Compositions and methods for treating biofilm and neutrophil extracellular trap formation

Abstract

Provided herein are synthetic polypeptides derived from the high mobility group box 1 (HMGB1) host protein that can disrupt bacterial biofilms and prevent the formation of neutrophil extracellular traps (NETs). Also provided herein are methods for disrupting abnormal or excessive NET formation, particularly suitable for treating high-risk populations, such as those infected with SARS-CoV-2, sepsis, autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and small vessel vasculitis), autoinflammatory diseases (such as gout and inflammatory bowel disease), and metabolic diseases (such as type 2 diabetes and obesity).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Patent Application No. 63 / 424,851, filed November 11, 2022, and U.S. Provisional Patent Application No. 63 / 424,888, filed November 12, 2022, the entire contents of which are incorporated herein by reference.

[0002] Government support statement This invention was made with government support under AI155501 & DC011818 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] background Neutrophils (polymorphonuclear leukocytes or PMNs) are specialized cells that are part of the innate immune system. Part of their function is to migrate to sites of infection to clear pathogens. One method PMNs can employ is a process known as NETosis, in which they first create elaborate structures of released extracellular DNA (eDNA) (neutrophil extracellular traps or NETs) that capture individual bacteria and interrupt bacterial aggregations (biofilms), and then use the deployed eDNA tendrils to localize and concentrate NET-associated antimicrobial substances to the captured microorganisms, limiting pathogen growth.

[0004] Despite this important host innate immune function, widespread release of NETs can occur in NETs' attempts to clear bacterial and viral pathogens, including pathological thrombi, which can lead to morbidity or even death. Furthermore, NETs can induce anti-inflammatory immune responses (e.g., autoimmune diseases). Therefore, developing a means to prevent or neutralize the eDNA structure of NETs is desirable to prevent or halt these potentially damaging coagulation or excessive inflammatory events. The present disclosure fulfills this need and provides related advantages. Summary of the Invention [Means for solving the problem]

[0005] Summary of the Disclosure Applicants have discovered that a polypeptide derived from the high mobility group box 1 (HMGB1) host protein can disrupt bacterial biofilms and prevent the formation of neutrophil extracellular traps (NETs). This polypeptide, designated "mB Box-97" by Applicants, consists essentially of or consists of amino acids 80-176 from the coding sequence of the native human HMGB1 protein, with a cysteine-to-serine point mutation at amino acid 106 that abrogates the polypeptide's ability to induce an inflammatory response. Native HMGB1 is known to be variably altered by the host through post-translational modifications, and these changes have been shown to affect HMGB1 function. Similarly, mB Box-97 should also be subject to post-translational modifications when recombinantly expressed by bacteria. In response, Applicants have synthesized mB Box-97 and found that both recombinant and synthetic mB Box-97 possess indistinguishable biofilm disruption and NET-inhibitory activity. Thus, synthetic mB Box-97 can be synthesized for therapeutic use, improving yield and quality control over recombinant mB Box-97 and mutated wild-type polypeptides without loss of therapeutic activity. Thus, synthetically produced mB Box-97 with a point mutation of the amino acid cysteine ​​to serine is referred to herein as "synthetic mB Box-97 or smB Box-97."

[0006] Applicant's disclosure also addresses and resolves problems associated with aberrant or excessive NET formation, particularly in high-risk populations, such as those infected with SARS-CoV-2, sepsis, autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and small-vessel vasculitis), autoinflammatory diseases (such as gout and inflammatory bowel disease), and metabolic diseases (such as type 2 diabetes and obesity). Without being bound by theory, mB Box 97 can prevent NET-mediated disease or prevent the progression of NET-mediated disease. Second, synthetic mB Box-97 can be used to prevent or treat bacterial biofilms because the mechanism of action of this peptide is different from other known similar peptides.

[0007] In another aspect of the present disclosure, Applicant has discovered that DNA-binding proteins that aggregate DNA can also condense the DNA tendrils of NETs, ​​either preventing NET formation or inducing the regression of existing NETs. For example, a family of homologous bacterial histone-like nucleoid structuring proteins (H-NS) proteins, known to function as nucleoid-associated proteins intracellularly, have additional, previously unknown effects outside the cell. These H-NS proteins are released from bacterial biofilms and not only prevent NET formation but also disrupt existing NETs. While the ability to cross-link adjacent DNA duplexes is a known function of H-NS, this level of DNA aggregation / condensation was not previously known to be a characteristic of H-NS. Therefore, any agent that causes DNA aggregation / condensation can also be used to effectively counteract and limit the symptoms of NETosis, such as those associated with SARS-CoV-2 infection.

[0008] There are no FDA-approved preventatives or treatments for pathological NETosis. There are treatments that can prevent NETosis, inactivate NETs, ​​or promote NET clearance (reviewed in Mutua and Gershwin (2020) Clinical Reviews in Allergy and Immunology 61:194-211; https: / / pubmed.ncbi.nlm.nih.gov / 32740860 / , incorporated herein by reference). The biggest drawback of inactivating NETs is that NETs also have beneficial functions. Virtually all approaches to treating NETs cannot compensate for the loss of beneficial functions. However, DNA clumping agents can be used to treat existing NETs, ​​so that only the pathology of the NETs is affected.

[0009] The present disclosure solves problems associated with aberrant or excessive NET formation, particularly in high-risk populations, such as those infected with SARS CoV-2, sepsis, autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and small-vessel vasculitis), autoinflammatory diseases (such as gout and inflammatory bowel disease), and metabolic diseases (such as type 2 diabetes and obesity). These DNA-aggregating agents can be used to prevent or treat NET-mediated diseases or to prevent the progression of NET-mediated diseases. [Brief explanation of the drawings]

[0010] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]Figures 1A-1C. mB Box-97 (also known as mB Box-97 peptide) inhibits phorbol myristate acetate (PMA)-induced NETosis. (A) Graphical representation of recombinant or synthetic full-length HMGB1, A Box (amino acids 1-89), AB Box (amino acids 1-176), B Box-97 (amino acids 80-176), recombinant and synthetic mB Box-97 (identical to B Box-97 except for a single amino acid change, C106S), and B Box-87 (amino acids 90-176) polypeptides. Specific cysteines / C and their respective amino acid numbers are indicated; in mB Box-97, the corresponding C at position 106 in HMGB1 is mutated to serine / S. Recombinantly produced HMGB1 polypeptides (e.g., full-length HMGB1, ABox, ABBox, BBox-97, mBBox-97, BBox-87) may have post-translational modifications, whereas synthetic HMGB1 polypeptides do not have any post-translational modifications. (B) Quantification of fluorescence studies shows that mBBox-97, BBox-87, and ABox do not induce NETosis. Each construct was used to induce NETs in isolated neutrophils (5*103) (200 nm, 4 h), and the released DNA was quantified using the cell-impermeable dye SYTOX Green (1 μM) in a fluorometer. Of all the constructs tested, mBBox-97, BBox-87, and ABox were unable to induce NET formation in isolated human neutrophils. (C) Quantification of the % NETs in total cells in different treatment conditions shows that only mBBox-97 has significant inhibition of PMA-induced NETosis. Formed NETs were quantified using ImageJ, and the % NET formation relative to total neutrophils was calculated for each image. Analysis from four separate experiments is shown, and data are presented as mean ± standard deviation (SD). *P<0.05 as assessed by unpaired t-test. mB Box-97 was the only HMGB1 construct that showed inhibition of PMA-induced NETosis.

[0011] [Figure 2-1]Figures 2A-2F. Because Ca2+ mediates NETosis via a pathway distinct from that of PMA or LPS, Applicants examined the effect of mB Box-97 on Ca2+-mediated NET formation. Neutrophils were allowed to form NETs for 6-8 hours in the presence of the Ca2+ ionophore A23187 (500 nM) with or without mB Box-97 or BBox-97 (200 nM). DNA, plasma membrane, NE, or MPO were probed as described in Materials and Methods, and NETs were visualized by CLSM at 63x magnification. (A) Changes in mean fluorescence intensity (MFI) of NET-associated proteins during PMA-stimulated NETosis. (B) Changes in mean fluorescence intensity (MFI) of NET-associated proteins during NTHI-stimulated NETosis. (C) Changes in mean fluorescence intensity (MFI) of NET-associated proteins during ionophore-stimulated NETosis. (D) mB Box-97 inhibits the secretion of NETs-associated proteins, along with the inhibition of PMA- or NTHI-induced NETosis. (E) Secreted nuclear elastase (NE) assay. (F) DNA-bound NE assay. [Figure 2-2] Same as above.

[0012] [Figure 3]Figures 3A-3B. Effect of mB Box-97 on reactive oxygen species (ROS) generation and p47phox phosphorylation. (A) Effect of mB Box-97 on ROS generation. Neutrophils preincubated with luminol (400 μM) were incubated with PMA, PMA with or without protein, protein, or buffer alone. Luminescence, as a measure of generated ROS, was detected at 5-minute intervals for 2 hours. N = 3, bars represent SEM. *P < 0.007 or less as assessed by multiple unpaired t-test. (B) Effect of mB Box-97 on p47phox phosphorylation. Neutrophils (1 × 107 cells / ml) were incubated with buffer, 200 nM PMA, or PMA with or without mB Box-97 or BBox-97 (1 μM) for 30 minutes, lysed, and proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Proteins were detected using immunoblotting with anti-phospho-p47phox antibodies, which detect phosphorylation on Ser-370, or anti-p47phox or GAPDH. Western blots from different experiments were scanned. Phosphorylated and total p47phox and GAPDH were quantified by densitometry. The intensity of phosphorylated p47phox was corrected for the amount of p47phox after also correcting for the amount of GAPDH. Results are expressed as mean ± SD (n = 4). ****P < 0.0001 as assessed by unpaired t-test. mB Box-97 significantly inhibited ROS production and phosphorylation of the key NOX protein p47phox in human neutrophils upon induction of NETosis with PMA.

[0013] [Figure 4]Figure 4A-4B. (A) Neutrophil-mediated killing is inactivated by mB Box-97. Sixteen-hour-old NTHI biofilms were challenged with 10 human neutrophils (B+N) and treated with 1 μM recombinant DNA BII protein HUNTHI (B+N+HU) positive control, 1 μM mB Box-97 (B+N+mB Box-97), or 1 μM B Box-97 (B+N+B Box-97) for 4 hours. NTHI biofilms without neutrophils were used as controls. Neutrophil-challenged bacteria (B+N) (mean = 50.79%) and B Box-97-treated groups (B+N+B Box-97) (mean = 40.86%) showed increased relative % killing compared to biofilm controls. Neutrophils treated with HUNTHI (B+N+HU) (mean = 4.83%) and mB Box-97 (B+N+mB Box-97) (mean = 13.09%) showed no difference in the relative % killing of bacteria by neutrophils. The results suggest that mB Box-97 treatment inactivated neutrophil-mediated bacterial killing. Replicates in the graph plots were derived from six healthy donors ± SEM. Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparison test (**p<0.01). mB Box-97 inhibited neutrophil-mediated bacterial killing comparable to that by the NTHI-derived DNABII protein HUNTHI. (B) ROS production measurement. Luminol-preloaded neutrophils were incubated with PMA, PMA with or without the respective proteins, proteins, or buffer alone, and luminescence, as a measure of ROS, was detected at 5-minute intervals over 2 hours.

[0014] [Figure 5] Interaction and inhibition of PKC with mB Box-97. mB Box-97 inhibits PKC activity. PKC activity was tested in the presence of histone H1, mB Box-97, and B Box-97 at two different concentrations (200 nM and 1000 nM) using a PKC activity assay kit according to the manufacturer's instructions. mB Box-97 shows inhibition of PKC activity at increasing concentrations.

[0015] [Figure 6] Graphical representation of the potential mechanism of mB Box-97 action. mB Box-97 interacts with PKC, resulting in inhibition of its activity. This inhibition results in the inhibition of p47phox phosphorylation, thus inhibiting the active assembly of NOX, thereby reducing ROS generation. Reduction of ROS leads to reduced release of NE and MPO, ultimately resulting in the inhibition of NET formation. mB Box-97 also inhibits Ca2+-induced NETosis in part, likely through inhibition of PKC activity, as Ca2+ also affects PKC activity.

[0016] [Figure 7] Figure 7A-7B. (A) Biofilm disruption ability of HMGB1-derived peptides. (B) mB Box-97syn significantly disrupts four additional high-priority ESKAPEE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli) regardless of biofilm age.

[0017] [Figure 8] Figures 8A-8B. mB Box-97 prevents pulmonary infection. Adult C57BL / 6 mice were intratracheally (it) challenged with (A) 10 or (B) 10 CFU of B. cenocepacia (Bc) along with 200 nM mB Box-97 or a negative control.

[0018] [Figure 9]Figures 9A-9B. (A) mB Box97 or HuTipMab ("HuTipMab" is an antibody described in Kurbatfinski, N. et al. Antimicrobial Agents and Chemotherapy 66.3(2022):e01877-21 and WO 2021007260) significantly inhibits biofilm growth by both NTHI and S. aureus compared to negative controls. (B) mB Box-97 (P = 0.03-P < 0.0001) or HuTipMab (P = 0.02-P < 0.0001) prevents biofilm growth over 16 h of incubation with P. aeruginosa (Pa), Enterobacteriacea sp. (Esp), E. faecium (Ef), uropathogenic E. coli (UPEC), A. baumannii (Ab), or B. cenocepacia (Bc) compared to the negative control.

[0019] [Figure 10] Figures 10A-10B. mB Box97 or HuTipMab demonstrate preventive activity against the pathogen K. pneumoniae. (A) Biomass measurements and (B) corresponding representative confocal scanning laser microscope (CSLM) images.

[0020] [Figure 11] Figures 11A-11B. mB Box-97 and HuTipMab show synergistic effects in inhibiting biofilm formation. (A) Prevention of NTHI biofilms by diluting mB Box-97, HuTipMab, or both together. (B) Prevention of S. aureus biofilms by diluting mB Box-97 or HuTipMab.

[0021] [Figure 12] Graphical representation showing that H-NS prevents bacterial killing by neutrophil extracellular traps.

[0022] [Figure 13A]Figures 13A-B. H-NS protein levels decrease in NTHI, S. pneumoniae, and UPEC biofilms as the biofilms mature. (A) Representative confocal images of H-NS (gray) in Streptococcus pneumoniae, NTHI, and UPEC biofilms of various ages, from 24 hours to 1 week. (B) Fluorescence intensity ratios of H-NS to NTHI, UPEC, and S. pneumoniae cells in 24-, 40-, 72-, and 1-week-old biofilms. An increase was observed from 24 hours to 40 hours, followed by a decrease in the fluorescence intensity ratio. [Figure 13B] Same as above.

[0023] [Figure 14] H-NS exits the biofilm and enters the bulk medium. H-NS concentrations were quantified using Western blots relative to CFU within the biofilm. Concentrations were found in the supernatant and within the biofilm at 16 hours and 1 week. H-NS concentrations significantly increased in the supernatant from 16 hours to 1 week, whereas a decrease in H-NS was observed in the biofilm.

[0024] [Figure 15] Figures 15A-15B. H-NS prevents PMA-induced NET formation and induces condensation of NET-evolving DNA. (A) Human neutrophils were induced with PMA for 3.5 hours with or without H-NS NTHI or (B) induced with PMA for 16 hours and then incubated with H-NS for 2 hours. NETs were incubated with wheat germ agglutinin, α-β-DNA, and α-NE antibodies and visualized by IF CLSM. H-NS prevented PMA-induced NET formation (A) and induced condensation of NET-evolving DNA (B).

[0025] [Figure 16]H-NS prevented NET killing of NTHI bacteria, similar to HU. Sixteen-hour-old NTHI biofilms were incubated with human neutrophils for 3 hours, and the percentage of bacteria killed was compared to the CFU present in NTHI without added neutrophils. The three proteins added to NTHI biofilms along with neutrophils were HU, CbpA, and H-NS. Prevention of bacterial killing by NETs was achieved by the presence of HU and H-NS. N = 5, P < 0.05. DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, preferred methods, devices, and materials are described herein. All technical publications and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure by prior disclosure.

[0027] 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, Sambrook and Russell eds.(2001)molecular cloning:a laboratory manual,3rd edition;the series ausubel et al.eds.(2007)current protocols in molecular biology;the series methods in enzymology(academic press,inc.,ny);macpherson et al.(1991)pcr 1:a practical approach(irl press at oxford university press); al.(1995)pcr 2:a practical approach;harlow and lane eds.(1999)antibodies,a laboratory manual;freshney(2005)culture of animal cells:a manual of basic technique,5th edition;gait ed.(1984)oligonucleotide synthesis;uspatent no.4,683,195;hames and higgins eds.(1984)nucleic acid hybridization;anderson(1999)nucleic acid hybridization;hames and higgins eds.(1984)transcription and translation;immobilized cells and enzymes(irl press(1986));perbal(1984)a practical guide to molecular cloning;miller and calos eds.(1987)gene transfer vectors for mammalian cells(cold spring harbor laboratory);makrides ed.(2003) Gene transfer and expression in mammalian cells; Mayer and Walker eds. (1987) Immunochemical methods in cell and molecular biology (Academic Press, London); and Herzenberg et al. eds. (1996) Weir's handbook of experimental immunology.

[0028] All numerical designations, including ranges, e.g., pH, temperature, time, concentration, and molecular weight, are approximations that vary (+) or (-) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variance of + / - 15%, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is understood, although not always expressly stated, that all numerical designations are preceded by the word "about." It is also understood, although not always expressly stated, that the reagents described herein are merely exemplary and that equivalents of such reagents are known in the art.

[0029] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "polypeptide" includes multiple polypeptides, including mixtures thereof.

[0030] As used herein, the term "comprising" is intended to mean that compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that are essential to the combination for the intended use. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from isolation and purification methods, as well as pharmaceutically acceptable carriers such as phosphate-buffered saline, preservatives, and the like. "Consisting of" is intended to mean excluding more than trace amounts of other component elements and substantial method steps for administering the compositions of the present disclosure. Embodiments defined by each of these transition terms are within the scope of the present disclosure.

[0031] As used herein, the term "comprising" is intended to mean that the peptides recited herein include the recited amino acid sequence but do not exclude other amino acids. "Consisting essentially of," when used to define a sequence, refers to a core sequence surrounded by additional amino acids as needed. Thus, peptides consisting essentially of a sequence defined herein do not exclude additional amino acids at the C- or N-terminus. In some embodiments, peptides consisting essentially of a sequence defined herein include up to 10 additional amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) at the N-terminus. In some embodiments, peptides consisting essentially of a sequence defined herein include up to 10 additional amino acids at the C-terminus. In some embodiments, peptides consisting essentially of a sequence defined herein include up to 10 additional amino acids at the N-terminus and up to 10 additional amino acids at the C-terminus. When used to refer to a peptide, "consisting of" a sequence means that the peptide does not contain any additional sequences. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0032] "Biofilm" refers to a thin layer or organized collection of microorganisms that can adhere to the surface of a structure, which can be organic or inorganic, or that can be concentrated at an interface (usually solid / liquid), surrounded by an extracellular polymeric slime matrix that contains polymers, such as DNA, secreted and / or released by the microorganisms. Biofilms can contain many different types of microorganisms, such as bacteria, archaea, protozoa, fungi, and algae. Biofilms are highly resistant to microbiotics and antimicrobial agents. They survive on gum tissue, teeth, and restorations, causing caries and periodontal disease, also known as periodontal plaque disease. They also cause chronic middle ear infections. Biofilms can also form on the surfaces of dental implants, stents, catheter lines, and contact lenses. They grow on pacemakers, heart valve replacements, artificial joints, and other surgical implants. The Centers for Disease Control estimate that over 65% of hospital-acquired infections are caused by biofilms. Fungal biofilms also frequently contaminate medical devices. They cause chronic vaginal infections and can lead to life-threatening systemic infections in people with compromised immune systems. Biofilms are also involved in a number of diseases. For example, cystic fibrosis patients often have Pseudomonas infections, which result in antibiotic-resistant biofilms.

[0033] As used herein, "treatment of a biofilm-associated disorder," also referred to herein as "preventing or treating a bacterial biofilm," refers to reducing the severity and / or duration of the disorder and / or reducing the severity and / or duration of symptoms from the disorder, particularly the symptoms of infection. In some embodiments, treatment results in the restoration of the individual's health. Preferably, the individual has less severe or shorter-lasting disease symptoms. Disease symptoms can be monitored using conventional techniques.

[0034] In some embodiments, the compositions of the present disclosure can be used to prevent or reduce biofilm formation or growth in vitro, ex vivo, or in vivo. As used herein, "preventing or reducing biofilm formation or growth" refers to preventing, slowing, or reducing biofilm formation or growth in vitro, ex vivo, or in vivo. As will be understood by those skilled in the art, such reduction in biofilm formation or growth can slow biofilm growth compared to untreated biofilm growth.

[0035] In some embodiments, the compositions disclosed herein are useful for disintegrating or reducing biofilms. As used herein, "disintegrating or reducing biofilms" refers to the partial or complete elimination of biofilms in vitro, ex vivo, or in vivo. As will be understood by those skilled in the art, after such treatment, planktonic bacteria (i.e., free-living bacteria suspended in liquid) may still exist.

[0036] "DNABII polypeptide or protein" refers to a DNA-binding protein or polypeptide that is composed of a DNA-binding domain and therefore has specific or general affinity for DNA. In one embodiment, they bind to DNA in the minor groove. Non-limiting examples of DNABII proteins are the integration host factor (IHF) protein and the histone-like protein (HU) from Escherichia coli U93 strain. Other DNA-binding proteins that may be associated with biofilms include DPS (Genbank accession number: CAA49169), H-NS (Genbank accession number: CAA47740), Hfq (Genbank accession number: ACE63256), CbpA (Genbank accession number: BAA03950) and CbpB (Genbank accession number: NP_418813).

[0037] By "tip fragment" of a DNABII polypeptide is intended a DNABII polypeptide that forms the two arms of the protein, using IHF alpha and IHF beta as examples. Non-limiting examples of such include IhfA, A tip fragment: NFELRDKSSRPGRNPKTGDVV, SEQ ID NO: 7 and IhfB, B tip fragment: SLHHRQPRLGRNPKTGDSVNL, SEQ ID NO: 8.

[0038] As used herein, "tip-chimeric peptide" or "IhfA5-mIhfB4 NTHI The term "apical chimera" or "apical chimera" refers to [ka] IhfA5-mIhfB4 comprising, consisting essentially of, or even consisting of the polypeptide sequence of (SEQ ID NO: 38) NTHI Peptide chimeras are contemplated, wherein "X" is an optional amino acid linker sequence, optionally comprising, consisting essentially of, or even consisting of between 1 and 20 amino acids; wherein "X1" is any amino acid, or alternatively, "X1" is selected from the amino acids Q, R, K, S, or T. In a further aspect, "X1" is K or Q. In a further embodiment, the tip-chimeric peptide IhfA5-mIhfB4 NTHI teeth, [ka] (SEQ ID NO: 39), where "X" is an optional amino acid linker sequence optionally comprising, consisting essentially of, or even consisting of between 1 and 20 amino acids. In yet a further embodiment, the tip-chimeric peptide IhfA5-mIhfB4 NTHI teeth, [ka] (SEQ ID NO: 40).

[0039] In certain embodiments, the tail-chimeric peptide IhfA3-IhfB2 NTHI comprises, consists essentially of, or even consists of the polypeptide sequence of FLEEIRLSLESGQDVKLSGF-X-TLSAKEIENMVKDILEFISQ (SEQ ID NO: 41), where "X" is an optional amino acid linker sequence optionally comprising, consisting essentially of, or even consisting of between 1 and 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 comprises, consists essentially of, or even consists of FLEEIRLSLESGQDVKLSGFGPSLTLSAKEIENMVKDILEFISQ (SEQ ID NO: 50).

[0040] "Integration host factor" or "IHF" proteins are bacterial proteins used by bacteriophages to integrate their DNA into host bacteria. These are DNA-binding proteins that function in genetic recombination and transcriptional and translational regulation. They also bind to extracellular microbial DNA. The genes encoding the IHF protein subunits in E. coli are the himA (Genbank accession number: POA6X7.1) and himD (POA6Y1.1) genes.

[0041] "HMGB1" is the high mobility group box (HMGB) 1 protein, which has been reported to bind to and distort the minor groove of DNA, and is an example of an interference factor. Recombinant or isolated proteins and polypeptides are commercially available from Atgenglobal, ProSpecBio, Protein1, and Abnova. The sequences of wild-type mouse HMGB1 and human HMGB1 proteins are provided in the Sequence Listing as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0042] "HU" or "histone-like protein from E. coli strain U93" refers to a class of heterodimeric proteins typically associated with E. coli. HU proteins are known to bind to DNA junctions. Related proteins have been isolated from other microorganisms. The complete amino acid sequence of E. coli HU was reported by Laine et al. (1980) Eur. J. Biochem. 103(3):447-481. Antibodies against the HU protein are commercially available from Abcam.

[0043] "Linker" or "peptide linker" refers to a peptide sequence linked to either the N-terminus or C-terminus of a polypeptide sequence. In one embodiment, the linker is about 1 to about 20 amino acid residues in length, or alternatively 2 to about 10, or about 3 to about 5 amino acid residues in length. Examples of peptide linkers are Gly-Pro-Ser-Leu-Lys-Leu (SEQ ID NO: 3) and PPKGETKKKF (SEQ ID NO: 4).

[0044] The term "Haemophilus influenzae" refers to a pathogenic bacterium that can cause many different infections, such as ear infections, eye infections, and sinusitis. Many different strains of Haemophilus influenzae have been isolated and contain the IhfA gene or protein. Some non-limiting examples of different strains of Haemophilus influenzae include Rd KW20, 86-028NP, R2866, PittGG, PittEE, R2846, and 2019.

[0045] By "microbial DNA" is intended single-stranded or double-stranded DNA derived from biofilm-producing microorganisms.

[0046] As used herein, the term "label" or "detectable label" refers to a directly or indirectly detectable compound or composition, such as an N-terminal histidine tag (N-His), a magnetically active isotope, e.g., a nucleotide sequence that is directly or indirectly conjugated to the composition to be detected to produce a "labeled" composition. 115 Sn, 117 Sn and 119 Sn, non-radioactive isotopes, e.g. 13 C and 15The term also contemplates a label that can be a polynucleotide, polynucleotide, or protein, such as an antibody. The term also includes sequences conjugated to a polynucleotide that provide a signal upon expression of the inserted sequence, such as green fluorescent protein (GFP). The label may be detectable by itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical alteration of a detectable substrate compound or composition. The label may be suitable for small-scale detection or may be more suitable for high-throughput screening. Suitable labels therefore include, but are not limited to, magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or quantified. A simply detected response generally includes a response whose presence is merely confirmed, while a quantified response generally includes a response having a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other property. In luminescent or fluorescent assays, the detectable response can be generated directly using a luminophore or fluorophore associated with the assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of signal-producing luminescent labels include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent response generally involves a change or occurrence of a luminescent signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art, see, for example, Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0047] " Gene delivery vehicle " is defined as any molecule that can carry inserted polynucleotide into host cell.Examples of gene delivery vehicle include liposome, micelle biocompatible polymers, including natural polymers and synthetic polymers; lipoprotein; polypeptide; polysaccharide; lipopolysaccharide; artificial virus envelope; metal particle; and bacteria, or viruses such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vector, and other recombinant vehicles that are typically used in the art, and can be used for gene therapy and simple protein expression in various eukaryotic and prokaryotic hosts.

[0048] The polynucleotides of the present disclosure can be delivered to cells or tissues using a gene delivery vehicle. As used herein, "gene delivery," "gene transfer," "transduction," and the like refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for transfer. Such methods include various well-known techniques, such as vector-mediated gene transfer (e.g., via viral infection / transfection or various other protein- or lipid-based gene delivery complexes), as well as techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques used for polynucleotide transfer). The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide contain an origin of replication compatible with the host cell or be integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid), or into the nuclear or mitochondrial chromosome. As known in the art and described herein, many vectors are known that can mediate the transfer of genes into mammalian cells.

[0049] As used herein, the term "eDNA" refers to extracellular DNA found as a component of pathogenic biofilms.

[0050] As used herein, ESKAPE pathogens include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. These pathogens are the leading cause of hospital-acquired infections worldwide.

[0051] A "plasmid" is an extrachromosomal DNA molecule that can replicate independently of chromosomal DNA and is often circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and typically confer a selective advantage under given environmental conditions. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively produced proteins may act as toxins under similar circumstances.

[0052] Plasmids used in genetic engineering are called "plasmid vectors." Many plasmids are commercially available for such purposes. The gene to be replicated is inserted into a copy of the plasmid, which contains a gene that confers resistance to a specific antibiotic and a multiple cloning site (MCS, or polylinker), a short region containing several commonly used restriction sites that allows for easy insertion of DNA fragments into this location. Another major use of plasmids is to produce large amounts of proteins. In this case, researchers grow bacteria containing a plasmid with the gene of interest. Similar to bacteria producing proteins that confer antibiotic resistance, bacteria can also be induced to produce large amounts of proteins from the inserted gene. This is an inexpensive and easy way to mass-produce a gene or the protein it encodes.

[0053] "Yeast artificial chromosome" or "YAC" refers to a vector used to clone large DNA fragments (greater than 100 kb and up to 3000 kb). It is an artificially constructed chromosome that contains telomere, centromere, and origin of replication sequences necessary for replication and storage in yeast cells. It is constructed using an initial circular plasmid, which is linearized using restriction enzymes. DNA ligase can then be used to add sequences or genes of interest within the linear molecule using the cohesive ends. Yeast expression vectors such as YAC, YIps (yeast integrating plasmid), and YEps (yeast episomal plasmid) are extremely useful because yeast itself is a eukaryotic cell, allowing for the production of eukaryotic protein products with post-translational modifications. However, YACs are more unstable than BACs and have been shown to produce chimeric effects.

[0054] A "viral vector" is defined as a recombinantly produced virus or virus particle that contains a polynucleotide delivered to a host cell either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, alphavirus vectors, and the like. Proteins can be produced using infectious tobacco mosaic virus (TMV)-based vectors, and it has been reported that they express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15):6099-6104). Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. In embodiments where gene transfer is mediated by a retroviral vector, vector construct refers to a polynucleotide comprising the retroviral genome or a portion thereof and a therapeutic gene.

[0055] As used herein, "retroviral-mediated gene transfer" or "retroviral transduction" have the same meaning and refer to the process by which a virus enters a cell and integrates its genome into the host cell genome, thereby stably introducing a gene or nucleic acid sequence into a host cell. The virus can enter the host cell through its normal infection mechanism, or it can be modified to bind to a different host cell surface receptor or ligand and enter the cell. As used herein, retroviral vector refers to a viral particle that can introduce exogenous nucleic acid into a cell via a viral or viral-like entry mechanism.

[0056] Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse transcribed into a DNA form and integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0057] In embodiments in which gene transfer is mediated by a DNA virus vector, such as adenovirus (Ad) or adeno-associated virus (AAV), the vector construct refers to a polynucleotide comprising the viral genome or a portion thereof and the transgene. Adenoviruses (Ad) are a relatively well-characterized, homogeneous group of viruses, including more than 50 serotypes. See, for example, International PCT Application No. WO95 / 27071. Ad does not require integration into the host cell genome. Recombinant Ad-derived vectors have also been constructed, particularly those that reduce the possibility of recombination and generation of wild-type viruses. See, International PCT Application Nos. WO95 / 00655 and WO95 / 11984. Wild-type AAV has high infectivity and specificity for integration into the genome of host cells. See Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0058] Vectors containing both a promoter and a cloning site into which a polynucleotide can be operably linked are known in the art. Such vectors can transcribe RNA in vitro or in vivo and are commercially available from suppliers such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to eliminate additional, potentially inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression at either the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' of the initiation codon to enhance expression.

[0059] Gene delivery vehicles also include DNA / liposome complexes, micelles, and targeting viral protein-DNA complexes. Liposomes containing targeting antibodies or fragments thereof can also be used in the methods of the present disclosure. In addition to the delivery of polynucleotides to cells or cell populations, direct introduction of the proteins described herein into cells or cell populations can be achieved by, but is not limited to, protein transfection techniques, or culture conditions that can enhance the expression and / or activity of the proteins of the present disclosure are other, but not limited to, techniques.

[0060] "Inhibiting, preventing, or disrupting" a biofilm contemplates prophylactic or therapeutic reduction of biofilm structure. In one aspect, this definition excludes biofilm prevention. In another aspect, the term "inhibiting, competing, or titrating" contemplates reduction of the formation of the DNA / protein matrix that is a component of microbial biofilms. In one aspect, prevention is excluded from treatment.

[0061] By "bent polynucleotide" is intended a double-stranded polynucleotide containing a small loop on one strand that is not paired with the other strand, and any polynucleotide that reduces the end-to-end distance beyond natural thermal fluctuations, i.e., bends beyond the 150 bp persistence length of native B-form double-stranded DNA. In some embodiments, the loop is 1 base to about 20 bases in length, or alternatively 2 bases to about 15 bases in length, or alternatively about 3 bases to about 12 bases in length, or alternatively about 4 bases to about 10 bases in length, or alternatively has about 4, 5, or 6, or 7, or 8, or 9, or 10 bases.

[0062] A "subject" of diagnosis or treatment is a cell or animal, such as a mammal or human. Non-human animals that are subjects of diagnosis and treatment include animals that are subject to infectious diseases or animal models, such as apes, rats, mice, chinchillas, canines, rabbits, livestock, sport animals, and pets.

[0063] The terms "protein," "peptide," and "polypeptide" are used interchangeably and, in their broadest sense, refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may comprise the sequence of a protein or peptide. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0064] The terms "isolated" or "recombinant," as used herein with respect to nucleic acids, such as DNA or RNA, refer to molecules separated from other DNA or RNA, respectively, present in the natural source of the macromolecule or polypeptide. The term "isolated or recombinant nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in their natural state. The term "isolated" is also used herein to refer to polynucleotides, polypeptides, and proteins that are isolated from other cellular proteins and is intended to encompass both purified and recombinant polypeptides. In other embodiments, the term "isolated or recombinant" refers to being separated from components, cells, and other things with which it is normally associated in nature, including cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof. For example, an isolated cell is one that is separated from tissues or cells of a different phenotype or genotype. An isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on a chromosome. As will be apparent to one of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof does not require "isolation" to be distinguished from its naturally occurring counterpart.

[0065] When the present disclosure relates to polypeptides, proteins, polynucleotides, or antibodies, it should be assumed without explicit recitation that equivalents or biological equivalents of such are intended within the scope of the present disclosure, unless otherwise intended. As used herein, the term "biological equivalent thereof" when referring to a reference protein, antibody, polypeptide, or nucleic acid is intended to be synonymous with "equivalent thereof," and is intended to be synonymous with "equivalent thereof," and to be one that has minimal homology while still maintaining the desired structure or function. Unless specifically recited herein, any polynucleotide, polypeptide, or protein referred to herein is intended to also include its equivalent. For example, an equivalent is intended to have at least about 70% homology or identity, or alternatively about 80% homology or identity, and alternatively at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively 98% percent homology or identity, and exhibit substantially the same biological activity as the reference protein, polypeptide, or nucleic acid. In another aspect, the term contemplates a polynucleotide that hybridizes to a reference polynucleotide or its complement under conditions of high stringency.

[0066] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are identical when comparing the two sequences. Alignment and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST using default parameters. Particularly preferred programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort criteria = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0067] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for purposes of comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 30% identity, or alternatively less than 25% identity, less than 20% identity, or alternatively less than 10% identity with one of the sequences of the present disclosure.

[0068] "Homology" or "identity" or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions to a reference polynucleotide or its complement.

[0069] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonds between the bases of nucleotide residues. The hydrogen bonds can occur through Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific manner. The complex can include two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction can constitute a step in a more extensive process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0070] Examples of stringent hybridization conditions include an incubation temperature of about 25°C to about 37°C, a hybridization buffer concentration of about 6xSSC to about 10xSSC, a formamide concentration of about 0% to about 25%, and a wash solution of about 4xSSC to about 8xSSC. Examples of moderate hybridization conditions include an incubation temperature of about 40°C to about 50°C, a buffer concentration of about 9xSSC to about 2xSSC, a formamide concentration of about 30% to about 50%, and a wash solution of about 5xSSC to about 2xSSC. Examples of high stringency conditions include an incubation temperature of about 55°C to about 68°C, a buffer concentration of about 1xSSC to about 0.1xSSC, a formamide concentration of about 55% to about 75%, and a wash solution of about 1xSSC, 0.1xSSC, or deionized water. Generally, hybridization incubation times range from 5 minutes to 24 hours, with one, two, or more wash steps, with wash incubation times of approximately 1, 2, or 15 minutes. SSC is a 0.15M NaCl and 15mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be used.

[0071] A "subject" of diagnosis or treatment can be a cell or an animal, such as a mammal or a human. Non-human animals that are subjects of diagnosis and treatment include animals subject to infectious diseases and animal models, such as apes, rats, mice, and chinchillas; canines, such as dogs; lagomorphs, such as rabbits; livestock; sport 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), livestock (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal can be of any age or stage of development (e.g., adult, teen, child, infant, or in utero mammal). The mammal can be male or female. In some embodiments, the subject is a human.

[0072] By host cell is intended a eukaryotic or prokaryotic cell that contains an exogenous agent. "Eukaryotic cell" encompasses all kingdoms of life except Monera. These can be easily distinguished by their membrane-bound nuclei. Animals, plants, fungi, and protists are eukaryotic organisms, i.e., organisms in which cells are organized into complex structures by internal membranes and a cytoskeleton. The most distinctive membrane-bound structure is the nucleus. Unless otherwise specified, the term "host" includes eukaryotic hosts, including, for example, yeast, higher plants, insects, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include apes, cows, pigs, mice, rats, birds, reptiles, and humans.

[0073] Prokaryotic cells typically lack a nucleus or any other membrane-bound organelles and are divided into two domains: bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in circular loops called episomes. Bacterial cells are very small, roughly the size of animal mitochondria (approximately 1-2 μm in diameter and 10 μm in length). Prokaryotic cells are characterized by three main shapes: rod-shaped, spherical, and spiral. Instead of undergoing an elaborate replication process like eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, Bacillus, Escherichia, and Salmonella.

[0074] As used herein, the terms "treat," "treatment," and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents a disorder or its signs or symptoms, and / or may be therapeutic, in that it partially or completely cures a disorder and / or adverse effects resulting from the disorder. In one embodiment, "treatment" excludes prevention.

[0075] By "prevent" is intended to prevent the disorder or effect in vitro or in vivo in a system or subject susceptible to the disorder or effect. Such an example would be preventing biofilm formation in a system infected with a microorganism known to produce biofilms.

[0076] "Pharmaceutically acceptable carrier" refers to any diluent, excipient, or carrier that can be used in the compositions of the present disclosure. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, saturated vegetable fatty acids, water, salts, or partial glyceride mixtures of electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference in this field. They are preferably selected based on the intended form of administration, i.e., oral tablets, capsules, elixirs, syrups, etc., and consistent with conventional pharmaceutical practice.

[0077] "Administration" can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods for determining the most effective means and dosage of administration are known to those skilled in the art and vary depending on the composition used for treatment, the purpose of the treatment, the target cells being treated, and the subject being treated. Single or multiple administrations can be performed, with the dose level and pattern selected by the treating physician. Appropriate dosage formulations and methods for administering drugs are known in the art. The 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 vary depending on the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and the target cells or tissues. Non-limiting examples of administration routes include oral administration, nasal administration, injection, and topical application.

[0078] The term "encoding," when applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide when, in its native state, or when manipulated by methods well known to those of skill in the art, it is capable of being transcribed and / or translated to produce mRNA for the polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

[0079] The term "effective amount" refers to an amount sufficient to achieve a beneficial or desired result or effect. In the context of therapeutic or prophylactic applications, the effective amount depends on the type and severity of the condition at issue, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. In the context of immunogenic compositions, in some embodiments, an effective amount is an amount sufficient to produce a protective response against a pathogen. In other embodiments, an effective amount of an immunogenic composition is an amount sufficient to produce antibody production against an antigen. In some embodiments, an effective amount is the amount necessary to confer passive immunity to a subject in need thereof. With respect to immunogenic compositions, in some embodiments, the effective amount depends on the intended use, the degree of immunogenicity of the particular antigenic compound, and the health / responsiveness of the subject's immune system, in addition to the factors described above. Those skilled in the art can determine the appropriate amount depending on these and other factors.

[0080] For in vitro applications, in some embodiments, the effective amount depends on the size and nature of the application in question. It also depends on the nature and sensitivity of the in vitro target and the method being used. Those skilled in the art can determine the effective amount based on these and other considerations. The effective amount may include one or more administrations of the composition depending on the embodiment.

[0081] The agents and compositions can be used in the manufacture of medicinal products and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as the active ingredients in pharmaceutical compositions.

[0082] The agents of the present disclosure can be administered for treatment by any suitable route of administration. It will also be understood that the preferred route will vary depending on the condition and age of the recipient and the disease being treated.

[0083] Examples of solid supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. The nature of the support can be either soluble to some extent or insoluble. The support material can have virtually any possible structural configuration so long as the coupled molecule can bind to a polynucleotide, polypeptide, or antibody. Thus, the support configuration can be spherical, such as a bead, or cylindrical, such as the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface can be flat, such as a sheet, test strip, or polystyrene beads. Those skilled in the art will know of many other suitable supports for binding antibodies or antigens, or can ascertain them by routine experimentation.

[0084] As used herein, "antibody" includes whole antibodies and any antigen-binding fragments or single chains thereof. Thus, the term "antibody" includes any protein- or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule. Examples of such include, but are not limited to, a heavy or light chain complementarity-determining region (CDR) or ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, or at least a portion of a binding protein. Antibodies can be polyclonal or monoclonal and can be isolated from any suitable biological source, such as mice, rats, sheep, or dogs.

[0085] The terms "antibody," "antibodies," and "immunoglobulin" also include immunoglobulins of any isotype, fragments of antibodies that retain specific binding to an antigen, including, but not limited to, Fab, Fab', F(ab)2, Fv, scFv, dsFv, Fd fragments, dAb, VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappabodies; and multispecific antibody fragments formed from antibody fragments. Examples of such fragments include, but are not limited to, heavy or light chain complementarity-determining regions (CDRs) or ligand-binding portions thereof, heavy or light chain variable regions (also referred to herein as variable domains), heavy or light chain constant regions (also referred to herein as constant domains), framework (FR) regions, or any portion thereof, at least a portion of a binding protein, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The variable regions of the heavy and light chains of immunoglobulin molecules contain binding domains that interact with antigens. The constant region of an antibody (Ab) mediates the binding of the immunoglobulin to host tissues. The term "anti-" when used before a protein name, such as anti-DNABII, anti-IHF, anti-HU, or anti-tip chimera, refers to a monoclonal or polyclonal antibody that binds to and / or has affinity for a specific protein. For example, "anti-IHF" refers to an antibody that binds to the IHF protein. A specific antibody may have affinity for or bind to proteins other than the protein to which it was raised. For example, anti-IHF is specifically raised against the IHF protein but may also bind to other proteins related through either sequence or structural homology.

[0086] Complementarity-determining regions (CDRs) are portions of the variable regions of antibodies or T-cell receptors, produced by B cells and T cells, respectively, that bind to their specific antigens (also called epitopes). In certain embodiments, the terms "variable region" and "variable domain" are used interchangeably and refer to polypeptides in the light or heavy chains of an antibody that vary significantly in sequence of amino acid residues from one antibody to another and that determine the conformation of the binding site that confers the antibody's specificity for a particular antigen. In further embodiments, the variable region is between about 90 and about 200 amino acids in length, including, but not limited to, about 100 amino acids in length, or alternatively about 110 amino acids in length, or alternatively about 120 amino acids in length, or alternatively about 130 amino acids in length, or alternatively about 140 amino acids in length, or alternatively about 150 amino acids in length, or alternatively about 160 amino acids in length, or alternatively about 170 amino acids in length, or alternatively about 180 amino acids in length, or alternatively about 190 amino acids in length. In certain embodiments, a variable region of an amino acid sequence, as used herein, refers to the first about 100 amino acids, or alternatively about 110 amino acids, or alternatively about 120 amino acids, or alternatively about 130 amino acids, or alternatively about 140 amino acids, or alternatively about 150 amino acids (including or excluding the signal peptide, if applicable) of an amino acid sequence being the variable region.

[0087] A set of CDRs constitutes a paratope, also called an antigen-binding site, which is the part of an antibody that recognizes and binds to an antigen. Three CDRs (CDR1, CDR2, and CDR3) are present in the amino acid sequence of the variable region of an antigen receptor, such as a heavy or light chain, non-contiguously arranged from the amino terminus to the carboxyl terminus, as appropriate. As used herein, CDRn refers to CDRn in or derived from an immunoglobulin chain, where the number n is selected from 1 to 3. In one embodiment, CDRLn refers to CDRn in or derived from a light chain, where the number n is selected from 1 to 3; while CDRHn refers to CDRn in or derived from a heavy chain, where the number n is selected from 1 to 3. In certain embodiments, framework region (FR) refers to the portion of the variable region that is not a CDR. In certain embodiments, FRn refers to FR in or derived from a heavy or light chain, where the number n is selected from 1 to 4. In certain embodiments, the variable region comprises, consists essentially of, or even consists of the following (optionally in the order provided, and optionally from amino terminus to carboxyl terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0088] The variable regions and / or CDRs of an antibody or fragment thereof can be determined by one of skill in the art, for example, using publicly or commercially available tools. Non-limiting examples of such tools include IgBlast (accessible at www.ncbi.nlm.nih.gov / igblast / ), Scaligner (available from drugdesigntech at www.scaligner.com / ), IMGT rules and / or tools (see, e.g., www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html, also accessible at www.imgt.org / ), Chothia Canonical Assignment (accessible at www.bioinf.org.uk / abs / chothia.html), Antigen Receptor Numbering And Receptor Classification (ANARCI, accessible at opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / anarci / ), Kabat numbering method / scheme (e.g., Kabat, E.A., et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242), or the Paratome web server (accessible at www.ofranlab.org / paratome / ; see Vered Kunik, et al, Nucleic Acids Research, Volume 40, Issue W1, 1 July 2012, Pages W521-W524).

[0089] Antibodies can be polyclonal, monoclonal, multispecific (e.g., bispecific) and antibody fragments, so long as they exhibit the desired biological activity. Antibodies can be isolated from any suitable biological source, such as mice, rats, sheep and dogs.

[0090] As used herein, the term "polyclonal antibody" or "polyclonal antibody composition" refers to a preparation of antibodies derived from different B-cell lines, which are a mixture of immunoglobulin molecules secreted against a particular antigen, each recognizing a different epitope.

[0091] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. Because each monoclonal antibody is directed against a single determinant on the antigen, monoclonal antibodies are highly specific. Antibodies can be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, or the like. Antibodies can be further conjugated to other moieties, such as a member of a specific binding pair, for example, biotin (a member of the biotin-avidin specific binding pair). Antibodies can also be bound to a solid support, including, but not limited to, a polystyrene plate or beads.

[0092] Monoclonal antibodies can be produced using hybridoma technology or recombinant DNA methods known in the art. Hybridomas are cells produced in the laboratory from the fusion of antibody-producing lymphocytes with non-antibody-producing cancer cells, usually myelomas or lymphomas. The hybridomas grow and produce continuous samples of specific monoclonal antibodies. Alternative techniques for producing or selecting antibodies include in vitro exposure of lymphocytes to the antigen of interest and screening antibody display libraries in cell, phage, or similar systems.

[0093] 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 by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Thus, as used herein, the term "human antibody" refers to an antibody comprising substantially all portions of the protein (e.g., CDRs, framework, CDRs ... L , C H Domain (e.g., C H1 , C H2 , C H3 A human antibody refers to an antibody in which the base sequences (VL, VH) are substantially non-immunogenic in humans and have only minor sequence changes or variations. Similarly, antibodies designated for primates (monkeys, baboons, chimpanzees, etc.), rodents (mouse, rats, rabbits, guinea pigs, hamsters, etc.), or other mammals designate such species-, subgenus-, genus-, subfamily-, or family-specific antibodies. Furthermore, chimeric antibodies include any combination of the above. Such changes or variations optionally retain or reduce immunogenicity in humans or other species compared to unmodified antibodies. Thus, a human antibody differs from a chimeric antibody or a humanized antibody. 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 and / or light chain) genes. Furthermore, when a human antibody is a single-chain antibody, it can include a linker peptide not found in native human antibodies. For example, an Fv can include a linker peptide, such as 2 to about 8 glycine or other amino acid residues, connecting the heavy chain variable region and the light chain variable region, and such a linker peptide is believed to be of human origin.

[0094] As used herein, a human antibody is "derived" from a particular germline sequence if the antibody is obtained from a system that uses human immunoglobulin sequences, for example, by immunizing a transgenic mouse carrying human immunoglobulin genes or by screening a human immunoglobulin gene library. A human antibody "derived" from a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequence of a human germline immunoglobulin. The selected human antibody typically has at least 90% amino acid sequence identity to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as human when compared to the germline immunoglobulin amino acid sequence of another species (e.g., a mouse germline sequence). In certain cases, a human antibody may have at least 95%, or even at least 96%, 97%, 98%, or 99% amino acid sequence identity to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence displays no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0095] As used herein, the term "humanized antibody" or "humanized immunoglobulin" refers to a human / non-human chimeric antibody that contains minimal sequence derived from non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a variable region or fragment thereof (e.g., one, two, three, four, five, or all six CDRs) of the recipient are replaced by residues from a variable region or fragment thereof (e.g., one, two, three, four, five, or all six CDRs) of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. Humanized antibodies may include residues that are not found in the recipient antibody or donor antibody. Humanized antibodies can also include non-human antibodies, optionally containing at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin, with one or more amino acids in the framework region, constant region, or CDR substituted with correspondingly positioned amino acids from a human antibody. Without wishing to be bound by theory, humanized antibodies produce a reduced immune response in a human host compared to a non-humanized version of the same antibody. Humanized antibodies may have conservative amino acid substitutions that do not substantially affect antigen binding or other antibody functions. Conservative substitution groups include glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serine-threonine, and asparagine-glutamine. Specifically, the humanized antibodies disclosed herein have a DNABII polypeptide or a fragment thereof (such as a tip chimeric peptide or a tail chimeric peptide) that has an EC 50 , K. on , K. off , K. A and / or K DSpecifically binds to one or more specific ranges of the DNABII polypeptide, inhibiting or releasing specific cytokines when treating a subject. In a further embodiment, a humanized antibody that specifically binds to the tip region (such as the tip chimeric peptide) of the DNABII polypeptide destroys biofilms both in vivo and in vitro. Furthermore, although the humanization process is a rational design process, it may result in unexpected changes (positive or negative) in binding affinity, antigen specificity, or physical properties such as solubility or aggregation. Therefore, the properties of a humanized antibody are inherently unpredictable from the properties of the starting non-human antibody.

[0096] In one embodiment, an antibody as used herein may be a recombinant antibody. As used herein, the term "recombinant antibody" includes all antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed to express the antibody, e.g., from transfectomas, antibodies isolated from recombinant combinatorial antibody libraries, and antibodies prepared, expressed, generated, or isolated by any other means, including splicing of immunoglobulin (Ig) gene sequences to other DNA sequences. However, in certain embodiments, such recombinant antibodies can be subjected to in vitro mutagenesis (or, when using animals transgenic for Ig sequences, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that may not naturally occur within the antibody germline repertoire in vivo. Methods for producing these antibodies are known in the art.

[0097] In one embodiment, the antibody used herein may be a chimeric antibody. As used herein, a chimeric antibody is an antibody whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species.

[0098] Additionally, the antibodies disclosed herein can be engineered to contain modifications within the Fc region to alter one or more functional properties of the antibody (e.g., serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity, etc.). Such modifications include, but are not limited to, changing the number of cysteine ​​residues in the hinge region to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody (U.S. Patent No. 5,677,425), and mutating amino acids in the Fc hinge region to decrease the biological half-life of the antibody (U.S. Patent No. 6,165,745).

[0099] In addition, the antibodies disclosed herein can be chemically modified. The glycosylation of an antibody can be altered, for example, by modifying one or more glycosylation sites within the antibody sequence to increase the affinity of the antibody for an antigen (U.S. Patent Nos. 5,714,350 and 6,350,861). Alternatively, to increase antibody-dependent cell-mediated cytotoxicity, hypofucosylated antibodies with reduced amounts of fucosyl residues or increased bisecting GlcNac structures can be obtained by expressing the antibody in a host cell with an altered glycosylation machinery (Shields, R. Lett. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-180).

[0100] The antibodies disclosed herein can be pegylated to increase biological half-life by reacting the antibody or antibody fragment with polyethylene glycol (PEG) or a reactive ester or aldehyde derivative of PEG under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. Antibody PEGylation may be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono(C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated can be an aglycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies disclosed herein (EP 0154316 and EP 0401384).

[0101] Furthermore, antibodies can be chemically modified by conjugating or fusing the antigen-binding region of the antibody to a serum protein such as human serum albumin to increase the half-life of the resulting molecule. Such approaches are described, for example, in EP0322094 and EP0486525.

[0102] The antibodies or fragments thereof of the present disclosure can be conjugated to diagnostic agents and used for diagnostic purposes, for example, to monitor the onset or progression of disease and determine the effectiveness of a given treatment regimen. Examples of diagnostic agents include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals using various positron emission tomography techniques, and non-radioactive paramagnetic metal ions. The detectable substance can be coupled or conjugated directly to the antibody or fragment thereof or indirectly via a linker using techniques known in the art. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include: 125 I, 131I, Indium-111, Lutetium-171, Bismuth-212, Bismuth-213, Astatine-211, Copper-62, Copper-64, Copper-67, Yttrium-90, Iodine-125, Iodine-131, Phosphorus-32, Phosphorus-33, Scandium-47, Silver-111, Gallium-67, Praseodymium-142, Samarium-153, Terbium-161, Dysprosium-166, Holmium-16 Radioactive metal ions include rhenium-186, rhenium-188, rhenium-189, lead-212, radium-223, actinium-225, iron-59, selenium-75, arsenic-77, strontium-89, molybdenum-99, rhodium-1105, palladium-109, praseodymium-143, promethium-149, erbium-169, iridium-194, gold-198, gold-199, and lead-211. Monoclonal antibodies can be indirectly conjugated to radioactive metal ions by using a bifunctional chelating agent covalently attached to the antibody. Chelators can be attached via amities (Meares et al. (1984) Anal. Biochem. 142:68-78); sulfhydryl groups of amino acid residues (Koyama (1994) Chem. Abstr. 120:217-262) and carbohydrate groups (Rodwell et al. (1986) PNAS USA 83:2632-2636; Quadri et al. (1993) Nucl. Med. Biol. 20:559-570).

[0103] Additionally, the antibodies or fragments thereof of the present disclosure may be conjugated to a therapeutic agent. Suitable therapeutic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin. anti-inflammatory drugs (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (e.g., mechlorethamine, thioepa, chloramfucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dapagliflozin ... carbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, e.g., carboplatin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), diphtheria toxin and related molecules (diphtheria A chain and its active fragments and hybrid molecules), ricin toxin (such as ricin A or deglycosylated ricin A chain toxin), cholera toxin, Shiga-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, soapwortofficinalis) inhibitors, gelonin, mitogenin, lystocin, phenomycin, enomycin toxin and mixed toxins.

[0104] Additional suitable conjugate molecules include ribonucleases (RNases), DNase I, antisense nucleic acids, inhibitory RNA molecules such as siRNA molecules, immunostimulatory nucleic acids, aptamers, ribozymes, triplex-forming molecules, and external guide sequences. Aptamers are small nucleic acids ranging from 15 to 50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets, and can bind to small molecules such as ATP (U.S. Pat. No. 5,631,146) and theophylline (U.S. Pat. No. 5,580,737), as well as large molecules such as reverse transcriptase (U.S. Pat. No. 5,786,462) and thrombin (U.S. Pat. No. 5,543,293). Ribozymes are nucleic acid molecules that can catalyze chemical reactions, either intramolecularly or intermolecularly. Ribozymes typically cleave nucleic acid substrates by recognizing and binding to the target substrate followed by cleavage. Triplex-forming functional nucleic acid molecules can interact with double-stranded or single-stranded nucleic acids by forming triplexes, in which the three DNA strands form a complex that relies on both Watson-Crick and Hoogsteen base pairing. Triplex molecules can bind to target regions with high affinity and specificity.

[0105] Functional nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or functional nucleic acid molecules can have de novo activity independent of any other molecule.

[0106] Therapeutic agents can be linked to antibodies directly or indirectly using any of a number of available methods. For example, drugs can be attached to the hinge region of a reduced antibody component via disulfide bond formation using cross-linking agents such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP) or via carbohydrate moieties in the Fc region of the antibody (Yu et al. 1994 Int. J. Cancer 56:244; Upeslacis et al., "Modification of Antibodies by Chemical Methods," in Monoclonal antibodies: principles and applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal antibodies: Production, engineering and clinical application, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995)).

[0107] Techniques for conjugating therapeutic agents to antibodies are well known (Amon et al. "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy; Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al. "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.); Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospects Of The Therapeutic Use Of Radiolabeled Antibodies" ... Antibody in Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al. “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,” (1982) Immunol. Rev. 62:119-58).

[0108] The antibodies or antigen-binding regions thereof disclosed herein can be linked to another functional molecule, such as another antibody or ligand for a receptor, to generate bispecific or multispecific molecules that bind to at least two or more different binding sites or target molecules. Linking of the antibody to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, can be achieved, for example, by chemical coupling, genetic fusion, or noncovalent association. Multispecific molecules can further include a third binding specificity in addition to the first and second target epitopes.

[0109] Bispecific and multispecific molecules can be prepared using methods known in the art. For example, each binding unit of a hi-specific molecule can be produced separately and then conjugated to each other. When the binding molecule is a protein or peptide, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitroberyllium acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (sulfo-SMCC) (Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). When the binding molecule is an antibody, it can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains.

[0110] The antibodies of the present disclosure or fragments thereof can be linked to a moiety that is toxic to the cells to which the antibody binds to form "depleting" antibodies. These antibodies are particularly useful in applications in which it is desired to deplete NK cells.

[0111] The antibodies disclosed herein can also be attached to a solid support, which is particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0112] Antibodies can also be bound to many different carriers. Thus, the present disclosure also provides compositions containing antibodies and other active or inactive substances. Examples of known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes disclosed herein. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or can ascertain such carriers using routine experimentation.

[0113] As used herein, the term "antibody derivative" encompasses a full-length antibody or a fragment of an antibody in which one or more amino acids have been chemically modified, for example, by alkylation, pegylation, acylation, ester formation, or amide formation, to link the antibody to a second molecule. This includes, but is not limited to, pegylated antibodies, cysteine-pegylated antibodies, and variants thereof.

[0114] As used herein, the term "immunoconjugate" includes an antibody or antibody derivative associated or linked to a second agent, such as a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semi-synthetic antibody, or a multispecific antibody.

[0115] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th (ed.).

[0116] In another embodiment, the fluorescent label is functionalized to facilitate covalent attachment to cellular components present in or on the surface of cells or tissues, such as cell surface markers. Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which can be used to attach the fluorescent label to a second molecule. The choice of functional group on the fluorescent label depends on the site of attachment to either a linker, drug, marker, or second labeling agent.

[0117] "Immune response" broadly refers to an antigen-specific response of lymphocytes to a foreign substance. Any substance capable of eliciting an immune response is said to be "immunogenic" and called an "immunogen." All immunogens are antigens, but not all antigens are immunogenic. The immune response of the present disclosure can be humoral (through antibody activity) or cell-mediated (through T-cell activation).

[0118] The term "modulating an immune response" includes inducing (increasing, eliciting) an immune response; and decreasing (suppressing) an immune response. An immunomodulatory method (or protocol) is a method of modulating an immune response in a subject.

[0119] "HMG domain" or "high mobility group (HMG) box domain" refers to an amino acid sequence involved in DNA binding (Stros et al., Cell Mol Life Sci. 64(19-20):2590-606(2007)). In one embodiment, the structure of an HMG-box domain consists of an irregular array of three helices. In another embodiment, the HMG-box domain allows the protein to bind with high affinity to non-B-form DNA conformations (twisted or unwound). HMG-box domains can be found in high mobility group proteins involved in regulating DNA-dependent processes such as transcription, replication, and DNA repair, all of which require altering the conformation of chromatin (Thomas (2001) Biochem. Soc. Trans. 29(Pt4):395-401).

[0120] Compositions used according to the present disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of the composition calculated to produce the desired response in conjunction with its administration, i.e., an appropriate route and regimen. The amount administered will depend on the outcome and / or desired protection, depending on both the number of treatments and the unit dose. The precise amount of the composition also depends on the judgment of the practitioner and is unique to each individual. Factors affecting dosage include the subject's physical and clinical condition, the route of administration, the intended treatment goal (alleviation of symptoms versus cure), and the efficacy, stability, and toxicity of the particular composition. Upon formulation, a solution will be administered in a manner compatible with the dosage formulation and in an amount that is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described herein.

[0121] The term "contacting" refers to a direct or indirect binding or interaction between two or more entities. A specific example of a direct interaction is binding. A specific example of an indirect interaction is when one entity acts on an intermediate molecule, which then acts on a second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell, and in vivo. In vivo contacting can be referred to as administering or administration.

[0122] As used herein, "recombinant peptide" refers to a peptide produced in a host cell (e.g., E. coli, yeast) using exogenous / recombinant nucleic acid. As used herein, "synthetic peptide" refers to a peptide synthesized by synthetic or chemical means without the use of a host cell. Recombinant peptides may have post-translational modifications, while synthetic peptides do not have any post-translational modifications.

[0123] In certain embodiments, the biofilm is derived from (i.e., produced by) Gram-negative or Gram-positive biofilm-producing bacteria. In certain embodiments, the biofilm comprises a DNABII protein. In further embodiments, the biofilm comprises a histone-like protein (HU) or integration host factor (IHF) binding protein from Escherichia coli U93 strain. In certain embodiments, the DNABII peptide is an IHF peptide. Additionally or alternatively, the DNABII peptide is an HU peptide. In certain embodiments, the apical region of the DNABII peptide is the apical region of IHFA and / or the apical region of IHFB. In further embodiments, the apical region of the DNABII peptide is the IHFA apical region conjugated directly or indirectly (e.g., via a linker) to the IHFB apical region. In yet further embodiments, the apical region of the DNABII peptide is IhfA5-mIhfB4 NTHI It is a tip chimeric peptide.

[0124] As used herein, "EC 50The term "antibody or fragment thereof" refers to the concentration of an antibody or fragment thereof that induces a response (e.g., binding between an antibody or fragment thereof and its target) intermediate between baseline and maximum after a specific exposure time.

[0125] Several parameters are used herein to describe the binding and non-binding reactions of receptor (R, e.g., an antibody or fragment thereof) and ligand (L, e.g., the target of the antibody or fragment thereof) molecules, which are formalized as follows:

number

[0126] Modes for Carrying Out the Disclosure Synthetic or Recombinant mB Box-97 Polypeptides Provided herein are synthetic or recombinant polypeptides that comprise, consist essentially of, or consist of amino acids 90-176 or amino acids 80-176 from the coding sequence of native human HMGB1 protein (set forth in SEQ ID NO:2), with a cysteine ​​to serine point mutation at amino acid 106 of mB Box-97 that abolishes the ability of the polypeptide to induce an inflammatory response, as well as equivalents thereof that consist of amino acids 90-176 or amino acids 80-176 from the coding sequence of native human HMGB1 protein (native / wild-type human HMGB1 sequence set forth in SEQ ID NO:2), with a cysteine ​​to serine point mutation at amino acid 106 of mB Box-97 that abolishes the ability of the polypeptide to induce an inflammatory response. Synthetic polypeptides are produced by synthetic or chemical means and are not wild-type or recombinantly produced.

[0127] As used herein, an equivalent of a mB Box-97 polypeptide, recombinant mB Box-97, or synthetic mB Box-97 refers to a sequence that is at least about 70%, or alternatively 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 mB Box-97 polypeptide, recombinant mB Box-97, or synthetic mB Box-97, and in one embodiment retains the mutated amino acid of the cysteine ​​to serine point mutation at amino acid 106. In one embodiment, percent identity is determined using the BLAST alignment program using default parameters. Particularly preferred programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort criteria = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0128] In some respects, equivalents of mB Box-97 polypeptides retain the intended function and / or structural characteristics of mB Box-97 polypeptides. In another embodiment, equivalents of mB-Box-97 polypeptides include mB Box-97 or recombinant mB Box-97 or synthetic mB Box-97 polypeptides that retain a C to S amino acid substitution at amino acid 106 and further contain, independently, at least two, or alternatively at least three, or alternatively at least four, or alternatively at least five, or at least six, or alternatively at least seven, or alternatively at least eight, or alternatively at least nine, or alternatively at least ten amino acids at the amino and / or carboxyl termini of the polypeptide.

[0129] In a further embodiment, the mB Box-97 HMGB1 polypeptide further comprises, consists essentially of, or even consists of one or more linker polypeptides. Exemplary peptide linkers are GPSLKL (SEQ ID NO: 3) or PPKGETKKKF (SEQ ID NO: 4) on the amine and / or carboxy termini.

[0130] The polypeptide can be detectably labeled and / or combined with a carrier, eg, a pharmaceutically acceptable carrier.

[0131] The disclosed mB Box-97 HMGB1 polypeptides are used to treat or prevent abnormal or excessive NET formation in a subject in need thereof by administering an effective amount of the mB Box-97 HMGB1 polypeptide to the subject. In one embodiment, the subject suffers from one or more of SARS-CoV-2 infection, sepsis, an autoimmune disease such as systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, small-vessel vasculitis, an autoinflammatory disease such as gout, inflammatory bowel disease, and a metabolic disease such as type 2 diabetes and obesity. Also provided are methods for preventing NET-mediated disease or the progression of NET-mediated disease in a subject in need thereof by administering an effective amount of the mB Box-97 HMGB1 polypeptide to the subject. Further provided are methods for preventing and treating bacterial biofilms by administering an effective amount of the mB Box-97 HMGB1 polypeptide to a subject.

[0132] Synthetic or Recombinant mB Box-97 Polypeptides and Compositions Comprising Them One aspect of the present disclosure relates to a synthetic polypeptide comprising, consisting essentially of, or even consisting of mB Box-97, or an equivalent thereof, consisting of amino acids 90 to 176 from the coding sequence of the native human HMGB1 protein set forth as SEQ ID NO:2, with a cysteine ​​to serine point mutation at amino acid 106.

[0133] Another aspect of the present disclosure relates to a recombinant polypeptide comprising, consisting essentially of, or even consisting of mB Box-97, or an equivalent thereof, consisting of amino acids 90 to 176 from the coding sequence of the native human HMGB1 protein set forth as SEQ ID NO:2, with a cysteine ​​to serine point mutation at amino acid 106.

[0134] As used herein, an equivalent of recombinant mB Box-97 or synthetic mB Box-97 refers to a sequence that is at least about 70%, or alternatively 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 recombinant mB Box-97 or reference synthetic mB Box-97, and in one embodiment retains the mutated amino acid of the cysteine ​​to serine point mutation at amino acid 106. In one embodiment, percent identity is determined using a BLAST alignment program using default parameters. Particularly preferred programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort criteria = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0135] In some respects, equivalents of recombinant polypeptides retain the intended function and / or structural characteristics of mB Box-97 polypeptides. In another embodiment, equivalents of mB-Box-97 include recombinant mB Box-97 polypeptides that retain a C to S amino acid substitution at amino acid 106 and further contain, independently, at least two, or alternatively at least three, or alternatively at least four, or alternatively at least five, or at least six, or alternatively at least seven, or alternatively at least eight, or alternatively at least nine, or alternatively at least ten amino acids at the amino and / or carboxyl termini of the polypeptide.

[0136] In a further embodiment, the recombinant mB Box-97 HMGB1 polypeptide further comprises, consists essentially of, or even consists of one or more linker polypeptides. Exemplary peptide linkers are GPSLKL (SEQ ID NO: 3) or PPKGETKKKF (SEQ ID NO: 4) on the amine and / or carboxy termini.

[0137] In some embodiments, the recombinant polypeptide consists of SEQ ID NO:5.

[0138] Another aspect of the present disclosure relates to a synthetic polypeptide comprising mB Box-97, or an equivalent thereof, consisting of amino acids 80 to 176 from the coding sequence of the native human HMGB1 protein shown as SEQ ID NO: 2, with a point mutation from cysteine ​​to serine at amino acid 106.

[0139] As used herein, "equivalent of synthetic mB Box-97" refers to a sequence that is at least about 70%, or alternatively 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 the reference synthetic mB Box-97, and in one embodiment, retains the mutated amino acid of the cysteine ​​to serine point mutation at amino acid 106. In one embodiment, percent identity is determined using the BLAST alignment program using default parameters. Particularly preferred programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort criteria = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0140] In some respects, equivalents of synthetic mB Box-97 polypeptides retain the intended function and / or structural characteristics of the synthetic mB Box-97 polypeptides. In another embodiment, equivalents of synthetic mB-Box-97 retain the C to S amino acid substitution at amino acid 106 and independently further contain at least two, or alternatively at least three, or alternatively at least four, or alternatively at least five, or at least six, or alternatively at least seven, or alternatively at least eight, or alternatively at least nine, or alternatively at least ten amino acids at the amino and / or carboxyl termini of the polypeptide.

[0141] In a further embodiment, the synthetic mB Box-97 HMGB1 polypeptide or equivalent thereof further comprises, consists essentially of, or even consists of one or more linker polypeptides. Exemplary peptide linkers are GPSLKL (SEQ ID NO: 3) or PPKGETKKKF (SEQ ID NO: 4) on the amine and / or carboxy termini.

[0142] Another aspect of the present disclosure relates to a recombinant or synthetic polypeptide comprising, consisting essentially of, or even consisting of a recombinant or synthetic mB Box-97 consisting of amino acids 80-176 from the coding sequence of the native human HMGB1 protein set forth as SEQ ID NO:2, or an equivalent thereof, with a cysteine ​​to serine point mutation at amino acid 106.

[0143] In some embodiments, the synthetic or recombinant polypeptide consists of SEQ ID NO:6.

[0144] In some embodiments, equivalents include amino acid sequences having at least about 80% homology or amino acid identity thereto, or amino acids encoded by a polynucleotide that hybridizes to a polynucleotide encoding the amino acid sequence or its complement under high stringency conditions, where high stringency conditions include an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1x SSC to about 0.1x SSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1x SSC, 0.1x SSC, or deionized water, and an equivalent of a cysteine ​​to serine point mutation at amino acid 106 of SEQ ID NO:2 is a serine to alanine mutation at amino acid 106 of SEQ ID NO:2.

[0145] In some embodiments, the synthetic or recombinant polypeptide includes a detectable label.

[0146] In some embodiments, the synthetic or recombinant polypeptide or their respective equivalents comprises a linker polypeptide, optionally comprising GPSLKL (SEQ ID NO: 3) or PPKGETKKKF (SEQ ID NO: 4). In some embodiments, the linker is at the C-terminus of the synthetic or recombinant polypeptide. In some embodiments, the linker is at the N-terminus of the synthetic or recombinant polypeptide.

[0147] Another aspect of the disclosure pertains to a plurality of synthetic or recombinant polypeptides of the disclosure. In some embodiments, the members of the plurality are the same or different from one another.

[0148] Another aspect of the present disclosure relates to a composition comprising, consisting essentially of, or even consisting of a synthetic or recombinant polypeptide of the present disclosure or a plurality of synthetic or recombinant polypeptides of the present disclosure and a carrier, in some embodiments, the carrier is a pharmaceutically acceptable carrier.

[0149] The isolated polypeptides disclosed herein are intended to include polypeptides and proteins recombinantly produced from prokaryotic and eukaryotic host cells, as well as muteins, analogs and fragments thereof, examples of such cells being described above.

[0150] It is understood that functional equivalents or variants of the wild-type polypeptides or proteins, for example, those having conservative amino acid substitutions, are also within the scope of this disclosure.

[0151] In a further embodiment, the polypeptide is conjugated or linked to a detectable label or agent, such as PEGylated PEG mimic, polysialylated, HESylated or glycosylated, to increase the half-life of the polypeptide. Suitable labels are known in the art and are described herein.

[0152] Proteins and polypeptides can be obtained by several processes known to those skilled in the art, including purification, chemical synthesis, and recombinant methods. Accordingly, the present disclosure also provides methods for producing mB Box-97 polypeptides using these methods and the polynucleotides and polypeptides disclosed herein and the methods disclosed herein. For example, recombinant mB Box-97 can be produced in a host cell system that includes a polynucleotide encoding the polypeptide and cultures host cells under conditions favorable for recombinant production of the polypeptide. The polypeptide can be isolated from the host cell system by methods such as immunoprecipitation with antibodies, as well as standard techniques such as gel filtration, ion exchange, reverse-phase, and affinity chromatography. For such methodologies, see, for example, Deutscher et al. (1999) Guide to Protein Purification: Methods in Enzymology (Vol. 182, Academic Press). Accordingly, the present disclosure also provides processes for obtaining these polypeptides, as well as products that can be obtained by these processes and the resulting products.

[0153] Polypeptides can also be obtained by chemical synthesis using a commercially available automated peptide synthesizer, such as one manufactured by Perkin / Elmer / Applied Biosystems, Inc., Model 430A or 431A, Foster City, Calif., USA. The synthesized polypeptides can be precipitated and further purified, for example, by high performance liquid chromatography (HPLC). Thus, the present disclosure also provides protein sequences and reagents, such as amino acids and enzymes, and a process for chemically synthesizing the proteins disclosed herein by linking the amino acids to each other in the appropriate orientation and linear sequence.

[0154] The present disclosure also provides the polypeptides described herein conjugated to detectable agents for use in diagnostic methods.For example, detectably labeled polypeptides can be attached to a column and used for antibody detection and purification.They are also useful as immunogens for antibody production.The polypeptides disclosed herein are useful in in vitro assay systems for screening agents or drugs that regulate cellular processes.

[0155] It is well known to those skilled in the art that the peptides disclosed herein can be modified to provide modified properties.As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including glycine and both D or L optical isomers, as well as amino acid analogs and peptidomimetics.When the peptide chain is short, a peptide of 3 or more amino acids is generally called an oligopeptide.When the peptide chain is long, a peptide is generally called a polypeptide or protein.

[0156] The peptides disclosed herein can be modified to include unnatural amino acids. Thus, peptides may incorporate combinations of D-amino acids, L-amino acids, and various "designer" amino acids (e.g., beta-methyl amino acids, C-alpha-methyl amino acids, and N-alpha-methyl amino acids) to impart special properties to the peptide. Furthermore, by assigning specific amino acids in specific coupling steps, peptides with alpha helices, beta turns, beta sheets, gamma turns, and cyclic peptides can be generated. It is generally believed that alpha-helical or random secondary structures may be particularly useful.

[0157] The polypeptides disclosed herein can also be combined with various solid-phase carriers, such as implants, stents, pastes, gels, dental implants, or medical implants, or liquid-phase carriers, such as beads, sterile or aqueous solutions, pharmaceutically acceptable carriers, pharmaceutically acceptable polymers, liposomes, micelles, suspensions, and emulsions. Examples of non-aqueous solvents include propyl ethylene glycol, polyethylene glycol, and vegetable oils. When used to prepare antibodies or induce an immune response in vivo, the carrier can also contain an adjuvant useful for nonspecifically enhancing a specific immune response. Those skilled in the art can easily determine whether an adjuvant is necessary and select one. However, by way of example only, suitable adjuvants include, but are not limited to, Freund's complete and incomplete, mineral salts, and polynucleotides. Other suitable adjuvants include monophosphoryl lipid A (MPL), mutant derivatives of Escherichia coli heat-labile enterotoxin, mutant derivatives of cholera toxin, CPG oligonucleotides, and squalene-derived adjuvants.

[0158] The present disclosure also provides pharmaceutical compositions comprising, or alternatively consisting essentially of, or even consisting of, any of the polypeptides, analogs, muteins, or fragments disclosed herein, alone or in combination with each other or other agents, such as antibiotics and acceptable carriers or solid supports or antibodies or fragments thereof described herein, which compositions are useful in the various diagnostic and therapeutic methods described herein.

[0159] Isolated polynucleotides, vectors, and isolated host cells Another aspect of the present disclosure relates to synthetic or recombinant polypeptides of the present disclosure, such as synthetic or recombinant polypeptides comprising, consisting essentially of, or consisting of amino acids 80-176 from the coding sequence of native human HMGB1 protein, with a cysteine-to-serine point mutation at amino acid 106 of mB Box-97 that abolishes the ability of the polypeptide to induce an inflammatory response, as well as isolated polynucleotides encoding equivalents thereof consisting of amino acids 80-176 from the coding sequence of native human HMGB1 protein (the native / wild-type human HMGB1 sequence is set forth in SEQ ID NO:2), with a cysteine-to-serine point mutation at amino acid 106 of mB Box-97 that abolishes the ability of the polypeptide to induce an inflammatory response. Complementary polynucleotides to polynucleotides encoding the polypeptides are also provided. The polynucleotides can be DNA, RNA, mRNA, or interfering RNA such as siRNA, miRNA, or dsRNA.

[0160] The present disclosure also provides polynucleotides encoding equivalents of mB Box-97 polypeptides, recombinant mB Box-97, or synthetic mB Box-97, which refer to sequences that are at least about 70%, or alternatively 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 mB Box-97 polypeptide, recombinant mB Box-97, or synthetic mB Box-97, and in one embodiment, retain the mutated amino acid of the cysteine ​​to serine point mutation at amino acid 106. In one embodiment, percent identity is determined using the BLAST alignment program using default parameters. Particularly preferred programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort criteria = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. The polynucleotide can be DNA, RNA, mRNA, or an interfering RNA such as siRNA, miRNA, or dsRNA.

[0161] In some respects, the polynucleotide encodes a polypeptide equivalent that retains the intended function and / or structural characteristics of a synthetic or recombinant mB Box-97 polypeptide. In another embodiment, the synthetic or recombinant mB-Box-97 equivalent includes a recombinant mB-Box-97 or recombinant mB Box-97 polypeptide that retains a C to S amino acid substitution at amino acid 106 and further contains, independently, at least two, or alternatively at least three, or alternatively at least four, or alternatively at least five, or alternatively at least six, or alternatively at least seven, or alternatively at least eight, or alternatively at least nine, or alternatively at least ten amino acids at the amino and / or carboxyl termini of the polypeptide. The polynucleotide can be DNA, RNA, mRNA, or an interfering RNA such as siRNA, miRNA, or dsRNA.

[0162] In a further embodiment, the polynucleotide encodes an mB Box-97 HMGB1 polypeptide that further comprises, consists essentially of, or even consists of one or more linker polypeptides. Examples of peptide linkers are GPSLKL (SEQ ID NO: 3) or PPKGETKKKF (SEQ ID NO: 4) on the amine and / or carboxy terminus. The polynucleotide can be DNA, RNA, mRNA, or an interfering RNA such as siRNA, miRNA, or dsRNA.

[0163] The polynucleotides can be conjugated to detectable markers, such as enzyme labels or radioisotopes, for detecting nucleic acids and / or gene expression in cells. A wide variety of suitable detectable markers are known in the art, including fluorescent, radioactive, enzymatic, or other ligands (such as avidin / biotin) that can provide detectable signals. In one embodiment, it may be desirable to use fluorescent labels or enzyme tags, such as urease, alkaline phosphatase, or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates can be used to provide a means visible to the human eye or to identify specific hybridization with complementary nucleic acid-containing samples spectrophotometrically. Therefore, the present disclosure further provides a method for detecting single-stranded polynucleotide or its complement by contacting target single-stranded polynucleotide with a labeled single-stranded polynucleotide (probe) that is a part of the polynucleotide disclosed herein under conditions that allow hybridization of complementary single-stranded polynucleotide (optionally under moderately stringent hybridization conditions), or optionally under highly stringent hybridization conditions.Hybridized polynucleotide pairs are separated from unhybridized single-stranded polynucleotides.Hybridized polynucleotide pairs are detected by methods known to those skilled in the art, for example, as described in Sambrook et al. (1989) above.

[0164] The polynucleotide embodied in the present disclosure can be obtained by chemical synthesis, recombinant cloning, PCR, or any combination thereof.The method of chemical polynucleotide synthesis is known in the art, and does not need to be described in detail herein.Those skilled in the art can use the sequence data provided herein to obtain desired polynucleotide by using DNA synthesizer or ordering from commercial service.

[0165] The polynucleotides disclosed herein can be isolated or replicated using PCR. PCR technology is the subject of U.S. Patent Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202, and is described in "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., Birkhauser Press, Boston (1994)) or MacPherson et al. (1991) and (1995) supra, and in the references cited therein. Alternatively, one skilled in the art can replicate DNA using the sequences provided herein and a commercially available DNA synthesizer. Thus, the present disclosure also provides the linear sequence of the polynucleotide, nucleotides, appropriate primer molecules, chemicals such as enzymes, and instructions for their replication, and a process for obtaining the polynucleotides disclosed herein by chemically replicating or linking the nucleotides in the appropriate orientation to obtain a polynucleotide. In a separate embodiment, these polynucleotides are further isolated. Furthermore, those skilled in the art can insert the polynucleotide into a suitable replicating vector, and then insert the vector into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification.The DNA thus amplified can be isolated from the cell by methods known to those skilled in the art.The process for obtaining polynucleotides by this method, as well as the polynucleotides thus obtained, are further provided herein.

[0166] RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell. DNA can be delivered by any suitable method, for example, by using a suitable gene delivery vehicle (e.g., liposome, plasmid, or vector) or by electroporation. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods known to those skilled in the art, for example, as described in Sambrook et al. (1989) supra. For example, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedure described in Sambrook et al. (1989) supra, or extracted using nucleic acid-binding resins according to the accompanying instructions provided by the manufacturer.

[0167] Polynucleotides exhibiting sequence complementarity or homology to the polynucleotides disclosed herein are useful as hybridization probes or as equivalents of the specific polynucleotides identified herein. Because the complete coding sequence of the transcript is known, any portion of this sequence or a homologous sequence can be used in the methods disclosed herein.

[0168] It is known in the art that a "perfectly matched" probe is not required for specific hybridization. Minor changes in probe sequence achieved by substitution, deletion, or insertion of a small number of bases do not affect hybridization specificity. Generally, as much as 20% base pair mismatch (when optimally aligned) can be tolerated. In some embodiments, probes useful for detecting the above-mentioned mRNAs are at least about 80% identical to the homologous region. In some embodiments, the probe is 85% identical to the corresponding gene sequence after alignment of the homologous region; in some embodiments, it exhibits 90% identity.

[0169] These probes can be used in radioactive assays (e.g., Southern blot analysis and Northern blot analysis) for detecting, prognosing, diagnosing, or monitoring various cells or tissues containing these cells. The probes can also be attached to arrays such as solid supports or chips for use in high-throughput screening assays to detect the expression of genes corresponding to the polynucleotides disclosed herein. Thus, the present disclosure also provides probes comprising or corresponding to the polynucleotides disclosed herein, or their equivalents, or their complements, or fragments, attached to solid supports for use in high-throughput screening.

[0170] The total size of the fragment, as well as the size of the complementary stretch, will depend on the intended use or application of a particular nucleic acid segment. Smaller fragments generally find use in hybridization embodiments, and the length of the complementary region can vary, for example, between at least 5 to 10 to about 100 nucleotides, or even the full length, depending on the complementary sequence desired to be detected.

[0171] Nucleotide probes with complementary sequences spanning stretches greater than 5-10 nucleotides in length are generally well suited for increasing hybrid stability and selectivity, thereby improving the specificity of the resulting hybrid molecules. In certain embodiments, polynucleotides can be designed with gene-complementary stretches of 10 or more, or even 50 nucleotides in length, or even longer if desired. Such fragments can be readily prepared, for example, by directly synthesizing the fragments by chemical means, by applying nucleic acid regeneration techniques such as PCR using two priming oligonucleotides as described in U.S. Pat. No. 4,603,102, or by introducing selected sequences into a recombinant vector for recombinant production. In one aspect, the probes are about 50-75 or more, or even 50-100 nucleotides in length.

[0172] The polynucleotides of the present disclosure can be used as primers for detecting genes or gene transcripts expressed in the cells described herein.In this context, amplification refers to any method using a primer-dependent polymerase that can replicate target sequences with reasonable fidelity.Amplification can be carried out by natural or recombinant DNA polymerases such as T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase.For illustrative purposes only, the primers are the same length as those identified for the probe.

[0173] One method for amplifying polynucleotides is PCR, and kits for PCR amplification are commercially available. After amplification, the resulting DNA fragments can be detected by any suitable method known in the art, such as agarose gel electrophoresis, followed by visualization with ethidium bromide staining and ultraviolet illumination.

[0174] Methods for administering an effective amount of a gene delivery vector or vehicle to cells have been developed and are known to those skilled in the art, and are described herein.Methods for detecting gene expression in cells are known in the art, and include techniques such as hybridization to DNA microarrays, in situ hybridization, PCR, RNase protection assays, and Northern blot analysis.Such methods are useful for detecting and quantifying gene expression in cells.Alternatively, the expression of encoded polypeptides can be detected by various methods.In particular, it is useful to prepare polyclonal or monoclonal antibodies that specifically react with target polypeptides.Such antibodies are useful for visualizing cells expressing polypeptides using techniques such as immunohistology, ELISA, and Western blotting.These techniques can be used to determine the expression level of expressed polynucleotides.

[0175] In some embodiments, the isolated polynucleotide is in a composition comprising a carrier or a pharmaceutically acceptable carrier.

[0176] In some embodiments, the isolated polynucleotide includes a detectable label and, optionally, a carrier or a pharmaceutically acceptable carrier.

[0177] Also provided are multiple polynucleotides, which may be the same or different from one another, as well as compositions containing multiple polynucleotides and a carrier, such as a pharmaceutically acceptable carrier.

[0178] Another aspect of the present disclosure relates to a vector comprising, consisting essentially of, or even consisting of an isolated polynucleotide of the present disclosure, and optionally a carrier or pharmaceutically acceptable carrier. The vector can be, for example, a lipid nanoparticle, a plasmid, or a viral vector.

[0179] Also provided are compositions containing multiple vectors, which may be the same or different, as well as multiple polynucleotides and a carrier, such as a pharmaceutically acceptable carrier.

[0180] In some embodiments, the isolated polynucleotide or vector further comprises a heterologous promoter sequence, and optionally a carrier or pharmaceutically acceptable carrier.

[0181] Another aspect of the present disclosure pertains to an isolated host cell containing one or more of a synthetic or recombinant polypeptide of this disclosure, a plurality of synthetic or recombinant polypeptides of this disclosure, an isolated polynucleotide of this disclosure, or a vector of this disclosure, and optionally a carrier or a pharmaceutically acceptable carrier.

[0182] In some embodiments, the host cell is a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell.

[0183] Further provided is a method for producing a recombinant polypeptide of the present disclosure by a method comprising delivering an isolated polynucleotide of the present disclosure or a vector of the present disclosure to a host cell under conditions that allow expression of the polypeptide of the present disclosure. In some embodiments, the host cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is an E. coli cell.

[0184] Further provided are methods for producing the synthetic polypeptides of the disclosure by methods including solid phase peptide synthesis or solution phase peptide synthesis.

[0185] Compositions Comprising Antibodies or Fragments Thereof Another aspect of the disclosure relates to a composition comprising an antibody or fragment thereof, wherein the antibody or fragment thereof is an antibody or fragment thereof that binds to a tip region of a DNABII peptide or a tip chimera (e.g., IhfA5-mIhfB4 as described herein). NTHI IhfA5-mIhfB4 recognizes and binds to the IhfA5-mIhfB4 chimera. NTHI Compositions including one or more antibodies that recognize and bind to the tip chimeras are also provided.

[0186] In one embodiment, the antibody is a monoclonal antibody, a humanized antibody, or an antigen-binding fragment thereof.

[0187] In some embodiments, an antibody or fragment thereof that binds to the tip region of a DNABII peptide or tip chimera, The antibody or fragment thereof may be (i) a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or even consisting of the sequence of amino acids (aa) 25 to aa 144 of SEQ ID NO: 21, or an equivalent thereof; and (ii) a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or even consisting of the sequence from aa21 to aa132 of SEQ ID NO: 22, or an equivalent thereof; contains, or The antibody or fragment thereof may be (i) a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or even consisting of the sequence from aa25 to aa144 of SEQ ID NO: 24, or an equivalent thereof; and (ii) a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or even consisting of the sequence from aa21 to aa132 of SEQ ID NO: 25, or an equivalent thereof; Includes.

[0188] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide is a heavy chain complementarity-determining region 1 (CDRH1) comprising, consisting essentially of, or even consisting of the sequence of GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 9, 10, 11, or 24); a heavy chain complementarity-determining region 2 (CDRH2) comprising, consisting essentially of, or even consisting of the sequence of GSDRRH (aa76 to aa81 of SEQ ID NO: 9 or 10 or 11 or 24); a heavy chain complementarity-determining region 3 (CDRH3) comprising, consisting essentially of, or even consisting of the sequence VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 9, 10, 11, or 24); a light chain complementarity-determining region 1 (CDRL1) comprising, consisting essentially of, or even consisting of the sequence QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 15, 16, 17, or 25); A light chain complementarity-determining region 2 (CDRL2) comprising, consisting essentially of, or even consisting of the sequence of LVS (aa75 to aa77 of SEQ ID NO: 15, 16, 17, or 25); and It includes a light chain complementarity-determining region 3 (CDRL3) that comprises, consists essentially of, or even consists of the sequence of WQGTHFP (aa114 to aa120 of SEQ ID NO: 15, 16, 17, or 25).

[0189] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide is a heavy chain complementarity-determining region 1 (CDRH1) comprising, consisting essentially of, or even consisting of the sequence of GFTFSRYG (aa 50 to aa 57 of SEQ ID NO: 12, 13, or 14); a heavy chain complementarity-determining region 2 (CDRH2) comprising, consisting essentially of, or even consisting of the sequence of ISSGGSYT (aa75 to aa82 of SEQ ID NO: 12, 13, or 14); a heavy chain complementarity-determining region 3 (CDRH3) comprising, consisting essentially of, or even consisting of the sequence ERHGGDGYWYFDV (aa121 to aa133 of SEQ ID NO: 12, 13, or 14); a light chain complementarity-determining region 1 (CDRL1) comprising, consisting essentially of, or even consisting of the sequence QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 15, 16, 17, or 25); A light chain complementarity-determining region 2 (CDRL2) comprising, consisting essentially of, or even consisting of the sequence of LVS (aa75 to aa77 of SEQ ID NO: 15, 16, 17, or 25); and The present invention also includes a light chain complementarity-determining region 3 (CDRL3) comprising, consisting essentially of, or even consisting of the sequence of WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 15, 16, 17, or 25).

[0190] In some embodiments, an antibody or fragment thereof that binds to the tip region or tip chimera of a DNABII peptide comprises, essentially consists of, or further consists of, or further consists of, a heavy chain (HC) immunoglobulin variable domain sequence consisting of, essentially consists of, or even consists of a sequence selected from the group of aa25 to aa144 of SEQ ID NO: 21 or 24, or an equivalent of each thereof, and / or a light chain (LC) immunoglobulin variable domain sequence consisting of, essentially consists of, or even consists of a sequence selected from the group of aa21 to aa132 of SEQ ID NO: 22 or 25, or an equivalent of each thereof. In certain embodiments, the antibody or fragment thereof binds to the tip region or tip chimera of a DNABII peptide (e.g., the tip region of IHF or HU, the tip region of IHFA or IHFB, and / or the tip-chimera peptide IfA5-mIhfB4). NTHI In one embodiment, the antibody or fragment thereof binds to the tip-chimeric peptide IhfA5-mIhfB4 described herein. NTHI In some embodiments, the tip-chimeric peptide IhfA5-mIhfB4 NTHI comprises, consists essentially of, or even consists of an amino acid sequence selected from SEQ ID NOs: 26-28.

[0191] In some embodiments, an antibody or fragment thereof that binds to the distal region of a DNABII peptide comprises, consists essentially of, or even consists of any one or any two or three heavy chain (HC) CDRs that comprise, consist essentially of, or even consist of a sequence selected from SEQ ID NOs: 9-14, or their respective equivalents, and / or any one or any two or three light chain (LC) CDRs that comprise, consist essentially of, or even consist of a sequence selected from SEQ ID NOs: 15-20, or their respective equivalents. In certain embodiments, the antibody or fragment thereof binds to the distal region of a DNABII peptide (e.g., the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal-chimeric peptide IhfA5-mIhfB4 described herein). NTHI In one embodiment, the antibody or fragment thereof binds to the tip-chimeric peptide IhfA5-mIhfB4 described herein. NTHI In some embodiments, the tip-chimeric peptide IhfA5-mIhfB4 NTHI comprises, consists essentially of, or even consists of an amino acid sequence selected from SEQ ID NOs: 26-28.

[0192] In some embodiments, an antibody or fragment thereof that binds to the distal region of a DNABII peptide comprises, consists essentially of, or even consists of all three heavy chain (HC) CDRs that comprise, consist essentially of, or even consist of a sequence selected from SEQ ID NOs: 9-11 or their respective equivalents, and / or all three light chain (LC) CDRs that comprise, consist essentially of, or even consist of a sequence selected from SEQ ID NOs: 15-17 or their respective equivalents. In certain embodiments, the antibody or fragment thereof binds to the distal region of a DNABII peptide (e.g., the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal-chimeric peptide IhfA5-mIhfB4). NTHIIn one embodiment, the antibody or fragment thereof binds to the tip-chimeric peptide IhfA5-mIhfB4 NTHI In some embodiments, the tip-chimeric peptide IhfA5-mIhfB4 NTHI comprises, consists essentially of, or even consists of an amino acid sequence selected from SEQ ID NOs: 26-28.

[0193] In some embodiments, an antibody or fragment thereof that binds to the distal region of a DNABII peptide comprises, consists essentially of, or even consists of all three heavy chain (HC) CDRs that comprise, consist essentially of, or even consist of a sequence selected from SEQ ID NOs: 12-14 or their respective equivalents, and / or all three light chain (LC) CDRs that comprise, consist essentially of, or even consist of a sequence selected from SEQ ID NOs: 18-20 or their respective equivalents. In certain embodiments, the antibody or fragment thereof binds to the distal region of a DNABII peptide (e.g., the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal-chimeric peptide IhfA5-mIhfB4). NTHI In one embodiment, the antibody or fragment thereof binds to the tip-chimeric peptide IhfA5-mIhfB4 NTHI In some embodiments, the tip-chimeric peptide IhfA5-mIhfB4 NTHI comprises, consists essentially of, or even consists of an amino acid sequence selected from SEQ ID NOs: 26-28.

[0194] Table 1 shows IhfA5-mIhfB4 NTHIRelative biofilm disruption data using a humanized monoclonal antibody designed to target the apical-chimeric peptide are presented. Biofilm disruption: NTHI86-028NP colonies were harvested from overnight cultures on chocolate agar and suspended in brain heart infusion broth (sBHI) supplemented with 2 μg of β-NAD and heme per ml of medium. The optical density at 490 nm was then adjusted to 0.65, and the culture was diluted 1:6 with sBHI and then incubated stationary at 37°C with 5% CO2 for 3 hours. The culture was then diluted 1:2500 with fresh sBHI, and 200 μl of the suspension was dispensed into each well of an 8-well chamber slide. The slide was then incubated stationary at 37°C with 5% CO2 for 3 hours. After 16 hours, 200 μl of fresh sBHI was added to each well, and the slide was incubated for an additional 8 hours. At this point, the medium was aspirated from each well, and 5 μg of monoclonal antibody was added per well. Biofilms were incubated for an additional 16 hours. Biofilms were then washed, stained with FM1-43FX bacterial cell membrane stain (Invitrogen), and fixed overnight at 4°C in 16% paraformaldehyde, 2.5% glutaraldehyde, and 4.0% acetic acid in 0.1 M phosphate buffer (pH 7.4). Before viewing the biofilms with a Zeiss 800 Meta laser scanning confocal microscope, the fixative was aspirated, and 200 μl of Hank's balanced salt solution was added to each well. Images were edited using Zeiss Zen Black software, and biofilm biomass was calculated using COMSTAT 2.1 software. IhfA5-mIhfB4 NTHI K of the tip chimeric peptide A (1 / M) is approximately 4E+05 to approximately 2E+08.

[0195] [Table 1]

[0196] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 21 and a light chain sequence comprising SEQ ID NO: 22.

[0197] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO:9 and a light chain sequence comprising SEQ ID NO:15.

[0198] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO:9 and a light chain sequence comprising SEQ ID NO:16.

[0199] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO:9 and a light chain sequence comprising SEQ ID NO:17.

[0200] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 10 and a light chain sequence comprising SEQ ID NO: 15.

[0201] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 10 and a light chain sequence comprising SEQ ID NO: 16.

[0202] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 10 and a light chain sequence comprising SEQ ID NO: 17.

[0203] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO:11 and a light chain sequence comprising SEQ ID NO:15.

[0204] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO:11 and a light chain sequence comprising SEQ ID NO:16.

[0205] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO:11 and a light chain sequence comprising SEQ ID NO:17.

[0206] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 12 and a light chain sequence comprising SEQ ID NO: 18.

[0207] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 12 and a light chain sequence comprising SEQ ID NO: 19.

[0208] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 12 and a light chain sequence comprising SEQ ID NO: 20.

[0209] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 13 and a light chain sequence comprising SEQ ID NO: 18.

[0210] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 13 and a light chain sequence comprising SEQ ID NO: 19.

[0211] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 13 and a light chain sequence comprising SEQ ID NO: 20.

[0212] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 14 and a light chain sequence comprising SEQ ID NO: 18.

[0213] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 14 and a light chain sequence comprising SEQ ID NO: 19.

[0214] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 14 and a light chain sequence comprising SEQ ID NO: 20.

[0215] The antibodies or fragments thereof provided herein can be monospecific or bispecific. In one embodiment, the antibodies or fragments thereof are trispecific, tetraspecific, or pentaspecific. Additionally or alternatively, the antibodies are selected from the group of IgA (such as IgA1 or IgA2), IgD, IgE, IgG (such as IgG1, IgG2, IgG3, or IgG4), or IgM antibodies. In one embodiment, the antibody further comprises a constant region selected from the group of an IgA constant region (such as an IgA1 constant region or an IgA2 constant region), an IgD constant region, an IgE constant region, an IgG constant region (such as an IgG1 constant region, an IgG2 constant region, an IgG3 constant region, or an IgG4 constant region), or an IgM constant region. In some embodiments, the constant region of the antibody comprises an amino acid sequence selected from the group of SEQ ID NOs: 23 and 35-42.

[0216] In another embodiment, the antibody can be modified by conventional techniques, such as PEGylation, PEG mimetics, polysialylation, HESylation, or glycosylation, which in one embodiment may increase the half-life of the antibody.

[0217] Further provided herein is a composition comprising, consisting essentially of, or even consisting of an antibody or fragment thereof as described herein, an mB Box-97 peptide, and optionally a carrier such as a pharmaceutically acceptable carrier. The mB Box peptide is a synthetic or recombinant polypeptide comprising, consisting essentially of, or consisting of mB Box-97 consisting of amino acids 90-176 or amino acids 80-176 from the coding sequence of native human HMGB1 protein (set forth in SEQ ID NO: 2), with a point mutation from cysteine ​​to serine at amino acid 106 that abolishes the ability of the polypeptide to induce an inflammatory response, as well as equivalents thereof consisting of amino acids 90-176 or amino acids 80-176 from the coding sequence of native human HMGB1 protein (native / wild-type human HMGB1 sequence set forth in SEQ ID NO: 2), with the point mutation from cysteine ​​to serine at amino acid 106 of mB Box-97 retaining the point mutation. Synthetic polypeptides are produced by synthetic or chemical means and are not wild-type or recombinantly produced.

[0218] Methods for producing antibodies and fragments thereof are known in the art and are described in US Pat. No. 11,104,723, which is incorporated herein in its entirety.

[0219] composition Compositions are also provided. The compositions include a carrier and one or more of the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the isolated host cells disclosed herein, the small molecules or antibodies, and / or the antigen-binding fragments disclosed herein. The carrier may be one or more of a solid support or a pharmaceutically acceptable carrier. The composition may further include an adjuvant or other component suitable for administration as a vaccine. In one aspect, the composition is formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, and / or adjuvants. In addition, embodiments of the compositions of the present disclosure include one or more of the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the small molecules, the isolated host cells disclosed herein, or the antibodies disclosed herein, formulated with one or more pharmaceutically acceptable substances.

[0220] For oral preparations, any one or more of the isolated or recombinant polypeptides described herein, the isolated or recombinant polynucleotides described herein, the vectors described herein, the isolated host cells described herein, the small molecules described herein, or the antibodies or fragments thereof can be used alone or in the pharmaceutical formulations disclosed herein, which comprise or consist essentially of the compound in combination with suitable additives to produce tablets, powders, granules, or capsules, for example, with conventional additives such as lactose, mannitol, cornstarch, or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, cornstarch, or gelatin; with disintegrants such as cornstarch, potato starch, or sodium carboxymethylcellulose; with lubricants such as talc or magnesium stearate; and, if necessary, with diluents, buffers, wetting agents, preservatives, and flavoring agents. Pharmaceutically compatible binders and / or adjuvants can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0221] Pharmaceutical formulations and unit dosage forms suitable for oral administration are particularly useful for treating chronic conditions, infectious diseases, and therapies where the patient self-administers the drug. In one embodiment, the formulation is specific for pediatric administration.

[0222] The present disclosure provides pharmaceutical formulations that can be formulated into injectable preparations in accordance with the present disclosure by dissolving, suspending or emulsifying one or more of an isolated polypeptide disclosed herein, an isolated polynucleotide disclosed herein, a vector disclosed herein, an isolated host cell disclosed herein, or an antibody disclosed herein in an aqueous or non-aqueous solvent such as a vegetable oil or other similar oil, a synthetic aliphatic acid glyceride, an ester of a higher fatty acid, or propylene glycol; optionally with conventional additives, such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives or other antimicrobial agents. Non-limiting examples of such include antimicrobial agents, other vaccine components such as surface antigens, e.g., OMP P5, OMP26, OMP P2, or type IV pilin proteins (see Jurcisek and Bakaletz (2007) J. of Bacteriology 189(10):3868-3875 and Murphy, TF, Bakaletz, LO and Smeesters, PR (2009) The Pediatric Infectious Disease Journal, 28:S121-S126), and antimicrobial agents. For intravenous administration, suitable carriers include physiological bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, compositions for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists.

[0223] The aerosol formulations provided by the present disclosure can be administered by inhalation and can be based on propellants or non-propellants.For example, the embodiments of the pharmaceutical formulations disclosed herein include the compounds disclosed herein formulated in pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, etc. For administration by inhalation, the compounds can be delivered in the form of aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer.A non-limiting example of a non-propellant is a pump spray that is expelled from a sealed container by mechanical force (i.e., by pressing down the plunger with one's finger, or by compressive force applied to the wall of the container or elastic force applied by the wall itself, such as by compressing the container with an elastic bladder, etc.).

[0224] The suppository disclosed herein can be prepared by mixing the compound disclosed herein with any of various bases, such as emulsifying base or water-soluble base.The pharmaceutical formulation embodiment of the compound disclosed herein can be administered rectally via suppository.Suppository can contain a vehicle such as cocoa butter, carbowax and polyethylene glycol, which melts at body temperature but solidifies at room temperature.

[0225] Oral or rectal administration unit dosage forms, such as syrups, elixirs and suspensions, can be provided, and each dosage unit, such as teaspoon, table spoon, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds disclosed herein.Similarly, injection or intravenous administration unit dosage forms can include the compound disclosed herein in the composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

[0226] The embodiments of the pharmaceutical preparations disclosed herein include the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the small molecules used in the present disclosure, the isolated host cells disclosed herein, or the antibodies or fragments thereof disclosed herein, which are formulated into injectable compositions.The injectable pharmaceutical preparations disclosed herein are prepared as liquid solutions or suspensions; or as solid forms suitable for dissolving or suspending in liquid vehicles before injection.The preparations can also be emulsified, or the active ingredient can be encapsulated in liposome vehicles, according to other embodiments of the pharmaceutical preparations disclosed herein.

[0227] In one embodiment, one or more of the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the isolated host cells disclosed herein, or the antibodies disclosed herein are formulated for delivery by a continuous delivery system. The term "continuous delivery system" is used interchangeably herein with "controlled delivery system" and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, etc., a wide variety of which are known in the art.

[0228] Mechanical or electromechanical infusion pumps can also be suitable for use in the present disclosure.Examples of such devices include those described in, for example, U.S. Patent Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966, etc. Generally, the delivery of the compounds disclosed herein can be achieved using any of a variety of rechargeable pump systems.The pump provides consistent controlled release over time.In some embodiments, the compounds disclosed herein are in a liquid formulation in a drug-impermeable reservoir and are continuously delivered to an individual.

[0229] In one embodiment, the drug delivery system is at least partially an implantable device. The implantable device can be implanted at any suitable implantation site using methods and devices known in the art. An implantation site is a site within a subject's body where the drug delivery device is introduced and placed. Implantation sites include, but are not necessarily limited to, subdermal, subcutaneous, intramuscular, or other suitable sites within a subject's body. In some embodiments, a subcutaneous implantation site is used for convenience in implanting and removing the drug delivery device.

[0230] Drug release devices suitable for use in the present disclosure may be based on any of a variety of modes of operation, for example, polymers such as poly(glycolide-co-lactide) (PGLA), which are commercially available from several vendors, including BioDegmer and Sigma-Aldrich. For example, drug release devices can be based on diffusion systems, convection systems, or erodible systems (e.g., erosion-based systems). For example, drug release devices can be electrochemical pumps, osmotic pumps, electroosmotic pumps, vapor pressure pumps, or osmotic burst matrices, where the drug is incorporated into a polymer (e.g., PGLA), which provides for release of the drug formulation upon degradation of the drug-impregnated polymeric material (e.g., a biodegradable drug-impregnated polymeric material). In other embodiments, drug release devices are based on electrodiffusion systems, electrolytic pumps, effervescent pumps, piezoelectric pumps, hydrolytic systems, and the like.

[0231] Drug release devices based on mechanical or electromechanical infusion pumps can also be suitable for use in the present disclosure.Examples of such devices include those described in, for example, U.S. Patent No. 4,692,147; U.S. Patent No. 4,360,019; U.S. Patent No. 4,487,603; U.S. Patent No. 4,360,019; U.S. Patent No. 4,725,852, etc. Generally, the treatment method of the subject can be achieved using any of a variety of rechargeable, non-replaceable pump systems. Pumps and other convective systems can be utilized because they generally have more consistent controlled release over time. Osmotic pumps are used in some embodiments due to their combined advantages of more consistent controlled release and relatively small size (e.g., PCT International Application Publication No. WO97 / 27840 and U.S. Patent No. 5,985,305 and 5,728,396). Exemplary osmotically driven devices suitable for use in the present disclosure include, but are not limited to, U.S. Patent Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202; Nos. 3, 4,203,440, 4,203,442, 4,210,139, 4,327,725, 4,627,850, 4,865,845, 5,057,318, 5,059,423, 5,112,614, 5,137,727, 5,234,692, 5,234,693, and 5,728,396. A further exemplary device that can be adapted for the present disclosure is the Synchromed infusion pump (Medtronic).

[0232] In some embodiments, the drug delivery device is an implantable device. The drug delivery device can be implanted at any suitable implantation site using methods and devices known in the art. As described herein, an implantation site is a site within a subject's body where the drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to, subdermal, subcutaneous, intramuscular, or other suitable sites within a subject's body.

[0233] Suitable excipient vehicles for the compounds disclosed herein are, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof.In addition, if necessary, vehicle may contain small amounts of auxiliary substances, such as wetting or emulsifying agents or pH buffering agents.Methods for preparing such dosage forms are known to those skilled in the art or will be clear in light of the present disclosure.See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985.In any case, the composition or formulation to be administered contains a sufficient amount of compound to achieve the desired state in the treated subject.

[0234] Compositions of the present disclosure include those that incorporate sustained-release or controlled-release matrices. Furthermore, embodiments of the present disclosure can be used in conjunction with other treatments that utilize sustained-release formulations. As used herein, a sustained-release matrix is ​​a matrix made of a material, usually a polymer, that can be degraded by enzymatic or acid-based hydrolysis or dissolution. Once inserted into the body, the matrix is ​​acted upon by enzymes and bodily fluids. The sustained-release matrix is ​​preferably selected from biocompatible materials such as liposomes, polylactides (polylactic acids), polyglycolides (polymers of glycolic acid), polylactide-co-glycolides (copolymers of lactic and glycolic acids), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, and isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicones. Exemplary biodegradable matrices include polylactide matrices, polyglycolide matrices, and polylactide-co-glycolide (copolymers of lactic and glycolic acid) matrices.

[0235] In another embodiment, the polypeptide, antibody, or fragment thereof (as well as the combination composition) is delivered in a controlled release system. For example, the compounds disclosed herein can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574). In another embodiment, a polymeric material is used. In yet another embodiment, the controlled release system is placed near the therapeutic target, i.e., the liver, thus requiring only a fraction of the systemic dose. In yet another embodiment, the controlled release system is placed near the therapeutic target, thus requiring only a fraction of the systemic dose. Other controlled release systems are discussed in the review by Langer (1990) Science 249:1527-1533.

[0236] In another embodiment, the compositions of the present disclosure (as well as the combined compositions, separately or together) include those formed by impregnating an inhibitor described herein into absorbent materials, such as sutures, bandages, and gauze, or coated onto solid materials, such as surgical staples, zippers, and catheters, to deliver the composition. Other delivery systems of this type will be readily apparent to those skilled in the art in view of the present disclosure.

[0237] The present disclosure provides methods and compositions for administering one or more interfering factors to a host (e.g., a human) to treat a microbial infection. In various embodiments, the methods disclosed herein extend to nearly any available method and route suitable for drug delivery, including in vivo and ex vivo methods, and systemic and local administration routes.

[0238] Methods of Using mB Box-97 Polypeptides Another aspect of the present disclosure relates to a method of treating a subject in need thereof, comprising, consisting essentially of, or even consisting of administering to the subject an effective amount of one or more of: (i) a synthetic or recombinant polypeptide of this disclosure or an equivalent of each thereof; (ii) a plurality of synthetic or recombinant polypeptides of this disclosure; (iii) a composition of this disclosure; (iv) an isolated polynucleotide of this disclosure; or (iv) a vector of this disclosure.

[0239] Another aspect of the present disclosure relates to a method for treating or preventing aberrant or excessive neutrophil extracellular trap (NET) formation in a subject in need thereof, or for preventing a neutrophil extracellular trap (NET)-mediated disease in a subject in need thereof, or for preventing the progression of a NET-mediated disease in a subject in need thereof, comprising, consisting essentially of, or even consisting of administering to a subject an effective amount of one or more of: (i) a synthetic or recombinant polypeptide of this disclosure or its respective equivalent; (ii) a plurality of synthetic or recombinant polypeptides of this disclosure; (iii) a composition of this disclosure; (iv) an isolated polynucleotide of this disclosure; or (iv) a vector of this disclosure.

[0240] In some embodiments, the subject is suffering from one or more of the following: a pulmonary disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-associated acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, or small vessel vasculitis; an autoinflammatory disease selected from gout or inflammatory bowel disease, and / or a metabolic disease selected from type 2 diabetes or obesity.

[0241] In some embodiments, an effective amount is between 50 nM and 2 μM (e.g., 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 150 nM, 170 nM, 200 nM, 220 nM, 250 nM, 270 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 85 ... 0 nM, 950 nM, 1000 nM (1 μM), 1050 nM, 1100 nM, 1150 nM, 1200 nM, 1250 nM, 1300 nM, 1350 nM, 1400 nM, 1450 nM, 1500 nM, 1550 nM, 1600 nM, 1650 nM, 1700 nM, 1750 nM, 1800 nM, 1850 nM, 1900 nM, 1950 nM, or 2000 nM (2 μM).

[0242] In some embodiments, the NET-mediated disease comprises, consists essentially of, or even consists of a pulmonary disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-associated acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, or small vessel vasculitis; an autoinflammatory disease selected from gout or inflammatory bowel disease, or a metabolic disease selected from type 2 diabetes or obesity.

[0243] Another aspect of the present disclosure relates to a method of preventing or treating a bacterial biofilm in a subject in need thereof, comprising, consisting essentially of, or even consisting of administering to the subject an effective amount of one or more of: (i) a synthetic or recombinant polypeptide of the present disclosure or an equivalent of each thereof; (ii) a plurality of synthetic or recombinant polypeptides of the present disclosure; (iii) a composition of the present disclosure; (iv) an isolated polynucleotide of the present disclosure; or (iv) a vector of the present disclosure.

[0244] In some embodiments, an effective amount is between 50 nM and 2 μM (e.g., 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 150 nM, 170 nM, 200 nM, 220 nM, 250 nM, 270 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 85 ... 0 nM, 950 nM, 1000 nM (1 μM), 1050 nM, 1100 nM, 1150 nM, 1200 nM, 1250 nM, 1300 nM, 1350 nM, 1400 nM, 1450 nM, 1500 nM, 1550 nM, 1600 nM, 1650 nM, 1700 nM, 1750 nM, 1800 nM, 1850 nM, 1900 nM, 1950 nM, or 2000 nM (2 μM).

[0245] In some embodiments, the method further comprises, consists essentially of, or even consists of administering to a subject an antibody or fragment thereof, wherein the antibody or fragment thereof binds to a distal region of a DNABII peptide, e.g., (IhfA5-mIhfB4 NTHI In one embodiment, the antibody is a monoclonal antibody, a humanized antibody, or an antigen-binding fragment thereof.

[0246] In some embodiments, an antibody or fragment thereof that binds to the tip region of a DNABII peptide or tip chimera, The antibody or fragment thereof may be (iii) a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or even consisting of amino acids (aa) 25 to aa 144 of SEQ ID NO: 21, or an equivalent thereof; and (iv) a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or even consisting of the sequence from aa21 to aa132 of SEQ ID NO: 22, or an equivalent thereof; contains, or The antibody or fragment thereof may be (iii) a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or even consisting of the sequence from aa25 to aa144 of SEQ ID NO: 24, or an equivalent thereof; and (iv) a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or even consisting of the sequence from aa21 to aa132 of SEQ ID NO: 25 or an equivalent thereof. Includes.

[0247] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide is a heavy chain complementarity-determining region 1 (CDRH1) comprising, consisting essentially of, or even consisting of the sequence of GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 9, 10, 11, or 24); a heavy chain complementarity-determining region 2 (CDRH2) comprising, consisting essentially of, or even consisting of the sequence of GSDRRH (aa76 to aa81 of SEQ ID NO: 9 or 10 or 11 or 24); a heavy chain complementarity-determining region 3 (CDRH3) comprising, consisting essentially of, or even consisting of the sequence VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 9, 10, 11, or 24); a light chain complementarity-determining region 1 (CDRL1) comprising, consisting essentially of, or even consisting of the sequence QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 15, 16, 17, or 25); A light chain complementarity-determining region 2 (CDRL2) comprising, consisting essentially of, or even consisting of the sequence of LVS (aa75 to aa77 of SEQ ID NO: 15, 16, 17, or 25); and It includes a light chain complementarity-determining region 3 (CDRL3) that comprises, consists essentially of, or even consists of the sequence of WQGTHFP (aa114 to aa120 of SEQ ID NO: 15, 16, 17, or 25).

[0248] In some embodiments, an antibody or fragment thereof that binds to the distal region of a DNABII peptide comprises, essentially consists of, or further consists of, or further consists of, a heavy chain (HC) immunoglobulin variable domain sequence consisting of, essentially consists of, or further consists of a sequence selected from the group of aa25 to aa144 of SEQ ID NO: 21 or 24, or an equivalent of each thereof, and / or a light chain (LC) immunoglobulin variable domain sequence consisting of, essentially consists of, or further consists of a sequence selected from the group of aa21 to aa132 of SEQ ID NO: 22 or 25, or an equivalent of each thereof. In certain embodiments, the antibody or fragment thereof binds to the distal region of a DNABII peptide (e.g., the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal-chimeric peptide IhfA5-mIhfB4). NTHI In one embodiment, the antibody or fragment thereof binds to the tip-chimeric peptide IhfA5-mIhfB4 NTHI As used herein, the tip-chimeric peptide IhfA5-mIhfB4 NTHI comprises, consists essentially of, or even consists of an amino acid sequence selected from SEQ ID NOs: 26-28.

[0249] In some embodiments, an antibody or fragment thereof that binds to the distal region of a DNABII peptide comprises, consists essentially of, or even consists of any one or any two or three heavy chain (HC) CDRs that comprise, consist essentially of, or even consist of a sequence selected from SEQ ID NOs: 9-14 or their respective equivalents, and / or any one or any two or three light chain (LC) CDRs that comprise, consist essentially of, or even consist of a sequence selected from SEQ ID NOs: 15-20 or their respective equivalents. In certain embodiments, the antibody or fragment thereof binds to the distal region of a DNABII peptide (e.g., the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal-chimeric peptide IhfA5-mIhfB4). NTHI In one embodiment, the antibody or fragment thereof binds to the tip-chimeric peptide IhfA5-mIhfB4 NTHI Combine with.

[0250] In some embodiments, an antibody or fragment thereof that binds to the distal region of a DNABII peptide comprises, consists essentially of, or even consists of all three heavy chain (HC) CDRs that comprise, consist essentially of, or even consist of a sequence selected from SEQ ID NOs: 9-11 or their respective equivalents, and / or all three light chain (LC) CDRs that comprise, consist essentially of, or even consist of a sequence selected from SEQ ID NOs: 15-17 or their respective equivalents. In certain embodiments, the antibody or fragment thereof binds to the distal region of a DNABII peptide (e.g., the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal-chimeric peptide IhfA5-mIhfB4). NTHI In one embodiment, the antibody or fragment thereof binds to the tip-chimeric peptide IhfA5-mIhfB4 NTHI Combine with.

[0251] In some embodiments, an antibody or fragment thereof that binds to the distal region of a DNABII peptide comprises, consists essentially of, or even consists of all three heavy chain (HC) CDRs that comprise, consist essentially of, or even consist of a sequence selected from SEQ ID NOs: 12-14 or their respective equivalents, and / or all three light chain (LC) CDRs that comprise, consist essentially of, or even consist of a sequence selected from SEQ ID NOs: 18-20 or their respective equivalents. In certain embodiments, the antibody or fragment thereof binds to the distal region of a DNABII peptide (e.g., the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal-chimeric peptide IhfA5-mIhfB4). NTHI In one embodiment, the antibody or fragment thereof binds to the tip-chimeric peptide IhfA5-mIhfB4 NTHI Combine with.

[0252] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 21 and a light chain sequence comprising SEQ ID NO: 22.

[0253] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO:9 and a light chain sequence comprising SEQ ID NO:15.

[0254] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO:9 and a light chain sequence comprising SEQ ID NO:16.

[0255] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO:9 and a light chain sequence comprising SEQ ID NO:17.

[0256] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 10 and a light chain sequence comprising SEQ ID NO: 15.

[0257] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 10 and a light chain sequence comprising SEQ ID NO: 16.

[0258] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 10 and a light chain sequence comprising SEQ ID NO: 17.

[0259] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO:11 and a light chain sequence comprising SEQ ID NO:15.

[0260] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO:11 and a light chain sequence comprising SEQ ID NO:16.

[0261] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO:11 and a light chain sequence comprising SEQ ID NO:17.

[0262] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 12 and a light chain sequence comprising SEQ ID NO: 18.

[0263] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 12 and a light chain sequence comprising SEQ ID NO: 19.

[0264] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 12 and a light chain sequence comprising SEQ ID NO: 20.

[0265] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 13 and a light chain sequence comprising SEQ ID NO: 18.

[0266] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 13 and a light chain sequence comprising SEQ ID NO: 19.

[0267] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 13 and a light chain sequence comprising SEQ ID NO: 20.

[0268] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 14 and a light chain sequence comprising SEQ ID NO: 18.

[0269] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 14 and a light chain sequence comprising SEQ ID NO: 19.

[0270] In some embodiments, the antibody or fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 14 and a light chain sequence comprising SEQ ID NO: 20.

[0271] The antibodies or fragments thereof provided herein can be monospecific or bispecific. In one embodiment, the antibodies or fragments thereof are trispecific, tetraspecific, or pentaspecific. Additionally or alternatively, the antibodies are selected from the group of IgA (such as IgA1 or IgA2), IgD, IgE, IgG (such as IgG1, IgG2, IgG3, or IgG4), or IgM antibodies. In one embodiment, the antibody further comprises a constant region selected from the group of an IgA constant region (such as an IgA1 constant region or an IgA2 constant region), an IgD constant region, an IgE constant region, an IgG constant region (such as an IgG1 constant region, an IgG2 constant region, an IgG3 constant region, or an IgG4 constant region), or an IgM constant region. In some embodiments, the constant region of the antibody comprises an amino acid sequence selected from the group of SEQ ID NOs: 23 and 35-42.

[0272] In another embodiment, the antibody can be modified by conventional techniques, such as PEGylation, PEG mimetics, polysialylation, HESylation, or glycosylation, which in one embodiment may increase the half-life of the antibody.

[0273] How to condense eDNA strands in neutrophil extracellular traps (NETs) Another aspect of the present disclosure relates to a method of condensing eDNA tendrils of neutrophil extracellular traps (NETs), comprising, consisting essentially of, or even consisting of, contacting the NETs with an effective amount of a DNA-binding agent.

[0274] Another aspect of the present disclosure relates to a method of preventing or neutralizing eDNA formation in neutrophil extracellular traps (NETs), comprising, consisting essentially of, or even consisting of, contacting the NETs with an effective amount of a DNA-binding agent.

[0275] Another aspect of the present disclosure relates to a method of preventing neutrophil extracellular trap (NET) formation or inducing regression of existing NETs comprising, consisting essentially of, or even consisting of contacting the NETs with an effective amount of a DNA-binding agent.

[0276] In some embodiments, the DNA binding agent is an agent that aggregates or condenses DNA.

[0277] In some embodiments, an effective amount is between 50 nM and 2 μM (e.g., 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 150 nM, 170 nM, 200 nM, 220 nM, 250 nM, 270 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 85 ... 0 nM, 950 nM, 1000 nM (1 μM), 1050 nM, 1100 nM, 1150 nM, 1200 nM, 1250 nM, 1300 nM, 1350 nM, 1400 nM, 1450 nM, 1500 nM, 1550 nM, 1600 nM, 1650 nM, 1700 nM, 1750 nM, 1800 nM, 1850 nM, 1900 nM, 1950 nM, or 2000 nM (2 μM).

[0278] In some embodiments, the DNA binding agent comprises, consists essentially of, or even consists of a histone-like nucleoid structuring protein (H-NS), a polyamine, or a polycation.

[0279] In some embodiments, the H-NS is derived from a gram-negative or gram-positive bacterium. In some embodiments, the H-NS is derived from a bacterium of the genus Escherichia, Haemophilus, Streptococcus, Mycobacteria, Klebsiella, or Pseudomonas; optionally, the H-NS is derived from Escherichia coli (E. coli), nontypeable Haemophilus influenzae (NTHI), S. pneumoniae, K. pneumoniae, Mycobacterium tuberculosis, or Pseudomonas aeruginosa.

[0280] In some embodiments, H-NS comprises, consists essentially of, or even consists of an amino acid sequence having at least 60% (e.g., at least 60, 65, 70, 75, 80, 85, 90, 95, 99% or more) identity to an amino acid sequence selected from SEQ ID NOs: 29-34, where the equivalent is identical to the reference polypeptide. In one aspect, percent identity is determined using the BLAST alignment program with default parameters. Particularly preferred programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort criteria = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0281] In some embodiments, the contacting is in vitro or in vivo.

[0282] Another aspect of the present disclosure relates to a method of halting damaging coagulation in a subject in need comprising, consisting essentially of, or even consisting of administering to a subject in need an effective amount of a DNA binding agent.

[0283] In some embodiments, the DNA binding agent comprises, consists essentially of, or even consists of a histone-like nucleoid structuring protein (H-NS), a polyamine, or a polycation.

[0284] In some embodiments, the H-NS is derived from a gram-negative or gram-positive bacterium. In some embodiments, the H-NS is derived from a bacterium of the genus Escherichia, Haemophilus, Streptococcus, Mycobacteria, Klebsiella, or Pseudomonas; optionally, the H-NS is derived from Escherichia coli (E. coli), nontypeable Haemophilus influenzae (NTHI), S. pneumoniae, K. pneumoniae, Mycobacterium tuberculosis, or Pseudomonas aeruginosa.

[0285] In some embodiments, H-NS comprises, consists essentially of, or even consists of an amino acid sequence having at least 60% (e.g., at least 60, 65, 70, 75, 80, 85, 90, 95, 99% or more) identity to an amino acid sequence selected from SEQ ID NOs: 29-34, where the equivalent is identical to the reference polypeptide. In one aspect, percent identity is determined using the BLAST alignment program with default parameters. Particularly preferred programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort criteria = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0286] Another aspect of the present disclosure relates to a method of stopping excessive inflammation in a subject in need thereof, comprising, consisting essentially of, or even consisting of administering to a subject in need thereof an effective amount of a DNA binding agent, optionally wherein the effective amount is between 50 nM and 2 μM.

[0287] Another aspect of the present disclosure relates to a method of preventing, treating, or preventing the progression of a neutrophil extracellular trap (NET)-mediated disease in a subject in need thereof, comprising, consisting essentially of, or even consisting of, administering to the subject in need thereof an effective amount of a DNA binding agent. In some embodiments, the effective amount is between 50 nM and 2 μM (e.g., 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 150 nM, 170 nM, 200 nM, 220 nM, 250 nM, 270 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 85 ... 0 nM, 950 nM, 1000 nM (1 μM), 1050 nM, 1100 nM, 1150 nM, 1200 nM, 1250 nM, 1300 nM, 1350 nM, 1400 nM, 1450 nM, 1500 nM, 1550 nM, 1600 nM, 1650 nM, 1700 nM, 1750 nM, 1800 nM, 1850 nM, 1900 nM, 1950 nM, or 2000 nM (2 μM).

[0288] In some embodiments, the DNA binding agent is an agent that aggregates or condenses DNA. In some embodiments, an effective amount is between 50 nM and 2 μM (e.g., 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 150 nM, 170 nM, 200 nM, 220 nM, 250 nM, 270 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 85 ... 0 nM, 950 nM, 1000 nM (1 μM), 1050 nM, 1100 nM, 1150 nM, 1200 nM, 1250 nM, 1300 nM, 1350 nM, 1400 nM, 1450 nM, 1500 nM, 1550 nM, 1600 nM, 1650 nM, 1700 nM, 1750 nM, 1800 nM, 1850 nM, 1900 nM, 1950 nM, or 2000 nM (2 μM).

[0289] In some embodiments, the DNA binding agent comprises, consists essentially of, or even consists of a histone-like nucleoid structuring protein (H-NS), a polyamine, or a polycation.

[0290] In some embodiments, the H-NS is derived from a gram-negative or gram-positive bacterium. In some embodiments, the H-NS is derived from a bacterium of the genus Escherichia, Haemophilus, Streptococcus, Mycobacteria, Klebsiella, or Pseudomonas; optionally, the H-NS is derived from Escherichia coli (E. coli), nontypeable Haemophilus influenzae (NTHI), S. pneumoniae, K. pneumoniae, Mycobacterium tuberculosis, or Pseudomonas aeruginosa.

[0291] In some embodiments, the H-NS comprises, consists essentially of, or even consists of an amino acid sequence having at least 60% (e.g., at least 60, 65, 70, 75, 80, 85, 90, 95, 99% or more) identity to an amino acid sequence selected from SEQ ID NOs: 29-34.

[0292] In some embodiments, H-NS comprises, consists essentially of, or even consists of an amino acid sequence having at least 60% (e.g., at least 60, 65, 70, 75, 80, 85, 90, 95, 99% or more) identity to an amino acid sequence selected from SEQ ID NOs: 29-34, where the equivalent is identical to the reference polypeptide. In one aspect, percent identity is determined using the BLAST alignment program with default parameters. Particularly preferred programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort criteria = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0293] In some embodiments, the subject is a mammal or a human patient.

[0294] In some embodiments, the subject is suffering from one or more of the following: a pulmonary disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-associated acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, or small vessel vasculitis; an autoinflammatory disease selected from gout or inflammatory bowel disease, and / or a metabolic disease selected from type 2 diabetes or obesity.

[0295] Combination treatment The compositions and related methods of the present disclosure may be used in combination with the administration of other therapies, including, but not limited to, the administration of DNase enzymes, antibiotics, antibacterial agents, anti-infective agents, antifungal agents, anti-parasitic agents, antiviral agents, or other antibodies.

[0296] In some embodiments, the methods and compositions include a deoxyribonuclease (DNase) enzyme that acts synergistically with an anti-DNABII antibody. DNase is any enzyme that catalyzes the cleavage of phosphodiester bonds in the DNA backbone. Three non-limiting examples of DNase enzymes known to target not only cruciform structures but also various DNA secondary structures include DNAse I, T4 Endo VII, T7 Endo I, RuvABC, and RusA. In certain embodiments, the effective amount of anti-DNABII antibody required to destabilize a biofilm is reduced when combined with DNase. When administered in vitro, DNase can be added directly to the assay or in an appropriate buffer known to stabilize the enzyme. The effective unit dose of DNase and assay conditions can vary and can be optimized according to procedures known in the art.

[0297] In other embodiments, the methods and compositions can be combined with antibiotics and / or antimicrobial agents. Antimicrobial agents are substances that kill or inhibit the growth of microorganisms, such as bacteria, fungi, or protozoa. While biofilms are generally resistant to the action of antibiotics, the compositions and methods described herein can be used to sensitize biofilm-related infections to conventional therapeutic methods for treating infections. In other embodiments, the use of antibiotics or antimicrobial agents in combination with the methods and compositions described herein allows for a reduction in the effective amount of the antimicrobial agent and / or biofilm-reducing agent. Some non-limiting examples of antimicrobial agents and antibiotics useful in combination with the methods of the present disclosure include amoxicillin, amoxicillin-clavulanate, cefdinir, azithromycin, and sulfamethoxazole-trimethoprim. The therapeutically effective dose of the antimicrobial agent and / or antibiotic in combination with the biofilm-reducing agent can be readily determined by conventional methods. In some embodiments, the dose of the antimicrobial agent in combination with the biofilm-reducing agent is the average effective dose shown to be effective in other bacterial infections, for example, bacterial infections whose etiology does not involve biofilms. In other embodiments, the dose is 0.1, 0.15, 0.2, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.8, 0.85, 0.9, 0.95, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, or 5 times the average effective dose. The antibiotic or antimicrobial agent can be added before, simultaneously with, or after the addition of the anti-DNABII antibody.

[0298] In other embodiments, the methods and compositions can be combined with antibodies to treat bacterial infections. One example of an antibody useful in combination with the methods and compositions described herein is an antibody against an unrelated outer membrane protein (i.e., OMP P5). Treatment with this antibody alone does not reduce biofilms in vitro. Combination treatment of this antibody with a biofilm-reducing agent produces a greater effect than can be achieved by either agent used alone at the same concentration. Other antibodies that may produce a synergistic effect when combined with a biofilm-reducing agent or method for reducing biofilms include anti-rsPilA, anti-OMP26, anti-OMP P2, and anti-total OMP preparations.

[0299] The compositions and methods described herein can be used to sensitize bacterial infections involving biofilms to common therapeutic modalities that are effective in treating bacterial infections that do not involve biofilms but are ineffective in treating different bacterial infections involving biofilms. In other embodiments, the compositions and methods described herein can be used in combination with therapeutic modalities that are effective in treating bacterial infections involving biofilms, but the combination of such additional treatments with biofilm-reducing agents or methods produces a synergistic effect such that the effective dose of either the biofilm-reducing agent or the additional therapeutic agent can be reduced. In other examples, the combination of such additional treatments with biofilm-reducing agents or methods produces a synergistic effect such that treatment is enhanced. Enhanced treatment can be evidenced by a shorter time required to treat the infection.

[0300] The additional therapeutic treatment can be added before, simultaneously with, or after the method or composition used to reduce the biofilm, and can be contained within the same formulation / composition or as a separate formulation / composition.

[0301] kit Also disclosed are kits containing one or more of the polypeptides, antibodies or fragments thereof, or agents and instructions necessary to carry out the in vitro and in vivo methods described herein. Accordingly, the present disclosure provides kits for carrying out these methods, which may include antibodies, antibody fragments, polypeptides, polynucleotides, vectors, or host cells, as well as instructions for carrying out the methods disclosed herein, for example, for collecting tissue and / or conducting screening and / or analyzing the results, and / or administering an effective amount of the antibodies, antibody fragments, polypeptides, polynucleotides, vectors, or host cells as defined herein. These can be used alone or in combination with other suitable antimicrobial agents.

[0302] For example, the kit can include, alternatively consist essentially of, or even consist of, any one or more of the above-identified agents, such as antibodies, antibody fragments, polypeptides, polynucleotides, vectors, or host cells, and instructions for use. The kit can further include one or more of an adjuvant, an antigenic peptide, or an antimicrobial agent. Examples of carriers include liquid carriers, pharmaceutically acceptable carriers, solid carriers, pharmaceutically acceptable carriers, pharmaceutically acceptable polymers, liposomes, micelles, implants, stents, pastes, gels, dental implants, or medical implants. [Example]

[0303] Example The following examples are intended to illustrate, but not limit, the scope of the present disclosure.

[0304] Example 1: mB Box-97 peptide and inhibition of NETosis Among all HMGB1-derived constructs, only mB Box-97 shows inhibition of NETosis in isolated human neutrophils. HMGB1 is a known inducer of inflammation and NETosis. However, we previously demonstrated that an engineered point mutation (C45S; mHMGB-1) strongly attenuates the pro-inflammatory function of HMGB1. Indeed, mHMGB1 can reduce neutrophil migration into the peritoneal cavity upon induction of peritoneal inflammation with thioglucolate, suggesting that mHMGB1 induces an attenuated inflammatory neutrophil response. mHMGB1 can also reduce the number of neutrophils in bronchoalveolar lavage (BAL) of mice infected with B. cenocepacia. Another study showed that a synthetic peptide encompassing only Box A of HMGB1 can affect macrophage function and attenuate liver injury in a mouse model of cirrhosis. These findings suggest that HMGB1 has the potential to affect neutrophils and that some of these functions reside in specific domains. Indeed, Applicant generated a series of truncated constructs of HMGB1 and found that only the extended B-Box domain (B Box 97) possessed antibiofilm activity comparable to that of the whole protein. Furthermore, Applicant demonstrated that a single point mutation (C106S) in the B Box, known to reduce the pro-inflammatory activity of HMGB1, possessed the same antibiofilm activity. Here, Applicant used various truncated constructs of HMGB1 to determine whether any portion of the protein specifically affected neutrophil function. Recombinant full-length HMGB1, A Box (amino acids 1–89), AB Box (amino acids 1–176), B Box-97 (amino acids 80–176), recombinant and synthetic mB Box-97 (identical to B Box-97 except for the single amino acid change C106S), and B Box-87 (amino acids 90–176) (Figure 1A) were each tested for their ability to induce NETosis in isolated human neutrophils. Isolated human neutrophils were incubated with 200 nM constructs for 3–4 h, and the plasma membrane was stained with Alexa Fluor 488-labeled wheat germ agglutinin (WGA) and an anti-double-stranded DNA mouse antibody cross-stained with Alexa Fluor 594-labeled anti-mouse secondary antibody. Cells were then visualized using a confocal microscope.All constructs except mB Box-97, BBox-87, and ABox induced NETs in isolated PMNs. This finding was confirmed by a plate-based assay in which released DNA was stained with the cell-impermeable fluorescent dye SYTOX Green (Figure 1B). Applicants also tested whether any of these constructs had any effect on PMA-induced NETosis. To do so, isolated neutrophils were treated with PMA alone or with PMA and various constructs for 3–4 h, stained for DNA and plasma membrane, and observed using confocal microscopy. Of the six constructs tested, only mB Box-97 showed inhibition of PMA-induced NETosis. Upon quantification of the % NETs in total cells in different treatment conditions, only mB Box-97 showed significant inhibition of PMA-induced NETosis (Figure 1C).

[0305] mB Box-97 inhibits PMA- and LPS-induced NET formation and the release of NET-associated proteins. PMA directly activates protein kinase C (PKC), while LPS binds to its respective cellular receptor, leading to PKC activation. Activated PKC phosphorylates component proteins of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) ensemble, resulting in its activation. Activated NOX generates reactive oxygen species (ROS), which in turn activates a cascade of events that leads to chromatin decondensation and the release of NETs. DNA and protein markers released during this process can be visualized or quantified using specific antibodies. Applicant investigated whether inhibition of NETs using mB Box-97 affects the release of NET-associated proteins such as NE and MPO. Isolated human neutrophils (2 × 10 5 ) with or without BBox-97 or mBBox-97 (200 nM), PMA (100 nM, 3.5 h) or heat-inactivated nontypeable Haemophilus influenzae (NTHI) (1*10 6, 12–16 h). NETs were stained for plasma membrane (Alexa fluor-488-labeled WGA), α-ds-DNA (Alexa fluor-594), and protein markers (Alexa fluor-405) for NETs—neutrophil elastase (NE), mitogen peroxidase (MPO), or citrullinated histone (H3-Cyt). The effect of treatment on NET formation was observed by capturing z-stack images using confocal microscopy. mB Box-97 inhibited PMA- and NTHI-induced NETosis, whereas B Box-97 had no effect. Applicants also calculated the mean fluorescence intensity (MFI) of NE, MPO, and H3-Cyt (Figures 2A–C). The MFI of NE and MPO in the mB Box-97-treated condition was significantly lower than that in the PMA-only or PMA-plus-B Box-97-treated condition. No differences in the MFI of citrullinated histones were observed across treatment conditions (Figures 2A-C).

[0306] To quantify NETosis, Applicant used the cell-impermeant fluorescent DNA stain SYTOX Green. Isolated neutrophils (5 × 10 3Neutrophils were treated with PMA with or without mB Box-97 and B Box-97 in 96-well plates for 3.5 hours; medium alone served as a control. Released extracellular DNA was stained with SYTOX Green (1 μM) for 10 minutes, washed with PBS, and fluorescence was measured using a fluorometer. mB Box-97 showed significant inhibition of PMA-induced NET formation, while B Box-97 showed no significant effect (Figure 2D). We also investigated the release of nuclear elastase (NE) in the medium and NE bound to DNA after PMA-induced NETosis. Quantification was performed using a human NE-specific ELISA kit. Neutrophils were allowed to form NETs in 6-well plates using PMA for 3–4 hours in the presence or absence of mB Box-97 or B Box-97, or no treatment. The medium was collected, and the formed NETs were treated with DNA to isolate DNA-bound NE. NE released into the medium and bound to DNA were measured by ELISA. mB Box-97 treatment significantly reduced NE release into the medium (secretion) (Fig. 2E) as well as NE bound to DNA (DNA binding) (Fig. 2F).

[0307] mB Box 97 is Ca 2+ partially inhibits mediated NETosis. PMA- and LPS-mediated NETosis induces Ca 2+ It is altered by ionophore A23187-induced NETosis. PMA-induced NETosis does not require PAD4, but A23187-mediated NET formation does. NETs formed upon calcium efflux induced by A23187 involve activation of PAD4, which citrullinates histones and leads to chromosome uncoiling. This pathway largely bypasses PKC-NOX2-ROS-mediated NETosis. Therefore, the applicant believes that Ca 2+We investigated the effect of mB Box-97 on ionophore-mediated NETosis. Isolated PMNs were treated with either mB Box-97 or B Box-97 for 4–6 h with A23187. NETs were visualized by confocal microscopy. We stained for the NET-associated marker proteins NE, MPO, or citrullinated histone H3. mB Box-97 inhibited Ca 2+ Although mB Box-97 inhibited ionophore-induced NETosis, the inhibition was not as significant as that observed in PMA / LPS-mediated NETosis. The MFI of citrullinated histones indicates that mB Box-97 had no effect on histone citrullination in Ca2+-induced NETosis, whereas a similar effect was observed in PMA / LPS-mediated NETosis. Visual inspection and MFI values ​​suggested an increase in H3-cyt staining upon induction with A23187 (Figure 2C). mB Box-97 partially inhibited the Ca2+-induced histone citrullination. 2+ Ionophore-induced NETosis can be inhibited by Ca 2 These findings suggest that the inhibition of NETosis by mB Box-97 is related to the activation of PKC by Ca. 2+ This may indicate that this may be mediated by the PKC / NOX-ROS pathway rather than the -PAD4 pathway.

[0308] ROS production in activated human neutrophils is mediated by p47 phox Regulated by mB Box-97 through inhibition of phosphorylation. PMA, a diacylglycerol mimetic, inhibits p47 phox , p67 phox and p40 phoxmB Box-97 activates PKC, which phosphorylates key components of the NOX complex, such as β-glucanase (ROS). Upon phosphorylation, these component proteins assemble into the active NOX complex, which generates ROS. ROS production is important for microbial death and the breakdown of the nuclear and granule membranes, ultimately leading to NETosis. Given the importance of ROS in NETosis, Applicant tested the effect of mB Box-97 on ROS production by neutrophils. Isolated neutrophils were preincubated with luminol, a chemiluminescent indicator of ROS. Cells were then activated with different treatment conditions, and ROS was measured over a 2-hour period, with readings taken at 5-minute intervals. mB Box-97 significantly inhibited PMA-induced ROS production in human neutrophils (Figure 3A). ROS levels are an indicator of NOX activity, which depends on the phosphorylation of its component proteins. Therefore, Applicant investigated the phosphorylation of the key NOX component protein p47 for serine 370 phosphorylation. phox The phosphorylation of p47 at ser-370 was investigated. Phosphorylation of this residue has been shown to be important for the assembly of the active NOX complex. Proteins from treated neutrophils were separated by SDS-PAGE and Western blotting was used to identify p47 at ser-370 with a fluorophore-specific antibody. phox Probed for phosphorylation of total p47 phox and GAPDH were used as controls. After densitometric analysis, mB Box-97 was found to be significantly reduced in p47 phox It was revealed that BBox-97 significantly inhibited the phosphorylation of BBox-1, whereas its non-mutated counterpart BBox-97 had no effect (Fig. 3B).

[0309] Inhibition of NETosis, ROS generation, and antimicrobial release by mB Box-97 can regulate bacterial killing by PMNs. In addition to causing NET formation, ROS produced by neutrophils contribute to microbial killing. Applicant has previously demonstrated that the bacterial DNA B1I protein HU (HU) from NTHI is involved in ROS production. NTHI(2013) demonstrated that inhibition of NETs by mB Box-97 attenuates bacterial killing by neutrophils. Because mB Box-97 was able to inhibit NETosis, ROS production, and the release of antimicrobial proteins such as NE, applicants tested whether it could demonstrate any effect on bacterial killing. Toward that goal, applicants tested 16-hour-old NTHI biofilms treated with buffer alone, mB Box-97, Bbox-97, and HU. NTHI Neutrophils were incubated with mB Box-97 for 4 hours. Neutrophils were lysed with Triton® X-100 to allow recovery of viable intracellular bacteria, and total CFU NTHI were calculated for each condition to determine the relative percent bacterial killing due to PMN NETosis compared to the buffer-only control in the system. mB Box-97 inhibited bacterial killing by neutrophils compared to B Box-97, and this inhibition was confirmed by HU Box-97, which was used as a positive control. NTHI The inhibition was comparable to that by α-glucan (Fig. 4A).

[0310] mB Box-97 is localized to the cytoplasm and plasma membrane of human neutrophils. Although the inhibition of NETosis by mB Box-97 was evident through various observations by the applicants, the specific target of the peptide was not. To gain more insight into the target, applicants localized the peptide in neutrophils. To accomplish this, applicants synthesized an N-terminal His-tagged recombinant mB Box-97 (mBBox-97-His) peptide. Applicants tested the peptide for its ability to inhibit NETosis and ROS production (Figure 4B). After confirming the NET-inhibiting ability of his-tagged mB Box-97, applicants performed a time-course study to identify the intracellular localization of the peptide. Neutrophils were incubated with his-tagged mB Box-97 for 10, 30, 60, 120, or 180 minutes, fixed in 10% formalin, and probed for the peptide using an anti-his rabbit primary antibody followed by detection using an Alexa fluor-405-conjugated goat anti-rabbit secondary antibody. DNA was detected with a mouse anti-double-stranded DNA primary antibody, which was detected with a goat Alexa fluor-595-tagged anti-mouse secondary antibody, and the plasma membrane was stained with Alexa fluor-488-labeled WGA. Images were captured using confocal microscopy. Applicant observed that the mB Box-97 signal remained strong in most neutrophils up to 120 minutes after treatment. The majority of the peptides were localized in the cytoplasm and plasma membrane of the cells. At 180 minutes, some peptides appeared to be localized in the nucleus.

[0311] Protein kinase C is a target of mB Box-97. PKC is a key kinase in NETosis, whose inhibition leads to the inhibition of NETosis. PKC is involved in the phosphorylation and subsequent assembly of the active NOX complex. The observation of cytoplasmic localization of mB Box-97 and p47 phox Based on the inhibition of phosphorylation, Applicant investigated whether mB Box-97 has any effect on PKC activity. Toward that goal, Applicant used a PKC activity kit. Two different concentrations of mB Box-97 were incubated with PKC for 10 minutes, and then the mixture was incubated with the substrate (Figure 5).

[0312] The formation of NETs is a key function of an active immune system. Research demonstrating the importance of NETs in the treatment of various pathologies is gaining momentum, as is research demonstrating the adverse effects of excessive NET formation. Essentially, NET formation must be maintained in balance between its production and clearance. In vitro, various upstream stimulators, including LPS, TNF, IL-8, and PKC agonists, as well as several pro-inflammatory molecules such as HMGB1, can activate NET formation. During NETosis, various bioactive molecules are released, leading to microbial death. However, due to their nonspecific nature, NET components can cause injury to surrounding tissues by themselves or by increasing the pro-inflammatory response. They can also play a role in the enhanced inflammation seen in autoimmune diseases such as psoriasis, rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE). Furthermore, autoinflammatory diseases such as gout are associated with NETosis. Excessive NETs can also cause a physical barrier to blood flow, leading to atherosclerosis and / or stroke. Cancer cells can increase NETosis by priming platelets in pancreatic cancer. NET-mediated platelet activation can promote several negative outcomes associated with late-stage metastatic breast cancer, including venous thromboembolism (VTE).

[0313] Recent studies on COVID-19 have demonstrated the significant role of enhanced neutrophil infiltration and release of NETs, ​​complement activation, and vascular thrombosis during necroinflammation in COVID-19. The formation of NETs in microvasculature increases the inflammatory response and vascular microthrombosis, which leads to ARDS in patients' lungs. Serum levels of NETosis markers are elevated in patients receiving intensive care, suggesting NETosis. Mechanical ventilation rather than room air in patients receiving ventilation may be associated with COVID-19 disease severity. Inhibition of NETs may reduce the severity of many such diseases, thereby improving survival. Herein, we describe an HMGB1-based mutant peptide, mB Box-97, which inhibits NET formation induced by different stimuli.

[0314] Although several direct or indirect inhibitors of NET formation have been investigated in preclinical studies, none of them have been approved for the treatment of NET-related complications. Some of these, such as hydroxychloroquine, methotrexate, and prednisolone (the active metabolite of prednisone), are molecules that have been used in the clinic for many years. Some are humanized antibodies, such as rituximab (an anti-CD20 mAb), belimumab (a fully human IgG1λ recombinant mAb), and tocilizumab (a mAb against the IL-6 receptor). Others are molecules that can inhibit the function of NE, MPO, or PAD4. Most of these act as indirect inhibitors of NETosis. Although several inhibitors have been tested, there remains a need for efficient inhibitors that do not cause unwanted side effects or interfere with normal neutrophil functions other than NETosis.

[0315] In a previous study, the applicant showed that mutant HMGB1 loses its pro-inflammatory activity while retaining its anti-biofilm activity (Devaraj A. et al., 2021, J Clin Invest 131(16)). In this disclosure, the applicant constructed a mutant peptide mB Box-97 based on HMGB1, which retained its anti-biofilm ability against multiple microorganisms without exhibiting any pro-inflammatory activity. The applicant demonstrated that this peptide inhibits Ca 2+ We observed that induced NET formation can be inhibited using PMA and LPS, while showing partial inhibition when tested against inducible NET formation. The pathways activated by these stimuli are different, hence the outcome of inhibition using mB Box-97. PMA- and LPS-mediated NETosis is often accompanied by activation of PKC, which leads to phosphorylation of key components of NADPH oxidase, ultimately aiding in the formation of the NOX complex and the generation of ROS. Applicant has demonstrated that p47 phox We investigated the effect of mB Box-97 on the phosphorylation of p47 and found that the phosphorylation was inhibited by the peptide (Figure 3B). phox We observed inhibition of ROS generation, consistent with the inhibition of phosphorylation. ROS generation is important for the release of antimicrobial agents and decondensed chromatin from granulocytes. ROS trigger the dissociation of NE from membrane-associated complexes into the cytosol and activate its proteolytic activity in a myeloperoxidase (MPO)-dependent manner. Activated NE translocates to the nucleus, cleaves chromatin, and releases it into the cytoplasm, thereby completing NET formation. We observed a reduction in NE release from neutrophils upon treatment with mB Box-97 (Figure 2E). NET formation, antimicrobial release, and ROS generation all affect NET-mediated microbial killing. Because mB Box-97 can inhibit all of these, we tested its effect on microbial killing and found that it could inhibit neutrophil-mediated bacterial killing (Figure 4A).

[0316] PKC is a key kinase in the process of NETosis and its inhibition is impaired. The majority of evidence in this study pointed to the involvement of mB Box-97 in the inhibition of PKC activity. For example, the inhibition of p47phox phosphorylation (Figure 3B) and the localization of the peptide in the cytoplasm and plasma membrane. Therefore, we used a standard PKC assay kit to examine the effect of mB Box-97 on PKC activity and found that mB Box-97 inhibited PKC activity (Figure 5). 2+ In addition to activating PAD4, the influx of Ca(2+) can also activate PKC, suggesting that the Ca(2+) influx via mB Box-97 2+ The partial inhibition of mediated NETosis may be due to the inhibition of PKC.

[0317] Without being bound by any particular theory, the HMGB1-based peptide mB Box-97 is thought to inhibit the activity of protein kinase C, resulting in the inhibition of phosphorylation of key NADPH oxidase component proteins. This inhibits the active assembly of NOX and reduces ROS production. Reduced ROS production prevents the release of granulocyte-associated proteins such as NE and ultimately the production of NETs (Figure 6). mB Box-97 does not induce inflammation while retaining the ability to inhibit biofilm formation. mB Box-97 has excellent therapeutic potential.

[0318] Treatment of rheumatoid arthritis with mB Box-97. Abnormal and excessive NETosis is believed to be the underlying cause of autoimmune diseases such as rheumatoid arthritis. A preclinical animal model of rheumatoid arthritis (RA) is used to demonstrate that subjects suffering from RA benefit from inhibiting abnormal and excessive NETosis using mB Box-97 peptide. Exemplary animal models of RA are found in Meehan, Gavin R., et al. (Annals of the Rheumatic Diseases 80.10(2021):1268-1277) and Zhao, Ting, et al. (Frontiers in Immunology 13(2022):887460), which are incorporated herein in their entirety. In some embodiments, the animal model of RA is a rodent model or a non-human primate model. In some embodiments, the animal model of RA is selected from a collagen-induced arthritis (CIA) model, an adjuvant-induced arthritis (AA) model, a delayed-type hypersensitivity arthritis (DTHA) model, an anti-citrullinated peptide antibody (ACPA)-mediated arthritis model, or a spontaneous model.

[0319] Animal models of RA are treated with mB Box-97 peptide as described herein at between 50 nM and 2 μM, and diagnostic blood markers for RA (e.g., C-reactive protein (CRP), rheumatoid factor (RF), or anti-cyclic citrullinated peptide (anti-CCP)) are measured periodically (e.g., every 3 hours, 6 hours, 12 hours, 24 hours, every other day, every 3 days, every 4 days, every 5 days, every 6 days, or once a week). Reductions in diagnostic blood markers for RA are observed over time in animals treated with mB Box-97 compared to controls administered with scrambled control peptide. Reductions in RA symptoms (e.g., joint swelling, fluid accumulation in the ankles, joint pain, and joint inflammation) are also observed with mB Box-97 compared to controls administered with scrambled control peptide.

[0320] Example 2: mB Box-97 disrupts and prevents biofilm formation by diverse human pathogens HMGB1-derived peptides containing the B-Box and mB Box-97 retain biofilm-disrupting activity in vitro First, we tested the disruptive ability of HMGB1-derived peptides. Addition of the A Box to established biofilms (UPEC, B. cenocepacia, NTHI, or K. pneumoniae) did not significantly affect the measured biofilm parameters (Figure 7A). While B Box-87 had limited antibiofilm activity, AB Box and B Box-97 retained antibiofilm activity (Figure 7A). Because only the B Box can regulate DNA bending, we hypothesize that HMGB1 disrupts biofilms at least in part through DNA binding / bending. Because the B Box has been reported to contain pro-inflammatory activity mediated primarily through interaction with TLR4-MD2, dependent on residue C106, we generated a modified recombinant B Box-97 variant (mB Box-97) with a C106S mutation (Figure 1A). The mB Box-97 variant disrupted biofilms formed by UPEC, NTHI, B. cenocepacia , and K. pneumoniae in vitro comparable to those induced by B Box-97.

[0321] mB Box-97 disrupts biofilms in vivo To determine whether mB Box-97 can prevent pulmonary infection and limit inflammation as intended, Applicant administered 10 mg of mB Box-97 to adult C57BL / 6 mice. 7 (Figure 8A) or 10 8(Figure 8B) CFU of B. cenocepacia (Bc) was challenged intratracheally (it) with either 200 nM mB Box-97 or a negative control. 18 hours later, mice were euthanized to collect both BAL fluid and lung tissue. Lung tissue was homogenized, diluted, and plated to determine relative CFU, while BAL fluid was similarly diluted and plated. Mice administered either rHMGB1 or mB Box-97 contained significantly less Bc in both BAL and lung tissue compared to control mice, indicating that mB Box-97 significantly reduced the number of Bc in the high bacterial challenge dose (10 8 Our results suggest that mB Box-97 inhibited aggregate biofilm formation in mouse airways, even at high bacterial loads (CFU), further demonstrating that this approach promoted bacterial clearance, thereby suggesting preventative efficacy. Furthermore, despite the use of potent therapeutic doses of mB Box-97, none of the mice showed signs of sepsis requiring euthanasia before the study endpoint. While LPS alone induced >100 pg / ml of TNF-α (the gold-standard surrogate for sepsis induction), neither rHMGB1 nor mB Box-97 induced detectable TNF-α or further pro-inflammatory signaling when administered to LPS-primed mice. Here, Applicants demonstrate that mB Box-97 retains its antibiofilm activity without inducing inflammation, both by limiting bacterial load and / or preventing the formation of biofilm aggregates in mouse lungs and by not inducing TNF-α.

[0322] Synthetic mB Box-97(mB Box-97 syn ) is equivalent to recombinant mB Box-97 in vitro Native HMGB1 is heavily post-translationally modified by these modifications, which affect various HMGB1 functions. Indeed, even recombinant proteins expressed in bacteria often have post-translational modifications. To determine whether these modifications play a role in anti-biofilm disruption, Applicant synthesized an otherwise identical 97 amino acid peptide based on the coding sequence of recombinant mB Box-97 (hereafter referred to as mB Box-97). syn (This is called mB Box-97) syn To determine whether mB Box-97 retains DNA binding activity, applicants performed electromobility shift assays using Holliday Junction DNA substrates, which mimic the cross-strands of the eDNA lattice in bacterial biofilms and are also natural substrates for HMGB1, and demonstrated the binding activity of all mB Box-97 to various truncated forms of HMGB1. syn The ability of mB Box-97 and mB Box-97 was compared. syn All truncated recombinant versions containing the complete domain structure were also able to bind to Holliday junction DNA. As an additional test of stability, recombinant mB Box-97 and mB Box-97 syn mB Box-97 was incubated in human serum and showed similar stability, consistent with the fact that post-translational modifications present on recombinant mB Box-97 did not affect protein stability. As a final test, Applicants also demonstrated that mB Box-97 remained stable regardless of biofilm age. synWe demonstrated that mB Box-97 significantly destroyed four additional high-priority ESKAPEE pathogens (Figure 7B). Here, Applicants also utilized a humanized monoclonal antibody against the binding tip of the DNABII protein ("HuTipMab"), previously shown to have broad biofilm-disrupting activity, as a positive control (Figure 7B). Biofilms formed for 24 hours and then treated with either 1.2 μg of mB Box-97 or 5.0 μg of HuTipMab for 2 hours were significantly disrupted compared to treatment with medium alone (P. aeruginosa; 30.5% and 29.8%, respectively; S. aureus; 12.2% and 24.8%, respectively; E. faecium; 39.2% and 29.1%, respectively; A. baumannii; 16.7% and 27.8%, respectively (P = 0.03 - P < 0.0001).

[0323] The age of older biofilms is mB Box-97 syn To determine whether incubation with a mouse monoclonal antibody (MsTipMab) against a non-protected domain of the bacterial DNABII protein (MsTailMab) could affect the ability of HIV-1 to disrupt bacterial biofilms, as these biofilms contain increasingly high concentrations of eDNA, Applicant tested this hypothesis using biofilms formed by the respiratory pathogen NTHI. To this end, Applicant treated 48- and 72-h NTHI biofilms with either 1.2 μg of mB Box-97 or 5.0 μg of a mouse monoclonal antibody (MsTipMab) (directed against the same epitope as HuTipMab). These older NTHI biofilms were not disrupted by incubation with a mouse monoclonal antibody (MsTailMab) against a non-protected domain of the bacterial DNABII protein, but were significantly disrupted by both mB Box-97 and MsTipMab compared to medium alone (48 h: 47.3% and 47.2%; 72 h: 46.4% and 34.4%). Biofilms grown for 96 h showed similar disruption (e.g., 49% and 37%, respectively) compared with mB Box-97, as expected given that eDNA and DNABII concentrations increase with biofilm maturation. synThe results were statistically significant (P=0.02, P=0.005, respectively). From this point on, applicants used mB Box-97 for the remainder of the experiment. syn Only the 100% hydroxybenzoate was used.

[0324] mB Box-97 syn prevents biofilm formation in vitro Applicant now hypothesized that mB Box-97 may also prevent bacterial biofilm formation, as this hypothesis is consistent with the currently demonstrated ability of domain variants to disrupt established biofilms in vitro. To test this hypothesis, Applicant first tested the ability of mB Box-97 to prevent the Gram-negative and Gram-positive pathogens NTHI and S. aureus, respectively, from forming biofilms by using the same two concentrations of mB Box-97 previously used for the in vitro disruption assay. rHMGB1 and mB Box-97 rec Incorporation of mB Box 97 served as a positive control, and medium alone and A Box were used as negative controls. Applicants also wanted to test two concentrations of HuTipMab, previously shown to have broad biofilm-disrupting activity, for the possibility of possibly also preventing biofilm formation. After 16 hours of incubation, both the positive control and all tested concentrations of mB Box 97 or HuTipMab significantly inhibited biofilm growth by both NTHI and S. aureus compared to the negative control (Figure 9A) (P=0.01-P<0.0001). The highest concentrations tested of mB Box-97 and HuTipMab (e.g., 1.8 μg and 7.5 μg, respectively) restricted growth to a bacterial monolayer or less (biomass <1.0 μM). 3 / μm 2 , P<0.0001), and the characteristic 3D biofilm architecture was not present.

[0325] Next, Applicants began to assay the breadth of mB Box-97 and / or HuTipMab's ability to prevent biofilm formation. To do so, Applicants used the most effective concentrations of mB Box-97 or HuTipMab in preventing biofilm growth by NTHI and S. aureus to similarly prevent biofilm formation by the remaining high-priority ESKAPEE pathogens or B. cenocepacia (Figure 9B). Applicants observed significant biofilm prevention after 16 hours of incubation with either P. aeruginosa, Enterobacteriacea sp., E. faecium, uropathogenic E. coli, A. baumannii, or B. cenocepacia with either mB Box-97 (P = 0.03 - P < 0.0001) or HuTipMab (P = 0.02 - P < 0.0001), compared to the negative control. To provide further evidence of the observed preventive activity, data generated with the pathogen K. pneumoniae (Figure 10A) are presented along with corresponding representative CSLM images (Figure 10B). These differences were statistically significant (P = 0.03 - P < 0.0001).

[0326] mB Box-97 syn synergizes with a humanized monoclonal antibody against the protective domain of the DNABII protein to prevent biofilm formation As shown in Figures 4 and 5, both mB Box-97 and HuTipMab, when tested individually, significantly prevented biofilm formation by nine pathogens, including seven high-priority ESKAPEEs. Here, Applicant evaluated whether mB Box-97 and HuTipMab could act additively or perhaps synergistically to prevent biofilm formation. To assess this, Applicant again used NTHI and S. aureus as model pathogens. Applicant first incubated NTHI or S. aureus with serial 1:2 dilutions of the highest dose of either mB Box 97 or HuTipMab used in the previous prevention assay to determine relative preventive activity.

[0327] As shown, prevention of NTHI biofilms by dilutions of HuTipMab (top row, Figure 11A) ranged from 0 to 72%, while prevention by dilutions of mB Box 97 (last column, Figure 11A) ranged from 0 to 43%. For S. aureus, prevention by dilutions of HuTipMab (top row, Figure 11B) similarly ranged from 0 to 74%, while prevention by dilutions of mB Box 97 (last column, Figure 11B) ranged from 0 to 60%. To assess the potential additive or synergistic preventive activity of these two biologics, Applicant then incubated NTHI or S. aureus with the mB Box-97 + HuTipMab cocktail at multiple similar 1:2-fold decreasing dilutions for 16 hours and determined the relative biomass (see values ​​in the diagonal boxes in both panels of Figure 11). For NTHI (Panel A, diagonal box), the relative percent prevention values ​​were 1 / 16 of each th The doses of each biologic exceeded those of any individual component of the cocktail, encompassing the full combined dose. th When used at doses of 100 mg / kg / day, the outcome was synergistic (66% in combination compared to 20% or 5% individually). For S. aureus (Panel B, diagonal box), the relative percent prevention value was again 1 / 8 of each other. th The combined full dose of the cocktail exceeded that of any individual component alone. Mathematically, no clear synergistic results were observed, although applicants each achieved 1 / 4 of the combined dose. th Dose and 1 / 8 th When prevention was assessed using a cocktail of doses of each biologic, there was additive prevention (69% compared with 41% or 26% individually; 57% compared with 38% or 18% individually, respectively).

[0328] Applicants found that when tested in combination against both pathogens, mB Box-97 and HuTipMab provided greater prevention at sub-maximal test concentrations (half the maximum concentration of both treatments prevented 75% of NTHI biofilms compared to the negative control, and half the maximum concentration of both treatments prevented 79% of S. aureus biofilms compared to the negative control) compared to the prevention at the highest tested concentration of either treatment administered separately (compared to the negative control, the highest concentration of HuTipMab tested prevented NTHI biofilm growth by 72% and the highest concentration of mB Box-97 prevented NTHI biofilm growth by 43%; compared to the negative control, the highest concentration of HuTipMab tested prevented S. aureus biofilm growth by 74% and the highest concentration of mB Box-97 prevented S. aureus biofilm growth by 60%).

[0329] Applicant also provides a combination index (CI,

number

[0330] material and method Humanized monoclonal antibody against a tip chimeric peptide designed to mimic the immunoprotective domain of the DNABII protein integration host factor (IHF). A humanized monoclonal antibody (of IgG isotype) against the tip-chimeric peptide (HuTipMab) was engineered from a murine monoclonal antibody.

[0331] Synthetic mB Box-97.(mB Box-97 syn;LifeTein®, LLC; for ease of large-scale production and greater than 95% homogeneity)

[0332] Bacterial species and sources. NTHI strain 86-028NP was isolated from the nasopharynx of a child with chronic otitis media at Nationwide Children's Hospital. Enterobacter spp. and K. pneumoniae were isolated. S. aureus strain 29213, A. baumannii strain 17978, and P. aeruginosa strain 27853 were obtained from ATCC. E. faecium strain Com12 was isolated from the feces of a healthy human volunteer. Strains not obtained from ATCC were kept frozen at low passage numbers in liquid nitrogen.

[0333] Disruption of bacterial biofilms. NTHI and S. aureus were cultured on chocolate agar for 18–24 h at 37°C in a humidified atmosphere containing 5% CO2. NTHI was then resuspended in brain heart infusion broth (sBHI) supplemented with heme [2 μg / mL] and β-NAD [2 μg / mL] broth to an OD at 490 nm of 0.1. S. aureus was resuspended in brain heart infusion broth (BHI) to an OD at 490 nm of 0.1. 490nm The cultures were then diluted in their respective medium to approximately 2 x 10 5 CFU / mL, and 200 μL of this suspension was inoculated into each well of an 8-well chambered cover glass slide (Thermo Fisher Scientific, Waltham, MA). P. aeruginosa and E. faecium were cultured on tryptic soy agar (TSA) as described above and then inoculated into tryptic soy broth (TSB) to an OD of 0.1. 490nm The cultures were then diluted with their respective media as described above, and 200 μL of this suspension was used to inoculate each well of an 8-well chambered cover glass slide. After incubating each bacterial species for 16 hours at 37°C with 5% CO2, the media was replaced with their respective fresh media and incubated for an additional 8 hours. At 24 hours, the media was replaced with their respective fresh media (control) or mB Box-97syn The medium was replaced with fresh medium containing (1.2 μg / 200 μL), A Box or (1.2 μg / 200 μL), or HuTipMab (5.0 μg / 200 μL) and incubated at 37°C with 5% CO for 2 hours. All biofilms were then washed twice with 1x Dulbecco's phosphate-buffered saline (DPBS) without calcium or magnesium (Corning, Corning, NY) and stained with LIVE / DEAD stain (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. The biofilms were washed again and then fixed with 1.6% paraformaldehyde, 0.025% glutaraldehyde, and 4% acetic acid in 0.1 M phosphate buffer, pH 7.4. Biofilms were imaged with a 63x objective on a Zeiss 800 confocal laser scanning microscope (CLSM; Zeiss) and analyzed with COMSTAT2. Biomass value (μm 3 / μm 2 ) were calculated by COMSTAT2 and represent the mean ± SEM of three biological replicates.

[0334] Prevention of bacterial biofilm formation. NTHI and S. aureus were cultured and resuspended as above, then diluted in their respective media to approximately 5 × 10 3 CFU / mL or approximately 1 x 10 3 CFU / mL, and 200 μL of this suspension was inoculated into each well of an 8-well chambered cover glass slide. UPEC strains UTI89, B. cenocepacia, K. pneumoniae, Enterobacter spp., and A. baumanii were cultured and resuspended as described above, after which approximately 1 × 10 3 -1×10 5 P. aeruginosa and E. faecium were cultured and suspended as described above, and then diluted in LB broth to contain approximately 1 × 10 CFU / mL. 200 μL of this suspension was inoculated into each well of an 8-well chambered cover glass slide. 3The suspension was diluted to contain CFU / mL, and 200 μL of this suspension was inoculated into each well of an 8-well chambered cover glass slide. After 16 hours of incubation at 37°C and 5% CO, the HMGB1 isoforms rHMGB1 (1.2 μg / 200 μL), A Box (1.2 μg / 200 μL), and mB Box-97 were added. rec (1.2 or 1.8 μg / 200 μL), mB Box-97 syn (1.2 or 1.8 μg / 200 μL) or HuTipMab (5.0 or 7.5 μg / 200 μL) was added to each well for all pathogens tested. After incubation, all biofilms were washed once with DPBS and fixed as described above. Biofilms were imaged and analyzed as described above. All assays were repeated a minimum of three times on separate days. Data are presented as mean + / - SEM. The HuTipMab antibody is as described in U.S. Pat. No. 11,104,723 and comprises a heavy chain complementarity determining region 1 (CDRH1) comprising the sequence GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 9); a heavy chain complementarity determining region 2 (CDRH2) comprising the sequence GSDRRH (aa 76 to aa 81 of SEQ ID NO: 9); a heavy chain complementarity determining region 3 (CDRH3) comprising the sequence VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 9); a light chain complementarity determining region 1 (CDRL1) comprising the sequence QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 15); a light chain complementarity determining region 2 (CDRL2) comprising the sequence LVS (aa 75 to aa 77 of SEQ ID NO: 15); and a light chain complementarity determining region 3 (CDRL3) comprising the sequence WQGTHFP (aa 114 to aa 120 of SEQ ID NO: 15).

[0335] Determination of synergy between mB Box-97 and HuTipMab. NTHI and S. aureus were cultured on chocolate agar as described, then resuspended and diluted in their respective media to yield approximately 5 × 10 3 or about 1 x 10 3CFU / mL, and 200 μL of this suspension was inoculated into each well of an 8-well chambered cover glass slide. mB Box-97 and HuTipMab were added to these bacterial solutions to obtain concentrations of 0.05–1.8 and 0.23–7.5 μg / 200 μL, respectively. After 16 h of incubation at 37°C and 5% CO2, biofilms were washed once with sterile saline and fixed, imaged, and analyzed as described above. All in vitro biofilm assays were repeated a minimum of three times on separate days. Data are presented as mean + / - SEM.

[0336] Statistical Analysis. For all in vitro assays, graph results and statistical tests were performed using GraphPad Prism 9. Statistical significance of in vitro assays was assessed by one-way ANOVA with multiple comparisons. A P value of less than 0.05 was considered significant.

[0337] Example 3: H-NS neutralizes neutrophil extracellular traps H-NS is released into the bulk medium as the biofilm matures H-NS is a B-DNA-binding protein found on the outside of bacterial cells within biofilms. Depletion of H-NS within biofilms did not affect biomass or biofilm thickness, indicating that H-NS is not required for biofilm structure like DNABII proteins. Measuring the ratio of H-NS fluorescence intensity relative to cellular fluorescence intensity revealed an increase from 24 to 40 hours in UPEC, NTHI, and S. pneumoniae biofilms; these levels then declined at 72 hours and continued to decrease as biofilms reached 1 week of age. In S. pneumoniae biofilms, the FI ratio of H-NS to cells at 1 week was too low to quantify (Figures 13A-B).

[0338] To confirm that H-NS was leaving the biofilm and entering the bulk medium, the concentrations of H-NS in both the biofilm and the medium were measured using Western blot. Comparison of 16-hour- and 1-week-old biofilms revealed a shift of the majority of the H-NS present in the biofilm to the medium, indicating that H-NS levels were decreasing within the biofilm as it matured (Figure 14).

[0339] To further confirm that levels of H-NS were reduced in biofilms due to entry into the bulk medium and not due to changes in gene expression, RT-PCR was performed.

[0340] H-NS prevents neutrophil DNA release and condenses previously released NET eDNA In vivo, NET eDNA is released by neutrophils upon stimulation by pathogens, but NETosis can also be stimulated in vitro using PMA. The strands of DNA exiting the cell result from the unwinding of nuclear DNA when histones are citrullinated and can be visualized using confocal microscopy. Considering that both H-NS and CbpA bind to curved DNA, but CbpA does not condense DNA, we utilized CbpA, a DnaJ homolog that preferentially binds to curved DNA, as a negative control. H-NS prevented the release of neutrophil DNA, but CbpA did not (Figure 15A-B). When neutrophils were stimulated using PMA and formed NETs, ​​H-NS was also able to compact neutrophil eDNA in the case of preformed NETs. Further staining of the antimicrobial agent bound to NET eDNA in addition to eDNA indicated that the antimicrobial agent was still bound to eDNA and was not displaced by H-NS.

[0341] H-NS prevents bacterial NET killing While NETs have limited ability to invade and kill bacteria within biofilms, they can kill free bacteria and inhibit biofilm growth. Because NETs rely on their released DNA tentacles to kill bacteria, and because H-NS causes these tentacles to condense, H-NS potentially prevented bacterial NET killing in addition to altering NET morphology. HU has been shown to convert NET eDNA to Z-DNA and prevent NET killing, and was therefore used as a positive control and comparison to determine the extent to which H-NS could prevent bacterial NET killing. Similar to HU, H-NS significantly reduced the amount of bacterial killing caused by NETs (Figure 16). CbpA was unable to prevent NET-induced killing of free bacteria, correlating with its inability to alter NET morphology and prevent NETosis. The ability of H-NS to prevent NET clearance of free bacteria confirms that by condensing NET eDNA, H-NS may act as a defense on behalf of the biofilm against the host immune system.

[0342] material and method Bacterial strains and plasmids. NTHI strain 86-028NP, a clinical isolate recovered from the nasopharynx of a child undergoing tympanostomy tube insertion, was streaked onto chocolate agar and grown overnight in an incubator at 37°C with 5% CO2. Uropathogenic E. coli (UPEC) (SG1019), a clinical isolate from a febrile UTI, renal abscess, was streaked and incubated overnight at 37°C with 5% CO2. Streptococcus pneumoniae 1121 (SG1241) was streaked onto blood agar and incubated overnight at 37°C with 5% CO2.

[0343] Expression and Purification of NAPs. The expression and purification of NTHI H-NS, NTHI CbpA, and NTHI HU have been previously described. Briefly, H-NS, CbpA, and HU were all cloned into the pTXB1 vector and transformed into the ER2566 E. coli expression strain. Growth occurred in LB containing 100 μg / mL ampicillin, and then protein overexpression was induced using 100 mM IPTG. Purification of each protein was performed on a chitin resin column, followed by further purification using FPLC on a HiTrap Heparin HP column (GE Healthcare).

[0344] Quantification of H-NS using immunofluorescence. Polyclonal anti-H-NS purified from rabbit serum was previously produced against recombinant NTHI H-NS and used to label H-NS in NTHI, S. pneumoniae, and UPEC biofilms of various ages. All biofilms were initiated in 8-well glass-bottom chamber slides, and the medium was aspirated and then fresh medium was added every 8 and 16 hours, except for the 1-week-old biofilms, in which the medium was changed every 12 hours after 5 days. NTHI colonies were resuspended in brain heart infusion (sBHI) broth (BD Diagnostic Systems) supplemented with 2 μg / mL β-NAD (NAD+) and 2 μg / mL heme, UPEC in LB, and S. pneumoniae in Todd-Hewitt broth (THB) supplemented with 0.2% yeast extract (BD Diagnostic Systems). All resuspensions were diluted to 2 × 10 5Biofilms were added to chamber slides at a concentration of 1000 cells / mL. Biofilms were grown for 24, 40, 72, and 1 week. Once the biofilms reached the desired age, they were washed twice with phosphate-buffered saline (PBS) and rabbit anti-H-NS (diluted 1:200 in 5% bovine serum albumin (BSA)) was added for 2 hours. After 2 hours of incubation at room temperature, the wells were washed once with PBS, and goat anti-rabbit Alexa Fluor 488 and FM 4-64 (both diluted 1:200 in 5% BSA in PBS) were added. The biofilms were then incubated in the dark for 1 hour at room temperature. The biofilms were then washed once more with PBS and visualized under a Zeiss 800 Light Scanning Microscope (LSM). The mean fluorescence intensity for both H-NS and cells was determined using ImageJ software.

[0345] Visualization of NETs. NETs were isolated from freshly collected blood from healthy donors using the EasySep™ Human Neutrophil Isolation Kit from StemCell Technologies (Cambridge, MA). Isolated neutrophils were quantified, and 200,000 cells were added to each well of an 8-well chamber slide. Neutrophils were then allowed to adhere to the bottom of the well for 30 minutes while incubating at 37°C and 5% CO2. After incubation, 100 nM phorbol 12-myristate 13-acetate (PMA) was added to the neutrophils to induce NETosis. To determine whether H-NS could prevent NETosis, 200 nM NTHI H-NS was added simultaneously with PMA. To determine whether H-NS could condense previously released NET eDNA, H-NS was added 16 hours after NETosis had been induced. After PMA addition, neutrophils were incubated for 3.5 hours at 37°C and 5% CO2. Neutrophils were then fixed with 0.4% formalin and 0.1% Triton® X-100 was added for cell permeabilization. Cells were then blocked with 5% normal goat serum for 30 minutes at 37°C and 5% CO2, after which primary antibodies were added overnight and incubated at 4°C. Primary antibodies utilized α-neutrophil elastase rabbit, α-citrullinated H3 rabbit, and α-DNA mouse antibodies, diluted 1:500 in PBS. The following day, secondary antibodies were added for 1 hour at 37°C and 5% CO2. Slides were then visualized using a Zeiss 800 confocal laser scanning microscope.

[0346] Quantification of NET killing. NET killing was initially measured at 2 × 10 5 The number of NTHI cells was determined by seeding 16-h NTHI biofilms at 2 × 10 cells / mL. Human neutrophils were then isolated from freshly collected human blood from healthy donors using the StemCell EasySep Neutrophil Isolation Kit. The 16-h NTHI biofilms were washed twice with PBS and then plated at 2 × 10 cells / mL. 5Neutrophils were added together with the following proteins: 1 μM H-NS, 1 μM HU, or 1 μM CbpA. To one biofilm, no protein was added, and to the other, no protein or neutrophils were added. The biofilms, proteins (if applicable), and neutrophils (if applicable) were then incubated at 37°C for 3 hours. After the 3-hour incubation, 0.1% Triton® X-100 was added for 5 minutes, and each sample was then serially diluted, plated on chocolate agar, and placed in a 37°C incubator containing 5% CO2. Colony-forming units (CFUs) were then counted after 16 hours, and the percentage of bacteria killed was calculated compared to a control without added neutrophils.

[0347] RT-PCR to determine H-NS expression levels. NTHI biofilms were cultured in BioLite 25 cm tissue flasks (ThermoFisher) at an initial concentration of 2 × 10 in 6 mL of sBHI medium. 5The cells / mL were grown for 16 hours and 1 week. Prior to RNA isolation, all surfaces and pipettes were cleaned with RNAaseZap™ (ThermoFisher). The medium was removed by inverting the flask and pouring it off. The biofilm was then resuspended in PBS and centrifuged at 4000 rpm for 5 minutes at 4°C to pellet the bacteria. The bacterial pellet was then resuspended in 0.75 mL of Trizol™ (ThermoFisher) for 0.25 mL samples, and the homogenized bacteria were incubated in Trizol™ for 5 minutes at room temperature. After 5 minutes, 200 μL of chloroform was added per mL of Trizol™, and the tube containing all reagents and bacteria was vigorously shaken for 15 seconds. After shaking, the tube was incubated at room temperature for 12 minutes, after which the tube was centrifuged at 4000 rpm for 5 minutes at 4°C. The upper clear phase formed after centrifugation was then removed and placed in a new 1.5 mL capped centrifuge tube. RNA purification was then performed using the Qiagen RNeasy mini kit (Qiagen, Germantown, MD). On-column DNAase I digestion (Sigma-Aldrich, St. Louis, MO) was used during isolation to remove residual DNA.

[0348] equivalent While the present disclosure has been described in conjunction with the above embodiments, it should be understood that the above descriptions and examples are intended to be illustrative and not limiting of the scope of the present disclosure. Other aspects, advantages and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which this disclosure pertains.

[0349] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All nucleotide sequences provided herein are presented in the 5' to 3' direction.

[0350] The embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, or limitation or limitations, not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," and "containing" are to be read expansively and without limitation. Furthermore, the terms and expressions used herein are used as terms of description rather than limitation, and there is no intention in the use of such terms and expressions to exclude equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present disclosure.

[0351] Thus, while the present disclosure has been specifically disclosed by certain embodiments and optional features, it should be understood that alterations, improvements, and variations of the embodiments disclosed herein may occur to those skilled in the art, and that such alterations, improvements, and variations are deemed to be within the scope of the present disclosure. The materials, methods, and examples provided herein are representative of particular embodiments and are illustrative and are not intended as limitations on the scope of the disclosure.

[0352] The scope of the present disclosure is described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of this disclosure. This includes general descriptions with a condition or negative limitation that removes any subject matter from the genus, regardless of whether the excluded material is specifically set forth herein.

[0353] Furthermore, when features or aspects of the present disclosure are described in terms of a Markush group, those skilled in the art will recognize that embodiments of the present disclosure may also be described thereby in terms of any individual member or subgroup of members of the Markush group.

[0354] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0355] Non-limiting embodiments of the present disclosure Embodiment 1. A synthetic polypeptide comprising mB Box-97, consisting of amino acids 90-176 from the coding sequence of the native human HMGB1 protein shown as SEQ ID NO:2, or an equivalent thereof, with a cysteine ​​to serine point mutation at amino acid 106.

[0356] Embodiment 2. A recombinant polypeptide comprising mB Box-97, consisting of amino acids 90-176 from the coding sequence of the native human HMGB1 protein shown as SEQ ID NO:2, or an equivalent thereof, having a cysteine ​​to serine point mutation at amino acid 106.

[0357] Embodiment 3. The synthetic or recombinant polypeptide of embodiment 1 or embodiment 2, consisting of SEQ ID NO:5.

[0358] Embodiment 4. A synthetic polypeptide comprising mB Box-97, consisting of amino acids 80-176 from the coding sequence of the native human HMGB1 protein shown as SEQ ID NO:2, or an equivalent thereof, with a cysteine ​​to serine point mutation at amino acid 106.

[0359] Embodiment 5. A recombinant polypeptide comprising mB Box-97, consisting of amino acids 80 to 176 from the coding sequence of the native human HMGB1 protein shown as SEQ ID NO: 2, or an equivalent thereof, having a cysteine ​​to serine point mutation at amino acid 106.

[0360] Embodiment 6. The synthetic or recombinant polypeptide of embodiment 4 or embodiment 5, consisting of SEQ ID NO:6.

[0361] Embodiment 7. The synthetic or recombinant polypeptide of any one of embodiments 1-5, wherein the equivalent comprises an amino acid sequence having at least about 80% homology or amino acid identity thereto, or an amino acid encoded by a polynucleotide that hybridizes to a polynucleotide encoding the amino acid sequence or its complement under high stringency conditions, wherein the high stringency conditions comprise an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1x SSC to about 0.1x SSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1x SSC, 0.1x SSC, or deionized water, wherein the equivalent of the cysteine ​​to serine point mutation at amino acid 106 of SEQ ID NO:2 is a serine to alanine mutation at amino acid 106 of SEQ ID NO:2.

[0362] Embodiment 8. A synthetic or recombinant polypeptide according to any one of embodiments 1 to 7, and a detectable label.

[0363] Embodiment 9. A synthetic or recombinant polypeptide according to any one of embodiments 1 to 8, and a linker polypeptide, wherein the linker polypeptide optionally comprises GPSLKL (SEQ ID NO: 3) or PPKGETKKKF (SEQ ID NO: 4).

[0364] Embodiment 10. A plurality of synthetic or recombinant polypeptides according to any one of embodiments 1 to 9, whereby the members of the plurality are the same or different from one another, as appropriate.

[0365] Embodiment 11. A composition comprising a plurality of synthetic or recombinant polypeptides according to any one of embodiments 1 to 9 or embodiment 10, and a carrier, optionally a pharmaceutically acceptable carrier.

[0366] Embodiment 12. An isolated polynucleotide encoding the synthetic or recombinant polypeptide of any one of embodiments 1 to 9, and optionally a carrier or pharmaceutically acceptable carrier.

[0367] Embodiment 13. An isolated polynucleotide and a detectable label according to embodiment 12, and optionally a carrier or a pharmaceutically acceptable carrier.

[0368] Embodiment 14. A vector comprising the isolated polynucleotide of embodiment 12 or embodiment 13, and optionally a carrier or a pharmaceutically acceptable carrier.

[0369] Embodiment 15. The isolated polynucleotide of embodiment 12 or embodiment 13, or the vector of embodiment 14, further comprising a heterologous promoter sequence, and optionally a carrier or a pharmaceutically acceptable carrier.

[0370] Embodiment 16. An isolated host cell comprising one or more of the synthetic or recombinant polypeptides of any one of embodiments 1 to 9, a plurality of synthetic or recombinant polypeptides of embodiment 10, an isolated polynucleotide of any one of embodiments 12 or 13, or a vector of embodiment 14, and optionally a carrier or a pharmaceutically acceptable carrier.

[0371] Embodiment 17. The isolated host cell of embodiment 16, wherein the host cell is a prokaryotic or eukaryotic cell.

[0372] Embodiment 18. The isolated host cell of embodiment 17, wherein the host cell is a eukaryotic cell, optionally wherein the eukaryotic cell is a mammalian cell.

[0373] Embodiment 19. A method for treating a subject in need thereof, comprising administering to the subject an effective amount of one or more of: (i) a synthetic or recombinant polypeptide of any one of embodiments 1 to 9; (ii) a plurality of synthetic or recombinant polypeptides of embodiment 10; (iii) a composition of embodiment 11; (iv) an isolated polynucleotide of embodiment 12 or embodiment 13; or (iv) a vector of embodiment 14.

[0374] Embodiment 20. A method for treating or preventing aberrant or excessive neutrophil extracellular trap (NET) formation in a subject in need thereof, or for preventing a neutrophil extracellular trap (NET)-mediated disease in a subject in need thereof, or for preventing the progression of a NET-mediated disease in a subject in need thereof, comprising administering to the subject an effective amount of one or more of: (i) a synthetic or recombinant polypeptide of any one of embodiments 1 to 9; (ii) a plurality of synthetic or recombinant polypeptides of embodiment 10; (iii) a composition of embodiment 11; (iv) an isolated polynucleotide of embodiment 12 or embodiment 13; or (iv) a vector of embodiment 14.

[0375] Embodiment 21. The method of embodiment 19 or 20, wherein the subject is suffering from one or more of: a pulmonary disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-associated acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, or small vessel vasculitis; an autoinflammatory disease selected from gout or inflammatory bowel disease, and / or a metabolic disease selected from type 2 diabetes or obesity, and wherein optionally the effective amount is between 50 nM and 2 μM.

[0376] Embodiment 22. The method of embodiment 20, wherein the NET-mediated disease comprises a pulmonary disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-associated acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, or small vessel vasculitis; an autoinflammatory disease selected from gout or inflammatory bowel disease, or a metabolic disease selected from type 2 diabetes or obesity.

[0377] Embodiment 23. A method for preventing or treating a bacterial biofilm in a subject in need thereof, comprising administering to the subject an effective amount of one or more of: (i) a synthetic or recombinant polypeptide of any one of embodiments 1-9; (ii) a plurality of synthetic or recombinant polypeptides of embodiment 10; (iii) a composition of embodiment 11; (iv) an isolated polynucleotide of embodiment 12 or embodiment 13; or (iv) a vector of embodiment 14, wherein optionally the effective amount is between 50 nM and 2 μM.

[0378] Embodiment 24. The method of any one of embodiments 19 to 23, further comprising administering to the subject an antibody or fragment thereof, wherein the antibody or fragment thereof binds to the tip region of the DNABII peptide.

[0379] Embodiment 25. The antibody or fragment thereof A heavy chain (HC) immunoglobulin variable domain sequence comprising the sequence of amino acids (aa) 25 to aa 144 of SEQ ID NO: 21, or an equivalent thereof; and a light chain (LC) immunoglobulin variable domain sequence comprising the sequence from aa21 to aa132 of SEQ ID NO: 22, or an equivalent thereof; contains, or The antibody or fragment thereof A heavy chain (HC) immunoglobulin variable domain sequence comprising the sequence from aa25 to aa144 of SEQ ID NO: 24, or an equivalent thereof; and A light chain (LC) immunoglobulin variable domain sequence comprising the sequence of aa21 to aa132 of SEQ ID NO: 25 or an equivalent thereof. 25. The method of embodiment 24, comprising:

[0380] Embodiment 26. The method of embodiment 24, wherein the antibody or fragment thereof is: heavy chain complementarity-determining region 1 (CDRH1) comprising the sequence of GFTFRTY (aa50 to aa56 of SEQ ID NO: 9, 10, 11, or 24); heavy chain complementarity-determining region 2 (CDRH2) comprising the sequence of GSDRRH (aa76 to aa81 of SEQ ID NO: 9, 10, 11, or 24); heavy chain complementarity-determining region 3 (CDRH3) comprising the sequence VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 9, 10, 11, or 24); light chain complementarity-determining region 1 (CDRL1) comprising the sequence QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 15, 16, 17, or 25); Light chain complementarity-determining region 2 (CDRL2) comprising the sequence of LVS (aa75 to aa77 of SEQ ID NO: 15, 16, 17, or 25); and Light chain complementarity-determining region 3 (CDRL3) comprising the sequence of WQGTHFP (aa114 to aa120 of SEQ ID NO: 15, 16, 17, or 25) The method includes:

[0381] Embodiment 27. A method for condensing eDNA strands of neutrophil extracellular traps (NETs), comprising contacting the NETs with an effective amount of a DNA-binding agent.

[0382] Embodiment 28. A method of preventing or neutralizing eDNA formation in neutrophil extracellular traps (NETs), comprising contacting the NETs with an effective amount of a DNA-binding agent.

[0383] Embodiment 29. A method of preventing neutrophil extracellular trap (NET) formation or inducing regression of existing NETs, ​​comprising contacting the NETs with an effective amount of a DNA-binding agent.

[0384] Embodiment 30. The method of any one of embodiments 27 to 29, wherein the DNA binding agent is an agent that aggregates or condenses DNA, and optionally the effective amount is between 50 nM and 2 μM.

[0385] Embodiment 31. The method of any one of embodiments 27 to 30, wherein the DNA binding agent comprises a histone-like nucleoid structuring protein (H-NS), a polyamine, or a polycation.

[0386] Embodiment 32. The method of embodiment 31, wherein the H-NS is derived from a Gram-negative or Gram-positive bacterium.

[0387] Embodiment 33. The method of embodiment 32, wherein the H-NS is derived from a bacterium of the genus Escherichia, Haemophilus, Streptococcus, Mycobacteria, Klebsiella, or Pseudomonas; optionally, the H-NS is derived from Escherichia coli (E. coli), nontypeable Haemophilus influenzae (NTHI), S. pneumoniae, K. pneumoniae, Mycobacterium tuberculosis, or Pseudomonas aeruginosa.

[0388] Embodiment 34. The method of any one of embodiments 27 to 33, wherein H-NS comprises an amino acid sequence having at least 60% identity to an amino acid sequence selected from SEQ ID NOs: 29 to 34.

[0389] Embodiment 35. The method of any one of embodiments 27 to 34, wherein the contacting is in vitro or in vivo.

[0390] Embodiment 36. A method of halting damaging coagulation in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a DNA binding agent.

[0391] Embodiment 37. A method of stopping excessive inflammation in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of a DNA binding agent, wherein optionally the effective amount is between 50 nM and 2 μM.

[0392] Embodiment 38. A method for preventing, treating, or preventing the progression of a neutrophil extracellular trap (NET)-mediated disease in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of a DNA binding agent, wherein optionally the effective amount is between 50 nM and 2 μM.

[0393] Embodiment 39. The method of any one of embodiments 36 to 38, wherein the DNA binding agent is an agent that aggregates or condenses DNA.

[0394] Embodiment 40. The method of any one of embodiments 36 to 39, wherein the DNA binding agent comprises a histone-like nucleoid structuring protein (H-NS).

[0395] Embodiment 41 The method of embodiment 40, wherein the H-NS is derived from a Gram-negative or Gram-positive bacterium.

[0396] Embodiment 42. The method of embodiment 41, wherein the H-NS is derived from a bacterium of the genus Escherichia, Haemophilus, Streptococcus, Mycobacteria, Klebsiella, or Pseudomonas; optionally, the H-NS is derived from Escherichia coli (E. coli), nontypeable Haemophilus influenzae (NTHI), S. pneumoniae, K. pneumoniae, Mycobacterium tuberculosis, or Pseudomonas aeruginosa.

[0397] Embodiment 43. The method of any one of embodiments 40 to 42, wherein H-NS comprises an amino acid sequence having at least 60% identity to an amino acid sequence selected from SEQ ID NOs: 29 to 34.

[0398] Embodiment 44. The method of any one of embodiments 36 to 43, wherein the subject is a mammal or a human patient.

[0399] Embodiment 45. The method of any one of embodiments 36 to 44, wherein the subject is suffering from one or more of the following: a pulmonary disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-associated acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, or small vessel vasculitis; an autoinflammatory disease selected from gout or inflammatory bowel disease, and / or a metabolic disease selected from type 2 diabetes or obesity. (Sequence Listing) SEQ ID NO: 1 Mus musculus wild-type HMGB1 protein [ka] SEQ ID NO: 2 Homo sapiens wild-type HMGB1 protein [ka] (Human, reproduced from GenBank accession number CAE48262.1) HMGB1 is a small 215-amino acid protein (approximately 30 Kda) composed of three domains: two positively charged domains, A and B Boxes (each encompassing 80 amino acids), and a negatively charged carboxyl terminus (an acidic C-tail consisting of approximately 30 consecutive aspartic acid and glutamic acid residues). Bolded amino acids (amino acids 1-70) indicate the A Box domain. Italicized amino acids (approximately amino acids 88-164) indicate the B Box domain. Underlined amino acids (amino acids 186-215) indicate the C-tail domain. SEQ ID NO: 3, artificial sequence, linker sequence [ka] SEQ ID NO: 4, artificial sequence, linker sequence [ka] SEQ ID NO: 5, human, mB Box-97 peptide [ka] SEQ ID NO: 6, human, mB Box-97 peptide with C-terminal linker sequence [ka] SEQ ID NO: 7, human IhfA, A tip fragment [ka] SEQ ID NO: 8, human, IhfB, B tip fragment [ka] SEQ ID NO: 9 (H10210 (1F8.F1 humanized HC1)) Bold font indicates exemplary variable regions, and bold, italic and underlined font indicates exemplary CDRs. [ka] SEQ ID NO: 10 (H10211 (1F8.F1 humanized HC2)) Bold font indicates exemplary variable regions, and bold, italic and underlined font indicates exemplary CDRs. [ka] [ka] SEQ ID NO: 11 (H10212 (1F8.F1 humanized HC3)) Bold font indicates exemplary variable regions, and bold, italic and underlined font indicates exemplary CDRs. [ka] SEQ ID NO: 12 (H10213 (11E7.C7 humanized HC1)) Bold font indicates exemplary variable regions, and bold, italic and underlined font indicates exemplary CDRs. [ka] SEQ ID NO: 13 (H10214 (11E7.C7 humanized HC2)) Bold font indicates exemplary variable regions, and bold, italic and underlined font indicates exemplary CDRs. [ka] SEQ ID NO: 14 (H10215 (11E7.C7 humanized HC3)) Bold font indicates exemplary variable regions, and bold, italic and underlined font indicates exemplary CDRs. [ka] SEQ ID NO: 15 (L10210 (1F8.F1 humanized LC1)) Bold font indicates exemplary variable regions, and bold, italic and underlined font indicates exemplary CDRs. [ka] SEQ ID NO: 16 (L10211 (1F8.F1 humanized LC2)) Bold font indicates exemplary variable regions, and bold, italic and underlined font indicates exemplary CDRs. [ka] SEQ ID NO: 17 (L10212 (1F8.F1 humanized LC3)) Bold font indicates exemplary variable regions, and bold, italic and underlined font indicates exemplary CDRs. [ka] SEQ ID NO: 18 (L10213 (11E7.C7 humanized LC1)) Bold font indicates exemplary variable regions, and bold, italic and underlined font indicates exemplary CDRs. [ka] [ka] SEQ ID NO: 19 (L10214 (11E7.C7 humanized LC2)) Bold font indicates exemplary variable regions, and bold, italic and underlined font indicates exemplary CDRs. [ka] SEQ ID NO: 20 (L10215 (11E7.C7 humanized LC3)) Bold font indicates exemplary variable regions, and bold, italic and underlined font indicates exemplary CDRs. [ka] SEQ ID NO: 21 (heavy chain consensus sequence) [ka] [ka] wherein X and a lowercase letter may be substituted at the corresponding position with any amino acid, or alternatively with an amino acid selected from SEQ ID NOs: 9 to 14. In one embodiment, X may also represent the absence of an amino acid residue. SEQ ID NO: 22 (light chain consensus sequence) [ka] In the formula, X and a lowercase letter may be substituted at the corresponding position with any amino acid, or alternatively with an amino acid selected from SEQ ID NOs: 15 to 20. In one embodiment, X may also indicate the absence of an amino acid residue. SEQ ID NO: 23 human IgG1 constant region, Uniprot: P01857 [ka] SEQ ID NO: 24 (top heavy chain consensus sequence) [ka] [ka] In the formula, lowercase letters may be substituted with an amino acid selected from SEQ ID NOs: 9 to 11 at the corresponding position. SEQ ID NO: 25 (apical light chain consensus sequence) [ka] In the formula, lowercase letters may be substituted at the corresponding position with an amino acid selected from SEQ ID NOs: 15 to 17. SEQ ID NO: 26 (Tip-chimeric peptide IhfA5-mIhfB4NTHI) [ka] wherein "X" is an optional amino acid linker sequence, optionally comprising, consisting essentially of, or even consisting of between 1 and 20 amino acids. wherein "X1" is any amino acid, or alternatively "X1" is selected from the amino acids Q, R, K, S, or T. SEQ ID NO: 27 (Tip-chimeric peptide IhfA5-mIhfB4NTHI) [ka] wherein "X" is an optional amino acid linker sequence, optionally containing between 1 and 20 amino acids. SEQ ID NO: 28 (Tip-chimeric peptide IhfA5-mIhfB4NTHI) [ka] SEQ ID NO: 29, E. coli, H-NS protein [ka] SEQ ID NO: 30, NTHI, H-NS protein [ka] SEQ ID NO: 31, Mycobacterium tuberculosis, H-NS [ka] SEQ ID NO: 32, Streptococcus pneumoniae H-NS protein [ka] SEQ ID NO: 33, Klebsiella pneumoniae H-NS protein [ka] SEQ ID NO: 34, Pseudomonas aeruginosa H-NS protein [ka] SEQ ID NO: 35 Human IgD constant region, Uniprot:P01880 [ka] SEQ ID NO: 36 SEQ ID NO: 36 Human IgG2 constant region, Uniprot: P01859 [ka] SEQ ID NO: 37 Human IgG3 constant region, Uniprot: P01860 [ka] [ka] SEQ ID NO: 38 Human IgM constant region, Uniprot:P01871 [ka] SEQ ID NO: 39 Human IgG4 constant region, Uniprot: P01861 [ka] SEQ ID NO: 40 Human IgA1 constant region, Uniprot: P01876 [ka] SEQ ID NO: 41 Human IgA2 constant region, Uniprot: P01877 [ka] SEQ ID NO: 42 Human Ig kappa constant region, Uniprot: P01834 [ka] (Item 1) A synthetic polypeptide comprising mB Box-97, or its equivalent, consisting of amino acids 90 to 176 from the coding sequence of the native human HMGB1 protein shown as SEQ ID NO: 2, with a point mutation from cysteine ​​to serine at amino acid 106. (Item 2) A recombinant polypeptide comprising mB Box-97, or an equivalent thereof, consisting of amino acids 90 to 176 from the coding sequence of the native human HMGB1 protein shown as SEQ ID NO: 2, with a point mutation from cysteine ​​to serine at amino acid 106. (Item 3) 3. The synthetic or recombinant polypeptide according to item 1 or 2, consisting of SEQ ID NO:5. (Item 4) A synthetic polypeptide comprising mB Box-97, or its equivalent, consisting of amino acids 80 to 176 from the coding sequence of the native human HMGB1 protein shown as SEQ ID NO: 2, with a point mutation from cysteine ​​to serine at amino acid 106. (Item 5) A recombinant polypeptide comprising mB Box-97, or an equivalent thereof, consisting of amino acids 80 to 176 from the coding sequence of the native human HMGB1 protein shown as SEQ ID NO: 2, with a point mutation from cysteine ​​to serine at amino acid 106. (Item 6) 6. The synthetic or recombinant polypeptide according to item 4 or 5, consisting of SEQ ID NO:6. (Item 7) 6. The synthetic or recombinant polypeptide of any one of Items 1 to 5, wherein the equivalent comprises an amino acid sequence having at least about 80% homology or amino acid identity thereto, or an amino acid sequence encoded by a polynucleotide that hybridizes to a polynucleotide encoding said amino acid sequence or its complement under high stringency conditions, wherein high stringency conditions comprise an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1xSSC to about 0.1xSSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1xSSC, 0.1xSSC, or deionized water, wherein the equivalent of the cysteine ​​to serine point mutation at amino acid 106 of SEQ ID NO:2 is a serine to alanine mutation at amino acid 106 of SEQ ID NO:2. (Item 8) 8. A synthetic or recombinant polypeptide according to any one of items 1 to 7, and a detectable label. (Item 9) 9. The synthetic or recombinant polypeptide and a linker polypeptide according to any one of items 1 to 8, wherein the linker polypeptide optionally comprises GPSLKL (SEQ ID NO: 3) or PPKGETKKKF (SEQ ID NO: 4). (Item 10) 10. A plurality of synthetic or recombinant polypeptides according to any one of items 1 to 9, optionally wherein the members of the plurality are the same or different from one another. (Item 11) A composition comprising the synthetic or recombinant polypeptide according to any one of items 1 to 9 or a plurality of synthetic or recombinant polypeptides according to item 10, and a carrier, optionally a pharmaceutically acceptable carrier. (Item 12) 10. An isolated polynucleotide encoding the synthetic or recombinant polypeptide according to any one of items 1 to 9, and optionally a carrier or a pharmaceutically acceptable carrier. (Item 13) 13. An isolated polynucleotide according to item 12 and a detectable label, and optionally a carrier or a pharmaceutically acceptable carrier. (Item 14) 14. A vector comprising the isolated polynucleotide of item 12 or 13, and optionally a carrier or a pharmaceutically acceptable carrier. (Item 15) 15. The isolated polynucleotide of item 12 or item 13, or the vector of item 14, further comprising a heterologous promoter sequence, and optionally a carrier or a pharmaceutically acceptable carrier. (Item 16) 15. An isolated host cell comprising one or more of the synthetic or recombinant polypeptides of any one of Items 1 to 9, the plurality of synthetic or recombinant polypeptides of Item 10, the isolated polynucleotide of any one of Items 12 or 13, or the vector of Item 14, and optionally a carrier or a pharmaceutically acceptable carrier. (Item 17) 17. The isolated host cell of item 16, wherein the host cell is a prokaryotic or eukaryotic cell. (Item 18) 18. The isolated host cell of item 17, wherein the host cell is a eukaryotic cell, optionally wherein the eukaryotic cell is a mammalian cell. (Item 19) 10. A method for treating a subject in need thereof, comprising administering to the subject an effective amount of one or more of: (i) the synthetic or recombinant polypeptide of any one of items 1 to 9; (ii) a plurality of the synthetic or recombinant polypeptides of item 10; (iii) the composition of item 11; (iv) the isolated polynucleotide of item 12 or item 13; or (iv) the vector of item 14. (Item 20) 10. A method for treating or preventing aberrant or excessive neutrophil extracellular trap (NET) formation in a subject in need thereof, or for preventing a neutrophil extracellular trap (NET)-mediated disease in a subject in need thereof, or for preventing the progression of a NET-mediated disease in a subject in need thereof, comprising administering to the subject an effective amount of one or more of: (i) the synthetic or recombinant polypeptide of any one of items 1-9; (ii) a plurality of the synthetic or recombinant polypeptides of item 10; (iii) the composition of item 11; (iv) the isolated polynucleotide of item 12 or item 13; or (iv) the vector of item 14. (Item 21) 21. The method of claim 19 or 20, wherein the subject is suffering from one or more of: a pulmonary disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-associated acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, or small vessel vasculitis; an autoinflammatory disease selected from gout or inflammatory bowel disease, and / or a metabolic disease selected from type 2 diabetes or obesity, and optionally the effective amount is between 50 nM and 2 μM. (Item 22) 21. The method of item 20, wherein the NET-mediated disease is a pulmonary disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-associated acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, or small vessel vasculitis; an autoinflammatory disease selected from gout or inflammatory bowel disease, or a metabolic disease selected from type 2 diabetes or obesity. (Item 23) 10. A method for preventing or treating a bacterial biofilm in a subject in need thereof, comprising administering to the subject an effective amount of one or more of: (i) the synthetic or recombinant polypeptide of any one of items 1-9; (ii) a plurality of the synthetic or recombinant polypeptides of item 10; (iii) the composition of item 11; (iv) the isolated polynucleotide of item 12 or item 13; or (iv) the vector of item 14; wherein optionally the effective amount is between 50 nM and 2 μM. (Item 24) 24. The method according to any one of items 19 to 23, further comprising administering to the subject an antibody or a fragment thereof, wherein the antibody or a fragment thereof binds to the tip region of the DNABII peptide. (Item 25) the antibody or fragment thereof (v) a heavy chain (HC) immunoglobulin variable domain sequence comprising the sequence of amino acids (aa) 25 to aa 144 of SEQ ID NO: 21, or an equivalent thereof; and (vi) a light chain (LC) immunoglobulin variable domain sequence comprising the sequence from aa21 to aa132 of SEQ ID NO: 22, or an equivalent thereof; contains, or the antibody or fragment thereof (v) a heavy chain (HC) immunoglobulin variable domain sequence comprising the sequence from aa25 to aa144 of SEQ ID NO: 24, or an equivalent thereof; and (vi) a light chain (LC) immunoglobulin variable domain sequence comprising the sequence of aa21 to aa132 of SEQ ID NO: 25 or an equivalent thereof Item 25. The method according to Item 24, comprising: (Item 26) the antibody or fragment thereof (i) heavy chain complementarity-determining region 1 (CDRH1) comprising the sequence of GFTFRTY (aa50 to aa56 of SEQ ID NO: 9, 10, 11, or 24); (ii) heavy chain complementarity-determining region 2 (CDRH2) comprising the sequence of GSDRRH (aa76 to aa81 of SEQ ID NO: 9, 10, 11, or 24); (iii) heavy chain complementarity-determining region 3 (CDRH3) comprising the sequence VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 9, 10, 11, or 24); (iv) light chain complementarity-determining region 1 (CDRL1) comprising the sequence QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 15, 16, 17, or 25); (v) light chain complementarity-determining region 2 (CDRL2) comprising the sequence of LVS (aa75 to aa77 of SEQ ID NO: 15, 16, 17, or 25); and (vi) Light chain complementarity-determining region 3 (CDRL3) comprising the sequence of WQGTHFP (aa114 to aa120 of SEQ ID NO: 15, 16, 17, or 25) Item 25. The method according to Item 24, comprising: (Item 27) A method for condensing DNA strands of neutrophil extracellular traps (NETs), comprising contacting the NETs with an effective amount of a DNA-binding agent. (Item 28) A method for preventing or neutralizing eDNA formation in neutrophil extracellular traps (NETs), comprising contacting the NETs with an effective amount of a DNA-binding agent. (Item 29) A method for preventing neutrophil extracellular trap (NET) formation or inducing regression of existing NETs, ​​comprising contacting the NETs with an effective amount of a DNA binding agent. (Item 30) 30. The method according to any one of items 27 to 29, wherein the DNA binding agent is an agent that aggregates or condenses DNA, and optionally the effective amount is between 50 nM and 2 μM. (Item 31) 31. The method of any one of items 27 to 30, wherein the DNA binding agent comprises a histone-like nucleoid structuring protein (H-NS), a polyamine, or a polycation. (Item 32) 32. The method of claim 31, wherein the H-NS is derived from a gram-negative or gram-positive bacterium. (Item 33) 33. The method of claim 32, wherein the H-NS is derived from a bacterium of the genus Escherichia, Haemophilus, Streptococcus, Mycobacteria, Klebsiella, or Pseudomonas; optionally, the H-NS is derived from Escherichia coli (E. coli), nontypeable Haemophilus influenzae (NTHI), S. pneumoniae, K. pneumoniae, Mycobacterium tuberculosis, or Pseudomonas aeruginosa. (Item 34) 34. The method according to any one of Items 27 to 33, wherein the H-NS comprises an amino acid sequence having at least 60% identity to an amino acid sequence selected from SEQ ID NOs: 29 to 34. (Item 35) 35. The method of any one of items 27 to 34, wherein the contacting is in vitro or in vivo. (Item 36) A method for halting damaging coagulation in a subject in need thereof, comprising administering to said subject in need thereof an effective amount of a DNA binding agent. (Item 37) 1. A method for stopping excessive inflammation in a subject in need thereof, comprising administering to said subject in need thereof an effective amount of a DNA binding agent, wherein optionally said effective amount is between 50 nM and 2 μM. (Item 38) 1. A method for preventing, treating, or preventing the progression of a neutrophil extracellular trap (NET)-mediated disease in a subject in need thereof, comprising administering to said subject in need thereof an effective amount of a DNA binding agent, wherein optionally said effective amount is between 50 nM and 2 μM. (Item 39) 39. The method according to any one of items 36 to 38, wherein the DNA binding agent is an agent that aggregates or condenses DNA. (Item 40) 40. The method of any one of items 36 to 39, wherein the DNA binding agent comprises a histone-like nucleoid structuring protein (H-NS). (Item 41) 41. The method of item 40, wherein the H-NS is derived from a gram-negative or gram-positive bacterium. (Item 42) 42. The method of claim 41, wherein the H-NS is derived from a bacterium of the genus Escherichia, Haemophilus, Streptococcus, Mycobacteria, Klebsiella, or Pseudomonas; optionally, the H-NS is derived from Escherichia coli (E. coli), nontypeable Haemophilus influenzae (NTHI), S. pneumoniae, K. pneumoniae, Mycobacterium tuberculosis, or Pseudomonas aeruginosa. (Item 43) 43. The method according to any one of Items 40 to 42, wherein the H-NS comprises an amino acid sequence having at least 60% identity to an amino acid sequence selected from SEQ ID NOs: 29 to 34. (Item 44) 44. The method of any one of items 36 to 43, wherein the subject is a mammal or a human patient. (Item 45) 45. The method of any one of items 36 to 44, wherein the subject is suffering from one or more of: a pulmonary disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-associated acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, or small vessel vasculitis; an autoinflammatory disease selected from gout or inflammatory bowel disease, and / or a metabolic disease selected from type 2 diabetes or obesity.

Claims

1. A synthetic polypeptide comprising mB Box-97, or an equivalent thereof, consisting of amino acids 90 to 176 from the coding sequence of the native human HMGB1 protein shown as SEQ ID NO: 2, which has a point mutation from cysteine ​​to serine at amino acid 106, and which does not have any post-translational modifications.

2. The synthetic polypeptide of claim 1 consisting of SEQ ID NO:

5.

3. A synthetic polypeptide comprising mB Box-97, or an equivalent thereof, consisting of amino acids 80 to 176 from the coding sequence of the native human HMGB1 protein shown as SEQ ID NO: 2, which has a point mutation from cysteine ​​to serine at amino acid 106, and which does not have any post-translational modifications.

4. The synthetic polypeptide of claim 3 consisting of SEQ ID NO:

6.

5. 4. The synthetic polypeptide of any one of claims 1-3, wherein an equivalent comprises an amino acid sequence having at least about 80% homology or amino acid identity thereto, or an amino acid encoded by a polynucleotide that hybridizes to a polynucleotide encoding said amino acid sequence or its complement under high stringency conditions, wherein high stringency conditions comprise an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1x SSC to about 0.1x SSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1x SSC, 0.1x SSC, or deionized water, wherein the equivalent of the cysteine ​​to serine point mutation at amino acid 106 of SEQ ID NO:2 is a serine to alanine mutation at amino acid 106 of SEQ ID NO:

2.

6. A synthetic polypeptide according to any one of claims 1 to 5 and a detectable label.

7. 7. A synthetic polypeptide and a linker polypeptide according to any one of claims 1 to 6, wherein the linker polypeptide optionally comprises GPSLKL (SEQ ID NO: 3) or PPKGETKKKF (SEQ ID NO: 4).

8. A plurality of synthetic polypeptides according to any one of claims 1 to 7, optionally wherein members of said plurality are the same or different from one another.

9. A composition comprising a synthetic polypeptide according to any one of claims 1 to 7 or a plurality of synthetic polypeptides according to claim 8, and a carrier, optionally a pharmaceutically acceptable carrier.

10. An isolated polynucleotide encoding the synthetic polypeptide of any one of claims 1 to 7, and optionally a carrier or a pharmaceutically acceptable carrier.

11. 11. The isolated polynucleotide of claim 10 and a detectable label, and optionally a carrier or a pharmaceutically acceptable carrier.

12. 12. A vector comprising the isolated polynucleotide of claim 10 or 11, and optionally a carrier or a pharmaceutically acceptable carrier.

13. 13. The isolated polynucleotide of claim 10 or claim 11, or the vector of claim 12, further comprising a heterologous promoter sequence, and optionally a carrier or a pharmaceutically acceptable carrier.

14. 13. An isolated host cell comprising one or more of the synthetic polypeptide of any one of claims 1 to 7, the plurality of synthetic polypeptides of claim 8, the isolated polynucleotide of any one of claims 10 or 11, or the vector of claim 12, and optionally a carrier or a pharmaceutically acceptable carrier.

15. 15. The isolated host cell of claim 14, wherein the host cell is a prokaryotic or eukaryotic cell.

16. 16. The isolated host cell of claim 15, wherein the host cell is a eukaryotic cell, optionally wherein the eukaryotic cell is a mammalian cell.

17. 13. A method for treating a subject in need thereof, comprising administering to the subject an effective amount of one or more of: (i) the synthetic polypeptide of any one of claims 1-7; (ii) a plurality of the synthetic polypeptides of claim 8; (iii) the composition of claim 9; (iv) the isolated polynucleotide of claim 10 or claim 11; or (iv) the vector of claim 12.

18. 13. A method for treating or preventing aberrant or excessive neutrophil extracellular trap (NET) formation in a subject in need thereof, or for preventing a neutrophil extracellular trap (NET)-mediated disease in a subject in need thereof, or for preventing the progression of a NET-mediated disease in a subject in need thereof, comprising administering to the subject an effective amount of one or more of: (i) the synthetic polypeptide of any one of claims 1-7; (ii) a plurality of the synthetic polypeptides of claim 8; (iii) the composition of claim 9; (iv) the isolated polynucleotide of claim 10 or claim 11; or (iv) the vector of claim 12.

19. 19. The method of claim 17 or 18, wherein the subject is suffering from one or more of: a pulmonary disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-associated acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, or small vessel vasculitis; an autoinflammatory disease selected from gout or inflammatory bowel disease, and / or a metabolic disease selected from type 2 diabetes or obesity, and optionally the effective amount is between 50 nM and 2 μM.

20. 19. The method of claim 18, wherein the NET-mediated disease comprises a pulmonary disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-associated acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, or small vessel vasculitis; an autoinflammatory disease selected from gout or inflammatory bowel disease, or a metabolic disease selected from type 2 diabetes or obesity.

21. 13. A method for preventing or treating a bacterial biofilm in a subject in need thereof, comprising administering to the subject an effective amount of one or more of: (i) the synthetic polypeptide of any one of claims 1-7; (ii) a plurality of synthetic polypeptides of claim 8; (iii) the composition of claim 9; (iv) the isolated polynucleotide of claim 10 or claim 11; or (iv) the vector of claim 12; wherein optionally the effective amount is between 50 nM and 2 μM.

22. 22. The method of any one of claims 17 to 21, further comprising administering to the subject an antibody or fragment thereof, wherein the antibody or fragment thereof binds to the tip region of the DNABII peptide.

23. the antibody or fragment thereof (i) a heavy chain (HC) immunoglobulin variable domain sequence comprising the sequence of amino acids (aa) 25 to aa 144 of SEQ ID NO: 21, or an equivalent thereof; and (ii) a light chain (LC) immunoglobulin variable domain sequence comprising the sequence from aa21 to aa132 of SEQ ID NO: 22, or an equivalent thereof; contains, or the antibody or fragment thereof (i) a heavy chain (HC) immunoglobulin variable domain sequence comprising the sequence of aa25 to aa144 of SEQ ID NO: 24, or an equivalent thereof; and (ii) a light chain (LC) immunoglobulin variable domain sequence comprising the sequence of aa21 to aa132 of SEQ ID NO: 25 or an equivalent thereof 23. The method of claim 22, comprising:

24. the antibody or fragment thereof (i) a heavy chain complementarity-determining region 1 (CDRH1) comprising the sequence GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 9, 10, 11, or 24); (ii) a heavy chain complementarity-determining region 2 (CDRH2) comprising the sequence of GSDRRH (aa76 to aa81 of SEQ ID NO: 9, 10, 11, or 24); (iii) a heavy chain complementarity-determining region 3 (CDRH3) comprising the sequence VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 9, 10, 11, or 24); (iv) a light chain complementarity-determining region 1 (CDRL1) comprising the sequence QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 15, 16, 17, or 25); (v) a light chain complementarity-determining region 2 (CDRL2) comprising the sequence of LVS (aa75 to aa77 of SEQ ID NO: 15, 16, 17, or 25); and (vi) light chain complementarity-determining region 3 (CDRL3) comprising the sequence of WQGTHFP (aa114 to aa120 of SEQ ID NO: 15, 16, 17, or 25); 23. The method of claim 22, comprising:

25. A method for condensing the eDNA strands of neutrophil extracellular traps (NETs), comprising contacting the NETs with an effective amount of a DNA-binding agent.

26. A method for preventing or neutralizing eDNA formation in neutrophil extracellular traps (NETs), comprising contacting the NETs with an effective amount of a DNA-binding agent.

27. A method for preventing neutrophil extracellular trap (NET) formation or inducing regression of existing NETs, ​​comprising contacting the NETs with an effective amount of a DNA binding agent.

28. 28. The method of any one of claims 25 to 27, wherein the DNA binding agent is an agent that aggregates or condenses DNA, and optionally the effective amount is between 50 nM and 2 μM.

29. The method of any one of claims 25 to 28, wherein the DNA binding agent comprises a histone-like nucleoid structuring protein (H-NS), a polyamine, or a polycation.

30. 30. The method of claim 29, wherein the H-NS is derived from a gram-negative or gram-positive bacterium.

31. 31. The method of claim 30, wherein the H-NS is derived from a bacterium of the genus Escherichia, Haemophilus, Streptococcus, Mycobacteria, Klebsiella, or Pseudomonas; optionally, the H-NS is derived from Escherichia coli (E. coli), non-typeable Haemophilus influenzae (NTHI), S. pneumoniae, K. pneumoniae, Mycobacterium tuberculosis, or Pseudomonas aeruginosa.

32. 32. The method of any one of claims 25 to 31, wherein the H-NS comprises an amino acid sequence having at least 60% identity to an amino acid sequence selected from SEQ ID NOs: 29 to 34.

33. The method of any one of claims 25 to 32, wherein the contacting is in vitro or in vivo.

34. A method for halting damaging coagulation in a subject in need thereof, comprising administering to said subject in need thereof an effective amount of a DNA binding agent.

35. 1. A method for stopping excessive inflammation in a subject in need thereof, comprising administering to said subject in need thereof an effective amount of a DNA binding agent, wherein optionally said effective amount is between 50 nM and 2 μM.

36. 1. A method for preventing, treating, or preventing the progression of a neutrophil extracellular trap (NET)-mediated disease in a subject in need thereof, comprising administering to said subject in need thereof an effective amount of a DNA binding agent, wherein optionally said effective amount is between 50 nM and 2 μM.

37. The method of any one of claims 34 to 36, wherein the DNA binding agent is an agent that aggregates or condenses DNA.

38. 38. The method of any one of claims 34 to 37, wherein the DNA binding agent comprises a histone-like nucleoid structuring protein (H-NS).

39. 39. The method of claim 38, wherein the H-NS is derived from a gram-negative or gram-positive bacterium.

40. 40. The method of claim 39, wherein the H-NS is derived from a bacterium of the genus Escherichia, Haemophilus, Streptococcus, Mycobacteria, Klebsiella, or Pseudomonas; optionally, the H-NS is derived from Escherichia coli (E. coli), nontypeable Haemophilus influenzae (NTHI), S. pneumoniae, K. pneumoniae, Mycobacterium tuberculosis, or Pseudomonas aeruginosa.

41. 41. The method of any one of claims 38 to 40, wherein the H-NS comprises an amino acid sequence having at least 60% identity to an amino acid sequence selected from SEQ ID NOs: 29 to 34.

42. 42. The method of any one of claims 34 to 41, wherein the subject is a mammal or a human patient.

43. 43. The method of any one of claims 34 to 42, wherein the subject is suffering from one or more of: a pulmonary disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-associated acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, or small vessel vasculitis; an autoinflammatory disease selected from gout or inflammatory bowel disease, and / or a metabolic disease selected from type 2 diabetes or obesity.

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