Antibody compositions and methods for disrupting nontuberculous mycobacterial biofilms - Patent Application 20070122999

Abstract

Provided herein is a method for preventing or treating infection caused by nontuberculous mycobacterium (NTM) species in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof that binds to the tip region of a DNABII peptide. Also provided is a method for sensitizing a biofilm to an antibiotic, the biofilm comprising a nontuberculous mycobacterium (NTM) species, the method comprising contacting the biofilm with an antibody or antigen-binding fragment thereof that binds to the tip region of a DNABII peptide.

Description

[Technical Field]

[0001] Cross-reference to related patent applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 425,261, filed November 14, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] background Nontuberculous mycobacteria (NTM), distant relatives of Mycobacterium tuberculosis, cause chronic infections in people with muco-obstructive pulmonary diseases, such as chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia, or CF. NTM are classified as slow-growing (e.g., Mycobacterium avium) or fast-growing (e.g., Mycobacterium abscessus). In addition to genotypic / phenotypic variability, NTM prevalence varies with the underlying lung disease. People with CF (PwCF) are highly susceptible to pulmonary infections, and NTM is endemic in both adults and children in the United States and Europe, with NTM prevalence increasing by 5% annually. One in five PwCF individuals is culture-positive for NTM, depending on geographic region, increasing age, and NTM species-related acquisition.

[0003] Established NTM infections are extremely difficult to treat and require long-term antibiotic therapy. Recommended treatment for PwCF who are culture-positive for the M. avium complex (M. avium, M. intracellulare, and M. chimaera) involves a regimen of oral and intravenous antibiotics, with duration ranging up to 17 months, depending on disease severity. M. abscessus presents an even greater challenge, with treatment regimens typically involving oral, intravenous, and inhaled antibiotics for up to 18–24 months. Despite these intensive regimens, failure rates are high, with 50–60% of these individuals failing to convert from NTM-positive to NTM-negative sputum cultures or maintaining this conversion for more than 12 months. Crucially, 30–60% of patients had to discontinue at least one of the prescribed antibiotics due to serious treatment sequelae, such as drug-related toxicity (e.g., nephrotoxicity or otovestibular toxicity).

[0004] Reasons for these difficulties in treating NTM infections include poor drug uptake due to the thick, hydrophobic mycobacterial cell wall, drug export by efflux pumps, the development of antibiotic resistance, and biofilm formation by NTM. Biofilms are aggregated bacterial communities embedded in extracellular polymeric substances (EPS) of proteins, carbohydrates, and extracellular DNA (eDNA). They are phenotypically distinct from planktonic bacteria and are found in the lungs and / or sputum of PwCF or COPD. Biofilm-resident bacteria are well protected from antibiotics, chemicals, mechanical stress, and immune effectors through a variety of mechanisms. Furthermore, they can tolerate antibiotic concentrations many times higher than those required to kill their planktonic counterparts. Typically, clinical isolates of M. abscessus exhibit intracellular survival with increased aggregation and induce greater inflammation compared to the laboratory-passaged reference strain, M. abscessus 19977, and are therefore considered to be more virulent and more representative of the disease-causing pathogen than their laboratory-passaged counterparts. This makes it highly recommended that clinical isolates be considered for testing in addition to laboratory-passaged strains. Summary of the Invention [Means for solving the problem]

[0005] There is an urgent need for new and more effective approaches to combat refractory NTM infections. The present disclosure fulfills this need and provides related advantages as well.

[0006] Summary of the Disclosure Applicant has surprisingly discovered that antibodies or antigen-binding fragments thereof that bind to the tip region of the DNABII peptide can disrupt or prevent biofilms formed by or containing nontuberculous mycobacterium (NTM) species. Applicant has also discovered that such anti-DNABII tip antibodies reduce antibiotic resistance in NTM biofilms and reduce the dose of antibiotics required to treat such biofilms and infections.

[0007] Certain aspects of the present disclosure are directed to a method for preventing or treating an infection caused by a nontuberculous mycobacterium (NTM) species in a subject, the method comprising, consisting essentially of, or consisting of administering to the subject an effective amount of an antibody or antigen-binding fragment thereof that binds to the tip region of a DNABII peptide.

[0008] Another aspect of the present disclosure is directed to a method for sensitizing a biofilm or a microorganism in a biofilm to an antibiotic, wherein the biofilm comprises, consists essentially of, or consists of a nontuberculous mycobacterium (NTM) species, and the method comprises, consists essentially of, or consists of contacting the biofilm with an antibody or antigen-binding fragment thereof that binds to the apical region of a DNABII peptide. The contacting step can be performed in vitro or in vivo. When performed in vivo, the method provides a method for sensitizing a biofilm produced by a nontuberculous mycobacterium (NTM) species to an antibiotic in a subject in need thereof, the method comprising, consists essentially of, or consists of administering to the subject an antibody or antigen-binding fragment thereof that binds to the apical region of a DNABII peptide. In one aspect, the method reduces the antibiotic dose required to treat such biofilms and associated infections.

[0009] In another aspect, a method for disrupting biofilms produced by nontuberculous mycobacteria (NTM), such as Mycobacterium abscessus (Mab) or Mycobacterium avium (Mav), is provided. The method comprises, consists essentially of, or even consists of contacting a biofilm produced by nontuberculous mycobacteria (NTM), such as Mycobacterium abscessus (Mab) or Mycobacterium avium (Mav), with one or more of the antibodies, fragments thereof, polypeptides, or CDRs disclosed herein. In one embodiment, the antibody, fragment, polypeptide, or CDR is a fragment of a DNABII peptide (the distal region of IHF or HU, the distal region of IHFA or IHFB, the distal chimeric peptide IhfA5-mIhfB4, or the distal region of IhfA5-mIhfB4). NTHI In a further embodiment, the antibody, fragment thereof, polypeptide or CDR binds to the tip chimeric peptide IhfA5-mIhfB4 NTHI In still further embodiments, the contacting step is performed at or.

[0010] In one aspect, a method is provided for detecting a biofilm-producing nontuberculous mycobacterium (NTM), e.g., Mycobacterium abscessus (Mab) or Mycobacterium avium (Mav) microbial infection in a subject. The method comprises, alternatively consists of, or even consists of contacting one or more of the antibodies, fragments thereof, polypeptides, or CDRs disclosed herein with a biological sample isolated from the subject suspected of containing a biofilm, and detecting binding of the antibody, fragment thereof, polypeptide, or CDR to any biofilm in the sample. The amount can be determined by the treating physician or veterinarian, and can be, for example, an amount effective for the subject. In one embodiment, the antibody, fragment thereof, polypeptide, or CDR is selected from the group consisting of the tail region or tip region of a DNABII peptide (the tip region of IHF or HU, the tip region of IHFA or IHFB, the tip chimeric peptide IhfA5-mIhfB4, and the like). NTHI , the tail region of IHF or HU, the tail region of IHFA or IHFB, and / or the tail-chimeric peptide IhfA3-IhfB2 NTHI In a further embodiment, the contacting step is or.

[0011] In another aspect, there is provided a method for screening a subject for a biofilm-producing nontuberculous mycobacterial infection (NTM), e.g., Mycobacterium abscessus (Mab) or Mycobacterium avium (Mav), comprising, alternatively consisting of, or even consisting of contacting one or more of the antibodies, fragments thereof, polypeptides, or CDRs disclosed herein with a biological sample isolated from the subject, the biological sample containing a biofilm, and detecting binding of the antibody, fragment thereof, polypeptide, or CDR to any biofilm in the sample. In one embodiment, the antibody, fragment thereof, polypeptide, or CDR is selected from the tail or tip region of a DNABII peptide (the tip region of IHF or HU, the tip region of IHFA or IHFB, the tip chimeric peptide IhfA5-mIhfB4 ... NTHI , the tail region of IHF or HU, the tail region of IHFA or IHFB, and / or the tail-chimeric peptide IhfA3-IhfB2 NTHI In a further embodiment, the subject detected by binding is selected for administration with one or more of the antibodies, fragments thereof, polypeptides or CDRs disclosed herein, and / or one or more polynucleotides or vectors encoding the antibodies, fragments thereof, polypeptides or CDRs, wherein the antibody, fragment, polypeptide or CDR is selected from the distal region of a DNABII peptide (the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal chimeric peptide IhfA5-mIhfB4 NTHI In yet a further embodiment, the contacting step is or.

[0012] Provided herein are methods for detecting a nontuberculous mycobacterial infection (NTM) in a subject, e.g., a biofilm produced by Mycobacterium abscessus (Mab) or Mycobacterium avium (Mav), by administering to the subject one or more of the antibodies or fragments thereof disclosed herein and detecting binding of any of the antibodies, fragments thereof, polypeptides, or CDRs disclosed herein to the biofilm. In one aspect, the antibody, fragment thereof, polypeptide, or CDR binds to the tail or tip region of a DNABII peptide (the tip region of IHF or HU, the tip region of IHFA or IHFB, the tip chimeric peptide IhfA5-mIhfB4, or the tip region of a DNABII peptide IhfA5-mIhfB4). NTHI , the tail region of IHF or HU, the tail region of IHFA or IHFB, and / or the tail chimeric peptide IhfA3-IhfB2 NTHI In another embodiment, the method further comprises detecting binding of the antibody, fragment thereof, polypeptide or CDR to the biofilm.

[0013] In one aspect, a method for preventing or disrupting a nontuberculous mycobacterial infection (NTM) in a subject, e.g., a biofilm produced by Mycobacterium abscessus (Mab) or Mycobacterium avium (Mav), comprises, or alternatively consists of, or even consists of, administering to a subject one or more of the antibodies, fragments thereof, polypeptides or CDRs, and / or one or more polynucleotides or vectors encoding the antibodies, fragments thereof, polypeptides or CDRs disclosed herein, wherein such antibody or the like is / are an antibody or antibody fragment that binds to a distal region of a DNABII peptide (e.g., the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal chimeric peptide IhfA5-mIhfB4). NTHIThe present invention provides a method for detecting a biofilm by contacting a sample suspected of containing a biofilm with one or more of the antibodies, fragments thereof, polypeptides, or CDRs disclosed herein, and detecting binding of the biofilm to the antibody, fragments thereof, polypeptides, or CDRs, wherein the antibody, fragments thereof, polypeptides, or CDRs bind to a tip region or tail region of a DNABII peptide (the tip region of IHF or HU, the tip region of IHFA or IHFB, the tip chimeric peptide IhfA5-mIhfB4, NTHI , the tail region of IHF or HU, the tail region of IHFA or IHFB, and / or the tail chimeric peptide IhfA3-IhfB2 NTHI (including but not limited to)

[0014] In another aspect, a method for inhibiting, preventing, or treating a nontuberculous mycobacterial infection (NTM), e.g., a microbial infection produced by Mycobacterium abscessus (Mab) or Mycobacterium avium (Mav), comprises, or alternatively consists of, or even consists of administering to a subject one or more of the antibodies, fragments thereof, polypeptides, or CDRs, and / or one or more polynucleotides or vectors encoding the antibodies, fragments thereof, polypeptides, or CDRs disclosed herein, wherein such antibody or the like is / are an antibody or antibody fragment that binds to a distal region of a DNABII peptide (e.g., the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal chimeric peptide IhfA5-mIhfB4). NTHIThe present invention provides a method for detecting a biofilm by contacting a sample suspected of containing a biofilm with one or more of the antibodies, fragments thereof, polypeptides, or CDRs disclosed herein, and detecting binding of the biofilm to the antibody, fragments thereof, polypeptides, or CDRs, wherein the antibody, fragments thereof, polypeptides, or CDRs bind to a tip region or tail region of a DNABII peptide (the tip region of IHF or HU, the tip region of IHFA or IHFB, the tip chimeric peptide IhfA5-mIhfB4, NTHI , the tail region of IHF or HU, the tail region of IHFA or IHFB, and / or the tail chimeric peptide IhfA3-IhfB2 NTHI In a further embodiment, an effective amount of amikacin and / or azithromycin is administered to a subject prior to, concurrently with, or after one or more of the antibodies, fragments thereof, polypeptides, or CDRs disclosed herein, and / or one or more polynucleotides or vectors encoding the antibodies, fragments thereof, polypeptides, or CDRs, wherein such antibodies or the like bind to the distal region of a DNABII peptide (the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal chimeric peptide IhfA5-mIhfB4 NTHI The amount can be determined by a treating physician or veterinarian, and can be, for example, an effective amount for the subject. In one embodiment, the subject is a human patient suffering from or predisposed to developing cystic fibrosis.

[0015] In yet another aspect, there is provided a method for preventing or treating a condition caused by a nontuberculous mycobacterial (NTM) infection characterized by biofilm formation, e.g., a Mycobacterium abscessus (Mab) infection or a Mycobacterium avium (Mav) infection, in a subject, by administering to the subject one or more of the antibodies, fragments thereof, polypeptides or CDRs, and / or one or more polynucleotides or vectors encoding the antibodies, fragments thereof, polypeptides or CDRs disclosed herein, wherein such antibody or other antibody is or is not specifically mutated. NTHI The present invention provides a method for administering an effective amount of amikacin and / or azithromycin to a subject, the method comprising ... NTHIThe amount can be determined by a treating physician or veterinarian, and can be, for example, an effective amount for the subject. In one aspect, the subject is a human patient suffering from or predisposed to developing cystic fibrosis. In one embodiment, the method further comprises detecting a biofilm by contacting one or more of the antibodies, fragments thereof, polypeptides, or CDRs disclosed herein with a sample suspected of containing a biofilm and detecting binding of the biofilm to the antibody, fragments thereof, polypeptides, or CDRs, wherein the antibody, fragments thereof, polypeptides, or CDRs bind to a tip or tail region of a DNABII peptide (the tip region of IHF or HU, the tip region of IHFA or IHFB, the tip chimeric peptide IhfA5-mIhfB4, NTHI , the tail region of IHF or HU, the tail region of IHFA or IHFB, and / or the tail chimeric peptide IhfA3-IhfB2 NTHI (including but not limited to)

[0016] A method for conferring passive immunity in a subject suffering from a non-tuberculous mycobacterial infection, such as an infection due to Mycobacterium abscessus (Mab) infection or Mycobacterium avium (Mav) infection, comprising, or alternatively consisting essentially of, or even consisting of, administering to a subject one or more of the antibodies, fragments thereof, polypeptides or CDRs disclosed herein, and / or one or more polynucleotides or vectors encoding the antibodies, fragments thereof, polypeptides or CDRs, wherein the antibody, fragment, polypeptide or CDR is selected from the group consisting of a distal region of a DNABII peptide (the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal chimeric peptide IhfA5-mIhfB4). NTHI Also provided are methods for binding to a cystic fibrosis antibody, including but not limited to, a cystic fibrosis antibody. The amount can be determined by a treating physician or veterinarian, and can be, for example, an effective amount for the subject. In one embodiment, the subject is a human patient suffering from cystic fibrosis.

[0017] In one embodiment, the therapeutic method is combined with a diagnostic method for detecting and / or monitoring biofilm formation and disruption using the antibodies, fragments thereof, polypeptides or CDRs disclosed herein.

[0018] Further provided are pharmaceutical compositions comprising, consisting essentially of, or even consisting of, as an active agent, an antibody or antigen-binding fragment thereof as described herein in combination with an effective amount of an antibiotic and / or an mB box-97 polypeptide and / or a DNA-binding agent, each as described herein, for treating an infection that is causing or has caused a biofilm as described herein. In one embodiment, the effective amount of antibiotic is provided in an amount that is less than the accepted MIC for the antibiotic for treatment of the infection.

[0019] Kits are also provided that include one or more of the antibodies, antigen-binding fragments thereof, a polypeptide (e.g., mB box-97 polypeptide), a DNA binding agent, an effective amount of an antibiotic to treat an infection, or one or more of the compositions disclosed herein, and, optionally, instructions for use.

[0020] Applicant's disclosure addresses and resolves issues associated with NTM infections and NTM-containing biofilms. NTM species belong to the class Actinomycetia and are distinguished by their thick, mycolic acid-rich cell walls. NTM biofilms are particularly resistant to antibiotics. Prior to this disclosure, it was unknown whether NTM biofilms incorporated DNABII proteins within the eDNA-rich biofilm matrix, and it was also unknown whether antibodies generated against specific defensive domains of traditional DNABII proteins (anti-DNABII tip antibodies disclosed herein) would recognize unique mycobacterial DNABII homologs and actively disrupt NTM biofilms. Applicant has shown that anti-DNABII tip antibodies disrupt NTM biofilms, rendering them susceptible to antibiotic treatment. The disclosed methods enhance antibiotic susceptibility and / or immune system clearance in infected individuals. The disclosed methods also benefit patients by reducing either the length of antibacterial treatment or the amount of antibiotic required. [Brief explanation of the drawings]

[0021] [Figure 1]HuTipMab ("HuTipMab" is an antibody described in Kurbatfinski, N. et al. Antimicrobial Agents and Chemotherapy 66.3 (2022): e01877-21 and WO2021007260A2) recognizes the isolated NTM DNA-binding protein HupB (amino acid sequence shown in SEQ ID NO: 57). (Figure 1A) A silver-stained gel of SDS-PAGE-separated proteins revealed a pure protein isolate with a band at the expected location based on the predicted molecular mass of the protein monomer: HupB = 28 kDa. (Figure 1B) The specificity of HuTipMab for the tail chimeric peptide (negative control, shown in SEQ ID NO: 43), HupB, and tip chimeric peptide (positive control) was determined by ELISA. In a representative image of an ELISA plate, dark wells indicated HuTipMab reactivity with HupB and tip chimeric peptide. (Figure 1C) The color development in the wells of the ELISA plate was quantified using a plate reader by measuring the optical density at 650 nm. The specificity of HuTipMab for HupB and the tip chimeric peptide was significantly greater than that for the tail chimeric peptide, which showed no reactivity. Statistically significant differences in optical density are reported as ***, P ≤ 0.001; ****, P < 0.0001.

[0022] [Figure 2]Average biomass values ​​are plotted on the graph for each treatment. HuTipMab disrupted 72-hour M. abscessus 19977 biofilms in a dose- and time-dependent manner. M. abscessus 19977 biofilms were incubated with medium alone, 5 μg human IgG ("HuIgG"), or 5, 7.5, or 10 μg of HuTipMab, stained with FM 1-43FX, fixed, and visualized by confocal laser scanning microscopy ("CLSM"). Biomass was calculated using COMSTAT2. All treatments were incubated for 30 minutes or with 5 μg HuTipMab for 60 minutes. M. abscessus 19977 biofilms treated with HuTipMab exhibited a significant relative reduction in biomass and height compared to biofilms incubated with medium or HuIgG. There was no significant difference in biomass between wells incubated with medium alone or 5 μg HuIgG. Compared to HuIgG incubation, all wells incubated with HuTipMab showed a significant reduction in biomass, ranging from 53% to 89% biofilm disruption. Statistical differences in biomass are reported as **, P ≤ 0.01; ****, P < 0.0001.

[0023] [Figure 3]The graph depicts the average biomass values ​​of biofilms incubated with each treatment condition. HuTipMab-induced disruption of 2-week-old M. avium biofilms was dose- and time-dependent. After incubation of M. avium biofilms with medium alone, 5 μg HuIgG, or 5, 7.5, or 10 μg HuTipMab for 30 minutes, or with 5 μg HuTipMab for 60 minutes, the biofilms were stained with FM 1-43FX, fixed, and visualized by CLSM. The displayed 3D biofilm images are representative of biofilms after each treatment. Biomass was calculated using COMSTAT2, and the average post-incubation biomass values ​​for each treatment are shown in the lower right portion of each image. The biomass and height of M. avium biofilms incubated with HuTipMab were significantly reduced compared to those found in wells incubated with medium alone or 5 μg HuIgG. Biomass values ​​from wells incubated with medium alone or 5 μg HuIgG were not significantly different. However, when biofilms were incubated with HuTipMab, biomass values ​​were significantly reduced compared to those incubated with HuIgG alone, with percent biomass destruction ranging from 51% to 80%. Statistical differences in biomass are reported as **, P ≤ 0.01; ***, P ≤ 0.001; ****, P < 0.0001.

[0024] [Figure 4]Disruption of 72-h biofilms formed by three isolates of M. abscessus cultured from PwCF by HuTipMab was also dose- and time-dependent. Biofilms of M. abscessus clinical isolates 1, 2, and 3 were incubated with medium alone, 5 μg HuIgG, or 5, 7.5, or 10 μg HuTipMab for 30 min, or with 5 μg HuTipMab for 60 min. Biofilms were stained with FM 1-43FX, fixed, and visualized by CLSM. Biomass was calculated using COMSTAT2. Incubation with HuTipMab significantly reduced biofilm biomass and height compared with incubation with medium alone or 5 μg HuIgG. (Figure 4A) Graphical representation of the average biomass values ​​for biofilms formed by M. abscessus clinical isolate 1 after incubation with each treatment condition. (Figure 4B) Graphical representation of the average biomass values ​​for biofilms formed by M. abscessus clinical isolate 1 after incubation with each treatment condition. (Figure 4C) Graphical representation of the average biomass values ​​for biofilms formed by M. abscessus clinical isolate 1 after incubation with each treatment condition. Disruption was significant for each HuTipMab treatment compared with treatment with HuIgG, which was not significantly different from wells treated with medium alone. Percent biomass destruction ranged from 58% to 91% for clinical isolate 1, 57% to 90% for clinical isolate 2, and 62% to 93% for clinical isolate 3. Statistically significant differences in biomass are reported as *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P < 0.0001.

[0025] [Figure 5-1]M. abscessus 19977 NRel (NRel = newly released bacteria) was significantly more susceptible to amikacin and azithromycin. Compared to planktonic killing, NRel killing was significantly greater for both (Figure 5A) amikacin and (Figure 5B) azithromycin, particularly at 1 / 4 and 1 / 2 MICs. Planktonic killing was 23% and 20%, respectively, while NRel killing was 61% and 42%, respectively. Statistically significant differences in percent killing are reported as **, P ≤ 0.01; ***, P ≤ 0.001. (Figure 5C) Graphical representation of NRel production in the non-biofilm-associated state (NBfA—also known as "planktonic") and after treatment with an antibody (HuTipMab) against the apical region of the DNABII protein. [Figure 5-2] Same as above.

[0026] [Figure 6] HuTipMab-induced M. avium NRel was significantly more susceptible to amikacin and azithromycin. When tested against both amikacin and azithromycin, two antibiotics used to treat NTM in PwCF, NRel was significantly more susceptible to antibiotic killing than planktonic M. avium (Figure 6A). When combined with 1 / 4 MIC of amikacin, planktonic M. avium was killed 17%, while M. avium NRel was killed 41% (Figure 6B). Similarly, incubation of planktonic M. avium with 1 / 2 MIC of azithromycin resulted in 19% killing, while incubation of M. avium NRel at the same concentration resulted in 36% killing. Statistically significant differences in percent mortality are reported as *, P≦0.05; **, P≦0.01; ***, P≦0.001; ****, P<0.0001.

[0027] [Figure 7]HuTipMab-induced NRel of M. abscessus clinical isolates 1 and 3 was significantly more susceptible to killing by amikacin and azithromycin than when grown in planktonic conditions (Figure 7A and 7B). When combined with amikacin, killing of planktonic M. abscessus clinical isolate 1 was 28%, while killing of HuTipMab-induced NRel of this clinical isolate was 45%. Similarly, incubation of planktonic M. abscessus clinical isolate 1 with azithromycin resulted in 28% killing, while killing of M. abscessus clinical isolate 1 NRel at the same concentration was 47% (Figure 7C and 7D). Killing of planktonic M. abscessus clinical isolate 3 by amikacin was 21%, while killing of the corresponding NRel was 47% (Figure 7C and 7D). Similarly, azithromycin killed 45% of M. abscessus clinical isolate 3 NRel at the same concentration that killed 25% of the planktonic M. abscessus clinical isolate 3. Statistically significant differences in percent killing are reported as *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P < 0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0028] Detailed Description 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 invention, the preferred methods, devices, and materials are described herein. All technical and patent publications cited herein are incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.

[0029] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3 rd edition;the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology;the series Methods in Enzymology (Academic Press, Inc., NY);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press);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. (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. Press, London); and Herzenberg et al. eds (1996) See Weir's Handbook of Experimental Immunology.

[0030] All numerical designations, including ranges, e.g., pH, temperature, time, concentration, and molecular weight, are approximations that are subject to variation (+) or (-) by increments of 1.0 or 0.1, as appropriate, or alternatively by variations of + / - 15%, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." It is also understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0031] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a polypeptide" includes multiple polypeptides, including mixtures thereof.

[0032] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding any significant other elements from the combination for the intended use. Thus, a composition consisting essentially of the elements defined herein would not exclude trace amounts of contaminants from isolation and purification methods, as well as pharmaceutically acceptable carriers, e.g., phosphate-buffered saline, preservatives, and the like. "Consisting of" is intended to mean excluding more than trace amounts of other ingredients and substantial method steps for administering the compositions of the present disclosure. Embodiments defined by each of these transitional phrases are within the scope of the present disclosure.

[0033] As used herein, the term "comprising" is intended to mean that the peptides recited herein include the recited amino acid sequence but do not exclude other amino acids. "Consisting essentially of," when used to define a sequence, refers to the core sequence, optionally surrounded by additional amino acids. Thus, a peptide consisting essentially of a sequence defined herein will not exclude additional amino acids at the C- or N-terminus. In some embodiments, a peptide consisting essentially of a sequence defined herein includes up to 10 additional amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) at the N-terminus. In some embodiments, a peptide consisting essentially of a sequence defined herein includes up to 10 additional amino acids at the C-terminus. In some embodiments, a peptide consisting essentially of a sequence defined herein includes up to 10 additional amino acids at the N-terminus and up to 10 additional amino acids at the C-terminus. When used in reference to a peptide, "consisting of" a sequence means that the peptide does not contain any additional sequences. Embodiments defined by each of these transitional phrases are within the scope of the present disclosure.

[0034] The term "biofilm" refers to a thin layer or organized community of microorganisms that can adhere to the surface of structures, which can be organic or inorganic, or that can be concentrated at an interface (usually solid / liquid) surrounded by a mucous matrix of extracellular polymers, including 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 antibiotics (microbiotics) and antimicrobial agents. Biofilms live on gum tissue, teeth, and restoration surfaces, causing dental caries and periodontal disease (also known as periodontal plaque disease). Biofilms also cause chronic middle ear infections. Biofilms can also form on the surfaces of dental implants, stents, catheter lines, and contact lenses. Biofilms grow on pacemakers, heart valve replacements, artificial joints, and other surgical implants. The U.S. Centers for Disease Control estimates that more than 65% of nosocomial (hospital-acquired) infections are caused by biofilms. Fungal biofilms also frequently contaminate medical devices. They cause chronic vaginal infections and lead to life-threatening systemic infections in people with compromised immune systems. Biofilms are also involved in many diseases. For example, cystic fibrosis patients have Pseudomonas or nontuberculous mycobacterium (NTM) infections, which often result in antibiotic-resistant biofilms.

[0035] As used herein, "treatment of a biofilm-associated disorder," also referred to herein as "for preventing or treating a bacterial biofilm," refers to reducing the severity and / or duration of the disorder and / or reducing the severity and / or duration of symptoms resulting from the disorder, particularly symptoms of infection. In some embodiments, treatment results in restoration of the health of the individual. Preferably, the individual has less severe disease symptoms or has symptoms for a shorter period of time. Disease symptoms can be monitored using conventional techniques. In one aspect, the term "treatment" excludes prevention.

[0036] In some embodiments, the compositions of the present disclosure can be used to prevent or reduce the formation or growth of biofilms ex vivo or in vivo. As used herein, "preventing or reducing biofilm formation or growth" refers to the prevention, delay, or reduction of the formation or growth of biofilms ex vivo or in vivo. As will be understood by those skilled in the art, such reduction of biofilm formation or growth can slow the growth of biofilms compared to the growth of untreated (untreated) biofilms.

[0037] In some embodiments, the compositions disclosed herein are useful for decomposing or reducing biofilms. As used herein, "decomposing or reducing biofilms" refers to the partial or complete elimination of biofilms in vivo or in vivo. As will be understood by those skilled in the art, after such treatment, planktonic bacteria (i.e., free-living bacteria floating in the liquid) may still exist.

[0038] "DNABII polypeptide or DNABII protein" refers to a DNA-binding protein or polypeptide that is composed of a DNA-binding domain and thus has specific or general affinity for DNA. In one embodiment, it binds to DNA in the minor groove. Non-limiting examples of DNABII proteins are the integration host factor (IHF) protein and the histone-like protein (HU) from E. coli U93 strain. Other DNA-binding proteins that may be associated with biofilms include DPS (Genbank Accession No.: CAA49169), H-NS (Genbank Accession No.: CAA47740), Hfq (Genbank Accession No.: ACE63256), CbpA (Genbank Accession No.: BAA03950) and CbpB (Genbank Accession No.: NP_418813).

[0039] By "terminal fragment" of a DNABII polypeptide is intended a DNABII polypeptide that forms the two protein arms, using IHF alpha and IHF beta as examples. A non-limiting example of such is IhfA, A terminal fragment: [ka] (SEQ ID NO: 1) and IhfB, B tip fragment: [ka] (SEQ ID NO: 2).

[0040] As used herein, the term "tip chimeric peptide" or "IhfA5-mIhfB4 NTHI "Chimer" or "chimer" is a [ka] IhfA5-mIhfB4 comprising, consisting essentially of, or even consisting of, the polypeptide sequence of (SEQ ID NO: 3) NTHI Peptide chimeras are contemplated, where "X" is an optional amino acid linker sequence, optionally comprising, consisting essentially of, or even consisting of 1 to 20 amino acids; and "X1" is any amino acid, or alternatively, "X1" is selected from the amino acids Q, R, K, S, or T. In a further aspect, "X1" is K or Q. In a further embodiment, the apical chimeric peptide IhfA5-mIhfB4 NTHI teeth, [ka] (SEQ ID NO: 4), where "X" is an optional amino acid linker sequence, optionally comprising, consisting essentially of, or even consisting of 1 to 20 amino acids. In certain embodiments, the linker is selected from any one or more of SEQ ID NOs: 6-8 and 26-30. In yet further embodiments, the tip chimeric peptide IhfA5-mIhfB4 NTHI teeth, [ka] (SEQ ID NO: 5).

[0041] By "tail fragment" of a DNABII polypeptide is intended the region of the protein that is exposed to the bulk of the medium and that is not occluded by DNA or other polypeptides.

[0042] As used herein, "chimer" or "chimeric tail peptide" refers to a polypeptide comprising the IHFA tail region of a DNABII peptide conjugated directly or indirectly (e.g., via a linker) to the IHFB tail region of a DNABII peptide. In yet further embodiments, the tail region of the DNABII peptide is an IhfA3-IhfB2NTHI chimeric tail peptide. In some embodiments, the chimera comprises the amino acid sequence set forth in SEQ ID NO: 43.

[0043] "Tip region" as used herein refers to either an "tip chimer peptide" and / or an "tip fragment."

[0044] The "integration host factor" or "IHF" protein is a bacterial protein used by bacteriophages to incorporate their DNA into host bacteria. It is a DNA-binding protein that functions in genetic recombination and transcriptional and translational regulation. It also binds to extracellular microbial DNA. The genes encoding the IHF protein subunits in E. coli are the himA (Genbank accession number: POA6X7.1) and himD (POA6Y1.1) genes.

[0045] "HMGB1" refers to the high mobility group box (HMGB) 1 protein, which has been reported to bind to and distort the minor groove of DNA, and is an example of an interfering agent. Recombinant or isolated proteins and polypeptides are commercially available from Atgenglobal, ProSpecBio, Protein1, and Abnova. The sequences of wild-type mouse HMGB1 protein and human HMGB1 protein are set forth in SEQ ID NO: 31 and SEQ ID NO: 32, respectively.

[0046] "HU" or "histone-like protein from E. coli strain U93" refers to a class of heterodimeric proteins typically associated with E. coli. HU proteins are known to bind to DNA junctions. Related proteins have been isolated from other microorganisms. The complete amino acid sequence of E. coli HU was reported by Laine et al. (1980) Eur. J. Biochem. 103(3):447-481. Antibodies against the HU protein are commercially available from Abcam.

[0047] "Linker" or "peptide linker" refers to a peptide sequence linked to either the N-terminus or C-terminus of a polypeptide sequence. In one embodiment, the linker is about 1 to about 20 amino acid residues in length, or alternatively, 2 to about 10, or about 3 to about 5 amino acid residues in length. Examples of peptide linkers are set forth in SEQ ID NOS: 6-8 and 26-30.

[0048] As used herein, the term "DNA-binding agent" refers to an agent that binds to eDNA and prevents the formation of or disrupts extracellular structures containing eDNA (e.g., biofilms and neutrophil extracellular traps (NETs)).

[0049] The term "Haemophilus influenzae" refers to a pathogenic bacterium that can cause many different infections, such as ear infections, eye infections, and sinusitis. Many different strains of Haemophilus influenzae have been isolated and contain the IhfA gene or protein. Some non-limiting examples of different strains of Haemophilus influenzae include Rd KW20, 86-028NP, R2866, PittGG, PittEE, R2846, and 2019.

[0050] By "microbial DNA" is intended single-stranded or double-stranded DNA derived from biofilm-producing microorganisms.

[0051] As used herein, the term "label" or "detectable label" refers to a directly or indirectly detectable compound or composition, e.g., an N-terminal histidine tag (N-His), a magnetically active isotope, e.g., a nucleotide sequence, which is directly or indirectly conjugated to a composition to be detected, such that a "labeled" composition can be produced. 115 Sn, 117 Sn and 119 Sn, non-radioactive isotopes, e.g. 13 C and 15The term "label" contemplates a polynucleotide, polynucleotide, or protein, such as an antibody. The term also includes sequences conjugated to a polynucleotide that provide a signal after expression of the inserted sequence, such as green fluorescent protein (GFP). The label may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical alteration of a detectable substrate compound or substrate composition. The label may be suitable for small-scale detection or may be more suitable for high-throughput screening. As such, suitable labels include, but are not limited to, magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or quantified. A simply detected response generally includes a response whose presence is simply confirmed, while a quantified response generally includes a response having a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other property. In luminescent or fluorescent assays, the detectable response can be generated directly using a luminophore or fluorophore associated with the assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent labels that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent response generally involves a change in, or the appearance of, a luminescent signal. Methods and luminophores suitable for luminescently labeling assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0052] "Gene delivery vehicle" is defined as any molecule that can carry inserted polynucleotide into host cells. Examples of gene delivery vehicles include liposomes, micelles, biocompatible polymers (including natural and synthetic polymers); lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial virus envelopes; metal particles; and bacteria or viruses, such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors, and other recombinant vehicles typically used in the art, which have been described for expression in various eukaryotic and prokaryotic hosts and can be used for gene therapy and simple protein expression.

[0053] The polynucleotides of the present disclosure can be delivered to cells or tissues using gene delivery vehicles. As used herein, "gene delivery," "gene transfer," and "transduction" refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for the introduction. Such methods include various well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes), as well as techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques used to introduce polynucleotides). The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide contain a replication origin compatible with the host cell or be integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. As is known in the art and described herein, several vectors are known to be capable of mediating the transfer of genes into mammalian cells.

[0054] As used herein, the term "eDNA" refers to extracellular DNA found as a component of pathogenic biofilms.

[0055] As used herein, ESKAPE pathogens include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. These pathogens are the leading cause of hospital-acquired infections worldwide.

[0056] A "plasmid" is an extrachromosomal DNA molecule that is separate from chromosomal DNA and capable of replicating independently of chromosomal DNA. It is often circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and typically provide a selective advantage under given environmental conditions. Plasmids can carry genes that provide resistance to antibiotics that are naturally present in a competitive environmental niche, or alternatively, the proteins produced can act as toxins under similar circumstances.

[0057] "Plasmids" used in genetic engineering are called "plasmid vectors." Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of the plasmid, which contains a gene that makes the cell resistant to a particular antibiotic and a multiple cloning site (MCS, or polylinker), a short region containing several commonly used restriction sites that allows for easy insertion of DNA fragments at this location. Another major use of plasmids is to produce large amounts of proteins. In this case, researchers grow bacteria containing a plasmid with the gene of interest. Just as bacteria produce proteins to confer their antibiotic resistance, they can also be induced to produce large amounts of proteins from the inserted gene. This is an inexpensive and easy way to mass-produce a gene or the protein it encodes.

[0058] "Yeast artificial chromosome" or "YAC" refers to a vector used to clone large DNA fragments (greater than 100 kb and up to 3000 kb). It is an artificially constructed chromosome that contains telomere, centromere, and origin of replication sequences required for replication and storage in yeast cells. Once constructed using an initial circular plasmid, it is linearized using a restriction enzyme, and then DNA ligase can add a sequence or gene of interest into the linear molecule using the cohesive ends. Because yeast itself is a eukaryotic cell, yeast expression vectors such as YAC, YIp (yeast integrating plasmid), and YEp (yeast episomal plasmid) are extremely useful because they can produce eukaryotic protein products with post-translational modifications. However, YACs are more unstable than BACs and have been found to produce chimeric effects.

[0059] A "viral vector" is defined as a recombinantly produced virus or virus particle containing a polynucleotide to be delivered into a host cell, either ex vivo or in vivo. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and alphavirus vectors. Infectious tobacco mosaic virus (TMV)-based vectors can be used to produce proteins and have been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15):6099-6104). Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. In embodiments where gene transfer is mediated by a retroviral vector, vector construct refers to a polynucleotide comprising the retroviral genome or portion thereof and a therapeutic gene.

[0060] As used herein, "retroviral-mediated gene transfer" or "retroviral transduction" have the same meaning and refer to the process by which a gene or nucleic acid sequence is stably transferred into a host cell based on a virus that invades the cell and integrates 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 and enter the cell. As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell via a viral or viral-like entry mechanism.

[0061] Retroviruses carry their genetic information in the form of RNA; however, when the virus infects a cell, the RNA is reverse transcribed into a DNA form that is integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0062] In embodiments in which gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or an adeno-associated virus (AAV), a vector construct refers to a polynucleotide comprising a viral genome or a portion thereof and a transgene. Adenoviruses (Ad) are a relatively well-characterized, homogeneous group of viruses, including more than 50 serotypes. See, for example, International PCT Application No. WO95 / 27071. Ad does not require integration into the host cell genome. Recombinant Ad-derived vectors have also been constructed, particularly those that reduce the possibility of recombination and wild-type virus generation. See, International PCT Application Nos. WO95 / 00655 and WO95 / 11984. Wild-type AAV has high infectivity and specificity for integration into the genome of host cells. See Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0063] Vectors containing both a promoter and a cloning site to which a polynucleotide can be operably linked are known in the art. Such vectors are capable of transcribing RNA in vitro and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). To optimize expression and / or transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to eliminate redundant, potentially inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression at either the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' of the initiation codon to enhance expression.

[0064] Gene delivery vehicles also include DNA / liposome complexes, micelles, and targeting viral protein-DNA complexes. Liposomes containing targeting antibodies or fragments thereof can be used in the methods of the present disclosure. In addition to delivering polynucleotides to cells or cell populations, direct introduction of the proteins described herein into cells or cell populations can be achieved by, but is not limited to, protein transfection techniques, or culture conditions that can enhance the expression and / or activity of the proteins of the present disclosure are other, but not limited to, techniques.

[0065] "Inhibiting, preventing, or disrupting" a biofilm contemplates prophylactic or therapeutic reduction of biofilm structure. In one embodiment, this definition excludes biofilm prevention. In another embodiment, the term "inhibiting, competing, or titrating" contemplates reduction of the formation of the DNA / protein matrix that is a component of microbial biofilms.

[0066] By "curved polynucleotide" is intended a double-stranded polynucleotide containing a small loop in one strand that does not pair with the other strand, and any polynucleotide whose end-to-end distance is reduced beyond the natural thermal fluctuation, i.e., curved beyond the 150 bp persistence length for native B-form double-stranded DNA. In some embodiments, the loop is 1 base to about 20 bases in length, or alternatively 2 bases to about 15 bases in length, or alternatively about 3 bases to about 12 bases in length, or alternatively about 4 bases to about 10 bases in length, or alternatively has about 4, 5, or 6, or 7, or 8, or 9, or 10 bases.

[0067] A "subject" of diagnosis or treatment is a cell or an animal, e.g., a mammal or a human. Non-human animals that are the subject of diagnosis or treatment are susceptible non-human animals or animal models, e.g., monkeys, mice, e.g., rats, mice, chinchillas, canines, e.g., dogs, lagomorphs, e.g., rabbits, farm animals, sport animals, and pets.

[0068] The terms "protein," "peptide," and "polypeptide" are used interchangeably and, in their broadest sense, refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids; there is no limit to the maximum number of amino acids that can make up the sequence of a protein or peptide. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine and both the D and L optical isomers, amino acid analogs, and peptidomimetics.

[0069] The terms "isolated" or "recombinant," as used herein with respect to nucleic acids, such as DNA or RNA, refer to molecules separated from other DNAs or RNAs, respectively, present in the natural source of the macromolecule and polypeptide. The term "isolated or recombinant nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to polynucleotides, polypeptides, and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the terms "isolated or recombinant" refer to cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragment(s) thereof, that are separated from cellular and other components with which they are normally associated in nature. For example, an isolated cell is one that is separated from tissues or cells of a different phenotype or genotype. An isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on a chromosome. As will be apparent to one of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof does not require "isolation" to distinguish it from its naturally occurring counterpart.

[0070] Without explicit recitation or other intention, when the present disclosure relates to a polypeptide, protein, polynucleotide, or antibody, it should be inferred that equivalents or biological equivalents of such are intended within the scope of the present disclosure. As used herein, the term "biological equivalent thereof" is intended to be synonymous with "equivalent thereof," and when referring to a reference protein, antibody, polypeptide, or nucleic acid, it is intended to refer to one that has minimal homology while still maintaining the desired structure or functionality. Unless specifically recited herein, any polynucleotide, polypeptide, or protein referred to herein is intended to also include its equivalent. For example, an equivalent is intended to have at least about 70% homology or identity, or alternatively about 80% homology or identity, and alternatively at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively 98% homology or identity percentage to the reference protein, polypeptide, or nucleic acid, and exhibit substantially equivalent biological activity. In another aspect, the term contemplates a polynucleotide that hybridizes under high stringency conditions to a reference polynucleotide or its complement.

[0071] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. Alignment and percent homology or percent sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST using default parameters. Particularly preferred programs are BLASTN and BLASTP using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; filtering method = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0072] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 30% identity, or alternatively less than 25% identity, less than 20% identity, or alternatively less than 10% identity, with one of the sequences of the present disclosure.

[0073] "Homology" or "identity" or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions to a reference polynucleotide or its complement.

[0074] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized by hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonding can occur through Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific manner. The complex can comprise two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction can constitute a step in a more extensive process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0075] Examples of stringent hybridization conditions include an incubation temperature of about 25°C to about 37°C, a hybridization buffer concentration of about 6xSSC to about 10xSSC, a formamide concentration of about 0% to about 25%, and a wash solution of about 4xSSC to about 8xSSC. Examples of moderate hybridization conditions include an incubation temperature of about 40°C to about 50°C, a buffer concentration of about 9xSSC to about 2xSSC, a formamide concentration of about 30% to about 50%, and a wash solution of about 5xSSC to about 2xSSC. Examples of high stringency conditions include an incubation temperature of about 55°C to about 68°C, a buffer concentration of about 1xSSC to about 0.1xSSC, a formamide concentration of about 55% to about 75%, and a wash solution of about 1xSSC, 0.1xSSC, or deionized water. Generally, hybridization incubation times are from 5 minutes to 24 hours, with one, two, or more wash steps, with wash incubation times of about 1 minute, about 2 minutes, or about 15 minutes. SSC is a 0.15M NaCl and 15mM citrate buffer. It is understood that equivalents of SSC using other buffer systems may be used.

[0076] A "subject" of diagnosis or treatment can be a cell or an animal, e.g., a mammal or a human. Non-human animals that are the subject of diagnosis or treatment can be susceptible non-human animals or animal models, such as monkeys, mice, e.g., rats, mice, chinchillas, canines, e.g., dogs, lagomorphs, e.g., rabbits, livestock, sport animals, and pets. The terms "subject," "host," "individual," and "patient" are used interchangeably herein to refer to animals, typically mammals. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, and macaques), domestic animals (e.g., dogs and cats), livestock animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal can be of any age or stage of development (e.g., adult, teenage, child, infant, or in utero mammal). The mammal can be male or female, hi some embodiments, the subject is a human.

[0077] By host cell is intended a eukaryotic or prokaryotic cell containing exogenous factors. "Eukaryotic cell" includes all kingdoms of life except Monera. Eukaryotic cells can be easily distinguished by a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes, or organisms whose cells are organized into complex structures by internal membranes and cytoskeleton. The most distinctive membrane-bound structure is the nucleus. Unless specifically recited, the term "host" includes eukaryotic hosts, including, for example, yeast, higher plants, insects, and mammalian cells. Non-limiting examples of eukaryotic cells or eukaryotic hosts include monkeys, cows, pigs, mice, rats, birds, reptiles, and humans.

[0078] "Prokaryotic cells," which typically lack a nucleus or any other membrane-bound organelles, are divided into two domains: bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in circular loops called episomes. Bacterial cells are very small, roughly the size of animal mitochondria (about 1-2 μm in diameter and 10 μm in length). Prokaryotic cells feature three main shapes: rod-shaped, spherical, and spiral. Instead of undergoing an intricate replication process like eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, Bacillus, E. coli, and Salmonella.

[0079] As used herein, the terms "treating," "treatment," and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disorder or its signs or symptoms, and / or therapeutic, in terms of partially or completely curing a disorder and / or adverse effects that may result from the disorder. In one aspect, "treatment" excludes prevention.

[0080] By "prevent" is intended to prevent or prevent the disorder or effect in a system or subject predisposed to the disorder or effect. An example of such would be preventing biofilm formation in a system infected with a microorganism known to produce biofilms.

[0081] "Pharmaceutically acceptable carrier" refers to any diluent, excipient, or carrier that can be used in the compositions of the present disclosure. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They are preferably selected for the intended form of administration, ie, oral tablets, capsules, elixirs, syrups, and the like, and are consistent with conventional pharmaceutical practice.

[0082] "MIC" is an acronym for minimum inhibition and defines the in vitro level of susceptibility or resistance of a particular bacterial strain to an applied antibiotic. Methods for converting MIC to a therapeutic dose are known in the art and are described in Kowalska-Krochmal and Wicher, Pathogens (2021) Feb; 10(2):165.

[0083] "Administration" can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those skilled in the art and will vary with the composition used in the therapy, the purpose of the therapy, the target cells being treated, and the subject being treated. Single or multiple administrations can be carried out, with the dose level and pattern selected by the treating physician. Suitable dosage formulations and methods of administering drugs are known in the art. The route of administration can also be determined, and methods of determining the most effective route of administration are known to those skilled in the art and will vary with the composition used in the therapy, the purpose of the therapy, the health condition 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 application.

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

[0085] The term "effective amount" refers to a quantity sufficient to achieve a beneficial or desired result or effect. In the context of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the condition at issue, as well as the characteristics of the individual subject, such as overall health, age, sex, weight, and tolerance to the pharmaceutical composition. In the context of immunogenic compositions, in some embodiments, an effective amount is an amount sufficient to produce a protective response against a pathogen. In other embodiments, an effective amount of an immunogenic composition is an amount sufficient to produce antibody production against an antigen. In some embodiments, an effective amount is the amount required to confer passive immunity in a subject in need thereof. With respect to immunogenic compositions, in some embodiments, 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, in addition to the factors described above. Those skilled in the art will be able to determine the appropriate amount depending on these and other factors.

[0086] In some embodiments, the effective amount will depend on the size and nature of the application in question.The effective amount will also depend on the nature and sensitivity of the target and the method used.Those skilled in the art will be able to determine the effective amount based on these and other considerations.The effective amount can include one or more administrations of the composition depending on the embodiment.

[0087] The agents and compositions can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as the active ingredient in a pharmaceutical composition.

[0088] The agents of the present disclosure can be administered for therapy by any suitable route of administration. It will also be appreciated that the preferred route will vary with the condition and age of the recipient and the disease being treated.

[0089] Examples of solid supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. The nature of the support can be either soluble to some extent or insoluble. The support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a polynucleotide, polypeptide, or antibody. Thus, the support configuration can be spherical, such as a bead, or cylindrical, such as the interior surface of a test tube or the exterior surface of a rod. Alternatively, the surface can be flat, e.g., a sheet, test strip, or the like, or alternatively, polystyrene beads. Those skilled in the art will know of many other suitable supports for binding antibodies or antigens, or will be able to ascertain such by use of routine experimentation.

[0090] As used herein, "antibody" includes whole antibodies and any antigen-binding fragments or single chains thereof. Thus, the term "antibody" includes any protein- or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule. Examples of such include, but are not limited to, the complementarity-determining regions (CDRs) of a heavy or light chain or its ligand-binding portion, the variable region of a heavy or light chain, the constant region of a heavy or light chain, the framework (FR) region, or any portion thereof, or at least a portion of a binding protein. Antibodies can be polyclonal or monoclonal and can be isolated from any suitable biological source, such as mice, rats, sheep, or canines.

[0091] The terms "antibody," "antibodies," and "immunoglobulin" also include immunoglobulins of any isotype, fragments of antibodies that retain specific binding to an antigen, including, but not limited to, Fab, Fab', F(ab)2, Fv, scFv, dsFv, Fd fragments, dAb, VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappabodies; and multispecific antibody fragments formed from antibody fragments. Examples of such fragments include, but are not limited to, the complementarity-determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, the variable regions (also referred to herein as variable domains) of a heavy or light chain, the constant regions (also referred to herein as constant domains) of a heavy or light chain, framework (FR) regions, or any portion thereof, at least a portion of a binding protein, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The variable regions of the heavy and light chains of immunoglobulin molecules contain binding domains that interact with antigens. The constant region of an antibody (Ab) can mediate the binding of the immunoglobulin to host tissue. The term "anti-" when used before the name of a protein, such as anti-DNABII, anti-IHF, anti-HU, or anti-tip chimera, refers to a monoclonal or polyclonal antibody that can bind to and / or have affinity for a specific protein. For example, "anti-IHF" refers to an antibody that binds to the IHF protein. A specific antibody has affinity for or can bind to proteins other than the protein to which it was raised. For example, anti-IHF is specifically raised against the IHF protein, but can also bind to other proteins related by either sequence or structural homology.

[0092] Complementarity-determining regions (CDRs) are portions of the variable regions of antibodies or T-cell receptors produced by B cells and T cells, respectively, that bind to their specific antigens (also called epitopes). In certain embodiments, the terms "variable region" and "variable domain" are used interchangeably to refer to polypeptides in the light or heavy chains of an antibody, the sequence of which varies considerably from antibody to antibody and determines the conformation of the combining site that confers the antibody's specificity for a particular antigen. In further embodiments, the variable region is between about 90 amino acids in length and about 200 amino acids in length, including, but not limited to, about 100 amino acids in length, or alternatively about 110 amino acids in length, or alternatively about 120 amino acids in length, or alternatively about 130 amino acids in length, or alternatively about 140 amino acids in length, or alternatively about 150 amino acids in length, or alternatively about 160 amino acids in length, or alternatively about 170 amino acids in length, or alternatively about 180 amino acids in length, or alternatively about 190 amino acids in length. In certain embodiments, a variable region of an amino acid sequence, as used herein, refers to the first about 100, or alternatively about 110, or alternatively about 120, or alternatively about 130, or alternatively about 140, or alternatively about 150 amino acids of the amino acid sequence (including or excluding the signal peptide, if applicable) being the variable region.

[0093] A set of CDRs constitutes a paratope, also called an antigen-binding site, which is the part of an antibody that recognizes and binds to an antigen. In the amino acid sequence of the variable region of an antigen receptor, such as a heavy or light chain, there are three CDRs (CDR1, CDR2, and CDR3) arranged non-contiguously, optionally from the amino terminus to the carboxyl terminus. As used herein, CDRn refers to CDRn in or derived from an immunoglobulin chain, where the number n is selected from 1 to 3. In one embodiment, CDRLn refers to CDRn in or derived from a light chain, where the number n is selected from 1 to 3; while CDRHn refers to CDRn in or derived from a heavy chain, where the number n is selected from 1 to 3. In certain embodiments, framework region (FR) refers to the part of the variable region that is not a CDR. In certain embodiments, FRn refers to FR in or derived from a heavy or light chain, where the number n is selected from 1 to 4. In certain embodiments, the variable region comprises, consists essentially of, or even consists of (optionally in the order presented, and further optionally from amino terminus to carboxyl terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0094] The variable regions and / or CDRs of an antibody or fragment thereof can be determined by one of skill in the art using, for example, publicly available or commercially available tools. Non-limiting examples of such tools include IgBlast (accessible at www.ncbi.nlm.nih.gov / igblast / ), Scaligner (available at drugdesigntech at www.scaligner.com / ), IMGT rules and / or tools (see, e.g., www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html, also accessible at www.imgt.org / ), Chothia Canonical Assignment (accessible at www.bioinf.org.uk / abs / chothia.html), Antigen Receptor Numbering And Receptor Classification (ANARCI, accessible at opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / anarci / ), Kabat numbering method / scheme (see, e.g., Kabat, EA, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of of Health and Human Services, NIH Publication No. 91-3242) or the Paratome web server (accessible at www.ofranlab.org / paratome / ; see Vered Kunik, et al, Nucleic Acids Research, Volume 40, Issue W1, 1 July 2012, Pages W521-W524).

[0095] Antibodies can be polyclonal, monoclonal, multispecific (e.g., bispecific) and antibody fragments, so long as they exhibit the desired biological activity. Antibodies can be isolated from any suitable biological source, such as mice, rats, sheep and canines.

[0096] The terms "polyclonal antibody" or "polyclonal antibody composition," as used herein, refer to a preparation of antibodies derived from different B-cell lines. These are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope.

[0097] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. Because each monoclonal antibody is directed against a single determinant on an antigen, monoclonal antibodies are highly specific. Antibodies can be detectably labeled, for example, with radioisotopes, enzymes that generate detectable products, and fluorescent proteins. Antibodies can be further conjugated to other moieties, such as members of specific binding pairs, for example, biotin (a member of the biotin-avidin specific binding pair). Antibodies can be bound to solid supports, including, but not limited to, polystyrene plates or beads.

[0098] Monoclonal antibodies can be produced using hybridoma techniques or recombinant DNA methods known in the art. Hybridomas are cells produced in the laboratory from the fusion of antibody-producing lymphocytes with non-antibody-producing cancer cells (usually myeloma or lymphoma). The hybridomas grow and produce continuous samples of specific monoclonal antibodies. Alternative techniques for producing or selecting antibodies include exposing lymphocytes to the antigen of interest and screening antibody display libraries in cells, phage, or similar systems.

[0099] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis or by somatic mutation). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Thus, as used herein, the term "human antibody" refers to antibodies that contain proteins (e.g., CDRs, framework, CDRs ... L , C H Domain (e.g., C H1 , C H2 , C H3 The term "human antibody" refers to an antibody in which substantially all of the following portions are substantially non-immunogenic in humans: the base (VL, VH) of the antibody, the hinge (VL, VH), and the like. Similarly, antibodies designated as primates (monkeys, baboons, chimpanzees, etc.), rodents (mouse, rats, rabbits, guinea pigs, hamsters, etc.), and other mammals refer to antibodies specific to that species, subgenus, genus, subfamily, or family. Furthermore, chimeric antibodies include any combination of the above. Such changes or modifications optionally retain or reduce immunogenicity in humans or other species compared to the unmodified antibody. Thus, human antibodies are distinct from chimeric and humanized antibodies. It is noted that human antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. Furthermore, human antibodies, when single-chain antibodies, can include linker peptides not found in native human antibodies. For example, an Fv can include a linker peptide, such as two to about eight glycine or other amino acid residues, connecting the heavy chain variable region and the light chain variable region, and such a linker peptide is considered to be of human origin.

[0100] As used herein, a human antibody is "derived from" a particular germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, for example, by immunizing a transgenic mouse carrying human immunoglobulin genes or by screening a human immunoglobulin gene library. A human antibody "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequence of a human germline immunoglobulin. The selected human antibody typically has at least 90% amino acid sequence identity to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as human when compared to germline immunoglobulin amino acid sequences of other species (e.g., mouse germline sequences). In certain cases, a human antibody may have at least 95% or even at least 96%, 97%, 98%, or 99% amino acid sequence identity to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody can display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0101] As used herein, the term "humanized antibody" or "humanized immunoglobulin" refers to a human / non-human chimeric antibody that contains minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a variable region or fragment thereof (e.g., one, two, three, four, five, or all six CDRs) of the recipient are replaced by residues from a variable region or fragment thereof (e.g., one, two, three, four, five, or all six CDRs) of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. Humanized antibodies can contain residues that are not found in either the recipient antibody or the donor antibody. Humanized antibodies can also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin, a non-human antibody containing one or more amino acids in the framework regions, constant region, or CDRs substituted with amino acids at the corresponding positions from a human antibody. Without wishing to be bound by theory, humanized antibodies produce a reduced immune response in a human host compared to a non-humanized version of the same antibody. Humanized antibodies can have conservative amino acid substitutions that do not substantially affect antigen binding or other antibody functions. Conservative substitution groupings include glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serine-threonine, and asparagine-glutamine. Specifically, the humanized antibodies disclosed herein are EC 50 , K. on , K. off , K. A and / or K DSpecifically binds to the DNABII polypeptide or a fragment thereof (such as the tip chimeric peptide or the tail chimeric peptide) in one or more specific ranges (multiple) of inhibiting or releasing a specific cytokine (multiple) in the treatment of a subject. In a further embodiment, a humanized antibody that specifically binds to the tip region (such as the tip chimeric peptide) of the DNABII polypeptide destroys biofilms in both and. In addition, although the humanization process is a rational design process, it may result in unexpected changes (positive or negative) in, for example, binding affinity, antigen specificity or physical properties, such as solubility or aggregation; therefore, the properties of humanized antibodies are essentially not predictable from the properties of the starting non-human antibody.

[0102] In one embodiment, an antibody, as used herein, may be a recombinant antibody. The term "recombinant antibody," as used herein, includes any antibody prepared, expressed, generated, or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice) transgenic or transchromosomal for immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed to express the antibody (e.g., from transfectomas), antibodies isolated from recombinant combinatorial antibody libraries, and antibodies prepared, expressed, generated, or isolated by any other means, including splicing immunoglobulin (Ig) gene sequences to other DNA sequences. However, in certain embodiments, such recombinant antibodies may be subjected to mutagenesis (or somatic mutagenesis, if an animal transgenic for Ig sequences is used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that may not naturally exist within the antibody germline repertoire. Methods for producing these antibodies are known in the art.

[0103] In one embodiment, an antibody, as used herein, may be a chimeric antibody. As used herein, a chimeric antibody is an antibody whose light and heavy chain genes have been constructed, typically by genetic engineering, from variable and constant region genes of antibodies belonging to different species.

[0104] In addition, the antibodies disclosed herein can be engineered to contain 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 antigen-dependent cellular cytotoxicity. Such modifications include, but are not limited to, altering the number of cysteine ​​residues in the hinge region to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody (U.S. Patent No. 5,677,425) and mutating amino acids in the Fc hinge region to decrease the biological half-life of the antibody (U.S. Patent No. 6,165,745).

[0105] Furthermore, the antibodies disclosed herein may be chemically modified. The glycosylation of an antibody can be altered by modifying one or more glycosylation sites within the antibody sequence, for example, to increase the affinity of the antibody for an antigen (U.S. Patent Nos. 5,714,350 and 6,350,861). Alternatively, to increase antibody-dependent cell-mediated cytotoxicity, hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures can be obtained by expressing the antibody in a host cell with altered glycosylation machinery (Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-180).

[0106] The antibodies disclosed herein can be pegylated to increase biological half-life by reacting the antibody or antigen-binding 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 an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG used to derivatize other proteins, such as mono(C1-C10)alkoxy-polyethylene glycol or mono(C1-C10)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).

[0107] Furthermore, antibodies can be chemically modified by conjugating or fusing the antigen-binding region of the antibody to a serum protein, such as human serum albumin, to increase the half-life of the resulting molecule. Such approaches are described, for example, in EP 0322094 and EP 0486525.

[0108] The antibodies or fragments thereof of the present disclosure can be conjugated to diagnostic agents and used for diagnosis, for example, to monitor the onset or progression of a disease and determine the effectiveness of a given treatment regimen. Examples of diagnostic agents include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals using various positron emission tomography techniques, and non-radioactive paramagnetic metal ions. Detectable substances can be coupled or conjugated directly to antibodies or fragments thereof or indirectly via linkers using techniques known in the art. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include: 125 I, 131I, Indium-111, Lutetium-171, Bismuth-212, Bismuth-213, Astatine-211, Copper-62, Copper-64, Copper-67, Yttrium-90, Iodine-125, Iodine-131, Phosphorus-32, Phosphorus-33, Scandium-47, Silver-111, Gallium-67, Praseodymium-142, Samarium-153, Terbium-161, Dysprosium-166, Holmium-166 , rhenium-186, rhenium-188, rhenium-189, lead-212, radium-223, actinium-225, iron-59, selenium-75, arsenic-77, strontium-89, molybdenum-99, rhodium-1105, palladium-109, praseodymium-143, promethium-149, erbium-169, iridium-194, gold-198, gold-199, and lead-211. Monoclonal antibodies may be indirectly conjugated to radioactive metal ions by use of a bifunctional chelating agent covalently linked to the antibody. Chelating agents may be attached via amides (Meares et al. (1984) Anal. Biochem. 142:68-78); sulfhydryls 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).

[0109] Additionally, the antibodies or fragments thereof of the present disclosure may be conjugated to a therapeutic agent. Suitable therapeutic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione), mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, antimetabolites (methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine, etc.), alkylating agents (mechlorethamine, thiorphan, chlorambucil, melphalan, carmustine (BiCNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, e.g., carboplatin etc.), antibiotics (dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC), etc.), diphtheria toxin and related molecules (diphtheria A chain and its active fragments and hybrid molecules, etc.), ricin toxin (ricin A or deglycosylated ricin A chain toxin, etc.), cholera toxin, Shiga-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaccaamericana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrietocin, phenomycin, enomycin toxins and mixed toxins.

[0110] Additional suitable conjugated molecules include ribonucleases (RNases), 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 ranging in length from 15 to 50 bases that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets, and can bind to small molecules such as ATP (U.S. Pat. No. 5,631,146) and theophiline (U.S. Pat. No. 5,580,737), as well as large molecules such as reverse transcriptase (U.S. Pat. No. 5,786,462) and thrombin (U.S. Pat. No. 5,543,293). Ribozymes are nucleic acid molecules capable of catalyzing chemical reactions, either intramolecularly or intermolecularly. Ribozymes typically cleave nucleic acid substrates by recognizing and binding to the target substrate followed by cleavage. Triplex-forming functional nucleic acid molecules can interact with double- or single-stranded nucleic acids by forming triplexes, where three DNA strands form a complex that relies on both Watson-Crick and Hoogsteen base pairing. Triplex molecules can bind to target regions with high affinity and specificity.

[0111] Functional nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or functional nucleic acid molecules can possess a de novo activity independent of any other molecule.

[0112] Therapeutic agents can be linked to antibodies directly or indirectly using any of a number of available methods. For example, drugs can be attached to the hinge region of an antibody component by reducing disulfide bond formation using cross-linking agents such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP) or via carbohydrate moieties in the Fc region of the antibody (Yu et al. 1994 Int. J. Cancer 56: 244; Upeslacis et al., "Modification of Antibodies by Chemical Methods," in Monoclonal antibodies: principles and applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal antibodies: Production, engineering and clinical application, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995)).

[0113] Techniques for conjugating therapeutic agents to antibodies are well known (Amon et al. "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy; Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al. "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.); Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospects Of The Therapeutic Use Of Radiolabeled 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).

[0114] The antibodies or antigen-binding regions thereof disclosed herein can be linked to another functional molecule, such as another antibody or a receptor ligand, to generate bispecific or multispecific molecules that bind to at least two or more different binding sites or target molecules. Linking of the antibody to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, can be achieved, for example, by chemical coupling, genetic fusion, or noncovalent association. Multispecific molecules can further include a third binding specificity in addition to the first and second target epitopes.

[0115] Bispecific and multispecific molecules can be prepared using methods known in the art.For example, each binding unit of a bispecific molecule can be produced separately and then conjugated with each other.When the binding molecule is a protein or peptide, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (sulfo-SMCC) (Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). When the binding molecule is an antibody, it can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains.

[0116] The antibodies of the present disclosure, or fragments thereof, can be linked to a moiety that is toxic to the cells to which the antibody is bound, to form "depleting" antibodies. These antibodies are particularly useful in applications in which it is desired to deplete NK cells.

[0117] The antibodies disclosed herein may 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.

[0118] Antibodies may also be bound to many different carriers. Thus, the present disclosure also provides compositions containing antibodies and other active or inactive substances. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes disclosed herein. Those skilled in the art will know other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such using routine experimentation.

[0119] As used herein, the term "antibody derivative" includes a full-length antibody or a fragment of an antibody in which one or more of the amino acids have been chemically modified, for example, by alkylation, pegylation, acylation, ester formation, or amide formation, to link the antibody to a second molecule, including, but not limited to, pegylated antibodies, cysteine-pegylated antibodies, and variants thereof.

[0120] As used herein, the term "immunoconjugate" includes an antibody or antibody derivative associated or linked to a second agent, e.g., a cytotoxic agent, a detectable agent, a radioactive agent, a targeting agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semi-synthetic antibody, or a multispecific antibody.

[0121] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine (TRITC), eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6 th (ed.).

[0122] In another embodiment, the fluorescent label is functionalized to facilitate covalent attachment to cellular components present within or on the surface of cells or tissues, such as cell surface markers. Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which can be used to attach the fluorescent label to a second molecule. The choice of functional group on the fluorescent label will depend on the site of attachment, either to a linker, drug, marker, or second labeling agent.

[0123] "Immune response" refers broadly to an antigen-specific response of lymphocytes to a foreign substance. Any substance capable of eliciting an immune response is said to be "immunogenic" and is referred to as an "immunogen." While all immunogens are antigens, not all antigens are immunogenic. The immune responses of the present disclosure can be humoral (through antibody activity) or cell-mediated (through T-cell activation).

[0124] The term "modulating an immune response" includes inducing (increasing, eliciting) an immune response; and reducing (suppressing) an immune response. An immunomodulatory method (or protocol) is one that modulates an immune response in a subject.

[0125] "HMG domain" or "high mobility group (HMG) box domain" refers to an amino acid sequence involved in DNA binding (Stros et al., Cell Mol Life Sci. 64(19-20):2590-606 (2007)). In one embodiment, the structure of an HMG-box domain consists of a disordered triple helix. In another embodiment, the HMG-box domain enables the protein to bind to non-B-form DNA conformations (twisted or unwound) with high affinity. HMG-box domains can be found in high mobility group proteins involved in regulating DNA-dependent processes such as transcription, replication, and DNA repair, all of which require changes in chromatin conformation (Thomas (2001) Biochem. Soc. Trans. 29(Pt 4):395-401).

[0126] Compositions used in accordance with the present disclosure may be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of composition calculated to produce a desired response in association with its administration, i.e., an appropriate route and regimen. The amount to be administered, both according to the number of treatments and the unit dose, depends on the desired outcome and / or protection. The precise amount of the composition also depends on the judgment of the practitioner and is peculiar to each individual. Factors affecting dosage include the subject's physical and clinical condition, the route of administration, the intended goal of treatment (palliative or curative), and the efficacy, stability, and toxicity of the particular composition. After formulation, a solution is administered in a manner compatible with the dosage formulation and in an amount that is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described herein.

[0127] The term "contacting" refers to a direct or indirect binding or interaction between two or more entities. A specific example of a direct interaction is binding. A specific example of an indirect interaction is when one entity acts on an intermediate molecule, which in turn acts on a second mentioned entity. Contacting, as used herein, includes in solution, in solid phase, ex vivo, intracellularly, and the like. Contacting can also be referred to as administering or administration.

[0128] As used herein, "recombinant peptide" refers to a peptide produced in a host cell (e.g., E. coli, yeast) using exogenous / recombinant nucleic acid. As used herein, "synthetic peptide" refers to a peptide synthesized by synthetic or chemical means without the use of a host cell. Recombinant peptides may have post-translational modifications, but synthetic peptides do not have any post-translational modifications.

[0129] In certain embodiments, the biofilm is derived from (i.e., produced by) Gram-negative or Gram-positive biofilm-producing bacteria, hi certain embodiments, the biofilm is derived from (i.e., produced by) Nontuberculous Mycobacterium (NTM) species.

[0130] In certain embodiments, the biofilm comprises a DNABII protein. In further embodiments, the biofilm comprises a histone-like protein (HU) or integration host factor (IHF) binding protein from E. coli U93 strain.

[0131] In certain embodiments, the DNABII peptide is an IHF peptide. Additionally or alternatively, the DNABII peptide is an HU peptide. In certain embodiments, the tip region of the DNABII peptide is the tip region of IHFA and / or the tip region of IHFB. In further embodiments, the tip region of the DNABII peptide is the IHFA tip region conjugated directly or indirectly (e.g., via a linker) to the IHFB tip region. In yet further embodiments, the tip region of the DNABII peptide is IhfA5-mIhfB4 NTHI It is a tip chimeric peptide.

[0132] As used herein, the term "EC 50 " refers to the concentration of an antibody or fragment thereof that induces a response (e.g., binding between an antibody or fragment thereof and its target) halfway between baseline and maximum after a specified exposure time.

[0133]

number

[0134] As used herein, "nontuberculous mycobacterium (NTM)" refers to any Mycobacterium species other than M. tuberculosis. In some embodiments, NTM species include M. abscessus (Mab), M. avium (Mav), M. intracellulare, or M. chimaera.

[0135] As used herein, "antibiotic resistance" or "resistance" refers to the resistance of a microorganism to an antibiotic when the antibiotic is administered to a subject in need thereof in a dose sufficient to successfully eliminate the non-resistant form of the microorganism. Biofilms are known to have high antibiotic resistance.

[0136] As used herein, "susceptibility of a biofilm to an antibiotic" means reducing the antibiotic resistance of the biofilm and sensitizing the resident microorganisms within the biofilm to the antibiotic.

[0137] MODES FOR CARRYING OUT THE DISCLOSURE Applicant has discovered that antibodies or antigen-binding fragments thereof that bind to the apical region of the DNABII peptide described herein exhibit unexpected efficacy against infections and biofilms caused by nontuberculous mycobacterium (NTM) species by enhancing either antibiotic susceptibility or immune system clearance in infected individuals. The methods disclosed herein can benefit patients by reducing either the length of antibacterial treatment or the amount of antibiotic required.

[0138] Methods for preventing or treating nontuberculous mycobacterial (NTM) infections Certain aspects of the present disclosure relate to a method for preventing or treating an infection caused by a nontuberculous mycobacterium (NTM) species in a subject, comprising, consisting essentially of, or even consisting of administering to the subject an effective amount of an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to a distal region or distal chimera of a DNABII peptide (e.g., IhfA5-mIhfB4 as described herein). NTHI The present invention relates to a method for recognizing and binding to IhfA5-mIhfB4 (a chimera with a nucleotide sequence similar to that of IhfA5-mIhfB4). NTHI Compositions comprising one or more antibodies or antigen-binding fragments thereof that recognize and bind to the tip chimera are also provided.

[0139] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region or tip chimera of the DNABII peptide is (i) a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or even consisting of, the sequence of amino acids (aa) 25 to aa 144 of SEQ ID NO: 21 or an equivalent thereof; and (ii) a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or even consisting of the sequence from aa21 to aa132 of SEQ ID NO: 22 or an equivalent thereof. or the antibody or antigen-binding fragment thereof comprises (i) a heavy chain (HC) immunoglobulin variable domain sequence comprising, consisting essentially of, or even consisting of, the sequence from aa25 to aa144 of SEQ ID NO: 24 or an equivalent thereof; and (ii) a light chain (LC) immunoglobulin variable domain sequence comprising, consisting essentially of, or even consisting of the sequence from aa21 to aa132 of SEQ ID NO: 25 or an equivalent thereof. Includes.

[0140] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide is a heavy chain complementarity determining region 1 (CDRH1) comprising, consisting essentially of, or even consisting of the sequence of GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 9 or 10 or 11 or 24); a heavy chain complementarity determining region 2 (CDRH2) comprising, consisting essentially of, or even consisting of the sequence of GSDRRH (aa76 to aa81 of SEQ ID NO: 9 or 10 or 11 or 24); a heavy chain complementarity determining region 3 (CDRH3) comprising, consisting essentially of, or even consisting of the sequence VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 9 or 10 or 11 or 24); A light chain complementarity determining region 1 (CDRL1) comprising, consisting essentially of, or even consisting of the sequence QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 15 or 16 or 17 or 25), A light chain complementarity-determining region 2 (CDRL2) comprising, consisting essentially of, or even consisting of the sequence of LVS (aa75 to aa77 of SEQ ID NO: 15, 16, 17, or 25), and Light chain complementarity-determining region 3 (CDRL3) comprising, consisting essentially of, or even consisting of the sequence of WQGTHFP (aa114 to aa120 of SEQ ID NO: 15, 16, 17, or 25). Includes.

[0141] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide is A heavy chain complementarity determining region 1 (CDRH1) comprising, consisting essentially of, or even consisting of the sequence of GFTFSRYG (aa50 to aa57 of SEQ ID NO: 12 or 13 or 14); a heavy chain complementarity determining region 2 (CDRH2) comprising, consisting essentially of, or even consisting of the sequence of ISSGGSYT (aa75 to aa82 of SEQ ID NO: 12 or 13 or 14); A heavy chain complementarity determining region 3 (CDRH3) comprising, consisting essentially of, or even consisting of the sequence ERHGGDGYWYFDV (aa121 to aa133 of SEQ ID NO: 12 or 13 or 14); A light chain complementarity determining region 1 (CDRL1) comprising, consisting essentially of, or even consisting of the sequence QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 15 or 16 or 17 or 25), A light chain complementarity-determining region 2 (CDRL2) comprising, consisting essentially of, or even consisting of the sequence of LVS (aa75 to aa77 of SEQ ID NO: 15, 16, 17, or 25), and Light chain complementarity-determining region 3 (CDRL3) comprising, consisting essentially of, or even consisting of the sequence WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 15, 16, 17, or 25). Includes.

[0142] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises a heavy chain (HC) immunoglobulin variable domain comprising the amino acid sequence of amino acids 25 to 144 of SEQ ID NO: 9, and / or a light chain (LC) immunoglobulin variable domain comprising the amino acid sequence of amino acids 21 to 132 of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0143] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region or tip chimera of a DNABII peptide comprises, essentially consists of, or even further consists of, or essentially consists of, or even further consists of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, essentially consists of, or even further consists of, a sequence selected from the group of aa25 to aa144 of SEQ ID NO: 21 or 24, or their respective equivalents, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, essentially consists of, or even further consists of, a sequence selected from the group of aa21 to aa132 of SEQ ID NO: 22 or 25, or their respective equivalents. In certain embodiments, the antibody or antigen-binding fragment thereof binds to the tip region or tip chimera of a DNABII peptide (the tip region of IHF or HU, the tip region of IHFA or IHFB, and / or the tip chimera peptide IhfA5-mIhfB4). NTHI In one embodiment, the antibody or antigen-binding fragment thereof binds to the apical chimeric peptide IhfA5-mIhfB4 described herein. NTHI In some embodiments, the tip chimeric peptide IhfA5-mIhfB4 NTHI comprises, consists essentially of, or even consists of, an amino acid sequence selected from SEQ ID NOs: 26-28.

[0144] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the distal region of a DNABII peptide comprises, consists essentially of, or even consists of any one or any two or three heavy chain (HC) CDRs comprising, consisting essentially of, or even consisting of a sequence selected from SEQ ID NOs: 9-14 or their respective equivalents, and / or any one or any two or three light chain (LC) CDRs comprising, consisting essentially of, or even consisting of a sequence selected from SEQ ID NOs: 15-20 or their respective equivalents. In certain embodiments, the antibody or antigen-binding fragment thereof binds to the distal region of a DNABII peptide (the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal chimeric peptide IhfA5-mIhfB4 described herein). NTHI In one embodiment, the antibody or antigen-binding fragment thereof binds to the apical chimeric peptide IhfA5-mIhfB4 described herein. NTHI In some embodiments, the tip chimeric peptide IhfA5-mIhfB4 NTHI comprises, consists essentially of, or even consists of, an amino acid sequence selected from SEQ ID NOs: 26-28.

[0145] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the distal region of the DNABII peptide comprises, consists essentially of, or even consists of all three heavy chain (HC) CDRs comprising, consisting essentially of, or even consisting of a sequence selected from SEQ ID NOs: 9-11 or their respective equivalents, and / or all three light chain (LC) CDRs comprising, consisting essentially of, or even consisting of a sequence selected from SEQ ID NOs: 15-17 or their respective equivalents. In certain embodiments, the antibody or antigen-binding fragment thereof binds to the distal region of the DNABII peptide (the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal chimeric peptide IhfA5-mIhfB4).NTHI In one embodiment, the antibody or antigen-binding fragment thereof binds to the tip chimeric peptide IhfA5-mIhfB4. NTHI In some embodiments, the tip chimeric peptide IhfA5-mIhfB4 NTHI comprises, consists essentially of, or even consists of, an amino acid sequence selected from SEQ ID NOs: 3-5.

[0146] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the distal region of the DNABII peptide comprises, consists essentially of, or even consists of all three heavy chain (HC) CDRs comprising, consisting essentially of, or even consisting of a sequence selected from SEQ ID NOs: 12-14 or their respective equivalents, and / or all three light chain (LC) CDRs comprising, consisting essentially of, or even consisting of a sequence selected from SEQ ID NOs: 18-20 or their respective equivalents. In certain embodiments, the antibody or antigen-binding fragment thereof binds to the distal region of the DNABII peptide (the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal chimeric peptide IhfA5-mIhfB4). NTHI In one embodiment, the antibody or antigen-binding fragment thereof binds to the tip chimeric peptide IhfA5-mIhfB4. NTHI In some embodiments, the tip chimeric peptide IhfA5-mIhfB4 NTHI comprises, consists essentially of, or even consists of, an amino acid sequence selected from SEQ ID NOs: 26-28.

[0147] In some embodiments, equivalents of an amino acid sequence include polypeptides having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to the amino acid, or equivalents of an amino acid sequence include polypeptides encoded by polynucleotides that hybridize under high stringency conditions to the complement of a polynucleotide encoding the amino acid sequence.

[0148] Table 1 shows IhfA5-mIhfB4 NTHIRelative biofilm disruption data using a humanized monoclonal antibody designed to target the apical chimeric peptide are provided. Biofilm disruption: NTHI 86-028NP colonies were harvested from overnight cultures on chocolate agar and suspended in brain heart infusion broth (sBHI) supplemented with 2 μg β-NAD and heme per ml of medium. The optical density at 490 nm was then adjusted to 0.65, and the culture was diluted 1:6 into sBHI, followed by 3 hours of static incubation at 37°C with 5% CO2. The culture was then diluted 1:2500 into 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 with 5% CO2 for 3 hours. After 16 hours, 200 μl of fresh sBHI was added to each well, and the slide was incubated for an additional 8 hours. At this point, the medium was aspirated from each well, and 5 μg of monoclonal antibody was added per well. The biofilms were incubated for an additional 16 hours. The biofilms were then washed, stained with FM1-43FX bacterial membrane stain (Invitrogen), and fixed overnight at 4°C in 16% paraformaldehyde, 2.5% glutaraldehyde, and 4.0% acetic acid in 0.1 M phosphate buffer (pH 7.4). The fixative was aspirated, and 200 μL of Hank's balanced salt solution was added to each well. The biofilms were then observed using a Zeiss 800 Meta-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 K of the tip chimeric peptide A (1 / M units) is approximately 4E+05 to approximately 2E+08.

[0149] [Table 1]

[0150] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 21 and a light chain sequence comprising SEQ ID NO: 22.

[0151] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 9 and a light chain sequence comprising SEQ ID NO: 15.

[0152] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO:9 and a light chain sequence comprising SEQ ID NO:16.

[0153] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO:9 and a light chain sequence comprising SEQ ID NO:17.

[0154] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 10 and a light chain sequence comprising SEQ ID NO: 15.

[0155] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 10 and a light chain sequence comprising SEQ ID NO: 16.

[0156] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 10 and a light chain sequence comprising SEQ ID NO: 17.

[0157] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 11 and a light chain sequence comprising SEQ ID NO: 15.

[0158] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 11 and a light chain sequence comprising SEQ ID NO: 16.

[0159] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 11 and a light chain sequence comprising SEQ ID NO: 17.

[0160] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 12 and a light chain sequence comprising SEQ ID NO: 18.

[0161] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 12 and a light chain sequence comprising SEQ ID NO: 19.

[0162] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 12 and a light chain sequence comprising SEQ ID NO: 20.

[0163] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 13 and a light chain sequence comprising SEQ ID NO: 18.

[0164] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 13 and a light chain sequence comprising SEQ ID NO: 19.

[0165] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 13 and a light chain sequence comprising SEQ ID NO: 20.

[0166] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 14 and a light chain sequence comprising SEQ ID NO: 18.

[0167] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 14 and a light chain sequence comprising SEQ ID NO: 19.

[0168] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 14 and a light chain sequence comprising SEQ ID NO: 20.

[0169] 1. A method for preventing or treating an infection caused by a nontuberculous mycobacterium (NTM) species in a subject, comprising administering to a subject a heavy chain complementarity determining region 1 (CDRH1) comprising the sequence GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 9); a heavy chain complementarity determining region 2 (CDRH2) comprising the sequence GSDRRH (aa 76 to aa 81 of SEQ ID NO: 9); a heavy chain complementarity determining region 3 (CDRH3) comprising the sequence VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 9); Also provided is a method comprising, consisting essentially of, or even consisting of administering to a subject an effective amount of an antibody or antigen-binding fragment thereof comprising: a light chain complementarity-determining region 1 (CDRL1) comprising the sequence of KTF (aa47 to aa57 of SEQ ID NO: 15); a light chain complementarity-determining region 2 (CDRL2) comprising the sequence of LVS (aa75 to aa77 of SEQ ID NO: 15); and a light chain complementarity-determining region 3 (CDRL3) comprising the sequence of WQGTHFP (aa114 to aa120 of SEQ ID NO: 15).

[0170] In one embodiment of the disclosed method, the subject is a mammal, for example, a pet or animal model or a human patient.The subject may be suffering from an active infection, or alternatively, may be at risk of infection.When administered to animals, the method can be a useful treatment for pets and other non-human animals or mammals, or can serve as a useful animal model for testing combination therapy.

[0171] Antibodies or antigen-binding fragments thereof for use in the disclosed methods provided herein can be monospecific or bispecific. In one embodiment, the antibody or antigen-binding fragment thereof is trispecific, tetraspecific, or pentaspecific. Additionally or alternatively, the antibody is selected from the group of IgA antibodies (e.g., IgA1 or IgA2), IgD antibodies, IgE antibodies, IgG antibodies (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM antibodies. In one embodiment, the antibody further comprises a constant region selected from the group of an IgA constant region (e.g., an IgA1 constant region or an IgA2 constant region), an IgD constant region, an IgE constant region, an IgG constant region (e.g., an IgG1 constant region, an IgG2 constant region, an IgG3 constant region, or an IgG4 constant region), or an IgM constant region. In some embodiments, the constant region of the antibody comprises an amino acid sequence selected from the group of SEQ ID NOs: 23 and 33-40.

[0172] In another embodiment, the antibody or antigen-binding fragment thereof can be modified by conventional techniques, which in one embodiment can increase the half-life of the antibody, for example, pegylation, PEG mimetic, polysialylation, hydroxylation, or glycosylation.

[0173] Methods for generating antibodies or antigen-binding fragments thereof that bind to the tip and tail chimeric peptides are described in US Pat. No. 11,104,723, which is incorporated herein in its entirety.

[0174] In some embodiments, the antigen-binding fragment is selected from the group of Fab, F(ab')2, Fab', scFv, or Fv.

[0175] In some embodiments, the method further comprises administering to the subject an effective amount of at least one antibiotic (e.g., one, two, three, up to all of the antibiotics listed) selected from amikacin, azithromycin, dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, methicillin, vancomycin, daptomycin, mupirocin, penicillin, cloxacillin, erythromycin, clarithromycin, roxithromycin, telithromycin, spiramycin, fidaxomicin, rifampin, ethambutol, streptomycin, or anthramycin. In one aspect, the effective amount of the antibiotic is administered in an amount less than that which would be administered in the absence of the antibody or antigen-binding fragment thereof. Non-limiting examples of such amounts are provided herein and can range, for example, from less than about 20% to less than about 80% of the effective amount when the antibiotic is the only active agent, and ranges therebetween.

[0176] In some embodiments, the NTM species is M. abscessus (Mab), M. avium (Mav), M. intracellulare, or M. chimaera.

[0177] In some embodiments, administration comprises one or more of inhalation, inhalation using a nebulizer, oral administration, nasal administration, injection and / or topical application.

[0178] In some embodiments, the subject has chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia or cystic fibrosis (CF), SARS CoV-2, asthma, bacterial pneumonia, respiratory syncytial virus bronchiolitis, influenza virus infection, transfusion-associated acute lung injury, mechanical ventilation; sepsis; atherosclerosis; an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes mellitus or small vasculitis; an autoinflammatory disease selected from gout or inflammatory bowel disease, and / or a metabolic disease selected from type 2 diabetes or obesity. In one aspect, the subject has cystic fibrosis (CF).

[0179] In some embodiments, the effective amount of the antibody or antigen-binding fragment is between 0.2 mg / kg and 30 mg / kg (e.g., 0.2, 0.5, 1, 1.5, 3, 5, 7, 9, 10, 12, 15, 18, 20, 23, 25, 28, 30 mg / kg, or any value therebetween).

[0180] In some embodiments, the effective amount of antibiotic is less than 50 mg / kg, for example, between 0.2 mg / kg and 50 mg / kg (e.g., 0.2, 0.5, 1, 1.5, 3, 5, 7, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 mg / kg or any value therebetween).

[0181] Methods for preventing or treating nontuberculous mycobacterial (NTM) infections in combination with mB box 97 peptides and / or DNA binding agents In some embodiments, the method further comprises administering to the subject an effective amount of a synthetic or recombinant polypeptide comprising, consisting essentially of, or even consisting of mB box-97, or an equivalent thereof, consisting of amino acids 90-176 from the coding sequence of the native human HMGB1 protein set forth as SEQ ID NO: 45, with a cysteine ​​to serine point mutation at amino acid 106.

[0182] As used herein, a synthetic or recombinant mB Box-97 equivalent refers to a sequence that is at least about 70% identical to a reference recombinant mB Box-97 or a reference synthetic mB Box-97, or alternatively at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% identical to the reference recombinant mB Box-97 or reference synthetic mB Box-97, and in one embodiment, retains a mutated amino acid, a cysteine ​​to serine point mutation at amino acid 106. In one embodiment, percent identity is determined using a BLAST alignment program using default parameters. Particularly preferred programs are BLASTN and BLASTP, using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expectation=10; matrix=BLOSUM62; description=50 sequences; filtering method=high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0183] In some aspects, recombinant or synthetic polypeptide equivalents retain the intended function and / or structural characteristics of mB-box-97 polypeptides. In some embodiments, equivalents of mB-box-97 include recombinant or synthetic mB-box-97 polypeptides that retain a C to S amino acid substitution at amino acid 106 and further contain, independently, at least 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.

[0184] In some embodiments, the recombinant or synthetic mB box-97 HMGB1 polypeptide further comprises, consists essentially of, or even consists of one or more linker polypeptides. Exemplary peptide linkers are one of SEQ ID NOS: 6-8 and 26-30, or PPKGETKKKF (SEQ ID NO: 46) at the amino and / or carboxy terminus.

[0185] In some embodiments, the recombinant or synthetic polypeptide consists of SEQ ID NO:47.

[0186] In another aspect of the present disclosure, a recombinant or synthetic polypeptide comprising mB box-97 consists of amino acids 80-176, or an equivalent thereof, derived from the coding sequence of the native human HMGB1 protein set forth as SEQ ID NO:45, with a cysteine ​​to serine point mutation at amino acid 106. In some embodiments, the synthetic or recombinant polypeptide consists of SEQ ID NO:48.

[0187] In some embodiments, an effective amount of a synthetic or recombinant polypeptide is between 50 nM and 2 μM (e.g., 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 150 nM, 170 nM, 200 nM, 220 nM, 250 nM, 270 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 8 50nM, 900nM, 950nM, 1000nM (1µM), 1050nM, 1100nM, 1150nM, 1200nM, 1250nM, 1300nM, 1350nM, 1400nM, 1450nM, 1500nM, 1550nM, 1600nM, 1650nM, 1700nM, 1750nM, 1800nM, 1850nM, 1900nM, 1950nM or 2000nM (2µM).

[0188] In some embodiments, the method further comprises administering to the subject an effective amount of a DNA binding agent in combination with an antibody or fragment thereof described herein, or further in combination with an mB Box 97 peptide. The administration of the DNA binding agent in combination with an antibody or fragment thereof described herein, or further in combination with an mB Box 97 peptide described herein, can be performed simultaneously or sequentially, with the antibody or antigen-binding fragment thereof being administered prior to or after the mB Box 97 peptide or their respective equivalents and / or the DNA binding agent.

[0189] In some embodiments, the DNA binding agent comprises, consists essentially of, or even consists of histone-like nucleoid structuring protein (H-NS), polyamines, or polycations. Administration of the DNA binding agent in combination with an antibody or fragment thereof described herein, or further in combination with an mB box 97 peptide described herein, can be performed simultaneously or sequentially, with the antibody or antigen-binding fragment thereof being administered prior to or after the mB box 97 peptide or their respective equivalents and / or the DNA binding agent.

[0190] In some embodiments, an effective amount of a DNA binding agent is between 50 nM and 2 μM (e.g., 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 150 nM, 170 nM, 200 nM, 220 nM, 250 nM, 270 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, , 900nM, 950nM, 1000nM (1µM), 1050nM, 1100nM, 1150nM, 1200nM, 1250nM, 1300nM, 1350nM, 1400nM, 1450nM, 1500nM, 1550nM, 1600nM, 1650nM, 1700nM, 1750nM, 1800nM, 1850nM, 1900nM, 1950nM or 2000nM (2µM).

[0191] In some embodiments, the DNA binding agent comprises, consists essentially of, or even consists of, a histone-like nucleoid structuring protein (H-NS), a polyamine, or a polycation.

[0192] In some embodiments, the H-NS is derived from a gram-negative or gram-positive bacterium. In some embodiments, the H-NS is derived from a bacterium of the genus Escherichia, Haemophilus, Streptococcus, Mycobacteria, Klebsiella, or Pseudomonas; optionally, the H-NS is derived from E. coli, non-encapsulated Haemophilus influenzae (NTHI), S. pneumoniae, K. pneumoniae, Mycobacterium tuberculosis, or Pseudomonas aeruginosa.

[0193] In some embodiments, H-NS comprises, consists essentially of, or even consists of an amino acid sequence having at least 60% identity (e.g., at least 60, 65, 70, 75, 80, 85, 90, 95, 99% or more) to an amino acid sequence selected from SEQ ID NOs: 51-56, where the equivalent is identical to the reference polypeptide. In one aspect, percent identity is determined using a BLAST alignment program using default parameters. Particularly preferred programs are BLASTN and BLASTP using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; filtering method = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. In a further embodiment, the subject is a mammal, for example, a mouse, a rat, or a human patient. In one embodiment of the disclosed method, the subject is a mammal, for example, a pet or an animal model, or a human patient. The subject may be suffering from an active infection, or alternatively, may be at risk of infection. When administered to animals, the method can be a useful treatment for pets and other non-human animals or mammals, or can serve as a useful animal model for testing combination therapies.

[0194] Methods for sensitizing nontuberculous mycobacterium (NTM) biofilms to antibiotics or disrupting NTM biofilms Another aspect of the present disclosure is a method for sensitizing a biofilm to an antibiotic or disrupting a biofilm, wherein the biofilm comprises a nontuberculous mycobacterium (NTM) species, and the method comprises, consists essentially of, or consists of contacting the biofilm with an effective amount of an antibody or fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to a distal region of a DNABII peptide or distal chimera (e.g., IhfA5-mIhfB4 as described herein). NTHI The method of the present invention is directed to a method for detecting and binding to a target molecule (e.g., a chimera) that recognizes and binds to the target molecule. The contacting step can be performed with or without a chimera.

[0195] Also provided is a method for treating a subject infected with or suffering from a disease or disorder caused by a nontuberculous mycobacterium (NTM) species, comprising, consisting essentially of, or consisting of administering to the subject an effective amount of an antibody or antigen-binding fragment thereof that binds to the apical region of the DNABII peptide and an antibiotic that inhibits replication or infectivity of the NTM species in the subject, wherein the antibiotic is administered at between about 15% and about 75% of the antibiotic's MIC. Alternatively, the antibiotic is administered at between 20% and about 70%, or alternatively at between 20% and about 70%, between 25% and about 60%, or between 25% and about 50%, or alternatively at about 25% or about 50% of the antibiotic's MIC. In one embodiment, the antibiotic is azithromycin or amikacin, and the subject is a mammal, e.g., a human patient. In one embodiment, the antibiotic is administered to the subject's lungs by inhalation therapy, e.g., using a nebulizer.

[0196] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region or tip chimera of the DNABII peptide comprises a heavy chain (HC) immunoglobulin variable domain sequence comprising, essentially consisting of, or even consisting of, the sequence of amino acids (aa) 25 to aa144 of SEQ ID NO: 21 or an equivalent thereof; and a light chain (LC) immunoglobulin variable domain sequence comprising, essentially consisting of, or even consisting of the sequence of aa21 to aa132 of SEQ ID NO: 22 or an equivalent thereof; or a heavy chain (HC) immunoglobulin variable domain sequence comprising, essentially consisting of, or even consisting of the sequence of aa25 to aa144 of SEQ ID NO: 24 or an equivalent thereof; and a light chain (LC) immunoglobulin variable domain sequence comprising, essentially consisting of, or even consisting of the sequence of aa21 to aa132 of SEQ ID NO: 25 or an equivalent thereof.

[0197] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, or essentially consists of, or even consists of the sequence of GFTFRTY (aa50 to aa56 of SEQ ID NO: 9, 10, 11, or 24); heavy chain complementarity determining region 1 (CDRH1) comprising, essentially consists of, or even consists of the sequence of GSDRRH (aa76 to aa81 of SEQ ID NO: 9, 10, 11, or 24); heavy chain complementarity determining region 2 (CDRH2) comprising, essentially consists of, or even consists of the sequence of VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 9, 10, 11, or 24). and light chain complementarity determining region 1 (CDRL1) comprising, or consisting essentially of, or even consisting of the sequence of QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 15, or 16, or 17, or 25); light chain complementarity determining region 2 (CDRL2) comprising, or consisting essentially of, or even consisting of the sequence of LVS (aa75 to aa77 of SEQ ID NO: 15, or 16, or 17, or 25); and light chain complementarity determining region 3 (CDRL3) comprising, or consisting essentially of, or even consisting of the sequence of WQGTHFP (aa114 to aa120 of SEQ ID NO: 15, or 16, or 17, or 25).

[0198] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises a heavy chain complementarity determining region 1 (CDRH1) comprising, essentially consisting of, or even consisting of the sequence of GFTFSRYG (aa50 to aa57 of SEQ ID NO: 12, 13, or 14); a heavy chain complementarity determining region 2 (CDRH2) comprising, essentially consisting of, or even consisting of the sequence of ISSGGSYT (aa75 to aa82 of SEQ ID NO: 12, 13, or 14); and light chain complementarity determining region 1 (CDRL1) comprising, consisting essentially of, or even consisting of the sequence QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 15, or 16, or 17, or 25); light chain complementarity determining region 2 (CDRL2) comprising, consisting essentially of, or even consisting of the sequence LVS (aa75 to aa77 of SEQ ID NO: 15, or 16, or 17, or 25); and light chain complementarity determining region 3 (CDRL3) comprising, consisting essentially of, or even consisting of the sequence WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 15, or 16, or 17, or 25).

[0199] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises a heavy chain (HC) immunoglobulin variable domain comprising the amino acid sequence of amino acids 25 to 144 of SEQ ID NO: 9; and / or a light chain (LC) immunoglobulin variable domain comprising the amino acid sequence of amino acids 21 to 132 of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0200] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region or tip chimera of the DNABII peptide comprises, essentially consists of, or even further consists of, or essentially consists of, or even further consists of, a heavy chain (HC) immunoglobulin variable domain sequence comprising, essentially consists of, or even further consists of, a sequence selected from the group of aa25 to aa144 of SEQ ID NO: 21 or 24, or their respective equivalents, and / or a light chain (LC) immunoglobulin variable domain sequence comprising, essentially consists of, or even further consists of, a sequence selected from the group of aa21 to aa132 of SEQ ID NO: 22 or 25, or their respective equivalents. In certain embodiments, the antibody or antigen-binding fragment thereof binds to the tip region or tip chimera of the DNABII peptide (the tip region of IHF or HU, the tip region of IHFA or IHFB, and / or the tip chimera peptide IhfA5-mIhfB4). NTHI In one embodiment, the antibody or antigen-binding fragment thereof binds to the apical chimeric peptide IhfA5-mIhfB4 described herein. NTHI In some embodiments of the method, the tip chimeric peptide IhfA5-mIhfB4 NTHI comprises, consists essentially of, or even consists of, an amino acid sequence selected from SEQ ID NOs: 26-28.

[0201] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the distal region of a DNABII peptide comprises, consists essentially of, or even consists of any one or any two or three heavy chain (HC) CDRs comprising, consisting essentially of, or even consisting of a sequence selected from SEQ ID NOs: 9-14 or their respective equivalents, and / or any one or any two or three light chain (LC) CDRs comprising, consisting essentially of, or even consisting of a sequence selected from SEQ ID NOs: 15-20 or their respective equivalents. In certain embodiments of the method, the antibody or antigen-binding fragment thereof binds to the distal region of a DNABII peptide (the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal chimeric peptide IhfA5-mIhfB4 described herein). NTHI In one embodiment of the method, the antibody or antigen-binding fragment thereof binds to the tip region of the DNABII peptide, including but not limited to, the tip chimeric peptide IhfA5-mIhfB4 described herein. NTHI In some embodiments, the tip chimeric peptide IhfA5-mIhfB4 NTHI comprises, consists essentially of, or even consists of, an amino acid sequence selected from SEQ ID NOs: 26-28.

[0202] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the distal region of the DNABII peptide comprises, consists essentially of, or even consists of all three heavy chain (HC) CDRs comprising, consisting essentially of, or even consisting of a sequence selected from SEQ ID NOs: 9-11 or their respective equivalents, and / or all three light chain (LC) CDRs comprising, consisting essentially of, or even consisting of a sequence selected from SEQ ID NOs: 15-17 or their respective equivalents. In certain embodiments of the method, the antibody or antigen-binding fragment thereof binds to the distal region of the DNABII peptide (the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal chimeric peptide IhfA5-mIhfB4). NTHI In one embodiment of the method, the antibody or antigen-binding fragment thereof binds to the tip chimeric peptide IhfA5-mIhfB4 NTHI In some embodiments, the tip chimeric peptide IhfA5-mIhfB4 NTHI comprises, consists essentially of, or even consists of, an amino acid sequence selected from SEQ ID NOs: 3-5.

[0203] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the distal region of the DNABII peptide comprises, consists essentially of, or even consists of all three heavy chain (HC) CDRs comprising, consisting essentially of, or even consisting of a sequence selected from SEQ ID NOs: 12-14 or their respective equivalents, and / or all three light chain (LC) CDRs comprising, consisting essentially of, or even consisting of a sequence selected from SEQ ID NOs: 18-20 or their respective equivalents. In certain embodiments, the antibody or antigen-binding fragment thereof binds to the distal region of the DNABII peptide (the distal region of IHF or HU, the distal region of IHFA or IHFB, and / or the distal chimeric peptide IhfA5-mIhfB4). NTHIIn one embodiment of the method, the antibody or antigen-binding fragment thereof binds to the tip chimeric peptide IhfA5-mIhfB4 NTHI In some embodiments of the method, the tip chimeric peptide IhfA5-mIhfB4 NTHI comprises, consists essentially of, or even consists of, an amino acid sequence selected from SEQ ID NOs: 26-28.

[0204] In some embodiments of the method, equivalents of an amino acid sequence include polypeptides having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to the amino acid, or equivalents of an amino acid sequence include polypeptides encoded by polynucleotides that hybridize under high stringency conditions to the complement of a polynucleotide encoding the amino acid sequence.

[0205] Table 1 referenced above shows IhfA5-mIhfB4 NTHI Relative biofilm disruption data using humanized monoclonal antibodies designed to target the apical chimeric peptides are provided. The antibodies disclosed therein are useful in the methods of the present disclosure, specific embodiments of which are provided below.

[0206] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 21 and a light chain sequence comprising SEQ ID NO: 22.

[0207] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 9 and a light chain sequence comprising SEQ ID NO: 15.

[0208] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 9 and a light chain sequence comprising SEQ ID NO: 16.

[0209] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 9 and a light chain sequence comprising SEQ ID NO: 17.

[0210] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 10 and a light chain sequence comprising SEQ ID NO: 15.

[0211] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 10 and a light chain sequence comprising SEQ ID NO: 16.

[0212] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 10 and a light chain sequence comprising SEQ ID NO: 17.

[0213] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 11 and a light chain sequence comprising SEQ ID NO: 15.

[0214] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 11 and a light chain sequence comprising SEQ ID NO: 16.

[0215] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 11 and a light chain sequence comprising SEQ ID NO: 17.

[0216] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 12 and a light chain sequence comprising SEQ ID NO: 18.

[0217] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 12 and a light chain sequence comprising SEQ ID NO: 19.

[0218] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 12 and a light chain sequence comprising SEQ ID NO: 20.

[0219] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 13 and a light chain sequence comprising SEQ ID NO: 18.

[0220] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 13 and a light chain sequence comprising SEQ ID NO: 19.

[0221] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 13 and a light chain sequence comprising SEQ ID NO: 20.

[0222] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 14 and a light chain sequence comprising SEQ ID NO: 18.

[0223] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 14 and a light chain sequence comprising SEQ ID NO: 19.

[0224] In some embodiments of the method, the antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide comprises, consists essentially of, or even consists of a heavy chain sequence comprising SEQ ID NO: 14 and a light chain sequence comprising SEQ ID NO: 20.

[0225] Another aspect of the present disclosure is a method for sensitizing a biofilm to an antibiotic, wherein the biofilm comprises a nontuberculous mycobacterium (NTM) species, the method comprising: 1) administering to the host a heavy chain complementarity determining region 1 (CDRH1) comprising the sequence of GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 9); 2) administering to the host a heavy chain complementarity determining region 2 (CDRH2) comprising the sequence of GSDRRH (aa 76 to aa 81 of SEQ ID NO: 9); 3) administering to the host a heavy chain complementarity determining region 3 (CDRH3) comprising the sequence of VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 9); , a light chain complementarity determining region 1 (CDRL1) comprising the sequence of QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 15), a light chain complementarity determining region 2 (CDRL2) comprising the sequence of LVS (aa75 to aa77 of SEQ ID NO: 15), and a light chain complementarity determining region 3 (CDRL3) comprising the sequence of WQGTHFP (aa114 to aa120 of SEQ ID NO: 15).

[0226] Antibodies or antigen-binding fragments thereof for use in the disclosed methods provided herein can be monospecific or bispecific. In one embodiment, the antibody or antigen-binding fragment thereof is trispecific, tetraspecific, or pentaspecific. Additionally or alternatively, the antibody is selected from the group of IgA antibodies (e.g., IgA1 or IgA2), IgD antibodies, IgE antibodies, IgG antibodies (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM antibodies. In one embodiment, the antibody further comprises a constant region selected from the group of an IgA constant region (e.g., an IgA1 constant region or an IgA2 constant region), an IgD constant region, an IgE constant region, an IgG constant region (e.g., an IgG1 constant region, an IgG2 constant region, an IgG3 constant region, or an IgG4 constant region), or an IgM constant region. In some embodiments of the methods, the constant region of the antibody comprises an amino acid sequence selected from the group of SEQ ID NOs: 23 and 33-40.

[0227] In another embodiment, the antibody can be modified by conventional techniques, which in one embodiment can increase the half-life of the antibody, for example, pegylation, PEG mimetic, polysialylation, HESylation, or glycosylation.

[0228] Methods for producing antibodies and antigen-binding fragments thereof are known in the art and are described in US Pat. No. 11,104,723, which is incorporated herein in its entirety.

[0229] In some embodiments of the method, the antigen-binding fragment is selected from the group of Fab, F(ab')2, Fab', scFv, or Fv.

[0230] In some embodiments of the method, the antibiotic comprises amikacin, azithromycin, dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, methicillin, vancomycin, daptomycin, mupirocin, penicillin, cloxacillin, erythromycin, clarithromycin, roxithromycin, telithromycin, spiramycin, fidaxomicin, rifampin, ethambutol, streptomycin, or anthramycin.

[0231] In some embodiments of the method, the effective amount of the antibody or antigen-binding fragment is between 0.2 mg / kg and 30 mg / kg (e.g., 0.2, 0.5, 1, 1.5, 3, 5, 7, 9, 10, 12, 15, 18, 20, 23, 25, 28, 30 mg / kg or any value therebetween).

[0232] In some embodiments of the method, the effective amount of the antibiotic is less than 50 mg / kg, for example, between 0.2 mg / kg and 50 mg / kg (e.g., 0.2, 0.5, 1, 1.5, 3, 5, 7, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 mg / kg or any value therebetween).

[0233] In some embodiments of the method, the contacting step is carried out at or.

[0234] In one embodiment of the disclosed in vivo method, the subject is a mammal, for example, a pet or animal model or a human patient.The subject may be suffering from an active infection, or alternatively, may be at risk of infection.When the method is carried out in vitro, the method is carried out in cell or tissue culture according to the animal system described herein.

[0235] In some embodiments of the method, the sensitized biofilm can be treated using less antibiotic, for example, 100 mg / kg or more IC 50 A biofilm that is resistant to an antibiotic having a minimum inhibitory concentration (ICM) will respond to 50 mg / kg or less of the antibiotic (e.g., 40, 30, 20, 10, 5, 1, 0.1 mg / kg or less of the antibiotic) after treatment with the claimed antibodies. Thus, the method can further include administering less than an ICM (minimum inhibitory concentration) of the antibiotic, wherein the amount or effective amount administered is simultaneous or sequential with the antibody or antigen-binding fragment thereof, and wherein the antibody or antigen-binding fragment is administered prior to, instead of, or after the antibody or antigen-binding fragment.

[0236] Combination with mB box 97 peptide In some embodiments, the method further comprises contacting the biofilm with an effective amount of a synthetic or recombinant polypeptide comprising, consisting essentially of, or even consisting of mB box-97, or an equivalent thereof, consisting of amino acids 90-176 from the coding sequence of the native human HMGB1 protein set forth as SEQ ID NO:45, with a cysteine ​​to serine point mutation at amino acid 106.

[0237] As used herein, a synthetic or recombinant mB Box-97 equivalent refers to a sequence that is at least about 70% identical to a reference recombinant mB Box-97 or a reference synthetic mB Box-97, or alternatively at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% identical to the reference recombinant mB Box-97 or reference synthetic mB Box-97, and in one embodiment, retains a mutated amino acid, a cysteine ​​to serine point mutation at amino acid 106. In one embodiment, percent identity is determined using a BLAST alignment program using default parameters. Particularly preferred programs are BLASTN and BLASTP, using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expectation=10; matrix=BLOSUM62; description=50 sequences; filtering method=high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0238] In some aspects, recombinant or synthetic polypeptide equivalents retain the intended function and / or structural characteristics of mB-box-97 polypeptides. In some embodiments, equivalents of mB-box-97 include recombinant or synthetic mB-box-97 polypeptides that retain a C to S amino acid substitution at amino acid 106 and further contain, independently, at least 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.

[0239] In some embodiments, the recombinant or synthetic mB box-97 HMGB1 polypeptide further comprises, consists essentially of, or even consists of one or more linker polypeptides. Exemplary peptide linkers are one of SEQ ID NOS: 6-8 and 26-30, or PPKGETKKKF (SEQ ID NO: 46) at the amino and / or carboxy terminus.

[0240] In some embodiments, the recombinant or synthetic polypeptide consists of SEQ ID NO:47.

[0241] In some embodiments, the method further comprises contacting the biofilm with an effective amount of a synthetic or recombinant polypeptide comprising, consisting essentially of, or even consisting of mB box-97, or an equivalent thereof, consisting of amino acids 80-176 from the coding sequence of the native human HMGB1 protein set forth as SEQ ID NO: 45, with a cysteine ​​to serine point mutation at amino acid 106.

[0242] In some embodiments, the synthetic or recombinant polypeptide consists of SEQ ID NO:48.

[0243] In some embodiments, an effective amount of a synthetic or recombinant polypeptide is between 50 nM and 2 μM (e.g., 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 150 nM, 170 nM, 200 nM, 220 nM, 250 nM, 270 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 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), and the amount or effective amount administered is concurrent or sequential with the antibody or antigen-binding fragment thereof, administered prior to, instead of, or after the antibody or antigen-binding fragment thereof.

[0244] Combination with DNA binding agents In some embodiments, the method further comprises contacting the biofilm with an effective amount of a DNA-binding agent.

[0245] In some embodiments, the DNA binding agent comprises, consists essentially of, or even consists of, a histone-like nucleoid structuring protein (H-NS), a polyamine, or a polycation.

[0246] In some embodiments, the H-NS is derived from a gram-negative or gram-positive bacterium. In some embodiments, the H-NS is derived from a bacterium of the genus Escherichia, Haemophilus, Streptococcus, Mycobacteria, Klebsiella, or Pseudomonas; optionally, the H-NS is derived from E. coli, non-encapsulated Haemophilus influenzae (NTHI), S. pneumoniae, K. pneumoniae, Mycobacterium tuberculosis, or Pseudomonas aeruginosa.

[0247] In some embodiments, H-NS comprises, consists essentially of, or even consists of an amino acid sequence having at least 60% (e.g., at least 60, 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: 51-56. In one aspect, percent identity is determined using a BLAST alignment program using default parameters. Particularly preferred programs are BLASTN and BLASTP using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; filtering method = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0248] In some embodiments, an effective amount of a DNA binding agent is between 50 nM and 2 μM (e.g., 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 150 nM, 170 nM, 200 nM, 220 nM, 250 nM, 270 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1000 nM (1 μM), 1050 nM, 1100 nM, 1150 nM, 1200 nM, 1300 nM, 1400 nM, 1500 nM, 1600 nM, 1700 nM, 1800 nM, 1900 nM, 2000 nM, 2100 nM, 2200 nM, 2300 nM, 2400 nM, 250 nM, 2600 nM, 2700 nM, 2800 nM, 2900 nM, 3000 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, 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), and the amount or effective amount administered is concurrent or sequential with the antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment being administered prior to, or instead of, the antibody or antigen-binding fragment being administered subsequently. Administration of the DNA-binding agent in combination with an antibody or fragment thereof described herein, or further in combination with an mB Box 95 peptide described herein, can be concurrent or sequential.

[0249] In a further embodiment, the subject to be treated is a mammal, eg, a mouse, rat, or a human patient.

[0250] composition Compositions are also provided. The compositions comprise a carrier and the antibody and / or antigen-binding fragment disclosed herein, alone or in combination with an additional agent, such as an antibiotic described herein and / or an mB box-97 peptide described herein and / or a DNA binder described herein. The carrier may be one or more of a solid support or a pharmaceutically acceptable carrier. The composition may further comprise an adjuvant or other component suitable for administration as a vaccine. In one aspect, the composition is formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, and / or adjuvants. Additionally, embodiments of the compositions of the present disclosure comprise an antibody of the present disclosure formulated with one or more pharmaceutically acceptable substances.

[0251] For oral preparations, any one or more of the antibodies or fragments thereof described herein can be used alone or in a pharmaceutical formulation as disclosed herein that comprises or consists essentially of the compound in combination with suitable additives for making tablets, powders, granules, or capsules, such as conventional additives such as lactose, mannitol, corn starch, or potato starch; binders such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch, or gelatin; disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose; lubricants such as talc or magnesium stearate; and, if desired, diluents, buffers, humectants, preservatives, and flavoring agents. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0252] Pharmaceutical formulations and unit dosage forms suitable for oral administration are particularly useful in treating chronic conditions, treating infections, and in therapeutic regimens in which the patient self-administers the drug. In one aspect, the formulation is specific for pediatric administration.

[0253] The present disclosure provides pharmaceutical formulations in which the antibodies disclosed herein, alone or in combination with additional agents, e.g., antibiotics described herein and / or mB box peptides described herein and / or DNA binding agents described herein, can be formulated into injectable preparations in accordance with the present disclosure by dissolving, suspending or emulsifying the antibodies in an aqueous or non-aqueous solvent (e.g., vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol), along with conventional additives, if desired, such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers and preservatives or other antimicrobial agents. Non-limiting examples of such agents include antimicrobial agents, such as other vaccine components, e.g., surface antigens such as OMP P5, OMP 26, OMP P2, or type IV pilin proteins (see Jurcisek and Bakaletz (2007) J. of Bacteriology 189(10):3868-3875 and Murphy, TF, Bakaletz, LO, and Smeesters, PR (2009) The Pediatric Infectious Disease Journal, 28:S121-S126), and antibacterial agents. For intravenous administration, suitable carriers include physiological bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, compositions for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists.

[0254] The aerosol formulations provided by the present disclosure can be administered by inhalation, and can be propellant-based or non-propellant-based.For example, the pharmaceutical formulations disclosed herein include the compounds disclosed herein that are formulated into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, and nitrogen.For administration by inhalation, the compounds can be delivered in the form of aerosol spray from a pressurized container or dispenser that contains a suitable propellant, for example, gas, such as carbon dioxide, or a nebulizer.A non-limiting example of a non-propellant is a pump spray that is expelled from a sealed container using mechanical force (i.e., by pressing down the plunger with a finger, or by compressing the container, for example, by compressive force applied to the container wall, or by the resilience exerted by the wall itself, for example, by an elastic sac).

[0255] The suppositories disclosed herein can be prepared by mixing the compounds disclosed herein with any of a variety of bases, such as emulsifying bases or water-soluble bases.The pharmaceutical formulations of the compounds disclosed herein can be administered rectally via suppositories.Suppositories can contain a medium that melts at body temperature but solidifies at room temperature, such as cocoa butter, carbowax and polyethylene glycol.

[0256] Unit dosage forms for oral or rectal administration, such as syrups, elixirs and suspensions, can be provided, and each dosage unit, for example, a teaspoon, a tablespoon, a tablet or a suppository, contains a predetermined amount of a composition containing one or more compounds disclosed herein.Similarly, unit dosage forms for injection or intravenous administration can contain the compounds disclosed herein in a composition as a solution in sterile water, physiological saline or another pharmaceutically acceptable carrier.

[0257] The pharmaceutical formulations disclosed herein include those in which the antibodies or fragments thereof disclosed herein are formulated into injectable compositions.The injectable pharmaceutical formulations disclosed herein are prepared as liquid solutions or suspensions; or as solid forms suitable for dissolution or suspension in a liquid medium prior to injection.In other embodiments of the pharmaceutical formulations disclosed herein, the preparation may be emulsified, or the active ingredient may be encapsulated in a liposomal vehicle.

[0258] In certain embodiments, the antibodies disclosed herein are formulated for delivery by a continuous delivery system. The term "continuous delivery system" is used interchangeably herein with "controlled delivery system" and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.

[0259] Mechanical or electromechanical infusion pumps can also be suitable for use according to the present disclosure.Examples of such devices include those described in, for example, U.S. Patent Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; and 6,198,966.Generally, the delivery of the compounds disclosed herein can be achieved using any of a variety of refillable pump systems.The pump provides consistent controlled release over time.In some embodiments of the method, the compounds disclosed herein are in a liquid formulation in a drug-impermeable reservoir and are delivered to an individual in a continuous manner.

[0260] In one embodiment, the drug delivery system is at least partially an implantable device. The implantable device can be implanted at any suitable implantation site using methods and devices known in the art. An implantation site is a site within a subject's body where the drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to, subdermal, subcutaneous, intramuscular, or other suitable sites within a subject's body. In some embodiments, a subcutaneous implantation site is used for convenience in implanting and removing the drug delivery device.

[0261] Drug release devices suitable for use in the present disclosure can be based on any of a variety of mechanisms of operation, including polymers such as poly(glycolide-co-lactide) (PGLA), commercially available from several vendors, e.g., BioDegmer and Sigma-Aldrich. For example, the drug release device can be based on a diffusion system, a convective system, or an erosion system (e.g., an erosion-based system). For example, the drug release device can be an electrochemical pump, an osmotic pump, an electroosmotic pump, a vapor pressure pump, or an osmotic burst matrix (e.g., a drug is incorporated into a polymer (e.g., PGLA), which releases the drug formulation concomitantly with degradation of the drug-impregnated polymeric material (e.g., a biodegradable drug-impregnated polymeric material)). In other embodiments of the method, the drug release device is based on an electrodiffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolysis system, or the like.

[0262] Drug release devices based on mechanical or electromechanical infusion pumps can also be suitable for use according to the present disclosure.Examples of such devices include those described in, for example, U.S. Patent No. 4,692,147; U.S. Patent No. 4,360,019; U.S. Patent No. 4,487,603; U.S. Patent No. 4,360,019; and U.S. Patent No. 4,725,852.Generally, the subject treatment method can be achieved using any of a variety of refillable and non-replaceable pump systems.Pumps and other convective systems can be utilized because of their generally more consistent controlled release over time.In some embodiments, osmotic pumps are used because of their combined advantages of more consistent controlled release and relatively small size (see, for example, PCT International Application Publication No. WO97 / 27840 and U.S. Patent No. 5,985,305 and 5,728,396). Exemplary osmotically driven devices suitable for use in the present disclosure are described in U.S. Patent Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202; 4,111,203; and 4,203,440. Nos. 4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; and 5,728,396. A further exemplary device that may be adapted for the present disclosure is the SynchroMed infusion pump (Medtronic).

[0263] In some embodiments of the method, the drug delivery device is an implantable device. The drug delivery device can be implanted at any suitable implantation site using methods and devices known in the art. As described herein, an implantation site is a site within a subject's body where the drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to, subdermal, subcutaneous, intramuscular, or other suitable sites within a subject's body.

[0264] Suitable excipient media for the compounds disclosed herein are, for example, water, saline, dextrose, glycerol, or ethanol, and combinations thereof. In addition, if desired, the media can contain minor amounts of auxiliary substances, such as wetting or emulsifying agents or pH buffering agents. Methods for preparing such dosage forms are known to those skilled in the art or will become apparent in light 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 case, contain an amount of compound appropriate to achieve the desired state in the subject being treated.

[0265] Compositions of the present disclosure include those containing sustained-release or controlled-release matrices. In addition, embodiments of the present disclosure can be used in conjunction with other treatments that use sustained-release formulations. As used herein, a sustained-release matrix is ​​a matrix made of a material (usually a polymer) that can be degraded by enzymatic or acid-based hydrolysis or by dissolution. Once inserted into the body, the matrix is ​​acted upon by enzymes and bodily fluids. The sustained-release matrix is ​​desirably selected from biocompatible materials, such as liposomes, polylactide (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide-co-glycolide (copolymer of lactic and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicones. Illustrative biodegradable matrices include polylactide matrices, polyglycolide matrices, and polylactide-co-glycolide (copolymers of lactic and glycolic acid) matrices.

[0266] In another embodiment, the polypeptide, antibody, or fragment thereof (as well as the combination composition) is delivered in a controlled release system. For example, the compounds disclosed herein can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other administration mechanisms. In one embodiment, a pump may be used (Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574). In another embodiment, a polymeric material is used. In yet another embodiment, the controlled release system is placed near the therapeutic target, i.e., the liver, thereby requiring a small fraction of the systemic dose. In yet another embodiment, the controlled release system is placed near the therapeutic target, thereby requiring a small fraction of the systemic dose. Other controlled release systems are described in the review by Langer (1990) Science 249:1527-1533.

[0267] In another embodiment, the compositions of the present disclosure (as well as the combination compositions, separately or together) include those formed by impregnation of absorbent materials, such as sutures, bandages, and gauze, with the inhibitory agents described herein, or coated onto solid materials, such as surgical staples, zippers, and catheters, to deliver the compositions. Other delivery systems of this type will be readily apparent to those skilled in the art in view of the present disclosure.

[0268] In various embodiments of the methods disclosed herein, the methods cover nearly all available methods and routes suitable for drug delivery, including ex vivo methods and systemic and local routes of administration.

[0269] Combination therapy The compositions and related methods of the present disclosure can be used in combination with the administration of other therapies, including, but not limited to, the administration of DNase enzymes, antibiotics, antimicrobials, anti-infectives, anti-fungals, anti-parasitics, anti-virals, or other antibodies. In one embodiment, the antibiotic is selected from amikacin and azithromycin, which in one embodiment are administered at about 50% or alternatively about 25% of their MICs, which in one embodiment translates to use in animals, e.g., human patients.

[0270] In some embodiments, the methods and compositions include a deoxyribonuclease (DNase) enzyme that acts synergistically with an anti-DNABII antibody. DNase is any enzyme that catalyzes the cleavage of phosphodiester bonds in the DNA backbone. Three non-limiting examples of DNase enzymes known to target not only DNA cruciform structures but also various secondary structures include DNAse I, T4 Endo VII, T7 Endo I, RuvABC, and RusA. In certain embodiments, the effective amount of anti-DNABII antibody required to destabilize a biofilm is reduced when combined with DNase. When administered at 1000 kJ / mL, DNase can be added to the assay directly or in a suitable buffer known to stabilize the enzyme. The effective unit dose of DNase and assay conditions can vary and can be optimized according to procedures known in the art.

[0271] In other embodiments, the methods and compositions can be combined with antibiotics and / or antimicrobials. Antimicrobials are substances that kill or inhibit the growth of microorganisms, such as bacteria, fungi, or protozoa. Biofilms are generally resistant to the action of antibiotics, but the compositions and methods described herein can be used to make biofilm-associated infections susceptible to traditional therapeutic methods for treating the infection. In other embodiments, the use of antibiotics or antimicrobials in combination with the methods and compositions described herein allows for a reduction in the effective amount of the antimicrobial and / or biofilm-reducing agent. Some non-limiting examples of antimicrobials and antibiotics useful in combination with the methods of the present disclosure include amoxicillin, amoxicillin-clavulanate, cefdinir, azithromycin, and sulfamethoxazole-trimethoprim. Therapeutically effective doses of antimicrobials and / or antibiotics in combination with biofilm-reducing agents can be readily determined by traditional methods. In some embodiments, the dose of the antimicrobial agent in combination with the biofilm reducing agent is the average effective dose shown to be effective in other bacterial infections, for example, bacterial infections where the etiology of the infection does not involve 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 average effective dose. The antibiotic or antimicrobial agent can be added prior to, simultaneously with, or after the addition of the anti-DNABII antibody.

[0272] In other embodiments, the methods and compositions can be combined with antibodies to treat bacterial infections. One example of an antibody useful in combination with the methods and compositions described herein is an antibody against an unrelated outer membrane protein (i.e., OMP P5). Treatment with this antibody alone does not reduce biofilms. Combined therapy with this antibody and a biofilm-reducing agent provides a greater effect than can be achieved with either agent used alone at the same concentration. Other antibodies that may produce a synergistic effect when combined with a biofilm-reducing agent or method for reducing biofilms include anti-rsPilA, anti-OMP26, anti-OMP P2, and anti-total OMP preparations.

[0273] The compositions and methods described herein can be used to sensitize biofilm-associated bacterial infections to common therapies that are effective in treating non-biofilm bacterial infections but are otherwise ineffective in treating biofilm-associated bacterial infections. In other embodiments, the compositions and methods described herein can be used in combination with a therapy that is effective in treating biofilm-associated bacterial infections, but the combination of such additional therapy with a biofilm-reducing agent or method produces a synergistic effect such that the effective dose of either the biofilm-reducing agent or the additional therapeutic agent can be reduced. In other instances, the combination of such additional therapy with a biofilm-reducing agent or method produces a synergistic effect such that treatment is enhanced. Enhanced treatment can be evidenced by a shorter amount of time required to treat the infection.

[0274] The additional therapeutic treatment can be added prior to, simultaneously with, or after the method or composition used to reduce the biofilm, and can be contained within the same formulation / composition or as a separate formulation / composition.

[0275] kit Also disclosed are kits containing one or more of the antibodies or fragments thereof or agents, mB box-97 peptides, DNA binders, compositions, and instructions necessary to carry out the methods described herein. Accordingly, the present disclosure provides kits for carrying out these methods, which may include antibodies, antibody fragments, polypeptides, polynucleotides, vectors, or host cells, as well as instructions for carrying out the methods disclosed herein, such as collecting tissue and / or performing screening and / or analyzing the results and / or administering an effective amount of the antibodies, antibody fragments, polypeptides, polynucleotides, vectors, or host cells, as defined herein. These may be used alone or in combination with other suitable antimicrobial agents.

[0276] For example, the kit can comprise, alternatively consist essentially of, or even consist of any one or more of the above-identified agents, such as antibodies, antibody fragments, polypeptides, polynucleotides, vectors, or host cells, and instructions for use. The kit can further comprise one or more of an adjuvant, an antigenic peptide, or an antimicrobial agent. Examples of carriers include liquid carriers, pharmaceutically acceptable carriers, solid carriers, pharmaceutically acceptable carriers, pharmaceutically acceptable polymers, liposomes, micelles, implants, stents, pastes, gels, dental implants, or medical implants. [Example]

[0277] The following examples are intended to illustrate, but not limit, the scope of the present disclosure.

[0278] Example 1 material and method

[0279] Antibodies. HuTipMab is an IgG isotype and was described (Kurbatfinski et al., 2022). Preservative-free human IgG (HuIgG) was used as a negative isotype control (Thermo Fisher Scientific, Inc., Waltham, MA). The HuTipMab antibody is claimed in U.S. Pat. No. 11,104,723 and comprises a heavy chain complementarity determining region 1 (CDRH1) comprising the sequence GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 9); a heavy chain complementarity determining region 2 (CDRH2) comprising the sequence GSDRRH (aa 76 to aa 81 of SEQ ID NO: 9); a heavy chain complementarity determining region 3 (CDRH3) comprising the sequence VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 9); a light chain complementarity determining region 1 (CDRL1) comprising the sequence QSLLDSDGKTF (aa 47 to aa 57 of SEQ ID NO: 15); a light chain complementarity determining region 2 (CDRL2) comprising the sequence LVS (aa 75 to aa 77 of SEQ ID NO: 15); and a light chain complementarity determining region 3 (CDRL3) comprising the sequence WQGTHFP (aa 114 to aa 120 of SEQ ID NO: 15).

[0280] Antibiotics. Amikacin sulfate and azithromycin dihydrate were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA) and stored according to the manufacturer's instructions. Amikacin was suspended and diluted in Middlebrook 7H9 broth (BD Difco™, Franklin Lakes, NJ) with 0.2% glycerol and 10% albumin-dextrose-catalase (ADC, BD BBL™, Franklin Lakes, NJ) immediately before use. Azithromycin was suspended in dimethyl sulfoxide (Fisher Scientific International, Inc., Hampton, NH) and then further diluted 1:1000 in 7H9 with 0.2% glycerol and 10% ADC immediately before use.

[0281] Bacterial stains and sources. M. abscessus 19977 (smooth morphology) was originally isolated from an individual with a knee infection. M. avium 25291 was originally isolated from an infected chicken liver. Both isolates were sourced from the American Type Culture Collection. M. abscessus clinical isolates 1, 2, and 3 (smooth morphology) were recovered from sputum of people with cystic fibrosis (PwCF).

[0282] Isolation and purification of recombinant HupB. M. tuberculosis HupB was PCR amplified using the following oligonucleotides: 5'-GCGTGCATATGAACAAAGCAGAGCTCATTGACGT-3' (SEQ ID NO: 41) and 5'-CGTGGCTCTTCCGCACGCTTTGCGACCCCGCCGAG-3' (SEQ ID NO: 42). Recombinant HupB was purified using a previously described protocol (Novotny et al., 2016, EBioMedicine 10, 33-44), concentrated using a centrifugal filter (3000 MWCO), dialyzed against storage buffer (50 mmol / L Tris pH = 7.4, 600 mmol / L KCl, 1 mmol / L EDTA, 10% glycerol), and then stored at -80°C until use. Approximately 200 ng of recombinant HupB was separated by SDS-PAGE using a 4-20% gradient gel at 5.6 V / cm for 1 hour. The expected molecular mass of HupB = 28 kDa. The relative purity of HupB was determined by silver staining (Pierce™ Silver Staining Kit, Thermo Fisher Scientific, Inc., Waltham, MA).

[0283] Recognition of HupB by HuTipMab by ELISA. Purified recombinant HupB, a tip chimeric peptide (a chimeric peptide mimicking a protective epitope of DNABII protein and used to generate HuTipMab; positive control), and a tail chimeric peptide (Novotny et al., 2016, EBioMedicine 10, 33-44) (a chimeric peptide mimicking a non-protective epitope of DNABII protein; negative control) were suspended in phosphate-buffered saline (PBS) (pH = 7.4). 1 μg of each was added in duplicate to wells of a Falcon® 96-well plate and incubated at 37°C for 1 hour. The solution was removed, and the wells were washed twice with PBS containing 1:2000 v / v Tween®-20 (PBS-T). The wells were blocked with 3% milk powder in PBS-T for 1 hour at 37°C and then washed twice with PBS-T. One set of samples received 0.1 μg HuTipMab / well, while the other set received PBS-T alone (background control), and then all were incubated at 37°C for 1 hour. The wells were washed three times with PBS-T, followed by the addition of goat anti-human IgG (1:5000 dilution) conjugated to horseradish peroxidase in PBS-T (Novus Biologicals LLC, Centennial, CO) and incubation at 37°C for 1 hour. The wells were washed three times with PBS-T and developed with 1-Step™ Ultra TMB (Pierce™) for 15 minutes at room temperature. The plate was read at 650 nm on a FLUOstar Omega plate reader (BMG Labtech, Cary, NC) and subsequently visualized on a Fluorchem M gel reader (ProteinSimple, Inc., Santa Clara, CA) using trans-UV light and a 593 nm filter. The assay was repeated three times on separate days.

[0284] Biofilm formation by M. abscessus and M. avium. M. abscessus or M. avium stocks were kept frozen at 2 x 10 CFU / mL in 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase (OADC, Hardy Diagnostics, Santa Maria, CA), 30% glycerol, and 0.05% Tween®-80 and stored at -80°C. The stocks were gently thawed on ice, and the bacteria were pelleted by centrifugation at 21,100 x g for 5 minutes at room temperature. The supernatant was discarded, and the pellet was resuspended and centrifuged again. After centrifugation, the bacteria were suspended in 7H9 containing 10% OADC and 0.05% Tween®-80 to a final volume of 1 mL. The stocks were diluted 10-fold in 7H9 / OADC / Tween®-80 to obtain 2 x 10 CFU / mL. 7 A final concentration of CFU / mL was obtained, and 200 μL was used to inoculate each well of an 8-well chamber cover glass slide (Cellvis, Mountainview, CA). Biofilms of M. abscessus or M. avium were allowed to form at 37°C and 5% CO2 in a humidified atmosphere until they grew to a height of approximately 30 μm, as determined by confocal laser scanning microscopy (CLSM) and assessed by COMSTAT2. These incubation times were determined to be 72 hours for M. abscessus and 2 weeks for the slower-growing M. avium. Biofilms of M. abscessus clinical isolates 1, 2, and 3 were also grown for an additional 24 hours (96 hours total) to assess the ability of HuTipMab to disrupt these more mature biofilms.

[0285] Disruption of M. abscessus and M. avium biofilms by HuTipMab. The medium was aspirated from M. abscessus or M. avium biofilms, which were then gently washed twice with 200 μL equilibrated 1× Dulbecco's phosphate-buffered saline (DPBS) without calcium or magnesium (Corning, Corning, NY). The biofilms were then incubated with either 7H9 alone, 5 μg HuIgG, or 5, 7.5, or 10 μg HuTipMab at 37°C in a humidified atmosphere with 5% CO for 30 minutes. To assay time-dependent disruption, additional wells were incubated with 5 μg HuTipMab for 60 minutes. After incubation, the biofilms were gently washed once with 200 μL equilibrated DPBS.

[0286] The remaining biofilms were stained with FM 1-43FX (Invitrogen) by incubation in the dark for 15 minutes. The stain was removed, and the biofilms were gently washed twice with DPBS and then fixed for ≥3 hours (1.6% paraformaldehyde, 2.5% glutaraldehyde, and 4% acetic acid in 0.1 M phosphate buffer). The fixative was removed and replaced with DPBS, and the biofilms were then visualized and imaged using a ZEISS CLSM800 microscope. Images were analyzed by COMSTAT2 to calculate relative biomass values ​​(μm3 / μm2). Values ​​represent the average of three biological replicates. Percent destruction was calculated as [mean biomass in HuIgG-treated wells - mean biomass in HuTipMab-treated wells] / [mean biomass in HuIgG-treated wells] × 100.

[0287] Antibiotic-mediated killing of freshly released M. abscessus or M. avium. M. abscessus or M. avium biofilms were incubated with medium alone (to recover bacteria growing / residing in planktonic state in the fluid above the biofilm) or 5 μg HuTipMab (to generate NRel), both with and without antibiotics. The concentrations of each antibiotic used were predetermined to limit planktonic bacterial killing to approximately 25% to facilitate detection of any enhanced relative killing of NRel. For assays of relative killing of clinical M. abscessus isolates, applicants chose to test isolates 1 and 3 because they exhibited greater antibiotic resistance than laboratory-passaged isolates and required higher concentrations of amikacin and azithromycin to achieve approximately 25% killing, thereby representing a more clinically relevant situation.

[0288] To determine the relative susceptibility of planktonic NTM to amikacin, biofilms were prepared as described above, gently washed twice with DPBS, and then treated with 200 μL of either 7H9 (growth control); 7H9 + 0.5 μg amikacin / mL for M. abscessus 19977; 7H9 + 12 μg amikacin / mL for M. avium; 7H9 + 0.7 μg amikacin / mL for M. abscessus clinical isolate 1; or 7H9 + 1 μg amikacin / mL for M. abscessus clinical isolate 3. To determine the relative susceptibility of NTM NRel, biofilms were treated with 200 μL of 7H9 + 5 μg HuTipMab alone (NRel growth control) or 7H9 + 5 μg HuTipMab + each of the amikacin concentrations used above. The treated biofilms were incubated statically at 37°C in a humidified atmosphere with 5% CO2 for 2 hours, after which 150 μL from each well was collected and dispensed into a 1.5 mL Eppendorf microcentrifuge tube. Then, 150 μL was collected from each well and pulse-vortexed with two sterile 3 mm glass beads (Fisher Scientific International, Inc., Hampton, NH) to disrupt aggregates. The suspension was gently sonicated in a water bath sonicator (Ultrasonic Bath 2.8 L, Fisher Scientific) for 2 minutes to further disrupt any aggregates. After sonication, the samples were diluted, plated on Middlebrook 7H10 agar, and incubated at 37°C in a humidified atmosphere with 5% CO2 to assess relative CFU.

[0289] To determine azithromycin-mediated killing, the same protocol as above was used with one adjustment. To avoid acidification of the medium due to bacterial growth in wells treated with azithromycin, which is unstable at lower pH (Johnson et al., 1999), these cultures were incubated at 37°C without 5% CO for 2 hours. Wells were treated with 200 μL of either 7H9 (growth control); 7H9 + 5 μg azithromycin / mL for M. abscessus 19977; 7H9 + 16 μg azithromycin / mL for M. avium; 7H9 + 10 μg azithromycin / mL for M. abscessus clinical isolate 1; or 7H9 + 8 μg azithromycin / mL for M. abscessus clinical isolate 3. The respective azithromycin concentrations used on 7H9 + 5 μg HuTipMab (NRel growth control) or 7H9 + 5 μg HuTipMab + Percent killing was then calculated as [growth control CFU / mL − NRel or planktonic CFU / mL] / [growth control CFU / mL].

[0290] Statistical analysis. Results are expressed as the mean ± SD of three biological replicates with three technical replicates each. Comparisons between groups were performed by unpaired t-test. All statistical analyses were performed with Graphpad (Prism) software V9.

[0291] Example 2 result

[0292] Verification of HuTipMab specificity. The purity of the isolated recombinant HupB was confirmed by silver staining (Figure 1A). Recognition of HupB by HuTipMab was demonstrated by ELISA, which showed that HuTipMab recognized both HupB and the tip chimeric peptide; no color development occurred in the absence of HuTipMab (P < 0.001-0.0001) (Figure 1B and Figure 1C). HuTipMab did not recognize the tail chimeric peptide.

[0293] Evaluation of relative HuTipMab-induced NTM biofilm destruction by CLSM. We next determined the biofilm-disrupting ability of HuTipMab by incubating M. abscessus 19977 biofilms for 72 hours or M. avium biofilms for 2 weeks with increasing concentrations of HuTipMab or for increasing incubation periods with HuTipMab. After incubation, newly released bacteria ("NRel") were removed, and the remaining biofilms were analyzed by confocal laser scanning microscopy (CLSM). Image analysis revealed that HuTipMab destroyed significantly more M. abscessus 19977 biofilms than biofilms incubated with medium alone or HuIgG (P < 0.01-0.0001) (Figure 2). Moreover, M. abscessus 19977 biofilms were disrupted in a dose- and time-dependent manner, as incubation with 5, 7.5, or 10 μg of HuTipMab for 30 min resulted in 53%, 79%, or 88% disruption, respectively, while incubation with 5 μg of HuTipMab for 60 min resulted in 89% disruption.

[0294] Similarly, M. avium biofilms were significantly disrupted by incubation with HuTipMab (P < 0.01-0.0001) (Figure 3). M. avium biofilms were also disrupted in a dose- and time-dependent manner, with disruption by 5, 7.5, or 10 μg of HuTipMab over 30 min being 51%, 68%, and 76%, respectively, while incubation with 5 μg HuTipMab for 60 min resulted in 80% disruption.

[0295] Significant dose- and time-dependent destruction of 72-h biofilms formed by all three clinical isolates of M. abscessus was also evident (P < 0.001–0.0001) (Figures 4A–4C). Destruction by 5, 7.5, or 10 μg of HuTipMab over 30 min was 57%–62%, 77%–88%, or 89%–93%, respectively, whereas incubation with 5 μg HuTipMab for 60 min resulted in 90%–92% destruction. More mature 96-h biofilms formed by these three clinical isolates were similarly significantly disrupted by HuTipMab, with relative mean percent destruction of 57%–59%, 76%–89%, or 89%–94% after 30-min incubation with 5, 7.5, or 10 μg, respectively, and 90%–93% after 60-min incubation with 5 μg HuTipMab (P<0.05–0.0001) (data not shown).

[0296] Enhanced Killing of NTM NRel by Antibiotics Commonly Used to Treat NTM Infections. To determine whether NTM NRel demonstrated enhanced antibiotic susceptibility, Applicants evaluated relative killing by amikacin and azithromycin. While killing of planktonic M. abscessus 19977 by amikacin or azithromycin was limited to 23% and 20%, respectively, M. abscessus 19977 NRel was significantly more susceptible to both antibiotics, with 61% and 42% killing, respectively (Figures 5A and 5B) (P<0.001 or 0.01, respectively). Notably, this enhanced killing occurred when amikacin and azithromycin were used at ¼ and ½ of the reported minimum inhibitory concentrations (MICs), respectively.

[0297] Similarly, M. avium NRel was significantly more susceptible to antibiotic killing than its isogenic planktonic counterpart; percent killing of planktonic M. avium by amikacin and azithromycin was limited to 17% and 19%, respectively, while percent killing of M. avium NRel was 41% and 36%, respectively (P < 0.01) (Figures 6A and 6B). This significantly enhanced susceptibility of M. avium NRel to killing by amikacin and azithromycin was observed when used at 1 / 4 and 1 / 2 of their respective MICs.

[0298] NRel from disrupted 72-h biofilms formed by M. abscessus clinical isolates 1 and 3 was also significantly more susceptible to killing by amikacin and azithromycin than its isogenic planktonic counterpart (P < 0.05-0.001) (Figures 7A-7D). Percent killing of planktonic M. abscessus by amikacin and azithromycin was limited to 28% and 28%, respectively, for clinical isolate 1, and 21% and 25%, respectively, for clinical isolate 3, whereas NRel was killed by amikacin and azithromycin at 45% and 47%, respectively, for clinical isolate 1, or 47% and 45%, respectively. It should be noted that, as might be expected, despite the higher concentrations of antibiotic required to achieve similar levels of killing as either the planktonic-grown clinical isolate or the laboratory-passaged M. abscessus 19977, the higher susceptibility of these anti-DNABII-induced NRel to killing was achieved at the same concentrations that killed only approximately 25% of the corresponding planktonic populations.

[0299] Antibiotic therapy for PwCF with positive cultures for NTM is associated with prolonged regimens with multiple antibiotics, the potential for multiple sequelae, and an unacceptably high rate of clinical failure. Modulator therapy reduces both symptoms and the frequency of NTM-positive cultures, although it remains to be determined whether this latter outcome is due to a reduced ability to collect sputum or a true reduction in NTM prevalence. Furthermore, modulator treatment remains out of reach for the majority of PwCF due to both cost and availability, which is currently limited to the United States, Europe, Australia, and New Zealand. Therefore, new strategies to enhance the efficacy of existing antibiotic therapies are needed, especially given the somewhat limited investment currently being made in new antibiotic discovery. Novel approaches, including those that target the biofilm matrix to release resident bacteria for elimination by either antibiotics or host immune effectors, are a high priority.

[0300] Example 3

[0301] This experiment provides a pig model for preclinical testing of antibodies and their antigen-binding fragments, alone or in combination with additional disclosed active agents, to treat cystic fibrosis. See Stoltz et al. (2010) Science Translational Medicine 2(29):29-31. Cystic fibrosis is an autosomal recessive disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) anion channel. In this model, pigs, called CF pigs, specifically bred to carry defects in the gene called "CFTR," spontaneously develop hallmarks of CF lung disease, including infection of the lower respiratory tract with multiple bacterial species. The pigs can receive the antibodies or their antigen-binding fragments, alone or in combination with antibiotics, which, in one embodiment, are administered to the animals' lungs by nebulization to evaluate the amelioration of disease and associated pathological symptoms.

[0302] Example 4

[0303] Applicants also provide a preclinical model for tuberculosis (TB). See Ordway et al. (2010) Anti. Agents and Chemotherapy 54:1820. The microorganism Mycobacterium tuberculosis is responsible for an ever-growing global epidemic. Current figures suggest that there are approximately 8 million new cases of TB and approximately 2.7 million deaths from TB each year. Particularly troubling, in addition to this microorganism's role as a co-infection in individuals with HIV (it is estimated that of the nearly 45 million people infected with HIV, nearly one-third are also co-infected with M. tuberculosis), is that isolates have become highly resistant to multiple drugs, and no new drugs for TB have been introduced in a quarter century. In this animal model, SPF guinea pigs are maintained in a barrier colony and infected with an aerosolized spray to deliver approximately 20 cfu of M. tuberculosis Erdman K01 bacilli to their lungs. At 25, 50, 75, 100, 125, and 150 days after challenge, animals are sacrificed and tissues are harvested for bacterial load determination and histopathological evaluation. Unlike mice, which do not develop classical signs of TB, guinea pigs challenged in this manner develop well-organized granulomas with central necrosis, a hallmark of human disease. Furthermore, similar to humans, guinea pigs develop severe pyogenic granulomas and necrotizing lymphadenitis in the draining lymph nodes as part of the primary lesion complex. Use of this model provides preclinical screening for the validation and identification of therapeutic agents, as well as preventative strategies for the reduction and / or elimination of the resulting M. tuberculosis biofilm, which has been observed to form in the lungs of these animals after challenge and is thought to contribute to both the pathogenesis and chronicity of disease.

[0304] Experimental Considerations

[0305] The applicant has developed such a strategy using epitope-targeting monoclonal antibodies against the DNABII protein. Although this approach has been shown to effectively disrupt biofilms formed by Gammaproteobacteria (Brockson et al., 2014, Mol Microbiol 93, 1246-1258; Freire et al., 2017, Mol Oral Microbiol 32, 74-88; Goodman et al., 2011, Mucosal Immunol 4, 625-637; Kurbatfinski et al., 2022, Antimicrob Agents Chemother., 66, e0187721; Kurbatfinski et al., 2023, Front Microbiol 14, 1202215), it was unclear whether HuTipMab treatment would also be effective against NTM, which belong to the class Actinomycetia and are distinguished by their thick, mycolic acid-rich cell walls.

[0306] Comparative genomics has shown that NTM can express DNABII homologs, and in silico analysis has revealed that mycobacterial HU has two domains. Its N-terminal 106 amino acid (AA) domain bears high similarity to other DNABII proteins (all approximately 90–105 AA in length), while its 108-amino acid C-terminal domain contains a eukaryotic H1 histone-like motif. Furthermore, 104 of 106 AA within the translated sequence of the N-terminal DNABII-like domain are identical between the HU proteins expressed by M. tuberculosis and M. avium, while 99 of 106 AA are a perfect match for HU expressed by M. abscessus. All three DNABII homologs expressed by these mycobacterial species share at least 37 consecutive identical AA, including the tip region targeted by HuTipMab, which may explain its ability to recognize M. tuberculosis HupB by ELISA. Both M. abscessus and M. avium form biofilms that incorporate eDNA into an EPS matrix, but it was unclear whether these biofilms also incorporated DNABII proteins within their eDNA-rich biofilm matrix, and whether HuTipMabs generated against specific defense domains of traditional DNABII proteins would recognize unique mycobacterial DNABII homologs and actively disrupt NTM biofilms. Applicants have shown here that HuTipMabs effectively disrupted biofilms formed by both M. abscessus and M. avium (including biofilms formed by an M. abscessus isolate recovered from PwCF, which may be more representative of M. abscessus found at disease sites than laboratory-passaged M. abscessus 19977) in a dose- and time-dependent manner, indicating that HuTipMabs retained their ability to recognize and disrupt NTM biofilms.

[0307] Upon disruption, NRel of M. abscessus 19977, NRel of M. avium, and NRel of M. abscessus clinical isolates 1 and 3 exhibited increased susceptibility to two highly clinically relevant antibiotics. Compared with their isogenic planktonic counterparts, NTM NRel was significantly more susceptible to amikacin and azithromycin, two antibiotics that are ineffective when NTM is within a biofilm (Clary et al., 2018, Antimicrob Agents Chemother., 62). Notably, the enhanced antibiotic susceptibility of NRel occurred within a fraction of the planktonic MIC of M. abscessus 19977 and M. avium. Similarly, NRel of both clinical isolates was more susceptible to antibiotic-mediated killing at the same concentrations than its planktonic counterparts. This result may be aided by the fact that amikacin and azithromycin have better access to their targets after NTM release from its protective biofilm.

[0308] Our data support a combinatorial treatment strategy for individuals infected with NTM, where biofilms significantly contribute to diminished lung function and poor quality of life. In one aspect, a method is provided for nebulizing HuTipMab into the lungs of these individuals to disrupt biofilm aggregates and release NTM from antibiotic-resistant biofilms into an NRel state, so that co-delivered antibiotics can rapidly kill the induced NTM NRel. This strategy would enhance existing antibiotics, improve clinical outcomes, and reduce the length of antibiotic treatment for PwCF, as well as the growing population of people without CF who have refractory NTM infections.

[0309] equivalent While the present disclosure has been described in conjunction with the above embodiments, it should be understood that the foregoing description and examples are intended to be illustrative and not limiting of the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure pertains.

[0310] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All nucleotide sequences provided herein are presented in the 5' to 3' direction.

[0311] The embodiments illustratively described herein can, where appropriate, be practiced in the absence of any element(s), limitation(s) not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc., should be interpreted expansively and without limitation. Moreover, the terms and expressions used herein are used as terms of description rather than limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present disclosure.

[0312] Thus, while the present disclosure has been specifically disclosed by specific embodiments and optional features, it should be understood that alterations, improvements, and variations of the embodiments therein disclosed herein may occur to those skilled in the art, and that such alterations, improvements, and variations are considered to be within the scope of the present disclosure. The materials, methods, and examples provided herein are representative and exemplary of particular embodiments and are not intended as limitations on the scope of the present disclosure.

[0313] The scope of the disclosure is described broadly and generically herein. Each of the narrower species and subgeneric groupings that fall within the generic disclosure also form part of this disclosure. This includes conditional generic descriptions or negative limitations that remove any subject matter from a genus, regardless of whether the removed material is specifically recited herein.

[0314] Additionally, where features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that embodiments of the disclosure may also be described in terms of any individual member or subgroup of members of the Markush group.

[0315] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0316] Non-limiting embodiments of the present disclosure Embodiment 1. A method for preventing or treating an infection caused by a nontuberculous mycobacterium (NTM) species in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide.

[0317] Embodiment 2. A method for sensitizing a biofilm to an antibiotic or for disrupting a biofilm, wherein the biofilm comprises a nontuberculous mycobacterium (NTM) species, and the method comprises contacting the biofilm with an antibody or antigen-binding fragment thereof that binds to the tip region of the DNABII peptide.

[0318] Embodiment 3. The antibody or antigen-binding fragment thereof a. 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; b. a heavy chain (HC) immunoglobulin variable domain sequence comprising the sequence from aa25 to aa144 of SEQ ID NO: 24 or an equivalent thereof, and a light chain (LC) immunoglobulin variable domain sequence comprising the sequence from aa21 to aa132 of SEQ ID NO: 25 or an equivalent thereof; c. Heavy chain complementarity-determining region 1 (CDRH1) comprising the sequence of GFTFRTY (aa 50 to aa 56 of SEQ ID NO: 9, 10, 11, or 24), heavy chain complementarity-determining region 2 (CDRH2) comprising the sequence of GSDRRH (aa 76 to aa 81 of SEQ ID NO: 9, 10, 11, or 24), and heavy chain complementarity-determining region 3 (CDRH3) comprising the sequence of VGPYDGYYGEFDY (aa 121 to aa 133 of SEQ ID NO: 9, 10, 11, or 24). a light chain complementarity-determining region 1 (CDRL1) comprising the sequence QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 15, 16, 17, or 25), a light chain complementarity-determining region 2 (CDRL2) comprising the sequence LVS (aa75 to aa77 of SEQ ID NO: 15, 16, 17, or 25), and a light chain complementarity-determining region 3 (CDRL3) comprising the sequence WQGTHFP (aa114 to aa120 of SEQ ID NO: 15, 16, 17, or 25); d. Heavy chain complementarity determining region 1 (CDRH1) comprising, consisting essentially of, or even consisting of the sequence of GFTFSRYG (aa 50 to aa 57 of SEQ ID NO: 12, 13, or 14); heavy chain complementarity determining region 2 (CDRH2) comprising, consisting essentially of, or even consisting of the sequence of ISSGGSYT (aa 75 to aa 82 of SEQ ID NO: 12, 13, or 14); heavy chain complementarity determining region 3 (CDRH3) comprising, consisting essentially of, or even consisting of the sequence of ERHGGDGYWYFDV (aa 121 to aa 133 of SEQ ID NO: 12, 13, or 14); QSLLDSDGKT a light chain complementarity determining region 1 (CDRL1) comprising, consisting essentially of, or even consisting of the sequence of F (aa47 to aa57 of SEQ ID NO: 15, or 16, or 17, or 25), a light chain complementarity determining region 2 (CDRL2) comprising, consisting essentially of, or even consisting of the sequence of LVS (aa75 to aa77 of SEQ ID NO: 15, or 16, or 17, or 25), and a light chain complementarity determining region 3 (CDRL3) comprising, consisting essentially of, or even consisting of the sequence of WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 15, or 16, or 17, or 25); and / or e. a heavy chain (HC) immunoglobulin variable domain comprising the amino acid sequence of amino acids 25 to 144 of SEQ ID NO: 9; and a light chain (LC) immunoglobulin variable domain comprising the amino acid sequence of amino acids 21 to 132 of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. 3. The method of embodiment 1 or embodiment 2, comprising one or more of:

[0319] Embodiment 4. The method of any one of embodiments 1-3, wherein the antibody or antigen-binding fragment thereof further comprises a constant region selected from the group consisting of an IgA constant region, an IgD constant region, an IgE constant region, an IgG constant region, or an IgM constant region.

[0320] Embodiment 5. The method of embodiment 4, wherein the constant region is an IgG1 constant region.

[0321] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the antigen-binding fragment is selected from the group of Fab, F(ab')2, Fab', scFv or Fv.

[0322] Embodiment 7. The method of any one of embodiments 3-6, wherein the equivalent of the amino acid sequence comprises a polypeptide having at least 80% amino acid identity to the amino acid, or the equivalent of the amino acid sequence comprises a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions to the complement of a polynucleotide encoding the amino acid sequence.

[0323] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the antibody or antigen-binding fragment thereof further comprises a modification.

[0324] Embodiment 9. The method of embodiment 8, wherein the modification is selected from the group of PEGylation, PEG mimetic, polysialylation, HESylation or glycosylation.

[0325] Embodiment 10. The method of any one of embodiments 2-9, wherein the antibiotic comprises amikacin, azithromycin, dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, methicillin, vancomycin, daptomycin, mupirocin, penicillin, cloxacillin, erythromycin, clarithromycin, roxithromycin, telithromycin, spiramycin, fidaxomicin, rifampin, ethambutol, streptomycin, or anthramycin, optionally administered in an amount of about 25% to about 50% of the MIC of the antibiotic. In a further embodiment, the antibody or antigen-binding fragment thereof and the antibiotic are administered simultaneously with each other or after each other, e.g., the antibiotic is administered after or instead precedes the antibody or antigen-binding fragment thereof.

[0326] Embodiment 11. The method of any one of embodiments 2 to 10, further comprising contacting the biofilm with an effective amount of a synthetic or recombinant polypeptide comprising, consisting essentially of, or even consisting of mB box-97, or an equivalent thereof, consisting of amino acids 90 to 176 from the coding sequence of native human HMGB1 protein set forth as SEQ ID NO: 45, with a cysteine ​​to serine point mutation at amino acid 106. The contacting steps can be simultaneous or sequential.

[0327] Embodiment 12. The method of any one of embodiments 2 to 10, further comprising contacting the biofilm with an effective amount of a synthetic or recombinant polypeptide comprising, consisting essentially of, or even consisting of mB box-97, or an equivalent thereof, consisting of amino acids 80 to 176 from the coding sequence of the native human HMGB1 protein set forth as SEQ ID NO: 45, with a cysteine ​​to serine point mutation at amino acid 106.

[0328] Embodiment 13 The method of embodiment 11 or embodiment 12, wherein the synthetic or recombinant polypeptide consists of SEQ ID NO: 48.

[0329] Embodiment 14. The method of any one of embodiments 2 to 13, further comprising contacting the biofilm with an effective amount of a DNA-binding agent.

[0330] Embodiment 15. The method of embodiment 14, wherein the DNA binding agent comprises a histone-like nucleoid structuring protein (H-NS), a polyamine, or a polycation.

[0331] Embodiment 16. The method of embodiment 15, wherein 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: 51-56.

[0332] Embodiment 17. The method of any one of embodiments 1 and 3-10, further comprising administering to the subject an effective amount of at least one antibiotic selected from amikacin, azithromycin, dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, methicillin, vancomycin, daptomycin, mupirocin, penicillin, cloxacillin, erythromycin, clarithromycin, roxithromycin, telithromycin, spiramycin, fidaxomicin, rifampin, ethambutol, streptomycin, or anthramycin, optionally administered in an amount of about 25% to about 50% of the antibiotic's MIC. In a further embodiment, the antibody or antigen-binding fragment thereof and the antibiotic are administered simultaneously with each other or after each other, e.g., the antibiotic is administered after or instead precedes the antibody or antigen-binding fragment thereof.

[0333] Embodiment 18. The method of any one of embodiments 1, 3-10, and 17, further comprising administering to the subject an effective amount of a synthetic or recombinant polypeptide comprising, consisting essentially of, or even consisting of, amino acids 90 to 176 from the coding sequence of native human HMGB1 protein set forth as SEQ ID NO: 45, with mB box-97 having a cysteine ​​to serine point mutation at amino acid 106, or an equivalent thereof.

[0334] Embodiment 19. The method of any one of embodiments 1, 3-10, and 17, further comprising administering to the subject an effective amount of a synthetic or recombinant polypeptide comprising, consisting essentially of, or even consisting of, amino acids 80 to 176 from the coding sequence of native human HMGB1 protein set forth as SEQ ID NO: 45, and mB box-97 with a cysteine ​​to serine point mutation at amino acid 106, or an equivalent thereof.

[0335] Embodiment 20. The method of embodiment 18 or embodiment 19, wherein the synthetic or recombinant polypeptide consists of SEQ ID NO: 48.

[0336] Embodiment 21. The method of any one of embodiments 1, 3-10 and 17-20, further comprising contacting the biofilm with an effective amount of a DNA-binding agent.

[0337] Embodiment 22. The method of embodiment 21, wherein the DNA binding agent comprises a histone-like nucleoid structuring protein (H-NS), a polyamine, or a polycation.

[0338] Embodiment 23. The method of embodiment 22, wherein 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: 51-56.

[0339] Embodiment 24. The method of any one of embodiments 1 to 23, wherein the NTM species is M. abscessus (Mab), M. avium (Mav), M. intracellulare, or M. chimaera.

[0340] Embodiment 25. The method of any of embodiments 1, 3-10, and 17, wherein administering comprises one or more of inhalation, oral administration, nasal administration, injection, and / or topical application.

[0341] Embodiment 26. The method of any one of embodiments 1, 3-10, and 15-25, wherein the subject has chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia, or cystic fibrosis (CF).

[0342] Embodiment 27. The method of any one of embodiments 2 to 16 and 24, wherein the contacting step is carried out with or. (Sequence Listing) SEQ ID NO: 1, human IhfA, A-tip fragment [ka] SEQ ID NO: 2, human IhfB, B tip fragment [ka] SEQ ID NO: 3 (tip chimeric peptide IhfA5-mIhfB4NTHI) [ka] wherein "X" is an optional amino acid linker sequence comprising, consisting essentially of, or even consisting of, optionally 1 to 20 amino acids; wherein "X1" is any amino acid, or alternatively "X1" is selected from the amino acids Q, R, K, S, or T. SEQ ID NO: 4 (tip chimeric peptide IhfA5-mIhfB4NTHI) [ka] wherein "X" is an optional amino acid linker sequence optionally containing 1 to 20 amino acids. SEQ ID NO: 5 (tip chimeric peptide IhfA5-mIhfB4NTHI) [ka] SEQ ID NO: 6: Non-limiting exemplary linker: [ka] SEQ ID NO: 7: Non-limiting exemplary linker: [ka] SEQ ID NO: 8: Non-limiting exemplary linker: [ka] SEQ ID NO: 9 (H10210 (1F8.F1 humanized HC1)) Bold font indicates exemplified variable regions, while bold, italic and underlined font indicates exemplified CDRs. [ka] SEQ ID NO: 10 (H10211 (1F8.F1 humanized HC2)) Bold font indicates exemplified variable regions, while bold, italic and underlined font indicates exemplified CDRs. [ka] SEQ ID NO: 11 (H10212 (1F8.F1 humanized HC3)) Bold font indicates exemplified variable regions, while bold, italic and underlined font indicates exemplified CDRs. [ka] [ka] SEQ ID NO: 12 (H10213 (11E7.C7 humanized HC1)) Bold font indicates exemplified variable regions, while bold, italic and underlined font indicates exemplified CDRs. [ka] SEQ ID NO: 13 (H10214 (11E7.C7 humanized HC2)) Bold font indicates exemplified variable regions, while bold, italic and underlined font indicates exemplified CDRs. [ka] [ka] SEQ ID NO: 14 (H10215 (11E7.C7 humanized HC3)) Bold font indicates exemplified variable regions, while bold, italic and underlined font indicates exemplified CDRs. [ka] SEQ ID NO: 15 (L10210 (1F8.F1 humanized LC1)) Bold font indicates exemplified variable regions, while bold, italic and underlined font indicates exemplified CDRs. [ka] SEQ ID NO: 16 (L10211 (1F8.F1 humanized LC2)) Bold font indicates exemplified variable regions, while bold, italic and underlined font indicates exemplified CDRs. [ka] [ka] SEQ ID NO: 17 (L10212 (1F8.F1 humanized LC3)) Bold font indicates exemplified variable regions, while bold, italic and underlined font indicates exemplified CDRs. [ka] SEQ ID NO: 18 (L10213 (11E7.C7 humanized LC1)) Bold font indicates exemplified variable regions, while bold, italic and underlined font indicates exemplified CDRs. [ka] SEQ ID NO: 19 (L10214 (11E7.C7 humanized LC2)) Bold font indicates exemplified variable regions, while bold, italic and underlined font indicates exemplified CDRs. [ka] SEQ ID NO: 20 (L10215 (11E7.C7 humanized LC3)) Bold font indicates exemplified variable regions, while bold, italic and underlined font indicates exemplified CDRs. [ka] SEQ ID NO: 21 (heavy chain consensus sequence) [ka] wherein X and the lowercase letter may be substituted with any amino acid, or alternatively with an amino acid at the corresponding position selected from SEQ ID NOs: 9 to 14. In one embodiment, X may also indicate the absence of an amino acid residue. SEQ ID NO: 22 (light chain consensus sequence) [ka] [ka] wherein X and the lowercase letter may be substituted with any amino acid, or alternatively, with an amino acid at the corresponding position selected from SEQ ID NOs: 15 to 20. In one embodiment, X may also indicate the absence of an amino acid residue. SEQ ID NO: 23 Human IgG1 constant region, Uniprot: P01857 [ka] SEQ ID NO: 24 (top heavy chain consensus sequence) [ka] In the formula, the lowercase letters may be substituted with an amino acid selected from SEQ ID NOs: 9 to 11 at the corresponding position. SEQ ID NO: 25 (apical light chain consensus sequence) [ka] In the formula, lowercase letters may be substituted with an amino acid selected from SEQ ID NOs: 15 to 17 at the corresponding position. SEQ ID NO: 26: Non-limiting exemplary linker: [ka] SEQ ID NO: 27: Non-limiting exemplary linker: [ka] SEQ ID NO: 28: Non-limiting exemplary linker: [ka] SEQ ID NO: 29: Non-limiting exemplary linker: [ka] SEQ ID NO: 30: Non-limiting exemplary linker: [ka] SEQ ID NO: 31 Mus musculus wild-type HMGB1 protein [ka] SEQ ID NO: 32 Homo sapiens wild-type HMGB1 protein [ka] HMGB1 (copied from human, GenBank accession number CAE48262.1) is a small, 215-amino acid protein (approximately 30 Kda) composed of three domains: two positively charged domains, the A-box and the B-box, each containing 80 amino acids, and a negatively charged, carboxyl-terminal, 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. Italicized amino acids (approximately amino acids 88-164) depict the B-box domain. Underlined amino acids (amino acids 186-215) depict the C-tail domain. SEQ ID NO: 33 Human IgD constant region, Uniprot:P01880 [ka] SEQ ID NO: 34 SEQ ID NO: 36 Human IgG2 constant region, Uniprot:P01859 [ka] SEQ ID NO: 35 Human IgG3 constant region, Uniprot: P01860 [ka] SEQ ID NO: 36 Human IgM constant region, Uniprot: P01871 [ka] SEQ ID NO: 37 Human IgG4 constant region, Uniprot:P01861 [ka] SEQ ID NO: 38 Human IgA1 constant region, Uniprot: P01876 [ka] [ka] SEQ ID NO: 39 Human IgA2 constant region, Uniprot: P01877 [ka] SEQ ID NO: 40 Human Ig kappa constant region, Uniprot: P01834 [ka] SEQ ID NO: 41: DNA, artificial sequence (M. tuberculosis HupB primer 1) [ka] SEQ ID NO: 42: DNA, artificial sequence (M. tuberculosis HupB primer 2) [ka] SEQ ID NO: 43 (tail chimeric peptide IhfA3-IhfB2 NTHI ) [ka] SEQ ID NO: 44: Mus musculus wild-type HMGB1 protein [ka] SEQ ID NO: 45 Homo sapiens wild-type HMGB1 protein [ka] HMGB1 (copied from human, GenBank accession number CAE48262.1) is a small, 215-amino acid protein (approximately 30 Kda) composed of three domains: two positively charged domains, the A-box and the B-box, each containing 80 amino acids, and a negatively charged, carboxyl-terminal, 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. Italicized amino acids (approximately amino acids 88-164) depict the B-box domain. Underlined amino acids (amino acids 186-215) depict the C-tail domain. SEQ ID NO: 46, artificial sequence, linker sequence [ka] SEQ ID NO: 47, human, mB box-97 peptide [ka] SEQ ID NO: 48, human, mB box-97 peptide, with C-terminal linker sequence [ka] SEQ ID NO: 49, human, IhfA, A tip fragment [ka] SEQ ID NO: 50, human IhfB, B tip fragment [ka] SEQ ID NO: 51, E. coli, H-NS protein [ka] SEQ ID NO: 52, NTHI, H-NS protein [ka] SEQ ID NO: 53, Mycobacterium tuberculosis, H-NS [ka] SEQ ID NO: 54, Streptococcus pneumoniae H-NS protein [ka] SEQ ID NO: 55, Klebsiella pneumoniae H-NS protein [ka] SEQ ID NO: 56, Pseudomonas aeruginosa H-NS protein [ka] SEQ ID NO: 57, (E. coli HupB, Genbank accession number: AP_001090.1, last accessed March 21, 2011) [ka]

Claims

1. A method for preventing or treating infection caused by a nontuberculous mycobacterium (NTM) species in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof that binds to the tip region of a DNABII peptide.

2. 1. A method for sensitizing a biofilm to an antibiotic or disrupting a biofilm, wherein the biofilm comprises a nontuberculous mycobacterium (NTM) species, the method comprising contacting the biofilm with an antibody or antigen-binding fragment thereof that binds to an apical region of a DNABII peptide.

3. the antibody or the antigen-binding fragment thereof, a. (i) a heavy chain (HC) immunoglobulin variable domain sequence comprising the sequence of amino acids (aa) 25 to aa 144 of SEQ ID NO: 21 or an equivalent thereof; and (ii) a light chain (LC) immunoglobulin variable domain sequence comprising the sequence from aa21 to aa132 of SEQ ID NO: 22 or an equivalent thereof; b. (i) a heavy chain (HC) immunoglobulin variable domain sequence comprising the sequence of aa25 to aa144 of SEQ ID NO: 24 or an equivalent thereof; and (ii) a light chain (LC) immunoglobulin variable domain sequence comprising the sequence of aa21 to aa132 of SEQ ID NO: 25 or an equivalent thereof; c. (i) a heavy chain complementarity-determining region 1 (CDRH1) comprising the sequence GFTFRTY (aa50 to aa56 of SEQ ID NO: 9, 10, 11, or 24); (ii) a heavy chain complementarity-determining region 2 (CDRH2) comprising the sequence of GSDRRH (aa76 to aa81 of SEQ ID NO: 9, 10, 11, or 24); (iii) a heavy chain complementarity-determining region 3 (CDRH3) comprising the sequence VGPYDGYYGEFDY (aa121 to aa133 of SEQ ID NO: 9, 10, 11, or 24); (iv) a light chain complementarity-determining region 1 (CDRL1) comprising the sequence QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 15, 16, 17, or 25); (v) a light chain complementarity-determining region 2 (CDRL2) comprising the sequence of LVS (aa75 to aa77 of SEQ ID NO: 15, 16, 17, or 25); and (vi) a light chain complementarity-determining region 3 (CDRL3) comprising the sequence WQGTHFP (aa114 to aa120 of SEQ ID NO: 15, 16, 17, or 25); d. (i) a heavy chain complementarity-determining region 1 (CDRH1) comprising, consisting essentially of, or even consisting of the sequence GFTFSRYG (aa50 to aa57 of SEQ ID NO: 12, 13, or 14); (ii) a heavy chain complementarity-determining region 2 (CDRH2) comprising, consisting essentially of, or even consisting of the sequence ISSGGSYT (aa75 to aa82 of SEQ ID NO: 12, 13, or 14); (iii) a heavy chain complementarity-determining region 3 (CDRH3) comprising, consisting essentially of, or even consisting of the sequence of ERHGGDGYWYFDV (aa121 to aa133 of SEQ ID NO: 12, 13, or 14); (iv) a light chain complementarity-determining region 1 (CDRL1) comprising, consisting essentially of, or even consisting of the sequence QSLLDSDGKTF (aa47 to aa57 of SEQ ID NO: 15, 16, 17, or 25); (v) a light chain complementarity-determining region 2 (CDRL2) comprising, consisting essentially of, or even consisting of the sequence of LVS (aa75 to aa77 of SEQ ID NO: 15, 16, 17, or 25); and (vi) a light chain complementarity-determining region 3 (CDRL3) comprising, consisting essentially of, or even consisting of the sequence WQGTHFPYT (aa114 to aa122 of SEQ ID NO: 15, 16, 17, or 25); and / or e. A heavy chain (HC) immunoglobulin variable domain comprising the amino acid sequence of amino acids 25 to 144 of SEQ ID NO:9; and A light chain (LC) immunoglobulin variable domain comprising the amino acid sequence of amino acids 21 to 132 of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:

17.

3. The method of claim 1 or claim 2, comprising one or more of:

4. The method of any one of claims 1 to 3, wherein the antibody or the antigen-binding fragment thereof further comprises a constant region selected from the group consisting of an IgA constant region, an IgD constant region, an IgE constant region, an IgG constant region, or an IgM constant region.

5. The method of claim 4, wherein the constant region is an IgG1 constant region.

6. The antigen-binding fragment is Fab, F(ab') ２ 6. The method of any one of claims 1 to 5, wherein the antibody is selected from the group consisting of Fab', scFv or Fv.

7. 7. The method of any one of claims 3 to 6, wherein said equivalent of an amino acid sequence comprises a polypeptide having at least 80% amino acid identity to said amino acid, or wherein said equivalent of an amino acid sequence comprises a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions to the complement of a polynucleotide encoding said amino acid sequence.

8. The method of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof further comprises a modification.

9. The method of claim 8, wherein the modification is selected from the group of PEGylation, PEG mimetic, polysialylation, HESylation or glycosylation.

10. 10. The method of any one of claims 2 to 9, wherein the antibiotic comprises amikacin, azithromycin, dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, methicillin, vancomycin, daptomycin, mupirocin, penicillin, cloxacillin, erythromycin, clarithromycin, roxithromycin, telithromycin, spiramycin, fidaxomicin, rifampin, ethambutol, streptomycin, or anthramycin.

11. 11. The method of any one of claims 2 to 10, further comprising contacting the biofilm with an effective amount of a synthetic or recombinant polypeptide comprising, consisting essentially of, or even consisting of mB box-97, consisting of amino acids 90 to 176 from the coding sequence of the native human HMGB1 protein shown as SEQ ID NO: 45, with a point mutation from cysteine ​​to serine at amino acid 106, or an equivalent thereof.

12. 11. The method of any one of claims 2 to 10, further comprising the step of contacting the biofilm with an effective amount of a synthetic or recombinant polypeptide comprising, consisting essentially of, or even consisting of mB box-97, consisting of amino acids 80 to 176 from the coding sequence of the native human HMGB1 protein as set forth as SEQ ID NO: 45, with a point mutation from cysteine ​​to serine at amino acid 106, or an equivalent thereof.

13. 13. The method of claim 11 or claim 12, wherein the synthetic or recombinant polypeptide consists of SEQ ID NO:

48.

14. The method of any one of claims 2 to 13, further comprising contacting the biofilm with an effective amount of a DNA-binding agent.

15. The method of claim 14, wherein the DNA binding agent comprises a histone-like nucleoid structuring protein (H-NS), a polyamine, or a polycation.

16. 16. The method of claim 15, wherein 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: 51-56.

17. 11. The method of any one of claims 1 and 3-10, further comprising administering to the subject an effective amount of at least one antibiotic selected from amikacin, azithromycin, dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, methicillin, vancomycin, daptomycin, mupirocin, penicillin, cloxacillin, erythromycin, clarithromycin, roxithromycin, telithromycin, spiramycin, fidaxomicin, or anthramycin.

18. 18. The method of any one of claims 1, 3 to 10 and 17, further comprising the step of administering to the subject an effective amount of a synthetic or recombinant polypeptide comprising, consisting essentially of, or even consisting of, mB box-97 consisting of amino acids 90 to 176 from the coding sequence of native human HMGB1 protein as set forth as SEQ ID NO: 45, with a point mutation from cysteine ​​to serine at amino acid 106, or an equivalent thereof.

19. 18. The method of any one of claims 1, 3 to 10 and 17, further comprising the step of administering to the subject an effective amount of a synthetic or recombinant polypeptide comprising, consisting essentially of, or even consisting of, mB box-97 consisting of amino acids 80 to 176 from the coding sequence of native human HMGB1 protein as set forth as SEQ ID NO: 45, with a point mutation from cysteine ​​to serine at amino acid 106, or an equivalent thereof.

20. 20. The method of claim 18 or claim 19, wherein the synthetic or recombinant polypeptide consists of SEQ ID NO:

48.

21. 21. The method of any one of claims 1, 3-10 and 17-20, further comprising contacting the biofilm with an effective amount of a DNA-binding agent.

22. 22. The method of claim 21, wherein the DNA binding agent comprises a histone-like nucleoid structuring protein (H-NS), a polyamine, or a polycation.

23. 23. The method of claim 22, wherein 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: 51-56.

24. 24. The method of any one of claims 1 to 23, wherein the NTM species is M. abscessus (Mab), M. avium (Mav), M. intracellulare, or M. chimaera.

25. 24. The method of any of claims 1, 3-10 and 17-23, wherein the administration comprises one or more of oral administration, nasal administration, injection and / or topical application.

26. 27. The method of any one of claims 1, 3-10 and 17-26, wherein the subject has chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia or cystic fibrosis (CF).

27. The method of any one of claims 2 to 16 and 24, wherein the contacting step is carried out in vitro or in vivo.

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