embodiments, an HMGB1 equivalent comprises, consists essentially of, or consists of an HMG-box domain, such as those disclosed herein, consists essentially of, or consists of. In further embodiments, an HMGB1 equivalent comprises, consists essentially of, or consists of one or more of HMGB2, HMGB3, or HMGB4. Additionally or alternatively, the HMGB1 equivalent comprises, consists essentially of, or consists of one or more mutations disclosed herein. In some embodiments, the HMGB1 equivalent comprises, consists essentially of, or consists of any one or more of SEQ ID NOs: 52-58, 68-74, 84-90, or 100-114, or their respective equivalents.

[0190] Exemplary sequences of polypeptides containing an HMG-box structure include NP_002119 (human HMGB1), NP_001124160 (human HMGB2), NP_005333 (human HMGB3), and NP_660206 (human HMGB4). For example, amino acid residues from about 9 to about 76 in human HMGB1 form an HMG-box domain, and amino acid residues from about 90 to about 138 form another HMG-box domain. For example, an HMGB1 fragment containing either of these two HMG-box structures also constitutes a polypeptide containing an HMG-box structure within the meaning of the present disclosure. In the examples described herein, recombinant HMGB1 (derived from humans and recombinantly expressed and purified in Escherichia coli) was used as a comparator for mHMGB1 (C45S), and its sequence is:

[0191] MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYR PKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKKKKEEEEGEEDEEDEEEEEDEEDEDEEEDDDDE(SEQID NO:51).

[0192] 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 indicates the mutated amino acid residue. For example, an HMGB1 polypeptide comprising a C45S mutation indicates that cysteine ​​(C), the 45th amino acid residue of HMGB1, is mutated to serine (S). In further embodiments, the full-length sequence (e.g., SEQ ID NO: 51) can serve as a reference, for example, when identifying amino acid positions in fragments. An example is that a B box fragment of an HMGB1 polypeptide can comprise a C106S mutation, wherein the number 106 refers to the amino acid residue position in the full-length HMGB1 polypeptide rather than the B box fragment position. In some embodiments, the reference is SEQ ID NO: 51. In some embodiments, the second letter can be omitted when referring to a position.

[0193] As used herein, amino acid positions in a sequence are identified by letters followed by numbers. The letters refer to the amino acid residue at that position, while the numbers indicate the position of the mutation in the reference sequence. For example, C45 represents 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 amino acid positions in a fragment. One example is that a B box fragment of an HMGB1 polypeptide can include a C106S mutation, where the number 106 refers to the amino acid residue position in the full-length HMGB1 polypeptide rather than the B box fragment. In some embodiments, the reference is SEQ ID NO: 51.

[0194] As used herein, the terms "modified high mobility group-box 1 domain" and mHMGB1 refer to HMGB1 that has been mutated, for example, by substitution of cysteine ​​residues at positions 23, 45 and / or 106 based on the consensus sequence polypeptide of HMGB1 derived from human: SEQ ID NO: 51.

[0195] Non-limiting example 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 a mutation of C23S, i.e., SEQ ID NO:53; and mHMGB1(C45S), which is SEQ ID NO:51 further comprising a mutation of C45S, i.e., SEQ ID NO:52, consisting of the following sequence: MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKSSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKKKKEEEEGEEDEEDEEEEEDEEDEDEEEDDDDE (SEQ ID NO:53). ID NO: 52); mHMGB1 (C106S), which is SEQ ID NO: 51 further comprising a mutation of C106S, i.e., SEQ ID NO: 54; mHMGB1 (C23S, C45S-double mutant), which is SEQ ID NO: 51 further comprising two mutations of C23S and C45S, i.e., SEQ ID NO: 55; mHMGB1 (C23S, C106S-double mutant), which is SEQ ID NO: 51 further comprising two mutations of C23S and C106S, i.e., SEQ ID NO: 56; mHMGB1 (C45S, C106S-double mutant), which is SEQ ID NO: 51 further comprising two mutations of C45S and C106S, i.e., SEQ ID NO: NO: 57; and mHMGB1 (C23S, C45S, C106S-triple mutant), which is SEQ ID NO: 51 further comprising three mutations of C23S, C45S, and C106S, namely, SEQ ID NO: 58.

[0196] Exemplary nucleic acid sequences encoding HMGB1 are provided below: ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCATCATATGCATTTTTTGTGCAAACTTGTCGGGAGGAGCATAAGAAGAAGCACCCAGATGCTTCAGTCAACTTCTCAGAGTTTTCTAAGAAGTGCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAGAAAGGAAAATTTGAAGATATGGCAAAGGCGGACAAGGCCCGTTATGAAAGAGAAATGAAAACCTATATCCCTCCCAAAGGGGAGACAAAAAAGAAGTTCAAGGATCCCAATGCACCCAAGAGGCCTCCTTCGGCCTTCTTCCTCTTCTGCTCTGAGTATCGCCCAAAAATCAAAGGAGAACATCCTGGCCTGTCCATTGGTGATGTTGCGAAGAAACTGGGAGAGATGTGGAATAACACTGCTGCAGATGACAAGCAGCCTTATGAAAAGAAGGCTGCGAAGCTGAAGGAAAAATACGAAAAGGATATTGCTGCATATCGAGCTAAAGGAAAGCCTGATGCAGCAAAAAAGGGAGTTGTCAAGGCTGAAAAAAGCAAGAAAAAGAAGGAAGAGGAGGAAGGTGAGGAAGATGAAGAGGATGAGGAGGAGGAGGAAGATGAAGAAGATGAAGATGAAGAAGAAGATGATGATGATGAA(SEQ ID NO:59).

[0197] Also provided are nucleic acid sequences encoding these modified high mobility group-box 1 domains (i.e., mHMGB1): mHMGB1(C23S), which is SEQ ID NO:59 further comprising AGT encoding C23S, i.e., SEQ ID NO:59 further comprising a 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 a T to A mutation at nucleotide residue 133 of SEQ ID NO:59 and a C to T mutation at nucleotide residue 135 of SEQ ID NO:59, see SEQ ID NO:61; mHMGB1(C106S), which is SEQ ID NO:59 further comprising AGC encoding C106S, i.e., SEQ ID NO:59 further comprising SEQ ID NO:59, a mutation from T to A at nucleotide residue 316, see SEQ ID NO:62; mHMGB1 (C23S, C45S-double mutant), which is SEQ ID NO:59 further comprising AGT encoding C23S and AGT encoding C45S, i.e., SEQ ID NO:59 further comprises a mutation from T to A at nucleotide residue 67 of SEQ ID NO:59, a mutation from T to A at nucleotide residue 133 of SEQ ID NO:59, and a mutation from C to T at nucleotide residue 135 of SEQ ID NO:59, see SEQ ID NO:63; mHMGB1 (C23S, C106S-double mutant), which is SEQ ID NO:59 further comprising AGT encoding C23S and AGC encoding C106S, i.e., SEQ ID NO:59 further comprises a mutation from T to A at nucleotide residue 67 of SEQ ID NO:59 and SEQ ID a T to A mutation at nucleotide residue 316 of SEQ ID NO:59, see SEQ ID NO:64; mHMGB1 (C45S, C106S-double mutant), which is SEQ ID NO:59 further comprising AGT encoding C45S and AGC encoding C106S, i.e., SEQ ID NO:59 further comprises a T to A mutation at nucleotide residue 133 of SEQ ID NO:59, a C to T mutation at nucleotide residue 135 of SEQ ID NO:59, and a T to A mutation at nucleotide residue 316 of SEQ ID NO:59, see SEQ ID NO:65;and mHMGB1 (C23S, C45S, C106S triple mutant), which is SEQ ID NO:59 further comprising AGT encoding C23S, AGT encoding C45S, and AGC encoding C106S, i.e., SEQ ID NO:59 further comprises a T to A mutation at nucleotide residue 67 of SEQ ID NO:59, a T to A mutation at nucleotide residue 133 of SEQ ID NO:59, a C to T mutation at nucleotide residue 135 of SEQ ID NO:59, and a T to A mutation at nucleotide residue 316 of SEQ ID NO:59, see SEQ ID NO:66.;

[0198] It will be understood that the term "modified high mobility group-box 1 domain" further encompasses equivalents having 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, as well as those comprising the same substitutions at corresponding positions in the equivalent sequence based on an alignment with the HMGB1 consensus sequence or with any one of SEQ ID NOs: 51-58.

[0199] It is understood that among equivalents of a modified HMG-box 1 domain, the equivalents are modified HMG-box 2, modified HMG-box 3, and modified HMG-box 4 comprising a cysteine ​​to serine substitution at one or more amino acid positions corresponding to C23S, C45S, and C106S of modified HMG-box 1, or their equivalents. As will be understood by those skilled in the art, mutations in one or more HMG-box proteins are considered equivalent if the positions of the mutations align with one another in a sequence alignment of one or more HMG-box proteins (e.g., HMGB1, HMGB2, HMGB3, or HMGB4). Additionally and optionally, in equivalent mutations, the original amino acid residue to be mutated is the same. Additionally and optionally, in equivalent mutations, the amino acid residue to be mutated is the same. Thus, an equivalent of an HMGB1 polypeptide may be a wild-type HMGB1 mutated at one or more positions (e.g., C23S, C45S, C106S, or any combination thereof, as disclosed herein). In addition, an equivalent of an HMGB1 polypeptide may be an HMG-box protein other than HMGB1, for example, HMGB2, HMGB3, or HMGB4 comprising an equivalent mutation. A non-limiting example is that an HMGB1 C45S mutant is an equivalent of an HMGB2 C45S mutant and an HMGB3 C45S or HMGB4 C45S. Alternatively, regardless of their position, the C to S mutations specified herein may be considered equivalent to each other, because, without being bound by theory, the applicants believe that the modified HMGB1 fragments disclosed herein will behave similarly.

[0200] Based on the following consensus sequence polypeptide derived from human HMGB2, corresponding amino acid substitutions can be made at cysteine ​​residues 23, 45, and / or 106 to obtain modified high mobility group-box 2:

[0201] MGKGDPNKPRGKMSSYAFFVQTCREEHKKKHPDSSVNFAEFSKKCSERWKTMSAKEKSKFEDMAKSDKARYDREMKNYVPPKGDKKGKKKDPNAPKRPPSAFFLFC SEHRPKIKSEHPGLSIGDTAKKLGEMWSEQSAKDKQPYEQKAAKLKEKYEKDIAAYRAKGKSEAGKKGPGRPTGSKKKNEPEDEEEEEEEEEDEDEEEEDEDEE(SEQ ID NO:67).

[0202] 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 a mutation of C23S, i.e., SEQ ID NO: 68; mHMGB2(C45S), which is SEQ ID NO: 67 further comprising a mutation of C45S, i.e., SEQ ID NO: 69; mHMGB2(C106S), which is SEQ ID NO: 67 further comprising a mutation of C106S, i.e., SEQ ID NO: 70; mHMGB2(C23S, C45S-double mutant), which is SEQ ID NO: 67 further comprising two mutations of C23S and C45S, i.e., SEQ ID NO: 71; mHMGB2(C23S, C106S-double mutant), which is SEQ ID NO: 67 further comprising two mutations of C23S and C106S, i.e., SEQ ID NO: 72. NO: 72; mHMGB2 (C45S, C106S-double mutant), which is SEQ ID NO: 67 further comprising two mutations of C45S and C106S, i.e., SEQ ID NO: 73; and mHMGB2 (C23S, C45S, C106S-triple mutant), which is SEQ ID NO: 67 further comprising three mutations of C23S, C45S and C106S, i.e., SEQ ID NO: 74.

[0203] Examples of nucleic acid sequences encoding HMGB2 are provided below: ATGGGTAAAGGAGACCCCAACAAGCCGCGGGGCAAAATGTCCTCGTACGCCTTCTTCGTGCAGACCTGCCGGGAAGAGCACAAGAAGAAACACCCGGACTCTTCCGTCAATTTCGCGGAATTCTCCAAGAAGTGTTCGGAGAGATGGAAGACCATGTCTGCAAAGGAGAAGTCGAAGTTTGAAGATATGGCAAAAAGTGACAAAGCTCGCTATGACAGGGAGATGAAAAATTACGTTCCTCCCAAAGGTGATAAGAAGGGGAAGAAAAAGGACCCCAATGCTCCTAAAAGGCCACCATCTGCCTTCTTCCTGTTTTGCTCTGAACATCGCCCAAAGATCAAAAGTGAACACCCTGGCCTATCCATTGGGGATACTGCAAAGAAATTGGGTGAAATGTGGTCTGAGCAGTCAGCCAAAGATAAACAACCATATGAACAGAAAGCAGCTAAGCTAAAGGAGAAATATGAAAAGGATATTGCTGCATATCGTGCCAAGGGCAAAAGTGAAGCAGGAAAGAAGGGCCCTGGCAGGCCAACAGGCTCAAAGAAGAAGAACGAACCAGAAGATGAGGAGGAGGAGGAGGAAGAAGAAGATGAAGATGAGGAGGAAGAGGATGAAGATGAAGAATAA(SEQ ID NO:75).

[0204] Also provided are nucleic acid sequences encoding these modified high mobility group-box 2 domains: mHMGB2(C23S), which is SEQ ID NO:75 further comprising AGT encoding C23S, i.e., SEQ ID NO:75 further comprises a T to A mutation at nucleotide residue 67 of SEQ ID NO:75 and a 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 comprises a 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 comprises a T to A mutation at nucleotide residue 316 of SEQ ID NO:75, see SEQ ID NO:78. ID NO: 78; mHMGB2 (C23S, C45S-double mutant), which is SEQ ID NO: 75 further comprising AGT encoding C23S and AGT encoding C45S, i.e., SEQ ID NO: 75 further comprises a mutation from T to A at nucleotide residue 67 of SEQ ID NO: 75, a mutation from C to T at nucleotide residue 69 of SEQ ID NO: 75, and a mutation from T to A at nucleotide residue 133 of SEQ ID NO: 75, see SEQ ID NO: 79; mHMGB2 (C23S, C106S-double mutant), which is SEQ ID NO: 75 further comprising AGT encoding C23S and AGC encoding C106S, i.e., SEQ ID NO: 75 further comprises a mutation from T to A at nucleotide residue 67 of SEQ ID NO: 75, a mutation from C to T at nucleotide residue 69 of SEQ ID NO: 75, and a mutation from T to A at nucleotide residue 133 of SEQ ID NO: 75, see SEQ ID NO: 79. a T to A mutation at nucleotide residue 316 of SEQ ID NO:75, see SEQ ID NO:80; mHMGB2 (C45S, C106S-double mutant), which is SEQ ID NO:75 further comprising AGT encoding C45S and AGC encoding C106S, i.e., SEQ ID NO:75 further comprising a T to A mutation at nucleotide residue 133 of SEQ ID NO:75 and a T to A mutation at nucleotide residue 316 of SEQ ID NO:75, see SEQ ID NO:81;and mHMGB2 (C23S, C45S, C106S triple mutant), which is SEQ ID NO:75 further comprising AGT encoding C23S, AGT encoding C45S, and AGC encoding C106S, i.e., SEQ ID NO:75 further comprises a T to A mutation at nucleotide residue 67 of SEQ ID NO:75, a C to T mutation at nucleotide residue 69 of SEQ ID NO:75, a T to A mutation at nucleotide residue 133 of SEQ ID NO:75, and a T to A mutation at nucleotide residue 316 of SEQ ID NO:75, see SEQ ID NO:82. ;

[0205] Based on the following consensus sequence polypeptide derived from human HMGB3, corresponding amino acid substitutions can be made at cysteine ​​residues 23, 45, and / or 104 to obtain a modified high mobility group-box 3 (mHMGB3): MAKGDPKKPKGKMSAYAFFVQTCREEHKKKNPEVPVNFAEFSKKCSERWKTMSGKEKSKFDEMAKADKVRYDREMKDYGPAKGGKKKKDPNAPKRPPSGFFLFCSEFRPKIKSTNPGISIGDVAKKLGEMWNNLNDSEKQPYITKAAKLKEKYEKDVADYKSKGKFDGAKGPAKVARKKVEEEDEEEEEEEEEEEEEEDE (SEQ ID NO: 83). It will be understood by those skilled in the art that mHMGB3 (C104S) is an equivalent of mHMGB1 (C106S) or mHMGB2 (C106S).

[0206] 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 a mutation of C23S, i.e., SEQ ID NO: 84; mHMGB3(C45S), which is SEQ ID NO: 83 further comprising a mutation of C45S, i.e., SEQ ID NO: 85; mHMGB3(C104S), which is SEQ ID NO: 83 further comprising a mutation of C104S, i.e., SEQ ID NO: 86; mHMGB3(C23S, C45S-double mutant), which is SEQ ID NO: 83 further comprising two mutations of C23S and C45S, i.e., SEQ ID NO: 87; mHMGB3(C23S, C104S-double mutant), which is SEQ ID NO: 83 further comprising two mutations of C23S and C104S, i.e., SEQ ID NO: 88. NO: 88; mHMGB3 (C45S, C104S-double mutant), which is SEQ ID NO: 83 further comprising two mutations of C45S and C104S, i.e., SEQ ID NO: 89; and mHMGB3 (C23S, C45S, C104S-triple mutant), which is SEQ ID NO: 83 further comprising three mutations of C23S, C45S and C104S, i.e., SEQ ID NO: 90.

[0207] Examples of nucleic acid sequences encoding HMGB3 are provided below: ATGGCTAAAGGTGACCCCAAGAAACCAAAGGGCAAGATGTCCGCTTATGCCTTCTTTGTGCAGACATGCCAGAGAAGAACATAAGAAGAAAAACCCAGAGGTCCCTGTCAATTTTGCGGAATTTTCCAAGAAGTGCTCTGAGAGGTGGAAGACGATGTCCGGGAAAGAGAAATCTAAATTTGATGAAATGGCAAAGGCAGATAAAGTGCGCTATGATCGGGAAATGAAGGATTATGGACCAGCTAAGGGAGGCAAGAAGAAGAAGGATCCTAATGCTCCCAAAAGGCCACCGTCTGGATTCTTCCTGTTCTGTTCAGAATTCCGCCCCAAGATCAAATCCACAAACCCCGGCATCTCTATTGGAGACGTGGCAAAAAAGCTGGGTGAGATGTGGAATAATTTAAATGACAGTGAAAAGCAGCCTTACATCACTAAGGCGGCAAAGCTGAAGGAGAAGTATGAGAAGGATGTTGCTGACTATAAGTCGAAAGGAAAGTTTGATGGTGCAAAGGGTCCTGCTAAAGTTGCCCGGAAAAAGGTGGAAGAGGAAGATGAAGAAGAGGAGGAGGAAGAAGAGGAGGAGGAGGAGGAGGAGGATGAATAA(SEQ ID NO:91).

[0208] Also provided below are nucleic acid sequences encoding these modified high mobility group-box 3 domains: mHMGB3(C23S), which is SEQ ID NO:91 further comprising AGT encoding C23S, i.e., SEQ ID NO:91 further comprises a mutation from T to A at nucleotide residue 67 of SEQ ID NO:91 and a mutation from C to T 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 comprises a mutation from T to A at nucleotide residue 133 of SEQ ID NO:91 and a mutation from C to T at nucleotide residue 135 of SEQ ID NO:91, see SEQ ID NO:93; mHMGB3(C104S), which is SEQ ID NO:91 further comprising AGC encoding C104S, i.e., SEQ ID NO:91 further comprises SEQ ID NO:91 from T to A mutation at nucleotide residue 310 of SEQ ID NO:91 and from T to C mutation at nucleotide residue 312 of SEQ ID NO:91, see SEQ ID NO:94; mHMGB3 (C23S, C45S-double mutant), which is SEQ ID NO:91 further comprising AGT encoding C23S and AGT encoding C45S, i.e., SEQ ID NO:91 further comprises a mutation at nucleotide residue 67 of SEQ ID NO:91 from T to A, a mutation at nucleotide residue 69 of SEQ ID NO:91 from C to T, a mutation at nucleotide residue 133 of SEQ ID NO:91 from T to A mutation, and a mutation at nucleotide residue 135 of SEQ ID NO:91 from C to T mutation, see SEQ ID NO:95; mHMGB3 (C23S, C104S-double mutant), which is SEQ ID NO:91 further comprising AGT encoding C23S and AGC encoding C104S, i.e., SEQ ID NO:91 further comprises a mutation from T to A at nucleotide residue 67 of SEQ ID NO:91, a mutation from C to T at nucleotide residue 69 of SEQ ID NO:91, a mutation from T to A at nucleotide residue 310 of SEQ ID NO:91, and a mutation from T to C at nucleotide residue 312 of SEQ ID NO:91, see SEQ ID NO:96;mHMGB3 (C45S, C104S-double mutant), which is SEQ ID NO:91 further comprising AGT encoding C45S and AGC encoding C104S, i.e., SEQ ID NO:91 further comprises a mutation from T to A at nucleotide residue 133 of SEQ ID NO:91, a mutation from C to T at nucleotide residue 135 of SEQ ID NO:91, a mutation from T to A at nucleotide residue 310 of SEQ ID NO:91, and a mutation from T to C at nucleotide residue 312 of SEQ ID NO:91, see SEQ ID NO:97; mHMGB3 (C23S, C45S, C104S-triple mutant), which is SEQ ID NO:91 further comprising AGT encoding C23S, AGT encoding C45S and AGC encoding C104S, i.e., SEQ ID NO:91 further comprises a mutation from T to A at nucleotide residue 67 of SEQ ID NO:91, a mutation from C to T at nucleotide residue 135 of SEQ ID NO:91, a mutation from T to A at nucleotide residue 310 of SEQ ID NO:91, and a mutation from T to C at nucleotide residue 312 of SEQ ID NO:91, see SEQ ID NO:97. a mutation from C to T at nucleotide residue 69 of SEQ ID NO:91, a mutation from T to A at nucleotide residue 133 of SEQ ID NO:91, a mutation from C to T at nucleotide residue 135 of SEQ ID NO:91, a mutation from T to A at nucleotide residue 310 of SEQ ID NO:91, a mutation from T to C at nucleotide residue 312 of SEQ ID NO:91, see SEQ ID NO:98.;

[0209] Based on the following consensus sequence polypeptide derived from human HMGB4, corresponding amino acid substitutions can be made at cysteine ​​residues at positions 45, 104, 164 and / or 178 to obtain a modified high mobility group-box 4 (mHMGB4): MGKEIQLKPKANVSSYVHFLLNYRNKFKEQQPNTYVGFKEFSRKCSEKWRSISKHEKAKYEALAKLDKARYQEEMMNYVGKRKKRRKRDPQEPRRPPSSFLLFCQDHYAQLKRENPNWSVVQVAKATGKMWSTATDLEKHPYEQRVALLRAKYFEELELYRKQCNARKKYRMSARNRCRGKRVRQS (SEQ ID NO: 99).

[0210] 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 further comprising a mutation of C45S, i.e., SEQ ID NO: 100; mHMGB4(C104S), which is SEQ ID NO: 99 further comprising a mutation of C104S, i.e., SEQ ID NO: 101; mHMGB4(C164S), which is SEQ ID NO: 99 further comprising a mutation of C164S, i.e., SEQ ID NO: 102; mHMGB4(C178S), which is SEQ ID NO: 99 further comprising a mutation of C178S, i.e., SEQ ID NO: 103; mHMGB4(C45S, C104S-double mutant), which is SEQ ID NO: 99 further comprising two mutations of C45S and C104S, i.e., SEQ ID NO: 104. NO: 104; mHMGB4 (C45S, C164S-double mutant), which is SEQ ID NO: 99 further comprising two mutations of C45S and C164S, i.e., SEQ ID NO: 105; mHMGB4 (C45S, C178S-double mutant), which is SEQ ID NO: 99 further comprising two mutations of C45S and C178S, i.e., SEQ ID NO: 106; mHMGB4 (C104S, C164S-double mutant), which is SEQ ID NO: 99 further comprising two mutations of C104S and C164S, i.e., SEQ ID NO: 107; mHMGB4 (C104S, C178S-double mutant), which is SEQ ID NO: 99 further comprising two mutations of C104S and C178S, i.e., SEQ ID NO: 108. NO: 108; mHMGB4 (C164S, C178S-double mutant), which is SEQ ID NO: 99 further comprising two mutations of C164S and C178S, i.e., SEQ ID NO: 109; mHMGB4 (C45S, C104S, C164S-triple mutant), which is SEQ ID NO: 99 further comprising three mutations of C45S, C104S and C164S, i.e., SEQ ID NO: 110; mHMGB4 (C45S, C104S, C178S-triple mutant), which is SEQ ID NO: 99 further comprising three mutations of C45S, C104S and C178S, i.e., SEQ ID NO: 111; mHMGB4 (C45S, C164S, C178S-triple mutant), which is SEQ ID NO: 99 further comprising three mutations of C45S, C164S, and C178S, i.e., SEQ ID NO: 112;mHMGB4 (C104S, C164S, C178S-triple mutant), which is SEQ ID NO: 99 further comprising three mutations of C104S, C164S, and C178S, i.e., SEQ ID NO: 113; and mHMGB4 (C45S, C104S, C164S, C178S-quadruple mutant), which is SEQ ID NO: 99 further comprising four mutations of C45S, C104S, C164S, and C178S, i.e., SEQ ID NO: 114. ;

[0211] An example of a nucleic acid sequence encoding human HMGB4 is provided below: (SEQ ID NO: 115).

[0212] Nucleic acid sequences encoding these modified high mobility group-box 4 domains are also provided below: mHMGB4(C45S), which is SEQ ID NO: 115 further comprising an AGT encoding C45S, i.e., SEQ ID NO: 115 further comprises a T to A mutation at nucleotide residue 133 of SEQ ID NO: 115, see SEQ ID NO: 116; mHMGB4(C104S), which is SEQ ID NO: 115 further comprising an AGT encoding C104S, i.e., SEQ ID NO: 115 further comprises a T to A mutation at nucleotide residue 310 of SEQ ID NO: 115 and a C to T mutation at nucleotide residue 312 of SEQ ID NO: 115, see SEQ ID NO: 117; mHMGB4(C164S), which is SEQ ID NO: 115 further comprising an AGT encoding C164S, see SEQ ID NO: NO: 118; mHMGB4 (C178S), which is SEQ ID NO: 115 further comprising AGC encoding C178S, see SEQ ID NO: 119; mHMGB4 (C45S, C104S-double mutant), which is SEQ ID NO: 115 further comprising AGT encoding C45S and AGT encoding C104S, see SEQ ID NO: 120; mHMGB4 (C45S, C164S-double mutant), which is SEQ ID NO: 115 further comprising AGT encoding C45S and AGT encoding C164S, see SEQ ID NO: 121; mHMGB4 (C45S, C178S-double mutant), which is SEQ ID NO: 115 further comprising AGT encoding C45S and AGC encoding C178S, see SEQ ID NO: 122; mHMGB4 (C104S, C164S-double mutant), which is SEQ ID NO: 115 further comprising AGT encoding C104S and AGT encoding C164S, see SEQ ID NO: 123; mHMGB4 (C104S, C178S-double mutant), which is SEQ ID NO: 115 further comprising AGT encoding C104S and AGC encoding C178S, see SEQ ID NO: 124; mHMGB4 (C164S, C178S-double mutant), which is SEQ ID NO: 115 further comprising AGT encoding C164S and AGC encoding C178S, see SEQ ID NO: 125;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. NO: 128; mHMGB4 (C104S, C164S, C178S-triple mutant), which is 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), which is SEQ ID NO: 115 further comprising AGT encoding C45S, AGT encoding C104S, AGT encoding C164S, and AGC encoding C178S, see SEQ ID NO: 130.;

[0213] Another exemplary nucleic acid sequence encoding HMGB4 is provided below: ATGGGAAAAGAAATCCAGCTAAAGCCTAAGGCAAATGTCTCTTCTTACGTTCACTTTTTGCTGAATTACAGAAACAAATTCAAGGAGCAGCAGCCAAATACCTATGTTGGCTTTAAAGAGTTCTCTAGAAAGTGTTCGGAAAAATGGAGATCCATCTCAAAGCATGAAAAGGCCAAATATGAAGCCCTGGCCAAACTCGACAAAGCCCGATACCAGGAAGAAATGATGAATTATGTTGGCAAGAGGAAGAAACGGAGAAAGCGGGATCCCCAGGAACCCAGACGGCCTCCATCATCCTTCCTACTCTTCTGCCAAGACCACTATGCTCAGCTGAAGAGGGAGAACCCGAACTGGTCGGTGGTGCAGGTGGCCAAGGCCACAGGGAAGATGTGGTCAACAGCGACAGACCTGGAGAAGCACCCTTATGAGCAAAGAGTGGCTCTCCTGAGAGCTAAGTACTTCGAGGAACTTGAACTCTACCGTAAACAATGTAATGCCAGGAAGAAGTACCGAATGTCAGCTAGAAACCGGTGCAGAGGGAAAAGAGTCAGGCAGAGCTGA (SEQ ID NO:135).

[0214] Nucleic acid sequences encoding these modified high mobility group-box 4 domains are also provided below: mHMGB4(C45S), which is SEQ ID NO: 135 further comprising an AGT encoding C45S, i.e., SEQ ID NO: 135 further comprises a 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 an AGT encoding C104S, i.e., SEQ ID NO: 135 further comprises a T to A mutation at nucleotide residue 310 of SEQ ID NO: 135 and optionally a C to T mutation at nucleotide residue 312 of SEQ ID NO: 135, see SEQ ID NO: 137; mHMGB4(C164S), which is SEQ ID NO: 135 further comprising an AGT encoding C164S, i.e., SEQ ID NO: 135 further comprises SEQ ID NO: 135, a mutation from T to A at nucleotide residue 490, see SEQ ID NO: 138; mHMGB4 (C178S), which is SEQ ID NO: 135 further comprising AGC encoding C178S, i.e., SEQ ID NO: 135 further comprises a mutation from T to A at nucleotide residue 532 of SEQ ID NO: 135, see SEQ ID NO: 139; mHMGB4 (C45S, C104S-double mutant), which is SEQ ID NO: 135 further comprising AGT encoding C45S and AGT encoding C104S, i.e., SEQ ID NO: 135 further comprises a mutation from T to A at nucleotide residue 133 of SEQ ID NO: 135, a mutation from T to A at nucleotide residue 310 of SEQ ID NO: 135, and optionally a mutation from C to T at nucleotide residue 312 of SEQ ID NO: 135, see SEQ ID NO: 140; mHMGB4 (C45S, C164S-double mutant), which is SEQ ID NO: 135 further comprising AGT encoding C45S and AGT encoding C164S, i.e., SEQ ID NO: 135 further comprises a T to A mutation at nucleotide residue 133 of SEQ ID NO: 135 and a T to A mutation at nucleotide residue 490 of SEQ ID NO: 135, see SEQ ID NO: 141;mHMGB4 (C45S, C178S-double mutant), which is SEQ ID NO: 135 further comprising AGT encoding C45S and AGC encoding C178S, i.e., SEQ ID NO: 135 further comprises a T to A mutation at nucleotide residue 133 of SEQ ID NO: 135, and a T to A mutation at nucleotide residue 532 of SEQ ID NO: 135, see SEQ ID NO: 142; mHMGB4 (C104S, C164S-double mutant), which is SEQ ID NO: 135 further comprising AGT encoding C104S and AGT encoding C164S, i.e., SEQ ID NO: 135 further comprises a T to A mutation at nucleotide residue 310 of SEQ ID NO: 135, optionally a C to T mutation at nucleotide residue 312 of SEQ ID NO: 135, and a T to A mutation at nucleotide residue 532 of SEQ ID NO: 135, see SEQ ID NO: 142. NO: 135, a mutation from T to A at nucleotide residue 490, see SEQ ID NO: 143; mHMGB4 (C104S, C178S-double mutant), which is SEQ ID NO: 135 further comprising AGT encoding C104S and AGC encoding C178S, i.e., SEQ ID NO: 135 further comprises a mutation from T to A at nucleotide residue 310 of SEQ ID NO: 135, optionally a mutation from C to T at nucleotide residue 312 of SEQ ID NO: 135, and a mutation from T to A at nucleotide residue 532 of SEQ ID NO: 135, see SEQ ID NO: 144; mHMGB4 (C164S, C178S-double mutant), which is SEQ ID NO: 135 further comprising AGT encoding C164S and AGC encoding C178S, i.e., SEQ ID NO: 135 further comprises SEQ a mutation from T to A at nucleotide residue 490 of SEQ ID NO: 135 and a mutation from T to A at nucleotide residue 532 of SEQ ID NO: 135, see SEQ ID NO: 145;mHMGB4 (C45S, C104S, C164S-triple mutant), which is SEQ ID NO: 135 further comprising AGT encoding C45S, AGT encoding C104S, and AGT encoding C164S, i.e., SEQ ID NO: 135 further comprises a T to A mutation at nucleotide residue 133 of SEQ ID NO: 135, a T to A mutation at nucleotide residue 310 of SEQ ID NO: 135, optionally a C to T mutation at nucleotide residue 312 of SEQ ID NO: 135, and a T to A mutation at nucleotide residue 490 of SEQ ID NO: 135, see SEQ ID NO: 146; mHMGB4 (C45S, C104S, C178S-triple mutant), which is SEQ ID NO: 1 NO: 135 further comprises AGT encoding C45S, AGT encoding C104S, and AGC encoding C178S, i.e., SEQ ID NO: 135 further comprises a T to A mutation at nucleotide residue 133 of SEQ ID NO: 135, a T to A mutation at nucleotide residue 310 of SEQ ID NO: 135, and optionally a T to A mutation at nucleotide residue 532 of SEQ ID NO: 135, see SEQ ID NO: 147; mHMGB4 (C45S, C164S, C178S-triple mutant), which is SEQ ID NO: 135 further comprising AGT encoding C45S, AGT encoding C164S, and AGC encoding C178S, i.e., SEQ ID NO: 135 further comprises a T to A mutation at nucleotide residue 133 of SEQ ID NO: 135, a T to A mutation at nucleotide residue 490 of SEQ ID NO: 135, and optionally a T to A mutation at nucleotide residue 532 of SEQ ID NO: 135, see SEQ ID NO: 147; mHMGB4 (C45S, C164S, C178S-triple mutant), which is SEQ ID NO: 135 further comprising AGT encoding C45S, AGT encoding C164S, and AGC encoding C178S, i.e., SEQ ID NO: 135 further comprises a T to A mutation at nucleotide residue 133 of SEQ ID NO: 135, a T to A mutation at nucleotide residue 490 of SEQ ID NO: 135, and a T to A mutation at nucleotide residue 532 of SEQ ID NO: 135, see SEQ ID NO: 148; mHMGB4 (C104S, C164S, C178S-triple mutant), which is SEQ ID NO: 135 further comprising AGT encoding C104S, AGT encoding C164S, and AGC encoding C178S, i.e., SEQ ID NO: 135 further comprises a T to A mutation at nucleotide residue 310 of SEQ ID NO: 135, optionally a C to T mutation at nucleotide residue 312 of SEQ ID NO: 135, a T to A mutation at nucleotide residue 490 of SEQ ID NO: 135, and a T to A mutation at nucleotide residue 532 of SEQ ID NO: 135, see SEQ ID NO: 149;mHMGB4 (C45S, C104S, C164S, C178S-quadruple mutant), which is SEQ ID NO: 135 further comprising AGT encoding C45S, AGT encoding C104S, AGT encoding C164S, and AGC encoding C178S, e.g., a T to A mutation at nucleotide residue 133 of SEQ ID NO: 135, SEQ ID NO: 135 further comprising a T to A mutation at nucleotide residue 310 of SEQ ID NO: 135, optionally a C to T mutation at nucleotide residue 312 of SEQ ID NO: 135, a T to A mutation at nucleotide residue 490 of SEQ ID NO: 135, and a T to A mutation at nucleotide residue 532 of SEQ ID NO: 135, see SEQ ID NO: 150.;

[0215] It will be appreciated that among the equivalents of the modified high mobility group box 1 domain, the equivalents are modified high mobility group box 2, modified high mobility group box 3 and modified high mobility group box 4 comprising a cysteine ​​to serine substitution or its equivalent at one or more amino acid positions corresponding to C23S, C45S and C106S of modified high mobility group box 1. For modified high mobility group box 2, the corresponding cysteine ​​residues are at positions 23, 45 and 106; for modified high mobility group box 3, the corresponding cysteine ​​residues are at positions 23, 45 and 104; and for modified high mobility group box 4, the corresponding cysteine ​​residues are at positions 45, 104, 164 and 178. Thus, Applicants believe that for a modified high mobility group-box 1 comprising one or more substitutions selected from C23S, C45S, and C106S, the same applies to the above-mentioned high mobility group-box species having one or more cysteine ​​to serine substitutions at said positions, such as a modified high mobility group-box 2 comprising one or more substitutions selected from C23S, C45S, and C106S; a modified high mobility group-box 3 comprising one or more substitutions selected from C23S, C45S, and C104S; and a modified high mobility group-box 4 comprising one or more substitutions selected from 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 more (as described herein), but with the proviso that the specific substituted amino acids (e.g., C23S, C45S, and / or C106S) are retained.

[0216] An "A box" polypeptide refers to a polypeptide comprising the A box structure 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. Point mutations of one or more amino acids K12, C23, and C45 may be introduced. In some embodiments, the A box polypeptide comprises, consists essentially of, or consists of aa 9 to aa 79 of SEQ ID NO: 51, or an equivalent thereof (e.g., a fragment of an HMGB1 polypeptide aligned with aa 9 to aa 79 of SEQ ID NO: 51 disclosed herein). In some embodiments, the A box polypeptide comprises, consists essentially of, or consists of aa 1 to aa 79 of SEQ ID NO: 51, or an equivalent thereof (e.g., a fragment of an HMGB1 polypeptide aligned with aa 1 to aa 79 of SEQ ID NO: 51 disclosed herein). In some embodiments, the A box polypeptide comprises, consists essentially of, or consists of aa 1 to aa 70 of SEQ ID NO: 51, or an equivalent thereof (e.g., a fragment of an HMGB1 polypeptide disclosed herein that aligns with aa 1 to aa 70 of SEQ ID NO: 51). Examples of A box polypeptides comprise, consist essentially of, or consist of the following sequence:

[0217] MGKGDPKKPRRKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKF (rat) (aa 1 to aa of SEQ ID NO: 132 89) or MGKGDPKKPRRKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKAR (aa 1 to aa70 of SEQ ID NO: 132); or

[0218] MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKF (human) (aa 1 to aa of SEQ ID NO: 51 89) or MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKAR (aa 1 to aa 70 of SEQ ID NO: 51).

[0219] A "B box" polypeptide refers to a polypeptide comprising the B box structure of an HMGB1 protein. The B box polypeptide may be mutated or comprise additional sequences, such as a linker sequence, a signal sequence, or a secretory sequence. Point mutations of amino acids K114 or C106 may be introduced to affect DNA binding, inflammatory properties, and anti-biofilm activity. In some embodiments, the B box polypeptide comprises, consists essentially of, or consists of aa 95 to aa 163 of SEQ ID NO: 51, or an equivalent thereof (e.g., a fragment of an HMGB1 polypeptide aligned with aa 95 to aa 163 of SEQ ID NO: 51 disclosed herein). In some embodiments, the B box polypeptide comprises, consists essentially of, or consists of aa 88 to aa 164 of SEQ ID NO: 51, or an equivalent thereof (e.g., a fragment of an HMGB1 polypeptide aligned with aa 88 to aa 164 of SEQ ID NO: 51 disclosed herein). In some embodiments, the B box polypeptide comprises, consists essentially of, or consists of aa 80 to aa 164 of SEQ ID NO: 51, or an equivalent thereof (e.g., a fragment of an HMGB1 polypeptide aligned with aa 80 to aa 164 of SEQ ID NO: 51 disclosed herein). In some embodiments, the B box polypeptide comprises, consists essentially of, or consists of aa 80 to aa 176 of SEQ ID NO: 51, or an equivalent thereof (e.g., a fragment of an HMGB1 polypeptide aligned with aa 80 to aa 176 of SEQ ID NO: 51 disclosed herein). In some embodiments, the B box polypeptide comprises, consists essentially of, or consists of aa 90 to aa 176 of SEQ ID NO: 51, or an equivalent thereof (e.g., a fragment of an HMGB1 polypeptide aligned with aa 90 to aa 176 of SEQ ID NO: 51 disclosed herein). In some embodiments, the B box polypeptide comprises, consists essentially of, or consists of aa 89 to aa 162 of SEQ ID NO: 51, or an equivalent thereof (e.g., a fragment of an HMGB1 polypeptide disclosed herein that aligns with aa 89 to aa 162 of SEQ ID NO: 51). Examples of B box polypeptides comprise, consist essentially of, or consist of the following sequence:

[0220] KDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAEKLKEKYEKDIAAYRAKGKPDAAKKGVV (mouse) (aa 90 to aa of SEQ ID NO: 132 176); or KDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVV (human) (aa 90 to aa 176 of SEQ ID NO: 51).

[0221] A "C box" polypeptide refers to a polypeptide comprising the C-tail domain of an HMGB1 protein. The C-tail polypeptide may be mutated or comprise 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 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 that aligns with aa 186 to aa 215 of SEQ ID NO: 51). In some embodiments, the C box polypeptide comprises, consists essentially of, or consists of EEEDEEDEEDEE EDEEDEEE DDDDE (SEQ ID NO: 133).

[0222] By "AB box" polypeptide is meant a polypeptide comprising the A and B box domains of an HMGB1 protein fused together, but lacking the amino acids corresponding to 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 of the amino acids described herein (e.g., at amino acids K12, C23, C45, C106, and / or K114) may be introduced to affect DNA binding, inflammatory properties, and anti-biofilm activity. In some embodiments, the AB box polypeptide comprises, consists essentially of, or consists of aa 1 to aa 176 of SEQ ID NO: 51, or an equivalent thereof (e.g., a fragment of an HMGB1 polypeptide disclosed herein that aligns with aa 1 to aa 176 of SEQ ID NO: 51). In some embodiments, the AB box polypeptide comprises, consists essentially of, or consists of aa 1 to aa 162 of SEQ ID NO: 51, or an equivalent thereof (e.g., a fragment of an HMGB1 polypeptide disclosed herein that aligns with aa 1 to aa 162 of SEQ ID NO: 51). In some embodiments, the AB box polypeptide comprises, consists essentially of, or consists of aa 1 to aa 164 of SEQ ID NO: 51, or an equivalent thereof (e.g., a fragment of an HMGB1 polypeptide disclosed herein that aligns with aa 1 to aa 164 of SEQ ID NO: 51).

[0223] In some embodiments, an equivalent of an HMGB1 polypeptide described herein may comprise, consist essentially of, or consist of an HMG-box domain truncation and / or mutant, or a protein or fragment thereof containing one or more HMG-box structures, truncations, mutants, or equivalents of a protein or fragment having the disclosed amino acid substitutions. In some embodiments, a fragment of an HMGB1 polypeptide comprises, consists essentially of, or consists of one or more of an A box polypeptide, a B box polypeptide, or an AB box polypeptide as disclosed herein.

[0224] In some embodiments, the A box polypeptide may further comprise, consist essentially of, or consist of one or more amino acid mutations selected from K12, C23, and C45 (e.g., native K or C is modified to an amino acid selected from serine, glycine, alanine, valine, isoleucine, or threonine) or its equivalent, the equivalent comprising one or more amino acid mutations selected from K12, C23, and C45, for example, native K or C is modified to an amino acid selected from serine, glycine, alanine, valine, isoleucine, or threonine. In one aspect, the mutation is a C45S mutation. The A box polypeptide may further comprise a linker or peptide sequence located at one or both ends. An example of a peptide linker is PPKGETKKKF (SEQ ID NO: 131).

[0225] When recombinantly produced, the A box polypeptide can be partially or fully acetylated, oxidized, or phosphorylated using methods known in the art, such as, for example, 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. al. (2007) JTH, 5(1): 109-16. doi: 10.1111 / j.1538-7836.2006.02255.x. PubMed PMID: 17239166. In one aspect, the A box polypeptide comprises, consists essentially of, or consists of amino acids 1 to 70 of a wild-type HMGB1 polypeptide carrying the above-mentioned mutations.

[0226] In some embodiments, the B box polypeptide may comprise, consist essentially of, or further consist of a mutation at amino acid C106 or K114 or both (e.g., a native cysteine ​​is changed to an amino acid selected from the group consisting of serine, glycine, alanine, valine, isoleucine, or threonine), or an equivalent thereof (e.g., a native cysteine ​​is changed to an amino acid selected from the group consisting of serine, glycine, alanine, valine, isoleucine, or threonine). In one aspect, the B box polypeptide comprises amino acids about 80 to about 176, or about 88 to about 164, or about 89 to about 162, or further about 80 to about 164 of a wt HMGB1 polypeptide carrying the above-mentioned mutations.

[0227] The B box polypeptide may further comprise a linker or peptide sequence located at one or both ends. An example of a peptide linker is PPKGETKKKF (SEQ ID NO: 131). When recombinantly produced, the disclosed B box polypeptides can be partially or fully acetylated, oxidized, or phosphorylated using methods known in the art, such as, for example, 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. al.(2007)JTH,5(1):109-16.doi:10.1111 / j.1538-7836.2006.02255.x.PubMed PMID:17239166.

[0228] In some embodiments, the AB box polypeptide may comprise one or more amino acid mutations selected from K12, C23, C45, C106 or K114 (e.g., native K or C is modified to an amino acid selected from serine, glycine, alanine, valine, isoleucine or threonine) or an equivalent thereof, the equivalent comprising, consisting essentially of, or also consisting of one or more amino acid mutations selected from K12, C23, C45, C106 or K114 (e.g., native K or C is modified to an amino acid selected from serine, glycine, alanine, valine, isoleucine or threonine). In one aspect, the mutation is a C45S mutation. In another aspect, the polypeptide comprises a mutation at amino acid C106 (e.g., a native cysteine ​​is changed to an amino acid selected from serine, glycine, alanine, valine, isoleucine or threonine) or its equivalent, the equivalent comprising one or more amino acid mutations selected from K12, C23, C45 and a mutation at amino acid C106 (e.g., a native cysteine ​​is changed to an amino acid selected from serine, glycine, alanine, valine, isoleucine or threonine). In one aspect, the AB box polypeptide comprises C45S and C106S mutations and equivalents that retain these mutations. In one aspect, the AB box polypeptide comprises, or consists essentially of, or consists of, amino acids 1 to 176, or 1 to 162, or further 1 to 164 of a wild-type HMGB1 polypeptide carrying the above mutations.

[0229] In some embodiments, the AB box polypeptide further comprises a linker polypeptide positioned between the A box polypeptide and the B box polypeptide, and in one aspect, a second linker linking the B box and C box polypeptides. 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, the isolated mutant HMGB1 polypeptide provides one or more amino acid substitutions as described herein in the A and / or B box domains, which can be optionally partially or fully acetylated, oxidized or phosphorylated using methods known in the art, such as, for example, 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.

[0230] Examples of AB box polypeptides comprise, consist essentially of, or consist of the following sequence with the aforementioned mutations: MGKGDPKKPRRKMSSYAFFVQTCREEHKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYKEREMKTYIPPKGETKKFKDPNKRAPPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAEKLKEKKYEKDIAAYRAKGKPDAAKKGVV (murine) (SEQ ID NO: 132); or

[0231] MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVV (Human) (aa 1 to aa 176 of SEQ ID NO: 51).

[0232] In another aspect, the AB box polypeptide further comprises a linker polypeptide located between the A box polypeptide and the B box polypeptide.

[0233] In some embodiments, the HMGB1 polypeptide or fragment thereof comprises, consists essentially of, or consists of the A, B, and C domains, wherein the polypeptide comprises one or more amino acid mutations selected from K12, C23, C45, C106, or K114, or consists essentially of, or consists of, an equivalent thereof, the equivalent thereof comprising one or more amino acid mutations selected from K12, C23, C45, C106, or K114.

[0234] As used herein, an equivalent of a polypeptide refers to a sequence that is 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% identical to a reference polypeptide, which in one aspect retains the mutated amino acid. In certain aspects, an equivalent of a polypeptide retains the expected function and / or structural features of the polypeptide, such as containing an HMG-box domain and optionally not inducing a pro-inflammatory response. In one aspect, the equivalent polypeptide comprises a domain that is at least about 70%, 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% identical to the HMG-box domain, which in one aspect retains the mutated amino acid. In certain aspects, such an equivalent domain retains the function and / or structural features of the HMB-box domain, such as binding to an HMB-box binding target, but optionally not inducing a pro-inflammatory response. In one aspect, the equivalent polypeptide is encoded by a polynucleotide capable of hybridizing to a polynucleotide encoding a polypeptide of the HMB-box domain under stringent conditions.

[0235] In some embodiments, the HMGB1 Box polypeptide further comprises a linker polypeptide positioned 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).

[0236] Immunodominant antigens are regions of proteins that are recognized and bound by antibodies with high affinity.

[0237] Immunoprotective antigens are regions of proteins that are recognized and bound with high affinity by antibodies, interfering with the protein's function; antibodies raised against immunoprotective antigens are characterized by their ability to enhance or optimize their effect on the targeted indication—in this case, the ability to clear biofilms—due to interference with protein function.

[0238] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, which can be deoxynucleotides or ribonucleotides or their analogs. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST 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. Modifications to the nucleotide structure (if any) can be given before or after assembly of the polynucleotides. Non-nucleotide components can interrupt the sequence of nucleotides. Polynucleotides can be further modified after polymerization, for example, by joining with labeling components. The term also refers to double-stranded and single-stranded molecules. Unless otherwise specified or required, any polynucleotide embodiment disclosed herein encompasses the double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0239] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, when the polynucleotide is RNA, uracil (U) for thymine. Thus, the term "polynucleotide sequence" is an alphabetic representation of a polynucleotide molecule. This alphabetic representation can be entered into a database in a computer with a central processing unit and used in bioinformatics applications (e.g., functional genomics and homology searching).

[0240] As used herein, the terms "isolated" or "recombinant" with respect to nucleic acids (e.g., DNA or RNA) refer to molecules that are separated from other DNA or RNA and polypeptides, respectively, that are present in the natural macromolecular source. The term "isolated or recombinant nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring fragments and are not found in nature. The term "isolated" is also used herein to refer to polynucleotides, polypeptides, and proteins that have been separated from other cellular proteins, and is intended to encompass purified and recombinant polypeptides. In other embodiments, the term "isolated or recombinant" refers to separation from cells and other components, wherein cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof, are normally associated in nature. For example, an isolated cell is a cell that is separated from a tissue or cell of a different phenotype or genotype. An isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated in its natural or native environment (e.g., on a chromosome). It will be apparent to one skilled in the art that a non-naturally occurring polynucleotide, polypeptide, peptide, protein, antibody, or fragment thereof does not require "isolation" to distinguish it from its naturally occurring counterpart.

[0241] As used herein, the term "isolated" refers to a molecule, organism, cellular material, cell, or biological sample that is substantially free of other materials. In one aspect, the term "isolated" refers to a nucleic acid, such as DNA or RNA, or a protein or polypeptide (e.g., an antibody or derivative thereof), or a cell or organelle, or a tissue or organ, that is separated from other DNA or RNA, or protein or polypeptide, or cell or organelle, or tissue or organ, respectively, present in its natural source.

[0242] In some embodiments, the term "engineered" refers to comprising at least one modification not normally found in a naturally occurring counterpart, wild type, or parent. In some embodiments, the term "engineered" is used interchangeably with "recombinant" to refer to synthesized by humans.

[0243] Unless otherwise specified, it is not necessary to explicitly state that when the present disclosure relates to polypeptides, proteins, polynucleotides or antibodies, it is intended that equivalents or biological equivalents of this type are also within the scope of the present disclosure. As used herein, when referring to a reference protein, antibody, fragment, polypeptide or nucleic acid, the term "biological equivalent thereof" is intended to be synonymous with "equivalent thereof," i.e., those proteins, antibodies, fragments, polypeptides or nucleic acids that have minimal homology and still retain the desired structure or function. Unless specifically enumerated herein, any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. In one aspect, an equivalent polynucleotide is a polynucleotide that hybridizes under stringent conditions to a polynucleotide for the method as described herein or to a complementary sequence of a polynucleotide. In another aspect, an equivalent antibody or antigen-binding polypeptide refers to a polynucleotide that binds to a reference antibody or antigen-binding fragment with at least 70%, or at least 75%, or at least 80%, or to 85%, or at least 90%, or at least 95% affinity or higher. In another aspect, the equivalents compete with the binding of the antibody or antigen-binding fragment to its antigen in a competitive ELISA assay. In another aspect, the equivalents are intended to have at least about 80% homology or identity, or at least about 85%, or at least about 90%, or at least about 95%, or 98% homology or identity to the reference protein, polypeptide, or nucleic acid, and exhibit substantially equivalent biological activity. Table 9 of WO 2011 / 123396 provides examples of biologically equivalent polypeptides that recognize conservative amino acid substitutions of the disclosed amino acid sequences.

[0244] In some aspects, the equivalent of an amino acid sequence comprises a polypeptide having at least 80% amino acid identity with the amino acid sequence, and / or wherein the equivalent of an amino acid sequence comprises a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with the complement of the polynucleotide encoding the amino acid sequence. In another aspect, the equivalent of an amino acid sequence comprises a polypeptide having at least 90% amino acid identity with the amino acid sequence, and / or wherein the equivalent of an amino acid sequence comprises a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with the complement of the polynucleotide encoding the amino acid sequence. In another aspect, the equivalent of an amino acid sequence comprises a polypeptide having at least 95% amino acid identity with the amino acid sequence, and / or wherein the equivalent of an amino acid sequence comprises a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with the complement of the polynucleotide encoding the amino acid sequence. In one aspect, the equivalent of an amino acid sequence comprises a polypeptide having at least 96% amino acid identity with the amino acid sequence, and / or wherein the equivalent of an amino acid sequence comprises a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with the complement of the polynucleotide encoding the amino acid sequence. In another aspect, an equivalent of an amino acid sequence comprises a polypeptide having at least 97% amino acid identity with the amino acid sequence, and / or wherein the equivalent of the amino acid sequence comprises a polypeptide encoded by a polynucleotide that hybridizes under high stringency to the complement of the polynucleotide encoding the amino acid sequence. In another aspect, an equivalent of an amino acid sequence comprises a polypeptide having at least 98% amino acid identity with the amino acid sequence, and / or wherein the equivalent of the amino acid sequence comprises a polypeptide encoded by a polynucleotide that hybridizes under high stringency to the complement of the polynucleotide encoding the amino acid sequence. In one aspect, an equivalent of an amino acid sequence comprises a polypeptide having at least 99% amino acid identity with the amino acid sequence, and / or wherein the equivalent of the amino acid sequence comprises a polypeptide encoded by a polynucleotide that hybridizes under high stringency to the complement of the polynucleotide encoding the amino acid sequence.

[0245] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" to another sequence, which means that, when aligned, the bases (or amino acids) of that percentage are the same when comparing the two sequences. Alignment and homology or percentage of sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. In certain embodiments, default parameters are used for alignment. A non-limiting exemplary comparison program is BLAST, using default parameters. In particular, exemplary programs include BLASTN and BLASTP, using the following default parameters. Genetic code = standard; filter = none; strand = duplex; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; ranking = high score; databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following web address: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percent identity are determined by incorporating it into clustalW (available at align.genome.jp, last accessed March 7, 2011). In some embodiments, Clustal Omega, available at www.ebi.ac.uk / Tools / msa / clustalo / In the example of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 are used for sequence alignment or to determine percent identity. In a further embodiment, the default settings are applied.

[0246] "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 single position in each sequence, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then 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 has less than 40% identity, or less than 25% identity, to one of the sequences of the present disclosure.

[0247] "Homology" or "identity" or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions.

[0248] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonding between the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific manner. The complex can include two chains that form a double-stranded structure, three or more chains that form a multi-chain complex, a single self-hybridizing chain, or any combination of these. The hybridization reaction can constitute a step in a broader process (e.g., the initiation of a PCR reaction or the digestion of a polynucleotide by a ribozyme).

[0249] Examples of stringent hybridization conditions include: an incubation temperature of about 25°C to about 37°C; a hybridization buffer concentration of about 6×SSC to about 10×SSC; a formamide concentration of about 0% to about 25%; and a wash solution of about 4×SSC to about 8×SSC. Examples of moderate hybridization conditions include: an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9×SSC to about 2×SSC; a formamide concentration of about 30% to about 50%; and a wash solution of about 5×SSC to about 2×SSC. Examples of highly stringent conditions include: an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1×SSC to about 0.1×SSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1×SSC, 0.1×SSC, or deionized water. Generally, hybridization incubation times range from 5 minutes to 24 hours, with one, two, or more wash steps, and wash incubation times of about 1, 2, or 15 minutes. SSC is a buffer containing 0.15M NaCl and 15mM citrate. It will be appreciated that the equivalent of SSC using other buffer systems may be employed.

[0250] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in a eukaryotic cell.

[0251] The term "encoding" as applied to a polynucleotide means that if the polynucleotide, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA and / or fragments thereof for a polypeptide, then the polynucleotide is said to "encode" a polypeptide. The antisense strand is the complementary sequence of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0252] As used herein, the terms "treat," "treat," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive, i.e., preventing a disorder or its signs or symptoms in whole or in part, and / or may be therapeutic, i.e., curing a disorder and / or the adverse effects attributable to the disorder in part or in whole. As used herein, "treating" or "managing" a subject's disease may also refer to (1) preventing symptoms or the disease from occurring in a subject who is predisposed to or who does not yet show symptoms of the disease; (2) inhibiting the disease or arresting its progression; or (3) ameliorating or causing regression of the disease or disease symptoms. As understood in the art, "treatment" is a method of obtaining a beneficial or desired result (including a clinical result). For purposes of the present technology, a beneficial or desired result may include one or more, but is not limited to: alleviation or improvement of one or more symptoms, a decrease in the extent of a condition (including a disease), stabilization (i.e., non-worsening) of a condition (including a disease), a delay or slowing of a condition (including a disease), progression, improvement, or alleviation of a condition (including a disease) state, and alleviation (whether partial or complete), whether detectable or undetectable. In one aspect, treatment does not include prevention.

[0253] Prevention refers to the prevention of a disorder or effect, in vitro or in vivo, in a system or subject susceptible to the disorder or effect. An example of this is preventing biofilm formation in a system infected with microorganisms known to produce biofilms.

[0254] "Composition" means a combination of an active ingredient and another compound or composition, which is inert (e.g., a detectable agent or label) or active (e.g., an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant or the like), 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, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides and oligosaccharides; derivatized sugars (e.g., aldehydes, aldonic acids, esterified sugars, etc.); and polysaccharides or carbohydrate polymers), which can be present alone or in combination, comprising 1-99.99% by weight or volume alone or in combination. Exemplary protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, etc. Representative amino acid / antibody components that can also play a buffering role include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. Carbohydrate excipients are also within the scope of the present 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 (glucose), and inositol.

[0255] "Pharmaceutical composition" means a composition comprising the active ingredients in combination with an inert or active carrier, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0256] "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 (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glycerol mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, a standard reference in this field by Mack Publishing Company. They can be selected according to the desired form of administration, i.e., oral tablets, capsules, elixirs, syrups, etc., and in accordance with common pharmaceutical practice.

[0257] The compositions used according to the present disclosure can be packaged into dosage unit forms for the purpose of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to a physically discontinuous unit suitable for use in an object, each unit comprising the amount of a predetermined composition that is calculated to produce the desired reaction associated with its administration, i.e., appropriate approach and course of treatment. Depending on the number of treatments and the unit dose, the amount administered depends on the desired result and / or protection. The precise amount of the composition also depends on the doctor's judgment and is specific to each individual. Factors affecting dosage include the physical and clinical state of the object, route of administration, expected therapeutic goals (symptom relief and cure), and the effectiveness, stability, and toxicity of the specific ingredients. When formulated, the solution is administered in a manner compatible with the dosage formulation and in an amount having a therapeutic or preventive effect. The formulation can be easily administered in various dosage forms (e.g., injection solution types as described herein).

[0258] As used herein, a combination means that the individual active ingredients of the composition are formulated separately for use in combination and may be packaged separately with or without specific dosages. The active ingredients of the combination may be administered simultaneously or sequentially.

[0259] "Biologically active agent" or active agent disclosed herein means one or more of an isolated or recombinant polypeptide, an isolated or recombinant polynucleotide, a vector, an isolated host cell, or an antibody, as well as compositions comprising one or more of these.

[0260] "Administration" can be carried out in the form of a single dose, continuously or intermittently, throughout the course of treatment. Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for treatment, the purpose of treatment, the target cell being treated, and the subject being treated. Single or multiple administrations can be performed, with the dosage level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administration are known in the art. Route of administration can also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and will vary with the composition used for treatment, the purpose of treatment, the health condition or disease stage of the subject being treated, and the target cell or tissue. Non-limiting examples of route of administration include oral administration, nasal administration, injection, and topical application.

[0261] The agents of the present disclosure may be administered by any suitable route of administration. It will also be appreciated that the optimal route will vary with the condition and age of the recipient and the disease being treated.

[0262] As used herein, the term "contacting" refers to the direct or indirect binding or interaction between two or more molecules. A specific example of a direct interaction is binding. A specific example of an indirect interaction is one entity acting on an intermediary molecule, which in turn acts on a second reference entity. As used herein, contacting includes contacting in solution, in a solid phase, in vitro, ex vivo, in a cell, and in vivo. In vivo contacting can be referred to as administration or dosing.

[0263] The term "effective amount" refers to an amount sufficient to achieve the desired effect. In the case of therapeutic or preventive applications, the effective amount will depend on the type and severity of the condition in question and the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. In the case of immunogenic compositions, in some embodiments, the effective amount is an amount sufficient to cause a protective response to the pathogen. In other embodiments, the effective amount of the immunogenic composition is an amount sufficient to cause the production of antibodies against the antigen. In some embodiments, the effective amount is the amount required to confer passive immunity to a subject in need. With regard to immunogenic compositions, in some embodiments, in addition to the factors mentioned above, the effective amount will depend on the intended use, the degree of immunogenicity of the specific antigenic compound, and the health / reactivity of the subject's immune system. Those skilled in the art will be able to determine the appropriate amount based on these and other factors.

[0264] In the case of in vitro applications, in some embodiments, the effective amount will depend on the size and nature of the application. It will also depend on the nature and sensitivity of the in vitro target and the method used. Those skilled in the art will be able to determine the effective amount based on these and other considerations. The effective amount can include one or more applications of the composition, depending on the embodiment.

[0265] The term "contacting" refers to the direct or indirect binding or interaction between two or more molecules. A specific example of a direct interaction is binding. A specific example of an indirect interaction is one entity acting on an intermediary molecule, which in turn acts on a second reference entity. As used herein, contacting includes contacting in solution, in a solid phase, in vitro, ex vivo, in a cell, and in vivo. In vivo contacting can be referred to as administration or dosing.

[0266] The term "conjugate moiety" refers to a group 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 bound to the residue of the chimeric polypeptide, or it can form a covalent bond with a linker that in turn forms a covalent bond with a residue of the chimeric polypeptide.

[0267] "Peptide conjugate" refers to the association of one or more polypeptides with another chemical or biological compound through covalent or non-covalent bonding. In one non-limiting example, "conjugation" of a polypeptide to a chemical compound results in increased stability or efficacy of the polypeptide for its intended purpose. In one embodiment, the peptide is conjugated to a carrier, wherein the carrier is a liposome, micelle, or pharmaceutically acceptable polymer.

[0268] "Liposomes" are microscopic vesicles composed of concentric lipid bilayers. Structurally, liposomes vary in size and shape from long tubes to spheres, and in dimensions from a few hundred angstroms to a fraction of a millimeter. The lipids that form the vesicles are selected to achieve a specific degree of fluidity or rigidity in the final complex, thereby providing the lipid composition of the outer layer. These are neutral (cholesterol) or bipolar, including phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and sphingomyelin (SM)) and other types of bipolar lipids, including but not limited to dioleoylphosphatidylethanolamine (DOPE), with hydrocarbon chain lengths between 14 and 22, saturated or with one or more double C═C bonds. Examples of phospholipids that can generate stable liposomes alone or in combination with other lipid components are phospholipids such as hydrogenated soybean phosphatidylcholine (HSPC), phosphatidylcholine, phosphatidylethanolamine, lysolecithin, lysolecithin, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, distearoylphosphatidylethan-olamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimido-triethyl)cyclohexane-1-carboxylate (DOPE-mal). Other non-phosphorus-containing lipids that can be incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl aryl sulfate, acetyl palmitate, glyceryl ricinoleate, stearic acid hexadecyl ester, amphoteric acrylic polymers, polyethoxylated fatty acid amides and above-mentioned cationic lipids (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol). Negatively charged lipids include phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), disulfide phosphatidylglycerol and (DOPG) and hexacosyl phosphate that can form vesicles. Usually, liposomes can be divided into three classes according to the character of their overall size and lamellar structure. According to the December 1977 New York Academy Sciences Meeting, "Liposomes and Their Use in Biology and Medicine," the three classifications are multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), and large unilamellar vesicles (LUVs).Biologically active agents can be encapsulated therein for administration according to the methods described herein.

[0269] A "micelle" is a collection of surfactant molecules dispersed in a liquid colloid. Typical micelles in aqueous solution form aggregates with a hydrophilic "head" region in contact with the surrounding solvent, isolating the hydrophobic tail region in the center of the micelle. This type of micelle is called a normal micelle (oil-in-water micelle). A reversed micelle has a head in the center and a tail extending outward (water-in-oil micelle). Micelles can be used to link polynucleotides, polypeptides, antibodies, or compositions described herein to facilitate efficient delivery to target cells or tissues.

[0270] The phrase "pharmaceutically acceptable polymer" refers to a group of compounds that can be conjugated to one or more polypeptides described herein. It is expected that conjugation of polymers to polypeptides can extend the half-life of the polypeptides in vivo and in vitro. Non-limiting examples include polyethylene glycol, polyvinyl pyrrolidone, 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.

[0271] A "gene delivery vector" is defined as any molecule that can carry an inserted polynucleotide into a host cell. Examples of gene delivery vectors are liposomes, micelles, biocompatible polymers, including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria or viruses, such as baculoviruses, adenoviruses, and retroviruses, phages, cosmids, plasmids, fungal vectors, and other recombinant vectors commonly used in the art that have been described for expression in a variety of eukaryotic and prokaryotic hosts and can be used for gene therapy as well as simple protein expression.

[0272] The polynucleotides disclosed herein can be delivered to cells or tissues or objects using gene delivery vectors. As used herein, "gene delivery," "gene transfer," "transduction," etc. refer to the terminology of introducing exogenous polynucleotides (sometimes referred to as "transgenes") into host cells, regardless of the method of introduction. This method includes a variety of well-known techniques, such as vector-mediated gene transfer (by, for example, viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes) and the technology (such as electroporation, "gene gun" delivery, and various other technologies for introducing polynucleotides) that promotes the delivery of "naked" polynucleotides. The introduced polynucleotides can be stably or transiently maintained in the host cell. Stable maintenance generally requires that the introduced polynucleotides contain a replication origin compatible with the host cell or be integrated into the replicon of the host cell, such as an extrachromosomal replicon (such as a plasmid) or a nuclear or mitochondrial chromosome. As known in the art and described herein, many vectors are known to be able to mediate the transfer of genes to mammalian cells.

[0273] As used herein, the terms "regulatory sequence," "regulatory element," "expression control element," or "promoter" refer 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.

[0274] As used herein, the term "promoter" refers to any sequence that regulates the expression of a coding sequence (e.g., a gene). For example, a promoter can be constitutive, inducible, repressible, or tissue-specific. A "promoter" is a control sequence in a region where the initiation and rate of transcription of a polynucleotide sequence are controlled. It can contain regulatory proteins and molecules that can bind to genetic elements such as RNA polymerase and other transcription factors. Non-limiting examples of promoters include the cytomegalovirus CMV promoter or the retroviral long terminal repeat (LTR) promoter. See, for example, Weber et al. Hum Gene Ther. 2007 Sep; 18(9): 849-60.

[0275] Enhancers are regulatory elements that increase 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" or "exogenous" or "heterologous." An "endogenous" enhancer / promoter is naturally connected to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is arranged in parallel with a gene by genetic manipulation (i.e., molecular biology techniques) so that the connected enhancer / promoter guides the transcription of the gene.

[0276] As used herein, the term "cDNA" refers to extracellular DNA found as a component of pathogenic biofilms.

[0277] A "plasmid" is an extrachromosomal DNA molecule that is separate from chromosomal DNA and capable of replicating independently of it. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a microbial population and often provide a selective advantage under given environmental conditions. Plasmids can carry genes that confer resistance to naturally occurring antibiotics in competitive environments or produce proteins that can act as toxins under similar circumstances.

[0278] A "plasmid" used in genetic engineering is called a "plasmid vector." Many plasmids are commercially available for this purpose. The gene to be replicated is inserted into multiple copies of a plasmid containing a gene that makes the cell resistant to a specific antibiotic and a multiple cloning site (MCS or polylinker), a short region containing several commonly used restriction sites that allows DNA fragments to be easily inserted at this location. Another major use of plasmids is to produce large quantities of proteins. In this case, researchers grow bacteria that contain a plasmid with the gene of interest. Just as the bacterium produces proteins to confer antibiotic resistance, it can also be induced to produce large quantities of protein from the inserted gene. This is a cheap and simple way to mass-produce a gene or encoded protein.

[0279] "Yeast artificial chromosome" or "YAC" refers to a vector used to clone large DNA fragments (greater than 100kb and up to 3000kb). It is an artificially constructed chromosome that contains the telomere, centromere and replication origin sequences required for replication and maintenance in yeast cells. It is constructed using an initial circular plasmid, which is linearized using restriction endonucleases, and then DNA ligase can add the sequence or gene of interest within the linear molecule through the use of sticky ends. Yeast expression vectors such as YAC, YIp (yeast integrating plasmid) and YEp (yeast episomal plasmid) are very useful because yeast itself is a eukaryotic cell, so eukaryotic protein products with post-translational modifications can be obtained, but YACs have been found to be more unstable than BACs and produce chimeric effects.

[0280] "Viral vector" is defined as a recombinantly produced virus or viral particle containing a polynucleotide to be delivered into a host cell in vivo, in vitro or in vitro. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, alphaviral vectors, and the like. Vectors based on the infectious tobacco mosaic virus (TMV) can be used to produce proteins, and Griffithsin has been reported to be expressed in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15): 6099-6104). Alphaviral vectors, such as those based on Semliki Forest virus and those based on Sindbis virus, have also been developed for 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 the context of gene transfer mediated by retroviral vectors, a vector construct refers to a polynucleotide comprising the retroviral genome or a portion thereof and the therapeutic gene.

[0281] As used herein, "retroviral-mediated gene transfer" or "retroviral transduction" have the same meaning and refer to the process of stably transferring a gene or nucleic acid sequence into a host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. Viruses can enter host cells through their normal infection mechanisms or by being modified to bind to different host cell surface receptors or ligands. As used herein, a retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or virus-like entry mechanism.

[0282] Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into DNA and integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0283] In aspects where gene transfer is mediated by a DNA viral vector, such as adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to a polynucleotide comprising the viral genome or portion thereof and a transgene. Adenovirus (Ad) is a relatively well-characterized group of homologous viruses that includes more than 50 serotypes. See, for example, PCT International Application Publication No. WO 95 / 27071. Ad does not need to be integrated into the host cell genome. Recombinant Ad-derived vectors have also been constructed, particularly those that reduce the likelihood of recombination and production of wild-type virus. See, PCT International Application Publication Nos. WO 95 / 00655 and WO 95 / 11984, wild-type AAV has high infectivity and specificity for integration into the host cell genome. See, Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0284] Vectors containing promoters and cloning sites operably linked to polynucleotides are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). In order to optimize expression and / or in vitro transcription, it may be necessary to remove, increase, or alter the 5′ and / or 3′ untranslated portion of the clone to eliminate additional, potentially inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression at the transcription or translation level. Alternatively, a consensus ribosome binding site may be inserted immediately 5′ of the start codon to enhance expression.

[0285] Gene delivery vectors also include DNA / liposome complexes, micelles, and targeted viral protein-DNA complexes. Liposomes further comprising targeted antibodies or antigen-binding fragments thereof can be used in the methods disclosed herein. In addition to delivering polynucleotides to cells or cell populations, proteins described herein can be directly introduced into cells or cell populations by non-limiting techniques such as protein transfection, or, other non-limiting techniques, can be cultured under conditions that enhance expression of the proteins disclosed herein and / or promote activity of the proteins disclosed herein.

[0286] As used herein, the terms "antibody" and "immunoglobulin" include complete antibodies and any antigen-binding fragments or single chains thereof. Thus, the term "antibody" includes any protein- or peptide-containing molecule that comprises at least a portion of an immunoglobulin molecule. The terms "antibody" and "immunoglobulin" also include immunoglobulins of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fab', F(ab)2, Fv, scFv, dsFv, Fd fragments, dAb, VH, VL, VhH and V-NAR domains, minibodies, diabodies, triabodies, tetrabodies and kappa antibodies; and multispecific antibody fragments formed from one or more isolated antibody fragments. Such embodiments include but are not limited to the complementarity determining regions (CDRs) of heavy or light chains or their ligand-binding portions, heavy or light chain variable regions (also referred to herein as variable domains), and heavy or light chain constant regions (also referred to herein as constant domains). The term "anti-IHF" refers to a protein-binding domain comprising at least one of a chimeric antibody, a humanized antibody, a single-chain antibody, and a fusion protein comprising the antigen-binding portion thereof and non-antibody proteins. The heavy chain and light chain variable regions of immunoglobulin molecules comprise an interactive binding domain with the antigen. The constant domains of antibody (Ab) can mediate the combination of immunoglobulin and host tissue. For example, when using the term "anti-" before the protein name, for example, anti-DNABII, anti-IHF, anti-HU, anti-OMP P5, refer to monoclonal or polyclonal antibodies that bind to specific protein and / or to specific protein. For example, "anti-IHF" refers to an antibody that is bound to the IHF protein. Specific antibodies may have affinity or combination with the protein beyond the protein to which it is directed. For example, although anti-IHF produces at the IHF protein specificity, it may also be combined with other proteins related to sequence homology or structural homology.

[0287] Complementarity determining region (CDR) is a part of the variable region of the antibody or T cell receptor produced by B cells and T cells, respectively, wherein these molecules are combined with their specific antigen (also referred to as epitope). In certain embodiments, the terms "variable region" and "variable domain" are used interchangeably and refer to the polypeptide of the antibody light chain or heavy chain, the sequence of amino acid residues of which varies greatly from one antibody to another and determines the conformation of the binding site, which gives the antibody specificity for a particular antigen. In another embodiment, the variable region length is about 90 amino acids long to about 200 amino acids long, including but not limited to about 100 amino acids long, or about 110 amino acids long, or about 120 amino acids long, or about 130 amino acids long, or about 140 amino acids long, or about 150 amino acids long, or about 160 amino acids long, or about 170 amino acids long, or about 180 amino acids long, or about 190 amino acids long. In certain embodiments, the variable region of an amino acid sequence as used herein refers to the first about 100 amino acids, or about 110 amino acids, or, about 120 amino acids, or about 130 amino acids, or about 140 amino acids, or about 150 amino acids of an amino acid sequence (including or excluding the signal peptide, if applicable) are the variable region.

[0288] A group of CDRs constitutes a paratope, also known as an antigen binding site, which is the part of an antibody that recognizes and binds to an antigen. There are three non-contiguously arranged CDRs (CDR1, CDR2, and CDR3) on the variable region amino acid sequence of an antigen receptor, such as a heavy chain or a light chain, optionally from the amino terminus to the carboxyl terminus. As used herein, CDRn refers to a CDRn in an immunoglobulin chain or derived from an immunoglobulin chain, wherein the number n is selected from 1-3. In one embodiment, CDRLn refers to a CDRn in a light chain or derived from a light chain, wherein the number n is selected from 1-3; and CDRHn refers to a CDRn in a heavy chain or derived from a heavy chain, wherein the number n is selected from 1-3. In certain embodiments, a framework region (FR) refers to a portion of a variable region that is not a CDR. In certain embodiments, FRn refers to a FR in a heavy chain or a light chain or derived from a heavy chain or a light chain, and wherein the number n is selected from 1-4. In certain embodiments, the variable region comprises, consists essentially of, or consists of the following components (optionally in the order provided, and further optionally from amino-terminus to carboxyl-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0289] The variable regions and / or CDRs of antibodies or fragments thereof can be determined by one skilled in the art, for example, using published or commercially available tools. Non-limiting examples of such tools include: IgBlast (available at www.ncbi.nlm.nih.gov / igblast / Access), Scaligner (available from drugdesigntech at www.scaligner.com / ), IMGT rules and / or tools (see, for example, www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html, also available at www.imgt.org / ), Chothia Canonical Assignment (available at www.bioinf.org.uk / abs / chothia.html), Antigen receptor Numbering And Receptor Calculation (ANARCI, available 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 (available at www.ofranlab.org / paratome / For access, see Vered Kunik, et al, Nucleic Acids Research, Volume 40, Issue W1, 1 July 2012, Pages W521–W524).

[0290] Antibodies can be polyclonal, monoclonal, multispecific (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. Antibodies can be isolated from any suitable biological source, such as murine, rat, sheep, and dog.

[0291] As used herein, the term "polyclonal antibody" or "polyclonal antibody composition" refers to an antibody preparation derived from different B cell lines. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each molecule recognizing a different epitope.

[0292] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homologous antibody population. Monoclonal antibodies are highly specific because each monoclonal antibody is directed against a single determinant on the antigen. The antibody can be detectably labeled, for example, with a radioisotope, an enzyme that produces a detectable product, a fluorescent protein, etc. The antibody can be further conjugated to other moieties, such as members of a specific binding pair, such as biotin (a member of a biotin-avidin specific binding pair), etc. The antibody can also be bound to a solid support, including but not limited to polystyrene plates or beads, etc.

[0293] Monoclonal antibodies can be produced using hybridoma technology or recombinant DNA methods known in the art. Hybridomas are cells formed in the laboratory by the fusion of antibody-producing lymphocytes and non-antibody-producing cancer cells (usually myeloma or lymphoma). Hybridomas proliferate and produce continuous samples of a specific monoclonal antibody. Alternative techniques for producing or selecting antibodies include exposing lymphocytes to the antigen of interest in vitro and screening antibody display libraries in cells, phage or similar systems.

[0294] As used herein, the term "human antibody" 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 somatic mutation in vivo). However, as used herein, the term "human antibody" 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 antibodies in which substantially every portion of a protein (e.g., CDR, framework, C L 、C H Domain (e.g. C H1 、C H2 、C H3), hinge, (VL, VH)) are essentially non-immunogenic in humans and have only minor sequence changes or variations. Similarly, antibodies designated as primates (monkeys, baboons, chimpanzees, etc.), rodents (mice, rats, rabbits, guinea pigs, hamsters, etc.) and other mammals are designated such species, subgenus, genus, subfamily, family specific antibodies. In addition, chimeric antibodies include any combination of the above. Relative to unmodified antibodies, such changes or variations optionally retain or reduce immunogenicity in humans or other species. Therefore, human antibodies are different from chimeric antibodies or humanized antibodies. It should be 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 chain and / or light chain) genes. In addition, when the human antibody is a single-chain antibody, it can include a linker peptide that is not present in natural human antibodies. For example, Fv can include a linker peptide connecting the heavy chain variable region and the light chain variable region, such as 2 to 8 glycine or other amino acid residues. Such a linker peptide is considered to be of human origin.

[0295] 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, such as by immunizing transgenic mice carrying human immunoglobulin genes or by screening a human immunoglobulin gene library. Human antibodies that are "derived from" a human germline immunoglobulin sequence can be identified by comparing the amino acid sequence of the human antibody to the amino acid sequence of human germline immunoglobulins. The selected human antibody typically has at least 90% identity in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene, and the selected human antibody contains amino acid residues that identify the human antibody as human when compared to the germline immunoglobulin amino acid sequences of other species (e.g., mouse germline sequences). In some cases, the human antibody may have at least 95%, or even at least 96%, 97%, 98%, or 99% identity 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 differs from the amino acid sequence encoded by the human germline immunoglobulin gene by no more than 10 amino acids. In certain cases, the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0296] 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. In most cases, a humanized antibody is a human immunoglobulin (acceptor antibody) in which the residues of the variable region or fragment thereof (e.g., 1, 2, 3, 4, 5, or all 6 CDRs) of the acceptor are replaced with residues of the variable region or fragment thereof (e.g., 1, 2, 3, 4, 5, or all 6 CDRs) of a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) with the desired specificity, affinity, and ability. The humanized antibody may include residues not found in the acceptor antibody or the donor antibody. The humanized antibody may also optionally include at least a portion of an immunoglobulin (usually a human immunoglobulin) constant region (Fc), a non-human antibody in which one or more amino acids in the framework region, constant region, or CDR are replaced by amino acids from the corresponding positions of a human antibody. Without wishing to be bound by theory, humanized antibodies produce a reduced immune response in human hosts compared to the non-humanized version of the same antibody. Humanized antibodies can have conservative amino acid substitutions that are substantially unaffected by antigen binding or other antibody functions. Conservative substitution groupings include: glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serine-threonine and asparagine-glutamic acid. Specifically, humanized antibodies as disclosed herein specifically bind DNABII polypeptide or its fragment (such as head chimeric peptide or tail chimeric peptide) with one or more of the following specific ranges: EC 50 , K on , K off , K A and / or K D , and inhibit or release specific cytokines when treating a subject. In a further embodiment, a humanized antibody that specifically binds to the head region of a DNABII polypeptide (e.g., a head chimeric peptide) destroys biofilms both in vivo and in vitro. In addition, the humanization process, although a rational design process, may produce unexpected changes (positive or negative) in, for example, binding affinity, antigen specificity, or physical properties (e.g., solubility or aggregation); therefore, the properties of a humanized antibody cannot be inherently predicted from the properties of the starting non-human antibody.

[0297] In one embodiment, the antibody as used herein can be a recombinant antibody. As used herein, the term "recombinant antibody" includes all antibodies prepared, expressed, produced or separated by recombinant means, such as antibodies isolated from transgenic or transchromosomal animals (e.g., mice) transfected with immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells (e.g., transfectomas) transformed to express antibodies, antibodies isolated from recombinant combinatorial antibody libraries, and antibodies prepared, expressed, produced or separated by any other means involving splicing immunoglobulin (Ig) gene sequences to other DNA sequences. However, in certain embodiments, such recombinant antibodies may be subjected to in vitro mutagenesis (or, when using Ig sequence transgenic animals, in vivo somatic mutagenesis) so that the amino acid sequences of the VH and VL regions of the recombinant antibodies may be sequences that are not naturally present in the antibody germline library in vivo. Methods for making these antibodies are described herein.

[0298] In one embodiment, 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 are constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species.

[0299] As used herein, the term "antibody derivative" encompasses full-length antibodies or antibody fragments in which one or more amino acids are chemically modified by, for example, alkylation, pegylation, acylation, ester formation, or amide formation, for linking the antibody to a second molecule. This includes, but is not limited to, pegylated antibodies, cysteine ​​pegylated antibodies, and variants thereof.

[0300] As used herein, the term "label" means a directly or indirectly detectable compound or component that is conjugated directly or indirectly to the composition to be detected, such as an N-terminal histidine tag (N-His), a magnetically active isotope, e.g. 115 Sn, 117 Sn and 119 Sn, non-radioactive isotopes, e.g. 13 C and 15N, polynucleotide or protein, such as antibody, to produce "label" component. The term also includes sequences conjugated to polynucleotides that provide signals when the expression of the inserted sequence is expressed, such as green fluorescent protein (GFP) etc. The label itself (such as radioisotope labeling or fluorescent labeling) can be detectable, or in the case of enzyme labeling, can catalyze the chemical change of a detectable substrate compound or component. Labeling can be applicable to small-scale detection or more suitable for high-throughput screening. Therefore, suitable labels include but are not limited to magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes and proteins (including enzymes). Labeling can be simply detected or quantified. The response of simple detection generally includes a response in which its presence is only confirmed, while the response of quantification generally includes a response with a quantifiable (such as, numerical value can be reported) value, such as intensity, polarization and / or other characteristics. In luminescence or fluorescence determination, detectable response can be directly generated using a luminophore or fluorophore related to the assay component actually involved in the combination, or indirectly generated using a luminophore or fluorophore related to another (such as a reporter molecule or indicator) component. Examples of luminescent labels that generate a signal include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent response typically comprises a change or appearance of a luminescent signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (1996). th Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0301] As used herein, the term "immunoconjugate" includes an antibody or antibody derivative linked or attached to a second agent, such as a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semisynthetic antibody, or a multispecific antibody.

[0302] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue, TM and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th ed.).

[0303] In another aspect, fluorescent label is functionalized to promote the covalent attachment of cellular components (such as cell surface markers) present in or on the cell or tissue surface. Suitable functional groups include but are not limited to isothiocyanate groups, amino groups, haloacetyl groups, maleimide, succinimide esters and sulfonyl halides, all of which can be used to connect fluorescent label to the second molecule. The selection of fluorescent label functional group will depend on the attachment site with joints, reagents, markers or second labeling reagents.

[0304] "Eukaryotic cells" include all kingdoms of life except for the non-nuclear protozoa. They can be easily distinguished by the presence of a membrane-bound nucleus. Animals, plants, fungi, and protozoa are eukaryotic organisms or organisms whose cells are organized into a complex structure by an internal membrane and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless otherwise specified, the term "host" includes eukaryotic hosts, including, for example, yeast, higher plant, insect, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include apes, cows, pigs, mice, rats, birds, reptiles, and humans.

[0305] "Prokaryotic cells" generally 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 can also contain genetic information in circular loops called episomes. Bacterial cells are very small, about the size of animal mitochondria (about 1-2 μm in diameter and about 10 μm long). Prokaryotic cells come in three main shapes: rod-shaped, spherical, and spiral-shaped. Bacterial cells do not undergo the complex replication process like eukaryotic cells, but instead divide by binary fission. Examples include, but are not limited to, Bacillus, Escherichia coli, and Salmonella.

[0306] A "native" antigen refers to an epitope-containing polypeptide, protein or fragment isolated from a natural biological source, which can specifically bind to an antigen receptor in a subject, particularly a T cell antigen receptor (TCR).

[0307] The terms "antigen" and "antigenicity" refer to a molecule that can be recognized by an antibody or otherwise serve as a member of an antibody-ligand pair. "Specific binding" or "binding" refers to the interaction of an antigen with the variable regions of the heavy and light chains of an immunoglobulin. Antibody-antigen binding can occur in vivo or in vitro. Those skilled in the art will understand that macromolecules, including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides, and polysaccharides, have the potential to act as antigens. Those skilled in the art will further understand that a nucleic acid encoding a protein with the potential to serve as an antibody ligand necessarily encodes an antigen. Those skilled in the art will further understand that antigens are not limited to full-length molecules, but may also include partial molecules. The term "antigen" is an adjective for molecules that have antigenic properties. The term includes immunogenic substances, i.e., immunogens, as well as substances that induce immune unresponsiveness or anergy, i.e., allergens.

[0308] "Altered antigens" are antigens that have a primary sequence that is different from that of the corresponding wild-type antigen. Altered antigens can be made by synthetic or recombinant methods, including but not limited to antigenic peptides that are differentially modified during or after translation, such as by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, or attachment to antibody molecules, membrane molecules, or other ligands. (Ferguson et al. (1988) Ann. Rev. Biochem. 57: 285-320). Synthetic or altered antigens disclosed herein are intended to bind to the same TCR as the native epitope.

[0309] "Autoantigens," also referred to herein as natural or wild-type antigens, refer to antigenic peptides that induce little or no immune response in a subject due to self-tolerance to the antigen. An example of an autoantigen is the melanoma-specific antigen gp100.

[0310] "Immune response" generally refers to the antigen-specific response of lymphocytes to foreign substances. The terms "immunogen" and "immunogenicity" refer to molecules that have the ability to elicit an immune response. All immunogens are antigens, however, not all antigens are immunogenic. The immune response disclosed herein can be humoral (through antibody activity) or cell-mediated (through T cell activation). The response can occur in vivo or in vitro. Those skilled in the art will understand that a variety of macromolecules, 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 nucleic acids encoding molecules capable of eliciting an immune response necessarily encode immunogens. Those skilled in the art will further understand that immunogens are not limited to full-length molecules, but can include partial molecules.

[0311] The term "passive immunity" refers to the transfer of immunity from one subject to another through antibody transfer. Passive immunity may occur naturally, as 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. The antibody donor and recipient can be human or non-human subjects. Antibodies can be polyclonal or monoclonal, can be produced in vitro or in vivo, and can be purified, partially purified, or unpurified depending on the embodiment. In some embodiments described herein, passive immunity is conferred to a subject in need by administering an antibody or antigen-binding fragment that specifically recognizes or binds to a specific antigen. In some embodiments, passive immunity is conferred by administering an isolated or recombinant polynucleotide encoding an antibody or antigen-binding fragment that specifically recognizes or binds to a specific antigen.

[0312] In the context of this 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, antibodies against the protein, or DNA against the protein. In one aspect, a ligand site within a protein binds directly to a complementary binding site on an immune effector cell.

[0313] As used herein, "solid support" or "solid support" used interchangeably are not limited to a particular type of support. Rather, a large number of supports are known and available to those of ordinary skill in the art. Solid supports include silica gel, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gel. As used herein, "solid support" also includes synthetic antigen presenting matrices, cells, and liposomes. Suitable solid supports can be selected based on the desired end use and suitability for various protocols. For example, for peptide synthesis, a solid support can refer to a resin, such as polystyrene (e.g., PAM-resin available from Bachem Inc., Peninsula Laboratories, etc.), resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol ( Rapp Polymere, Tubingen, Germany) or polydimethylacrylamide resin (obtained from Milligen / Biosearch, CA).

[0314] 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 soluble or insoluble to some extent. As long as the connected molecule is capable of binding to a polynucleotide, polypeptide or antibody, the material of the support can actually have any possible structural configuration. Therefore, the structure of the support can be spherical, such as beaded, or cylindrical, such as the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface can be flat, such as a sheet, a test strip, etc., or a polystyrene bead. Those skilled in the art will know many other carriers suitable for binding antibodies or antigens, or will be able to determine these carriers by using routine experiments.

[0315] As used herein, a biological sample or sample can be obtained from a subject, cell line, or cultured cell or tissue. Exemplary samples include, but are not limited to, cell samples, tissue samples, liquid samples such as blood and other biologically derived liquid samples (including, but not limited to, eye 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, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid or pre-ejaculatory fluid, female ejaculation, sweat, tears, cyst fluid, pleural effusion, pericardial fluid, ascites, lymph, chyme, bile, interstitial fluid, menstruation, pus, sebum, vomitus, vaginal secretions / washing, synovial fluid, mucosal secretions, fecal water, pancreatic juice, lavage fluid from the sinus cavity, bronchopulmonary effusion, blastocyst cavity fluid, or umbilical cord blood. In one embodiment, the biological sample is suspected of having a biofilm. In another embodiment, the biological sample comprises a biofilm.

[0316] As used herein, the term "signal peptide" or "signal polypeptide" refers to an amino acid sequence that is typically present at the N-terminus of a newly synthesized secretory or membrane polypeptide or protein. Its function is to direct the polypeptide to a specific cellular location, such as through the cell membrane, into the cell membrane, or into the cell nucleus. In some embodiments, the signal peptide is removed after localization. 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.

[0317] As used herein, a cleavable peptide, also referred to as a cleavable linker, refers to a peptide that can be cleaved (e.g., cleaved by an enzyme). A translated polypeptide comprising such a cleavable peptide can produce two final products, thus allowing more than one polypeptide to be expressed from one open reading frame. An example of a cleavable peptide is a self-cleaving peptide (e.g., a 2A self-cleaving peptide). 2A self-cleaving peptides are a class of 18-22aa 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. SciRep. 2015; 5: 16273. Published on November 5, 2015.

[0318] As used herein, the terms "T2A" and "2A peptide" are used interchangeably and refer to any 2A peptide or fragment thereof, any 2A-like peptide or fragment thereof, or an artificial peptide comprising the essential amino acids in a relatively short peptide sequence (approximately 20 amino acids long, based on the native virus) containing the consensus polypeptide motif DV / IEXNPGP, where X refers to any amino acid generally believed to be self-cleaving (SEQ ID NO: 134).

[0319] As used herein, the term "chimera" or "chimeric peptide" refers to a recombinant polypeptide comprising two or more fragments or domains of a DNABII polypeptide that are directly or indirectly (e.g., via a linker) conjugated to each other, or consisting essentially of, or consisting of. In one embodiment, these domains are conformational head domains and / or conformational tail domains. In addition or alternatively, these two or more fragments or domains are derived from identical or different DNABII polypeptides. In one embodiment, the chimeric peptide comprises an IhfA head domain and an IhfB head domain that are directly or indirectly (e.g., via a linker) conjugated to each other, or consisting essentially of, or consisting of. In another embodiment, the chimeric peptide comprises an IhfA tail domain and an IhfB tail domain that are directly or indirectly (e.g., via a linker) conjugated to each other, or consisting essentially of, or consisting of. A "conformational head domain" of a polypeptide refers to a polypeptide comprising a primary amino acid sequence in which the structure has an antiparallel β band with a sharp turn typically mediated by a proline residue. The "head" of the IHF polypeptide is shown in WO2018 / 129078 Figure 1 middle.

[0320] As used herein, the phrase "derived from" or "derived from" means isolated from, purified from, or engineered from, or any combination thereof.

[0321] In certain embodiments, the antibody specifically binds to the head chimeric peptide IhfA5-mIhfB4 NTHI , comprising the following polypeptide sequence: RPGRNPX1TGDVVPVSARRVV-X-FSLHHRQPRLGRNPX1TGDSV (SEQ ID NO: 38), or consisting essentially of, or consisting of, wherein "X" is an optional amino acid linker sequence, optionally comprising, consisting essentially of, or consisting of 1 to 20 amino acids; and wherein "X1" is any amino acid, or "X1" is selected from amino acids Q, R, K, S, or T. In another aspect, "X1" is K or Q. In another embodiment, the head chimeric peptide IhfA5-mIhfB4 NTHIComprising, consisting essentially of, or consisting of the following polypeptide sequence: RPGRNPKTGDVVPVSARRVV-X-FSLHHRQPRLGRNPKTGDSV (SEQ ID NO: 39), wherein "X" is an optional amino acid linker sequence, optionally comprising, consisting essentially of, or consisting of 1 to 20 amino acids. In yet another embodiment, the head chimeric peptide IhfA5-mIhfB4 NTHI Comprising, consisting essentially of, or consisting of the following polypeptide sequence: RPGRNPKTGDVVPVSARRVGPSLFSLHHRQPRLGRNPKTGDSV (SEQ ID NO: 40).

[0322] In certain embodiments, the antibody specifically binds to the tail chimeric peptide IhfA3-IhfB2 NTHI , comprising, consisting essentially of, or consisting of the following polypeptide sequence: FLEEIRLSGQDVKLSGF-X-TLSAKEIENMVKDILEFISQ (SEQ ID NO: 41), wherein "X" is an optional amino acid linker sequence, optionally comprising, consisting essentially of, or consisting of 1 to 20 amino acids. In certain embodiments, the linker is selected from any one or more of SEQ ID NOs: 42-49. In one embodiment, the tail chimeric peptide IhfA3-IhfB2 NTHI Comprising, consisting essentially of, or consisting of FLEEIRLSGQDVKLSGFGPSLTLSAKEIENMVKDILEFISQ (SEQ ID NO: 50).

[0323] As used herein, the term "EC 50 ” refers to the concentration of an antibody or antigen-binding fragment thereof that induces a response (e.g., binding between the antibody or antigen-binding fragment thereof and its target) that is intermediate between baseline and maximum after a specified exposure time.

[0324] Several parameters are used herein to describe the binding and dissociation reactions of receptor (R, such as an antibody or antigen-binding fragment thereof) and ligand (L, such as the target of the antibody or antigen-binding fragment thereof) molecules, which are formalized as follows: The reaction is characterized by an association rate constant k on and the dissociation rate constant k off To represent, its units are M -1 s -1 and s -1 In equilibrium, the forward binding transition R+L→RL should be balanced by the reverse non-binding transition RL→R+L. 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 receptor-ligand complex. In addition, the equilibrium dissociation constant "K D ” can be calculated as k off / k on , that is, [R]x[L] / [RL], and the equilibrium binding constant "K A ” can be calculated as k on / k off , that is [RL] / ([R]x[L]).

[0325] As used herein, the term "cytokine" refers to small proteins (about 5-20 kDa) that are important in cell signaling, including but not limited to chemokines, interferons, interleukins (IL), lymphokines and tumor necrosis factors, but generally does not include hormones. Cytokines are peptides that cannot pass through the lipid bilayer of the cell to enter the cytoplasm. Inflammatory cytokines or proinflammatory cytokines are signaling molecules (cytokines) that are secreted by immune cells such as helper T cells (Th) and macrophages, as well as certain other cell types that promote inflammation. They include but are not limited to interleukin-1 (IL-1), IL-12 and IL-18, tumor necrosis factor α (TNF-α), interferon γ (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM-CSF), and play an important role in mediating innate immune responses. Inflammatory cytokines are mainly produced by and participate in the upregulation of inflammatory responses. The term "anti-inflammatory cytokines" includes immunomodulatory molecules that control proinflammatory cytokine responses. Cytokines work together with specific cytokine inhibitors and soluble cytokine receptors to regulate the human immune response. The main 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-α and IL-18 can also serve as pro-inflammatory cytokine inhibitors. Methods for measuring cytokine levels (including anti-inflammatory cytokines 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.

[0326] As used herein, the terms "proinflammatory response" and "inflammatory response" are used interchangeably and refer to the biological response of a subject to a pathogen (e.g., bacteria, viruses, or other microorganisms that can cause disease). In some embodiments, a proinflammatory response or inflammatory response refers to an immune response involving specific and nonspecific 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 nonspecific defense system response is an inflammatory response mediated by white blood cells (e.g., macrophages, eosinophils, and neutrophils) that generally have no immune memory capacity. In some embodiments, the immune response comprises, or consists essentially of, or consists of, the secretion of proinflammatory cytokines, or consists essentially of, or consists of, resulting in elevated levels of proinflammatory cytokines. Additionally or alternatively, the immune response comprises, or consists essentially of, or consists of, reducing the levels of anti-inflammatory cytokines. Therefore, a proinflammatory response can comprise, or consists essentially of, or consists of, the secretion of proinflammatory cytokines or a reduction in anti-inflammatory cytokines or both.

[0327] As used herein, the term "anti-infective" refers to a drug that can inhibit the spread of infectious organisms or completely kill infectious organisms. This term encompasses, but is not limited to, antibiotics, antifungals, anthelmintics, antimalarials, antiprotozoals, antituberculosis drugs, and antivirals. Antifungals are also known as antimycotics. They kill or inactivate fungi and are used to treat fungal infections (including yeast infections). As a non-limiting example, polyene antifungals are not absorbed when taken orally and are therefore used to treat fungal infections of the gastrointestinal tract, such as thrush. Another non-limiting example is the azole antifungal drug, which is a synthetic bacteriostatic agent with broad spectrum activity; Echinocandin is a lipopeptide molecule that non-competitively inhibits (1,3) β-d-glucan synthase and targets the fungal cell wall; 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). It can be used, for example, in www.drugbank.ca / categories / DBCAT000065 Find more anti-infective drugs at . The term "antiviral" refers to a class of drugs used to treat viral infections. Most antiviral drugs are directed against specific viruses, while broad-spectrum antivirals are effective against a wide range of viruses. Unlike most antibiotics, antiviral drugs do not destroy their target pathogen; instead, they inhibit its development. Some of the ways they may work include preventing viral replication by inhibiting viral DNA polymerase; binding to specific cell surface receptors and inhibiting viral penetration or uncoating; inhibiting viral protein synthesis; or blocking late stages of viral assembly. Non-limiting examples of antiviral agents can be found, for example, in www.drugbank.ca / categories / DBCAT000066 Found in.

[0328] As used herein, the term "antiparasitic drug" refers to a class of drugs used to treat parasitic diseases, such as those caused by helminths, amoebas, ectoparasites, parasitic fungi, and protozoa. Non-limiting examples of antiparasitic drugs can be found in, for example, www.drugbank.ca / categories / DBCAT000522 Found in.

[0329] Modes for carrying out the present disclosure

[0330] Although the components of extracellular polymeric substances (EPS) vary among different bacterial species, extracellular DNA (eDNA) is a common basic structural component of the entire different bacterial biofilm (Flemming et al., Nat Rev Microbiol, 2010.8 (9): p.623-33). The eDNA structure was further characterized and it was determined that the eDNA lattice is composed of Holliday junction (HJ)-like structures, which are indispensable for the stability of the bacterial biofilm EPS of eDNA dependence (Devaraj et al., Proc Natl Acad Sci USA, 2019 Dec 10; 116 (50): 25068-25077). The DNA binding proteins of the bacterial DNABII family include integration host factors (IHF) and histone-like proteins (HU), which bind to these HJ-like structures within the eDNA lattice and act as key proteins to maintain the structural integrity of the eDNA-dependent EPS (Devaraj et al.). Immobilization of free DNABII protein (by exposure to a specific antibody against the DNA binding domain of DNABII protein (α-DNABII)) shifts the equilibrium from the eDNA-bound to the unbound state, subsequently leading to bacterial biofilm disruption (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; Devarajet 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.).

[0331] Although there is no DNABII protein in vertebrates, eukaryotes possess a partially functional homologous gene sequence (orthologue), HMGB1, which binds to similar curved DNA structures (e.g., HJ DNA) (Bianchi et al., Science, 1989. 243(4894Pt 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): p.109-21). It functions as a monomer, consisting of two tandem DNA-binding domains and an acidic C-terminal tail (Bianchi et al., EMBO J, 1992. 11(3): p. 1055-63), and typically has post-translational modifications (PTMs) that determine its location (nuclear, cytoplasmic, or extracellular) and activity (reviewed in Kang et al., Mol Aspects Med, 2014. 40: p. 1-116). HMGB1 acts as an accessory protein in a variety of 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): p. 3289-301; Sutrias-Grau et al., J Biol Chem, 1999. 274(3): p. 1628-34; and Yuan et al., J Biol Chem, 2004. 279(20): p. 20935-40).HMGB1 also functions as a damage-associated molecular pattern (DAMP) molecule, which is released from eukaryotic cells into the extracellular environment through the NF-κB pathway by binding to TLR2, TLR4, TLR9 and RAGE, inducing a series of proinflammatory responses (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). Therefore, it serves as an alarmin with the potential to cause sepsis, resulting in devastating consequences for the host (Qin et al., J Cancer Prev, 2012. 13(4): p.1365-70). ExpMed, 2006.203(7):p.1637-42; and Diener et al., Immunol Cell Biol, 2013.91(7):p.443-50). Outside the cell, HMGB1 also has a wide range of functions, including tissue regeneration and wound healing, aging, and killing bacteria at very high concentrations (1.75μM-12μM) (Ranzato et al., Mol Cell Biochem, 2009.332(1-2):p.199-205; Gong et al., J Biomed Sci, 2009.16:p.83; Davalos et al., J Cell Biol, 2013.201(4):p.613-29; and Zetterstrom et al., Pediatr Res, 2002.52(2):p.148-54). Perhaps most importantly, extracellular HMGB1 is a component of the eDNA of neutrophil extracellular traps (NETs), the host's primary means of sequestering bacteria for further elimination (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; Brinkman et al., Science, 2004. 303(5663): p. 1532-5; and Peng et al., Sci Rep, 2017. 7(1): p. 16628), and as proposed herein, may also serve as a strategy to prevent the proliferation of bacterial biofilms.

[0332] The host innate immune effector HMGB1 of eukaryotes and the bacterial DNABII protein play similar roles in the nuclear protein affairs of the host and bacteria, respectively (reviewed in Kang et al. and Browning et al., Curr Opin Microbiol, 2010.13 (6): p.773-80). Although HMGB1 and DNABII proteins have no identifiable sequence identity or secondary structure, they can still replace each other's functions in in vitro affairs. Therefore, HMGB1 was originally considered to be a functional homologous gene sequence of DNABII protein (Paull et al., Genes Dev, 1993.7 (8): p.1521-34; and Segal et al., EMBO J, 1994.13 (19): p.4536-48). Although both proteins bind to DNA and bend it, they do so through different mechanisms. HMGB1 (as monomer) and DNABII protein (as dyad) are both bound to DNA through its minor groove, however, HMGB1 stabilizes DNA bending from the convex side, and DNABII protein stabilizes DNA bending from the concave side (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.1295-306). In view of their extraordinary functional similarities within the cell, and because HMGB1 and DNABII proteins are also found outside the cell, further disclosed herein is the interaction of HMGB1 within the EPS of eDNA-dependent bacterial biofilms.

[0333] Described and illustrated herein is a hitherto unknown extracellular function of the vertebrate high-mobility group protein 1 (HMGB1) in the proliferation of bacterial biofilms. Within host cells, HMGB1 acts as a DNA structural protein, similar to the ubiquitous DNABII family of bacterial proteins, although these proteins do not share the same amino acid sequence identity. Extracellularly, HMGB1 induces a proinflammatory immune response, while DNABII proteins stabilize the extracellular DNA-dependent matrix of bacterial biofilms. Without wishing to be bound by theory, when these two proteins converge on eDNA within bacterial biofilms, HMGB1, unlike DNABII proteins, disrupts biofilms in vitro (including against the high-priority ESKAPEE pathogen) and in vivo in two different animal models, despite inducing a robust inflammatory response that was attenuated by a single engineered amino acid change. Here, we propose a model in which extracellular HMGB1 balances the extent of induced inflammation and biofilm inhibition without excessive release of bacteria within the biofilm.

[0334] While interfering with the binding of DNABII protein to extracellular DNA (e.g., by administering anti-DNABII antibodies) and providing HMGB1 polypeptides, a surprising beneficial effect was demonstrated and observed, namely, complete elimination of biofilm. See, Examples 1 and Figure 16I and 16J It is further noted that repeated administration of HMGB1 polypeptide alone or anti-DNABII antibody alone did not achieve the level of biofilm reduction achieved by a single administration of the combination of HMGB1 polypeptide and anti-DNABII antibody, suggesting an effect that exceeds additive (i.e., synergistic). This observation is consistent with the mechanism proposed herein. Without being bound by theory, other combinations of HMGB polypeptides and anti-DNA antibodies (e.g., antibodies or fragments thereof that bind to the head polypeptide or head chimeric polypeptide) are also expected to produce such synergistic effects. ***

[0335] In one aspect, a composition or combination is provided, comprising: (a) a high mobility group protein 1 (HMGB1) polypeptide or a fragment thereof, said fragment comprising, consisting essentially of, or consisting of its B box, A box, or AB box; and (b) an anti-DNABII antibody or antigen-binding fragment thereof as disclosed herein, consisting essentially of, or consisting of.

[0336] In another aspect, a composition or combination comprises, consists essentially of, or consists of: (a) an HMGB1 polypeptide further comprising a C45S mutation; and (b) an anti-DNABII antibody or antigen-binding fragment thereof as disclosed herein.

[0337] In one aspect, a composition or combination is provided, comprising: (a) a high mobility group protein 1 (HMGB1) polypeptide or a fragment thereof; and (b) an antibody or an antigen-binding fragment thereof that specifically recognizes and binds to the head domain of a DNABII protein, provided that (i) the composition or combination 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 polyclonal antibody or a Fab of an antibody that does not comprise a polyclonal antibody, or both (i) and (ii) are essentially composed of, or consist of.

[0338] In one aspect, provided is a polypeptide comprising, consisting essentially of, or consisting of (a) a high mobility group protein 1 (HMGB1) polypeptide or a fragment thereof; and (b) an anti-DNABII antibody or an antigen-binding fragment thereof.

[0339] In one aspect, a polypeptide is provided, comprising: (a) a high mobility group protein 1 (HMGB1) polypeptide or a fragment thereof; and (b) an antibody or an antigen-binding fragment thereof that specifically recognizes and binds to the head domain of a DNABII protein, provided 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 polyclonal antibody or a Fab of an antibody that does not comprise a polyclonal antibody, or both (i) and (ii) or consist essentially of, or consist of.

[0340] In some embodiments, the polypeptide further comprises a cleavable peptide located between (a) and (b).

[0341] In another aspect, a polynucleotide encoding a polypeptide as disclosed herein, or a polynucleotide complementary thereto is provided.

[0342] In one aspect, provided are polynucleotides encoding (a) a high mobility group protein box 1 (HMGB1) polypeptide or a fragment thereof and (b) an anti-DNABII antibody or an antigen-binding fragment thereof, or polynucleotides complementary thereto.

[0343] In one aspect, provided are polynucleotides encoding or complementary to: (a) a high mobility group protein 1 (HMGB1) polypeptide or a fragment thereof; and (b) an antibody or an antigen-binding fragment thereof that specifically recognizes and binds to the head domain of a DNABII protein, provided that (i) the polynucleotide does not encode 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 polyclonal antibody or a Fab of an antibody that does not comprise a polyclonal antibody, or both (i) and (ii).

[0344] In some embodiments, (a) and (b) are encoded by a single contiguous polynucleotide, e.g., under the direction of the same regulatory sequence. 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.

[0345] In one aspect, a vector is provided, comprising, consisting essentially of, or consisting of a polynucleotide as disclosed herein.

[0346] In one aspect, a host cell is provided, comprising one or more of the following: a composition or combination as disclosed herein, a polypeptide as disclosed herein, a polynucleotide as disclosed herein, or a vector as 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 a composition or combination as disclosed herein, a polypeptide as disclosed herein, a polynucleotide as disclosed herein, or a vector as disclosed herein, for example, by culturing the host cell and collecting the composition or combination, polypeptide, polynucleotide, or vector.

[0347] In one aspect, a method for producing a composition or combination as disclosed herein is provided. The method comprises culturing a host cell comprising a polynucleotide as 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, or consisting essentially of, or consisting of. In another aspect, a method for producing a polypeptide as disclosed herein is provided. The method comprises culturing a host cell comprising a polynucleotide as disclosed herein and isolating the polypeptide from the cell culture, or consisting essentially of, or consisting of. In some embodiments, the method further comprises introducing the polynucleotide or a vector comprising the polynucleotide into the host cell.

[0348] In one aspect, a method is provided for one or more of: (A) preventing the formation of a biofilm or disrupting a biofilm in vitro or in vivo, (B) preventing the formation of a biofilm or disrupting a biofilm in a subject, (C) inhibiting, preventing, or treating an infection by a biofilm-producing microorganism in a subject, or (D) treating a condition characterized by the formation of a biofilm in a subject. The method comprises, consists essentially of, or consists of administering to a subject:

[0349] (a) a high mobility group protein box 1 (HMGB1) polypeptide or a fragment thereof, said fragment comprising, consisting essentially of, or consisting of a B box, A box, or AB box thereof; and

[0350] (b) an anti-DNABII antibody or antigen-binding fragment thereof as disclosed herein.

[0351] In one aspect, a method is provided for inducing or increasing the formation of neutrophil extracellular traps (NETs) in close proximity to a biofilm in a subject and disrupting the biofilm, optionally without inducing a proinflammatory response. The method comprises administering to the subject, or consists essentially of, or consists of administering to the subject:

[0352] (a) a high mobility group protein box 1 (HMGB1) polypeptide or a fragment thereof, the polypeptide comprising the amino acid sequence of any one or more of SEQ ID NOs: 51-58, the fragment comprising, consisting essentially of, or consisting of a B box, A box, or AB box thereof; and

[0353] (b) an anti-DNABII antibody or antigen-binding fragment thereof as disclosed herein.

[0354] In one aspect, a method is provided for one or more of: (A) preventing the formation of a biofilm or disrupting a biofilm in vitro or in vivo, (B) preventing the formation of a biofilm or disrupting a biofilm in a subject, (C) inhibiting, preventing, or treating an infection by a biofilm-producing microorganism in a subject, or (D) treating a condition characterized by the formation of a biofilm in a subject. The method comprises, consists essentially of, or consists of administering to a subject one or more of a composition or combination as disclosed herein, a polypeptide as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, or a host cell as disclosed herein.

[0355] In one aspect, a method of inducing or increasing the formation of neutrophil extracellular traps (NETs) in close proximity to a biofilm in a subject and disrupting the biofilm, optionally without inducing a proinflammatory response, is provided. The method comprises administering to the subject one or more of, or consisting essentially of, or consisting of a composition or combination as disclosed herein, a polypeptide as disclosed herein, a polynucleotide as disclosed herein, a vector as disclosed herein, or a host cell as disclosed herein, with the proviso that the HMGB1 polypeptide comprises, consists essentially of, or consists of any one or more of SEQ ID NOs: 51-58.

[0356] In some embodiments, the method further comprises administering to the subject one or more of the following: a DNase enzyme, an antibacterial agent, an antimicrobial agent, an anti-infective agent, an antifungal agent, an antiparasitic agent, an antiviral agent, or an antibody or antigen-binding fragment thereof that specifically recognizes or binds to OMP P5, rsPilA, OMP 26, OMP P2, or type IV Pilin.

[0357] In some embodiments, the condition characterized by the formation of a biofilm comprises, consists essentially of, or consists of one or more of: a chronic non-healing wound, a Burkholderia infection, a Burkholderia lung infection, a venous ulcer, a diabetic foot ulcer, an ear infection, a sinus infection, a urinary tract infection, a gastrointestinal disease, hospital-acquired pneumonia, ventilator-associated pneumonia, a surgical implant-associated infection, a lung infection, a respiratory tract infection, cystic fibrosis, chronic obstructive pulmonary disease, a catheter-associated infection, an indwelling device-associated infection, a prosthesis-associated infection, osteomyelitis, cellulitis, an abscess, or periodontal disease.

[0358] In some embodiments, the administration of (a) and the administration of (b) are performed 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.

[0359] In some aspects related to any of the methods disclosed herein, optionally comprising an administration step, the antibody or antigen-binding fragment thereof reduces one or more pro-inflammatory cytokines and increases one or more anti-inflammatory cytokines in a subject. Regarding the methods disclosed herein, applicants unexpectedly discovered that HMGB1 fragments disclosed herein retain DNA binding activity despite not undergoing post-translational modification. Without being bound by theory, applicants believe that modified HMGB1 fragments may exhibit similar performance.

[0360] In some aspects related to any aspect, any embodiment and / or method as disclosed herein, an HMGB1 polypeptide or fragment thereof as disclosed herein and an anti-DNABII antibody or antigen-binding fragment thereof as disclosed herein are combined and contained in the same composition (e.g., in the same pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier, and / or are contacted or administered simultaneously in one composition in a method as disclosed herein).

[0361] In other aspects related to any aspect, any embodiment and / or method as disclosed herein, an HMGB1 polypeptide or a fragment thereof as disclosed herein is a first composition (referred to herein as an HMGB1 composition, optionally a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier), and an anti-DNABII antibody or its antigen-binding fragment is provided as a second or separate discreet composition (referred to herein as an anti-DNABII antibody composition, which may be another pharmaceutical composition). In another aspect, the mixture of HMGB1 and anti-DNABII antibody compositions comprises, or consists essentially of, or consists of, an HMGB1 polypeptide or its fragment and an anti-DNABII antibody or its antigen-binding fragment as disclosed herein, for use in methods as disclosed herein. In one aspect, the HMGB1 composition is contacted or administered alone (i.e., in the absence of the other party) with an anti-DNABII antibody composition in a method as disclosed herein. Additionally or alternatively, the HMGB1 composition is contacted or administered simultaneously with an anti-DNABII antibody composition in a method as disclosed herein (e.g., within 0.5 hours). In yet another aspect, HMGB1 compositions is contacted or applied in order with anti-DNABII antibody compositions in the method as disclosed herein, with or without time interval in the middle.This interval can be a few minutes to a few weeks, optionally selected from but not limited to 0.5 hour, 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, HMGB1 compositions and anti-DNABII antibody compositions can be applied with identical route of administration (such as intravenous injection) or with different routes of administration in the method as disclosed herein. In yet another aspect, said composition is applicable to predetermined route of administration (such as with suitable pH value). Without wishing to be bound by theory, in one embodiment, such a composition, pharmaceutical composition and / or pharmaceutically acceptable carrier stabilizes the HMGB1 polypeptide or fragment thereof and / or anti-DNABII antibody or antigen-binding fragment thereof, and / or prevents degradation of the HMGB1 polypeptide or fragment thereof and / or anti-DNABII antibody or antigen-binding fragment thereof.

[0362] In some aspects related to the method as disclosed herein, the method further comprises contacting an antibody or an antigen-binding fragment of the antibody that binds to the DNABII polypeptide with a sample suspected of containing biofilm, and detecting the combination of the biofilm and the antibody or its fragment to detect the biofilm. In one aspect, the detection antibody or its antigen-binding fragment is bound to the head region of the DNABII polypeptide. In another aspect, the detection antibody or its antigen-binding fragment is bound to the tail region of the DNABII polypeptide.

[0363] A method for screening an object using a composition or method as described herein is also provided. The screening method comprises, or is essentially composed of, or is composed of the following steps: contacting an anti-DNABII antibody, anti-DNABII polypeptide, or an antigen-binding fragment of an antibody as disclosed herein with a biological sample containing a biomembrane and separated from the object, and detecting the binding of the antibody or its antigen-binding fragment to any biomembrane in the sample. In one aspect, the antigen-binding fragment of the antibody is selected from Fab, F(ab')2, Fab', scFv, or Fv. Additionally or alternatively, the antibody or its antigen-binding fragment specifically binds to the head region of the DNABII peptide. In one aspect, the DNABII peptide is an IHF peptide. In one aspect, screening is performed for antibodies or antigen-binding fragments that bind to the head region of the DNABII polypeptide. In another aspect, screening is performed for antibodies or antigen-binding fragments that bind to the tail region of the DNABII polypeptide.

[0364] In one aspect, a kit for use in a method as disclosed herein is provided. The kit comprises, consists essentially of, or consists of instructions for use and one or more selected from a composition or combination as disclosed herein, a polypeptide as disclosed herein, a polynucleotide as disclosed herein (e.g., a polynucleotide as disclosed herein), a vector as disclosed herein, or a host cell as disclosed herein.

[0365] HMGB composition

[0366] In some embodiments, the HMGB is human HMGB1 or murine HMGB1. In some embodiments, the HMGB polypeptide as used herein comprises, consists essentially of, or 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) as disclosed herein. In some embodiments, the HMGB polypeptide comprises, consists essentially of, or consists of any one or more of SEQ ID NOs: 51-58, 68-74, 84-90, or 100-114.

[0367] In some embodiments, HMGB1 is substituted with HMGB2, or HMGB3, or HMGB4, or other equivalents as disclosed herein.

[0368] In some embodiments, the HMGB1 polypeptide further comprises one or more mutations selected from 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 mutations at K12, C23, or C45. In some embodiments, the B box of the HMGB1 polypeptide further comprises one or two mutations at C106 or K114. In some embodiments, one or more of the mutations is 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 further embodiments, wherein the HMGB polypeptide is HMGB2, HMGB3, or HMGB4, or a modified version thereof, it has a mutation at the corresponding position of modified HMGB1, serine, glycine, alanine, valine, isoleucine, or threonine at K12, C23, or C45. Additionally or alternatively, the mutant HMGB (mHMGB) comprises one or more mutations at positions corresponding to C23S, C45S, and C106S. In another aspect, the polypeptide further comprises a mutation corresponding to C45S.

[0369] In some embodiments, one or more mutations of HMGB1 are selected from mutations at K12, C23, C45, C106 or K114. In another aspect, one or more mutations of HMGB1 are selected from mutations at K12, C23, C45, C106 or K114 to serine, glycine, alanine, valine, isoleucine or threonine. Additionally or alternatively, mutant HMGB1 (mHMGB1) comprises one or more mutations selected from C23S, C45S and C106S. In a further embodiment, wherein the HMGB polypeptide is HMGB2, HMGB3 or HMGB4 or a modified version thereof, it has a mutation at the corresponding position of the modified HMGB1, serine, glycine, alanine, valine, isoleucine or threonine at K12, C23, C45, C106 or K114. Additionally or alternatively, the mutant HMGB (mHMGB) comprises one or more mutations at positions corresponding to C23S, C45S and C106S.

[0370] In some embodiments, a fragment of an HMGB polypeptide (e.g., an HMGB1 polypeptide) comprises, consists essentially of, or consists of a B box, A box, or AB box thereof. In some embodiments, a fragment of an HMGB1 polypeptide or an HMGB1 polypeptide comprises, consists essentially of, or consists of a B box polypeptide as disclosed herein, an A box polypeptide as disclosed herein, or an AB box polypeptide as disclosed herein.

[0371] Antibody composition

[0372] In some embodiments, the antibody or its antigen-binding fragment as used herein comprises a heavy chain (HC) variable domain sequence and a light chain (LC) variable domain sequence, or is essentially composed of it, or is composed of it, wherein the heavy chain and light chain immunoglobulin variable domain sequence form an antigen binding site that is combined with the epitope of the DNABII protein. In certain embodiments, the antibody or its antigen-binding fragment binds to the head region of the DNABII peptide (such as DNABII peptide, including but not limited to: the head region or head chimera (chimer) of IHF or HU, the head region of IHFA or IHFB, and / or the head chimeric peptide IhfA5-IhfB4 NTHI and / or the 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 head chimeric peptide IhfA5-IhfB4 NTHI In another embodiment, the antibody or antigen-binding fragment thereof binds to the tail chimeric peptide IhfA3-IhfB2 NTHI .

[0373] In some embodiments, the antibody or antigen-binding fragment thereof binds to the head region (ie, head domain) or head chimera of the DNABII peptide. In one aspect, the DNABII peptide is an IHF peptide. In some embodiments, the head domain comprises, consists essentially of, or consists of one or more amino acid sequences selected from: NFELRDKSSRPGRNPKTGDVV (SEQ ID NO:31); SLHHRQPRLGRNPKTGDSVNL (SEQ ID NO:32); RPGRNPX1TGDVVPVSARRVV-X-FSLHHRQPRLGRNPX1TGDSV, wherein "X" is an optional amino acid linker sequence, wherein "X1" is any amino acid (SEQ ID NO:38); RPGRNPKTGDVVPVSARRV-X-FSLHHRQPRLGRNPKTGDSV, wherein "X1" is any amino acid (SEQ ID NO:39); or RPGRNPKTGDVVPVSARRVGPSLFSLHHRQPRLGRNPKTGDSV (SEQ ID NO:40).

[0374] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of: (i) a heavy chain complementary determining region 1 (CDRH1) comprising, consisting essentially of, or consisting of GFTFRTY (aa 50 to aa 56 in SEQ ID NO: 1 or 2 or 3 or 24); (ii) a heavy chain complementary determining region 2 (CDRH2) comprising, consisting essentially of, or consisting of GSDRRH (aa 76 to aa 81 in SEQ ID NO: 1 or 2 or 3 or 24); (iii) a heavy chain complementary determining region 3 (CDRH3) comprising, consisting essentially of, or consisting of VGPYDGYYGEFDY (aa 121 to aa 133 in SEQ ID NO: 1 or 2 or 3 or 24); (iv) a light chain complementary determining region 1 (CDRL1) comprising, consisting essentially of, or consisting of QSLLDSDGKTF (SEQ ID NO: 1 or 2 or 3 or 24); NO: 7 or 8 or 9 or 25); (v) a light chain complementary determining region 2 (CDRL2) comprising, consisting essentially of, or consisting of LVS (aa 75 to aa 77 of SEQ ID NO: 7 or 8 or 9 or 25); and (vi) a light chain complementary determining region 3 (CDRL3) comprising, consisting essentially of, or consisting of WQGTHFPYT (aa 114 to aa 122 of SEQ ID NO: 7 or 8 or 9 or 25).

[0375] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain. In further embodiments, the HC immunoglobulin variable domain comprises, consists essentially of, or consists of amino acids 25 to 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 consists of a light chain (LC) immunoglobulin variable domain. In further embodiments, the LC immunoglobulin variable domain comprises, consists essentially of, or consists of amino acids 21 to 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 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 consists of amino acids 25 to 144 of any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In further embodiments, the LC immunoglobulin variable domain comprises, consists essentially of, or consists of amino acids 21 to 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 consists of amino acids 25 to 144 of any one of SEQ ID NO: 1, and the LC immunoglobulin variable domain comprises, consists essentially of, or consists of amino acids 21 to 132 of SEQ ID NO: 7. In some embodiments, the HC immunoglobulin variable domain comprises, consists essentially of, or consists of amino acids 25 to 144 of any one of SEQ ID NO: 1, and the LC immunoglobulin variable domain comprises, consists essentially of, or consists of amino acids 21 to 132 of SEQ ID NO: 8. In some embodiments, the HC immunoglobulin variable domain comprises, consists essentially of, or consists of amino acids 25 to 144 of any one of SEQ ID NO: 1, and the LC immunoglobulin variable domain comprises, consists essentially of, or consists of amino acids 21 to 132 of SEQ ID NO: 9.

[0376] 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.

[0377] In some embodiments, the antigen-binding fragment thereof comprises Fab, F(ab')2, Fab', scFv, or Fv.

[0378] 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, polysialyation, HESylation or glycosylation.

[0379] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody or an antigen-binding fragment of a monoclonal antibody.

[0380] In some embodiments, the antibody or antigen-binding fragment thereof comprises a humanized or human framework.

[0381] In some embodiments, the antibody or antigen-binding fragment is derived from a mammal. In a further embodiment, 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 camel. In other embodiments, the antibody or antigen-binding fragment is derived from a human. In 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, and 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.

[0382] 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 consists of a sequence selected from amino acids (aa) 25 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 consists of a sequence selected from aa 21 to aa 132 of SEQ ID NO: 7-9, 14 or 25, aa21 to aa 126 of SEQ ID NO: 10-12 or 27, or an equivalent of each thereof.

[0383] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of a sequence selected from aa 25 to aa 473 of SEQ ID NO: 1-6, 13, 24 or 26, or an equivalent of each thereof; and / or a light chain (LC) comprising, consists essentially of, or consists of a sequence selected from aa 21 to aa 239 of SEQ ID NO: 7-9, 14 or 25, aa 21 to aa 233 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 consists of a heavy chain (HC) comprising, consists essentially of, or consists of a sequence selected from 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, consists essentially of, or consists of a sequence selected from 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.

[0384] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) and / or a light chain (LC), wherein the HC comprises, consists essentially of, or consists of a sequence selected from SEQ ID NO: 1-6, 13, 24 or 26, or an equivalent of each thereof; and the LC comprises, consists essentially of, or consists of a sequence selected from SEQ ID NO: 7-12, 14, 25 or 27, or an equivalent of each thereof.

[0385] In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of the following CDR sequences: any one or any two or all three CDRs selected from a sequence of SEQ ID NO: 1-6, 13, 24 or 26, or an equivalent of each thereof; and / or any one or any two or all three CDRs selected from a sequence of SEQ ID NO: 7-12, 14, 25 or 27, or an equivalent of each thereof.

[0386] 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 consists of a sequence selected from aa 25 to aa 144 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 consists of a sequence selected from aa 21 to aa 132 of SEQ ID NO: 14 or 25, aa 21 to aa 126 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 and / or a light chain (LC) immunoglobulin variable domain sequence, wherein the HC immunoglobulin variable domain sequence comprises, consists essentially of, or consists of a sequence selected from the group consisting of aa 25 to aa 144 of SEQ ID NO: 1-6, 13, 24 or 26, or an equivalent of each thereof; and the LC immunoglobulin variable domain sequence comprises, consists essentially of, or consists of a sequence selected from the group consisting 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.

[0387] 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 1, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 132 of any one of SEQ ID NOs: 7-9, 14, or 25, aa 21 to aa 126 of SEQ ID NOs: 10-12, or 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 2, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 132 of any one of SEQ ID NOs: 7-9, 14, or 25, aa 21 to aa 126 of SEQ ID NOs: 10-12, or 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 3, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 132 of any one of SEQ ID NOs: 7-9, 14, or 25, aa 21 to aa 126 of SEQ ID NOs: 10-12, or 27, or an equivalent thereof.

[0388] 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 4, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 132 of any one of SEQ ID NOs: 7-9, 14, or 25, aa 21 to aa 126 of SEQ ID NOs: 10-12, or 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 5, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 132 of any one of SEQ ID NOs: 7-9, 14, or 25, aa 21 to aa 126 of SEQ ID NOs: 10-12, or 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 6, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 132 of any one of SEQ ID NOs: 7-9, 14, or 25, aa 21 to aa 126 of SEQ ID NOs: 10-12, or 27, or an equivalent thereof.

[0389] 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 consists of the amino acid sequence of 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, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 132 of SEQ ID NO: 7, 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 consists of the amino acid sequence of 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, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 132 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 consists of the amino acid sequence of 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, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 132 of SEQ ID NO: 9, or an equivalent thereof.

[0390] 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 consists of the amino acid sequence of 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, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 126 of SEQ ID NO: 10, 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 consists of the amino acid sequence of aa25 to aa 144 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 comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 126 of SEQ ID NO: 11, 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 consists of the amino acid sequence of 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, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 126 of SEQ ID NO: 12, or an equivalent thereof.

[0391] 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 1, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa21 to aa 132 of SEQ ID NO: 7, 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 1, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 132 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 consists of the amino acid sequence of aa25 to aa 144 of SEQ ID NO: 1, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 132 of SEQ ID NO: 9, or an equivalent thereof.

[0392] 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 2, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa21 to aa 132 of SEQ ID NO: 7, 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 the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 2, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 132 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 2, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 132 of SEQ ID NO: 9, or an equivalent thereof.

[0393] 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 of aa 25 to aa 144 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 of aa 21 to aa 132 of SEQ ID NO:7, 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO:3, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 132 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 the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 3, or an equivalent thereof, or consists essentially of, or consists of a light chain (LC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 21 to aa 132 of SEQ ID NO: 9, or an equivalent thereof.

[0394] 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO:4, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 126 of SEQ ID NO:10, 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 4, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 126 of SEQ ID NO: 11, 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 4, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 126 of SEQ ID NO: 12, or an equivalent thereof.

[0395] 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 5, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 126 of SEQ ID NO: 10, 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 5, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 126 of SEQ ID NO: 11, 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 5, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 126 of SEQ ID NO: 12, or an equivalent thereof.

[0396] 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 6, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 126 of SEQ ID NO: 10, 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 6, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 126 of SEQ ID NO: 11, 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 6, or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 126 of SEQ ID NO: 12, or an equivalent thereof.

[0397] In one aspect, the antibody or its antigen-binding fragment comprises a heavy chain (HC) immunoglobulin variable domain sequence and / or a light chain (LC) immunoglobulin variable domain sequence, or consists essentially of, or consists of, the HC immunoglobulin variable domain sequence comprises an amino acid sequence of aa 25 to aa 144 of SEQ ID NO:24 or its equivalent, or consists essentially of, or consists of, the LC immunoglobulin variable domain sequence comprises an amino acid sequence of aa 21 to aa 132 of SEQ ID NO:25 or its equivalent, or consists essentially of, or consists of. In another embodiment, the antibody or its antigen-binding fragment binds to the head region of the DNABII peptide (including but not limited to: the head region of IHF or HU, the head region of IHFA or IHFB, and / or the head chimeric peptide IhfA5-mIhfB4 NTHIIn one embodiment, the antibody or antigen-binding fragment thereof binds to the head chimeric peptide IhfA5-mIhfB4 NTHI In another embodiment, the fragment is an antigen-binding fragment.

[0398] In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 1 or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of aa 21 to aa 132 of SEQ ID NO: 7 or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 1, or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of aa 21 to aa 132 of SEQ ID NO: 8, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 1, or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of aa 21 to aa 132 of SEQ ID NO: 9, or an equivalent thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 2, or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of aa 21 to aa 132 of SEQ ID NO: 7, or an equivalent thereof.In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 2, or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of aa 21 to aa 132 of SEQ ID NO: 8, or an equivalent thereof. In another embodiment, 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 consists of the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 2, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa21 to aa 132 of SEQ ID NO: 9, or an equivalent thereof. In one embodiment, 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 consists of the amino acid sequence of aa25 to aa 144 of SEQ ID NO: 3, or an equivalent thereof, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of aa 21 to aa 132 of SEQ ID NO: 7, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 3, or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of aa 21 to aa 132 of SEQ ID NO: 8, or an equivalent thereof.In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 3, or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of aa 21 to aa 132 of SEQ ID NO: 9, or an equivalent thereof.

[0399] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence, or consists essentially of, or consists of, the HC immunoglobulin variable domain sequence comprises an amino acid sequence of aa 25 to aa 144 of SEQ ID NO:26 or its equivalent, or consists essentially of, or consists of, the LC immunoglobulin variable domain sequence comprises an amino acid sequence of aa 21 to aa 126 of SEQ ID NO:27 or its equivalent, or consists essentially of, or consists of. In another embodiment, the antibody or its antigen-binding fragment binds to the tail region of the 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 another 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) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of the amino acid sequence of 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 consists of the amino acid sequence of 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, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 4, or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of aa 21 to a 126 of SEQ ID NO: 11, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO:4, or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of aa 21 to aa 126 of SEQ ID NO:12, or an equivalent thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 5, or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of 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, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 5, or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of aa 21 to a 126 of SEQ ID NO: 11, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 5, or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of aa 21 to aa 126 of SEQ ID NO: 12, or an equivalent thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 6, or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of 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, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 6, or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of aa 21 to aa 126 of SEQ ID NO: 11, or an equivalent thereof.In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) immunoglobulin variable domain sequence comprising the amino acid sequence of aa 25 to aa 144 of SEQ ID NO: 6, or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or consists of, the amino acid sequence of aa 21 to aa 126 of SEQ ID NO: 12, or an equivalent thereof.

[0400] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain (HC) and / or a light chain (LC), or consists essentially of it, or consists of it, the HC comprises the amino acid sequence of SEQ ID NO: 24 or its equivalent, or consists essentially of it, or consists of it, the LC comprises the amino acid sequence of SEQ ID NO: 25 or its equivalent, or consists essentially of it, or consists of it. In another embodiment, the antibody or its antigen-binding fragment binds to the head region of the DNABII peptide (including but not limited to: the head region of IHF or HU, the head region of IHFA or IHFB, and / or the head chimeric peptide IhfA5-mIhfB4 NTHI In one embodiment, the antibody or antigen-binding fragment thereof binds to the head chimeric peptide IhfA5-mIhfB4 NTHI. In another 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) and / or a light chain (LC), wherein the HC comprises the amino acid sequence of SEQ ID NO: 1 or an equivalent thereof, or consists essentially of, or consists of, and the LC comprises the amino acid sequence of SEQ ID NO: 7 or an equivalent thereof, or consists essentially of, or consists of. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) and / or a light chain (LC), wherein the HC comprises the amino acid sequence of SEQ ID NO: 1 or an equivalent thereof, or consists essentially of, or consists of, and the LC comprises the amino acid sequence of SEQ ID NO: 8 or an equivalent thereof, or consists essentially of, or consists of. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1 or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists 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 consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2 or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists 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 consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2 or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 8 or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2 or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists 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 consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3 or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists 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 consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3 or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 8 or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3 or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9 or an equivalent thereof.

[0401] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain (HC) and a light chain (LC), or consists essentially of it, or consists of it, the HC comprises the amino acid sequence of SEQ ID NO:26 or its equivalent, or consists essentially of it, or consists of it, the LC comprises the amino acid sequence of SEQ ID NO:27 or its equivalent, or consists essentially of it, or consists of it. In another embodiment, the antibody or its antigen-binding fragment binds to the tail region of the 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 NTHI. In another 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) and / or a light chain (LC), wherein the HC comprises the amino acid sequence of SEQ ID NO: 4 or an equivalent thereof, or consists essentially of, or consists of, and the LC comprises the amino acid sequence of SEQ ID NO: 10 or an equivalent thereof, or consists essentially of, or consists of. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) and / or a light chain (LC), wherein the HC comprises the amino acid sequence of SEQ ID NO: 4 or an equivalent thereof, or consists essentially of, or consists of, and the LC comprises the amino acid sequence of SEQ ID NO: 11 or an equivalent thereof, or consists essentially of, or consists of. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 4 or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists 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 consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5 or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or 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, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5 or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 11 or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5 or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists 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 consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6 or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or 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, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6 or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 11 or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 6 or an equivalent thereof, or consists essentially of, or consists of, and / or a light chain (LC) comprising, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 12 or an equivalent thereof.

[0402] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain (HC) and / or a light chain (LC), or consists essentially of it, or consists of it, the HC comprises an amino acid sequence of aa 25 to aa 473 of SEQ ID NO:24 or its equivalent, or consists essentially of it, or consists of it, the LC comprises an amino acid sequence of aa 21 to aa239 of SEQ ID NO:25 or its equivalent, or consists essentially of it, or consists of it. In another embodiment, the antibody or its antigen-binding fragment binds to the head region of the DNABII peptide (including but not limited to: the head region of IHF or HU, the head region of IHFA or IHFB, and / or the head chimeric peptide IhfA5-mIhfB4 NTHI In one embodiment, the antibody or antigen-binding fragment thereof binds to the head chimeric peptide IhfA5-mIhfB4 NTHI. In another 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, consists essentially of, or consists of an amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 1, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of an amino acid sequence of aa 21 to aa 239 of SEQ ID NO: 7, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) and / or a light chain (LC) comprising, consists essentially of, or consists of an amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 1, or an equivalent thereof, and / or consists essentially of, or consists of an amino acid sequence of aa 21 to aa 239 of SEQ ID NO: 8, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 1, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 239 of SEQ ID NO: 9, or an equivalent thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 2, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 239 of SEQ ID NO: 7, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 2, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 239 of SEQ ID NO: 8, or an equivalent thereof.In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 2, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 239 of SEQ ID NO: 9, or an equivalent thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 3, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 239 of SEQ ID NO: 7, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 3, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 239 of SEQ ID NO: 8, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 3, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 239 of SEQ ID NO: 9, or an equivalent thereof.

[0403] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain (HC) and a light chain (LC), or consists essentially of, or consists of, the HC comprises an amino acid sequence of aa 25 to aa 473 of SEQ ID NO:26 or its equivalent, or consists essentially of, or consists of, the LC comprises an amino acid sequence of aa 21 to aa 233 of SEQ ID NO:27 or its equivalent, or consists essentially of, or consists of. In another embodiment, the antibody or its antigen-binding fragment binds to the tail region of the 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 NTHIIn one embodiment, the antibody or antigen-binding fragment thereof binds to the tail chimeric peptide IhfA3-IhfB2 NTHIIn another 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, consists essentially of, or consists of an amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 4, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of an amino acid sequence of aa 21 to aa 233 of SEQ ID NO: 10, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) and / or a light chain (LC) comprising, consists essentially of, or consists of an amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 4, or an equivalent thereof, and / or consists essentially of, or consists of an amino acid sequence of aa 21 to aa 233 of SEQ ID NO: 11, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 4, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 233 of SEQ ID NO: 12, or an equivalent thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 5, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 233 of SEQ ID NO: 10, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 5, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 233 of SEQ ID NO: 11, or an equivalent thereof.In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 5, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 233 of SEQ ID NO: 12, or an equivalent thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 6, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 233 of SEQ ID NO: 10, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 6, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 233 of SEQ ID NO: 11, or an equivalent thereof. In another embodiment, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain (HC) comprising, consists essentially of, or consists of the amino acid sequence of aa 25 to aa 473 of SEQ ID NO: 6, or an equivalent thereof, and / or a light chain (LC) comprising, consists essentially of, or consists of the amino acid sequence of aa 21 to aa 233 of SEQ ID NO: 12, or an equivalent thereof.

[0404] In some embodiments, the antibody or its antigen-binding fragment comprises the following CDR sequences, or consists essentially of it, or consists of it: any one or any two or all three CDRs selected from the sequence of SEQ ID NO: 1-3 or 24 or the equivalents of each thereof; and / or any one or any two or all three CDRs selected from the sequence of SEQ ID NO: 7-9 or 25 or the equivalents of each thereof. In another embodiment, the antibody or its antigen-binding fragment binds to the head region of the DNABII peptide (including but not limited to: the head region of IHF or HU, the head region of IHFA or IHFB, and / or the head chimeric peptide IhfA5-mIhfB4 NTHI In one embodiment, the antibody or antigen-binding fragment thereof binds to the head chimeric peptide IhfA5-mIhfB4NTHI In another embodiment, the fragment is an antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of the following CDR sequences: all three CDRs selected from a sequence of SEQ ID NOs: 1-3 or 24, or an equivalent thereof; and / or all three CDRs selected from a sequence of SEQ ID NOs: 7-9 or 25, or an equivalent thereof.

[0405] In some embodiments, the antibody or its antigen-binding fragment comprises the following CDR sequences, or consists essentially of it, or consists of it: any one or any two or all three CDRs selected from the sequence of SEQ ID NO: 4-6 or 26 or the equivalents of each thereof; and / or any one or any two or all three CDRs selected from the sequence of SEQ ID NO: 10-12 or 27 or the equivalents of each thereof. In another embodiment, the antibody or its antigen-binding fragment binds to the tail region of the 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 and antigen-binding fragment thereof bind to the tail chimeric peptide IhfA3-IhfB2 NTHI In another embodiment, the fragment is an antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of the following CDR sequences: all three CDRs selected from a sequence of SEQ ID NOs: 4-6 or 26, or an equivalent thereof; and / or all three CDRs selected from a sequence of SEQ ID NOs: 10-12 or 27, or an equivalent thereof.

[0406] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise one or more signal peptides. In one embodiment, the signal peptide comprises, or consists essentially of, or consists of amino acids (aa) 1 to aa 24 of any one of SEQ ID NOs: 1-6, 13, 24, or 26. In another embodiment, the signal peptide comprises, or consists essentially of, or consists of aa 1 to aa 20 of any one of SEQ ID NOs: 7-12, 14, 25, and 27. In a further embodiment, the signal peptide is located at the amino terminus of the light chain variable region. Additionally or alternatively, the same signal peptide or a different signal peptide is located at the amino terminus of the heavy chain variable region.

[0407] The antibodies or their antigen-binding fragments provided herein can be monospecific or bispecific. In one embodiment, the antibody or its antigen-binding fragment is trispecific or tetraspecific or pentaspecific. Additionally or alternatively, the antibody is selected from IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4) or IgM antibodies. In one embodiment, the antibody further comprises a constant region selected from: IgA constant region (e.g., IgA1 constant region or IgA2 constant region), IgD constant region, IgE constant region, IgG constant region (e.g., IgG1 constant region, IgG2 constant region, IgG3 constant region, or IgG4 constant region) or IgM constant region.

[0408] In certain embodiments, an equivalent of an amino acid sequence comprises, consists essentially of, or consists of a polypeptide having at least about 80% (including about 80% to 100%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) amino acid identity with the amino acid sequence. Additionally or alternatively, an equivalent of an amino acid sequence comprises, consists essentially of, or consists of a polypeptide encoded by a polynucleotide that hybridizes under highly stringent conditions to the complement of a polynucleotide encoding the amino acid sequence. In further embodiments, equivalents of the amino acid sequence comprise, consist essentially of, or consist of a polypeptide that is at least 80% (including about 80% to 100%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) identical to the amino acid sequence. In certain embodiments, an equivalent of an amino acid sequence (e.g., an antibody or antigen-binding fragment thereof, or any one or more of SEQ ID NOs: 1-14 and 24-26 as disclosed herein, or a fragment thereof, including but not limited to aa25 to aa 144 of SEQ ID NO: 13, 24 or 26, aa 21 to aa 132 of SEQ ID NO: 14 or 25, aa 21 to aa 126 of SEQ ID NO: 27, aa 25 to aa 473 of SEQ ID NO: 13, 24 or 26, aa 21 to aa 239 of SEQ ID NO: 14 or 25, aa21 to aa 233 of SEQ ID NO: 27) comprises, consists essentially of, or consists of a polypeptide comprising one or more or all of the CDRs in the amino acid sequence. Additionally or alternatively, the polypeptide has at least about 80% (including about 80% to 100%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) amino acid identity with the amino acid sequence.

[0409] In certain embodiments, an equivalent of an amino acid sequence (e.g., an antibody, an antigen-binding fragment thereof, a complementarity determining region (CDR) thereof, or a polypeptide containing a CDR) lacks amino acid differences from the amino acid sequence in the CDRs. However, an equivalent of an amino acid sequence (e.g., an antibody, an antigen-binding fragment thereof, a CDR thereof, or a polypeptide containing a CDR) may comprise one or more amino acid differences in the non-CDR regions compared to the amino acid sequence, such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid differences, provided that the three-dimensional arrangement of the CDRs and / or the CDRs are maintained. In certain embodiments, an equivalent polypeptide of an amino acid sequence (e.g., an antibody, an antigen-binding fragment thereof, a CDR thereof, or a polypeptide containing a CDR) has at least about 80% (including about 80% to 100%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) amino acid identity with the amino acid sequence, provided that the three-dimensional arrangement of the CDRs and / or the CDRs are maintained.

[0410] Non-limiting examples of such non-CDR regions include framework regions (FRs), constant regions, Fc regions, pFc' regions, heavy chain (CH) constant domains (e.g., CH1, CH2, CH3, or CH4), light chain (CL) constant domains, or hinge regions. In one embodiment, such amino acid differences may be conservative amino acid substitutions and / or do not alter the three-dimensional arrangement of the antibody, its antigen-binding fragment, its CDR, or CDR-containing polypeptide. In another embodiment, equivalents may comprise conservative amino acid substitutions at the boundaries of the CDRs, such as one or two amino acids at the amino terminus, carboxyl terminus, or both of the CDRs.

[0411] In one aspect, one or more CDRs (e.g., any 1, or 2, or 3, or 4, or 5, or 6 CDRs) of an antibody or antigen-binding fragment thereof as disclosed herein are provided. In one embodiment, a set of CDRs is provided, comprising, consisting essentially of, or consisting of, one or more ...

Claims

1. A composition or combination comprising: (a) a high mobility group protein (HMGB) polypeptide or a fragment thereof comprising its B box, A box or AB box; and (b) an anti-DNABII antibody or an antigen-binding fragment thereof, comprising: (i) heavy chain complementarity determining region 1 (CDRH1) comprising GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 1 or 2 or 3 or 24); (ii) heavy chain complementarity determining region 2 (CDRH2), comprising GSDRRH (aa 76 to aa 81 of SEQ ID NO: 1 or 2 or 3 or 24); (iii) heavy chain complementarity determining region 3 (CDRH3) comprising VGPYDGYYGEFDY (aa121 to aa 133 of SEQ ID NO: 1 or 2 or 3 or 24); (iv) light chain complementarity determining region 1 (CDRL1) comprising QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7 or 8 or 9 or 25); (v) light chain complementarity determining region 2 (CDRL2) comprising LVS (aa 75 to aa 77 of SEQ ID NO: 7 or 8 or 9 or 25); and (vi) light chain complementarity determining region 3 (CDRL3) comprising WQGTHFPYT (aa 114 to aa 122 of SEQ ID NO: 7 or 8 or 9 or 25).

2. The composition or combination of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain (HC) immunoglobulin variable domain comprising amino acids 25-144 of any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

3. The composition or combination of claim 2, wherein the antibody or antigen-binding fragment thereof comprises a light chain (LC) immunoglobulin variable domain comprising amino acids 21-132 of any one of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:

9.

4. The composition or combination of claim 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 wherein the LC immunoglobulin variable domain comprises amino acids 21 to 132 of SEQ ID NO:

7.

5. The composition or combination of claim 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 wherein the LC immunoglobulin variable domain comprises amino acids 21 to 132 of SEQ ID NO:

8.

6. The composition or combination of claim 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 wherein the LC immunoglobulin variable domain comprises amino acids 21 to 132 of SEQ ID NO:

9.

7. The composition or combination according to any one of claims 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.

8. The composition or combination of claim 1, wherein the antigen-binding fragment thereof comprises Fab, F(ab')2, Fab', scFv or Fv.

9. The composition or combination of claim 1, wherein the antibody or antigen-binding fragment thereof is modified.

10. The composition or combination of claim 9, wherein the antibody or antigen-binding fragment thereof is modified by a method selected from PEGylation, polysialylation, HESylation or glycosylation.

11. The composition or combination of claim 1, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody or an antigen-binding fragment of the monoclonal antibody.

12. The composition or combination of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a humanized or human framework.

13. The composition or combination of claim 1, wherein the HMGB polypeptide further comprises one or more mutations selected from mutations at K12, C23, C45, C106, or K114; or an HMGB polypeptide selected from HMGB2, HMGB3, or HMGB4 polypeptides having mutations corresponding to HMGB1 polypeptides comprising one or more mutations selected from mutations at K12, C23, C45, C106, or K114.

14. The composition or combination according to claim 1, wherein the A box of the HMGB polypeptide further comprises one or more mutations selected from mutations at K12, C23 or C45, or corresponding mutations when the HMGB polypeptide is selected from HMGB2, HMGB3 or HMGB4 polypeptide and has a mutation corresponding to the mutated HMGB1 polypeptide.

15. The composition or combination according to claim 1, wherein the B box of the HMGB polypeptide further comprises one or two mutations at C106 or K114, or when the HMGB polypeptide is selected from HMGB2, HMGB3 or HMGB4 polypeptide, a mutation corresponding to the mutated HMGB1 polypeptide.

16. The composition or combination of claim 1, wherein the one or more mutations are to serine, glycine, alanine, valine, isoleucine, or threonine.

17. The composition or combination of claim 1, wherein the HMGB polypeptide further comprises one or more mutations selected from C23S, C45S and C106S, or the HMGB polypeptide is selected from HMGB2, HMGB3 or HMGB4 polypeptide and has one or more mutations corresponding to the mutated HMGB1 polypeptide.

18. The composition or combination of claim 1, wherein the HMGB polypeptide further comprises a C45S mutation, or the HMGB polypeptide is selected from an HMGB2, HMGB3, or HMGB4 polypeptide and has a mutation corresponding to the mutated HMGB1 polypeptide.

19. The composition or combination according to claim 1, comprising: (a) the HMGB polypeptide further comprises a C45S mutation; and (b) The anti-DNABII antibody or antigen-binding fragment thereof comprises a heavy chain (HC) immunoglobulin variable domain comprising amino acids 25 to 144 of SEQ ID NO: 1 and a 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.

20. A composition or combination comprising: (a) a high mobility group protein box 1 (HMGB1) polypeptide or a fragment thereof comprising its B box, A box or AB box; and (b) an antibody or an antigen-binding fragment thereof that specifically recognizes and binds to the head domain of the DNABII protein, provided that (i) the composition or combination does not contain SEQ ID NO: 52, or (ii) the antigen-binding fragment does not contain Fab, or both (i) and (ii).

21. The composition or combination of claim 20, wherein the head domain comprises one or more amino acid sequences selected from the group consisting of: NFELRDKSSRPGRNPKTGDVV (SEQ ID NO: 31); SLHHRQPRLGRNPKTGDSVNL (SEQ ID NO: 32); RPGRNPX1TGDVVPVSARRVV-X-FSLHHRQPRLGRNPX1TGDSV, wherein "X" is an amino acid linker sequence and wherein "X1" is any amino acid (SEQ ID NO: 38); RPGRNPKTGDVVPVSARRVV-X-FSLHHRQPRLGRNPKTGDSV, wherein "X" is any amino acid (SEQ ID NO: 39); or RPGRNPKTGDVVPVSARRVVGPSLFSLHHRQPRLGRNPKTGDSV (SEQ ID NO: 40).

22. A polypeptide comprising: (a) a high mobility group protein (HMGB) polypeptide, or a fragment thereof comprising its B box, A box, or AB box; and (b) an anti-DNABII antibody or an antigen-binding fragment thereof, comprising: (i) heavy chain complementarity determining region 1 (CDRH1) comprising GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 1 or 2 or 3 or 24); (ii) heavy chain complementarity determining region 2 (CDRH2), comprising GSDRRH (aa 76 to aa 81 of SEQ ID NO: 1 or 2 or 3 or 24); (iii) heavy chain complementarity determining region 3 (CDRH3) comprising VGPYDGYYGEFDY (aa121 to aa 133 of SEQ ID NO: 1 or 2 or 3 or 24); (iv) light chain complementarity determining region 1 (CDRL1) comprising QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7 or 8 or 9 or 25); (v) light chain complementarity determining region 2 (CDRL2) comprising LVS (aa 75 to aa 77 of SEQ ID NO: 7 or 8 or 9 or 25); and (vi) light chain complementarity determining region 3 (CDRL3) comprising WQGTHFPYT (aa 114 to aa 122 of SEQ ID NO: 7 or 8 or 9 or 25).

23. A polypeptide comprising: (a) a high mobility group protein (HMGB) polypeptide, or a fragment thereof comprising its B box, A box, or AB box; and (b) an antibody or an antigen-binding fragment thereof that specifically recognizes and binds to the head domain of the DNABII protein, provided that (i) the polypeptide does not comprise SEQ ID NO: 52, or (ii) the antigen-binding fragment does not comprise Fab, or both (i) and (ii).

24. The polypeptide of claim 23, wherein the head domain comprises one or more amino acid sequences selected from the group consisting of: NFELRDKSSRPGRNPKTGDVV (SEQ ID NO: 31); SLHHRQPRLGRNPKTGDSVNL (SEQ ID NO: 32); RPGRNPX1TGDVVPVSARRVV-X-FSLHHRQPRLGRNPX1TGDSV, wherein "X" is an amino acid linker sequence and wherein "X1" is any amino acid (SEQ ID NO: 38); RPGRNPKTGDVVPVSARRVV-X-FSLHHRQPRLGRNPKTGDSV, wherein "X" is any amino acid (SEQ ID NO: 39); or RPGRNPKTGDVVPVSARRVVGPSLFSLHHRQPRLGRNPKTGDSV (SEQ ID NO: 40).

25. The polypeptide of any one of claims 22 to 24, further comprising a cleavable peptide located between (a) and (b).

26. A polynucleotide encoding: (a) a high mobility group protein (HMGB) polypeptide, or a fragment thereof comprising its B box, A box, or AB box; and (b) an anti-DNABII antibody or an antigen-binding fragment thereof, comprising: (i) heavy chain complementarity determining region 1 (CDRH1) comprising GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 1 or 2 or 3 or 24); (ii) heavy chain complementarity determining region 2 (CDRH2), comprising GSDRRH (aa 76 to aa 81 of SEQ ID NO: 1 or 2 or 3 or 24); (iii) heavy chain complementarity determining region 3 (CDRH3) comprising VGPYDGYYGEFDY (aa121 to aa 133 of SEQ ID NO: 1 or 2 or 3 or 24); (iv) light chain complementarity determining region 1 (CDRL1) comprising QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7 or 8 or 9 or 25); (v) light chain complementarity determining region 2 (CDRL2) comprising LVS (aa 75 to aa 77 of SEQ ID NO: 7 or 8 or 9 or 25); and (vi) light chain complementarity determining region 3 (CDRL3) comprising WQGTHFPYT (aa 114 to aa 122 of SEQ ID NO: 7 or 8 or 9 or 25), or a polynucleotide complementary thereto.

27. A polynucleotide encoding: (a) a high mobility group protein (HMGB) polypeptide, or a fragment thereof comprising its B box, A box, or AB box; and (b) an antibody or antigen-binding fragment thereof that specifically recognizes and binds to the head domain of 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, or both (i) and (ii).

28. The polynucleotide of claim 27, wherein the head domain comprises one or more amino acid sequences selected from the group consisting of: NFELRDKSSRPGRNPKTGDVV (SEQ ID NO: 31); SLHHRQPRLGRNPKTGDSVNL (SEQ ID NO: 32); RPGRNPX1TGDVVPVSARRVV-X-FSLHHRQPRLGRNPX1TGDSV, wherein "X" is an amino acid linker sequence and wherein "X1" is any amino acid (SEQ ID NO: 38); RPGRNPKTGDVVPVSARRVV-X-FSLHHRQPRLGRNPKTGDSV, wherein "X" is any amino acid (SEQ ID NO: 39); or RPGRNPKTGDVVPVSARRVVGPSLFSLHHRQPRLGRNPKTGDSV (SEQ ID NO: 40).

29. A vector comprising the polynucleotide of any one of claims 26 to 28.

30. A host cell comprising one or more of the following: the composition or combination of any one of claims 1 to 21, the polypeptide of any one of claims 22 to 25, the polynucleotide of any one of claims 26 to 28, or the vector of claim 29.

31. The host cell of claim 30, wherein the host cell secretes the HMGB polypeptide, or a fragment thereof; and the anti-DNABII antibody or an antigen-binding fragment thereof.

32. Use of a combination for the preparation of a medicament for one or more of: (A) preventing biofilm formation or disrupting biofilm in vitro or ex vivo, (B) preventing the formation of a biofilm 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, The combination includes: (a) a high mobility group protein (HMGB) polypeptide, or a fragment thereof comprising its B box, A box, or AB box; and (b) an anti-DNABII antibody or an antigen-binding fragment thereof, comprising: (i) heavy chain complementarity determining region 1 (CDRH1) comprising GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 1 or 2 or 3 or 24); (ii) heavy chain complementarity determining region 2 (CDRH2), comprising GSDRRH (aa 76 to aa 81 of SEQ ID NO: 1 or 2 or 3 or 24); (iii) heavy chain complementarity determining region 3 (CDRH3) comprising VGPYDGYYGEFDY (aa121 to aa 133 of SEQ ID NO: 1 or 2 or 3 or 24); (iv) light chain complementarity determining region 1 (CDRL1) comprising QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7 or 8 or 9 or 25); (v) light chain complementarity determining region 2 (CDRL2) comprising LVS (aa 75 to aa 77 of SEQ ID NO: 7 or 8 or 9 or 25); and (vi) light chain complementarity determining region 3 (CDRL3) comprising WQGTHFPYT (aa 114 to aa 122 of SEQ ID NO: 7 or 8 or 9 or 25).

33. The use according to 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 Fab, or both (i) and (ii).

34. Use of a combination in the preparation of a medicament for inducing or increasing the formation of neutrophil extracellular traps (NETs) proximate to a biofilm in a subject and disrupting the biofilm without inducing a proinflammatory response, the combination comprising: (a) a high mobility group protein 1 (HMGB1) polypeptide comprising the amino acid sequence of SEQ ID NO: 52, or a fragment thereof, wherein the fragment comprises or consists of the B box, A box or AB box thereof; and (b) an anti-DNABII antibody or an antigen-binding fragment thereof, comprising: (i) heavy chain complementarity determining region 1 (CDRH1) comprising GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 1 or 2 or 3 or 24); (ii) heavy chain complementarity determining region 2 (CDRH2), comprising GSDRRH (aa 76 to aa 81 of SEQ ID NO: 1 or 2 or 3 or 24); (iii) heavy chain complementarity determining region 3 (CDRH3) comprising VGPYDGYYGEFDY (aa121 to aa 133 of SEQ ID NO: 1 or 2 or 3 or 24); (iv) light chain complementarity determining region 1 (CDRL1) comprising QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 7 or 8 or 9 or 25); (v) light chain complementarity determining region 2 (CDRL2) comprising LVS (aa 75 to aa 77 of SEQ ID NO: 7 or 8 or 9 or 25); and (vi) light chain complementarity determining region 3 (CDRL3) comprising WQGTHFPYT (aa 114 to aa 122 of SEQ ID NO: 7 or 8 or 9 or 25).

35. The use according to any one of claims 32 to 34, wherein the medicament is formulated for simultaneous or sequential administration of (a) and (b).

36. The use of claim 32, wherein the medicament is formulated for repeating the administration of (a) and (b) at least once, at least twice, at least three times, or more.

37. Use of one or more of the composition or combination of any one of claims 1 to 21, the polypeptide of any one of claims 22 to 25, the polynucleotide of any one of claims 26 to 28, the vector of claim 29, or the host cell of claim 30 or 31 in the preparation of a medicament for one or more of: (A) preventing biofilm formation or disrupting biofilm in vitro or ex vivo, (B) preventing the formation of a biofilm 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.

38. Use of one or more of the composition or combination of any one of claims 1 to 21, the polypeptide of any one of claims 22 to 25, the polynucleotide of any one of claims 26 to 28, the vector of claim 29, or the host cell of claim 30 or 31 in the preparation of a medicament for inducing or increasing the formation of neutrophil extracellular traps (NETs) adjacent to a biofilm in a subject and disrupting the biofilm without inducing a proinflammatory response, wherein the HMGB1 polypeptide comprises the amino acid sequence of SEQ ID NO: 52 or a fragment thereof, which comprises or consists of its B box, A box, or AB box.

39. The use according to claim 32, wherein the medicament is formulated for administration together with one or more of the following: a DNase enzyme, an antibiotic agent, an antimicrobial agent, an anti-infective agent, an antifungal agent, an antiparasitic agent, an antiviral agent, or an antibody or antigen-binding fragment thereof that specifically recognizes or binds to OMP P5, rsPilA, OMP 26, OMP P2 or type IV fimbriae protein.

40. The use according to claim 32, wherein the condition characterized by biofilm formation comprises one or more of the following: chronic non-healing wounds, venous ulcers, diabetic foot ulcers, ear infections, sinus infections, urinary tract infections, gastrointestinal diseases, hospital-acquired pneumonia, ventilator-associated pneumonia, surgical implant-related infections, lung infections, respiratory tract infections, cystic fibrosis, chronic obstructive pulmonary disease, catheter-related infections, indwelling device-related infections, implant-related infections, osteomyelitis, cellulitis, abscesses, or periodontal disease.

41. The use according to claim 40, wherein the lung infection is a lung infection caused by Burkholderia.

42. A kit for use in one or more of: (A) preventing biofilm formation or disrupting biofilm in vitro or ex vivo, (B) preventing the formation of a biofilm or disrupting a biofilm in a subject, (C) inhibiting, preventing or treating a biofilm-producing microbial infection in a subject, (D) treating a condition characterized by biofilm formation in a subject, or (E) inducing or increasing the formation of neutrophil extracellular traps (NETs) in close proximity to a biofilm in a subject and disrupting the biofilm without inducing a proinflammatory response, The kit comprises instructions for use and one or more of the following: a composition or combination of any one of claims 1 to 21, a polypeptide of any one of claims 22 to 25, a polynucleotide of any one of claims 26 to 28, a vector of claim 29, or a host cell of claim 30 or 31.

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