Compositions and methods for treating biofilm and neutrophil extracellular trap formation

Abstract

Provided herein is a synthetic polypeptide derived from a high mobility group cassette 1 (HMGB1) host protein that can both disrupt bacterial biofilm and prevent the formation of neutrophil extracellular traps (NETs). Also provided herein are methods of disrupting abnormal or excessive NET formation, which are particularly suitable for treating high risk populations, the composition can be used for people suffering from infection of SARS CoV-2, septicemia, autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, type I diabetes mellitus and small vasculitis), autoinflammatory diseases (such as gout and inflammatory bowel disease) and metabolic diseases (such as type 2 diabetes mellitus and obesity).

Description

Cross - reference to Related Patent Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 424,851, filed on November 11, 2022, and U.S. Provisional Application No. 63 / 424,888, filed on November 12, 2022, under 35 U.S.C.§119(e), the entire contents of which are incorporated herein by reference. Government Sponsorship Statement

[0002] This invention was made with government support under AI155501 and DC011818 awarded by the National Institutes of Health. The government has certain rights in the invention. Background of the Invention

[0003] 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 that PMNs can use is a process called NETosis to create elaborate structures of released extracellular DNA (eDNA) (neutrophil extracellular traps or NETs), which first entrap individual bacteria, sequester bacterial communities (biofilms), and use deployed eDNA tendrils to localize and focus NET - associated antimicrobial substances towards the entrapped microbes, thereby restricting pathogen proliferation.

[0004] Despite this crucial host innate immune function, widespread release of NETs can occur during the attempt of NETs to clear bacterial and viral pathogens, including pathological thrombi that can lead to morbidity or even death. In addition, NETs can induce anti - inflammatory immune responses (such as autoimmune diseases). Therefore, there is a need to develop a method to prevent or inactivate the eDNA structures of NETs to prevent or stop these potentially destructive coagulation or excessive inflammation events. This disclosure meets this need and provides related advantages. Summary of the Invention

[0005] The Applicant has found that a polypeptide derived from the host protein High Mobility Group Box 1 (HMGB1) can both disrupt bacterial biofilms and prevent the formation of neutrophil extracellular traps (NETs). This polypeptide, which the Applicant has designated "mB Box-97", consists essentially of, or consists of, amino acids 80 to 176 in the coding sequence of the native human HMGB1 protein, with a cysteine to serine point mutation at amino acid 106, which eliminates the ability of this polypeptide to induce an inflammatory response. It is known that the host variably modifies native HMGB1 by post-translational modification, and these changes have been shown to affect the function of HMGB1. Similarly, when recombinantly expressed by bacteria, mB Box-97 should also be post-translationally modified. To this end, the Applicant synthesized mB Box-97 and found that both recombinant and synthetic mB Box-97 have indistinguishable biofilm-disrupting and NET-inhibiting activities. Therefore, synthetic mB Box-97 can be synthesized for therapeutic use, improving the yield and quality control of recombinant mB Box-97 and mutant wild-type polypeptides without loss of therapeutic activity. Therefore, the synthetically produced mb Box-97 with a cysteine to serine amino acid point mutation is referred to herein as "synthetic mb Box-97 or smB Box-97".

[0006] The Applicant's disclosure also addresses problems associated with abnormal 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 I diabetes, small vessel vasculitis), autoinflammatory diseases (such as gout, 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 diseases or prevent the progression of NET-mediated diseases. Secondly, synthetic mB Box-97 can be used to prevent or treat bacterial biofilms because the mechanism of action of this peptide is different from that of other known similar peptides.

[0007] In another aspect of the present disclosure, the applicant has found that DNA-binding proteins that aggregate DNA can also condense the eDNA tendrils of NETs, preventing NET formation or inducing the contraction of existing NETs. For example, the homologous bacterial histone-like nucleoid structuring protein (H-NS) family, known to act as nucleoid-associated proteins intracellularly, has an additional hitherto unknown role extracellularly. These H-NS proteins, released from bacterial biofilms, can not only prevent NET formation but also disrupt existing NETs. While the ability to bridge adjacent DNA duplexes is a known function of H-NS, it has not been clear whether this level of DNA aggregation / condensation is also a property. Thus, any reagent that causes DNA aggregation / condensation can also be used to limit conditions that counter effective NETosis, such as those associated with SARS CoV-2 infection.

[0008] There are currently no FDA-approved drugs for the prevention or treatment of pathologic NETosis. There are some therapeutic approaches that can prevent NETosis, inactivate NETs, or promote the clearance of NETs (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. Almost all approaches to treating NETs fail to compensate for the loss of beneficial functions. However, DNA aggregating agents can be used to treat existing NETs, so that only NET pathology is affected.

[0009] The present disclosure addresses problems associated with abnormal 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 I diabetes, small-vessel vasculitis), autoinflammatory diseases (such as gout, 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 prevent the progression of NET-mediated diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1A - 1C. mB Box-97 (also known as mB Box-97 peptide) inhibits phorbol 12-myristate 13-acetate (PMA)-induced NETosis. (A) Graphic representation of recombinant or synthetic full-length HMGB1, ABox (amino acids 1 to 89), ABBox (amino acids 1-176), BBox-97 (amino acids 80 to 176), recombinant and synthetic mB Box-97 (identical to BBox-97 except for a single amino acid change C106S), and BBox-87 (amino acids 90 to 176) polypeptides. The specific cysteine (C) in mB Box-97 C and its respective amino acid number are shown mutated to serine (S) at position 106 at the corresponding position on HMGB1. Recombinant-produced HMGB1 polypeptides (such as full-length HMGB1, ABox, ABBox, BBox-97, mB Box-97, BBox-87) may have post-translational modifications, while synthetic HMGB1 polypeptides do not have any post-translational modifications. (B) Fluorescence quantitative studies showed that mB Box-97, BBox-87, and ABox did not induce NETosis. Net was induced in isolated neutrophils (5*10 3 ) (200 nM, 4 hours) using each construct, and the released DNA was quantified using a fluorometer with the cell-impermeable dye SYTOX Green (1 μM). Among all the tested constructs, mB Box-97, BBox-87, and ABox failed to induce NET formation in isolated human neutrophils. (C) Quantitative determination of the percentage of NETs in total cells under different treatment conditions showed that only mB Box-97 had a significant inhibitory effect on PMA-induced NETosis. NETs formed were quantified using ImageJ, and the percentage of NET formation relative to total neutrophils in each image was calculated. Analyses of 4 different experiments are shown, and the data are presented as mean ± standard deviation (SD). *P < 0.05, evaluated by unpaired t-test. mB Box-97 was the only HMGB1 construct that showed inhibition of PMA-induced NETosis.

[0011] Figures 2A-2F. Ca 2+ Mediates NETosis through a different pathway from PMA or LPS, so the applicant studied the effect of mBBox-97 on Ca 2+ -mediated NET formation. Neutrophils were allowed to form NETs in the presence or absence of mB Box-97 or BBox-97 (200 nM) in Ca 2+NETs were formed for 6 - 8 h in the presence of ionophore A23187 (500 nM). DNA, plasma membrane, NE or MPO were detected as described in "Materials and Methods" and NETs were observed by CLSM at 63-fold magnification. (A) Changes in mean fluorescence intensity (MFI) of NET-related proteins after PMA-stimulated NETosis. (B) Changes in mean fluorescence intensity (MFI) of NET-related proteins after NTHI-stimulated NETosis. (C) Changes in mean fluorescence intensity (MFI) of NET-related proteins after ionophore-stimulated NETosis. (D) mB Box-97 inhibits the secretion of NET-related proteins by inhibiting PMA- or NTHI-induced NETosis. (E) Secreted neutrophil elastase (NE) measurement. (F) DNA-bound NE measurement.

[0012] Figure 3A - 3B . Effects of mB Box-97 on reactive oxygen species (ROS) production and p47 phox phosphorylation. (A) Effects of mB Box-97 on ROS generation. Neutrophils pre-incubated with luminol (400 μM) were incubated with PMA, PMA with or without protein, protein or buffer alone, and luminescence was detected every 5 min for 2 h over time to measure the ROS produced. N = 3, bar graphs represent SEM. *P < 0.007 or lower, evaluated by multiple unpaired t-tests. (B) Effects of mBBox-97 on p47 phox phosphorylation. Neutrophils (1×10 7 cells / ml) were incubated with buffer, 200 nM PMA or PMA (with or without mB Box-97 or BBox-97 (1 μM)) for 30 min and lysed, proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Proteins were detected using immunoblotting, phosphorylation at Ser-370 was detected using anti-phospho-p47 phox antibody, or anti-p47 phox or GAPDH. Protein blots from different experiments were scanned; phosphorylation and total p47 phox and GAPDH were quantified by densitometry; after correcting for the amount of GAPDH, the intensity of phosphorylated p47 phox was corrected according to the amount of p47 phox . Results are expressed as mean ± standard deviation (n = 4). ****P < 0.0001, evaluated by unpaired t-tests. After inducing NETosis using PMA, mB Box-97 significantly inhibited ROS production and phosphorylation of the key NOX protein p47 phox in human neutrophils.

[0013] Figure 4A - 4B 。(A) Neutrophil-mediated killing was inactivated by mB Box-97. Human neutrophils (10 6 (B+N) were used to attack NTHI biofilms for 16 hours, and then treated with 1 μM recombinant DNABII protein HU NTHI (B+N+HU) as a positive control, 1 μM mB Box-97 (B+N+mB Box-97), or 1 μM BBox-97 (B+N+BBox-97) for 4 hours. NTHI biofilms without neutrophils were used as a control. Compared with the biofilm control, bacteria attacked by neutrophils (B+N) (average = 50.79%) and the group treated with BBox-97 (B+N+BBox-97) (average = 40.86%) showed an increase in the relative killing percentage. Neutrophils treated with HU NTHI (B+N+HU) (average = 4.83%) and mB Box-97 (B+N+mB Box-97) (average = 13.09%) showed no difference in the relative killing percentage of bacteria caused by neutrophils. The results indicate that mB Box-97 treatment inactivated neutrophil-mediated bacterial killing. The graphs represent data from 6 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, which was comparable to the inhibitory effect of the DNABII protein HU from NTHI NTHI . (B) Measurement of ROS production. Neutrophils pre-loaded with luminol were incubated with PMA, PMA with or without the corresponding protein, or alone with buffer, and the ROS luminescence was detected at 5-minute intervals for 2 hours over time.

[0014] Figure 5 . Interaction and inhibition of PKC by mB Box-97. mB Box-97 inhibited PKC activity. According to the manufacturer's instructions, a PKC activity assay kit was used to test the activity of PKC in the presence of histone H1, mB Box-97, and BBox-97 at two different concentrations (200 and 1000 nM). mB Box-97 showed an inhibitory effect on PKC activity as the concentration increased.

[0015] Figure 6 . Graphic representation of the potential mechanism of action of mB Box-97. mB Box-97 interacts with PKC, resulting in the inhibition of its activity. This inhibition leads to p47 phoxInhibition of phosphorylation, thereby inhibiting the active assembly of NOX, thereby reducing the production of ROS. The reduction of ROS leads to a decrease in the release of NE and MPO, ultimately resulting in the inhibition of NET formation. mB Box-97 may also partially inhibit Ca 2+ -induced NETosis because Ca2+ also affects PKC activity.

[0016] Figure 7A - 7B . (A) The biofilm-disrupting ability of HMGB1-derived peptides. (B) mB Box-97 syn significantly disrupts four additional high-priority ESKAPEE pathogens (abbreviations for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli), regardless of how long the biofilm has formed.

[0017] Figure 8A - 8B . mB Box-97 can prevent pulmonary infection. Adult C57BL / 6 mice were intratracheally injected with (A) 10 7 or (B) 10 8 CFU of Burkholderia cenocepacia (B.cenocepacia, Bc), along with 200 nM of mB Box-97 or a negative control.

[0018] Figure 9A - 9B . (A) mB Box 97 or HuTipMab (an antibody described in Kurbatfinski, N. et al. Antimicrobial Agents and Chemotherapy 66.3 (2022): e01877-21 and WO2021007260A2) significantly inhibited the biofilm growth of NTHI and Staphylococcus aureus compared to the negative control. (B) Compared to the negative control group, mB Box-97 (P = 0.03 - P < 0.0001) or HuTipMab (P = 0.02 - P < 0.0001) could prevent biofilm growth after 16 hours of incubation with Pseudomonas aeruginosa (Pa), Enterobacter spp. (Esp), Enterococcus faecalis (Ef), uropathogenic Escherichia coli (UPEC), Acinetobacter baumannii (Ab), or Burkholderia cenocepacia (Bc).

[0019] Figure 10A - 10B 。mB Box 97 or HuTipMab showed preventive activity against the pathogen Klebsiella pneumoniae. (A) Biomass measurements and (B) corresponding representative confocal scanning laser microscopy (CSLM) images.

[0020] Figure 11A - 11B 。mB Box-97 and HuTipMab showed a synergistic effect in inhibiting biofilm formation. (A) Prevention of NTHI biofilm by dilutions of mB Box-97, HuTipMab, or both. (B) Prevention of Staphylococcus aureus biofilm by dilutions of mB Box-97 or HuTipMab.

[0021] Figure 12 。The figure shows that H-NS can prevent neutrophil extracellular traps from killing bacteria.

[0022] Figure 13A - 13B 。As biofilms matured, the levels of H-NS protein within NTHI, Streptococcus pneumoniae, and UPEC biofilms decreased. (A) Representative confocal images of H-NS (gray) in Streptococcus pneumoniae, NTHI, and UPEC biofilms, with formation times ranging from 24 hours to 1 week. (B) Ratio of fluorescence intensity of H-NS to cells in NTHI, UPEC, and Streptococcus pneumoniae in 24-, 40-, 72-hour, and 1-week-old biofilms. There was an increase from 24 hours to 40 hours, after which the ratio of fluorescence intensity decreased.

[0023] Figure 14 。H-NS left the biofilm and entered the bulk culture medium. The concentration of H-NS was quantified using Western Blot relative to the CFU within the biofilm. Concentrations were found in the supernatant of the biofilm and within the biofilm at 16 hours and 1 week. The concentration of H-NS in the supernatant increased significantly from 16 hours to 1 week, but a decrease in H-NS was observed within the biofilm.

[0024] Figure 15A - 15B 。H-NS could both prevent PMA-induced NET formation and induce the condensation of eDNA in NET expansion. (A) Human neutrophils were induced with PMA for 3.5 hours in the presence or absence of 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-B-DNA, and α-NE antibodies and visualized by IF CLSM. H-NS could both prevent PMA-induced NET formation (A) and induce the condensation of eDNA in NET expansion (B).

[0025] Figure 16. H-NS, similar to HU, blocked NET killing of NTHI bacteria. NTHI biofilms formed for 16 hours were incubated with human neutrophils for 3 hours, and the percentage of bacteria killed was found to be relative to the CFU present in NTHI without added neutrophils. The three proteins added to the neutrophil-NTHI biofilms were HU, CbpA, and H-NS. Prevention of NET-mediated bacterial killing was achieved by the presence of HU and H-NS. N = 5, P < 0.05. Detailed Description

[0026] 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods, devices, and materials are now described. All technical and patent publications cited herein are hereby incorporated by reference in their entirety. Nothing herein shall be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.

[0027] Unless otherwise noted, the practice of the present disclosure will employ conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, for example, Sambrook and Russel eds. (2001) Molecular Cloning: A Laboratory Manual, 3 rd edition; Ausubel et al. eds. (2007) Current Protocols in Molecular Biology series; Methods in Enzymology (Academic Press, Inc., N.Y.) series; MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5 thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology.

[0028] All numerical values (including ranges), for example pH, temperature, time, concentration, and molecular mass, are approximations that vary by increments of plus or minus (+) or (-) 1.0 or 0.1, as appropriate, or vary by plus or minus 15%, or 10%, or 5%, or 2%. It should be understood that, although not always explicitly stated, all numerical values are preceded by the term “about”. It should also be understood that, although not always explicitly stated, the reagents described herein are merely exemplary and that their equivalents are known in the art.

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

[0030] As used herein, the term "comprising" or "including" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean excluding other elements that are of any substantial significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein will not exclude trace contaminants from separation and purification methods and pharmaceutically acceptable carriers (e.g., phosphate buffered saline, preservatives, etc.). "Consisting of" means excluding other ingredients and trace elements of substantial method steps for administering the compositions disclosed herein. Embodiments defined by each of these transitional terms are within the scope of the present invention.

[0031] As used herein, the term "comprising" or "including" is intended to mean that the peptides described herein include the recited amino acid sequences, but do not exclude other amino acids. When used to define a sequence, "consisting essentially of" means the core sequence, optionally surrounded by additional amino acids. Thus, a peptide consisting essentially of the sequence defined herein will not exclude additional amino acids at the C- or N-terminus. In some embodiments, a peptide consisting essentially of the sequence defined herein includes up to ten additional amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) at the N-terminus. In some embodiments, a peptide consisting essentially of the sequence defined herein includes up to ten additional amino acids at the C-terminus. In some embodiments, a peptide consisting essentially of the sequence defined herein includes up to ten additional amino acids at the N-terminus and up to ten additional amino acids at the C-terminus. When used with respect to a peptide, "consisting of a sequence" means that the peptide does not contain any other sequences. Embodiments defined by each of these transitional terms are within the scope of the present invention.

[0032] "Biofilm" means a thin or organized microbial community that can sometimes adhere to the surface of a structure, which can be organic or inorganic, or can be concentrated at an interface (usually solid / liquid), surrounded by an extracellular polymeric mucus matrix that contains polymers secreted and / or released by the microorganisms, such as DNA. Biofilms can contain many different types of microorganisms, such as bacteria, archaea, protozoa, fungi, and algae. Biofilms are highly resistant to microorganisms and antimicrobial agents. They live on gingival tissues, teeth, and restorations, causing dental caries and periodontal diseases (also known as periodontal plaque diseases). They also cause chronic middle ear infections. Biofilms can also form on the surfaces of dental implants, stents, catheters, and contact lenses. They grow on pacemakers, heart valve replacements, artificial joints, and other surgical implants. The Centers for Disease Control estimates that more than 65% of hospital (nosocomial) infections are caused by biofilms. Fungal biofilms also frequently contaminate medical devices. They cause chronic vaginal infections and life-threatening systemic infections in people with a weakened immune system. Biofilms are also involved in a variety of diseases. For example, patients with cystic fibrosis suffer from Pseudomonas infections, which often lead to the formation of antibiotic-resistant biofilms.

[0033] As used herein, "treatment of biofilm-related diseases", also referred to herein as "prevention or treatment of bacterial biofilms", refers to a reduction in the severity and / or duration of the disease and / or a reduction in the severity and / or duration of the symptoms of the disease, particularly the symptoms of the infection. In some embodiments, the treatment results in the restoration of the health of the individual. Preferably, the individual has milder disease symptoms or for a shorter duration. The 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 the formation or growth of biofilms in vitro, ex vivo, or in vivo. As used herein, "prevent or reduce biofilm formation or growth" means to prevent, delay, or reduce the formation or growth of biofilms in vitro, ex vivo, or in vivo. Those skilled in the art will understand that this reduction in biofilm formation or growth can slow down the growth of the biofilm compared to the growth of an untreated biofilm.

[0035] In some embodiments, the compositions disclosed herein can be used to degrade or reduce biofilms. As used herein, "degrade or reduce biofilms" means to partially or completely eliminate biofilms in vitro, ex vivo, or in vivo. Those skilled in the art will understand that after such treatment, planktonic bacteria (i.e., free-living bacteria suspended in a liquid) may still be present.

[0036] "DNABII polypeptide or protein" means a DNA-binding protein or polypeptide consisting of a DNA-binding domain and thus having specific or general affinity for DNA. In one aspect, they bind 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 strain U93. 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] The "tip fragment" of a DNABII polypeptide means a DNABII polypeptide which, taking IhfA and IhfB as examples, forms the two arms of the protein. Non-limiting examples of such include the IhfA, A tip fragment: NFELRDKSSRPGRNPKTGDVV, SEQ ID NO:7, and the IhfB, B tip fragment: SLHHRQPRLGRNPKTGDSVNL, SEQ ID NO:8.

[0038] As used herein, the term "tip chimeric peptide" or "IhfA5-mIhfB4 NTHI tip chimera" or "tip chimera" refers to a chimera of the IhfA5-mIhfB4 NTHI peptide which comprises or consists essentially of, or consists of, the polypeptide sequence of RPGRNPX1TGDVVPVSARRVV-X-FSLHHRQPRLGRNPX1TGDSV (SEQ ID NO:38), wherein "X" is an optional amino acid linker sequence optionally comprising 1 to 20 amino acids, or consists essentially of, or consists of, the same; and wherein "X1" is any amino acid, or "X1" is selected from the amino acids Q, R, K, S or T. In one aspect, "X1" is K or Q. In another embodiment, the tip chimeric peptide IhfA5-mIhfB4 NTHI comprises the polypeptide sequence of RPGRNPKTGDVVPVSARRVV-X-FSLHHRQPRLGRNPKTGDSV (SEQ ID NO:39), or consists essentially of, or consists of, the same, wherein "X" is an optional amino acid linker sequence optionally comprising 1 to 20 amino acids, or consists essentially of, or consists of, the same. In another embodiment, the tip chimeric peptide IhfA5-mIhfB4 NTHIA polypeptide sequence comprising RPGRNPKTGDVVPVSARRVVGPSLFSLHHRQPRLGRNPKTGDSV (SEQ ID NO:40), or consisting essentially of or consisting of the same.

[0039] In certain embodiments, the tail chimeric peptide IhfA3-IhfB2 NTHI A polypeptide sequence comprising FLEEIRLSLESGQDVKLSGF-X-TLSAKEIENMVKDILEFISQ (SEQ ID NO:41), or consisting essentially of or consisting of the same, wherein "X" is an optional amino acid linker sequence, optionally comprising 1 to 20 amino acids, or consisting essentially of or consisting of the same. In certain embodiments, the linker is selected from any one or more of SEQ ID NO:42-49. In one embodiment, the tail chimeric peptide IhfA3-IhfB2 NTHI Comprising FLEEIRLSLESGQDVKLSGFGPSLTLSAKEIENMVKDILEFISQ (SEQ ID NO:50), or consisting essentially of or consisting of the same.

[0040] The "integration host factor" or "IHF" protein is a bacterial protein that is used by bacteriophages to incorporate their DNA into the host bacterium. These are DNA-binding proteins that play a role in genetic recombination as well as transcriptional and translational regulation. They also bind extracellular microbial DNA. The genes encoding the IHF protein subunits in Escherichia coli are the himA (Genbank accession number: POA6X7.1) and himD (POA6Y1.1) genes.

[0041] "HMGB1" is the high-mobility group box 1 protein, which is reported to bind and distort the minor groove of DNA and is an example of an interferent. 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 of Escherichia coli strain U93" refers to a class of heterodimeric proteins that are commonly associated with Escherichia coli. The HU protein is known to bind DNA convergence points. Related proteins have been isolated from other microorganisms. Laine et al. (1980) Eur. J. Biochem 103(3) 447-481 reported the complete amino acid sequence of Escherichia coli HU. Antibodies to the HU protein are commercially available from Abeam.

[0043] "Linker" or "peptide linker" refers to a peptide sequence that is linked to the N-terminus or C-terminus of a polypeptide sequence. On the one hand, the linker has a length of about 1 to about 20 amino acid residues, or is 2 to about 10, about 3 to about 5 amino acid residues long. Examples of peptide linkers are Gly-Pro-Ser-Leu-Lys-Leu (SEQ ID NO:3) or 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 have the IhfA gene or protein. Some non-limiting examples of different Haemophilus influenzae strains include Rd KW20, 86-028NP, R2866, PittGG, PittEE, R2846, and 2019.

[0045] "Microbial DNA" means single-stranded or double-stranded DNA from a biofilm-producing microorganism.

[0046] As used herein, the term "label" or "detectable label" means a compound or composition that is directly or indirectly detectable and is directly or indirectly conjugated to the composition to be detected, such as an N-terminal histidine tag (N-His), a magnetoactive isotope (e.g., 115 Sn,[[]] 117 Sn and 119 Sn), a non-radioactive isotope (e.g., 13 C and 15N), polynucleotides or proteins (such as antibodies) to generate "labeled" components. The term also includes sequences conjugated to polynucleotides that will provide a signal when the inserted sequence is expressed, such as green fluorescent protein (GFP), etc. The label itself can be detectable (e.g., radioisotope label or fluorescent label), or in the case of an enzyme label, can catalyze a chemical change in a detectable substrate compound or composition. The label can be suitable for small-scale detection or more suitable for high-throughput screening. Thus, suitable labels include, but are not limited to, magnetoactive isotopes, non-radioactive isotopes, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins (including enzymes). The label can be simply detected or can be quantified. A response that is simply detected typically includes only a response that is confirmed to be present, while a response that is quantified typically includes a response with a quantifiable (e.g., digitally reportable) value such as intensity, polarization, and / or other properties. In luminescence or fluorescence assays, a luminophore or fluorophore associated directly with the assay component actually involved in binding can be used directly to generate a detectable reaction, or a luminophore or fluorophore associated with another (e.g., reporter molecule or indicator) component can be used indirectly to generate a detectable response. Examples of luminescent labels that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent response typically includes a change or appearance of a luminescent signal. Suitable methods and luminophores for labeling assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (Sixth Edition). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0047] A "gene delivery vector" is defined as any molecule that can carry an inserted polynucleotide into a host cell. Examples of gene delivery vectors are: liposomes, micelles, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial virus envelopes; metal particles; and bacteria or viruses, such as baculoviruses, adenoviruses, and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors, and other recombinant vectors commonly used in the art, which have been described for expression in various eukaryotic and prokaryotic hosts and can be used for gene therapy as well as simple protein expression.

[0048] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vectors. As used herein, "gene delivery", "gene transfer", "transduction", etc. are terms that refer to the introduction of exogenous polynucleotides (sometimes referred to as "transgenes") into host cells, regardless of the method of introduction. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (e.g., by 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 for introducing polynucleotides). The introduced polynucleotides can be maintained stably or transiently in the host cell. Stable maintenance generally requires that the introduced polynucleotide contain an origin of replication compatible with the host cell or be integrated into the replicon of the host cell, such as an episomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. As is known in the art and described herein, many vectors are known to be capable of mediating the transfer of genes into mammalian cells.

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

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

[0051] A "plasmid" is an extrachromosomal DNA molecule that is separate from chromosomal DNA and is capable of replicating independently of chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within microbial populations and generally confer a selective advantage under a given environmental state. Plasmids can carry genes that confer resistance to naturally occurring antibiotics in a competitive environment, or, under similar circumstances, the proteins produced can act as toxins.

[0052] The "plasmids" used in genetic engineering are called "plasmid vectors". Many plasmids are commercially available for such uses. The gene to be replicated is inserted into a copy of the plasmid, which contains genes that confer resistance to specific antibiotics in cells and multiple cloning sites (MCS or polylinkers), which is a short region containing several commonly used restriction enzyme cleavage sites where DNA fragments can be easily inserted. Another major use of plasmids is to produce large amounts of protein. In this case, researchers can grow bacteria that contain the plasmid with the gene of interest. Just as bacteria produce proteins that confer antibiotic resistance to them, they can also be induced to produce large amounts of protein from the inserted gene. This is a cheap and simple method for mass-producing a gene or the protein it subsequently encodes.

[0053] "Yeast artificial chromosome" or "YAC" refers to a vector used for cloning large DNA fragments (greater than 100 kb and up to 3000 kb). It is an artificially constructed chromosome that contains the telomere, centromere, and origin of replication sequences required for replication and preservation in yeast cells. It is constructed using an initial circular plasmid and then linearized using restriction enzymes, and then DNA ligase can add target sequences or genes within the linear molecule by using sticky ends. Yeast expression vectors, such as YAC, YIp (yeast integrating plasmid), and YEp (yeast episomal plasmid), are very useful because eukaryotic protein products with post-translational modifications can be obtained since yeast itself is a eukaryotic cell, but it has been found that YAC is more unstable than BAC and produces chimeric effects.

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

[0055] As used herein, "retrovirus-mediated gene transfer" or "retroviral transduction" have the same meaning and refer to the process of stably transferring a gene or nucleic acid sequence into a host cell by means of a virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell through its normal infection mechanism or can be modified to bind to a different host cell surface receptor or ligand to enter the cell. As used herein, a retroviral vector refers to a viral particle capable of introducing foreign nucleic acid into a cell through a viral or viroidal 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 the DNA form, which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0057] In the context of gene transfer mediated by DNA viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to a polynucleotide comprising a viral genome or a portion thereof and a transgene. Adenovirus (Ad) is a relatively well-characterized homogeneous virus, including over 50 serotypes. See, e.g., PCT International Application Publication No. WO 95 / 27071. Ad does not require integration into the host cell genome. Recombinant Ad-derived vectors have also been constructed, particularly those that reduce the likelihood of recombination and the production of wild-type virus. See PCT International Application Publication Nos. WO 95 / 00655 and WO 95 / 11984. Wild-type AAV has a high infectivity and specificity for integration into the host cell genome. See Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0058] Vectors that contain both a promoter and a cloning site to which a polynucleotide can be operably linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources 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 alter the cloned 5' and / or 3' untranslated portions to eliminate additional, potentially inappropriate alternative translation initiation codons that may interfere or other sequences that interfere with or reduce expression at the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' of the start codon to enhance expression.

[0059] Gene delivery vectors also include DNA / liposome complexes, micelles, and targeted virus protein-DNA complexes. Liposomes that also contain a targeting antibody or fragment thereof can be used in the methods disclosed herein. In addition to delivering polynucleotides to cells or cell populations, the direct introduction of the proteins described herein into cells or cell populations can also be accomplished by non-limiting techniques of protein transfection, or by culture conditions that enhance expression and / or promote the activity of the proteins disclosed herein are other non-limiting techniques.

[0060] "Inhibit, prevent, or disrupt a biofilm" is intended to prevent or therapeutically reduce the structure of a biofilm. In one aspect, this definition excludes the prevention of biofilms. In another aspect, the terms "inhibit, compete with, or titrate" are intended to reduce the formation of the DNA / protein matrix that is a component of microbial biofilms. In one aspect, prevention is excluded from treatment.

[0061] "Curved polynucleotide" means a double-stranded polynucleotide that contains a small loop on one strand that does not pair with the other strand and any polynucleotide, wherein the end-to-end distance is reduced beyond natural thermal fluctuations, i.e., the curvature exceeds the persistence length of native B-form double-stranded DNA of 150 bp. In some embodiments, the loop has a length of 1 base to about 20 bases, or alternatively is 2 bases to about 15 bases long, or alternatively is about 3 bases to about 12 bases long, or alternatively is about 4 bases to about 10 bases long, or alternatively has about 4, 5, or 6, or 7, or 8, 9, or 10 bases.

[0062] An "object" of diagnosis or treatment is a cell or an animal, such as a mammal or a human. A non-human animal that undergoes diagnosis or treatment is an animal that is subjected to infection or an animal model, such as a primate, a rodent (e.g., a rat, a mouse, a ground squirrel), a canine (e.g., a dog), a lagomorph (e.g., a rabbit), a livestock animal, a sport animal, and a pet.

[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 can be linked by peptide bonds. In another embodiment, the subunits can 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 can make up the protein or peptide sequence. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and D- and L-enantiomers, amino acid analogs, and peptidomimetics.

[0064] As used herein with respect to nucleic acids (e.g., DNA or RNA), the terms "isolated" or "recombinant" refer to molecules that are separated from other DNA or RNA, respectively, and that are present in macromolecules as well as in their natural sources of polypeptides. The term "isolated or recombinant nucleic acid" refers to nucleic acid fragments that are not naturally occurring as fragments and that are not found in their natural state. The term "isolated" is also used herein to refer to polynucleotides, polypeptides, and proteins that are separated from other cellular proteins and is intended to include purified and recombinant polypeptides. In other embodiments, the term "isolated or recombinant" refers to being separated from components, cells, or others, where the cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof are normally associated in nature. For example, an isolated cell is a cell that is separated from tissues or cells having different phenotypes or genotypes. An isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated in its natural or native environment (e.g., on a chromosome). It will be apparent to those skilled in the art that non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof do not need to be "isolated" to distinguish them from their naturally occurring counterparts.

[0065] It can be inferred that, without explicit recitation, unless otherwise stated, when the present disclosure relates to polypeptides, proteins, polynucleotides, or antibodies, their equivalents or biological equivalents should be within the scope of the present disclosure. As used herein, the term "its biological equivalent" is intended to be synonymous with "its equivalent" when referring to a reference protein, antibody, fragment, polypeptide, or nucleic acid, meaning those having minimal homology while still retaining the required structure or function. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide, or protein mentioned herein also includes its equivalents. For example, an equivalent means a percentage homology or identity of at least about 70%, at least about 80%, at least about 85%, alternatively at least about 90%, alternatively at least about 95%, or 98% and exhibits substantially equivalent biological activity to the reference protein, polypeptide, or nucleic acid. On the other hand, the term means a polynucleotide that hybridizes to a reference polynucleotide or its complementary sequence under high stringency conditions.

[0066] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" with another sequence means that when aligned, that percentage of bases (or amino acids) is the same when comparing the two sequences. Software programs known in the art can be used to determine the alignment and the percent homology or sequence identity, 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, the alignment is performed using default parameters. A preferred alignment program is BLAST, using default parameters. In particular, exemplary programs include BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort = HIGH SCORE; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0067] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for the purpose of comparison. When 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 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 to a reference polynucleotide or its complementary sequence under stringent conditions.

[0069] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonds between the bases of nucleotide residues. The hydrogen bonds can occur through Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex can include two strands that form a duplex structure, three or more strands that form a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. The hybridization reaction can constitute a step in a broader 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 6x SSC to about 10x SSC; a formamide concentration of about 0% to about 25%; and a wash solution of about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9x SSC to about 2x SSC; a formamide concentration of about 30% to about 50%; and a wash solution of about 5x SSC to about 2x SSC. Examples of high stringency hybridization 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 to about 0.1x SSC or deionized water. Generally, the hybridization incubation time is from 5 minutes to 24 hours, with 1, 2, or more wash steps, and the wash incubation time is about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It should be understood that equivalents of SSC using other buffer systems can be employed.

[0071] An "object" for diagnosis or treatment is a cell or an animal, such as a mammal or a human. Non-human animal objects for diagnosis or treatment are those that are infected or are animal models, such as, for example, anthropoid apes, murine (e.g., rats, mice, chinchillas), canines (e.g., dogs), lagomorphs (e.g., rabbits), livestock, sport animals, and pets. The terms "object", "host", "individual", and "patient" are used interchangeably herein to refer to an animal, typically a mammal. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal can be of any age or at any stage of development (e.g., adult, adolescent, child, infant, or a mammal in utero). The mammal can be male or female. In some embodiments, the object is a human.

[0072] A host cell refers to a eukaryotic or prokaryotic cell that contains an exogenous agent. "Eukaryotic cells" include all kingdoms of life except for the nucleated protozoans. They can be easily distinguished by a membrane-bound nucleus. Animals, plants, fungi, and protozoans are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and the cytoskeleton. The most characteristic 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, avians, reptiles, and humans.

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

[0074] As used herein, terms such as "treatment" are used herein to mean obtaining a desired pharmacological and / or physiological effect. In terms of completely or partially preventing a disease or its signs or symptoms, the effect can be prophylactic, and / or in terms of partial or complete cure of a disease and / or an adverse effect attributable to the disease, the effect can be therapeutic. In one aspect, "treatment" does not include prevention.

[0075] "Prevention" is intended to prevent a disease or effect in or on a system or subject that is susceptible to the disease or effect. One such example is preventing the formation of a biofilm 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 disclosed herein. Pharmaceutically acceptable carriers include: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffering substances (such as phosphates, glycine, sorbic acid, potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polypropylene-block polymers, polyethylene glycol and lanolin. Suitable pharmaceutical carriers are described in the standard reference work in the field, Remington's Pharmaceutical Sciences, Mack Publishing Company. They can be selected according to the intended form of administration, i.e., oral tablets, capsules, elixirs, syrups, etc., and are consistent with conventional pharmaceutical practice.

[0077] "Administer" can be carried out continuously or intermittently in a single dose throughout the course of treatment. Methods for determining the most effective mode of administration and dose are known to those skilled in the art and will vary depending on the composition used for treatment, the purpose of treatment, the target cells being treated and the subject being treated. The dose level and mode of administration can be selected by the treating physician for single or multiple administrations. Suitable dosage forms and methods of administering the agent are known in the art. The route of administration can also be determined, and the method for determining the most effective route of administration is known to those skilled in the art and will vary depending on the composition used for treatment, the purpose of treatment, the health status or disease stage of the subject being treated, and the target cells or tissues. Non-limiting examples of routes of administration include oral administration, nasal administration, injection and topical administration.

[0078] When applied to a polynucleotide, the term "encoding" means that a polynucleotide said to "encode" a polypeptide can be transcribed and / or translated to produce an mRNA of the polypeptide and / or its fragment in its native state or when manipulated by methods well known to those skilled in the art. The antisense strand is the complement of such a nucleic acid and the coding sequence can be deduced therefrom.

[0079] The term "effective amount" means an amount sufficient to achieve the desired effect. In the case of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the condition being discussed as well as the characteristics of the individual subject, such as general health, age, gender, weight, and tolerance to the pharmaceutical composition. In the case of immunogenic compositions, in some embodiments, the effective amount is an amount sufficient to elicit a protective response against a pathogen. In other embodiments, the effective amount of an immunogenic composition is an amount sufficient to result in the production of antibodies against an antigen. In some embodiments, the effective amount is the amount required to confer passive immunity to a subject in need thereof. With respect to immunogenic compositions, in some embodiments, in addition to the above factors, the effective amount will depend on the intended use, the degree of immunogenicity of the particular antigenic compound, and the health / responsiveness of the subject's immune system. One of ordinary skill in the art will be able to determine the appropriate amount based on these and other factors.

[0080] In the case of in vitro applications, in some embodiments, the effective amount will depend on the size and nature of the application being discussed. It will also depend on the nature and sensitivity of the in vitro target as well as the method used. One of ordinary skill in the art will be able to determine the effective amount based on these and other considerations. Depending on the embodiment, the effective amount can include one or more administrations of the composition.

[0081] The reagents and compositions can be used in the manufacture of medicaments and for the treatment of humans and other animals by administering them according to conventional methods (e.g., active ingredients in pharmaceutical compositions).

[0082] The reagents / agents of the present invention can be administered by any suitable route of administration for treatment. It should also be understood that the optimal route will vary with the condition and age of the subject as well as the disease being treated.

[0083] Examples of solid supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbro, and magnetite. The nature of the carrier can be somewhat soluble or insoluble. The carrier material can actually have any possible structural configuration as long as the coupled molecules are capable of binding polynucleotides, polypeptides, or antibodies. Thus, the carrier structure can be spherical, such as bead-like, or cylindrical, such as on 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, etc., or alternatively polystyrene beads. One of ordinary skill in the art will know many other suitable carriers for binding antibodies or antigens or will be able to determine them by using routine experimentation.

[0084] As used herein, the term "antibody" includes intact antibodies and any antigen-binding fragment thereof or single chains thereof. Thus, the term "antibody" includes any protein or peptide molecule that comprises at least a portion of an immunoglobulin molecule. Examples of such include, but are not limited to, complementarity determining regions (CDRs) of heavy or light chains or ligand-binding portions thereof, heavy or light chain variable regions, heavy or light chain constant regions, framework (FR) regions or any portion thereof, binding proteins, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising at least a portion of an antigen-binding portion of an antibody and a non-antibody protein. Antibodies can be polyclonal antibodies, monoclonal antibodies, and can be isolated from any suitable biological source (e.g., mouse, rat, sheep, and dog).

[0085] The terms "antibody" and "immunoglobulin" also include immunoglobulins of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fab′, F(ab)2, Fv, scFv, dsFv, Fd fragments, dAb, VH, VL, VhH, and V-NAR domains, minibodies, diabodies, triabodies, tetra-bodies, and κ bodies; and multispecific antibody fragments formed from one or more isolated antibody fragments. Examples of such include, but are not limited to, complementarity determining regions (CDRs) of heavy or light chains or ligand-binding portions thereof, heavy or light chain variable regions (which are also referred to herein as variable domains), heavy or light chain constant regions (which are 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 heavy and light chain variable regions of an immunoglobulin molecule contain binding domains that interact with an antigen. The constant region of an antibody (Ab) can mediate the binding of the immunoglobulin to host tissues. For example, when the term "anti-" is used before a protein name, such as anti-DNABII, anti-IHF, anti-HU, anti-head chimeric, it refers to a monoclonal or polyclonal antibody that binds to a specific protein and / or has an affinity for a specific protein. For example, "anti-IHF" refers to an antibody that binds to the IHF protein. A specific antibody may have an affinity for or bind to a protein other than the protein it is directed against. For example, anti-IHF, although specifically generated against the IHF protein, may also bind to other proteins related by sequence homology or structural homology.

[0086] Complementary determining regions (CDRs) are parts of the variable regions of antibodies or T cell receptors that are produced by B cells and T cells, respectively, where these molecules bind to their specific antigens (also called epitopes). In certain embodiments, the terms "variable region" and "variable domain" are used interchangeably and refer to the polypeptide of an antibody light chain or heavy chain, the amino acid residue sequence of which varies greatly from one antibody to another and determines the conformation of the binding site, which confers on the antibody specificity for a particular antigen. In another embodiment, the variable region is from about 90 amino acids long to about 200 amino acids long, including but not limited to about 100 amino acids long, or about 110 amino acids long, or about 120 amino acids long, or about 130 amino acids long, or about 140 amino acids long, or about 150 amino acids long, or about 160 amino acids long, or about 170 amino acids long, or about 180 amino acids long, or about 190 amino acids long. In certain embodiments, the variable region of an amino acid sequence as used herein refers to the first about 100 amino acids, or about 110 amino acids, or about 120 amino acids, or about 130 amino acids, or about 140 amino acids, or about 150 amino acids (including or not including the signal peptide if applicable) of the amino acid sequence as the variable region.

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

[0088] The variable regions and / or CDRs of an antibody or its fragment can be determined by those skilled in the art, for example, using publicly available or commercially available tools. Non-limiting examples of such tools include: IgBlast (accessible at www.ncbi.nlm.nih.gov / igblast / ), Scaligner (obtainable from drugdesigntech at www.scaligner.com / ), IMGT rules and / or tools (see, for example, 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 CalssificatiIon (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, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) or 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 antibodies) and antibody fragments, provided that they exhibit the desired biological activity. Antibodies can be isolated from any suitable biological source, such as mouse, rat, sheep and dog.

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

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

[0092] Monoclonal antibodies can be produced using hybridoma techniques or recombinant DNA methods known in the art. A hybridoma is a cell formed in the laboratory by the fusion of antibody-producing lymphocytes and cancer cells that do not produce antibodies (usually myelomas or lymphomas). Hybridomas proliferate and produce continuous samples of a specific monoclonal antibody. Alternative techniques for generating or selecting antibodies include exposing lymphocytes in vitro to an antigen of interest and screening antibody display libraries in cells, phages, 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 in vitro random or site-specific mutagenesis or in vivo somatic mutation). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences from the germline of another mammalian species (e.g., mouse) have been grafted onto a human framework sequence. Thus, as used herein, the term "human antibody" refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, C L 、C H domain (e.g., C H1 、C H2 、C H3)), hinge, (VL, VH)) are substantially non-immunogenic in humans and have only minor sequence variations or mutations. Similarly, antibodies designated for primates (monkeys, baboons, chimpanzees, etc.), rodents (mice, rats, rabbits, guinea pigs, hamsters, etc.) and other mammals are antibodies specific for such species, subgenera, genera, subfamilies, families. In addition, chimeric antibodies include any combination of the above. Such alterations or variations optionally retain or reduce immunogenicity in humans or other species relative to unmodified antibodies. Thus, human antibodies are different from chimeric or humanized antibodies. It should be noted that human antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes. In addition, when a human antibody is a single-chain antibody, it may contain a linker peptide not present in native human antibodies. For example, the Fv may contain a linking peptide connecting the heavy chain variable region and the light chain variable region, such as 2 to 8 glycine or other amino acid residues. Such linker peptides are considered to be of human origin.

[0094] As used herein, a human antibody "derives from" a particular germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, such as by immunizing transgenic mice carrying human immunoglobulin genes or by screening a human immunoglobulin gene library. A human antibody that "derives from" a human germline immunoglobulin sequence can be identified by comparing the amino acid sequence of the human antibody with the amino acid sequence of the human germline immunoglobulin. A selected human antibody typically has at least 90% identity in amino acid sequence with the amino acid sequence encoded by the human germline immunoglobulin gene, and when compared with the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences), the selected human antibody contains amino acid residues that identify the human antibody as human. In some cases, a human antibody can have at least 95%, even at least 96%, 97%, 98% or 99% identity in amino acid sequence with the amino acid sequence encoded by the germline immunoglobulin gene. Generally, a human antibody that derives from a particular human germline sequence differs from the amino acid sequence encoded by the human germline immunoglobulin gene by no more than 10 amino acids. In some cases, a human antibody may show a difference of no more than 5, even no more than 4, 3, 2 or 1 amino acid 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 the minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues of the variable region or a fragment thereof (e.g., 1, 2, 3, 4, 5, or all 6 CDRs) from the recipient are replaced with the residues of the variable region or a fragment thereof (e.g., 1, 2, 3, 4, 5, or all 6 CDRs) from a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) that has the desired specificity, affinity, and capacity. A humanized antibody may contain residues not found in the recipient antibody or the donor antibody. A humanized antibody may also optionally contain at least a portion of the immunoglobulin (usually a human immunoglobulin) constant region (Fc), a non-human antibody in which one or more amino acids in the framework region, constant region, or CDR are replaced with the amino acids at the corresponding positions from a human antibody. Without wishing to be bound by theory, a humanized antibody produces a reduced immune response in a human host as compared to the non-humanized version of the same antibody. A humanized antibody may have conservative amino acid substitutions that have substantially no effect on antigen binding or other antibody functions. Conservative substitution groupings include: glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serine-threonine, and asparagine-glutamic acid. Specifically, the humanized antibodies disclosed herein specifically bind to a DNABII polypeptide or a fragment thereof (e.g., a head chimeric peptide or a tail chimeric peptide) within a specific range of one or more of the following: EC 50 , K on , K off , K A and / or K D , and inhibits or releases specific cytokines when treating a subject. In a further embodiment, a humanized antibody that specifically binds to the head region of a DNABII polypeptide (e.g., a head chimeric peptide) disrupts biofilms both in vivo and in vitro. Additionally, although the humanization process is a rational design process, it may produce unexpected changes (positive or negative) in, for example, binding affinity, antigen specificity, or physical properties (e.g., solubility or aggregability); thus, the properties of a humanized antibody cannot be inherently predicted from the properties of the starting non-human antibody.

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

[0097] In one embodiment, an antibody as used herein can be a chimeric antibody. As used herein, a chimeric antibody is an antibody whose light and heavy chain genes are typically constructed by genetic engineering from antibody variable and constant region genes of different species.

[0098] Additionally, the antibodies disclosed herein can be engineered to include modifications in the Fc region to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cell-mediated cytotoxicity. Such modifications include, but are not limited to, altering the number of cysteine residues in the hinge region to facilitate light and heavy chain assembly or to increase or decrease the stability of the antibody (U.S. Patent No. 5,677,425), or amino acid mutations in the Fc hinge region to reduce the biological half-life of the antibody (U.S. Patent No. 6,165,745).

[0099] Furthermore, the antibodies disclosed herein can be chemically modified. The glycosylation of the 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, afucosylated antibodies with a reduced amount of fucose residues or antibodies with an increased bisecting GlcNAc structure can be obtained by expressing the antibody in host cells with an altered glycosylation machinery (Shields, R.L. 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 the biological half-life by reacting the antibody or a fragment thereof 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 can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has 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 pegylating proteins are known in the art and can be applied to the antibodies disclosed herein (EP 0154316 and EP 0401384).

[0101] Alternatively, the antibody can be chemically modified to increase the half-life of the resulting molecule by conjugating or fusing the antigen-binding region of the antibody to a serum protein, such as human serum albumin. Such methods are described, for example, in EP 0322094 and EP 0486525.

[0102] The antibodies or fragments thereof disclosed herein can be conjugated to a diagnostic agent and can be used diagnostically, for example, to monitor the development or progression of a disease and to determine the efficacy of a given treatment regimen. Examples of diagnostic agents include enzymes, cofactors, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions. Detectable substances can be directly conjugated or conjugated to the antibody or a fragment thereof, or indirectly conjugated or conjugated via a linker, using techniques known in the art. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable repair 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 substances include 125 I、 131I, indium 111, lutetium 171, bismuth 212, bismuth 213, astatine 211, copper 62, copper 64, copper 67, yttrium 90, iodine 125, iodine 131, phosphorus 32, phosphorus 33, scandium 47, silver 111, gallium 67, praseodymium 142, samarium 153, terbium 161, dysprosium 166, holmium 166, 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. By using a bifunctional chelator covalently linked to an antibody, monoclonal antibodies can be indirectly conjugated with radioactive metal ions. Chelators can be linked by affinity binding (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] In addition, the antibody or fragment thereof of the present invention can be conjugated with a therapeutic agent. Suitable therapeutic agents include: paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dactinomycin, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (such as nitrogen mustard, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives (such as carboplatin)), antibiotics (such as actinomycin, bleomycin, daunorubicin (formerly called daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), diphtheria toxin and related molecules (such as 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 toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, aloin, saponin, modeccin, gelanin, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin toxin and hybrid toxins.

[0104] Other suitable conjugate molecules include ribonuclease (RNase), DNase I, antisense nucleic acids, inhibitory RNA molecules (e.g., siRNA molecules), immunostimulatory nucleic acids, aptamers, ribozymes, triplex-forming molecules, and external guide sequences. Aptamers are small nucleic acids 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quadruplexes, and can bind small molecules (e.g., ATP (U.S. Patent No. 5,631,146) and theophylline (U.S. Patent No. 5,580,737)) as well as macromolecules (e.g., reverse transcriptase (U.S. Patent No. 5,786,462) and thrombin (U.S. Patent No. 5,543,293)). Ribozymes are nucleic acid molecules capable of catalyzing chemical reactions intramolecularly or intermolecularly. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate followed by cleavage. Nucleic acid molecules with triplex-forming functionality can interact with double-stranded or single-stranded nucleic acids by forming triplexes, where triplex DNA forms a complex that relies on 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, regulators, and stimulators of specific activities possessed by target molecules, or functional nucleic acid molecules can have de novo activity independent of any other molecule.

[0106] Therapeutic agents can be linked to the antibody directly or indirectly using any of a variety of available methods. For example, the reagent can be attached to the hinge region of the reduced antibody component through disulfide bond formation, using a cross-linking agent (such as N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP)), or through the sugar moiety 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 with 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 Prospective Of The Therapeutic Use Of Radiolabeled 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 (e.g., a ligand of another antibody or receptor) to produce a bispecific or multispecific molecule that binds to at least two or more different binding sites or target molecules. The linkage of an antibody to one or more other binding molecules (e.g., another antibody, antibody fragment, peptide, or binding mimetic) can be accomplished, for example, by chemical conjugation, genetic fusion, or non-covalent binding. In addition to the first and second target epitopes, the multispecific molecule can further include a third binding specificity.

[0109] Bispecific and multispecific molecules can be prepared using methods known in the art. For example, each binding unit of the multispecific molecule can be produced separately and then conjugated to each other. When the binding molecules are proteins or peptides, a variety of coupling agents or cross-linking agents can be used for covalent binding. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-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 molecules are antibodies, they can be bound through disulfide bonds in the C-terminal hinge regions of the two heavy chains.

[0110] The antibodies or fragments thereof disclosed herein can be linked to a moiety that is toxic to cells that bind the antibody to form a "depleting" antibody. These antibodies are particularly useful in applications where 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 conjugated to a number of different carriers. Accordingly, the present invention also provides compositions comprising an antibody and another active or inert substance. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnet. For the purposes disclosed herein, the nature of the carrier can be soluble or insoluble. Those skilled in the art will know other suitable carriers for conjugating monoclonal antibodies or will be able to determine such carriers using routine experimentation.

[0113] As used herein, the term "antibody derivative" includes full-length antibodies or fragments of antibodies, wherein one or more amino acids are chemically modified by alkylation, polyethylene glycolylation, acylation, ester formation, amide formation, etc. (e.g., for attaching the antibody to a second molecule). This includes, but is not limited to, polyethylene glycolylated antibodies, cysteine polyethylene glycolylated antibodies, and variants thereof.

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

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

[0116] In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to a cell or tissue surface or to a cellular component present on the surface (e.g., a cell surface marker). Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimide groups, 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 for the fluorescent label will depend on the site of attachment to the linker, reagent, label, or second labeling reagent.

[0117] An "immune response" broadly refers to an antigen-specific response of lymphocytes to a foreign substance. Any substance that can elicit an immune response is said to be "immunogenic" and is called an "immunogen". All immunogens are antigens; however, not all antigens are immunogenic. The immune responses disclosed herein can be humoral (through antibody activity) or cell-mediated (through T cell activation).

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

[0119] "HMG domain" or "high mobility group (HMG) box domain" refers to an amino acid sequence involved in binding to DNA (Stros et al., Cell Mol Life Sci. 64(19-20):2590-606(2007)). In one embodiment, the structure of the HMG-box domain consists of three helices in an irregular array. In another embodiment, the HMG-box domain enables the protein to bind non-B DNA conformations (kinks or unwound) with high affinity. The HMG-box domain is located in high mobility group proteins, which are involved in the regulation of DNA-dependent processes such as transcription, replication, and DNA repair, all of which require changes in chromatin conformation (Thomas (2001) Biochem. Soc. Trans. 29(Pt 4):395-401).

[0120] Compositions used according to the present disclosure can be packaged in unit dose form for ease of administration and uniformity of dosage. The term "unit dose" or "dose" refers to physically discrete units suitable for use in a subject, each unit containing a pre-determined amount of the composition, which amount is calculated to produce the desired response associated with its administration, i.e., the appropriate route and course of treatment. Depending on the number of treatments and the unit dose, the amount administered depends on the desired outcome and / or protection. The exact amount of the composition also depends on the judgment of the physician and is specific to each individual. Factors affecting the dose include the physical and clinical state of the subject, the route of administration, the intended therapeutic goal (symptom relief versus cure), and the potency, stability, and toxicity of the particular ingredient. In formulation, the solution is administered in a manner appropriate to the dosage formulation and in an amount having a therapeutic or prophylactic effect. The formulation can be readily administered in various dosage forms (such as the types of injection solutions described herein).

[0121] As used herein, the term "contact" refers to the direct or indirect binding or interaction between two or more molecules. Specific examples of direct interaction are binding. Specific examples of indirect interaction are one entity acting on an intermediate molecule, which in turn acts on a second reference entity. Contact as used herein includes occurring in solution, in the solid phase, in vitro, ex vivo, in cells, and in vivo. In vivo contact can be referred to as administration or dosing.

[0122] As used herein, a "recombinant peptide" refers to a peptide produced in a host cell (such as E. coli, yeast) using exogenous / recombinant nucleic acid. As used herein, a "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 DNABII proteins. In another embodiment, the biofilm comprises histone-like protein (HU) or integration host factor (IHF) binding proteins from Escherichia coli strain U93. In certain embodiments, the DNABII peptide is an IHF peptide. Additionally or alternatively, the DNABII peptide is an HU peptide. In certain embodiments, the head region of the DNABII peptide is the head region of IHFA and / or the head region of IHFB. In another embodiment, the head region of the DNABII peptide is the head region of IHFA that binds directly or indirectly (e.g., via a linker) to the head region of IHFB. In another embodiment, the head region of the DNABII peptide is hfA5-mIhfB4 NTHI Head chimeric peptide.

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

[0125] Several parameters are used herein to describe the binding and dissociation reactions of receptor (R, e.g., an antibody or antigen-binding fragment thereof) and ligand (L, e.g., the target of an antibody or antigen-binding fragment thereof) molecules, which are formalized as: The reaction is characterized by an association rate constant k on and a dissociation rate constant k off with units of M -1 s -1 and s -1 respectively. At equilibrium, the forward binding transition R + L → RL should be balanced by the reverse unbinding transition RL → R + L. That is, k on [R][L] = k off [RL], where [R], [L], and [RL] represent the concentrations of unbound free receptor, unbound free ligand, and receptor-ligand complex, respectively. Additionally, the equilibrium dissociation constant "K D " can be calculated as k off / k on , i.e., [R] x [L] / [RL], and the equilibrium association constant "K A " can be calculated as k on / k off , i.e., [RL] / ([R] x [L]). Modes for practicing the present invention Synthesize or recombinantly produce mB Box-97 polypeptide

[0126] The present invention provides a synthetic or recombinant polypeptide that comprises mB Box-97, or consists essentially of mB Box-97, or consists of mB Box-97, wherein mB Box-97 consists of amino acids 90 to 176 or amino acids 80 to 176 of the coding sequence of the native human HMGB1 protein (as shown in SEQ ID NO:2), with a cysteine to serine point mutation at amino acid 106, which eliminates the ability of the polypeptide to induce an inflammatory response, and equivalents thereof that retain the cysteine to serine point mutation at amino acid 106 and consist of amino acids 90 to 176 or amino acids 80 to 176 of the coding sequence of the native human HMGB1 protein (the native / wild-type human HMGB1 sequence is as shown in SEQ ID NO:2), and which eliminates the ability of the polypeptide to induce an inflammatory response. The synthetic polypeptide is produced by synthetic or chemical means and is not wild-type or recombinantly produced.

[0127] As used herein, an equivalent of an mB Box-97 polypeptide, recombinant mB Box-97, or synthetic mB Box-97 refers to a sequence that has 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% identity to a reference mB Box-97 polypeptide, recombinant mB Box-97, or synthetic mB Box-97, and in one aspect, retains the mutant amino acid of the cysteine to serine point mutation at amino acid 106. In one aspect, the percent identity is determined using the BLAST alignment program with default parameters. In particular, preferred programs include BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = HIGH SCORE; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following website: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0128] In some aspects, equivalents of the mB Box-97 polypeptide retain the expected functions and / or structural features of the mB Box-97 polypeptide. In another aspect, an equivalent of the mB-Box-97 polypeptide includes an mB Box-97 that retains the C to S amino acid substitution at amino acid 106, or a recombinant mB Box-97, or a synthetic mB Box-97 polypeptide, and further independently includes at least 2, or alternatively at least 3, or alternatively at least 4, or alternatively at least 5, or at least 6, or alternatively at least 7, or alternatively at least 8, or alternatively at least 9, or alternatively at least 10 amino acids at the amino and / or carboxyl terminus of the polypeptide.

[0129] In another aspect, the mB Box-97 HMGB1 polypeptide further comprises one or more linker polypeptides, or consists essentially of, or consists of. An example of a peptide linker is GPSLKL (SEQ ID NO:3) or PPKGETKKKF (SEQ ID ID:4) at the amine and / or carboxyl terminus.

[0130] The polypeptide can be detectably labeled and / or conjugated to a carrier (such as a pharmaceutically acceptable carrier).

[0131] The disclosed mB Box-97 HMGB1 polypeptide is 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-97HMGB1 peptide to the subject. In one aspect, the subject has one or more of the following: SARS CoV-2 infection, sepsis, autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, small vessel vasculitis), autoinflammatory diseases (such as gout, inflammatory bowel disease), and metabolic diseases (such as type 2 diabetes and obesity). Also provided is a method for preventing a NET-mediated disease or preventing the progression of a 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 is a method for preventing and treating bacterial biofilms by administering an effective amount of the mB Box-97 HMGB1 polypeptide to the subject. Synthetic or recombinant mB Box-97 polypeptides and compositions comprising the same

[0132] One aspect of the present disclosure relates to a synthetic polypeptide that comprises mB Box-97, or consists essentially of mB Box-97, or consists of mB Box-97, wherein the mB Box-97 consists of amino acids 90 to 176 of the coding sequence of the native human HMGB1 protein shown in SEQ ID NO:2 and has a cysteine to serine point mutation at amino acid 106, or an equivalent thereof.

[0133] Another aspect of the present disclosure relates to a recombinant polypeptide that comprises mB Box-97, or consists essentially of mB Box-97, or consists of mB Box-97, wherein mB Box-97 consists of amino acids 90 to 176 of the coding sequence of the native human HMGB1 protein shown in SEQ ID NO:2 and has a cysteine to serine point mutation at amino acid 106, or an equivalent thereof.

[0134] As used herein, an equivalent of recombinant mB Box-97 or synthetic mB Box-97 refers to a sequence that has 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% identity to a reference recombinant mBBox-97 or reference synthetic mB Box-97, and in one aspect retains the mutant amino acid of the cysteine to serine point mutation at amino acid 106. In one aspect, the percent identity is determined using the BLAST alignment program with default parameters. In particular, preferred programs include BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = HIGH SCORE; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0135] In some aspects, an equivalent of the recombinant polypeptide retains the expected functions and / or structural features of the mB Box-97 polypeptide. In another aspect, an equivalent of mB Box-97 includes a recombinant mBBox-97 polypeptide that retains the C to S amino acid substitution at amino acid 106 and further independently includes at least 2, or alternatively at least 3, or alternatively at least 4, or alternatively at least 5, or at least 6, or alternatively at least 7, or alternatively at least 8, or alternatively at least 9, or alternatively at least 10 amino acids at the amino and / or carboxyl terminus of the polypeptide.

[0136] In another aspect, the recombinant mB Box-97 HMGB1 polypeptide further comprises one or more linker polypeptides, or consists essentially of, or consists of, the same. An example of a peptide linker is GPSLKL (SEQ ID NO:3) or PPKGETKKKF (SEQ ID ID:4) at the amine and / or carboxyl terminus.

[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, wherein the mB Box-97 consists of amino acids 80 to 176 of the coding sequence of the native human HMGB1 protein shown in SEQ ID NO:2 and has a cysteine to serine point mutation at amino acid 106, or an equivalent thereof.

[0139] As used herein, an equivalent of synthetic mB Box-97 refers to a sequence that has 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% identity to the reference synthetic mB Box-97, and in one aspect retains the mutant amino acid of the cysteine to serine point mutation at amino acid 106. In one aspect, the percent identity is determined using the BLAST alignment program with default parameters. In particular, preferred programs include BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = HIGH SCORE; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0140] In some aspects, an equivalent of the synthetic polypeptide retains the expected functional and / or structural characteristics of the synthetic mB Box-97 polypeptide. In another aspect, an equivalent of synthetic mB Box-97 includes those that retain the C to S amino acid substitution at amino acid 106 and further independently include at least 2, or alternatively at least 3, or alternatively at least 4, or alternatively at least 5, or at least 6, or alternatively at least 7, or alternatively at least 8, or alternatively at least 9, or alternatively at least 10 amino acids at the amino and / or carboxyl terminus of the polypeptide.

[0141] In another aspect, the synthetic mB Box-97 HMGB1 polypeptide or an equivalent thereof further comprises one or more linker polypeptides, or consists essentially of, or consists of, the same. An example of a peptide linker is GPSLKL (SEQ ID NO:3) or PPKGETKKKF (SEQ ID ID:4) at the amine and / or carboxyl terminus.

[0142] Another aspect of the present disclosure relates to a recombinant or synthetic polypeptide that comprises, consists essentially of, or consists of recombinant or synthetic mB Box-97, wherein the mB Box-97 consists of amino acids 90 to 176 of the coding sequence of the native human HMGB1 protein shown in SEQ ID NO:2 and has a cysteine to serine point mutation at amino acid 106, or an equivalent thereof.

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

[0144] In some embodiments, 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 under high stringency conditions to a polynucleotide encoding the amino acid sequence or its complementary sequence, wherein the 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 wherein an 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.

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

[0146] In some embodiments, the synthetic or recombinant polypeptide or an equivalent thereof includes a linker polypeptide, optionally wherein the linker polypeptide includes GPSLKL (SEQ ID NO:3) or PPKGETKKKF (SEQ ID NO:4). In some embodiments, the linker is located at the C-terminus of the synthetic or recombinant polypeptide. In some embodiments, the linker is located at the N-terminus of the synthetic or recombinant polypeptide.

[0147] Another aspect of the present disclosure relates to a plurality of the synthetic or recombinant polypeptides of the present disclosure. In some embodiments, the members of the plurality of polypeptides are identical or different from each other.

[0148] Another aspect of the present disclosure relates to a composition that comprises, consists essentially of, or consists of the synthetic or recombinant polypeptide of the present disclosure or the plurality of 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 recombinantly produced polypeptides and proteins from prokaryotic and eukaryotic host cells, as well as mutant proteins, analogs, and fragments thereof, such as those described above for such cells.

[0150] It should be understood that functional equivalents or variants of wild-type polypeptides or proteins are also within the scope of the present disclosure. For example, polypeptides or proteins having conservative amino acid substitutions of amino acids.

[0151] In another aspect, polypeptides are conjugated or linked to detectable labels or reagents to increase the half-life of the polypeptides, such as PEGylation, PEG mimetics, polysialylation, HESylation or glycosylation. Suitable labels are known in the art and described herein.

[0152] Proteins and polypeptides can be obtained by many methods known to those skilled in the art, including purification, chemical synthesis and recombinant methods. Accordingly, the present disclosure also provides methods of using these methods as well as the polynucleotides and polypeptides disclosed herein to produce mBBox-97 polypeptides and the methods also disclosed herein. For example, for recombinant mBBox-97, it can be produced in a host cell system comprising a polynucleotide encoding the polypeptide, and the host cells are cultured under conditions favorable for the recombinant production of the polypeptide. The polypeptide can be isolated from the host cell system by methods such as immunoprecipitation with an antibody and standard techniques such as gel filtration, ion exchange, reverse phase and affinity chromatography. For such methods, see, for example, Deutscher et al. (1999) Guide To Protein Purification: Methods In Enzymology (Vol. 182, Academic Press). Accordingly, the present disclosure also provides methods of obtaining these polypeptides and the products obtainable and obtained by these methods.

[0153] Polypeptides can also be obtained by chemical synthesis using commercially available automated peptide synthesizers, such as those produced 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). Accordingly, the present disclosure also provides a method of chemically synthesizing the proteins of the present invention by providing the sequences of the proteins and reagents (such as amino acids and enzymes) and linking the amino acids together in the correct orientation and linear sequence.

[0154] The present disclosure also provides the polypeptides described herein conjugated to detectable reagents for use in diagnostic methods. For example, detectably labeled polypeptides can be bound to columns and used for the detection and purification of antibodies. They can also be used as immunogens for generating antibodies. The polypeptides of the present invention can be used in in vitro assay systems to screen for reagents or drugs that modulate cellular processes.

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

[0156] The peptides of the present invention can be modified to include unnatural amino acids. Thus, the peptides can contain combinations of D-amino acids, L-amino acids, and various "designed" amino acids (such as β-methyl amino acids, C-α-methyl amino acids, and N-α-methyl amino acids, etc.) to convey special properties of the peptides. Additionally, by assigning specific amino acids in specific coupling steps, peptides with α-helix, β-turn, β-sheet, γ-turn, and cyclic peptides can be produced. Generally, the α-helix secondary structure or random secondary structure is considered to be particularly useful.

[0157] The polypeptides of the present invention can also be combined with various solid-phase carriers (such as implants, scaffolds, 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 propylene glycol, polyethylene glycol, and vegetable oils. When used to prepare antibodies or induce an immune response in vivo, the carrier can also include adjuvants that can be used to non-specifically enhance the specific immune response. Those skilled in the art can easily determine whether an adjuvant is needed and select one. However, for illustrative purposes only, suitable adjuvants include, but are not limited to, Freund's complete and incomplete solutions, mineral salts, and polynucleotides. Other suitable adjuvants include monophosphoryl lipid A (MPL), mutant derivatives of the heat-labile enterotoxin of Escherichia coli, mutant derivatives of cholera toxin, CPG oligonucleotides, and adjuvants derived from squalene.

[0158] The present invention also provides a pharmaceutical composition comprising any polypeptide, analog, mutant protein, or fragment of the present invention, alone or in combination with each other or with other reagents (such as antibiotics and acceptable carriers or solid supports or antibodies or fragments thereof described herein), or consisting essentially of or consisting of the same. These compositions can be used in the various diagnostic and therapeutic methods described herein. Isolated polynucleotides, vectors, isolated host cells

[0159] Another aspect of the present disclosure relates to an isolated polynucleotide encoding a synthetic or recombinant polypeptide of the present disclosure, such as a synthetic or recombinant polypeptide that comprises mB Box-97, or consists essentially of mB Box-97, or consists of mB Box-97, wherein mB Box-97 consists of amino acids 80 to 176 of the coding sequence of the native human HMGB1 protein, with a cysteine to serine point mutation at amino acid 106, which eliminates the ability of the polypeptide to induce an inflammatory response, and equivalents thereof that retain the cysteine to serine point mutation at amino acid 106 of mB Box-97 and consist of amino acids 80 to 176 of the coding sequence of the native human HMGB1 protein (the native / wild-type human HMGB1 sequence is shown as SEQ ID NO:2), and which eliminates the ability of the polypeptide to induce an inflammatory response. Also provided are complementary polynucleotides of the polynucleotides encoding the polypeptides. The polynucleotide can be DNA, RNA, mRNA or interfering RNA, such as siRNA, miRNA or dsRNA.

[0160] The present disclosure also provides a polynucleotide encoding an equivalent of an mB Box-97 polypeptide, recombinant mB Box-97 or synthetic mB Box-97, the equivalent being 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 which, in one aspect, retains the mutant amino acid of the cysteine to serine point mutation at amino acid 106. In one aspect, the percent identity is determined using the BLAST alignment program with default parameters. In particular, preferred programs include BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = HIGH SCORE; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at: ncbi.nlm.nih.gov / cgi-bin / BLAST. The polynucleotide can be DNA, RNA, mRNA or interfering RNA, such as siRNA, miRNA or dsRNA.

[0161] In some aspects, the polynucleotide encodes an equivalent of the polypeptide, the equivalent retaining the expected functions and / or structural features of the synthetic or recombinant mB Box-97 polypeptide. In another aspect, an equivalent of the synthetic or recombinant mB Box-97 polypeptide includes a recombinant mB Box-97 that retains the C-to-S amino acid substitution at amino acid 106, or a synthetic mB Box-97 polypeptide, and further independently includes at least 2, or alternatively at least 3, or alternatively at least 4, or alternatively at least 5, or at least 6, or alternatively at least 7, or alternatively at least 8, or alternatively at least 9, or alternatively at least 10 amino acids at the amino and / or carboxyl termini of the polypeptide. The polynucleotide can be DNA, RNA, mRNA or interfering RNA, such as siRNA, miRNA or dsRNA.

[0162] In another aspect, the polynucleotide encodes an mB Box-97 HMGB1 polypeptide that further comprises one or more linker polypeptides, or consists essentially of, or consists of, the same. An example of a peptide linker is GPSLKL (SEQ ID NO:3) or PPKGETKKKF (SEQ ID ID:4) at the amine and / or carboxyl termini. The polynucleotide can be DNA, RNA, mRNA or interfering RNA, such as siRNA, miRNA or dsRNA.

[0163] The polynucleotide can be conjugated to a detectable label, such as an enzyme label or a radioisotope, to detect the expression of the nucleic acid and / or gene in cells. A variety of suitable detectable labels are known in the art, including fluorescent, radioactive, enzymatic or other ligands capable of giving a detectable signal, such as avidin / biotin. In one aspect, one may wish to use a fluorescent label or an enzyme label, such as urease, alkaline phosphatase or peroxidase, in place of radioactive or other environmentally unfriendly reagents. In the case of an enzyme label, a calorimetric indicator substrate can be used to provide a means visible to the human eye or spectrophotometrically to identify specific hybridization to a sample containing a complementary nucleic acid. Accordingly, the present invention further provides a method for detecting a single-stranded polynucleotide or its complementary sequence by contacting the target single-stranded polynucleotide with a labeled single-stranded polynucleotide (probe) that is part of the polynucleotide of the present invention under conditions that permit hybridization of the complementary single-stranded polynucleotides (preferably medium stringency hybridization conditions), or more preferably under highly stringent hybridization conditions. The hybridized polynucleotide pairs are separated from the unhybridized single-stranded polynucleotides. The hybridized polynucleotide pairs are detected using methods known to those skilled in the art, such as those described in Sambrook et al. (1989) (ibid).

[0164] The polynucleotides embodied in the present invention can be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof. Methods of chemical polynucleotide synthesis are known in the art and need not be described in detail herein. One of ordinary skill in the art can use the sequence data provided herein to obtain the desired polynucleotides by using a DNA synthesizer or from a commercial service.

[0165] The polynucleotides of the present disclosure 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) (ibid.), and the references cited therein. Alternatively, one of ordinary skill in the art can use the sequences provided herein and a commercial DNA synthesizer to replicate DNA. Accordingly, the present invention also provides methods for obtaining the polynucleotides of the present invention that provide the linear sequence of the polynucleotide, nucleotides, suitable primer molecules, chemicals (such as enzymes), and instructions for their replication and that chemically replicate or ligate the nucleotides in the appropriate orientation to obtain the polynucleotide. In a separate embodiment, the polynucleotides are further isolated. Still further, one of ordinary skill in the art can insert the polynucleotide into a suitable replication vector and insert the vector into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification. The so-amplified DNA can be isolated from the cells by methods known to one of ordinary skill in the art. Also provided herein are methods for obtaining polynucleotides by this method and the polynucleotides so obtained.

[0166] RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell. The DNA can be delivered by any suitable method, such as by using a suitable gene delivery vector (such as a liposome, plasmid, or vector) or by electroporation. When the cell replicates and the DNA is transcribed into RNA; then the RNA can be isolated using methods known to one of ordinary skill in the art, such as as described in Sambrook et al. (1989) (ibid.). For example, various lytic enzymes or chemical solutions can be used to isolate mRNA according to the methods described in Sambrook et al. (1989) (ibid.), or it can be extracted by nucleic acid binding resins according to the accompanying instructions provided by the manufacturer.

[0167] Polynucleotides that exhibit sequence complementarity or homology to the polynucleotides of the present invention can be used as hybridization probes or as equivalents of the specific polynucleotides identified herein. Since the complete coding sequences of the transcripts are known, any portion of such sequences or homologous sequences can be used in the methods of the present invention.

[0168] It is known in the art that specific hybridization does not require "perfect match" probes. Minor variations in the probe sequence achieved by substitution, deletion, or insertion of a small number of bases do not affect hybridization specificity. Generally, up to 20% base pair mismatches (when optimally aligned) can be tolerated. In some embodiments, the probes used to detect the above-mentioned mRNA are at least about 80% identical to the homologous region. In some embodiments, after alignment of the homologous regions, the probe has 85% identity with the corresponding gene sequence; in some embodiments, it exhibits 90% identity.

[0169] These probes can be used in radiological assays (such as Southern and Northern blot analyses) to detect, prognose, diagnose, or monitor various cells or tissues containing these cells. The probes can also be attached to solid supports or arrays (such as chips) for use in high-throughput screening assays to detect the expression of genes corresponding to the polynucleotides of the present invention. Accordingly, the present invention also provides probes that comprise or correspond to the polynucleotides of the present invention or their equivalents or their complementary sequences or fragments thereof, which are attached to a solid support for high-throughput screening.

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

[0171] Nucleotide probes having a complementary sequence over an extended sequence greater than 5 to 10 nucleotides in length are generally preferred to increase the stability and selectivity of the hybrid and thus improve the specificity of the particular hybrid molecules obtained. In certain embodiments, polynucleotides can be designed having a gene complementary sequence of 10 or more or greater than 50 nucleotides in length, or even longer if desired. Such fragments can be readily prepared, for example, by directly synthesizing the fragment chemically, by applying nucleic acid replication techniques such as the PCR technique with two primer oligonucleotides as described in U.S. Patent No. 4,603,102, or by introducing the selected sequence into a recombinant vector for recombinant production. In one aspect, the probe is about 50 - 75 or more nucleotides in length, alternatively 50 - 100 nucleotides.

[0172] The polynucleotides of the present invention can be used as primers for detecting genes or gene transcripts expressed in the cells described herein. As used herein, amplification refers to any method using a primer-dependent polymerase capable of replicating a target sequence with reasonable fidelity. Amplification can be carried out by natural or recombinant DNA polymerases such as T7 DNA polymerase, the Klenow fragment of Escherichia coli DNA polymerase, and reverse transcriptase. For illustrative purposes only, the length of the primers is the same as the length identified for the probes.

[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 by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet irradiation.

[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 can be used to detect and quantify gene expression in cells. Alternatively, the expression of the encoded polypeptide can be detected by a variety of methods. In particular, it is useful to prepare polyclonal or monoclonal antibodies that specifically react with the target polypeptide. Such antibodies can be used to visualize cells expressing the polypeptide using techniques such as immunohistology, ELISA, and Western blot. These techniques can be used to determine the expression level of the expressed polynucleotide.

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

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

[0177] Also provided are multiple polynucleotides that can be the same or different from each other, and compositions comprising multiple polynucleotides and a vector (such as a pharmaceutically acceptable carrier).

[0178] Another aspect of the present disclosure relates to a vector that comprises, consists essentially of, or consists of the isolated polynucleotide of the present disclosure and optionally a vector or a pharmaceutically acceptable carrier. For example, the vector can be, for example, a lipid nanoparticle, a plasmid, or a viral vector.

[0179] Also provided are multiple vectors that can be the same or different from each other, and compositions comprising multiple vectors 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 vector or a pharmaceutically acceptable carrier.

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

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

[0183] There is further provided a method for producing a recombinant polypeptide of the present disclosure, the method comprising: delivering an isolated polynucleotide of the present disclosure or a vector of the present disclosure to a host cell under conditions where the polypeptide of the present disclosure can be expressed. 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 Escherichia coli cell.

[0184] There is further provided a method for producing a synthetic polypeptide of the present disclosure by a method comprising solid-phase peptide synthesis or solution-phase peptide synthesis. A composition comprising an antibody or a fragment thereof

[0185] Another aspect of the present disclosure relates to a composition comprising an antibody or a fragment thereof, wherein the antibody or fragment thereof recognizes and binds to the head region of a DNABII peptide or a head chimera (such as IhfA5-mIhfB4 NTHI head chimera) as described herein. There is also provided a composition comprising one or more antibodies that recognize and bind to the NTHI IhfA5-mIhfB4 head chimera.

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

[0187] In some embodiments, the antibody or fragment thereof binds to the head region of a DNABII peptide or a head chimera, and the antibody or fragment thereof comprises: (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 consisting essentially of, or consisting of, or an equivalent thereof; and (ii) a light chain (LC) immunoglobulin variable domain sequence comprising the aa of SEQ ID NO:22 a sequence from aa 21 to aa 132, or consisting essentially of, or consisting of, or an equivalent thereof; or wherein said antibody or fragment thereof comprises: (i) a heavy chain (HC) immunoglobulin variable domain sequence comprising aa 25 to aa 144 of SEQ ID NO:24, or consisting essentially of, or consisting of, or an equivalent thereof; and (ii) a light chain (LC) immunoglobulin variable domain sequence comprising aa 21 to aa 132 of SEQ ID NO:25, or consisting essentially of, or consisting of, or an equivalent thereof.

[0188] In some embodiments, an antibody or fragment thereof that binds to the head region of the DNABII peptide comprises: heavy chain complementarity determining region 1 (CDRH1) comprising the sequence GFTFRTY (aa 50 to aa 56 of SEQ ID NO:9 or 10 or 11 or 24), or consisting essentially of, or consisting of; heavy chain complementarity determining region 2 (CDRH2) comprising the sequence GSDRRH (aa 76 to aa 81 of SEQ ID NO:9 or 10 or 11 or 24), or consisting essentially of, or consisting of; heavy chain complementarity determining region 3 (CDRH3) comprising the sequence VGPYDGYYGEFDY (SEQ ID NO: aa 121 to aa 133 of 9 or 10 or 11 or 24), or consisting essentially of, or consisting of; light chain complementarity determining region 1 (CDRL1) comprising the sequence QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO:15 or 16 or 17 or 25), or consisting essentially of, or consisting of; light chain complementarity determining region 2 (CDRL2) comprising the sequence LVS (aa 75 to aa 77 of SEQ ID NO:15 or 16 or 17 or 25), or consisting essentially of, or consisting of; and light chain complementarity determining region 3 (CDRL3) comprising the sequence WQGTHFP (aa114 to aa 120 of SEQ ID NO:15 or 16 or 17 or 25), or consisting essentially of, or consisting of.

[0189] In some embodiments, an antibody or fragment thereof that binds to the head region of the DNABII peptide comprises: Heavy chain complementarity determining region 1 (CDRH1), which comprises the sequence GFTFSRYG (aa 50 to aa 57 of SEQ ID NO: 12 or 13 or 14), or consists essentially of or consists of the same; Heavy chain complementarity determining region 2 (CDRH2), which comprises the sequence ISSGGSYT (aa 75 to aa 82 of SEQ ID NO: 12 or 13 or 14), or consists essentially of or consists of the same; Heavy chain complementarity determining region 3 (CDRH3), which comprises the sequence ERHGGDGYWYFDV (aa 121 to aa 133 of SEQ ID NO: 12 or 13 or 14), or consists essentially of or consists of the same; Light chain complementarity determining region 1 (CDRL1), which comprises the sequence QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 15 or 16 or 17 or 25), or consists essentially of or consists of the same; Light chain complementarity determining region 2 (CDRL2), which comprises the sequence LVS (aa 75 to aa 77 of SEQ ID NO: 15 or 16 or 17 or 25), or consists essentially of or consists of the same; and Light chain complementarity determining region 3 (CDRL3), which comprises the sequence WQGTHFPYT (aa 114 to aa 122 of SEQ ID NO: 15 or 16 or 17 or 25), or consists essentially of or consists further of the same.

[0190] In some embodiments, an antibody or fragment thereof that binds to the head region of the DNABII peptide or a head chimera comprises the following sequences, or consists essentially of or consists of the following sequences: a heavy chain (HC) immunoglobulin variable domain sequence that comprises a sequence selected from aa 25 to aa 144 of SEQ ID NO: 21 or 24 or their respective equivalents, or consists essentially of or consists of the same, and / or a light chain (LC) immunoglobulin protein variable domain sequence that comprises a sequence selected from aa 21 to aa 132 of SEQ ID NO: 22 or 25 or their respective equivalents, or consists essentially of or consists of the same. In certain embodiments, the antibody or fragment thereof binds to the DNABII peptide (e.g., the head region of the DNABII peptide, including but not limited to: the head regions of IHF or HU, the head regions of IHFA or IHFB, and / or the head chimeric peptide IhfA5-mIhfB4 NTHI ). In one embodiment, the antibody or fragment thereof binds to the head chimeric peptide IhfA5-mIhfB4 as described herein NTHI . In some embodiments, the head chimeric peptide IhfA5-mIhfB4NTHI comprises an amino acid sequence selected from SEQ ID NO: 26 - 28, or consists essentially of, or consists of the same.

[0191] In some embodiments, an antibody or fragment thereof that binds to the head region of the DNABII peptide comprises the following sequences, or consists essentially of, or consists of the same: any one or any two or three heavy chain (HC) CDRs, said CDRs comprising a sequence selected from SEQ ID NO: 9 - 14 or their respective equivalents, or consisting essentially of, or consisting of the same, and / or any one or any two or three light chain (LC) CDRs, said CDRs comprising a sequence selected from SEQ ID NO: 15 - 20 or their respective equivalents, or consisting essentially of, or consisting of the same. In certain embodiments, the antibody or fragment thereof binds to the DNABII peptide (e.g., the head region of the DNABII peptide, including but not limited to: the head region of IHF or HU, the head region of IHFA or IHFB, and / or the head chimeric peptide IhfA5 - mIhfB4 as described herein NTHI ). In one embodiment, the antibody or fragment thereof binds to the head chimeric peptide IhfA5 - mIhfB4 as described herein NTHI . In some embodiments, the head chimeric peptide IhfA5 - mIhfB4 NTHI comprises an amino acid sequence selected from SEQ ID NO: 26 - 28, or consists essentially of, or consists of the same.

[0192] In some embodiments, an antibody or fragment thereof that binds to the head region of the DNABII peptide comprises the following sequences, or consists essentially of, or consists of the same: all three heavy chain (HC) CDRs, said CDRs comprising a sequence selected from SEQ ID NO: 9 - 11 or their respective equivalents, or consisting essentially of, or consisting of the same, and / or all three light chain (LC) CDRs, said CDRs comprising a sequence selected from SEQ ID NO: 15 - 17 or their respective equivalents, or consisting essentially of, or consisting of the same. In certain embodiments, the antibody or fragment thereof binds to the DNABII peptide (e.g., the head region of the DNABII peptide, including but not limited to: the head region of IHF or HU, the head region of IHFA or IHFB, and / or the head chimeric peptide IhfA5 - mIhfB4 NTHI ). In one embodiment, the antibody or fragment thereof binds to the head chimeric peptide IhfA5 - mIhfB4 NTHI . In some embodiments, the head chimeric peptide IhfA5 - mIhfB4 NTHI comprises an amino acid sequence selected from SEQ ID NO: 26 - 28, or consists essentially of, or consists of the same.

[0193] In some embodiments, an antibody or fragment thereof that binds to the head region of a DNABII peptide comprises the following sequences, consists essentially of the following sequences, or consists of the following sequences: all three heavy chain (HC) CDRs, the CDRs comprising sequences selected from SEQ ID NO: 12-14 or their respective equivalents, or consisting essentially of or consisting of the same, and / or all three light chain (LC) CDRs, the CDRs comprising sequences selected from SEQ ID NO: 18-20 or their respective equivalents, or consisting essentially of or consisting of the same. In certain embodiments, the antibody or fragment thereof binds to a DNABII peptide (e.g., the head region of a DNABII peptide, including but not limited to: the head region of IHF or HU, the head region of IHFA or IHFB, and / or the head chimeric peptide IhfA5-mIhfB4 NTHI ). In one embodiment, the antibody or fragment thereof binds to the head chimeric peptide IhfA5-mIhfB4 NTHI . In some embodiments, the head chimeric peptide IhfA5-mIhfB4 NTHI comprises an amino acid sequence selected from SEQ ID NO: 26-28, or consists essentially of or consists of the same.

[0194] Table 1 provides the use of targeting IhfA5-mIhfB4 NTHIRelative biofilm disruption data of a humanized monoclonal antibody designed with a head chimeric peptide. Biofilm disruption: NTHI 86-028NP colonies were collected from a chocolate agar overnight culture and suspended in brain heart infusion broth (sBHI) supplemented with 2 μg β-NAD and hemin per milliliter of medium. The optical density at 490 nm was then adjusted to 0.65, and the culture was diluted 1:6 in sBHI and then incubated statically at 37 °C with 5% CO2 for 3 hours. Next, the culture was diluted 1:2500 in fresh sBHI, and 200 μl of the suspension was aliquoted into each well of an 8-well chamber slide. The slide was then incubated statically at 37 °C and 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 time point, the medium was aspirated from each well, and 5 μg of the monoclonal antibody was added to each well. The biofilm was incubated for an additional 16 hours. The biofilm was then washed, stained with the FM1-43FX bacterial cell membrane stain (Invitrogen), and fixed overnight at 4 °C in 0.1 M phosphate buffer (pH 7.4) containing 16% paraformaldehyde, 2.5% glutaraldehyde, and 4.0% acetic acid. The fixative was aspirated and 200 μl of Hank’s balanced salt solution was added to each well, and the biofilm was then observed on a Zeiss 800Meta laser scanning confocal microscope. Images were compiled using Zeiss Zen Black software, and biofilm biomass was calculated using COMSTAT 2.1 software. IhfA5-mIhfB4 NTHI The K A (1 / M) of the head chimeric peptide is approximately 4E+05 to 2E+08.

[0195] Table 1: Relative biofilm disruption data using a humanized monoclonal antibody designed with a targeting IhfA5-mIhfB4 NTHI head chimeric peptide

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0215] The antibodies or fragments thereof provided herein can be monospecific or bispecific. In one embodiment, the antibody or fragment thereof is trispecific, or tetra-specific or penta-specific. Additionally or alternatively, the antibody is selected from IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4) or IgM antibodies. In one embodiment, the antibody further comprises a constant region selected from: IgA constant region (e.g., IgA1 constant region or IgA2 constant region), IgD constant region, IgE constant region, IgG constant region (e.g., IgG1 constant region, IgG2 constant region, IgG3 constant region or IgG4 constant region) or IgM constant region. In some embodiments, the constant region of the antibody comprises an amino acid sequence selected from SEQ ID NO:23 and 35-42.

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

[0217] The present disclosure further provides a composition comprising the antibody or fragment thereof described herein and the mB Box-97 peptide and optionally a carrier (such as a pharmaceutically acceptable carrier), or consisting essentially of or consisting of the foregoing. The mB Box peptide is a synthetic or recombinant polypeptide that comprises mB Box-97, or consists essentially of or consists of mB Box-97, which is composed of amino acids 90 to 176 or amino acids 80 to 176 of the coding sequence of the native human HMGB1 protein (such as SEQ ID NO:2), with a cysteine to serine point mutation at amino acid 106, which eliminates the ability of the polypeptide to induce an inflammatory response, and equivalents thereof that retain the cysteine to serine point mutation at amino acid 106 of mB Box-97 and are composed of amino acids 90 to 176 or amino acids 80 to 176 of the coding sequence of the native human HMGB1 protein (the native / wild-type human HMGB1 sequence is as shown in SEQ ID NO:2), and that eliminate the ability of the polypeptide to induce an inflammatory response. The synthetic polypeptide is produced by synthetic or chemical means and is not wild-type or recombinantly produced.

[0218] Methods for preparing antibodies and their fragments are known in the art and are described in U.S. Patent No. 11,104,723, the entire content of which is incorporated herein by reference. Composition

[0219] The present disclosure further provides compositions. The compositions comprise a carrier and one or more of the following: the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the carriers disclosed herein, the isolated host cells disclosed herein, small molecules or antibodies and / or antigen-binding fragments disclosed herein. The carrier can be one or more solid supports or a pharmaceutically acceptable carrier. The compositions can further comprise an adjuvant or other components suitable for administration as a vaccine. In one aspect, the compositions are formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, and / or adjuvants. Additionally, embodiments of the compositions of the present disclosure include one or more of the following: the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the carriers disclosed herein, small molecules, the isolated host cells disclosed herein, or the antibodies of the present disclosure, formulated with one or more pharmaceutically acceptable substances.

[0220] For oral formulations, any one or more of the isolated or recombinant polypeptides described herein, the isolated or recombinant polynucleotides described herein, the carriers described herein, the isolated host cells described herein, the small molecules or antibodies or fragments thereof described herein can be used alone or in pharmaceutical formulations disclosed herein, which comprise (or consist of) the compound and a suitable combination of additives to prepare tablets, powders, granules, or capsules, for example, in combination with conventional additives such as lactose, mannitol, corn starch, or potato starch; in combination with binders such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch, or gelatin; in combination with disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose; in combination with lubricants such as talc or magnesium stearate; and, if desired, in combination with diluents, buffers, wetting agents, preservatives, and flavoring agents. Pharmaceutically compatible binder and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. can contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0221] Pharmaceutical formulations and unit dosage forms suitable for oral administration are particularly useful in the treatment of chronic diseases, infections, and therapies for self-administration by patients. In one aspect, the formulation is specifically for pediatric administration.

[0222] The present disclosure provides pharmaceutical formulations, wherein one or more of the isolated polypeptides, isolated polynucleotides, vectors, isolated host cells or antibodies disclosed herein can be formulated into injectable preparations by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent such as vegetable oil or other similar oils, synthetic fatty acid glycerides, higher fatty acid esters or propylene glycol; if desired, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives or other antimicrobial agents can be used. Non-limiting examples of such are antimicrobial agents, such as other vaccine components, such as surface antigens, such as OMP P5, OMP 26, OMP P2 or type IV fimbrial protein (see Jurcisek and Bakaletz (2007) J. of Bacteriology 189(10):3868-3875 and Murphy, T F, Bakaletz, L O and Smeesters, P R (2009) The Pediatric Infectious Disease Journal, 28:S121-S126) and antibacterial agents. For intravenous administration, suitable carriers include bacteriostatic water for injection, Cremophor EL TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the compositions for parenteral administration must be sterile and should be fluid to the extent that easy injection is possible.

[0223] The aerosol formulations provided by the present disclosure can be administered by inhalation and can be propellant-based or non-propellant. For example, embodiments of the pharmaceutical formulations disclosed herein include formulating the compounds disclosed herein into a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen, etc. For inhalation administration, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant such as a gas (such as carbon dioxide) or a spray. Non-limiting examples of non-propellant are pump sprays that are ejected from a closed container by mechanical force (i.e., by pushing a piston down with a finger or by compressing the container, such as by applying a compressive force to the container wall, or for example by the elastic bladder applying a spring force by the container wall itself).

[0224] The suppositories disclosed herein can be prepared by mixing the compounds disclosed herein with any of a variety of bases such as an emulsifying base or a water-soluble base. Embodiments of the pharmaceutical formulations of the compounds disclosed herein can be administered rectally by suppository. Suppositories can include carriers such as cocoa butter, carbowax and polyethylene glycol, which melt at body temperature but solidify at room temperature.

[0225] Unit dosage forms for oral or rectal administration can be provided, such as syrups, elixirs, and suspensions, wherein each dosage unit (e.g., teaspoon, tablespoon, tablet, or suppository) contains a predetermined amount of a composition comprising one or more of the compounds disclosed herein. Similarly, unit dosage forms for injection or intravenous administration can contain the compounds disclosed herein in a sterile aqueous solution, physiological saline, or another pharmaceutically acceptable carrier as a solution composition.

[0226] Embodiments of the pharmaceutical formulations disclosed herein include compositions in which one or more of the isolated polypeptides disclosed herein, the isolated polynucleotides disclosed herein, the vectors disclosed herein, the small molecules for the present disclosure, the isolated host cells disclosed herein, or the antibodies or fragments thereof as disclosed herein are formulated in an injectable composition. The injectable pharmaceutical formulations disclosed herein are prepared as liquid solutions or suspensions; or as solid forms suitable for dissolving or suspending in a liquid carrier prior to injection. According to other embodiments of the pharmaceutical formulations disclosed herein, the formulations can also be emulsified or the active ingredient encapsulated in a liposome carrier.

[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 includes continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, etc., and various types of such continuous delivery systems are known in the art.

[0228] Mechanical or electromechanical infusion pumps can also be suitable for the present invention. Examples of such devices include those described in, for example, U.S. Pat. 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, delivery of the compounds disclosed herein can be accomplished using any of a variety of refillable pump systems. The pump provides a consistent, controlled release over a long period of time. In some embodiments, the compounds disclosed herein are present in a liquid formulation in a drug-impermeable reservoir and are delivered to the individual in a continuous manner.

[0229] In one embodiment, the drug delivery system is an at least partially implantable device. The implantable device can be implanted at any suitable implantation site using methods and devices well known in the art. The implantation site is the site in the subject's body where the drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to, subcutaneous, intramuscular, or other suitable sites within the subject. Subcutaneous implantation sites are used in some embodiments because they facilitate the implantation and removal of the drug delivery device.

[0230] Drug delivery devices suitable for the present disclosure can be based on any of a variety of operating modes, with polymers such as poly(glycolide-co-lactide) (PGLA), which are commercially available from many suppliers, such as, for example, BioDegmer and Sigma-Aldrich. For example, the drug delivery device can be based on a diffusion system, a convection system, or an erodible system (e.g., a corrosion-based system). For example, the drug delivery device can be an electrochemical pump, an osmotic pump, an electroosmotic pump, a vapor pressure pump, or an osmotic burst matrix, e.g., where the drug is incorporated into a polymer (e.g., PGLA) and the polymer provides the release of the pharmaceutical formulation while the polymer material impregnated with the drug (e.g., a biodegradable drug-impregnated polymer material) degrades. In other embodiments, the drug delivery device is based on an electro-diffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolysis system, etc.

[0231] Drug delivery devices based on mechanical or electromechanical infusion pumps can also be suitable for the present invention. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147, 4,360,019, 4,487,603, 4,360,019, 4,725,852, etc. Generally, any of a variety of refillable, non-replaceable pump systems can be used to effect the subject treatment methods. Pumps and other convection systems can be used because they generally release more consistently and controllably over time. Osmotic pumps are used in some embodiments because they have the combined advantages of more consistent controlled release and relatively small size (see, e.g., PCT International Application Publication No. WO 97 / 27840 and U.S. Pat. Nos. 5,985,305 and 5,728,396). Exemplary osmotic-driven devices suitable for the present disclosure include, but are not necessarily limited to, those described in U.S. Pat. Nos. 3,760,984, 3,845,770, 3,916,899, 3,923,426, 3,987,790, 3,995,631, 3,916,899, 4,016,880, 4,036,228, 4,111,202, 4,111,203, 4,203,440, 4,203,442, 4,210,139, 4,327,725, 4,627,850, 4,865,845, 5,057,318, 5,059,423, 5,112,614, 5,137,727, 5,234,692, 5,234,693, 5,728,396, etc. Another exemplary device suitable for the present disclosure is a synchronous infusion pump (Medtronic).

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

[0233] Excipient carriers suitable for the compounds disclosed herein are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. Additionally, if desired, the carrier may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Methods of preparing such dosage forms are known to those of skill in the art or will be apparent upon consideration of the present disclosure. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985. The composition or formulation to be administered will in any event contain an amount of the compound sufficient to achieve the desired state in the subject being treated.

[0234] The compositions of the present disclosure include those that contain a sustained-release or controlled-release matrix. Additionally, the embodiments of the present disclosure can be used in combination with other therapies using sustained-release formulations. As used herein, a sustained-release matrix is a matrix made of a material that degrades by enzymatic or acid-base hydrolysis or by dissolution, typically a polymer. Once inserted into the body, the matrix is subject to the action of enzymes and body fluids. Desirable sustained-release matrices are selected from biocompatible materials such as liposomes, polylactic acid, polyglycolide (a polymer of glycolic acid), poly(lactide-co-glycolide) (a copolymer of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids (such as aniline, tyrosine, isoleucine), polynucleotides, polyethylene propylene, polyvinylpyrrolidone, and siloxanes. Exemplary biodegradable matrices include polylactide matrices, polyglycolide matrices, and poly(lactide-co-glycolide) (a copolymer of lactic acid and glycolic acid) matrices.

[0235] In another embodiment, the polypeptide, antibody, or fragment thereof (and combination components) is delivered in a controlled release system. For example, the compounds disclosed herein can be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes, or other modes of administration. In one embodiment, a pump can 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, polymeric materials are used. In yet another embodiment, the controlled release system is placed near the therapeutic target, i.e., the liver, and thus only a fraction of the systemic dose is required. In yet another embodiment, the controlled release system is placed near the therapeutic target, and thus only a fraction of the systemic administration is required. 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 (and combination components, alone or together) include compositions formed by impregnating an absorbent material, such as sutures, bandages, and gauze, with an inhibitor described herein, or coating the surface of a solid phase material, such as surgical staples, zippers, and catheters, to deliver the composition. Given the present disclosure, other delivery systems of this type will be apparent to those skilled in the art.

[0237] The present disclosure provides methods and compositions for administering one or more interfering agents to a host (e.g., a human) to treat a microbial infection. In various embodiments, the methods disclosed herein encompass almost any available method and route applicable to drug delivery, including in vivo and ex vivo methods, as well as systemic and local administration routes. Methods using the mB Box-97 polypeptide

[0238] Another aspect of the present disclosure relates to a method for treating a subject in need thereof, the method comprising the steps of, consisting essentially of, or consisting of: administering to the subject an effective amount of one or more of the following: (i) a synthetic or recombinant polypeptide of the present disclosure or an equivalent thereof, (ii) a plurality of 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.

[0239] Another aspect of the present disclosure relates to a method for treating or preventing abnormal or excessive neutrophil extracellular trap (NET) formation, or preventing NET-mediated diseases or preventing the progression of NET-mediated diseases in a subject in need thereof, the method comprising the steps of, consisting essentially of, or consisting of: administering to the subject an effective amount of one or more of the following: (i) a synthetic or recombinant polypeptide of the present disclosure or an equivalent thereof, (ii) a plurality of 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.

[0240] In some embodiments, the subject has one or more of the following: a lung disease, selected from SARS-CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-related acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease, selected from systemic lupus erythematosus, rheumatoid arthritis, type I 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, the effective amount is from 50 nM to 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, 850 nM, 900 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 diseases include, consist essentially of, or consist of the following diseases: lung diseases, selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-related acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; autoimmune diseases, selected from systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, or small vessel vasculitis; autoinflammatory diseases, selected from gout or inflammatory bowel disease; or metabolic diseases, selected from type 2 diabetes or obesity.

[0243] Another aspect of the present disclosure relates to a method for preventing or treating bacterial biofilms in a subject in need thereof, the method comprising the steps of, consisting essentially of, or consisting of: administering to the subject an effective amount of one or more of the following: (i) a synthetic or recombinant polypeptide of the present disclosure or an equivalent thereof, (ii) a plurality of 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, the effective amount is from 50 nM to 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, 850 nM, 900 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 the steps of, consisting essentially of, or consisting of: administering to the subject an antibody or a fragment thereof, wherein the antibody or the fragment thereof binds to the head region of the DNABII peptide, such as (IhfA5-mIhfB4 NTHI head chimera). In one aspect, the antibody is a monoclonal antibody, a humanized antibody, or an antigen-binding fragment thereof.

[0246] In some embodiments, the antibody or fragment thereof binds to the head region or head chimeric of the DNABII peptide, and the antibody or fragment thereof comprises: (iii) a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, consisting of, or being an equivalent of the sequence of amino acids (aa) 25 to aa 144 of SEQ ID NO: 21; and (iv) a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, consisting of, or being an equivalent of the aa 21 to aa 132 of SEQ ID NO: 22; or wherein the antibody or fragment thereof comprises: (iii) a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, consisting of, or being an equivalent of the aa 25 to aa 144 of SEQ ID NO: 24; and (iv) a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, consisting of, or being an equivalent of the aa 21 to aa 132 of SEQ ID NO: 25.

[0247] In some embodiments, the antibody or fragment thereof that binds to the head region of the DNABII peptide comprises: Heavy chain complementarity determining region 1 (CDRH1) comprising, consisting essentially of, or consisting of the sequence GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 9 or 10 or 11 or 24); Heavy chain complementarity determining region 2 (CDRH2) comprising, consisting essentially of, or consisting of the sequence GSDRRH (aa 76 to aa 81 of SEQ ID NO: 9 or 10 or 11 or 24); Heavy chain complementarity determining region 3 (CDRH3) comprising, consisting essentially of, or consisting of the sequence VGPYDGYYGEFDY (SEQ ID NO: 9 or 10 or 11 or 24, aa 121 to aa 133); Light chain complementarity determining region 1 (CDRL1) comprising, consisting essentially of, or consisting of the sequence QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 15 or 16 or 17 or 25); The light chain complementarity determining region 2 (CDRL2), which comprises the sequence LVS (aa 75 to aa 77 of SEQ ID NO: 15 or 16 or 17 or 25), or consists essentially of the same, or consists of the same; and The light chain complementarity determining region 3 (CDRL3), which comprises the sequence WQGTHFP (aa 114 to aa 120 of SEQ ID NO: 15 or 16 or 17 or 25), or consists essentially of the same, or consists of the same.

[0248] In some embodiments, an antibody or fragment thereof that binds to the head region of a DNABII peptide or a head chimera comprises the following sequences, or consists essentially of the following sequences, or consists of the following sequences: a heavy chain (HC) immunoglobulin variable domain sequence that comprises a sequence selected from aa 25 to aa 144 of SEQ ID NO: 21 or 24 or their respective equivalents, or consists essentially of the same, or consists of the same, and / or a light chain (LC) immunoglobulin variable domain sequence that comprises a sequence selected from aa 21 to aa 132 of SEQ ID NO: 22 or 25 or their respective equivalents, or consists essentially of the same, or consists of the same. In certain embodiments, the antibody or fragment thereof binds to a DNABII peptide (e.g., the head region of a DNABII peptide, including but not limited to: the head region of IHF or HU, the head region of IHFA or IHFB, and / or the head chimeric peptide IhfA5-mIhfB4 NTHI ). In one embodiment, the antibody or fragment thereof binds to the head chimeric peptide IhfA5-mIhfB4 NTHI . As used herein, the head chimeric peptide IhfA5-mIhfB4 NTHI comprises an amino acid sequence selected from SEQ ID NO: 26 - 28, or consists essentially of the same, or consists of the same.

[0249] In some embodiments, an antibody or fragment thereof that binds to the head region of a DNABII peptide comprises the following sequences, or consists essentially of the following sequences, or consists of the following sequences: any one or any two or three heavy chain (HC) CDRs, the CDRs comprising a sequence selected from SEQ ID NO: 9 - 14 or their respective equivalents, or consists essentially of the same, or consists of the same, and / or any one or any two or three light chain (LC) CDRs, the CDRs comprising a sequence selected from SEQ ID NO: 15 - 20 or their respective equivalents, or consists essentially of the same, or consists of the same. In certain embodiments, the antibody or fragment thereof binds to a DNABII peptide (e.g., the head region of a DNABII peptide, including but not limited to: the head region of IHF or HU, the head region of IHFA or IHFB, and / or the head chimeric peptide IhfA5-mIhfB4 NTHI)。In one embodiment, the antibody or fragment thereof binds to the head chimeric peptide IhfA5-mIhfB4 NTHI 。

[0250] In some embodiments, an antibody or fragment thereof that binds to the head region of a DNABII peptide comprises the following sequences, consists essentially of the following sequences, or consists of the following sequences: all three heavy chain (HC) CDRs, the CDRs comprising sequences selected from SEQ ID NO: 9-11 or their respective equivalents, consisting essentially of, or consisting of, and / or all three light chain (LC) CDRs, the CDRs comprising sequences selected from SEQ ID NO: 15-17 or their respective equivalents, consisting essentially of, or consisting of. In certain embodiments, the antibody or fragment thereof binds to a DNABII peptide (e.g., the head region of a DNABII peptide, including but not limited to: the head regions of IHF or HU, the head regions of IHFA or IHFB, and / or the head chimeric peptide IhfA5-mIhfB4 NTHI )。In one embodiment, the antibody or fragment thereof binds to the head chimeric peptide IhfA5-mIhfB4 NTHI 。

[0251] In some embodiments, an antibody or fragment thereof that binds to the head region of a DNABII peptide comprises the following sequences, consists essentially of the following sequences, or consists of the following sequences: all three heavy chain (HC) CDRs, the CDRs comprising sequences selected from SEQ ID NO: 12-14 or their respective equivalents, consisting essentially of, or consisting of, and / or all three light chain (LC) CDRs, the CDRs comprising sequences selected from SEQ ID NO: 18-20 or their respective equivalents, consisting essentially of, or consisting of. In certain embodiments, the antibody or fragment thereof binds to a DNABII peptide (e.g., the head region of a DNABII peptide, including but not limited to: the head regions of IHF or HU, the head regions of IHFA or IHFB, and / or the head chimeric peptide IhfA5-mIhfB4 NTHI )。In one embodiment, the antibody or fragment thereof binds to the head chimeric peptide IhfA5-mIhfB4 NTHI 。

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0271] The antibodies or fragments thereof provided herein can be monospecific or bispecific. In one embodiment, the antibody or fragment thereof is trispecific, or tetra - specific or penta - specific. Additionally or alternatively, the antibody is selected from IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4) or IgM antibodies. In one embodiment, the antibody further comprises a constant region selected from: IgA constant region (e.g., IgA1 constant region or IgA2 constant region), IgD constant region, IgE constant region, IgG constant region (e.g., IgG1 constant region, IgG2 constant region, IgG3 constant region or IgG4 constant region) or IgM constant region. In some embodiments, the constant region of the antibody comprises an amino acid sequence selected from SEQ ID NO:23 and 35 - 42.

[0272] In another aspect, antibodies can be modified by conventional techniques, which in one aspect can increase the half - life of the antibody, such as PEGylation, PEG mimetics, polysialylation, HESylation or glycosylation. Method for concentrating the eDNA tendrils of neutrophil extracellular traps (NETs)

[0273] Another aspect of the present disclosure relates to a method for concentrating the eDNA tendrils of neutrophil extracellular traps (NETs), the method comprising the steps of, or consisting essentially of, or consisting of: contacting the NET with an effective amount of a DNA - binding agent.

[0274] Another aspect of the present disclosure relates to a method for preventing or inactivating the eDNA structure of neutrophil extracellular traps (NETs), the method comprising the steps of, or consisting essentially of, or consisting of: contacting the NET with an effective amount of a DNA - binding agent.

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

[0276] In some embodiments, the DNA - binding agent is a reagent that aggregates or concentrates DNA.

[0277] In some embodiments, the effective amount is from 50 nM to 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, 850 nM, 900 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 consists of histone-like nucleoid structuring protein (H-NS), polyamines or polycations.

[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 wherein the H-NS is derived from Escherichia coli (E.Coli), nontypeable Haemophilus influenzae (NTHI), Streptococcus pneumoniae (S.pneumoniae), Klebsiella pneumoniae (K.pneumoniae), Mycobacterium tuberculosis or Pseudomonas aeruginosa.

[0280] In some embodiments, the H-NS comprises, consists essentially of, or 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 NO: 29-34. In one aspect, the percent identity is determined using the BLAST alignment program with default parameters. In particular, preferred programs include BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = HIGHSCORE; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at: ncbi.nlm.nih.gov / cgi-bin / BLAST.

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

[0282] Another aspect of the present disclosure relates to a method for preventing destructive blood clotting in a subject in need thereof, the method comprising, consisting essentially of, or consisting of the steps of: administering to the subject in need thereof an effective amount of a DNA binder.

[0283] In some embodiments, the DNA binder comprises, consists essentially of, or consists of histone-like nucleoid structuring protein (H-NS), polyamines or polycations.

[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 wherein the H-NS is derived from Escherichia coli (E.Coli), nontypeable Haemophilus influenzae (NTHI), Streptococcus pneumoniae (S.pneumoniae), Klebsiella pneumoniae (K.pneumoniae), Mycobacterium tuberculosis or Pseudomonas aeruginosa.

[0285] In some embodiments, the H-NS comprises 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 NO: 29-34, or consists essentially of, or consists of, such an amino acid sequence. In one aspect, the percent identity is determined using the BLAST alignment program with default parameters. In particular, preferred programs include BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = HIGHSCORE; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0286] Another aspect of the present disclosure relates to a method for arresting excessive inflammation in a subject in need thereof, the method comprising the steps of, consisting essentially of, or consisting of: administering to the subject in need thereof an effective amount of a DNA binder, optionally wherein the effective amount is from 50 nM to 2 μM.

[0287] Another aspect of the present disclosure relates to a method for preventing, treating, or preventing the progression of neutrophil extracellular trap (NET)-mediated diseases in a subject in need thereof, the method comprising the steps of, consisting essentially of, or consisting of: administering to the subject in need thereof an effective amount of a DNA binder. In some embodiments, the effective amount is from 50 nM to 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, 850 nM, 900 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 a reagent that aggregates or concentrates DNA. In some embodiments, the effective amount is from 50 nM to 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, 850 nM, 900 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 consists of histone-like nucleoid structuring protein (H-NS), polyamines or polycations.

[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 wherein the H-NS is derived from Escherichia coli (E.Coli), nontypeable Haemophilus influenzae (NTHI), Streptococcus pneumoniae (S.pneumoniae), Klebsiella pneumoniae (K.pneumoniae), Mycobacterium tuberculosis or Pseudomonas aeruginosa.

[0291] In some embodiments, the H-NS comprises 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 NO: 29-34, or consists essentially of, or consists of, such an amino acid sequence.

[0292] In some embodiments, the H-NS comprises 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, or consists essentially of, or consists of, such an equivalent, which is identical to the reference polypeptide. In one aspect, the percent identity is determined using the BLAST alignment program with default parameters. In particular, preferred programs include BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = HIGH SCORE; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at: ncbi.nlm.nih.gov / cgi-bin / BLAST.

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

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

[0295] The compositions and related methods of the present disclosure can be used in combination with the administration of other therapies. These include, but are not limited to, the administration of DNase enzymes, antibiotics, antimicrobials, anti-infectives, antifungals, antiparasitics, antivirals, or other antibodies.

[0296] In some embodiments, the methods and compositions include a deoxyribonuclease (DNase) that acts in concert with an anti-DNABII antibody. A DNase is any enzyme that catalyzes the cleavage of phosphodiester bonds in the DNA backbone. Three non-limiting examples of DNase enzymes that are known to target not only cruciform structures but also a variety of DNA secondary structures include DNAse I, T4 EndoVII, T7 EndoI, RuvABC, and RusA. In certain embodiments, when combined with a DNase, the effective amount of the anti-DNABII antibody required to destabilize the biofilm is reduced. When administered in vitro, the DNase can be added directly to the assay or to a suitable buffer known to stabilize the enzyme. The effective unit dose of the DNase and the 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 an antibiotic and / or an antimicrobial agent. An antimicrobial agent is a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, or protozoa. Although biofilms are generally resistant to the action of antibiotics, the compositions and methods described herein can be used to render infections involving biofilms sensitive to conventional methods of treating the infections. In other embodiments, the combination of an antibiotic or an antimicrobial agent with the methods and compositions described herein enables a reduction in the effective amount of the antimicrobial agent and / or the biofilm-reducing agent. Some non-limiting examples of antimicrobial agents and antibiotics that can be used in combination with the methods of the present disclosure include amoxicillin, amoxicillin-clavulanate, cefdinir, azithromycin, and sulfamethoxazole-trimethoprim. The therapeutically effective dose of the combination of an antimicrobial agent and / or an antibiotic with a biofilm-reducing agent can be readily determined by conventional methods. In some embodiments, the dose of the antimicrobial agent in combination with a biofilm-reducing agent is the mean effective dose that has been shown to be effective in other bacterial infections, such as bacterial infections where the etiology of the infection does not include a biofilm. 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 mean 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, these methods and compositions can be combined with antibodies for treating bacterial infections. An example of an antibody that can be used in combination with the methods and compositions described herein is an antibody against an unrelated outer membrane protein (i.e., OMPP5) or an antigen-binding fragment thereof. Treatment with this antibody alone does not reduce the volume of the biofilm in vitro. Combining this antibody with a biofilm-reducing agent produces an effect greater than that achievable with either reagent alone at the same concentration. Other antibodies that may produce a synergistic effect when combined with a biofilm-reducing agent or a method for reducing biofilms include anti-rsPilA antibodies, anti-OMP26 antibodies, anti-OMP P2 antibodies, and anti-total OMP antibody preparations.

[0299] The compositions and methods described herein can be used to render bacterial infections involving biofilms sensitive to common treatment modalities that are effective in treating bacterial infections in the absence of biofilms but ineffective in treating bacterial infections involving biofilms. In other embodiments, the compositions and methods described herein can be used in combination with treatment modalities that are effective in treating bacterial infections involving biofilms, but the combination of such additional therapies with a biofilm-reducing agent or method produces a synergistic effect such that the effective dose of the biofilm-reducing agent or the additional therapeutic agent can be reduced. In other cases, the combination of such additional therapies and a biofilm-reducing agent or method produces a synergistic effect, thereby enhancing the treatment. The enhancement of the treatment can be demonstrated by a shorter time required to treat the infection.

[0300] Additional therapeutic treatments can be added before, simultaneously with, or after the method or composition for reducing biofilms and can be included in the same formulation / composition or as a separate formulation / composition. Kit

[0301] Also disclosed herein are kits comprising one or more of the polypeptides, antibodies or fragments thereof, or reagents and instructions required to perform the in vitro and in vivo methods described herein. Accordingly, the present disclosure provides kits for performing these methods, which can include antibodies, fragments thereof, polypeptides, polynucleotides, vectors, or host cells, and instructions for performing the methods disclosed herein, such as collecting tissue, and / or performing screening, and / or analyzing results, and / or administering an effective amount of an antibody, antibody fragment, polypeptide, polynucleotide, vector, or host cell as defined herein. These can be used alone or in combination with other suitable antimicrobials.

[0302] For example, the kit may comprise any one or more of the above-identified reagents (e.g., antibodies, antibody fragments, polypeptides, polynucleotides, vectors or host cells), together with instructions for use, or consist essentially of, or consist of the same. The kit may further comprise one or more of an adjuvant, an antigenic peptide or an antimicrobial agent. Examples of vectors include liquid carriers, pharmaceutically acceptable carriers, solid-phase carriers, pharmaceutically acceptable carriers, pharmaceutically acceptable polymers, liposomes, micelles, implants, scaffolds, pastes, gels, dental implants or medical implants. Example

[0303] The following examples are intended to illustrate and not to limit the scope of the disclosure. Example 1: Inhibition of mB Box-97 Peptide and NETosis Among all HMGB1-derived constructs, only mB Box-97 showed inhibition of NETosis in isolated human neutrophils.

[0304] HMGB1 is a known inducer of inflammation and NETosis. However, the previous applicant demonstrated that an engineered point mutation (C45S; mHMGB-1) strongly reduced the pro-inflammatory function of HMGB1. In fact, mHMGB1 was able to reduce the migration of neutrophils into the peritoneal cavity during thioglycolate-induced peritoneal inflammation, indicating a reduced inflammatory neutrophil response induced by mHMGB1. mHMGB1 was also able to reduce the number of neutrophils in the bronchoalveolar lavage fluid (BAL) of mice infected with Burkholderia cepacia. In other studies, a synthetic peptide containing only BoxA of HMGB1 was shown to affect macrophage function and could reduce liver injury in a mouse model of cirrhosis. These observations suggest that HMGB1 has the potential to affect neutrophils, and that some of these functions reside in specific domains. In fact, the applicant has constructed a series of truncated constructs of HMGB1 and found that the extended B-Box domain alone (B Box 97) has anti-biofilm activity comparable to that of the whole protein. In addition, the applicant demonstrated that a single point mutation (C106S) in the B Box, which is known to reduce the pro-inflammatory activity of HMGB1, has the same anti-biofilm activity. Here, the applicant used various truncated constructs of HMGB1 to determine whether any part of the protein specifically affects neutrophil function. Recombinant full-length HMGB1, ABox (amino acids 1 to 89), ABBox (amino acids 1-176), BBox-97 (amino acids 80 to 176), recombinant and synthetic mB Box-97 (identical to BBox-97 except for a single amino acid change C106S), and BBox-87 (amino acids 90 to 176) (Figure 1A) were all tested peptides to evaluate their ability to induce NETosis of isolated human neutrophils. Isolated human neutrophils were incubated with 200 nM constructs for 3-4 hours 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 an Alexa fluor 594-labeled anti-mouse secondary antibody. The cells were then visualized using confocal microscopy. 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). The applicant also tested whether any of these constructs had any effect on PMA-induced NETosis. For this purpose, isolated neutrophils were treated with PMA alone or with PMA and various constructs for 3-4 hours, stained for DNA and plasma membrane, and observed using confocal microscopy.Among the six constructs tested, only mB Box-97 showed an inhibitory effect on PMA-induced NETosis. After quantifying the NET% of total cells under different treatment conditions, only mB Box-97 showed a significant inhibitory effect on PMA-induced NETosis (Figure 1C). mB Box-97 inhibits PMA- and LPS-induced NET formation and the release of NET-related proteins.

[0305] PMA directly activates protein kinase C (PKC), while the binding of LPS to the corresponding cell receptor leads to the activation of PKC. Activated PKC phosphorylates the component proteins of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) assembly, resulting in its activation. Activated NOX generates reactive oxygen species (ROS), which in turn activates a series of events leading to chromatin decondensation and NET release. The DNA and protein markers released during this process can be visualized or quantified using specific antibodies. The applicant investigated whether inhibiting NET using mB Box-97 would affect the release of NET-related proteins such as NE and MPO. Isolated human neutrophils (2*10 5 ) were treated with PMA (100 nM, 3.5 hours) or heat-inactivated nontypeable Haemophilus influenzae (NTHI) (1*10 6 , 12 - 16 hours), with or without BBox-97 or mB Box-97 (200 nM). NETs were stained for the plasma membrane (WGA labeled with Alexa fluoro-488), αds-DNA (Alexa fluoro-594), and the protein markers of NETs - neutrophil elastase (NE), myeloperoxidase (MPO), or citrullinated histone (H3 Cyt) (Alexa fluor-405). Z-stack images were taken by confocal microscopy to observe the effect of the treatment on NET formation. mB Box-97 inhibited PMA-induced NETosis and NTHI-induced NETesis, while B Box97 had no effect. The applicant also calculated the mean fluorescence intensity (MFI) of NE, MPO, and H3-Cyt ( Figure 2A - 2C ). Under the mB Box-97 treatment condition, the MFI of NE and MPO was significantly lower than that of PMA alone or PMA treated with BBox-97. There was no difference in the MFI of citrullinated histone observed under the two treatment conditions ( Figure 2A - 2C ).

[0306] To quantify NETosis, the applicant used the cell-impermeable fluorescent DNA stain SYTOX-Green. Isolated neutrophils (5*10 3) Treat for 3.5 hours with PMA in a 96-well plate, with or without mB Box-97 and BBox-97, using separate medium as a control. The released extracellular DNA was stained with SYTOX Green (1 μM) for 10 minutes, washed with PBS, and the fluorescence was measured with a fluorometer. mB Box 97 showed significant inhibition of PMA-induced NET formation, while BBox had no significant effect (Figure 2D). The applicant also studied the release of nuclear elastase (NE) into the medium after PMA-induced NETosis and its binding to DNA. Quantification was performed using a human NE-specific ELISA kit. PMA was used with or without mB Box-97 or BBox-97, or without treatment, to allow neutrophils to form NETs in a six-well plate for 3 - 4 hours. The medium was collected, and the NETs formed were treated with DNA to isolate DNA-bound NE. The NE released into the medium and bound to DNA was measured by ELISA. mBBox-97 treatment significantly reduced the release of NE (secreted) into the medium (Figure 2E) and NE bound to DNA (DNA-bound) (Figure 2F). mB Box 97 partially inhibits Ca 2+ -mediated NETosis.

[0307] PMA- and LPS-mediated NETosis varies with Ca 2+ -ionophore A23187-induced NETosis. PMA-induced NETosis does not require PAD4, but A23187-mediated NET formation does. The NETs formed by A23187-induced calcium efflux involve the activation of PAD4, which citrullinates histones and leads to chromatin unfolding. This pathway mainly bypasses PKC-NOX2-ROS-mediated NETosis. Therefore, the applicant studied the effect of mB Box-97 on Ca 2+ -ionophore-mediated NETosis. Isolated PMNs were treated with A23187 with mB Box-97 or BBox-97 for 4 - 6 hours. NETs were observed with a confocal microscope. The applicant stained for NET-related marker proteins NE, MPO, or citrullinated histone H3. mB Box-97 inhibited Ca 2+ -ionophore-induced NETosis, but the inhibition was not as significant as that observed in PMA / LPS-mediated NETosis. The MFI of citrullinated histones indicated that mB Box-97 had no effect on histone citrullination in Ca2+-induced NETosis, and a similar effect was observed in PMA / LPS-mediated NETosis. Visual inspection and MFI values indicated an increase in H3-cyt staining after induction with A23187 (Figure 2C). Although only partially, mB Box-97 can inhibit Ca2+ Ionophore-induced NETosis. This may be because the elevation of Ca 2+ can activate PKC. These observations may indicate that the inhibition of NETosis by mB Box-97 may be mediated by the PKC / NOX-ROS pathway rather than the Ca 2+ -PAD4 pathway. mB Box-97 regulates the production of ROS in activated human neutrophils by inhibiting the phosphorylation of p47 phox .

[0308] PMA is a diacylglycerol mimic that activates PKC, which phosphorylates key components of the NOX complex, such as p47 phox , p67 phox and p40 phox . After phosphorylation, these component proteins aggregate in the active NOX complex to produce ROS. The production of ROS is important for the killing of microorganisms and the final breakdown of the nuclear and granule membranes to complete NETosis. Given the importance of ROS in NETosis, the applicant tested the effect of mB Box-97 on the production of ROS by neutrophils. Isolated neutrophils were pre-incubated with the chemiluminescent indicator of ROS, luminol. The cells were then activated with different treatment conditions, and ROS was measured over 2 hours with readings taken every 5 minutes. mB Box-97 significantly inhibited the production of ROS in PMA-induced human neutrophils (Figure 3A). The ROS level is an indicator of NOX activity, which depends on the phosphorylation of its component proteins. Therefore, the applicant investigated the effect of the phosphorylation of the key NOX component protein p47 phox on the phosphorylation of serine 370. The phosphorylation of this residue has been shown to be crucial for the assembly of the active NOX complex. Proteins in the treated neutrophils were separated by SDS-PAGE and the phosphorylation of p47 phox on ser-370 was detected using a phosphor-ser-370 specific antibody by Western blotting. Total p47 phox and GAPDH were used as controls. After densitometric analysis, it was clear that mB Box-97 significantly inhibited the phosphorylation of p47 phox , while its non-mutated counterpart BBox-97 had no effect on this (Figure 3B). The inhibition of NETosis, ROS production, and antimicrobial release by mB Box-97 can modulate the bactericidal effect of PMNs.

[0309] The ROS produced by neutrophils not only trigger the formation of NETs but also contribute to killing microorganisms. The applicant previously showed that the bacterial DNABII protein HU (HU NTHI) Inhibition of NET reduced neutrophil killing of bacteria. Since mB Box-97 was able to inhibit NETosis, ROS production, and release of antibacterial proteins such as NE, the applicant tested whether it had any effect on bacterial killing. To achieve this goal, the applicant incubated neutrophils with buffer alone, mB Box-97, Bbox-97, and HU NTHI for 4 hours together with NTHI biofilms that had formed for 16 hours. Neutrophils were lysed with Triton X-100 to recover live intracellular bacteria, and the total CFU NTHI in each case was counted to measure the relative percentage of bacterial killing caused by PMN NETosis compared to the buffer control alone in the system. Compared with BBox-97, mBBox-97 inhibited neutrophil bacterial killing, and this inhibition was comparable to that of HU NTHI used as a positive control (Figure 4A). mB Box-97 localized to the cytoplasm and plasma membrane of human neutrophils.

[0310] The applicant's various observations showed that the inhibitory effect of mB Box-97 on NETosis was obvious, but the specific target of the peptide was not. To gain a deeper understanding of the target, the applicant localized the peptide in neutrophils. To achieve this goal, the applicant synthesized an N-terminal His-tagged recombinant mB Box-97 (mB Box-97-His) peptide. The applicant tested the ability of this peptide to inhibit NETosis and inhibit ROS production (Figure 4B). After confirming the NET inhibitory ability of His-tagged mB Box-97, the applicant conducted a time-course study to determine the subcellular localization of the peptide. Neutrophils were incubated with His-tagged mB Box-97 for 10, 30, 60, 120, or 180 minutes, fixed with 10% formalin, probed for the peptide with anti-His rabbit primary antibody, and then detected with Alexa fluor-405-labeled goat anti-rabbit secondary antibody. DNA was detected with mouse anti-double-stranded DNA primary antibody and then detected with goat Alexa fluor-595-labeled anti-mouse secondary antibody, while the plasma membrane was stained with Alexa fluor 488-labeled WGA. Images were taken using a confocal microscope. The applicant observed that within 120 minutes after treatment, the signal of mB Box-97 was still strong in most neutrophils. Most of the peptide localized to the cytoplasm and plasma membrane. At 180 minutes, some of the peptide seemed to localize to the nucleus. Protein kinase C is the target of mB Box-97.

[0311] PKC is a key kinase in NETosis, and its inhibition leads to the inhibition of NETosis. PKC is involved in the phosphorylation of the active NOX complex and thus in its assembly. Based on the observation of the cytoplasmic localization of mB Box-97 and the inhibition of p47 phox phosphorylation, the applicant investigated whether mB Box-97 has any effect on PKC activity. To achieve this goal, the applicant used a PKC activity kit. Two different concentrations of mB Box-97 were incubated with PKC for 10 minutes, and then the mixture of the two was incubated with the substrate.( Figure 5 )

[0312] The formation of NETs is an important function of the innate immune system. Studies showing the importance of NETs in the treatment of various diseases are increasing, as are studies showing the negative effects of excessive NET formation. Essentially, NET formation must maintain a balance between its production and clearance. In vitro, various upstream stimulants, including LPS, TNF, IL-8, and PKC agonists, as well as some pro-inflammatory molecules such as HMGB1, can activate the formation of NETs. During NETosis, various bioactive molecules are released, leading to the death of microorganisms. However, due to their non-specificity, the components of NETs themselves or by increasing the pro-inflammatory response can cause damage to surrounding tissues. They can also play a role in exacerbating inflammation in autoimmune diseases such as psoriasis, rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE). In addition, autoinflammatory diseases such as gout are associated with NETosis. Excessive NETs can also cause physical barriers in the bloodstream, leading to atherosclerosis and / or stroke. In pancreatic cancer, cancer cells can increase NETosis through activated platelets. NET-mediated platelet activation can promote several negative outcomes associated with advanced metastatic breast cancer, including venous thromboembolism (VTE).

[0313] Recent studies on COVID-19 have shown that neutrophil infiltration enhancement, NET release, complement activation, and vascular thrombosis play important roles during COVID-19 necrotic inflammation. The formation of NETs in the microvasculature increases the inflammatory response and vascular microthrombosis, leading to ARDS in the lungs of patients. The serum levels of NETosis markers in patients under intensive care and mechanical ventilation are higher than those in patients breathing room air, indicating that NETosis may also be related to the disease severity of COVID-19. Inhibiting NETs can reduce the severity of many such diseases, thus improving survival rates. Here, the applicant describes a HMGB1-based mutant peptide, mB Box-97, which inhibits the formation of NETs induced by different stimuli.

[0314] There are multiple direct or indirect NET formation inhibitors in preclinical studies, although none of them have been approved for the treatment of NET-related complications. Some of them are molecules that have been used clinically for many years, such as hydroxychloroquine, methotrexate, and prednisolone (the active metabolite of prednisone). Some are humanized antibodies, such as rituximab (anti-CD20 monoclonal antibody), belimumab (fully human IgG1λ recombinant monoclonal antibody), and tocilizumab (anti-IL-6 receptor monoclonal antibody). Others are molecules that can inhibit the functions of NE, MPO, or PAD4. Most of them are indirect inhibitors of NETosis. Although multiple inhibitors are being tested, there is still a need for effective inhibitors that do not cause unnecessary side effects and do not hinder the normal functions of neutrophils other than NETosis.

[0315] In a previous study, the applicant showed that mutant HMGB1 lost its pro-inflammatory activity while retaining its anti-biofilm activity (Devaraj A. et al., 2021, J Clin Invest 131(16)). In the present disclosure, the applicant constructed a mutant peptide mB Box-97 based on HMGB1, which retained the anti-biofilm ability against multiple microorganisms without showing any pro-inflammatory activity. The applicant observed that this peptide could inhibit NET formation induced by PMA and LPS, while showing partial inhibition when tested against Ca 2+ induced NET formation. These stimuli activate different pathways, so the results of inhibition using mB Box-97 are also different. PMA- and LPS-mediated NETosis usually involves the activation of PKC, leading to the phosphorylation of key components of NADPH oxidase, which ultimately contributes to the formation of the NOX complex and the production of ROS. The applicant studied the effect of mB Box-97 on p47 phox phosphorylation and found that its phosphorylation was inhibited by the peptide (Figure 3B). The applicant also observed the inhibition of ROS production, which was consistent with the inhibition of p47 phox phosphorylation. The production of ROS is very important for the release of antimicrobial agents by granulocytes and the release of decondensed chromatin. ROS triggers the dissociation of NE from the membrane-bound complex into the cytoplasm and activates its proteolytic activity in an MPO-dependent manner. Activated NE moves to the nuclear mitotic chromatin and releases it into the cytoplasm, thus completing the formation of NET. The applicant observed a decrease in the release of NE by neutrophils after treatment with mB Box-97 (Figure 2E). The formation of NET, the release of antimicrobial agents, and the production of ROS all affect the killing of microorganisms by NET. Since mB Box-97 can inhibit all of these, the applicant tested its effect on microbial killing and found that it was able to inhibit neutrophil-mediated bacterial killing (Figure 4A).

[0316] PKC is a key kinase in the process of NETosis, and its inhibition impairs NETosis. Most of the evidence in this study indicates that mB Box-97 is involved in the inhibition of PKC activity. For example, the inhibition of p47phox phosphorylation (Figure 3B), as well as the localization of the peptide in the cytoplasm and plasma membrane. Therefore, the applicant studied the effect of mB Box-97 on PKC activity using a standard PKC assay kit and found that mB Box-97 inhibits PKC activity( Figure 5 ). The partial inhibition of Ca 2+ -mediated NETosis by mB Box-97 can be attributed to the inhibition of PKC, because the influx of Ca 2+ can activate PKC in addition to activating PAD4.

[0317] Without being bound by a particular theory, it is believed that the HMGB1-based peptide mB Box-97 inhibits the activity of protein kinase C, thereby inhibiting the phosphorylation of key NADPH oxidase component proteins. This inhibits the active assembly of NOX and reduces the production of ROS. The reduction in ROS production hinders the release of granule-associated proteins such as NE and ultimately hinders the production of NETs( Figure 6 ). mBBox-97 does not cause inflammation while retaining the ability to inhibit biofilm formation. mB Box-97 has excellent therapeutic potential. Treat rheumatoid arthritis using mB Box-97.

[0318] Abnormal, excessive NETosis is considered a potential cause of autoimmune diseases such as rheumatoid arthritis. To demonstrate that subjects with rheumatoid arthritis (RA) will benefit from using the mB Box-97 peptide to inhibit abnormal, excessive NETosis, a preclinical animal model of RA will be used. 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 hereby incorporated by reference 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 arthropathy (AA) model, a delayed-type hypersensitivity arthritis (DTHA) model, an anti-citrullinated peptide antibody (ACPA)-mediated arthritis model, or a spontaneous model.

[0319] Treat an animal model of RA with 50 nM to 2 μM of the mB Box-97 peptide as described herein, and regularly measure the diagnostic blood markers of RA (such as C-reactive protein (CRP), rheumatoid factor (RF), or anti-cyclic citrullinated peptide (anti-CCP)) (such as once every 3 hours, once every 6 hours, once every 12 hours, once every 24 hours, once every other day, once every three days, once every four days, once every five days, or once every six days, or once a week). Compared with the control group administered with a scrambled control peptide, in the animals treated with mB Box-97, a decrease in the diagnostic blood markers of RA was observed over time. Compared with the control group administered with a scrambled control peptide, mB Box-97 also observed a reduction in RA symptoms (such as joint swelling, ankle effusion, joint pain, joint inflammation). Example 2: mB Box-97 disrupts and prevents biofilm formation of multiple human pathogens. The HMGB1-derived peptides containing B-Box and mB Box-97 retained the in vitro biofilm-disrupting activity.

[0320] First, the applicant tested the disrupting ability of the HMGB1-derived peptides. Adding A Box to the established biofilms (UPEC, Burkholderia cepacia, NTHI, or Klebsiella pneumoniae) had no significant effect on the measured biofilm parameters (Figure 7A). The anti-biofilm activity of BBox-87 was limited, while A-B Box and B Box-97 retained the anti-biofilm activity (Figure 7A). Since only B Box can regulate DNA bending, the applicant hypothesized that HMGB1 disrupts biofilms at least in part through DNA binding / bending. It has been reported that B Box contains pro-inflammatory activity, mainly mediated by interaction with TLR4-MD2 dependent on residue C106. The applicant created a modified recombinant B Box-97 variant (mB Box-97) with a C106S mutation (Figure 1A). The mB Box-97 variant disrupted the biofilms formed by UPEC, NTHI, Burkholderia cepacia, and Klebsiella pneumoniae in vitro, comparable to that induced by B Box-97. mB Box-97 disrupts biofilms in vivo

[0321] To determine whether mB Box-97 could prevent lung infections and limit inflammation as expected, the applicant used 10 7 (Figure 8A) or 10 8(Figure 8B) Adult C57BL / 6 mice were challenged by intratracheal injection (i.t.) with Burkholderia cenocepacia (Bc) CFU and 200 nM of mB Box-97 or a negative control. After 18 hours, the mice were euthanized to collect BAL fluid and lung tissue. The lung tissue was homogenized, diluted, and plated to determine the relative CFU, and the BAL fluid was similarly diluted and plated. Compared with control group mice, the Bc content in the BAL and lung tissues of mice administered rHMGB1 or mB Box-97 was significantly reduced, indicating that mB Box-97 can inhibit the formation of aggregated biofilms in the airways of mice even at a high bacterial challenge dose (10 8 CFU). In addition, this method promoted bacterial clearance, thus demonstrating a preventive effect. Furthermore, although a potent therapeutic dose of mB Box-97 was used, no mice showed signs of sepsis requiring euthanasia before the study endpoint. Although LPS alone induced more than 100 pg / ml of TNF-α (a gold standard surrogate for sepsis induction), neither rHMGB1 nor mBox-97 induced detectable TNF-β, nor did they induce additional proinflammatory signals when administered to mice pretreated with LPS. Here, the applicant demonstrated that mB Box-97 maintains its anti-biofilm activity without inducing inflammation by restricting bacterial load and / or preventing the formation of biofilm aggregates in the mouse lung and without inducing TNF-α. Synthetic mB Box-97 (mB Box-97 syn ) is equivalent to recombinant mB Box-97 in vitro

[0322] Native HMGB1 undergoes extensive modifications after translation, which affect various HMGB1 functions. In fact, even recombinant proteins expressed in bacteria often have post-translational modifications. To determine whether these modifications play a role in anti-biofilm disruption, the applicant synthesized an identical 97-amino acid peptide based on the coding sequence of recombinant mB Box-97, hereafter referred to as mB Box-97 syn . To determine whether mB Box-97 syn retains DNA-binding activity, the applicant performed electrophoretic mobility shift assays using Holiday junction DNA substrates that mimic the cross-strands of the bacterial biofilm eDNA lattice and are also native substrates of HMGB1, and compared the ability of all mB Box-97 syn to various truncated forms of HMGB1. Recombinant versions of all truncated forms containing the full domain structure, including mB Box-97 and mB Box-97 syn, can similarly bind to Holliday junction DNA. As an additional test of stability, recombinant mB Box-97 and mB Box-97 syn were incubated in human serum and showed similar stability, which is consistent with the fact that any post-translational modifications present on recombinant mB Box-97 do not affect protein stability. As a final test, the applicant also showed that mB Box-97 syn significantly disrupted four additional high-priority ESKAPEE pathogens, regardless of the biofilm formation time (Figure 7B). Here, the applicant also used a humanized monoclonal antibody against the DNA-binding head 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 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 (Pseudomonas aeruginosa: 30.5%, 29.8%, Staphylococcus aureus: 12.2%, 24.8%, Enterococcus faecalis: 39.2%, 29.1%, Acinetobacter baumannii: 16.7%, 27.8% (P = 0.03 - P < 0.0001).

[0323] To determine whether longer biofilm formation times would affect the ability of mB Box-97 syn to disrupt bacterial biofilms, as these biofilms contain increasing concentrations of eDNA, the applicant tested this hypothesis using biofilms formed by the respiratory pathogen NTHI. For this purpose, the applicant treated 48-hour and 72-hour NTHI biofilms with 1.2 μg of mB Box-97 or 5.0 μg of a mouse monoclonal antibody (MsTipMab) against the same epitope as HuTipMab. Although these longer-formed NTHI biofilms were not disrupted by incubation with a mouse monoclonal antibody (MsTailMab) against the non-protective domain of the bacterial DNABII protein, they were significantly disrupted by mB Box-97 and MsTipMab compared to medium alone (48 hours: 47.3% and 47.2%. 72 hours: 46.4% and 34.4%). Biofilms grown for 96 hours required twice the concentration of mB Box-97 syn or MsTipMab as above to produce similar disruption (e.g., 49% and 37% respectively), as expected since eDNA and DNABII concentrations increase as the biofilm matures. These results were statistically significant (P = 0.02 and P = 0.005 respectively). Thereafter, the applicant used only mB Box-97 in the remaining experiments syn . mB Box-97 synPrevents in vitro biofilm formation

[0324] The Applicant now hypothesizes that mB Box-97 may also prevent the formation of bacterial biofilms, as this hypothesis is consistent with the currently demonstrated ability of the domain variant to disrupt established biofilms in vitro. To test this hypothesis, the Applicant first tested the ability of mB Box-97 to prevent biofilm formation by NTHI and Staphylococcus aureus (Gram-negative and Gram-positive pathogens), respectively, using the same two concentrations of mB Box-97 as previously used for the in vitro disruption assay. rHMGB1 and mB Box-97 rec were used as positive controls, while only medium and A Box were used as negative controls. The Applicant now also wishes to test two concentrations of HuTipMab, which has previously been shown to have broad biofilm-disrupting activity, as it may also have the potential to prevent biofilm formation. After 16 hours of incubation, compared to the negative control, the positive control and all tested concentrations of mBBox 97 or HuTipMab significantly inhibited the biofilm growth of NTHI and Staphylococcus aureus (Figure 9A) (P = 0.01 - P < 0.0001). The highest tested concentrations of mB Box-97 and HuTipMab (e.g., 1.8 μg and 7.5 μg, respectively) limited the growth to a bacterial monolayer or less (biomass < 1.0 μm 3 / μm 2 ), with no characteristic 3D biofilm structure.

[0325] Next, the Applicant began to determine the range of the ability of mB Box-97 and / or HuTipMab to prevent biofilm formation. To this end, the Applicant used the most effective concentration of mB Box-97 or HuTipMab to prevent the biofilm growth of NTHI and Staphylococcus aureus, and similarly to prevent the formation of biofilms by the remaining high-priority ESKAPEE pathogens or Burkholderia cepacia (Figure 9B). The Applicant observed that compared to the negative control group, after incubation with mB Box-97 (P = 0.03 - P < 0.0001) or HuTipMab (P = 0.02 - P < 0.0001) for 16 hours, the biofilms of Pseudomonas aeruginosa, Enterobacter spp., Enterococcus faecalis, uropathogenic Escherichia coli, Acinetobacter baumannii, or Burkholderia cepacia were significantly prevented. To provide additional evidence for the observed preventive activity, the data generated by the pathogen Klebsiella pneumoniae (Figure 10A) were presented together with the corresponding representative CSLM images (Figure 10B). These differences were statistically significant (P = 0.03 - P < 0.0001). mB Box-97 syn acts synergistically with a humanized monoclonal antibody against the protective domain of the DNABII protein to prevent biofilm formation

[0326] As shown in FIG. 4 and Figure 5 as indicated, when tested individually, both mB Box-97 and HuTipMab significantly blocked biofilm formation by 9 pathogens, including 7 high-priority ESKAPEEs. Here, the Applicant now evaluated whether mB Box-97 and HuTipMab could act additively or synergistically to prevent biofilm formation. To evaluate this, the Applicant again used NTHI and Staphylococcus aureus as model pathogens. The Applicant first incubated NTHI or Staphylococcus aureus with 1:2 serial dilutions of the maximum doses of mB Box97 or HuTipMab used in the earlier prevention trials to determine relative preventive activity.

[0327] As shown, the preventive effects of the HuTipMab dilutions on NTHI biofilms (top row, FIG. 11A) ranged from 0 - 72%, while those of the mB Box 97 dilutions (last column, FIG. 11A) ranged from 0 - 43%. For Staphylococcus aureus, the preventive effects of the HuTipMab dilutions (top row, FIG. 11B) similarly ranged from 0 - 74%, while those of the mB Box97 dilutions (last column, FIG. 11B) ranged from 0 - 60%. To evaluate the potential additive or synergistic preventive activity of the two biologics, the Applicant then incubated NTHI or Staphylococcus aureus with mixtures of mB Box-97 plus HuTipMab in multiple similar 1:2-fold dilutions for 16 hours and determined the relative biomass (see the values within the diagonal boxes in the two insets of FIG. 11). For NTHI (inset A, diagonal box), for mixtures composed of the full combined dose down to mixtures containing each 1 / 16 dose, the relative percentage preventive values exceeded those of either individual component. When each biologic was used at a 1 / 8 dose, the result was synergistic (66% combined use compared to 20% or 5% alone). For Staphylococcus aureus (inset B, diagonal box), for mixtures composed of the full combined dose down to mixtures containing each 1 / 8 dose, the relative percentage preventive values again exceeded those of either individual component alone. Although no clear synergistic results were found mathematically, there was additive prevention when the Applicant evaluated prevention using mixtures of 1 / 4 and 1 / 8 doses of each biologic (69% compared to 41% or 26% alone; 57% compared to 38% or 18% alone).

[0328] The applicant found that, compared to the prevention with the highest tested concentration of each treatment administered alone (the maximum concentration of the tested HuTipMab prevented 72% of NTHI biofilm growth and the maximum concentration of mB Box-97 prevented 43% of NTHI biofilm growth compared to the negative control. The maximum concentration of the tested HuTipMab prevented 74% of Staphylococcus aureus biofilm growth and the maximum concentration of mB Box-97 prevented 60% of Staphylococcus aureus biofilm growth compared to the negative control), mBBox-97 and HuTipMab, when used in combination and tested against both pathogens, produced a greater preventive effect at sub-maximum tested concentrations (the 1 / 2 maximum concentration of both treatments prevented 75% of NTHI biofilm compared to the negative control, and the 1 / 2 maximum concentration of both treatments prevented 79% of Staphylococcus aureus biofilm compared to the negative control).

[0329] The applicant also determined the combination index This calculation was used to evaluate the overall synergy between two or more compounds. The scores for NTHI and Staphylococcus aureus were 0.4 and 0.5, respectively, indicating that mB Box-97 and HuTipMab have a synergistic effect when used in combination, as a CI score < 1 indicates synergy. Materials and Methods

[0330] A humanized monoclonal antibody against the head chimeric peptide, designed to mimic the immunoprotective domain of the DNABII protein integration host factor (IHF). The humanized monoclonal antibody (IgG isotype) against the head chimeric peptide (HuTipMab) was derived from a murine monoclonal antibody.

[0331] Synthesized mB Box-97. (mB Box-97 syn ; LLC; facilitating large-scale production and uniformity > 95%)

[0332] Bacterial species and sources. The NTHI strain 86-028NP was isolated from the nasopharynx of a child with chronic otitis media at Nationwide Children's Hospital in the United States. Enterobacter and Klebsiella pneumoniae were isolated therefrom. The Staphylococcus aureus strain 29213, Acinetobacter baumannii strain 17978, and Pseudomonas aeruginosa strain 27853 were obtained from ATCC. The Enterococcus faecalis Com12 strain was isolated from the feces of healthy human volunteers. Strains not obtained from ATCC were cryopreserved at low passage numbers in liquid nitrogen.

[0333] Disruption of bacterial biofilms. NTHI and Staphylococcus aureus were cultured on chocolate agar at 37 °C in a humidified atmosphere containing 5% CO2 for 18 - 24 hours. Then NTHI was resuspended in brain heart infusion broth (sBHI) supplemented with heme [2 μg / mL] and β-NAD [2 μg / mL] broth to an OD of 0.1 at 490 nm. Staphylococcus aureus was resuspended in brain heart infusion broth (BHI) to an OD 490nm of 0.1. Then the cultures were diluted in their respective media to approximately 2×10 5 CFU / mL, and 200 μL of this suspension was inoculated into each well of an 8-well chamber slide (Thermo Fisher Scientific, Waltham, MA). As described above, Pseudomonas aeruginosa and Enterococcus faecalis were cultured on tryptic soy agar (TSA) and then suspended in tryptic soy broth (TSB) to an OD 490nm of 0.1. Then, as described above, the cultures were diluted in their respective media and 200 μL of this suspension was inoculated into each well of an 8-well chamber slide. After culturing each bacterium for 16 hours at 37 °C in 5% CO2, the medium was replaced with the respective fresh medium and cultured for another 8 hours. After 24 hours, the medium was replaced with the respective fresh medium (control) or fresh culture medium containing mB Box-97 syn (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 in 5% CO2 for 2 hours. Then, all biofilms were washed twice with 1× Dulbecco’s phosphate-buffered saline (DPBS) without calcium or magnesium (Corning, Corning, NY) according to the manufacturer's instructions and stained with LIVE / DEAD stain (Thermo Fisher Scientific, Waltham, MA). The biofilms were washed again and then fixed in 1.6% paraformaldehyde, 0.025% glutaraldehyde, and 4% acetic acid in 0.1 M phosphate buffer at pH 7.4. The biofilms were imaged on a Zeiss 800 confocal laser scanning microscope (CLSM; Zeiss) with a ×63 objective and analyzed with COMSTAT2. The biomass values (μm 3 / μm 2 ) were calculated by COMSTAT2 and represent the mean ± SEM of 3 biological replicates.

[0334] Prevention of bacterial biofilm formation. As described above, NTHI and Staphylococcus aureus were cultured and resuspended, then diluted in their respective media until they contained approximately 5×10 3 CFU / mL or approximately 1×103 CFU / mL, and 200 μL of this suspension was inoculated into each well of an 8-well chamber slide. As described above, the UPEC strain UTI89, Burkholderia cepacia, Klebsiella pneumoniae, Enterobacter, and Acinetobacter baumannii were cultured and resuspended, and then diluted in LB broth to contain ~1×10 3 -1×10 5 CFU / mL, and 200 μL of this suspension was inoculated into each well of an 8-well chamber slide. As described above, Pseudomonas aeruginosa and Enterococcus faecalis were cultured and suspended, and then diluted to contain ~1×10 3 CFU / mL, and 200 μL of this suspension was inoculated into each well of an 8-well chamber slide. After incubation at 37 °C in 5% CO2 for 16 hours, the HMGB1 isoform rHMGB1 (1.2 μg / 200 μL), A Box (1.2 μg / 100 μL), mB Box-97 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) were added to the respective wells of all test pathogens. After incubation, all biofilms were then washed once with DPBS and fixed as described above. As described above, the biofilms were imaged and analyzed. On different days, all assays were repeated at least 3 times. Data are presented as mean + / - SEM. The HuTipMab antibody, as described in U.S. Patent 11,104,723, comprises a heavy-chain complementarity-determining region 1 (CDRH1) that comprises the sequence GFTFRTY (aa 50 to aa 56 of SEQ ID NO:9); a heavy-chain complementarity-determining region 2 (CDRH2) that comprises the sequence GSDRRH (aa 76 to aa 81 of SEQ ID NO:9); a heavy-chain complementarity-determining region 3 (CDRH3) that comprises the sequence VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO:9); a light-chain complementarity-determining region 1 (CDRL1) that comprises the sequence QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO:15); a light-chain complementarity-determining region 2 (CDRL2) that comprises the sequence LVS (aa 75 to aa 77 of SEQ ID NO:15); and a light-chain complementarity-determining region 3 (CDRL3) that comprises the sequence WQGTHFP (aa 114 to aa 120 of SEQ ID NO:15).

[0335] Determination of the synergistic effect of mB Box-97 with HuTipMab. As described above, NTHI and Staphylococcus aureus were cultured on chocolate agar, then resuspended and diluted in their respective media until they contained ~5×103 or ~1×10 3 CFU / mL, and 200 μL of this suspension was inoculated into each well of an 8-well chamber slide. mB Box-97 and HuTipMab were added to these bacterial solutions to obtain 0.05 - 1.8 and 0.23 - 7.5 μg / 200 μL. After incubation at 37 °C in 5% CO2 for 16 h, the biofilm was washed once with sterile saline and fixed, imaged, and analyzed as described above. All in vitro biofilm assays were repeated at least 3 times on different days. Data are presented as mean + / - SEM.

[0336] Statistical analysis. GraphPad Prism 9 was used for graphical results and statistical tests for all in vitro assays. Statistical significance of in vitro assays was evaluated by one-way ANOVA and multiple comparisons. A P value less than 0.05 was considered significant. Example 3: H-NS inactivates neutrophil extracellular traps As the biofilm matures, H-NS is released into the bulk medium

[0337] H-NS is a B-DNA binding protein found extracellularly in bacterial cells within the biofilm. When H-NS was depleted within the biofilm, it had no effect on the biomass and thickness of the biofilm, indicating that H-NS is not essential for biofilm structure like DNABII proteins. When measuring the ratio of H-NS fluorescence intensity to cell fluorescence intensity, it was found that in UPEC, NTHI, and Streptococcus pneumoniae biofilms, the H-NS fluorescence brightness increased from 24 h to 40 h, then decreased at 72 h, and continued to decrease as the biofilm reached 1 week old. In Streptococcus pneumoniae biofilms, the F.I. ratio of H-NS to cells at 1 week was too low to be quantified ( Figure 13A - 13B ).

[0338] To confirm that H-NS is leaving the biofilm and entering the bulk medium, the concentration of H-NS in the biofilm and medium was measured using Western blotting. Comparing biofilms at 16 h and 1 week old showed a shift of most of the H-NS present in the biofilm to the medium, indicating that the level of H-NS within the biofilm is decreasing due to the release of H-NS during biofilm maturation ( Figure 14 ).

[0339] To further confirm that the level of H-NS in the biofilm is decreasing to enter the bulk medium rather than due to changes in gene expression, RT-PCR was performed. H-NS blocks neutrophil DNA release and condenses previously released NET eDNA

[0340] In vivo, neutrophils release NET eDNA when stimulated by pathogens, but NETosis can also be stimulated in vitro using PMA. The DNA strands that leave the cell are due to the unwinding of nuclear DNA when histones are citrullinated, which can be observed by confocal microscopy. Considering that both H-NS and CbpA can bind to bent DNA, but CbpA does not condense DNA, the DnaJ homolog CbpA, which preferentially binds to bent DNA, was used as a negative control. H-NS prevents the release of neutrophil DNA, while CbpA does not ( Figure 15A - 15B ). When neutrophils are stimulated with PMA to form NETs, in the case of preformed NETs, H-NS is also able to condense neutrophil eDNA. In addition to eDNA, further staining of antimicrobial agents that bind to NET eDNA indicates that the antimicrobial agents remain bound to eDNA and are not displaced by H-NS. H-NS prevents NET killing of bacteria

[0341] Although NETs have limited ability to penetrate and kill bacteria within biofilms, they are able to kill free bacteria and can prevent biofilm growth. NETs rely on the DNA tentacles they release to kill bacteria, while H-NS causes these tentacles to condense. In addition to altering NET morphology, H-NS most likely also prevents NETs from killing bacteria. HU has been shown to convert NET eDNA into Z-DNA and prevent NET killing, and was therefore used as a positive control and comparison to determine to what extent H-NS can prevent NET killing of bacteria4. H-NS significantly reduced the killing of bacteria by NETs as much as HU ( Figure 16 ). CbpA cannot prevent NETs from killing free bacteria, which is related to CbpA's inability to alter NET morphology and prevent NETosis. The ability of H-NS to prevent NETs from clearing free bacteria confirms that by condensing the eDNA of NETs, H-NS may represent a defense of the biofilm against the host immune system. Materials and methods

[0342] Bacterial strains and plasmids. The NTHI strain 86-028NP is a clinical isolate obtained from the nasopharynx of a child undergoing tympanostomy tube insertion, streaked on chocolate agar, and cultured overnight in an incubator at 37 °C with 5% CO2. The uropathogenic Escherichia coli (UPEC) (SG1019) is a clinical isolate from a clinical isolate of febrile urinary tract infection and renal abscess, streaked and incubated overnight at 37 °C with 5% CO2. The Streptococcus pneumoniae 1121 (SG1241) was streaked on blood agar and incubated overnight at 37 °C with 5% CO2.

[0343] Expression and purification of NAP. The expression and purification of NTHI H-NS, NTHI CbpA, and NTHI HU have been described previously7. Briefly, H-NS, CbpA, and HU were all cloned into the pTXB1 vector and transformed into the ER2566 Escherichia coli expression strain. Growth occurred in LB containing 100 μg / mL ampicillin, and then protein overexpression was induced using 100 mM IPTG. Each protein was purified on a chitin resin column and then further purified 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 generated previously against recombinant NTHI H-NS and used to label H-NS in NTHI, Streptococcus pneumoniae, and UPEC biofilms of different formation times. All biofilms were initiated in 8-well glass-bottom chamber slides, the medium was aspirated, and then fresh medium was added in 8-hour and 16-hour increments, with the exception of the one-week biofilm, 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 hemin, UPEC was resuspended in LB, and Streptococcus pneumoniae was resuspended in Todd-Hewitt broth (THB) (BD Diagnostic Systems) supplemented with 0.2% yeast extract. All resuspensions were added to the chamber slides at a concentration of 2×10 5 cells / mL. Biofilms were grown for 24 hours, 40 hours, 72 hours, and one week. When 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 incubation at room temperature for 2 hours, the wells were washed once with PBS, and then goat anti-rabbit Alexa Fluor 488 and FM 4-64 (both diluted 1:20 in 5% BSA in PBS) were added, and then the biofilms were incubated for 1 hour at room temperature in the dark, and then the biofilms were washed once more with PBS and observed on a Zeiss 800 confocal laser scanning microscope (LSM). The mean fluorescence intensity of H-NS was determined, and cells were measured using ImageJ software.

[0345] Visualization of NETs. Using EasySep from StemCell Technologies (Cambridge, MA) TMThe human neutrophil isolation kit isolates NETs from freshly collected blood of healthy donors. The isolated neutrophils are quantified, and 200,000 cells are added to each well of an 8-well chamber slide. Then, the neutrophils are 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) is added to the neutrophils to induce NETosis. To determine whether H-NS can prevent NETosis, 200 nM NTHI H-NS is added simultaneously with PMA. When determining whether H-NS can condense previously released NET eDNA, H-NS is added 16 hours after inducing NETosis. After adding PMA, the neutrophils are incubated at 37 °C and 5% CO2 for 3.5 hours. Then the neutrophils are fixed with 0.4% formalin and 0.1% Triton X-100 is added for cell permeabilization. Then the cells are blocked with 5% normal goat serum at 37 °C and 5% CO2 for 30 minutes, after which the primary antibody is added and incubated overnight at 4 °C. The primary antibodies of α-neutrophil elastase rabbit, α-citrullinated H3 rabbit, and α-DNA mouse are diluted 1:500 in PBS. The next day, the secondary antibody is added for 1 hour at 37 °C and 5% CO2. Then the slides are visualized on a Zeiss 800 confocal laser scanning microscope.

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

[0347] RT-PCR was used to detect the expression level of H-NS. NTHI biofilms were grown in a BioLite 25 cm3 tissue flask (ThermoFisher) in 6 mL of sBHI medium for 16 hours and one week at an initial concentration of 2×10 5 cells / mL. Before RNA isolation, all surfaces and pipettes were cleaned with RNAaseZap TM (ThermoFacher). The medium was removed by inverting the flask and pouring out the medium. 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 TM (ThermoFisher) for a 0.25 mL sample, and the homogenized bacteria were incubated in Trizol TM for 5 minutes at room temperature. After 5 minutes, 200 μL of chloroform was added per 1 mL of Trizol TM , and the tube containing all the reagents and bacteria was shaken vigorously for 15 seconds. After shaking, the tube was incubated at room temperature for 12 minutes and then centrifuged at 4 °C and 4000 rpm for 5 minutes. The top clear phase formed after centrifugation was then removed and placed into a new 1.5 mL capped centrifuge tube. RNA purification was then performed using the Qiagen RNeasy Mini Kit (Qiagen, Germantown, MD). On-column DNase I digestion (Sigma-Aldrich, St. Louis, MO) was used during the isolation to eliminate residual DNA. equivalent

[0348] It should be understood that although the present disclosure has been described in conjunction with the above embodiments, the above description and examples are intended to illustrate rather than limit 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 the present 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 pertains. All nucleotide sequences provided herein are presented in the 5' to 3' direction.

[0350] The embodiments described herein by way of example can be suitably implemented without any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, the terms "comprising", "containing", "including", etc. should be understood broadly and without limitation. In addition, the terms and expressions used herein have been used as descriptive terms and not restrictive terms, and in using these terms and expressions, it is not intended to exclude any equivalents of the features shown and described or portions thereof, but it should be recognized that various modifications can be made within the scope of the present disclosure.

[0351] Accordingly, it should be understood that although the present disclosure has been specifically disclosed by way of specific embodiments and optional features, those skilled in the art can make modifications, improvements and variations to the embodiments disclosed herein, and such modifications, improvements and variations are considered to be within the scope of the present disclosure. The materials, methods and examples provided herein are representative of specific embodiments, are exemplary, and are not intended to limit the scope of the present disclosure.

[0352] The scope of the present disclosure has been described herein broadly and generally. Each narrower genus and subgenus grouping falling within the general disclosure also forms part of the present disclosure. This includes the general description with conditional or negative limitations deleting any subject matter, whether or not the deleted material is specifically recited herein.

[0353] In addition, where the 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 can thus also be described 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 hereby 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) shall control. Non-limiting embodiments of the present disclosure

[0355] Embodiment 1. A synthetic polypeptide comprising mB Box-97, wherein mB Box-97 consists of amino acids 90 to 176 of the coding sequence of the native human HMGB1 protein shown in SEQ ID NO:2 and has a cysteine to serine point mutation at amino acid 106, or an equivalent thereof.

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

[0357] Embodiment 3. The synthetic or recombinant polypeptide according to Embodiment 1 or 2, wherein the synthetic or recombinant polypeptide consists of SEQ ID NO:5.

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

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

[0360] Embodiment 6. The synthetic or recombinant polypeptide according to Embodiment 4 or 5, wherein the synthetic or recombinant polypeptide consists of SEQ ID NO:6.

[0361] Embodiment 7. The synthetic or recombinant polypeptide according to 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 the polynucleotide encoding the amino acid sequence or its complementary sequence under high stringency conditions, wherein the 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 washing solution of about 1x SSC, 0.1x SSC, or deionized water; and 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. The synthetic or recombinant polypeptide according to any one of Embodiments 1-7, and a detectable label.

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

[0364] Embodiment 10. A plurality of the synthetic or recombinant polypeptides according to any one of Embodiments 1-9, optionally wherein the members of the plurality of polypeptides are the same or different from each other.

[0365] Embodiment 11. A composition comprising the synthetic or recombinant polypeptide according to any one of Embodiments 1-9 or the polypeptides according to Embodiment 10, and a carrier, optionally the carrier is a pharmaceutically acceptable carrier.

[0366] Embodiment 12. An isolated polynucleotide encoding the synthetic or recombinant polypeptide according to any one of Embodiments 1-9, and an optional carrier or a pharmaceutically acceptable carrier.

[0367] Embodiment 13. The isolated polynucleotide according to Embodiment 12, and a detectable label, and an optional carrier or a pharmaceutically acceptable carrier.

[0368] Embodiment 14. A carrier comprising the isolated polynucleotide according to Embodiment 12 or 13, and an optional carrier or a pharmaceutically acceptable carrier.

[0369] Embodiment 15. The isolated polynucleotide according to Embodiment 12 or 13, or the carrier according to Embodiment 14, further comprising a heterologous promoter sequence, and an optional carrier or a pharmaceutically acceptable carrier.

[0370] Embodiment 16. An isolated host cell comprising one or more of the following: the synthetic or recombinant polypeptide according to any one of Embodiments 1-9, the polypeptides according to Embodiment 10, the isolated polynucleotide according to any one of Embodiments 12 or 13, or the carrier according to Embodiment 14, and an optional carrier or a pharmaceutically acceptable carrier.

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

[0372] Embodiment 18. The isolated host cell according to 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, comprising administering to the subject an effective amount of one or more of the following: (i) the synthetic or recombinant polypeptide according to any one of Embodiments 1-9, (ii) the polypeptides according to Embodiment 10, (iii) the composition according to Embodiment 11, (iv) the isolated polynucleotide according to Embodiments 12 or 13, or (iv) the carrier according to Embodiment 14.

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

[0375] Embodiment 21. The method according to Embodiment 19 or 20, wherein the subject suffers from one or more of the following: a lung disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-related acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type I 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, optionally wherein the effective amount is from 50 nM to 2 μM.

[0376] Embodiment 22. The method according to Embodiment 20, wherein the NET-mediated diseases include: a lung disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-related acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type I 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 the following: (i) a synthetic or recombinant polypeptide according to any one of Embodiments 1-9, (ii) a plurality of polypeptides according to Embodiment 10, (iii) a composition according to Embodiment 11, (iv) an isolated polynucleotide according to Embodiment 12 or 13, or (iv) a vector according to Embodiment 14, optionally wherein the effective amount is from 50 nM to 2 μM.

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

[0379] Embodiment 25. The method according to embodiment 24, wherein the antibody or the fragment thereof comprises: 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 of aa 21 to aa 132 of SEQ ID NO: 22 or an equivalent thereof; or wherein the antibody or the fragment thereof comprises: a heavy chain (HC) immunoglobulin variable domain sequence comprising the sequence of aa 25 to aa 144 of SEQ ID NO: 24 or an equivalent thereof; and a light chain (LC) immunoglobulin variable domain sequence comprising the sequence of aa 21 to aa 132 of SEQ ID NO: 25 or an equivalent thereof.

[0380] Embodiment 26. The method according to embodiment 24, wherein the antibody or the fragment thereof comprises: a heavy chain complementarity determining region 1 (CDRH1) comprising the sequence GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 9 or 10 or 11 or 24); a heavy chain complementarity determining region 2 (CDRH2) comprising the sequence GSDRRH (aa 76 to aa 81 of SEQ ID NO: 9 or 10 or 11 or 24); a heavy chain complementarity determining region 3 (CDRH3) comprising the sequence VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 9 or 10 or 11 or 24); a light chain complementarity determining region 1 (CDRL1) comprising the sequence QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 15 or 16 or 17 or 25); a light chain complementarity determining region 2 (CDRL2) comprising the sequence LVS (aa 75 to aa 77 of SEQ ID NO: 15 or 16 or 17 or 25); and a light chain complementarity determining region 3 (CDRL3) comprising the sequence WQGTHFP (aa 114 to aa 120 of SEQ ID NO: 15 or 16 or 17 or 25).

[0381] Embodiment 27. A method for concentrating eDNA tendrils of neutrophil extracellular traps (NETs), comprising contacting the NETs with an effective amount of a DNA binder.

[0382] Embodiment 28. A method for preventing or inactivating the eDNA structure of neutrophil extracellular traps (NETs), comprising contacting the NETs with an effective amount of a DNA binder.

[0383] Embodiment 29. A method for preventing the formation of neutrophil extracellular traps (NETs) or inducing the contraction of existing NETs, comprising contacting the NETs with an effective amount of a DNA binder.

[0384] Embodiment 30. The method according to any one of Embodiments 27-29, wherein the DNA binder is a reagent that aggregates or concentrates DNA, and optionally wherein the effective amount is from 50 nM to 2 μM.

[0385] Embodiment 31. The method according to any one of Embodiments 27-30, wherein the DNA binder comprises histone-like nucleoid structuring protein (H-NS), polyamines or polycations.

[0386] Embodiment 32. The method according to Embodiment 31, wherein the H-NS is derived from Gram-negative bacteria or Gram-positive bacteria.

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

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

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

[0390] Embodiment 36. A method for preventing destructive blood clotting in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a DNA binder.

[0391] Embodiment 37. A method for preventing excessive inflammation in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a DNA binder, optionally wherein the effective amount is from 50 nM to 2 μM.

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

[0393] Embodiment 39. The method according to any one of embodiments 36-38, wherein the DNA binder is a reagent that aggregates or concentrates DNA.

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

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

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

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

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

[0399] Embodiment 45. The method according to any one of embodiments 36 - 44, wherein the subject suffers from one or more of the following: lung diseases, selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-related acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; autoimmune diseases, selected from systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, or small vessel vasculitis; autoinflammatory diseases, selected from gout or inflammatory bowel disease; and / or metabolic diseases, selected from type 2 diabetes or obesity. Sequence Listing SEQ ID NO:1 Mus musculus wild-type HMGB1 protein SEQ ID NO:2 Homo sapiens wild-type HMGB1 protein (Human, copied from GenBank accession number CAE48262.1) HMGB1 is a small protein (about 30 Kda) of a 215 - amino acid protein, consisting of 3 domains: two positively charged domains, A and B box, each domain consisting of 80 amino acids; and a negatively charged carboxyl terminus - acidic C tail, consisting of approximately 30 consecutive aspartic acid and glutamic acid residues. Bold amino acids (amino acids 1 - 70) depict the A Box domain. Italic amino acids (approximately amino acids 88 - 164) depict the B Box domain. The underlined amino acids (amino acids 186 - 215) depict the C - tail domain . SEQ ID NO:3, artificial sequence, linker sequence GPSLKL SEQ ID NO:4, artificial sequence, linker sequence PPKGETKKKF SEQ ID NO:5, human, mB Box-97 peptide KDPNAPKRPPSAFFLFSSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAEKLKEKYEKDIAAYRAKGKPDAAKKGVV SEQ ID NO:6, human, mB Box-97 peptide, with C-terminal linker sequence PPKGETKKKFKDPNAPKRPPSAFFLFSSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAEKLKEKYEKDIAAYRAKGKPDAAKKGVV SEQ ID NO:7, Human, IhfA, A head fragment NFELRDKSSRPGRNPKTGDVV SEQ ID NO:8, Human IhfB, B head fragment SLHHRQPRLGRNPKTGDSVNL SEQ ID NO:9 (H10210(1F8.F1 humanized HC1)) Exemplary variable regions are shown in bold, while exemplary CDRs are shown in bold, italic, and underlined fonts. SEQ ID NO:10 (H10211(1F8.F1 humanized HC2)) Exemplary variable regions are shown in bold, while exemplary CDRs are shown in bold, italic, and underlined fonts. SEQ ID NO:11 (H10212(1F8.F1 humanized HC3)) Exemplary variable regions are shown in bold, while exemplary CDRs are shown in bold, italic, and underlined fonts. SEQ ID NO:12 (H10213(11E7.C7 humanized HC1)) Exemplary variable regions are shown in bold, while exemplary CDRs are shown in bold, italic, and underlined fonts. SEQ ID NO:13 (H10214(11E7.C7 humanized HC2)) Exemplary variable regions are shown in bold, while exemplary CDRs are shown in bold, italic, and underlined fonts. SEQ ID NO:14 (H10215(11E7.C7 humanized HC3)) Exemplary variable regions are shown in bold, while exemplary CDRs are shown in bold, italic, and underlined fonts. SEQ ID NO:15 (L10210(1F8.F1 humanized LC1)) Exemplary variable regions are shown in bold, while exemplary CDRs are shown in bold, italic, and underlined fonts. SEQ ID NO:16 (L10211 (1F8.F1 humanized LC2)). Exemplary variable regions are shown in bold, while exemplary CDRs are shown in bold, italic, and underlined fonts. SEQ ID NO:17 (L10212 (1F8.F1 humanized LC3)) Exemplary variable regions are shown in bold, while exemplary CDRs are shown in bold, italic, and underlined fonts. SEQ ID NO:18 (L10213 (11E7.C7 humanized LC1)) Exemplary variable regions are shown in bold, while exemplary CDRs are shown in bold, italic, and underlined fonts. SEQ ID NO:19 (L10214 (11E7.C7 humanized LC2)) Exemplary variable regions are shown in bold, while exemplary CDRs are shown in bold, italic, and underlined fonts. SEQ ID NO:20 (L10215 (11E7.C7 humanized LC3)) Exemplary variable regions are shown in bold, while exemplary CDRs are shown in bold, italic, and underlined fonts. SEQ ID NO:21 (Heavy chain consensus sequence) MDPKGSLSWR ILLFLSLAFE LSYGEVqLVe SGgglvXPGg SlrlSCaASG 50 FTFXXYXMSW VRQAPGkgLE WVaTIXSXXX XTYYXDsvkG RfTIsRDNaK 100 NtLYlqmnSL raEDTAVYYC XXXXXXXYXX FDXWGQGTXV TVSSASTKGP 150 SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV 200 LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT 250 HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV 300 KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV 350 SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY 400 PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF 450 SCSVMHEALH NHYTQKSLSL SPG**475 Wherein X and lowercase letters can be replaced by any amino acid at the corresponding positions, or by amino acids selected from SEQ ID NO: 9-14. In one embodiment, X can also represent the absence of an amino acid residue. SEQ ID NO:22 (light chain consensus sequence) METDTLLLWV LLLWVPGSTG DXvMTQSPXs LsvslGXrat isCrXSQXXX 50 XXXXXXXLNW XQQkPGqaXX XLIYXXSXlX SGvPdRFSGS GSGTDXTLtI 100 SslXXEDXav YyCXQGXXXX XTFGXGTKXE IKRTVAAPSV FIFPPSDEQL 150 KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL 200 SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC*240 Wherein X and lowercase letters can be replaced by any amino acid at the corresponding positions, or by amino acids selected from SEQ ID NO: 15-20. In one embodiment, X can also represent the absence of an amino acid residue. SEQ ID NO:23 human IgG1 constant region, Uniprot:P01857 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:24 (Common sequence of the heavy chain of the head) MDPKGSLSWR ILLFLSLAFE LSYGEVkLVe SGgglvqPGg SlrlSCaASG 50 FTFRTYAMSW VRQAPGkgLE WVATIGSDRR HTYYPDsvkG RfTIsRDNaK 100 NTLYlqmnSL RaEDTAVYYC VGPYDGYYGE FDYWGQGTLV TVSSASTKGP 150 SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV 200 LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT 250 HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV 300 KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV 350 SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY 400 PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF 450 SCSVMHEALH NHYTQKSLSL SPG**475 The lowercase letters can be substituted with amino acids selected from SEQ ID NO: 9-11 at the corresponding positions. SEQ ID NO:25 (common sequence of the head light chain) METDTLLLWV LLLWVPGSTG DVVMTQSPlS LpVtLGqpAs IsCrSSQSLL 50 DSDGKTFLNW LQQrPGQsPr RLIYLVSKlD SGVPDRFSGS GSGTDFTLkI 100 SrveAEDVgV YYCWQGTHFP YTFGQGTKLE IKRTVAAPSV FIFPPSDEQL 150 KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL 200 SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC**240 The lowercase letters can be substituted with amino acids selected from SEQ ID NO: 15-17 at the corresponding positions. SEQ ID NO:26 (chimeric peptide of the head IhfA5-mIhfB4NTHI) RPGRNPX1TGDVVPVSARRVV-X-FSLHHRQPRLGRNPX1TGDSV Wherein "X" is an optional amino acid linker sequence, optionally comprising 1 to 20 amino acids, or consisting essentially of or consisting of the same. Wherein "X1" is any amino acid, or "X1" is selected from the amino acids Q, R, K, S or T. SEQ ID NO:27 (chimeric peptide of the head IhfA5-mIhfB4NTHI) RPGRNPKTGDVVPVSARRVV-X-FSLHHRQPRLGRNPKTGDSV Wherein "X" is an optional amino acid linker sequence, optionally comprising 1 to 20 amino acids SEQ ID NO:28 (chimeric peptide of the head IhfA5-mIhfB4NTHI) RPGRNPKTGDVVPVSARRVVGPSLFSLHHRQPRLGRNPKTGDSV SEQ ID NO:29, E.Coli, H-NS protein MSEALKILNNIRTLRAQARECTLETLEEMLEKLEVVVNERREEESAAAAEVEERTRKLQQYREMLIADGIDPNELLNSLAAVKSGTKAKRAQRPAKYSYVDENGETKTWTGQGRTPAVIKKAMDEQGKSLDDFLIKQ SEQ ID NO:30, NTHI, H-NS protein MNELVRGLTNLRSLRAAVRELTLEQAENALEKLQTAIEEKRANEAELIKAETERKERLAKYKELMEKEGITPEELHKIFGTKTVSIQAKRAPRPAKYAFIDENGEHKTWTGQGRTPRPIQNALNKGKSLSDFEI SEQ ID NO:31, Mycobacterium tuberculosis, H-NS MPDPQDRPDSEPSDASTPPAKKLPAKKAAKKAPARKTPAKKAPAKKTPAKGAKSAPPKPAEAPVSLQQRIETNGQLAAAAKDAAAQAKSTVEGANDALARNASVPAPSHSPVPLIVAVTLSLLALLLIRQLRRR SEQ ID NO:32, Streptococcus pneumoniae H-NS protein MNELVRSLTNLRSLRAAVRELTLEQAENALEKLQTAIEEKRANEAELIKAETERKERLAKYKELMEKEGITPEELHEIFGTKTVSIRAKRPPRPAKYAFIDENGEHKTWTGQGRTPRPIQNALNKGKSLSDFEI SEQ ID NO:33, Klebsiella pneumoniae H-NS protein MSEALKILNNIRTLRAQARECTLETLEEMLEKLEVVVNERREEENAAAAEIEERTRKLQQYREMLIADGIDPNELLSTMAAVKAGTKTKRAARPAKYSYVDENGETKTWTGQGRTPAVIKKAMDEQGKSLDDFLI SEQ ID NO:34, Pseudomonas aeruginosa H-NS protein MSLINEYRNTEQTIKELQARLASLQQDGRMKAELEFDTKLRALMSEYNKSLRDVIILLDPQAQNRSSKTPPTSGRRERQLKRYLNPNTSEVVETKGGNHKILKEWKTQFGADVVESWLQS SEQ ID NO:35 Human IgD constant region, Uniprot:P01880 APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMKSEQ ID NO:36 SEQ ID NO:36 Human IgG2 constant region, Uniprot:P01859 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:37 Human IgG3 constant region, Uniprot:P01860 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK SEQ ID NO:38 Human IgM constant region, Uniprot:P01871 GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY SEQ ID NO:39 Human IgG4 constant region, Uniprot:P01861 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:40 Human IgA1 constant region, Uniprot: P01876 ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY SEQ ID NO:41 Human IgA2 constant region, Uniprot: P01877 ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY SEQ ID NO:42 Human Ig kappa constant region, Uniprot: P01834 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. A synthetic polypeptide comprising mB Box-97, wherein mB Box-97 consists of amino acids 90 to 176 of the coding sequence of the native human HMGB1 protein shown in SEQ ID NO:2 and has a cysteine to serine point mutation at amino acid 106, or an equivalent thereof.

2. A recombinant polypeptide comprising mB Box-97, wherein mB Box-97 consists of amino acids 90 to 176 of the coding sequence of the native human HMGB1 protein shown in SEQ ID NO:2 and has a cysteine to serine point mutation at amino acid 106, or an equivalent thereof.

3. The synthetic or recombinant polypeptide according to claim 1 or 2, wherein the synthetic or recombinant polypeptide consists of SEQ ID NO:

5.

4. A synthetic polypeptide comprising mB Box-97, wherein mB Box-97 consists of amino acids 80 to 176 of the coding sequence of the native human HMGB1 protein shown in SEQ ID NO:2 and has a cysteine to serine point mutation at amino acid 106, or an equivalent thereof.

5. A recombinant polypeptide comprising mB Box-97, wherein mB Box-97 consists of amino acids 80 to 176 of the coding sequence of the native human HMGB1 protein shown in SEQ ID NO:2 and has a cysteine to serine point mutation at amino acid 106, or an equivalent thereof.

6. The synthetic or recombinant polypeptide according to claim 4 or 5, wherein the synthetic or recombinant polypeptide consists of SEQ ID NO:

6.

7. The synthetic or recombinant polypeptide according to any one of claims 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 under high stringency conditions to a polynucleotide encoding the amino acid sequence or its complementary sequence, wherein the 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 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.

8. The synthetic or recombinant polypeptide according to any one of claims 1-7, and a detectable label.

9. The synthetic or recombinant polypeptide according to any one of claims 1-8, and a linker polypeptide, optionally wherein the linker polypeptide comprises GPSLKL (SEQ ID NO:3) or PPKGETKKKF (SEQ ID NO:4).

10. A plurality of the synthetic or recombinant polypeptides according to any one of claims 1-9, optionally wherein the members of the plurality of polypeptides are the same or different from each other.

11. A composition comprising the synthetic or recombinant polypeptide according to any one of claims 1-9 or the polypeptides according to claim 10, and a carrier, optionally wherein the carrier is a pharmaceutically acceptable carrier.

12. An isolated polynucleotide encoding the synthetic or recombinant polypeptide according to any one of claims 1-9, and optionally a carrier or a pharmaceutically acceptable carrier.

13. The isolated polynucleotide according to claim 12, and a detectable label, and optionally a carrier or a pharmaceutically acceptable carrier.

14. A carrier comprising the isolated polynucleotide according to claim 12 or 13, and optionally a carrier or a pharmaceutically acceptable carrier.

15. The isolated polynucleotide according to claim 12 or 13, or the carrier according to claim 14, further comprising a heterologous promoter sequence, and optionally a carrier or a pharmaceutically acceptable carrier.

16. An isolated host cell comprising one or more of the following: the synthetic or recombinant polypeptide according to any one of claims 1-9, the polypeptides according to claim 10, the isolated polynucleotide according to any one of claims 12 or 13, or the carrier according to claim 14, and optionally a carrier or a pharmaceutically acceptable carrier.

17. The isolated host cell according to claim 16, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

18. The isolated host cell according to claim 17, wherein the host cell is a eukaryotic cell, optionally wherein the eukaryotic cell is a mammalian cell.

19. A method for treating a subject in need thereof, comprising administering to the subject an effective amount of one or more of the following: (i) the synthetic or recombinant polypeptide according to any one of claims 1-9, (ii) the polypeptides according to claim 10, (iii) the composition according to claim 11, (iv) the isolated polynucleotide according to claim 12 or 13, or (iv) the carrier according to claim 14.

20. A method for treating or preventing abnormal or excessive neutrophil extracellular trap (NET) formation or preventing NET-mediated diseases or preventing the progression of NET-mediated diseases in a subject in need thereof, comprising administering to the subject an effective amount of one or more of the following: (i) the synthetic or recombinant polypeptide according to any one of claims 1-9, (ii) the polypeptides according to claim 10, (iii) the composition according to claim 11, (iv) the isolated polynucleotide according to claim 12 or 13, or (iv) the carrier according to claim 14.

21. The method according to claim 19 or 20, wherein the subject suffers from one or more of the following: a lung disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-related acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type I 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, optionally wherein the effective amount is from 50 nM to 2 μM.

22. The method according to claim 20, wherein the NET-mediated diseases include: A lung disease selected from SARS CoV-2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-related acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type I 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.

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 the following: (i) a synthetic or recombinant polypeptide according to any one of claims 1-9, (ii) a plurality of polypeptides according to claim 10, (iii) a composition according to claim 11, (iv) an isolated polynucleotide according to claim 12 or 13, or (iv) a vector according to claim 14, optionally wherein the effective amount is from 50 nM to 2 μM.

24. The method according to any one of claims 19-23, further comprising administering to the subject an antibody or a fragment thereof, wherein the antibody or the fragment thereof binds to the head region of the DNABII peptide.

25. The method according to claim 24, wherein the antibody or the fragment thereof comprises: (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 of aa 21 to aa 132 of SEQ ID NO: 22 or an equivalent thereof; Or wherein the antibody or the fragment thereof comprises: (v) a heavy chain (HC) immunoglobulin variable domain sequence comprising the sequence of amino acids (aa) 25 to aa 144 of SEQ ID NO: 24 or an equivalent thereof; and (vi) a light chain (LC) immunoglobulin variable domain sequence comprising the sequence of aa 21 to aa 132 of SEQ ID NO: 25 or an equivalent thereof.

26. The method according to claim 24, wherein the antibody or the fragment thereof comprises: (i) Heavy chain complementarity-determining region 1 (CDRH1), which comprises the sequence GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 9 or 10 or 11 or 24); (ii) Heavy chain complementarity-determining region 2 (CDRH2), which comprises the sequence GSDRRH (aa 76 to aa 81 of SEQ ID NO: 9 or 10 or 11 or 24); (iii) Heavy chain complementarity-determining region 3 (CDRH3), which comprises the sequence VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 9 or 10 or 11 or 24); (iv) Light chain complementarity-determining region 1 (CDRL1), which comprises the sequence QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 15 or 16 or 17 or 25); (v) Light chain complementarity-determining region 2 (CDRL2), which comprises the sequence LVS (aa 75 to aa 77 of SEQ ID NO: 15 or 16 or 17 or 25); and (vi) Light chain complementarity-determining region 3 (CDRL3), which comprises the sequence WQGTHFP (aa 114 to aa 120 of SEQ ID NO: 15 or 16 or 17 or 25).

27. A method for concentrating neutrophil extracellular trap (NET) eDNA tendrils, comprising contacting the NET with an effective amount of a DNA-binding agent.

28. A method for preventing or inactivating neutrophil extracellular trap (NET) eDNA structures, comprising contacting the NET with an effective amount of a DNA-binding agent.

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

30. The method according to any one of claims 27-29, wherein the DNA-binding agent is a reagent that aggregates or concentrates DNA, and optionally wherein the effective amount is from 50 nM to 2 μM.

31. The method according to any one of claims 27-30, wherein the DNA-binding agent comprises histone-like nucleoid structuring protein (H-NS), polyamines, or polycations.

32. The method according to claim 31, wherein the H-NS is derived from Gram-negative or Gram-positive bacteria.

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

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

35. The method according to any one of claims 27-34, wherein the contacting is carried out in vitro or in vivo.

36. A method for preventing destructive blood clotting in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a DNA binder.

37. A method for preventing excessive inflammation in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a DNA binder, optionally wherein the effective amount is from 50 nM to 2 μM.

38. A method for preventing, treating or preventing the progression of neutrophil extracellular trap (NET)-mediated diseases in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a DNA binder, optionally wherein the effective amount is from 50 nM to 2 μM.

39. The method according to any one of claims 36-38, wherein the DNA binder is a reagent that aggregates or condenses DNA.

40. The method according to any one of claims 36-39, wherein the DNA binder comprises histone-like nucleoid structuring protein (H-NS).

41. The method according to claim 40, wherein the H-NS is derived from a Gram-negative bacterium or a Gram-positive bacterium.

42. The method according to claim 41, wherein the H-NS is derived from a bacterium of the genus Escherichia, Haemophilus, Streptococcus, Mycobacteria, Klebsiella or Pseudomonas; optionally wherein the H-NS is derived from Escherichia coli, nontypeable Haemophilus influenzae (NTHI), Streptococcus pneumoniae, Klebsiella pneumoniae, Mycobacterium tuberculosis or Pseudomonas aeruginosa.

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

44. The method according to any one of claims 36 - 43, wherein the subject is a mammalian or human patient.

45. The method according to any one of claims 36 - 44, wherein the subject suffers from one or more of the following: a lung disease selected from SARS CoV - 2, cystic fibrosis, asthma, chronic obstructive pulmonary disease, tuberculosis, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion - related acute lung injury, and mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type I 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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