Compositions and methods for disrupting bacterial biofilms without associated inflammation

Abstract

To provide compositions and methods for disruption of bacterial biofilms without accompanying inflammation.SOLUTION: Methods of disrupting a biofilm or inhibiting, preventing or treating a microbial infection that produces a biofilm are disclosed, which involve administration of a polypeptide that has one or more modified HMG-box 1 domains to a subject suffering from the infection or having the biofilm. By competing with microbial proteins that bind to a DNA scaffold in the biofilm, these polypeptides destabilize the biofilm thereby disrupting and removing the biofilm by the immune system.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-references to related applications This application asserts the interest under Section 119(e) of the United States Patent Act in relation to U.S. Provisional Application No. 62 / 471,834, filed on 15 March 2017, the contents of which are incorporated herein by reference in their entirety. background

[0002] Bacteria residing in biofilms throughout the human body are responsible for approximately two-thirds of all chronic / recurrent diseases. These biofilms are often composed of bacteria protected from gaining access to the bacteria inside the biofilm by an outer "slime" consisting mainly of DNA, antibiotics, and other antibacterial agents that interfere with the innate and adaptive immune systems. Biofilms make it extremely difficult to clear infections from the body. Furthermore, biofilms can act as reservoirs for acute infections, often with fatal outcomes.

[0003] At least one protein derived from the DNABII protein family is found in all known Eubacteria and is naturally present outside bacterial cells. While these proteins induce a strong innate immune response, the host subject cannot naturally produce specific antibodies against family members as a result of infection. A major challenge associated with bacterial biofilms is that the host immune system and / or antibiotics and / or other antimicrobial substances cannot gain access to the bacteria protected within the biofilm.

[0004] Biofilms are also present in industrial environments. For example, biofilms affect a wide range of petroleum process challenges, from production sites to gas station storage tanks. At the production site, sulfate-reducing biofilm bacteria produce hydrogen sulfide (sour oil). In process pipelines, biofilm activity generates slime that clogs filters and openings. Biofilms and biofilm organisms also cause corrosion in pipelines and petroleum process equipment. These challenges can range from the entire oil or gas production facility to the point where adhesive and corrosive biofilm organisms are found on the surfaces of end-product storage tanks.

[0005] In homes, biofilms can be found in or on any surface that supports microbial growth (e.g., in drainpipes, on surfaces where food is prepared, in toilets, and in swimming pools and spas).

[0006] Biofilms affect a wide range of water treatment applications (both domestic and industrial). They can grow on the surfaces of equipment, interfering with its performance (e.g., reduced heat transfer or clogging of filters and membranes). Biofilms growing on cooling tower packing can add enough weight to cause the packing to collapse. Biofilms can corrode even highly specialized stainless steel. Biofilms in water treatment can reduce the value of the final product. Biofilms growing in drinking water distribution systems can become a habitat for potentially pathogenic organisms, scourges, or bacteria, degrading the aesthetic quality of the water.

[0007] Therefore, there is a need to break down the protective barrier of the biofilm, treat or kill the associated bacterial infection, and remove them from the surface and water system. The present invention satisfies this need and also provides related advantages. [Overview of the project] [Means for solving the problem]

[0008] Abstract HMGB1 (eukyotic protein) was discovered 40 years ago as a non-histone chromatin protein (Goodwin et al., 1973). Due to its ability to bind to DNA, HMGB1 is a ubiquitously expressed nuclear protein that functions as a component in the entire domain of nuclear protein interactions, including DNA replication, repair, and gene regulation. It also facilitates protein-protein interactions in various cancers, autophagy (Tang et al., 2011), and non-standard secretory pathways (Lee et al., 2010). Although HMGB1 is primarily found in the nucleus, it can be secreted and / or released into the extracellular space by several cell types (including activated immune cells (macrophages, monocytes, neutrophils, dendritic cells, and natural killer cells), epithelial cells, and fibroblasts) (Yang et al., 2007). Extracellular HMGB1 exhibits cytokine induction, chemokine-like, and pro-angiogenic functions (Melloni et al. 1995a; 1995b; Pistoia and Raffaghello 2011) (Ranzato et al. 2009) (Abraham et al. 2000; Agnello et al. 2002) (Mardente et al. 2012) (Wang et al. 1999). It is also involved in bacterial killing (Gong et al. 2009; Zetterstrom et al. 2006) and cellular senescence (Davalos et al. 2013). HMGB1 associates with neutrophil extracellular traps (NETs) released by neutrophils to combat microbial infections.

[0009] Antibiotics are the first-line treatment for bacterial infections. Biofilms are essential components of approximately 80% of all bacterial infections and strongly influence the chronic and recurrent nature of infectious diseases. Furthermore, bacteria within biofilms can exhibit antibiotic resistance up to 1000 times or more. The chronic and recurrent nature of biofilm-mediated bacterial infections necessitates the overuse of antibiotics, which in turn leads to the inevitable emergence of bacteria resistant to numerous antibiotics. This perplexing cascading damage resulting from the overuse of antibiotics leads to failure of antibiotic therapy and difficulty / inability to treat infections. Another significant side effect of antibiotics is their negative impact on the commensal microbiome, which can lead to secondary infections.

[0010] HMGB1 has two tandem DNA-binding domains (A and B boxes) and a highly acidic C-terminal tail. HMGB1 functions as a monomer, using its two tandem DNA-binding domains to bind to the DNA minor groove and bend the DNA. HMGB1 also has a C-terminal domain consisting of approximately 30 acidic amino acid residues that have antimicrobial activity against free-floating bacteria (Gong et al. 2009). The applicant has previously shown that DNABII proteins, which bind to and bend DNA, are essential for the structure of biofilms formed by numerous human pathogens (Brandstetter et al. 2013; Brockson et al. 2014; Devaraj et al. 2015; Freire et al. 2016; Goodman et al. 2011; Gustave et al. 2013; Idicula et al. 2016; Justice et al. 2012; Novotny et al. 2013a; Novotny et al. 2016; Rocco et al. 2016). HMGB1 does not share sequence or structural homology with DNABII proteins, but has been shown to replace DNABII proteins in vitro for functional purposes in some bacteria (Paull et al. 1993; Segall et al. 1994). The applicant demonstrated that DNABII proteins stabilize the eDNA-dependent extracellular matrix of biofilms, while recombinant HMGB1 (rHMGB1) disrupts pre-formed bacterial biofilms in vitro (Table 1), clears NTHI biofilms from the middle ear of chinchillas in an experimental model of otitis media (OM) (Figures 1A-1B), and inhibits Burkholderia cenocepacia biofilm formation in mouse airways (Figures 2A-2D), directly resulting in a reduced bacterial load.

[0011] While rHMGB1 has been shown to be effective in disrupting biofilms in vitro and in vivo, it has also been shown in detail to induce a strong inflammatory response that may potentially be harmful to the host. Several post-translational modifications (PTMs), including acetylation, phosphorylation, methylation, glycosylation, ADP-ribosylation, and oxidation of cysteine ​​residues, have been described as modulating the location (nuclear, cytoplasmic, or extracellular), function, and ability to bind to DNA of HMGB1 (review (Kang et al. 2014)). Human HMGB1 contains three cysteine ​​residues at positions 23, 45, and 106, and oxidation of these cysteine ​​residues affects its inflammatory properties (Kazama et al. 2008). HMGB1 containing a C106 thiol group and a C23-C45 disulfide bond triggers inflammation, while terminally oxidized cysteine ​​promotes the resolution of inflammation (Yang et al. 2012).

[0012] The aspects disclosed herein include modified high mobility, which involve substitutions of one or more or all of the following groups: C23S, C45S, and C106S. The present invention relates to a group-box 1 domain, an isolated or recombinant polynucleotide encoding the modified high mobility group-box 1 domain, a vector containing the isolated or recombinant polynucleotide, and a composition containing an effective amount of the modified high mobility group-box 1 domain and / or the aforementioned isolated or recombinant polynucleotide and / or the aforementioned vector. In some embodiments, the modified high mobility group-box 1 domain includes the substituted C45S. In some embodiments, the modified high mobility group-box 1 domain includes the substituted C23S, C45S, and C106S.

[0013] The applicant hereby indicates that C45S recombinant HMGB1 or modified HMGB1(C45S) ("mHMGB1(C45S)") disrupts existing biofilms both in vitro and in vivo (in two mammalian models). Dispersing large quantities of bacteria from biofilm to a suspension state in vivo may have a potential drawback in that these suspension bacteria may gain access to other sites that they did not have access to before potentially causing a secondary site infection. This dispersion effect may also lead to sepsis. None of these outcomes have been observed with mHMGB1, and in addition to the fact that these potential pitfalls may be debatable depending on the site of biofilm infection, there are means to circumvent these potential limitations.

[0014] A further aspect relates to a method for inhibiting, competing for, or titrating the binding of deoxyribonucleic acid B II (DNABII) polypeptides to microbial DNA in a biofilm, wherein the method comprises contacting the microbial DNA in the biofilm with one or more isolated polypeptides or recombinant polypeptides or compositions disclosed above, each comprising an effective amount of a modified high mobility group-box 1 domain containing one or more substitutions selected from the group C23S, C45S, and C106S, thereby inhibiting, competing for, or titrating the binding of DNABII polypeptides to the microbial DNA, or comprising essentially the same or even more than the same. In some embodiments, the modified high mobility group-box 1 domain contains the substitution C45S. In some embodiments, the modified high mobility group-box 1 domain contains one or more, or all, of the substitutions C23S, C45S, and C106S. In some embodiments, the contacting step is performed in vitro or in vivo. In some embodiments, the biofilm is formed by Burkholderia cenocepacia, Enterobacter spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterococcus faecium, urinary tract pathogenic Escherichia coli (UPEC), non-encapsulated Haemophilus influenzae (NTHI), and Aggregatibacter actinomycetemcomitans. In some embodiments, the method comprises the steps of administering an effective amount of antibiotic to a subject in need, further comprising, or otherwise essentially consisting of, or still comprising, the steps of coating the subject as needed.

[0015] Further aspects relate to methods for preventing the formation of biofilms on surfaces, the methods comprising the steps of contacting the surface with one or more isolated polypeptides or recombinant polypeptides or compositions disclosed above, which include an effective amount of a modified high mobility group-box 1 domain containing one or more substitutions selected from the group C23S, C45S, and C106S, and optionally coating the surface, thereby inhibiting, competing for, or titrating the binding of the DNABII polypeptide to microbial DNA during biofilm formation, or essentially consisting of the above steps instead. In some embodiments, the modified high mobility group-box 1 domain contains the substitution C45S. In some embodiments, the modified high mobility group-box 1 domain contains the substitutions C23S, C45S, and C106S. In some embodiments, the biofilm is formed by Burkholderia cenocepacia, Enterobacter spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterococcus faecium, urinary tract pathogenic Escherichia coli (UPEC), non-encapsulated Haemophilus influenzae (NTHI), and Aggregatibacter actinomycetemcomitans. In some embodiments, the method comprises the step of administering an effective amount of antibiotic to the surface, further comprising, or optionally, a coating step, or instead essentially consisting of, or still comprising, the above step.

[0016] A further aspect relates to a method for preventing or treating microbial infection in a subject that generates a biofilm, the method comprising administering an effective amount of an isolated polypeptide or recombinant polypeptide comprising a modified high mobility group-box 1 domain comprising one or more substitutions selected from the group consisting of C23S, C45S, and C106S, or any one or more of the compositions disclosed above, whereby inhibiting, competing with, or titrating the binding of a DNABII polypeptide to microbial DNA, or alternatively consisting essentially of or still further consisting of the above steps. In some embodiments, the modified high mobility group-box 1 domain comprises the substitution C45S. In some embodiments, the modified high mobility group-box 1 domain comprises the modifications C23S, C45S, and C106S. In some embodiments, the biofilm is formed by Burkholderia cenocepacia, Enterobacter spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterococcus faecium, uropathogenic Escherichia coli (UPEC), non-encapsulated Haemophilus influenzae (NTHI), Aggregatibacter actinomycetemcomitans. In some embodiments, the method further comprises administering an effective amount of an antibiotic, or alternatively consisting essentially of or still further consisting of the above steps.

[0017] It is recognized that among the equivalents to the modified high mobility group-box 1 domain are modified high mobility group-box 2, modified high mobility group-box 3, and modified high mobility group-box 4, which include cysteine-to-serine substitutions at one or more amino acid positions corresponding to C23S, C45S, and C106S of modified high mobility group box 1. For modified high mobility group-box 2, its corresponding cysteine ​​residues are found at positions 23, 45, and 106; modified high mobility With respect to group-box 3, the corresponding cysteine ​​residues are found at positions 23, 45, and 104; and with respect to modified high mobility group-box 4, the corresponding cysteine ​​residues are found at positions 45, 104, 164, and 178. Accordingly, the applicant states that the aforementioned aspect disclosed with respect to modified high mobility group-box 1, which includes one or more substitutions selected from the group C23S, C45S, and C106S, is equivalent to the aforementioned high mobility group-box species (e.g., modified high mobility group-box 1, which includes one or more substitutions selected from the group C23S, C45S, and C106S) having one or more cysteine-to-serine substitutions at the position mentioned. We consider this to be equally applicable to modified high mobility group-box 3, which includes one or more substitutions selected from the group C23S, C45S, and C104S, as well as modified high mobility group-box 4, which includes one or more substitutions selected from the group C45S, C104S, C164S, and C178S. In certain embodiments, for example, the following items are provided: (Item 1) A modified high mobility group-box 1 domain comprising one or more substitutions selected from the group consisting of C23S, C45S, and C106S. (Item 2) The modified high mobility group-box 1 domain according to item 1, wherein the modified high mobility group-box 1 domain comprises the substitution C45S. (Item 3) The modified high mobility group-box 1 domain according to item 1, wherein the modified high mobility group-box 1 domain comprises the substitutions C23S, C45S, and C106S. (Item 4) An isolated polynucleotide or recombinant polynucleotide encoding the modified high mobility group-box 1 domain according to any one of items 1 to 3. (Item 5) A vector comprising the isolated polynucleotide or recombinant polynucleotide according to item 4. (Item 6) A composition comprising an effective amount of the modified high mobility group-box 1 domain according to any one of items 1 to 3 and / or the isolated polynucleotide or recombinant polynucleotide according to item 4 and / or the vector according to item 5. (Item 7) A method for inhibiting, competing with, or titrating the binding of a deoxyribonucleic acid B II (DNABII) polypeptide to microbial DNA in a biofilm, the method comprising contacting the microbial DNA in the biofilm with an isolated polypeptide or recombinant polypeptide comprising an effective amount of a modified high mobility group-box 1 domain comprising one or more substitutions selected from the group consisting of C23S, C45S, and C106S, thereby inhibiting, competing with, or titrating the binding of the DNABII polypeptide to the microbial DNA. (Item 8) A modified high mobility group-box 1 domain is the method described in item 7, including the substitution C45S. (Item 9) The modified high mobility group-box 1 domain is the method described in item 7, comprising the substitutions C23S, C45S, and C106S. (Item 10) The method according to any one of items 7 to 9, wherein the contact step is performed in vitro or in vivo. (Item 11) The biofilm is formed by Burkholderia cenocepacia, Enterobacter spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterococcus faecium, urinary tract pathogenic Escherichia coli (UPEC), non-encapsulated Haemophilus influenzae (NTHI), and Aggregatibacter actinomycetemcomitans, as described in any one of items 7 to 10. (Item 12) The method according to any one of items 7 to 11, further comprising the step of administering an effective amount of antibiotic to a surface, and optionally coating it. (Item 13) A method for preventing the formation of a biofilm on a surface, the method comprising contacting the surface with an effective amount of an isolated polypeptide or recombinant polypeptide comprising one or more modified high-mobility group-box 1 domains selected from the group C23S, C45S, and C106S, and optionally coating the surface, thereby inhibiting, competing for, or measuring the titer of the binding of the DNABII polypeptide to microbial DNA exuded during biofilm formation. (Item 14) A modified high mobility group-box 1 domain is the method described in item 13, including the substitution C45S. (Item 15) The modified high mobility group-box 1 domain is the method described in item 13, which includes the substitutions C23S, C45S, and C106S. (Item 16) The method according to any one of items 13 to 15, wherein the contact step is performed in vitro or in vivo. (Item 17) The biofilm is formed by Burkholderia cenocepacia, Enterobacter spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterococcus faecium, urinary tract pathogenic Escherichia coli (UPEC), non-encapsulated Haemophilus influenzae (NTHI), and Aggregatibacter actinomycetemcomitans, as described in any one of items 13 to 16. (Item 18) The method according to any one of items 13 to 17, further comprising the step of administering an effective amount of antibiotic to the surface, and optionally coating the surface. (Item 19) A method for preventing or treating microbial infection in a subject that forms a biofilm, the method comprising administering an effective amount of an isolated polypeptide or recombinant polypeptide comprising a modified high-mobility group-box 1 domain containing one or more substitutions selected from the group C23S, C45S, and C106S, thereby inhibiting, competing for, or measuring the titer of the binding of the DNABII polypeptide to microbial DNA. (Item 20) A modified high mobility group-box 1 domain is the method described in item 19, including the substitution C45S. (Item 21) The modified high mobility group-box 1 domain is the method described in item 19, which includes the substitutions C23S, C45S, and C106S. (Item 22) The biofilm is formed by Burkholderia cenocepacia, Enterobacter spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterococcus faecium, urinary tract pathogenic Escherichia coli (UPEC), non-encapsulated Haemophilus influenzae (NTHI), and Aggregatibacter actinomycetemcomitans, as described in any one of items 19 to 21. (Item 23) The method according to any one of items 19 to 22, further comprising the step of administering an effective amount of antibiotic to the surface, and optionally coating the surface. [Brief explanation of the drawing]

[0018] [Figure 1] Figures 1A–1B show that rHMGB1 and mHMGB1(C45S) promote bacterial clearance in an established experimental OM animal model. Diluents, 5 μg rHMGB1, or 5 μg mHMGB1(C45S) were directly delivered to the middle ear of chinchillas 4 and 5 days after infection with NTHI. Animals were sacrificed 24 hours later, and their middle ears were imaged (Figure 1A) and scored blindly based on the criteria described below Figure 1A (Figure 1B). Bars represent SEM. ***P<0.001. The imaging and scoring show that HMGB1 was able to clear pre-formed NTHI biofilms in vivo.

[0019] [Figure 2-1] Figures 2A–2D show that mHMGB1(C45S) promotes bacterial clearance and does not increase the inflammatory response in the airways of mice infected with B. cenocepacia. C57BL / 6 mice were infected with 107 CFU(it) and simultaneously administered rHMGB1 or mHMGB1(C45S). After 18 hours, (Figure 2A) B. cenocepacia CFU was quantified in the BAL, (Figure 2B) total host cells infiltrating into the lung were counted in the BAL, and (Figure 2C) neutrophil migration was measured by staining BAL cells with anti-CD45, CD11b, and Ly-6G. Bars represent standard deviation. *P<0.05;**P<0.005. (Figure 2D) Lungs were collected at 72 hpi. Tissue was fixed, embedded in paraffin, sectioned, and stained with H&E. All panels show 10× magnification. Note that mHMGB1(C45S) treatment resulted in a significant reduction in B. cenocepacia CFU and in vivo inflammatory cell infiltration compared to rHMGB1. [Figure 2-2] Figures 2A–2D show that mHMGB1(C45S) promotes bacterial clearance and does not increase the inflammatory response in the airways of mice infected with B. cenocepacia. C57BL / 6 mice were infected with 107 CFU(it) and simultaneously administered rHMGB1 or mHMGB1(C45S). After 18 hours, (Figure 2A) B. cenocepacia CFU was quantified in the BAL, (Figure 2B) total host cells infiltrating into the lung were counted in the BAL, and (Figure 2C) neutrophil migration was measured by staining BAL cells with anti-CD45, CD11b, and Ly-6G. Bars represent standard deviation. *P<0.05;**P<0.005. (Figure 2D) Lungs were collected at 72 hpi. Tissue was fixed, embedded in paraffin, sectioned, and stained with H&E. All panels show 10× magnification. Note that mHMGB1(C45S) treatment resulted in a significant reduction in B. cenocepacia CFU and in vivo inflammatory cell infiltration compared to rHMGB1.

[0020] [Figure 3]Figure 3 shows that mHMGB1(C45S) does not promote neutrophil migration in the peritoneal cavity. C57BL / 6 mice were injected into the peritoneal cavity with 4% thioglycolate, rHMGB1, or mHMGB1(C45S). Cells in the peritoneal exudate were stained with anti-CD45, CD11b, and Ly-6G to determine the total neutrophil count in the peritoneal cavity (PC). Bars represent SEM. *P<0.05. Note that mHMGB1(C45S) lacks the inflammatory activity of rHMGB1.

[0021] [Figure 4] Figure 4 shows the antibiofilm effect of HMGB1 variants against bacterial pathogens. The HMGB1 isoforms shown (200 nM) were added to bacterial biofilms formed in vitro over 24 hours. Sixteen hours after inoculation, the biofilms were stained with LIVE / DEAD® and then visualized via CLSM. The images were analyzed by Comstat to calculate the average thickness and biomass. A representative image of UPEC is shown in Figure 4A. The percentage change in average thickness compared to the control is shown in Figure 4C. 800 nM rHMGB1 and 200 nM mHMGB1 (C45S) were used to treat S. aureus (ESKAPE) biofilms. 800 nM rHMGB1 and mHMGB1 (C45S) were used to treat E. faecium (ESKAPE) biofilms. These were incubated with their respective proteins for 1 hour, in contrast to the 16-hour incubation. Biomass (not shown) showed the same trend. Bars represent SEM. Statistical significance compared to the control was evaluated using an unpaired t-test (*P<0.05). HMGB1 and its variants were able to significantly disrupt established biofilms.

[0022] [Figure 5-1]Figure 5 shows that mHMGB1(C45S) enhances antibiotic-mediated killing. B. cenocepacia biofilms were pre-formed over 24 hours and then incubated for 16 hours with minocycline (1 mg / ml), mHMGB1(C45S) (200 nM), or mHMGB1(C45S) (200 nM) + minocycline (1 mg / ml). The biofilms were stained with LIVE / DEAD® and imaged via CLSM. Live cells are shown in the upper half of each image, and dead cells are shown in the lower half of each image. Note that dead cells were increased only in the presence of both mHMGB1(C45S) and minocycline. [Figure 5-2] Figure 5 shows that mHMGB1(C45S) enhances antibiotic-mediated killing. B. cenocepacia biofilms were pre-formed over 24 hours and then incubated for 16 hours with minocycline (1 mg / ml), mHMGB1(C45S) (200 nM), or mHMGB1(C45S) (200 nM) + minocycline (1 mg / ml). The biofilms were stained with LIVE / DEAD® and imaged via CLSM. Live cells are shown in the upper half of each image, and dead cells are shown in the lower half of each image. Note that dead cells were increased only in the presence of both mHMGB1(C45S) and minocycline.

[0023] [Figure 6] Figure 6 shows that rHMGB1 and mHMGB1(C45S) promote biofilm dissipation in an experimental animal model of omniostomy. Diluents or 5 μg of rHMGB1 or mHMGB1(C45S) were directly delivered to the middle ear of chinchillas 4 and 5 days after infection with NTHI. Animals were sacrificed 24 hours later, and their middle ears were imaged (Figure 6A) and blindly scored based on the criteria described below (Figure 6A) (Figure 6B). Bars represent SEM. ***P<0.001. Imaging and scoring show that HMGB1 promoted the clearance of pre-formed NTHI biofilms in vivo.

[0024] [Figure 7] Figure 7 shows that mHMGB1(C45S) promotes bacterial clearance but does not increase airway inflammation in mice infected with B. cenocepacia. C57BL / 6 mice were infected with 107 CFU(it) and simultaneously administered 5 mg rHMGB1 or mHMGB1(C45S). (Figure 7A) B. cenocepacia aggregates were observed by fluorescence microscopy in sections probed with α-B. cenocepacia antibody. After 18 hours, (Figure 7B) CFU was quantified in BAL cells, and (Figure 7C) BAL cells were stained with α-CD45, CD11b, and Ly-6G to measure neutrophil migration. Bars represent SD. *P<0.05. (Figure 7D) Lung tissue collected at 72 hpi was fixed, embedded in paraffin, sectioned, and stained with H&E. 10× magnification. mHMGB1(C45S) treatment, compared to rHMGB1, resulted in a significant reduction in B. cenocepacia CFU and inflammatory cell infiltration in vivo. [Modes for carrying out the invention]

[0025] Detailed explanation definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any materials and methods similar or equivalent to those described herein may be used in the practice or testing of the present invention, but preferred methods, devices, and materials are described herein. All technical publications and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein should be construed as an acknowledgment that the present invention does not have prior art rights prior to such disclosure. For example, Green and Sambrook (eds.) (2012) Molecular Cloning: A Laboratory Manual, 4th edition; Ausubel et al. (eds.) (2015) Current Protocols in Molecular Biology; Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (2015) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; McPherson et al. (2006) PCR: The Basics (Garland Science); edited by Harlow and Lane (1999) Antibodies, A Laboratory Manual; Edited by Greenfield (2014) Antibodies, A Laboratory Manual; Freshney (2010) Culture of Animal Cells: A Manual of Basic Technique, 6th edition; edited by Gait (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; edited by Hames and Higgins (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; edited by Herdewijn (2005) Oligonucleotide Synthesis: Methods and Applications; edited by Hames and Higgins (1984) Transcription and Translation; edited by Buzdin and Lukyanov (2007) Nucleic Acids Hybridization: Modern Applications; Immobilized Cells and Enzymes (IRL Press (1986)); edited by Grandi (2007) In Vitro Transcription and Translation Protocols, 2nd edition; edited by Guisan (2006) Immobilization of Enzymes and Cells; Perbal (1988) A Practical Guide to Molecular Cloning, 2nd edition; edited by Miller and Calos (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor See also: Laboratory; Makrides (eds.) (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker (eds.) (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Lundblad and Macdonald (eds.) (2010) Handbook of Biochemistry and Molecular Biology, 4th edition; and Herzenberg et al. (eds.) (1996) Weir's Handbook of Experimental Immunology, 5th edition; and the latest editions of each of these available at the time of filing.

[0026] All numerical specifications (e.g., pH, temperature, time, concentration, and molecular weight (including ranges)) are approximations that vary by increments of 1.0 or 0.1 in (+) or (-) where appropriate, or by ±15%, or 10%, or 5%, or 2% instead. It should be understood that, although not always explicitly stated, all numerical specifications are preceded by the term "about." It should also be understood that, although not always explicitly stated, the reagents described herein are illustrative and equivalents are known in the art.

[0027] As used herein and in the claims, the singular forms “a,” “an,” and “the” include a plural form unless the context clearly indicates otherwise. For example, the term “a polypeptide” includes multiple polypeptides, including mixtures thereof.

[0028] As used herein, the term “comprising” is intended to mean including the elements of the composition and method described, but not excluding others. “Consisting essentially of” as used to define a composition and method means excluding any other elements of essential importance to the combination relating to its intended use. Thus, a composition consisting essentially of the elements as defined herein does not exclude trace amounts of impurities and pharmaceutically acceptable carriers (e.g., phosphate-buffered saline, preservatives) derived from isolation and purification methods. “Consisting of” means excluding trace amounts of other components and steps beyond substantial steps of the method for administering the composition of the present invention. Embodiments defined by each of these transitional clauses are within the scope of the present invention.

[0029] A "biofilm" refers to a thin layer or organized community of microorganisms that, along with polymers they secrete and / or release (e.g., DNA), can sometimes adhere to the surface of structures (which can be organic or inorganic). These biofilms are highly resistant to microbiotics and antimicrobial agents. They reside on gingival tissue, teeth, and restorations, causing dental caries and periodontal disease (also known as periodontal plaque disease). They also cause chronic middle ear infections. Biofilms can also form on the surfaces of dental implants, stents, catheter lines, and contact lenses. They can grow on pacemakers, heart valve replacements, artificial joints, and other surgical implants. The U.S. Centers for Disease Control and Prevention estimates that more than 65% of 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, patients with cystic fibrosis often have Pseudomonas infections that lead to antibiotic-resistant biofilms.

[0030] A "DNABII polypeptide or protein" refers to a DNA-binding protein or polypeptide composed of a DNA-binding domain and therefore possessing specific or general affinity for DNA. In one aspect, they bind to DNA in the minor groove. Non-exclusive examples of DNABII proteins include integrated host factor (IHF) proteins and histone-like proteins (HU) derived from E. coli strain U93. Other DNA-binding proteins that may be associated with biofilms include DPS (Genbank accession number: CAA49169), H-NS (Genbank accession number: CAA47740), Hfq (Genbank accession number: ACE63256), CbpA (Genbank accession number: BAA03950), and CbpB (Genbank accession number: NP_418813).

[0031] Integration host factor (IHF) proteins are bacterial proteins used by bacteriophages to integrate their DNA into host bacteria. These are DNA-binding proteins that function in genetic recombination, as well as in transcription and translation regulation. They also bind to extracellular microbial DNA. The genes encoding IHF protein subunits in E. coli are the himA gene (Genbank accession number: POA6X7.1) and the himD gene (POA6Y1.1).

[0032] "HU," or "histone-like protein from E. coli strain U93," refers to a class of heterodimer 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 HU proteins are commercially available from Abcam.

[0033] "Microbial DNA" refers to single-stranded or double-stranded DNA derived from microorganisms that produce biofilms.

[0034] To inhibit, prevent, or destroy biofilms "Preventing or breaking down" refers to the intention of preventing or therapeutically reducing the structure of a biofilm. In one context, the term "inhibiting, competing, or titrating" refers to the intention of reducing the formation of the DNA / protein matrix, which is a component of a microbial biofilm (for example, as shown in Figure 1).

[0035] "Bent polynucleotide" refers to any double-stranded polynucleotide that contains a small loop on one strand which does not pair with the other strand, and any polynucleotide whose end-to-end distance is shorter than natural thermal fluctuations, i.e., bent beyond a duration of 150 bp with respect to native B-type double-stranded DNA. In some embodiments, the loop is 1 nucleotide to about 20 nucleotides long, or instead 2 nucleotides to about 15 nucleotides long, or instead about 3 nucleotides to about 12 nucleotides long, or instead about 4 nucleotides to about 10 nucleotides long, or instead about 4 nucleotides, 5 nucleotides, or 6 nucleotides, or 7 nucleotides, or 8 nucleotides, or 9 nucleotides, or 10 nucleotides long.

[0036] The “subject” for diagnosis or treatment is a cell or an animal (e.g., a mammal or a human). Non-human animals undergoing diagnosis or treatment are those that are infected or animal models (e.g., monkeys, rodents (e.g., rats, mice), chinchillas, canids (e.g., dogs), leporids (e.g., rabbits), livestock, sports animals, and pets).

[0037] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably and, in their broadest sense, refer to compounds of two or more subunit amino acids, amino acid analogs, or peptide mimics. These subunits may be linked by peptide bonds. In another embodiment, these subunits may be linked by other bonds (e.g., esters, ethers, etc.). A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may constitute a protein or peptide sequence. As used herein, the term “amino acid” may refer to any amino acid, whether natural and / or unnatural or synthetic (including glycine and both D and L optical isomers, amino acid analogs, and peptide mimics). The 20 amino acids naturally found in the human body are shown in the following table, along with their respective three-letter abbreviations, one-letter abbreviations, structures, and corresponding codons: [Table A-1] [Table A-2]

[0038] The terms "polynucleotide" and "oligonucleotide" refer to polymeric forms of nucleotides (deoxyribonucleotides or ribonucleotides, or analogs thereof) of any length that are interchangeable. Polynucleotides can have any three-dimensional structure and can perform any function, whether known or not. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides may include modified nucleotides (e.g., methylated nucleotides and nucleotide analogs). Modifications to the nucleotide structure, if present, may be conferred before or after the assembly of the polynucleotide. The sequence of a nucleotide may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization (for example, by conjugation with labeling components). The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or requested, any embodiment of the present invention that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or presumed to constitute that double-stranded form.

[0039] When used herein in relation to nucleic acids (e.g., DNA or RNA), the terms “isolated” and “recombinant” refer to molecules that are isolated from other DNA or RNA present in the natural sources of macromolecules and polypeptides, respectively. The term “isolated or recombinant nucleic acid” means that it includes nucleic acid fragments that do not exist naturally as fragments and are not found in their natural state. The term “isolated” is also used herein to refer to polynucleotides, polypeptides and proteins isolated from other cellular proteins, and means that it includes both purified polypeptides and recombinant polypeptides. In other embodiments, the terms “isolated or recombinant” mean that cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies or their fragments are isolated from components, cells and other things that are naturally and normally associated with them. For example, isolated cells are cells isolated from tissues or cells with dissimilar phenotypes or genotypes. Isolated polynucleotides are separated from the 3' and 5' consecutive nucleotides that are normally associated on chromosomes, for example, in their native or natural environment. As will be apparent to those skilled in the art, polynucleotides, peptides, polypeptides, proteins, antibodies or their fragments that do not exist in nature do not require "isolation" to distinguish them from their naturally occurring counterparts.

[0040] The terms “equivalent” or “biological equivalent” are used interchangeably when referring to specific molecules, biological or cellular substances, intended to have minimal homology while still maintaining the desired structure or functionality.

[0041] Unless otherwise explicitly stated and intended, if the invention relates to a polypeptide, protein, polynucleotide, or antibody, it should be inferred that equivalents or bioequivalents thereof are intended to be within the scope of the invention. As used herein, the terms “biolgically equivalent thereof” are intended to be synonymous with “equivalent thereof” when referring to a reference protein, antibody, polypeptide, or nucleic acid, and are intended to have minimal homology while still maintaining the desired structure or functionality. Unless specifically stated herein, any polynucleotide, polypeptide, or protein referred herein is also intended to include its equivalent. For example, an equivalent may be at least about 70% Homology or identity, or rather about 80% homology or identity, and instead at least about 85%, or instead at least about 90%, or instead at least about 95%, or instead 98%, is intended to be substantially equivalent to the biological activity of its reference protein, polypeptide, or nucleic acid. In other contexts, the term is intended to be a polynucleotide that hybridizes to a reference polynucleotide or its complement under high stringency conditions.

[0042] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" with respect to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same when comparing the two sequences. This alignment and percentage homology or sequence identity can be determined using software programs known in the art (e.g., those described in Current Protocols in Molecular Biology (Ausubel et al., 1987), Appendix 30, Section 7.7.18, Table 7.7.1). Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, which uses default parameters. Particularly preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expected value = 10; matrix = BLOSUM62; description = 50 sequences; sort by = HIGH SCORE; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. More details on these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0043] "Homology," "identity," or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homologousity can be determined by comparing the positions in each sequence that can be aligned for comparison purposes. If the positions in the sequences being compared are occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by those sequences. An "unrelated" or "non-homologous" sequence shares less than 30% identity with one of the sequences of the present invention, or instead less than 25% identity, less than 20% identity, or instead less than 10% identity.

[0044] "Homologousity," "identity," or "similarity" may also refer to two nucleic acid molecules that hybridize under stringent conditions to a reference polynucleotide or its complement.

[0045] Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonds between the bases of nucleotide residues. Hydrogen bonding can occur through Watson-Crick base pairing, Hougsteen bonds, or any other sequence-specific manner. The complex may consist of two strands forming a double helix, three or more strands forming a multihelix, one self-hybriding strand, or any combination thereof. Hybridization reactions can constitute steps in a broader range of processes (e.g., initiation of a PCR reaction or enzymatic cleavage of polynucleotides by ribosomes).

[0046] Examples of stringent hybridization conditions include: incubation temperature of approximately 25°C to 37°C; hybridization buffer concentration of approximately 6×SSC to 10×SSC; formamide concentration of approximately 0% to 25%; and washing solution of approximately 4×SSC to 8×SSC. Examples of moderate hybridization conditions include: incubation temperature of approximately 40°C to 50°C; buffer concentration of approximately 9×SSC to 2×SSC; formamide concentration of approximately 30% to 50%; and washing solution of approximately 5×SSC to 2×SSC. Examples of highly stringent conditions include: incubation temperature of approximately 55°C to 68°C; buffer concentration of approximately 1×SSC to 0.1×SSC; formamide concentration of approximately 55% to 75%; and washing solution of approximately 1×SSC, 0.1×SSC, or deionized water. Generally, hybridization incubation times range from 5 minutes to 24 hours, with one, two, or more washing steps, and washing incubation times of approximately 1 minute, 2 minutes, or 15 minutes. SSC is 0.15M The buffers are NaCl and 15 mM citrate buffer. It is understood that equivalent SSCs using other buffer systems may be used.

[0047] When applied to polynucleotides, the term “encode” refers to a polynucleotide that, in its native state or when manipulated by methods well known to those skilled in the art, is said to “encode” a polypeptide if it is transcribed and / or translated to produce mRNA for a polypeptide and / or its fragments. Its antisense strand is the complement of such nucleic acid, from which its encoding sequence can be derived.

[0048] As used herein, terms such as “treating” and “treatment” mean obtaining a desired pharmacological and / or physiological effect. This effect may be preventative in that it completely or partially prevents a disorder or its signs or symptoms, and / or therapeutic in that it partially or completely cures the disorder and / or any adverse effects attributable to it.

[0049] "Preventing" means preventing a system or subject that is susceptible to a disorder or effect, either in vitro or in vivo. An example of this is preventing the formation of a biofilm in a system infected with a microorganism known to form biofilms.

[0050] "Pharmacopoecitable carrier" refers to any diluent, excipient, or carrier that may be used in the compositions of the present invention. Examples of pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffers (e.g., phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g., 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 lanolin. Suitable pharmaceutically acceptable carriers are listed in Remington's Pharmaceutical Sciences, Mack Publishing Company (a standard reference textbook in this field). These are preferably selected in relation to the intended dosage form, i.e., oral tablets, capsules, elixirs, syrups, etc., and are consistent with conventional pharmaceutical practices.

[0051] "Administration" refers to a single dose that may be administered continuously or intermittently throughout the course of treatment. Methods and dosages for determining the most effective means are known to those skilled in the art and vary depending on the composition used in treatment, the therapeutic purpose, the target cells being treated, and the subject being treated. Single or multiple administrations may be given at dose levels and patterns selected by the treating physician. Appropriate dosage formulations and methods for administering drugs are known in the art. The route of administration may also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and vary depending on the composition used in treatment, the therapeutic purpose, the health status or disease stage of the subject being treated, and the target cells or tissues. Non-limiting examples of routes of administration include oral administration, nasal administration, injection, and topical application.

[0052] The term "effective amount" refers to a quantity sufficient to achieve a beneficial or desirable result or effect. In the context of therapeutic or prophylactic applications, the effective amount depends on the type and severity of the condition in question, as well as the characteristics of the individual subject (e.g., overall health, age, sex, weight, and tolerance to the pharmaceutical composition). In the context of immunogenic compositions, in some embodiments, the effective amount is sufficient to produce a protective response against a pathogen. In other embodiments, the effective amount of the immunogenic composition is sufficient to produce antibody production against an antigen. In some implementations, the effective amount is the amount required to confer passive immunity to a subject in need. With respect to immunogenic compositions, in some embodiments, the effective amount depends, in addition to the factors mentioned above, on the intended use, the degree of immunogenicity of the particular antigenic compound, and the health / responsiveness of the subject's immune system. A person skilled in the art can determine an appropriate amount depending on these and other factors.

[0053] In the case of in vitro application, in some embodiments, the effective amount depends on the size and nature of the application in question. It also depends on the nature and sensitivity of the in vitro target, as well as the method of use. Those skilled in the art can determine the effective amount based on these and other considerations. The effective amount may consist of one or more doses of the composition, depending on the embodiment.

[0054] Drugs and compositions may be used in the manufacture of pharmaceuticals and for the treatment of humans and other animals by administration according to conventional procedures (e.g., active ingredients in pharmaceutical compositions).

[0055] The agents of the present invention may be administered for therapeutic purposes by any suitable route of administration. It is also recognized that the preferred route may vary depending on the recipient's condition and composition, as well as the disease being treated.

[0056] Examples of solid-phase supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. The properties of the carrier may be either soluble or insoluble to some extent. The support material may have substantially any conceivable structural configuration, insofar as the molecule to be coupled can bind to a polynucleotide, polypeptide, or antibody. Thus, the structure of the support may be spherical, as in beads, or cylindrical, as in the inner surface of a test tube or the outer surface of a rod. Alternatively, its surface may be flat, such as in a sheet or test piece, or it may be a polystyrene bead instead. Those skilled in the art know of many other suitable carriers for binding antibodies or antigens, or can confirm similarity using conventional experiments.

[0057] As used herein, the terms “antibody,” “antibodies,” and “immunoglobulin” include the entire antibody and any antigen-binding fragment or a single chain thereof. Therefore, the term “antibody” includes any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule. The terms “antibody,” “antibodies,” and “immunoglobulin” also include any isotype of immunoglobulin, antibody fragments that hold specific binding to an antigen, including, but are not limited to, Fab, Fab', F(ab)2, Fv, scFv, dsFv, Fd fragment, dAb, VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and κbodies; multispecific antibody fragments formed from antibody fragments and one or more isolated ones. Examples of such components include, but are not limited to, the complementarity-determining region (CDR) or its ligand-binding portion of the heavy or light chain, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the framework (FR) region, or any portion thereof, at least one portion of a binding protein, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. The variable regions of the heavy and light chains of the immunoglobulin molecule contain binding domains that interact with the antigen. The constant region of an antibody (Ab) can mediate the binding of the immunoglobulin to host tissue. The term "anti-" when used before a protein name, e.g., anti-IHF, anti-HU, anti-OMP P5, refers to a monoclonal or polyclonal antibody that binds to and / or has affinity for a particular protein. For example, "anti-IHF" refers to an antibody that binds to the IHF protein. The specific antibody may have affinity for or bind to proteins other than the protein that evoked it.For example, anti-IHF receptors are specifically induced against IHF proteins, but they can also bind to other related proteins, either through sequence homology or structural homology.

[0058] The antibody may be a polyclonal antibody, a monoclonal antibody, a multispecific antibody (e.g., a bispecific antibody), or an antibody fragment, insofar as it exhibits the desired biological activity. The antibody can be isolated from any suitable biological source (e.g., mouse, rat, sheep, and canid).

[0059] As used herein, “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. Monoclonal antibodies are highly specific because each monoclonal antibody is directed to a single determinant on an antigen. The antibody may be labeled detectably with, for example, a radioisotope, an enzyme that produces a detectable product, or a fluorescent protein. The antibody may be further conjugated to other parts, such as members of a specific binding pair (e.g., biotin (a member of the biotin-avidin specific binding pair)). The antibody may also be bound to a solid support (such as, but not limited to, a polystyrene plate or beads).

[0060] Monoclonal antibodies can be produced using hybridoma techniques or recombinant DNA methods known in the art. Hybridomas are cells generated in the laboratory from the fusion of antibody-producing lymphocytes and non-antibody-producing cancer cells (usually myeloma or lymphoma). Hybridomas proliferate and produce serial samples of specific monoclonal antibodies. Alternative techniques for antibody production or selection include in vitro exposure of lymphocytes to the antigen of interest and screening of antibody display libraries in cells, phages, or similar systems.

[0061] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies disclosed herein may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random mutagenesis or site-directed mutagenesis, or by in vivo somatic mutation). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species (e.g., mouse) is grafted onto a human framework sequence. Therefore, as used herein, the term "human antibody" refers to substantially all parts of its protein (e.g., CDR, framework, C). L , C H Domain (for example, C H1 , C H2 , C H3 Chimeric antibodies are those that, despite having very minor sequence changes or variations, are substantially non-immunogenic in humans (VL, VH). Similarly, antibodies referring to primates (monkeys, baboons, chimpanzees, etc.), rodents (mice, rats, rabbits, guinea pigs, hamsters, etc.) and other mammals refer to antibodies specific to such species, subgenus, genus, subfamily, or family. Furthermore, chimeric antibodies include any combination of the above. Such changes or variations, as necessary, retain or reduce immunogenicity in humans or other species compared to unmodified antibodies. Thus, human antibodies are distinct from chimeric antibodies or humanized antibodies. It should be noted that human antibodies can be produced by cells of non-human animals or prokaryotes or eukaryotes capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes. Furthermore, if a human antibody is a single-chain antibody, it may contain linker peptides not found in native human antibodies. For example, Fv may contain a linker peptide (e.g., 2 to about 8 glycine or other amino acid residues) that connects the variable regions of the heavy chain and the light chain. Such a linker peptide is considered to be of human origin.

[0062] As used herein, a human antibody is "derived" from a specific germline sequence if the antibody is obtained from a system that uses human immunoglobulin sequences, for example, by immunizing transgenic mice having human immunoglobulin genes or by screening a human immunoglobulin gene library. Human antibodies "derived" from human germline immunoglobulin sequences can thus be identified by comparing the amino acid sequence of the human antibody with that of the human germline immunoglobulin. A selected human antibody typically contains amino acid residues that are at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene, and that, when compared with the germline immunoglobulin amino acid sequence of another species (e.g., mouse germline sequence), identify the human antibody as human. In certain cases, a human antibody may be at least 95%, or even more at least 96%, 97%, 98%, or 99%, identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will show no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody may exhibit five or fewer amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene, or even four or fewer, three, two, or one amino acid difference.

[0063] A "human monoclonal antibody" refers to a single-binding specific antibody that has a variable region and a constant region derived from a human germline immunoglobulin sequence. The term also refers to recombinant human antibodies. Methods for producing these antibodies are described herein.

[0064] The term “recombinant human antibody” as used herein includes all human antibodies prepared, expressed, produced or isolated by recombinant means (e.g., antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal to human immunoglobulin genes or from hybridomas prepared therefrom, antibodies isolated from host cells transformed to express from transfectomas, antibodies isolated from recombinant, combinatorial human antibody libraries, and antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences). Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, if transgenic animals for human Ig sequences are used, in vivo somatic mutagenesis), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibody may be derived from and related to human germline VH and VL sequences, but may not be naturally present in the human antibody germline repertoire in vivo. Methods for producing these antibodies are described herein.

[0065] As used herein, a chimeric antibody is an antibody in which its light chain and heavy chain genes are typically constructed from antibody variable region and constant region genes belonging to different species, through genetic engineering.

[0066] As used herein, the terms “humanized antibody” or “humanized immunoglobulin” refer to a human / non-human chimeric antibody that contains the smallest sequence derived from a non-human immunoglobulin. For the most part, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues derived from the variable region of the recipient are replaced by residues derived from the variable region (donor antibody) of a non-human species such as mouse, rat, rabbit, or non-human primate, which have the desired specificity, affinity, and ability. A humanized antibody may contain residues not found in either the recipient antibody or the donor antibody. The humanized antibody may also contain, if necessary, a non-human antibody containing one or more amino acids in the framework region, constant region, or CDR of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin, which are replaced with correspondingly located amino acids derived from the human antibody. In general, a humanized antibody is expected to produce a reduced immune response in the human host compared to a non-humanized version of the same antibody. These humanized antibodies may have conserved amino acid substitutions that do not substantially affect antigen binding or other antibody functions. Groups of conserved substitutions include: glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serine-threonine, and asparagine-glutamine.

[0067] The terms "polyclonal antibody" or "polyclonal antibody composition," as used herein, refer to preparations of antibodies derived from different B cell lines. They are mixtures of immunoglobulin molecules secreted in response to specific antigens, each recognizing a different epitope.

[0068] As used herein, the term "antibody derivative" refers to a full-length antibody or an antibody fragment, wherein one or more of its amino acids are chemically modified, such as by alkylation, pegylation, acylation, ester formation or amide formation, for example, to link the antibody to a second molecule. This includes, but is not limited to, pegylated antibodies, cysteine-pegylated antibodies, and variants thereof.

[0069] As used herein, the term "label" refers to a directly or indirectly detectable compound or composition (e.g., an N-terminal histidine tag (N-His), a magnetically active isotope (e.g., 115 Sn, 117 Sn and 119 Sn), a non-radioactive isotope (e.g., 13 C and 15N) refers to polynucleotides or proteins (e.g., antibodies for generating “labeled” compositions). The term also includes sequences that are conjugated to polynucleotides that provide a signal during the expression of an inserted sequence (e.g., green fluorescent protein (GFP)). The term “labeled” generally refers to a composition that is covalently attached to a composition to be detected, while specifically excluding naturally occurring nucleotides and amino acids known to fluoresce under certain conditions (e.g., temperature, pH, etc.), as well as any naturally occurring fluorescence that may be present in the composition to be detected. The label may be detectable on its own (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The label may be suitable for small-scale detection or for high-throughput screening. 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 that simply confirms the presence of a label, while a quantified response generally includes a response with a quantifiable (e.g., reportable numerical) value (e.g., intensity, polarization, and / or other properties). In luminescence or fluorescence assays, the detectable response may be generated by directly using a luminophore or fluorescent phenotype associated with the assay component actually involved in binding, or by indirectly using a luminophore or fluorescent phenotype associated with another component (e.g., a reporter or indicator). Examples of luminescence labels that generate a signal include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescence response generally includes a change in the luminescence signal or the generation of a luminescence signal. Suitable methods and luminophores for luminescence-labeled assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th edition).Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0070] As used herein, the term “immunoconjugate” includes an antibody or antibody derivative associated with or conjugated with a second agent (e.g., a cytotoxic agent, a detectable agent, a radioactive agent, a targeted agent, a human antibody, a humanized antibody, a chimeric antibody, a synthetic antibody, a semi-synthetic antibody, or a multispecific antibody).

[0071] Examples of suitable fluorescent labels include fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, and Cascade Blue. TM Examples include, but are not limited to, Texas Red. Other suitable optical dyes are also mentioned. (See Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals) (This is included in the 6th edition.)

[0072] In another context, the fluorescent label is functionalized to promote covalent adhesion to cellular components present on or on the surface of cells or tissues (e.g., 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 for the fluorescent label depends on the attachment site to the linker, drug, marker, or second labeled drug.

[0073] The term "eukaryotic cell" includes all life kingdoms except the Monera kingdom. They can be easily distinguished by their membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by an inner membrane and cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless otherwise specified, the term "host" includes eukaryotic hosts (e.g., yeast, higher plants, insects, and mammals). Non-limiting examples of eukaryotic cells or hosts include monkeys, cattle, pigs, mice, rats, birds, reptiles, and humans.

[0074] Prokaryotic cells, which typically lack a nucleus or any other membrane-bound organelles, are divided into two regions: 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 are characterized by three main shapes: rod-shaped, spherical, and spiral. Instead of undergoing the elaborate replication process of eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, Bacillus, E. coli, and Salmonella bacteria.

[0075] A “native” or “natural” antigen is a fragment containing a polypeptide, protein, or epitope that is isolated from a natural biological source and can specifically bind to antigen receptors, particularly T cell antigen receptors (TCRs), in a subject.

[0076] The terms “antigen” and “antigenic” refer to molecules that have the ability to be recognized by antibodies or other substances that act as members of an antibody-ligand pair. “Specific binding” refers to the interaction between an antigen and the variable regions of the heavy and light chains of immunoglobulins. Antibody-antigen binding may occur in vivo or in vitro. Those skilled in the art will understand that macromolecules (including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides, and polysaccharides) may act as antigens. Those skilled in the art will further understand that nucleic acids encoding proteins that may act as antibody ligands necessarily encode antigens. Those skilled in the art will further understand that antigens are not limited to full-length molecules but may also include partial molecules. The term “antigenic” is an adjective term referring to molecules that possess the properties of an antigen. The term encompasses substances that are immunogenic (i.e., immunogens) and substances that induce immunological non-responsiveness, or anergy (i.e., anergens).

[0077] An "altered antigen" is one that has a primary sequence different from that of its corresponding wild-type antigen. Altered antigens can be produced by synthetic or recombinant methods and include, but are not limited to, antigenic peptides that are differentially modified during or after translation by, for example, phosphorylation, glycosylation, crosslinking, acylation, proteolytic cleavage, or linkage to antibody molecules, membrane molecules, or other ligands (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320). The synthetic or altered antigens disclosed herein are intended to bind to the same TCR as the natural epitope.

[0078] In this specification, a “self-antigen,” also referred to as a native or wild-type antigen, is an antigenic peptide that, due to self-tolerance to that antigen, does little to no immune response in the subject. An example of a self-antigen is the melanoma-specific antigen gp100.

[0079] The term “passive immunity” refers to the transfer of immunity from one subject to another through the transfer of antibodies. Passive immunity can occur naturally, such as when a transfer of antibodies is transferred to a fetus. Passive immunity can also occur artificially, such as when an antibody composition is administered to a non-immune subject. Antibody donors and recipients may be human or non-human subjects. Antibodies may be polyclonal or monoclonal, produced in vitro or in vivo, and purified, partially purified or unpurified, depending on the embodiment. In some embodiments disclosed herein, passive immunity is conferred to a subject in need through the administration of an antibody or antigen-binding fragment that specifically recognizes or binds to a particular antigen. In some embodiments, passive immunity is conferred through the administration of an isolated polynucleotide or recombinant polynucleotide encoding an antibody or antigen-binding fragment that specifically recognizes or binds to a particular antigen.

[0080] "Immune response" broadly refers to the antigen-specific response of lymphocytes to foreign substances. The elements "immunogen" and "immunogenic" refer to molecules that have the ability to induce an immune response. All immunogens are antigens; however, not all antigens are immunogenic. The immune responses disclosed herein may be humoral (via antibody activity) or cell-mediated (via T cell activation). The response may occur in vivo or in vitro. Those skilled in the art will understand that various macromolecules (including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides, and polysaccharides) may be immunogenic. Those skilled in the art will further understand that nucleic acids encoding molecules capable of inducing an immune response necessarily encode immunogens. Those skilled in the art will further understand that immunogens are not limited to full-length molecules but may include partial molecules.

[0081] As used herein, the term "inducing an immune response in a subject" is a term well understood in the art and is intended to mean that an increase in the immune response to an antigen (or epitope) of at least about 2 times, more preferably at least about 5 times, more preferably at least about 10 times, more preferably at least about 100 times, even more preferably at least about 500 times, even more preferably at least about 1000 times, or higher, can be detected or measured after the introduction of the antigen (or epitope) into the subject compared to the immune response (if any) before the introduction of the antigen (or epitope) into the subject. An immune response to an antigen (or epitope) includes, but is not limited to, the production of antigen-specific (or epitope-specific) antibodies and the production of immune cells that express molecules on their surface that specifically bind to the antigen (or epitope). Methods for determining whether an immune response to a given antigen (or epitope) is being induced are well known in the art. For example, antigen-specific antibodies can be detected using any of the various immunoassays known in the art (including, but not limited to, ELISA, which detects the binding of antibodies in a sample to an immobilized antigen (or epitope) using a detectably labeled secondary antibody (e.g., enzyme-labeled mouse anti-human Ig antibody)).

[0082] The term "modulate an immune response" includes inducing (increasing, triggering) an immune response; and reducing (suppressing) an immune response. An immunomodulatory method (or protocol) modulates the immune response in a subject.

[0083] The term "vector" refers to a polynucleotide (usually DNA) used to artificially deliver foreign genetic material to another cell, which can be replicated or expressed. Non-exclusive exemplary vectors include plasmids, viral vectors, cosmids, and artificial chromosomes. Such vectors can originate from various sources (including bacterial and viral sources). A non-exclusive exemplary viral source for plasmids is adeno-associated virus.

[0084] As used herein, the term “recombinant expression system” refers to a genetic construct for the expression of certain genetic material formed by recombination; the term “construct” is in this respect interchangeable with the term “vector” as defined herein.

[0085] The terms "HMG domain," "high mobility group (HMG) box domain," or "HMGB" refer to amino acid sequences involved in DNA binding (Stros et al., Cell Mol Life Sci. 64(19-20):2590-606 (2007)). In one embodiment, the structure of the HMG-box domain consists of three helices in an irregular sequence. In another embodiment, the HMG-box domain enables the protein to bind non-B DNA configurations (twisted or untwisted) with high affinity. HMG-box domains are found in high mobility group proteins and are involved in the regulation of DNA-dependent processes (e.g., transcription, replication, and DNA repair, all of which require changes in chromatin conformation) (Thomas (2001) Biochem. Soc. Trans. 29(Pt 4):395-401). HMGB1 is a high mobility group box (HMGB) 1 protein that has been reported to bind to and bend the minor groove of DNA. Recombinant or isolated proteins and polypeptides are commercially available from Atgenglobal, ProSpecBio, Protein1, and Abnova.

[0086] HMG-box proteins are found in various eukaryotic organisms and can be broadly divided into two groups based on sequence-dependent and sequence-independent DNA recognition; the former typically contain one HMG-box motif, while the latter may contain multiple HMG-box motifs. Non-limiting examples of polypeptides containing HMG-box domains include: the chromatin non-histone components HMG1 (HMGB1), HMG2 (HMGB2), HMGB3, and HMGB4; SRY (sex-determining region Y protein) involved in differential gonadal development; and the SOX family of transcription factors (Harley et al. (2003)). Endocr. Rev. 24(4):466-87); Sequence-specific LEF1 (lymphoid enhancer-binding factor 1) and TCF-1 (T cell factor 1) involved in the regulation of organogenesis and thymocyte differentiation (Labbe et al. (2000) Proc. Natl. A cad. Sci. USA 97(15):8358-63); SSRP, a structure-specific recognition protein involved in transcription and replication; MTF1, a mitochondrial transcription factor; UBF 1 / 2 (upstream binding factors) involved in transcription by RNA polymerase I; Abf2, a yeast ARS binding factor (Cho et al. (2001) Biochim. Biophys. Acta. 1522(3):175-86); yeast transcription factors lxr1, Rox1, Nhp6b, and Spp41; mating type protein (MAT) involved in sexual reproduction in fungi (Barve et al. (2003) Fungal Genet. Biol. 39(2):151-67); and YABBY, a plant-specific transcription factor.

[0087] Exemplary sequences of polypeptides containing HMG-box domains include NP_002119 (human HMGB1), NP_001124160 (human HMGB2), NP_005333 (human HMGB3), and NP_660206 (human HMGB4). Approximately 9 to 76 amino acid residues of human HMGB1 form, for example, an HMG-box domain, and approximately 90 to 138 amino acid residues form another HMG-box domain. An HMGB1 fragment containing either of these two HMG-box domains also constitutes, for example, an HMG-box domain-containing polypeptide within the meaning of this disclosure. In the examples described herein, recombinant HMGB1 (derived from humans, recombinantly expressed in E. coli, and purified) is used as a comparer to mHMGB1(C45S) and has the following sequence: [ka]

[0088] The term "modified high mobility group-box 1 domain," as used herein, refers, for example, to the consensus sequence polypeptide of human-derived HMGB1: [ka] Based on this, it refers to HMGB1 that is mutated via substitutions of cysteine ​​residues at positions 23, 45, and / or 106.

[0089] Non-exclusive illustrative sequences of the modified high mobility group-box 1 domain include, but are not limited to, the following:

[0090] mHMGB1(C23S): [ka]

[0091] mHMGB1(C45S): [ka]

[0092] mHMGB1(C106S): [ka]

[0093] mHMGB1 (C23S, C45S - double mutant): [ka]

[0094] mHMGB1 (C23S, C106S - double mutant): [ka]

[0095] mHMGB1 (C45S, C106S - double mutant): [ka]

[0096] mHMGB1 (C23S, C45S, C106S - Triple mutant): [ka]

[0097] The nucleic acid sequences encoding these modified high mobility group-box 1 domains are also provided herein below:

[0098] mHMGB1(C23S): [ka]

[0099] mHMGB1(C45S): [ka]

[0100] mHMGB1(C106S): [ka] [ka]

[0101] mHMGB1 (C23S, C45S - double mutant): [ka]

[0102] mHMGB1 (C23S, C106S - double mutant): [ka]

[0103] mHMGB1 (C45S, C106S - double mutant): [ka]

[0104] mHMGB1 (C23S, C45S, C106S - Triple mutant): [ka]

[0105] The term “modified high mobility group-box 1 domain” further encompasses its equivalents that have at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%, identity with the HMGB1 consensus sequence, and it is recognized that these equivalent sequences contain the same substitutions at corresponding positions based on alignment with the HMGB1 consensus sequence.

[0106] It is recognized that among the equivalents of the modified high mobility group-box 1 domain are modified high mobility group-box 2, modified high mobility group-box 3, and modified high mobility group-box 4, which include cysteine-to-serine substitutions at one or more amino acid positions corresponding to C23S, C45S, and C106S of modified high mobility group box 1.

[0107] The corresponding amino acid substitutions that result in the modified high mobility group-box 2 are found in the human HMGB2 consensus sequence polypeptide: [ka] Based on this, it may be performed at cysteine ​​residues at positions 23, 45, and / or 106.

[0108] Non-exclusive illustrative sequences of modified high mobility group-box 2 domains include, but are not limited to, the following:

[0109] mHMGB2(C23S): [ka]

[0110] mHMGB2(C45S): [ka]

[0111] mHMGB2(C106S): [ka]

[0112] mHMGB2 (C23S, C45S - double mutant): [ka] [ka]

[0113] mHMGB2 (C23S, C106S - double mutant): [ka]

[0114] mHMGB2 (C45S, C106S - double mutant): [ka]

[0115] mHMGB2 (C23S, C45S, C106S - Triple mutant): [ka]

[0116] The nucleic acid sequences encoding these modified high mobility group-box 2 domains are also provided herein below:

[0117] mHMGB2(C23S): [ka]

[0118] mHMGB2(C45S): [ka]

[0119] mHMGB2(C106S): [ka]

[0120] mHMGB2 (C23S, C45S - double mutant): [ka] [ka]

[0121] mHMGB2 (C23S, C106S - double mutant): [ka]

[0122] mHMGB2 (C45S, C106S - double mutant): [ka]

[0123] mHMGB2 (C23S, C45S, C106S - Triple mutant): [ka]

[0124] The corresponding amino acid sequence substitutions that result in the modified high mobility group-box 3 are found in the human HMGB3 consensus sequence polypeptide: [ka] Based on this, it may be performed at cysteine ​​residues at positions 23, 45, and / or 104.

[0125] Non-exclusive illustrative sequences of modified high mobility group-box 2 domains include, but are not limited to, the following:

[0126] mHMGB3(C23S): [ka]

[0127] mHMGB3(C45S): [ka]

[0128] mHMGB3(C104S): [ka]

[0129] mHMGB3 (C23S, C45S - double mutant): [ka]

[0130] mHMGB3 (C23S, C104S - double mutant): [ka]

[0131] mHMGB3 (C45S, C104S - double mutant): [ka]

[0132] mHMGB3 (C23S, C45S, C104S - Triple mutant): [ka]

[0133] The nucleic acid sequences encoding these modified high mobility group-box 2 domains are also provided herein below:

[0134] mHMGB3(C23S): [ka] [ka]

[0135] mHMGB3(C45S): [ka]

[0136] mHMGB3(C104S): [ka] [ka]

[0137] mHMGB3 (C23S, C45S - double mutant): [ka]

[0138] mHMGB3 (C23S, C104S - double mutant): [ka]

[0139] mHMGB3 (C45S, C104S - double mutant): [ka] [ka]

[0140] mHMGB3 (C23S, C45S, C104S - Triple mutant): [ka]

[0141] The corresponding amino acid sequence substitutions that result in the modified high mobility group-box 2 are found in the human HMGB2 consensus sequence polypeptide: [ka] Based on this, it can be done at cysteine ​​residues at positions 45, 104, 164, and / or 178.

[0142] Non-exclusive illustrative sequences of modified high mobility group-box 2 domains include, but are not limited to, the following:

[0143] mHMGB4(C45S): [ka] [ka]

[0144] mHMGB4(C104S): [ka]

[0145] mHMGB4(C164S): [ka]

[0146] mHMGB4(C178S): [ka]

[0147] mHMGB4 (C45S, C104S - double mutant): [ka]

[0148] mHMGB4 (C45S, C164S - double mutant): [ka]

[0149] mHMGB4 (C45S, C178S - double mutant): [ka] [Chemical formula]

[0150] mHMGB4 (C104S, C164S - double mutant): [Chemical formula]

[0151] mHMGB4 (C104S, C178S - double mutant): [Chemical formula]

[0152] mHMGB4 (C164S, C178S - double mutant): [Chemical formula]

[0153] mHMGB4 (C45S, C104S, C164S - triple mutant): [Chemical formula]

[0154] mHMGB4 (C45S, C104S, C178S - triple mutant): [Chemical formula]

[0155] mHMGB4 (C45S, C164S, C178S - triple mutant): [Chemical formula]

[0156] mHMGB4 (C104S, C164S, C178S) - triple mutant): [Chemical formula]

[0157] mHMGB4 (C45S, C104S, C164S, C178S - quadruple mutant):

Chem.

[0158] The nucleic acid sequences encoding these modified high mobility group - box 2 domains are also provided herein below:

[0159] mHMGB4 (C45S):

Chem.

[0160] mHMGB4 (C104S):

Chem.

Chem.

[0161] mHMGB4 (C164S):

Chem.

[0162] mHMGB4 (C178S):

Chem.

[0163] mHMGB4 (C45S, C104S - double mutant):

Chem.

[0164] mHMGB4 (C45S, C164S - double mutant): [ka]

[0165] mHMGB4 (C45S, C178S - double mutant): [ka]

[0166] mHMGB4 (C104S, C164S - double mutant): [ka] [ka]

[0167] mHMGB4 (C104S, C178S - double mutant): [ka]

[0168] mHMGB4 (C164S, C178S - double mutant): [ka] [ka]

[0169] mHMGB4 (C45S, C104S, C164S - Triple mutant): [ka]

[0170] mHMGB4 (C45S, C104S, C178S - Triple mutant): [ka]

[0171] mHMGB4 (C45S, C164S, C178S - Triple mutant): [ka] [ka]

[0172] mHMGB4 (C104S, C164S, C178S - Triple mutant): [ka]

[0173] mHMGB4 (C45S, C104S, C164S, C178S - quadruple mutant): [ka]

[0174] Methods for implementing disclosure The applicant hereby demonstrates that DNABII proteins stabilize the eDNA-dependent extracellular matrix of biofilms, while recombinant HMGB1 (rHMGB1) disrupts pre-formed bacterial biofilms in vitro (Table 1), clearing NTHI biofilms from the middle ear of chinchillas in an experimental model of otitis media (OM) (Figures 1A-1B), and inhibiting Burkholderia cenocepacia biofilm formation in mouse airways, directly resulting in a reduced bacterial load (Figures 2A-2D).

[0175] While rHMGB1 has been shown to be effective in disrupting biofilms in vitro and in vivo, it is also well documented to induce a strong inflammatory response that can be potentially harmful to the host. Several post-translational modifications (PTMs), including acetylation, phosphorylation, methylation, glycosylation, ADP-ribosylation, and oxidation of cysteine ​​residues, have been described for HMGB1, modulating its location (nuclear, cytoplasmic, or extracellular), function, and ability to bind DNA (review (Kang et al. 2014)). Human HMGB1 contains three cysteine ​​residues at positions 23, 45, and 106, and oxidation of these cysteine ​​residues affects its inflammatory properties (Kazama et al. 2008). HMGB1 containing a C106 thiol group and a C23-C45 disulfide bond triggers inflammation, while terminally oxidized cysteine ​​promotes the resolution of inflammation (Yang et al. 2012).

[0176] In the disclosed study, the applicant generated a variant form of HMGB1 (mHMGB1) in which the applicant substituted serine at position 45 (C45S). The applicant showed that mHMGB1 (C45S) was effective in disrupting pre-formed biofilms in vitro formed by numerous human pathogens, clearing NTHI biofilms in an experimental model of OM, and reducing B. cenocepacia bacterial load in mouse respiratory tracts, while simultaneously not enhancing the inflammatory response. The applicant's data suggest that the applicant generated an HMGB1 variant (mHMGB1) that retains desirable anti-biofilm function against numerous human pathogens but is defective in enhancing an undesirable, strong pro-inflammatory response. Furthermore, this study suggests that other equivalent modifications of HMGB1 (e.g., C23S and C106S) may produce similar results. Accordingly, the applicant generates a triple mutant HMGB1 comprising modified C23S, C45S, and C106S, as well as further mutant HMGB1 comprising one or more modifications selected from the group C23S, C45S, and C106S.

[0177] The applicant's approach does not rely on compounds with bactericidal activity that put further pressure on bacteria to induce resistance mechanisms, but rather is an improvement over current technology in that it targets the extracellular matrix of biofilms and causes the disruption of biofilms, leading to disease resolution in the absence of excessive inflammation.

[0178] This method and composition are useful in the treatment of a wide variety of biofilm-borne infections, including, but not limited to, otitis media, urinary tract infections, pneumonia, gingivitis, peri-implantitis, periodontitis, cystic fibrosis, endocarditis, and burn wound infections.

[0179] This method and composition, when combined with antibiotics or other antimicrobial agents, are useful for "sterilizing" the target site of device insertion to prevent device-related infections.

[0180] C45S recombinant HMGB1 or modified HMGB1(C45S) ("mHMGB1(C45S)") has been shown to disrupt existing biofilms both in vitro and in vivo (in two mammalian models). Dispersing large quantities of bacteria from biofilm to a suspension state in vivo may have a potential drawback in that these suspension bacteria may gain access to other sites that they did not have access to before potentially causing secondary site infection. This dispersion effect may also lead to sepsis. None of these outcomes have been observed with mHMGB1, and in addition to the fact that these potential pitfalls may be debatable depending on the site of biofilm infection, there are means to circumvent these potential limitations. Indeed, the C45S mutation appears to increase the molecular affinity for branched DNA structures (extracellular DNA within bacterial biofilms is thought to constitute these structures), thus also weakening its inflammatory response and thus providing a better therapeutic response.

[0181] Accordingly, the aspects disclosed herein relate to a modified high mobility group-box 1 domain comprising one or more or all substitutions selected from the group C23S, C45S, and C106S; an isolated polynucleotide or recombinant polynucleotide encoding the modified high mobility group-box 1 domain; a vector comprising the isolated polynucleotide or recombinant polynucleotide; and a composition comprising an effective amount of the modified high mobility group-box 1 domain and / or the aforementioned isolated polynucleotide or recombinant polynucleotide and / or the aforementioned vector. In one aspect, the polynucleotide is DNA, and in another aspect, the polynucleotide is RNA. A recombinant method is also provided for producing the mHMGB1 polypeptide by expressing the polynucleotide in a prokaryotic system or a eukaryotic system. In a further aspect, the protein produced in the system is isolated. Accordingly, the disclosure also provides polypeptides produced in prokaryotic and eukaryotic host cell systems. In some embodiments, the modified high mobility group-box 1 domain comprises the substitution C45S. In some embodiments, the modified high mobility group-box 1 domain includes substitutions C23S, C45S, and C106S.

[0182] A further aspect relates to a method for inhibiting, competing for, or titrating the binding of deoxyribonucleic acid B II (DNABII) polypeptides to microbial DNA in a biofilm, the method comprising contacting the microbial DNA in the biofilm with one or more isolated polypeptides or recombinant polypeptides or compositions disclosed above, each containing an effective amount of a modified high mobility group-box 1 domain comprising one or more substitutions selected from the group C23S, C45S, and C106S, thereby inhibiting, competing for, or titrating the binding of the DNABII polypeptide to the microbial DNA. In some embodiments, the modified high mobility group-box 1 domain comprises the substitution C45S. In some embodiments, the modified high mobility group-box 1 domain comprises the substitutions C23S, C45S, and C106S. In some embodiments, the contacting step is in vitro or in vivo. In some embodiments, the biofilm is formed by Burkholderia cenocepacia, Enterobacter spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterococcus faecium, urinary tract pathogenic Escherichia coli (UPEC), non-encapsulated Haemophilus influenzae (NTHI), and / or Aggregatibacter actinomycetemcomitans. In some embodiments, the method further comprises the steps of administering an effective amount of antibiotic to the surface and optionally coating it.

[0183] Further aspects relate to methods for preventing the formation of biofilms on surfaces, comprising the steps of contacting the surface with an isolated polypeptide or recombinant polypeptide containing an effective amount of a modified high mobility group-box 1 domain containing one or more or all substitutions selected from the group C23S, C45S, and C106S, or with one or more of the compositions disclosed above, and optionally coating, thereby inhibiting, competing for, or titrating the binding of the DNABII polypeptide to microbial DNA during biofilm formation. In some embodiments, the modified high mobility group-box 1 domain contains the substitution C45S. In some embodiments, the modified high mobility group-box 1 domain contains the substitution C23S, C45S, and C106S. In some embodiments, the contacting step is in vitro or in vivo. In some embodiments, the biofilm is formed by Burkholderia cenocepacia, Enterobacter spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterococcus faecium, urinary tract pathogenic Escherichia coli (UPEC), non-encapsulated Haemophilus influenzae (NTHI), and / or Aggregatibacter actinomycetemcomitans. In some embodiments, the method further comprises the steps of administering an effective amount of antibiotic to its surface and, if necessary, coating it.

[0184] A further aspect relates to a method for preventing or treating microbial infection in a subject that forms a biofilm, the method comprising administering an effective amount of an isolated polypeptide or recombinant polypeptide, or one or more of the compositions disclosed above, comprising inhibiting, competing for, or titrating the binding of the DNABII polypeptide to microbial DNA. In some embodiments, the modified high mobility group-box 1 domain includes the substitution C45S. In some embodiments, the modified high mobility group-box 1 domain includes the substitutions C23S, C45S, and C106S. In some embodiments, the biofilm is formed by Burkholderia cenocepacia, Enterobacter spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterococcus faecium, urinary tract pathogenic Escherichia coli (UPEC), non-encapsulated Haemophilus influenzae (NTHI), and / or Aggregatibacter actinomycetemcomitans. In some embodiments, the method further comprises the step of administering an effective amount of antibiotic.

[0185] It is recognized that among the equivalents to the modified high mobility group-box 1 domain are modified high mobility group-box 2, modified high mobility group-box 3, and modified high mobility group-box 4, which include cysteine-to-serine substitutions at one or more amino acid positions corresponding to C23S, C45S, and C106S of modified high mobility group-box 1. For modified high mobility group-box 2, the corresponding cysteine ​​residues are found at positions 23, 45, and 106; for modified high mobility group-box 3, the corresponding cysteine ​​residues are found at positions 23, 45, and 104; and for modified high mobility group-box 4, the corresponding cysteine ​​residues are found at positions 45, 104, 164, and 178. Therefore, the applicant recognizes that modified high mobility group-box 4 includes one or more substitutions selected from the group C23S, C45S, and C106S. We believe that the aforementioned aspects disclosed with respect to mobility group-box 1 are equally applicable to the aforementioned high mobility group-box species having one or more cysteine-to-serine substitutions at the position referred to (e.g., modified high mobility group-box 2 including one or more substitutions selected from the group C23S, C45S, and C106S; modified high mobility group-box 3 including one or more substitutions selected from the group C23S, C45S, and C104S; and modified high mobility group-box 4 including one or more substitutions selected from the group C45S, C104S, C164S, and C178S).

[0186] Methods for implementing disclosure polypeptide The aspects disclosed herein relate to modified high mobility group-box 1 domains that include one or more substitutions selected from the group C23S, C45S, and C106S. In some embodiments, the modified high mobility group-box 1 domain includes the substitution C45S. In some embodiments, the modified high mobility group-box 1 domain includes one, two, or all three substitutions C23S, C45S, and C106S.

[0187] Equivalents to the modified high mobility group-box 1 domain include cysteine-to-serine substitutions at one or more amino acid positions corresponding to C23S, C45S, and C106S of the modified high mobility group box 1. 2. Modified high mobility group-box 3, and modified high It is recognized that there is a mobility group-box 4. With respect to the modified high mobility group-box 2, its corresponding cysteine ​​residues are found at positions 23, 45, and 106; with respect to the modified high mobility group-box 3, its corresponding cysteine ​​residues are found at positions 23, 45, and 104; and with respect to the modified high mobility group-box 4, its corresponding cysteine ​​residues are found at positions 45, 104, 164, and 178. Thus, the applicant has modified high mobility group-box 2 including one or more substitutions selected from the group C23S, C45S, and C106S. The aforementioned polypeptide aspect disclosed with respect to mobility group-box 1 is a modified high mobility group-box species having one or more cysteine-to-serine substitutions at the position referred to (e.g., a modified high mobility group-box 2 having one or more substitutions selected from the group C23S, C45S, and C106S; a modified high mobility group-box 2 having one or more substitutions selected from the group C23S, C45S, and C104S). We believe this is equally applicable to the modified high mobility group-box 4), which includes mobility group-box 3 and one or more substitutions selected from the group C45S, C104S, C164S, and C178S.

[0188] The proteins and polypeptides can be obtained by many processes known to those skilled in the art, including purification, chemical synthesis, and recombination. Polypeptides can be isolated from preparations such as host cell systems by methods such as immunoprecipitation with antibodies, as well as by standard techniques such as gel filtration, ion exchange, reversed-phase, and affinity chromatography. For such methodologies, see, for example, Deutscher et al. (1999) Guide To Protein Purification: Methods In Enzymology (Vol. 182, Academic Press). Thus, the present invention also provides processes for obtaining these polypeptides, as well as the products that can be obtained by these processes.

[0189] The polypeptide can also be obtained by chemical synthesis using a commercially available automated peptide synthesizer (e.g., Model 430A or 431A manufactured by Perkin / Elmer / Applied Biosystems, Inc., Foster City, CA, USA). The synthesized polypeptide can be precipitated and further purified, for example, by high-performance liquid chromatography (HPLC). Thus, the present invention also provides a protein sequence and reagents (amino acids and enzymes), and a process for chemically synthesizing the protein of the present invention by linking the amino acids together in appropriate orientation and linear sequence.

[0190] Alternatively, the proteins and polypeptides may be obtained by well-known recombinant or mutagenetic methods using the host cells and vector systems described herein, for example, as described in Sambrook et al. (2012) cited herein.

[0191] The polypeptides of the present invention can also be combined with various solid-phase carriers (e.g., implants, stents, pastes, gels, dental implants, or medical implants) or liquid-phase carriers (e.g., beads, sterile or aqueous solutions, pharmaceutically acceptable carriers, suspensions, or emulsions). Examples of non-aqueous solvents include propyl ethylene glycol, polyethylene glycol, and vegetable oils. When used to prepare antibodies or induce an immune response in vivo, the carrier may also include adjuvants useful for non-specifically enhancing a specific immune response. Those skilled in the art can easily determine whether an adjuvant is required and whether to select one. However, for illustrative purposes only, suitable adjuvants include, but are not limited to, Freund's complete and incomplete adjuvants, inorganic salts, and polynucleotides. Other suitable adjuvants include monophosphoryl lipid A (MPL), mutant derivatives of the heat-labile enterotoxin of E. coli, mutant derivatives of cholera toxin, CPG oligonucleotides, and adjuvants derived from squalene.

[0192] Polynucleotides and vectors The aspects disclosed herein relate to isolated polynucleotides or recombinant polynucleotides encoding a modified high mobility group-box 1 domain containing one or more substitutions selected from the group C23S, C45S, and C106S, and / or vectors containing such isolated polynucleotides or recombinant polynucleotides. In some embodiments, the modified high mobility group-box 1 domain contains the substitution C45S. In some embodiments, the modified high mobility group-box 1 domain contains the substitutions C23S, C45S, and C106S.

[0193] In general, methods for packaging genetic material (e.g., DNA or RNA) into one or more vectors are well known in the art. In some embodiments, the modified high mobility group-box 1 domain includes a substitution C45S. In some embodiments, the modified high mobility The group-box 1 domain includes substitutions C23S, C45S, and C106S. For example, its genetic material can be packaged using packaging vectors and cell lines and introduced via conventional recombination methods.

[0194] Equivalents to the modified high mobility group-box 1 domain include cysteine-to-serine substitutions at one or more amino acid positions corresponding to C23S, C45S, and C106S of the modified high mobility group box 1. 2. Modified high mobility group-box 3, and modified high The presence of mobility group-box 4 is recognized. (Modified high) With respect to mobility group-box 2, the corresponding cysteine ​​residues are found at positions 23, 45, and 106; with respect to modified high mobility group-box 3, the corresponding cysteine ​​residues are found at positions 23, 45, and 104; and with respect to modified high mobility group-box 4, the corresponding cysteine ​​residues are found at positions 45, 104, 164, and 178. Accordingly, the applicant states that isolated polynucleotides and vectors disclosed with respect to modified high mobility group-box 1, which includes one or more substitutions selected from the group C23S, C45S, and C106S, are equivalent to the aforementioned high mobility group-box species (e.g., modified high mobility group-box 1, which includes one or more substitutions selected from the group C23S, C45S, and C106S) having one or more cysteine-to-serine substitutions at the positions mentioned. 2; Modified high mobility group-box 3) containing one or more substitutions selected from the group C23S, C45S, and C104S; and modified high mobility group-box 4) containing one or more substitutions selected from the group C45S, C104S, C164S, and C178S are considered equally applicable.

[0195] In some embodiments, the packaging vector may be, but is not limited to, a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated virus vector (AAV8, if applicable). The packaging vector comprises elements and sequences that facilitate the delivery of genetic material to cells. For example, the retroviral construct is a packaging plasmid that trans-encodes all the virion proteins required to package a non-replicating retroviral vector and contains at least one retroviral helper DNA sequence derived from a non-replicating retroviral genome to generate virion proteins capable of packaging a high-titer, non-replicating retroviral vector without generating a replicating helper virus. The retroviral DNA sequence lacks the region encoding the native enhancer and / or promoter of the virus's viral 5' LTR and both the psi functional sequence and 3' LTR responsible for packaging the helper genome, but encodes an exogenous polyadenylation site (e.g., an SV40 polyadenylation site) and an exogenous enhancer and / or promoter directed toward efficient transcription in cell types where virogenesis is desired. The retrovirus is a leukemia virus (e.g., Moloney's mouse leukemia virus (MMLV)), human immunodeficiency virus (HIV), or gibbon leukemia virus (GALV). The exogenous enhancer and promoter may be the human cytomegalovirus (HCMV) immediate (IE) enhancer and promoter, the Moloney's mouse sarcoma virus (MMSV) enhancer and promoter (U3 region), the Rous sarcoma virus (RSV) U3 region, the Spleen Focus Forming Virus (SFFV) U3 region, or the HCMV IE enhancer conjugated to the native Moloney's mouse leukemia virus (MMLV) promoter.

[0196] The retroviral packaging plasmid may consist of two retroviral helper DNA sequences encoded by a plasmid-based expression vector, for example, where the first helper sequence contains cDNA encoding the gag and pol proteins of allotropic MMLV or GALV, and the second helper sequence contains cDNA encoding the env protein. The Env gene that determines its host range may be derived from heterotropic, bitropic, allotropic, multitropic (mink focus formation), or 10A1 mouse leukemia virus env protein, or gibbon leukemia virus (GALV) env protein, human immunodeficiency virus env(gp160) protein, vesicular stomatitis virus (VSV) G protein, a gene encoding human T-cell leukemia (HTLV) type I and II env gene products, a chimeric envelope gene derived from one or more combinations of the aforementioned env genes, or a chimeric envelope gene encoding the cytoplasmic and transmembrane portions of the aforementioned env gene products, as well as monoclonal antibodies directed against specific surface molecules on desired target cells. Similar vector-based systems may utilize other vectors (e.g., sleeping beauty vectors or transposon elements).

[0197] The resulting packaged expression system can then be introduced via a suitable route of administration, which will be discussed in detail with respect to the method aspects disclosed herein.

[0198] composition Further aspects relate to compositions comprising a modified high mobility group-box 1 domain containing an effective amount of one or more substitutions selected from the group C23S, C45S, and C106S, and / or the aforementioned isolated or recombinant polynucleotide and / or the aforementioned vector. In some embodiments, the modified high mobility group-box 1 domain contains the substitution C45S. In some embodiments, the modified high mobility group-box 1 domain contains the substitution C23S, C45S, and C106S.

[0199] Equivalents to the modified high mobility group-box 1 domain include cysteine-to-serine substitutions at one or more amino acid positions corresponding to C23S, C45S, and C106S of the modified high mobility group box 1. 2. Modified high mobility group-box 3, and modified high The presence of mobility group-box 4 is recognized. (Modified high) For mobility group-box 2, the corresponding cysteine ​​residues are found at positions 23, 45, and 106; for modified high mobility group-box 3, the corresponding cysteine ​​residues are found at positions 23, 45, and 104; and for modified high mobility group-box 4, the corresponding cysteine ​​residues are found at positions 45, 104, 164, and 178. Accordingly, the applicant believes that a compositional aspect disclosed with respect to modified high mobility group-box 1, which includes one or more substitutions selected from the group C23S, C45S, and C106S, is equally applicable to the aforementioned high mobility group-box species having one or more cysteine-to-serine substitutions at the position referred to (e.g., modified high mobility group-box 2, which includes one or more substitutions selected from the group C23S, C45S, and C106S; modified high mobility group-box 3, which includes one or more substitutions selected from the group C23S, C45S, and C104S; and modified high mobility group-box 4, which includes one or more substitutions selected from the group C45S, C104S, C164S, and C178S).

[0200] In short, the pharmaceutically acceptable compositions of this disclosure, including but not limited to one of the claimed compositions, may include a modified high mobility group-box 1 domain as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0201] Well-known examples of carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The properties of these carriers may be either soluble or insoluble for the purposes of disclosure. Those skilled in the art may know of other suitable carriers for binding antibodies, or may confirm such carriers using conventional experiments.

[0202] Such compositions may also include buffers (e.g., neutral buffered salt solutions, phosphate-buffered saline, etc.); carbohydrates (e.g., glucose, mannose, sucrose, or dextran, mannitol); proteins; polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA or glutathione); adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of this disclosure may be formulated for oral, intravenous, topical, enteral, and / or parenteral administration. In certain embodiments, the compositions of this disclosure are formulated for intravenous administration.

[0203] The administration of the composition may be given in a single dose, continuously or intermittently, throughout the course of treatment. Methods for determining the most effective means and doses of administration are known to those skilled in the art and vary depending on the composition used for treatment, the therapeutic purpose, and the subject being treated. Single or multiple doses may be given at dose levels and patterns selected by the treating physician. Appropriate formulations and methods of administering the drugs are known in the art. In further context, the cells and compositions of this disclosure may be administered in combination with other treatments.

[0204] Vectors, recombinant expression systems, and / or compositions are administered to a host using methods known in the art. Such administration of the compositions of this disclosure may be carried out to generate animal models of a desired disease, disorder, or condition for experimental and screening assays.

[0205] In short, the pharmaceutically acceptable compositions of the present disclosure, comprising but not limited to one of the claimed compositions, may comprise one or more vectors or recombinant expression systems as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers (e.g., neutral buffered salt solutions, phosphate-buffered saline, etc.); carbohydrates (e.g., glucose, mannose, sucrose, or dextran, mannitol); proteins; polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA or glutathione); adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present disclosure may be formulated for oral, intravenous, topical, enteral, and / or parenteral administration. In certain embodiments, the compositions of the present disclosure are formulated for intravenous administration.

[0206] The pharmaceutical compositions of this disclosure may be administered in a manner appropriate to the disease, disorder, or condition to be treated or prevented. The dosage and frequency of administration will be determined by factors such as the patient's condition, the type and severity of the patient's disease, but the appropriate dosage may be determined by clinical trials.

[0207] treatment The aspects disclosed herein relate to therapeutic methods. For example, the aspects relate to a method for inhibiting, competing for, or titrating the binding of deoxyribonucleic acid B II (DNABII) polypeptides to microbial DNA in a biofilm, the method comprising contacting the microbial DNA in the biofilm with one or more isolated polypeptides or recombinant polypeptides or compositions disclosed herein, each containing an effective amount of a modified high mobility group-box 1 domain comprising one or more substitutions selected from the group C23S, C45S, and C106S, thereby inhibiting, competing for, or titrating the binding of the DNABII polypeptide to the microbial DNA. In some embodiments, the modified high mobility group-box 1 domain comprises the substitution C45S. In some embodiments, the modified high mobility The group-box 1 domain includes substitutions C23S, C45S, and C106S. In some embodiments, the contact step is performed in vitro or in vivo.

[0208] A further aspect relates to a method for preventing the formation of biofilms on a surface, the method comprising contacting the surface with one or more isolated polypeptides or recombinant polypeptides or compositions disclosed above, which include an effective amount of a modified high mobility group-box 1 domain containing one or more substitutions selected from the group C23S, C45S, and C106S, and optionally coating the surface, thereby inhibiting, competing for, or titrating the binding of the DNABII polypeptide to microbial DNA during biofilm formation. In some embodiments, the modified high mobility group-box 1 domain contains the substitution C45S. In some embodiments, the modified high mobility group-box 1 domain contains the substitution C23S, C45S, and C106S. In some embodiments, the contacting step is in vitro or in vivo.

[0209] A further aspect relates to a method for preventing or treating microbial infection in a subject that forms a biofilm, the method comprising administering an effective amount of one or more isolated polypeptides or recombinant polypeptides or compositions disclosed above, comprising a modified high mobility group-box 1 domain containing one or more substitutions selected from the group C23S, C45S, and C106S, thereby inhibiting, competing for, or titrating the binding of the DNABII polypeptide to microbial DNA. In some embodiments, the modified high mobility group-box 1 domain contains the substitution C45S. In some embodiments, the modified high mobility group-box 1 domain contains the substitutions C23S, C45S, and C106S.

[0210] In some aspects, polypeptides containing a modified high-mobility group-box 1 domain with one or more substitutions selected from the C23S, C45S, and C106S groups contain, or instead essentially consist of, or even more specifically, a biological equivalent to any polypeptide described above.

[0211] Equivalents to the modified high mobility group-box 1 domain include cysteine-to-serine substitutions at one or more amino acid positions corresponding to C23S, C45S, and C106S of the modified high mobility group box 1. 2. Modified high mobility group-box 3, and modified high The presence of mobility group-box 4 is recognized. (Modified high) For mobility group-box 2, the corresponding cysteine ​​residues are found at positions 23, 45, and 106; for modified high mobility group-box 3, the corresponding cysteine ​​residues are found at positions 23, 45, and 104; and for modified high mobility group-box 4, the corresponding cysteine ​​residues are found at positions 45, 104, 164, and 178. Accordingly, the applicant believes that the method aspect disclosed with respect to modified high mobility group-box 1, which includes one or more substitutions selected from the group C23S, C45S, and C106S, is equally applicable to the aforementioned high mobility group-box species having one or more cysteine-to-serine substitutions at the position referred to (e.g., modified high mobility group-box 2, which includes one or more substitutions selected from the group C23S, C45S, and C106S; modified high mobility group-box 3, which includes one or more substitutions selected from the group C23S, C45S, and C104S; and modified high mobility group-box 4, which includes one or more substitutions selected from the group C45S, C104S, C164S, and C178S).

[0212] In some respects, the isolated or recombinant protein is a mammalian protein. In certain respects, the mammalian protein is a human protein.

[0213] Any of the above methods further includes, or instead essentially consists of, the step of administering to a subject one or more of the following: an effective amount of an antimicrobial substance, an antigenic peptide, or an adjuvant. The subject is, in one aspect, a non-human animal or a human patient.

[0214] The polypeptide is administered by methods including topical, percutaneous, sublingual, rectal, vaginal, oral administration, intramuscular, intraperitoneal, urethral, ​​nasal, inhalation, or orally.

[0215] In some cases, the subjects are pediatric patients, and the polypeptide is administered in a formulation for those pediatric patients.

[0216] In any of the embodiments described above, the biofilm may contain microbial DNA derived from the microorganisms identified in Table 1.

[0217] In one embodiment, the polypeptide is administered topically to a microbial infection.

[0218] In one embodiment, the Disclosure provides a method for inducing or providing an immune response in a subject in need, the method comprising, or instead essentially consisting of, or even more than, the step of administering to the subject a modified high mobility group-box 1 domain containing one or more substitutions selected from the group C23S, C45S, and C106S. In another embodiment, the administration is local to a site where the immune response is desired. Examples of modified high mobility group-box 1 domains containing one or more substitutions selected from the group C23S, C45S, and C106S are described above. In some embodiments, the modified high mobility group-box 1 domain contains the substitution C45S. In some embodiments, the modified high mobility group-box 1 domain contains the substitutions C23S, C45S, and C106S.

[0219] The isolated or recombinant protein may be a mammalian protein, or, in certain contexts, a human protein. The subject may, in some contexts, be a non-human animal or a human patient.

[0220] The agents and compositions of the present invention may be administered simultaneously with or in conjunction with other antimicrobial agents and / or surface antigens. In one particular context, administration is local to the site of infection. Other non-limiting examples of administration include one or more methods, including percutaneous, sublingual, rectal, vaginal, ocular, subcutaneous, intramuscular, intraperitoneal, urethral, ​​nasal, by inhalation, or orally.

[0221] In one embodiment, the use of any of the above polypeptides comprising, instead of essentially, or further comprising, a modified high mobility group-box 1 domain comprising one or more substitutions selected from the group C23S, C45S, and C106S is also provided for the manufacture of a pharmaceutical in which a biofilm is destroyed or a biofilm-forming microbial infection is inhibited, prevented, or treated. In some embodiments, the modified high The mobility group-box 1 domain includes substitution C45S. In some embodiments, the modified high mobility group-box 1 domain includes substitution C23S, C45S, and C106S.

[0222] In some of these methods, the contact step may be performed in vitro or in vivo. If the contact step is performed in vitro, the method may provide a means to determine the efficacy of the drug of the present invention before animal or clinical studies and may be used to determine whether the drug of the present invention acts synergistically with further antimicrobial substances. If performed in vivo in an animal model, the method may provide a means to determine the efficacy of the drug of the present invention before studies in human patients and may be used to determine whether the drug of the present invention acts synergistically with further antimicrobial substances (e.g., antibiotics).

[0223] Microbial infections and diseases that can be treated by the methods of the present invention include infections by organisms identified in Table 1, such as Burkholderia cenocepacia, Enterobacter spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterococcus faecium, urinary tract pathogenic Escherichia coli (UPEC), non-encapsulated Haemophilus influenzae (NTHI), or Aggregatibacter actinomycetemcomitans. These microbial infections may be present in the upper, middle, or lower respiratory tract (otitis, sinusitis, or bronchitis), but may also be present in complications and / or exacerbations of chronic obstructive pulmonary disease (COPD), chronic cough, cystic fibrosis (CF), and community-acquired pneumonia (CAP).

[0224] Infections can also occur in the oral cavity (caries, periodontitis), caused by Streptococcus mutans, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans. Infections can also be localized to the skin (abscesses, Staphylococcus infections, impetigo, secondary burns, Lyme disease), caused by Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Borrelia burdorferi. Urinary tract infections (UTIs) can also be treated, typically caused by Escherichia coli. Gastrointestinal (GI) infections (diarrhea, cholera, gallstones, gastric ulcers) are typically caused by Salmonella enterica serovar, Vibrio cholerae, and Helicobacter pylori. Genital infections are typically caused by Neisseria gonorrhoeae. Infections of the bladder or indwelling devices may be caused by Enterococcus faecalis. Infections typically caused by various bacteria associated with implanted prosthetic devices (e.g., artificial hip or knee joints) or dental implants or medical devices (e.g., pumps or monitoring systems) can be treated by the methods of the present invention. These devices may be coated with or conjugated with the agents described herein.

[0225] Infections caused by Streptococcus agalactiae are a major cause of bacterial sepsis in neonates. Such infections can also be treated by the methods of the present invention. Similarly, infections caused by Neisseria meningitidis, which can cause meningitis, can also be treated.

[0226] Accordingly, applicable routes of administration for the methods of the present invention include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical, intravenous, rectal, nasal, oral, and other enteral and parenteral routes of administration. The routes of administration may be combined (if desired) or modified depending on the drug and / or the desired effect. The active drug may be administered in a single dose or in multiple doses. Embodiments of these methods and routes of delivery suitable for delivery include systemic or topical routes. Generally, suitable routes of administration for the methods of the present invention include, but are not limited to, enteral, parenteral, or inhalation routes.

[0227] Other parenteral administration routes besides inhalation include, but are not limited to, local, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intrathecal, intrasternal, and intravenous routes, i.e., any route of administration other than through the gastrointestinal tract. Parenteral administration may be performed to provide systemic or local delivery of the inhibitor. When systemic delivery is desired, administration typically requires invasive or systemically absorbed local or mucosal administration of the pharmaceutical preparation.

[0228] The compounds of the present invention can also be delivered to subjects by enteral administration. Enteral administration routes include, but are not limited to, oral delivery and rectal delivery (e.g., using suppositories).

[0229] Methods for administering the active substance through the skin or mucous membranes include, but are not limited to, topical application of appropriate pharmaceutical preparations, transcutaneous transmission, transdermal transmission, injection, and epidermal administration. For transdermal transmission, absorption enhancers or iontophoresis are suitable methods. Iontophoresis can be achieved using commercially available "patches" that continuously deliver the products via electrical pulses through undamaged skin over several days or longer periods.

[0230] In various embodiments of the method of the present invention, the active ingredient is administered orally in a continuous, daily manner, at least once per day (QD), and in various embodiments, twice per day (BID), three times per day (TID), or four times per day. Typically, the therapeutically effective daily dose is at least about 1 mg, or at least about 10 mg, or at least about 100 mg or about 200 to about 500 mg, and sometimes, depending on the compound, up to about 1 g to about 2.5 g.

[0231] Administration may be achieved according to the methods of the present invention using capsules, tablets, oral suspensions, suspensions for intramuscular injection, suspensions for intravenous injection, gels or creams for topical application, or suspensions for intra-articular injection.

[0232] The dosage, toxicity, and therapeutic efficacy of the compositions described herein can be determined, for example, by standard pharmaceutical procedures in cell cultures or experimental animals to determine the LD50 (lethal dose for 50% of the population) and ED50 (therapeutably effective dose for 50% of the population). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions exhibiting a high therapeutic index are preferred. Compounds exhibiting toxic side effects may be used, but care should be taken to design a delivery system that targets such compounds to the site of the affected tissue in order to minimize potential damage to non-infected cells and thereby reduce side effects.

[0233] Data obtained from cell culture assays and animal studies can be used in formulating dosage ranges for human use. Doses of such compounds are preferably within a circulating concentration range including ED50 that is little to no toxicity. The dosage may vary within this range depending on the form of administration used and the route of administration utilized. For any compound used in the method, its therapeutically effective dose can be initially predicted from cell culture assays. The dose can be formulated in animal models to achieve a circulating plasma concentration range including IC50 (i.e., the concentration of the test compound that achieves half of the maximum symptom inhibition) when determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0234] In some embodiments, the effective amount of the composition sufficient to achieve a therapeutic or preventive effect ranges from about 0.000001 mg / kilogram body weight / administer to about 10,000 mg / kilogram body weight / administer. Preferably, the dosage range is from about 0.0001 mg / kilogram body weight / administer to about 100 mg / kilogram body weight / administer. The administration may be provided as an initial dose, followed by one or more “booster” doses. Booster doses may be provided 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months after the initial dose. In some embodiments, booster doses are administered after evaluation of the subject’s response to previous doses.

[0235] Those skilled in the art will recognize that certain factors may influence the dosage and timing required to effectively treat a subject (including, but not limited to, the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other pre-existing conditions). Furthermore, treatment of a subject with a therapeutically effective amount of the therapeutic composition described herein may consist of a single treatment or a series of treatments.

[0236] Combination therapy The compositions and related methods of the present invention may be used in combination with the administration of other therapeutic agents. These include, but are not limited to, the administration of DNase enzymes, antibiotics, antimicrobial substances, or other antibodies.

[0237] In some embodiments, the methods and compositions include a deoxyribonuclease (DNase) enzyme, e.g., DNase, which acts synergistically with the compositions of the Disclosure. DNase is any enzyme that catalyzes the cleavage of phosphodiester bonds in the DNA backbone. Three non-limiting examples of DNase enzymes known to target not only cruciform structures but also various secondary structures of DNA include DNAse I, T4 EndoVII, and T7 Endo I. In certain embodiments, the effective amount of anti-DNABII antibody required to destabilize the biofilm is reduced when combined with DNase. When administered in vitro, the DNase may be added directly to the assay or in a suitable buffer known to stabilize the enzyme. The effective unit dose of DNase and assay conditions may vary and may be optimized according to procedures known in the art.

[0238] In other embodiments, the methods and compositions may be combined with antibiotics and / or antimicrobial substances. Antimicrobial substances are substances that kill or inhibit the growth of microorganisms (e.g., bacteria, fungi, or protozoa). While biofilms are generally resistant to the action of antibiotics, the compositions and methods described herein may be used to make biofilm-related infections responsive to conventional treatments for treating the infection. In other embodiments, the use of antibiotics or antimicrobial substances in combination with the methods and compositions described herein allows for a reduction in the effective amount of antimicrobial substances and / or biofilm-reducing agents. Some non-limiting examples of antimicrobial substances and antibiotics useful in combination with the methods of the present invention include minocycline, amoxicillin, amoxicillin-clavulanate, cefdinir, azithromycin, and sulfamethoxazole-trimethoprim. The therapeutically effective dose of the antimicrobial substance and / or antibiotic in combination with the biofilm-reducing agent can be readily determined by conventional methods. In some embodiments, the dose of the antimicrobial agent in combination with the biofilm-reducing agent is the average effective dose that has been shown to be effective in other bacterial infections (e.g., bacterial infections in which the etiology of infection does not involve biofilm). In other embodiments, the dose is 0.1 times, 0.15 times, 0.2 times, 0.25 times, 0.30 times, 0.35 times, 0.40 times, 0.45 times, 0.50 times, 0.55 times, 0.60 times, 0.65 times, 0.70 times, 0.75 times, 0.8 times, 0.85 times, 0.9 times, 0.95 times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.5 times, 3.0 times, or 5 times the average effective dose. The antibiotic or antimicrobial substance may be added before the addition of the anti-DNABII antibody, simultaneously with the addition of this antibody, or after the addition of this antibody.

[0239] In other embodiments, the methods and compositions may be combined with antibodies that treat the bacterial infection. An example of an antibody useful in combination with the methods and compositions described herein is an antibody directed against an unrelated outer membrane protein (e.g., OMP P5). Treatment with this antibody alone does not debulk the biofilm in vitro. Combination therapy with this antibody and a biofilm-reducing agent produces a greater effect than that which can be achieved by either reagent used alone at the same concentration. Other antibodies that may produce synergistic effects when combined with biofilm-reducing agents or methods for reducing biofilms include anti-rsPilA, anti-OMP26, anti-OMP P2, and anti-complete OMP preparations.

[0240] The compositions and methods described herein may be used to make biofilm-related bacterial infections sensitive to general therapeutic modalities that are effective in treating biofilm-free bacterial infections but are otherwise ineffective in treating biofilm-related bacterial infections. In other embodiments, the compositions and methods described herein may be used in combination with therapeutic modalities that are effective in treating biofilm-related bacterial infections, such combinations of further treatments and biofilm-reducing agents or methods may produce a synergistic effect that reduces the effective dose of either the biofilm-reducing agent or the further treatment. In other cases, such combinations of further treatments and biofilm-reducing agents or methods may produce a synergistic effect that enhances the treatment. Enhancement of treatment may be demonstrated by a shorter amount of time required to treat the infection.

[0241] Further therapeutic measures may be included, either within the same formulation or as a separate formulation, before, simultaneously with, or after the methods or compositions used to reduce the biofilm.

[0242] Antibodies and their derivatives This disclosure also provides antibodies that conjugate and / or specifically recognize modified high-mobility group-box 1 containing one or more substitutions selected from the group of C23S, C45S, and C106S disclosed herein.

[0243] Equivalents to the modified high mobility group-box 1 domain include cysteine-to-serine substitutions at one or more amino acid positions corresponding to C23S, C45S, and C106S of the modified high mobility group box 1. 2. Modified high mobility group-box 3, and modified high The presence of mobility group-box 4 is recognized. (Modified high) For mobility group-box 2, the corresponding cysteine ​​residues are found at positions 23, 45, and 106; for modified high mobility group-box 3, the corresponding cysteine ​​residues are found at positions 23, 45, and 104; and for modified high mobility group-box 4, the corresponding cysteine ​​residues are found at positions 45, 104, 164, and 178. Accordingly, the applicant believes that the antibody surface disclosed with respect to modified high mobility group-box 1, which includes one or more substitutions selected from the group C23S, C45S, and C106S, is equally applicable to the aforementioned high mobility group-box species having one or more cysteine-to-serine substitutions at the position referred to (e.g., modified high mobility group-box 2, which includes one or more substitutions selected from the group C23S, C45S, and C106S; modified high mobility group-box 3, which includes one or more substitutions selected from the group C23S, C45S, and C104S; and modified high mobility group-box 4, which includes one or more substitutions selected from the group C45S, C104S, C164S, and C178S).

[0244] The antibody may be any of the various antibodies described herein, non-limiting examples of such include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, human antibodies, veneered antibodies, diabodies, humanized antibodies, antibody derivatives, recombinant humanized antibodies, or derivatives or fragments of any of these. In one aspect, the fragment comprises the CDR of the antibody, or instead consists essentially of the CDR, or further consists of the CDR, examples of which are provided herein. In one aspect, the antibody is detectably labeled, or further comprises a detectable label conjugated to the antibody. Hybridoma cell lines that produce the monoclonal antibodies disclosed herein are also provided. Compositions comprising one or more of the above embodiments, or instead consisting essentially of these embodiments, or further comprising these embodiments are further provided herein. Polynucleotides encoding the amino acid sequences of the antibody and fragment, as well as methods for recombinantly producing or chemically synthesizing the antibody polypeptide and its fragment, are further provided. The antibody polypeptide may be produced in eukaryotic or prokaryotic cells, or by other methods known in the art and described herein.

[0245] Antibodies can also be produced using prior art that is publicly known in the field and well documented in the literature. Several methodologies exist for the production of polyclonal antibodies. For example, polyclonal antibodies are typically produced by immunizing a suitable mammal (e.g., chickens, goats, guinea pigs, hamsters, horses, mice, rats, and rabbits, but not limited to these). The antigen is injected into the mammal to induce B lymphocytes to produce immunoglobulins specific to that antigen. The immunoglobulins can be purified from the serum of the mammal. Variations of this methodology include modifications of adjuvants, routes and sites of administration, injection volumes per site, and the number of sites per animal and humane treatment of the animals for optimal production. For example, adjuvants can typically be used to improve or enhance the immune response to an antigen. Most adjuvants provide injection-site antigen depots that allow for stow release of the antigen into influx-region lymph nodes. Other adjuvants include surfactants and immunostimulatory molecules that promote the concentration of protein antigen molecules over a large surface area. Non-limiting examples of adjuvants for the production of polyclonal antibodies include Freund's adjuvant, the Ribi adjuvant system, and Titermax. Polyclonal antibodies may be produced using methods known in the art, some of which are described in U.S. Patents No. 7,279,559; No. 7,119,179; No. 7,060,800; No. 6,709,659; No. 6,656,746; No. 6,322,788; No. 5,686,073; and No. 5,670,153.

[0246] Monoclonal antibodies can be produced using conventional hybridoma techniques that are well known in the field and have been adequately described in the literature. For example, hybridomas can be produced using appropriate immortalized cell lines (e.g., myeloma cell lines (e.g., Sp2 / 0, Sp2 / 0-AG14, NSO, NS1, NS2, AE-1, L.5, P3X63Ag8,653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U397, MIA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 313, HL-60, MLA 144, NAMAIWA, NEURO Examples include, but are not limited to, 2A, CHO, PerC.6, YB2 / O), or heteromyeloma, its fusion products, or any cells or fusion cells derived therefrom, or any other suitable cell line as known in the art (see the following web addresses, e.g., atcc.org, lifetech.com (last accessed November 26, 2007)), and antibody-producing cells (e.g., isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B-cell-containing cells, but are not limited thereto), or heavy or light chains. Antibody-producing cells can be generated by fusing the constant region or variable region or framework or CDR sequence of the chain with any other cell expressing (as endogenous or heterologous nucleic acids, recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptile, fish, mammalian, rodent, horse, sheep, goat, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single-stranded, double-stranded or triple-stranded, hybridized, or any combination thereof). Antibody-producing cells can also be obtained from peripheral blood, or, in certain embodiments, from the spleen or lymph nodes of a human or other suitable animal immunized with the antigen of interest.Any suitable host cell may also be used to express heterologous or endogenous nucleic acids encoding antibodies, their specific fragments, or variants thereof, as disclosed herein. Fused cells (hybridoms) or recombinant cells may be isolated using selective culture conditions or other suitable known methods and cloned by limiting dilution or cell sorting or other known methods.

[0247] Other suitable methods may be used to generate or isolate antibodies of essential specificity, and recombinant antibodies may be displayed in peptide or protein libraries (e.g., bacteriophages, ribosomes, oligonucleotides, cDNA, etc.), display libraries; for example, various commercial suppliers (e.g., MorphoSys (Martinsreid / Planegg, Del.), BioInvent (Lund, Methods of selection using methods known in the art (where available from Sweden, Affitech (Oslo, Norway)) include, but are not limited to, methods known in the art. Methods known in the art are described in patent documents, some of which include U.S. Patent Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; and 5,976,862. An alternative method relies on immunizing transgenic animals capable of producing a repertoire of human antibodies, as is known in the art and / or described herein (e.g., SCID mice, Nguyen et al. (1977) Microbiol. Immunol. 41:901-907 (1997); Sandhu et al. (1996) Crit, Rev. Biotechnol. 16:95-118; Eren et al. (1998) Mumma 93:154-161). Examples of such technologies include, but are not limited to, the following: ribosome display (e.g., Wanes et al. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998) Proc. Natl. Acad. Sci. USA 95:14130-14135); single-cell antibody generation techniques (e.g., selective lymphocyte antibody assay ("SLAM") (U.S. Patent No. 5,627,052, Wen et al., (1987) J. Immunol 17:887-892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848); gel microdrops and flow cytometry (Powell et al. (1990) Biotechnol. 8:333-337; single-cell systems (Cambridge, Mass.)); Gray et al. (1995) J. Imm. Meth. 182:155-163; and Kenny et al., (1995) Bio. Technol. 13:787-790); B cell selection (Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134).

[0248] The antibody derivatives of this disclosure may also be prepared by delivering polynucleotides encoding the antibodies or fragments thereof disclosed herein to a suitable host (for example, to a transgenic animal or mammal that produces such antibodies in its milk (e.g., goats, cattle, horses, sheep, etc.)). These methods are known in the art and are described, for example, in U.S. Patents 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.

[0249] The term “antibody derivative” includes post-translational modifications to the linear polypeptide sequence of the antibody or fragment. For example, U.S. Patent No. 6,602,684 B1 describes a method for producing a modified glycol form of an antibody (including a complete antibody molecule, an antibody fragment, or a fusion protein containing a region equivalent to the Fc region of an immunoglobulin and having enhanced Fc-mediated cytotoxicity, and a glycoprotein thus produced).

[0250] The antibodies disclosed herein also include derivatives modified by covalent attachment of any type of molecule to the antibody, such that the covalent attachment does not prevent the antibody from generating an anti-idiotype response. Examples of antibody derivatives include, but are not limited to, antibodies modified by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or linkage to cell ligands or other proteins. Furthermore, the derivatives may contain one or more non-classical amino acids.

[0251] Antibody derivatives can also be prepared by delivering the polynucleotides disclosed herein to provide transgenic plants and cultured plant cells (e.g., tobacco, maize, and duckweed) that produce such antibodies, specific parts of the variants in parts of plants or cells cultured therefrom. For example, Cramer et al. (1999) Curr. Top. Microbol. Immunol. 240:95-118 and the references cited therein describe, for example, the production of transgenic tobacco leaves expressing large amounts of recombinant proteins using inducible promoters. Transgenic maize has been used at commercial production levels to express mammalian proteins with equivalent biological activity to those produced in other recombinant systems or purified from natural sources. For example, see Hood et al. (1999) Adv. Exp. Med. Biol. 464:127-147 and the references cited therein. Antibody derivatives have also been produced in large quantities from transgenic plant seeds (such as tobacco seeds and potato tubers) containing antibody fragments (e.g., single-chain antibodies (scFv's)). See, for example, Conrad et al. (1998) Plant Mol. Biol. 38:101-109 and the references cited therein. Thus, antibodies can also be produced using transgenic plants according to known methods.

[0252] Antibody derivatives can also be produced by adding exogenous sequences, for example, to modify immunogenicity, or to reduce, enhance, or modify binding, affinity, on-rate, off-rate, binding strength, specificity, half-life, or any other suitable properties. Generally, some or all of a non-human or human CDR sequence is maintained while its variable and constant regions are replaced with non-human sequences of human or other amino acids.

[0253] Generally, these CDR residues are involved in directly, and mostly substantially, influencing antigen binding. The humanization or engineering of antibodies may be carried out using any known method (e.g., U.S. Patent Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567).

[0254] The general structure of antibodies is well known in the field and is only briefly summarized here. An immunoglobulin monomer contains two heavy chains and two light chains linked by disulfide bonds. Each heavy chain is paired with one of the light chains, which is directly linked via disulfide bonds. Each heavy chain contains a constant region (which varies depending on the antibody isotype) and a variable region. Its variable region contains three hypervariable regions (or complementarity-determining regions), which are designated CDRH1, CDRH2, and CDRH3, and are supported within a framework region. Each light chain contains a constant region and a variable region, and its variable region contains three hypervariable regions (designated CDRL1, CDRL2, and CDRL3) supported by a framework region in a manner similar to that of the variable region of the heavy chain.

[0255] The hypervariable regions of each pair of heavy and light chains cooperate with each other to provide antigen-binding sites capable of binding to target antigens. The binding specificity of a pair of heavy and light chains is determined by the sequences of their CDR1, CDR2, and CDR3. Thus, once a set of CDR sequences that produces a particular binding specificity (i.e., the sequences of its heavy and light chains' CDR1, CDR2, and CDR3) is determined, that set of CDR sequences can, in principle, be inserted into appropriate positions within any other antibody framework region linked to any antibody constant region to provide different antibodies with the same antigen-binding specificity.

[0256] In some aspects of the antibodies provided herein, the antibody targets the DNABII protein, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 Dissociation constant less than M (K D They are joined together using ).

[0257] In some aspects of the antibodies provided herein, the antibodies are soluble Fab.

[0258] In some of the antibody aspects provided herein, their HC and LC variable domain sequences are components of the same polypeptide chain. In some of the antibody aspects provided herein, their HC and LC variable domain sequences are components of different polypeptide chains.

[0259] In some aspects of antibodies provided herein, the antibodies are full-length antibodies.

[0260] In some aspects of antibodies provided herein, the antibody is a monoclonal antibody.

[0261] In some aspects of the antibodies provided herein, the antibodies are either chimeric or humanized.

[0262] In some aspects of the antibodies provided herein, the antibodies are Fab, F(ab)'2, Fab', scF v , and F v Selected from the group consisting of

[0263] In some aspects of the antibodies provided herein, the antibody contains an Fc domain. In some aspects of the antibodies provided herein, the antibody is a rabbit antibody. In some aspects of the antibodies provided herein, the antibody is a human or humanized antibody, or is non-immunogenic in humans.

[0264] In some aspects of the antibodies provided herein, the antibody includes a human antibody framework region.

[0265] In other contexts, one or more amino acid residues in the CDR of the antibodies provided herein are substituted with other amino acids. Substitutions can be "conservative" in the sense that they are substitutions within the same family of amino acids. The naturally occurring amino acids can be divided into four families, and conservative substitutions occur within these families.

[0266] 1) Amino acids with basic side chains: lysine, arginine, histidine.

[0267] 2) Amino acids with acidic side chains: aspartic acid, glutamic acid.

[0268] 3) Amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, tyrosine.

[0269] 4) Amino acids with nonpolar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine.

[0270] In another context, one or more amino acid residues are added to or deleted from one or more CDRs of the antibody. Such additions or deletions occur at the N-terminus or C-terminus of the CDR, or at some position within the CDR.

[0271] By altering the amino acid sequence of the antibody's CDR through the addition, deletion, or substitution of amino acids, various effects (e.g., increased binding affinity to the target antigen) can be obtained.

[0272] It should be recognized that antibodies of this disclosure containing such altered CDR sequences still bind with a similar specificity and sensitivity profile to the disclosed antibodies. This can be tested through binding assays.

[0273] The constant region of an antibody can also vary. For example, an antibody may be supplied with the Fc region of any isotype: IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), or IgM. Non-limiting examples of constant region sequences include:

[0274] Human IgD constant region, Uniprot: P01880, Sequence ID 37 [ka]

[0275] Human IgG1 constant region, Uniprot: P01857, SEQ ID NO: 38 [ka] [ka]

[0276] Human IgG2 constant region, Uniprot: P01859, SEQ ID NO: 39 [ka]

[0277] Human IgG3 constant region, Uniprot: P01860, SEQ ID NO: 40 [ka]

[0278] Human IgM constant region, Uniprot: P01871, Sequence ID 41 [ka]

[0279] Human IgG4 constant region, Uniprot: P01861, Sequence ID 42 [ka] [ka]

[0280] Human IgA1 constant region, Uniprot: P01876, Sequence ID 43 [ka]

[0281] Human IgA2 constant region, Uniprot: P01877, Sequence ID 44 [ka]

[0282] Human Igκ constant region, Uniprot: P01834, Sequence ID 45 [ka]

[0283] In some aspects of the antibodies provided herein, the antibodies include structural modifications to facilitate rapid binding and cellular uptake and / or slow release. In some aspects, the DNABII antibodies include deletions in the CH2 constant heavy chain region of the antibody to facilitate rapid binding and cellular uptake and / or slow release. In some aspects, Fab fragments are used to facilitate rapid binding and cellular uptake and / or slow release. In some aspects, F(ab)'2 fragments are used to facilitate rapid binding and cellular uptake and / or slow release.

[0284] Antibodies, fragments, and their equivalents may be combined with carriers (e.g., pharmaceutically acceptable carriers) or other agents to provide formulations for use and / or storage.

[0285] The chimeric, humanized, or primate-like antibodies of this disclosure may be prepared based on the sequence of a reference monoclonal antibody prepared using standard molecular biology techniques. The DNA encoding the heavy and light chain immunoglobulins may be obtained from the hybridoma of interest and may be manipulated using standard molecular biology techniques to include a non-reference (e.g., human) immunoglobulin sequence. For example, to produce a chimeric antibody, the mouse variable region may be ligated to the human constant region using methods known in the art (U.S. Patent No. 4,816,567). To produce a humanized antibody, the mouse CDR region may be inserted into the human framework using methods known in the art (U.S. Patents Nos. 5,225,539 and 5,530,101; 5,585,089; 5,693,762 and 6,180,370). Similarly, to produce primate-like antibodies, the mouse CDR region can be inserted into the primate framework using methods known in the art (WO 93 / 02108 and WO 99 / 55369).

[0286] Methods for producing partial to complete human antibodies are known in the art, and any such techniques may be used. According to one embodiment, complete human antibody sequences are produced in transgenic mice engineered to express human heavy and light chain antibody genes. Numerous lines of such transgenic mice have been produced, which can produce various antibody classes. B cells derived from transgenic mice producing the desired antibody can be fused to create hybridoma cell lines for the serial production of the desired antibody (e.g., Russell et al. (2000) Infection). and Immunity April 2000:1820-1826; Gallo et al. (2000) European J. of Immun. 30:534-540; Green (1999) J. of Immun. Methods 231:11-23; Yang et al. (1999A) J. of Leukocyte Biology 66:401-410; Yang (1999B) Cancer Research 59(6):1236-1243; Jakobovits (1998) Advanced Drug Reviews 31:33-42; Green and Jakobovits (1998) J. Exp. Med. 188(3):483-495; Jakobovits (1998) Exp. Opin. Invest. Drugs 7(4):607-614; Tsuda et al. (1997) Genomics 42:413-421; Sherman-Gold (1997) Genetic Engineering News 17(14); Mendez et al. (1997) Nature Genetics 15:146-156; Jakobovits (1996) Weir's Handbook of Experimental Immunology, The Integrated Immune System Vol. IV, 194.1-194.7; Jakobovits (1995) Current Opinion in Biotechnology 6:561-566; Mendez et al. (1995) Genomics 26:294-307; Jakobovits (1994) Current Biology 4(8):761-763; Arbones et al. (1994) Immunity 1(4):247-260; Jakobovits (1993) Nature 362(6417):255-258; Jakobovits et al. (See Proc. Natl. Acad. Sci. USA 90(6):2551-2555 (1993) and U.S. Patent No. 6,075,181).

[0287] The antibodies disclosed herein may also be modified to produce chimeric antibodies. A chimeric antibody is one in which various domains of the heavy and light chains of the antibody are encoded by DNA from one or more species. See, for example, U.S. Patent No. 4,816,567.

[0288] Alternatively, the antibodies disclosed herein may also be modified to produce veneering antibodies. Veneeering antibodies are those in which the outer amino acid residues of an antibody of one species are cleverly replaced or "veneered" with outer amino acid residues of a second species, resulting in the antibody of the first species being non-immunogenic in the second species, thereby reducing the immunogenicity of the antibody. Since the antigenicity of a protein depends primarily on the properties of its surface, the immunogenicity of an antibody can be reduced by replacing exposed residues that are different from those normally found in antibodies of another mammalian species. This clever substitution of outer residues should have little or no effect on the inner domain or on interdomain contact. Therefore, ligand-binding properties should remain unaffected as a consequence of qualitative changes limited to variable region framework residues. The process is called "veneering" because only the outer surface or skin of the antibody is qualitatively altered, while its supporting residues remain unimpeded.

[0289] The procedure for "venation" utilizes available sequence data of human antibody variable domains compiled by Kabat et al. (1987) Sequences of Proteins of Immunological Interest, 4th edition, Bethesda, Md., National Institutes of Health, updates to this database, and other accessible US and foreign databases (both nucleic acid and protein). Non-limiting examples of methods used to generate venated antibodies include those described in EP 519596; US Patent No. 6,797,492; and Padlan et al. (1991) Mol. Immunol. 28(4-5):489-498.

[0290] The term “antibody derivative” also includes “diabodies,” which are small antibody fragments having two antigen-binding sites, where the fragment contains a heavy-chain variable domain (VH) attached to a light-chain variable domain (VL) within the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with a complementary domain on another chain, creating two antigen-binding sites (see also U.S. Patent No. 6,632,926 (Chen et al.), which discloses an antibody variant having one or more amino acid residues inserted into the hypervariable region of the parent antibody and a binding affinity to a target antigen that is at least about twice as strong as the binding affinity of the parent antibody to that antigen).

[0291] The term “antibody derivative” further includes: manipulated antibody molecules, fragments and single domains (e.g., scFv, dAbs, nanobodies, minibodies, unibodies, and affibodies) as well as Hudson (2005) Nature Biotech 23(9):1126-36; U.S. Patent Application Publication US 2006 / 0211088; PCT Publication WO 2007 / 059782; U.S. Patent No. 5,831,012.

[0292] The term "antibody derivative" further includes "linear antibodies." The procedure for producing linear antibodies is well known in the field, as demonstrated by Zapata et al. (1995) This is described in Protein Eng. 8(10):1057-1062. Briefly, these antibodies have a pair of tandem Ed segments (V) that form a pair of antigen-binding regions. H -C H 1-VH-C H (1) includes. Linear antibodies may be bispecific or monospecific.

[0293] The antibodies disclosed herein may be recovered and purified from recombinant cell cultures by known methods, including but not limited to protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography ("HPLC") may also be used for purification.

[0294] The antibodies disclosed herein include naturally purified products, products of chemical synthesis procedures, and products produced from eukaryotic hosts (e.g., yeast, higher plants, insects, and mammalian cells) or, alternatively, from prokaryotic hosts as described above by recombinant technology. Many antibody production systems are described in Birch & Radner (2006) Adv. Drug Delivery Rev. 58:671-685.

[0295] If the antibody being tested binds to a protein or polypeptide, the antibody being tested and the antibody provided herein are equivalent. It is also possible to determine, without excessive experimentation, whether an antibody has the same specificity as the antibody disclosed herein by determining whether the antibody being tested prevents the antibody disclosed herein from binding to its protein or polypeptide (to which the antibody is normally reactive). If the antibody being tested competes with the antibody disclosed herein, as indicated by a decrease in binding by the monoclonal antibody disclosed herein, the two antibodies are likely to bind to the same or closely related epitopes. Alternatively, it is possible to pre-incubate the antibody disclosed herein with a protein to which it is normally reactive and determine whether the antibody being tested is inhibited in its ability to bind to that antigen. If the antibody being tested is inhibited, it is likely to have the same or closely related epitope specificity as the antibody disclosed herein.

[0296] The term “antibody” is also intended to include antibodies of all immunoglobulin isotypes and subclasses. Specific isotypes of monoclonal antibodies can be prepared directly by selection from the initial fusion, or secondarily by using sib selection techniques to isolate class-switched variants from parent hybridomas secreting different isotypes of monoclonal antibodies, using procedures described in Steplewski et al. (1985) Proc. Natl. Acad. Sci. USA 82:8653 or Spira et al. (1984) J. Immunol. Methods 74:307. Alternatively, recombinant DNA techniques may be used.

[0297] The isolation of other monoclonal antibodies having the specificity of the monoclonal antibodies described herein can also be achieved by those skilled in the art by generating anti-idiotype antibodies. Herlyn et al. (1986) Science 232:100. Anti-idiotype antibodies are antibodies that recognize specific determinants present on the monoclonal antibody of interest.

[0298] In several aspects disclosed herein, it is useful to make antibodies detectable or therapeutically labeled. Appropriate labeling is described above. Methods for conjugating antibodies to these agents are known in the art. For illustrative purposes only, antibodies may be labeled with a detectable moiety (e.g., a radioactive atom, chromophore, fluorescent phore, etc.). Such labeled antibodies may be used for diagnostic techniques either in vivo or in isolated test samples.

[0299] Coupling antibodies to low molecular weight haptens can increase the sensitivity of those antibodies in an assay. The haptens can then be specifically detected by a second reaction. For example, it is common to use haptens (e.g., biotin that reacts with avidin), or dinitrophenol, pyridoxal, and fluorescein (which can react with specific anti-hapten antibodies). See Harlow and Lane (1988) (cited above).

[0300] The variable regions of the antibodies disclosed herein can be modified by mutating amino acid residues within the CDR 1, CDR 2, and / or CDR 3 regions of the VH and / or VL regions to improve one or more binding properties (e.g., affinity) of the antibody. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and the effect on antibody binding or other functional properties of interest may be evaluated in appropriate in vitro or in vivo assays. In certain embodiments, conservative modifications are introduced, typically involving one, two, three, four, or five or fewer residues within the CDR region that are qualitatively altered. The mutations may be amino acid substitutions, additions, or deletions.

[0301] Framework modification can be performed on antibodies to reduce their immunogenicity, for example, by "backmutating" one or more framework residues into their corresponding germline sequences.

[0302] Furthermore, antibodies disclosed herein may be manipulated to include modifications within the Fc region to qualitatively alter one or more of the antibody's functional properties (e.g., serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cytotoxicity). Such modifications include, but are not limited to, altering the number of cysteine ​​residues in the hinge region to facilitate light- and heavy-chain assembly or to increase or decrease the antibody's stability (U.S. Patent No. 5,677,425) and altering the number of amino acid mutations in the Fc hinge region to reduce the antibody's biological half-life (U.S. Patent No. 6,165,745).

[0303] Furthermore, the antibodies disclosed herein can be chemically modified. Antibody glycosylation can be qualitatively altered, for example, by modifying one or more glycosylation sites in the antibody sequence to increase the antibody's affinity for an antigen (U.S. Patents 5,714,350 and 6,350,861). Alternatively, to increase antibody-dependent cell-mediated cytotoxicity, low-fucosylated antibodies with a reduced amount of fucosyl residues or antibodies with an increased bisected GlcNac structure can be obtained by expressing the antibody in host cells with a qualitatively altered glycosylation mechanism (Shields et al., 2002 J. Biol. Chem. 277:26733-26740; Umana et al., 1999 Nat. Biotech. 17:176-180).

[0304] Antibodies disclosed herein may be PEGylated to increase their biological half-life by reacting the antibody or its fragment with polyethylene glycol (PEG) or a reactive ester or aldehyde derivative of PEG under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. Antibody PEGylation may be carried out by acylation or alkylation reactions with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG used to derivatize other proteins (e.g., mono(C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide). The antibody to be PEGylated may be an unglycosylated antibody. Methods for PEGylation of proteins are known in the art and may be applied to the antibodies disclosed herein (EP 0154316 and EP 0401384).

[0305] Furthermore, antibodies can be chemically modified by conjugating or fusing their antigen-binding domain to a serum protein (e.g., human serum albumin) to increase the half-life of the resulting molecule. Such approaches are described, for example, in EP 0322094 and EP 0486525.

[0306] The antibodies or fragments thereof of this disclosure may be conjugated into diagnostic agents and used diagnostically, for example, to monitor the onset or progression of a disease and to determine the effectiveness of a given treatment regimen. Examples of diagnostic agents include enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances, positron-emitting metals, and non-radioactive ambient metal ions, which are used with various positron emission tomography techniques. The detectable substances may be coupled or conjugated either directly or indirectly through a linker to the antibody or its fragment using techniques known in the art. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansilchloride, or phycoerythrin. An example of a luminescent substance is luminol. Examples of bioluminescent substances include luciferase, luciferin, and aequorin. Examples of suitable radioactive substances include: 125 I, 131I, Indium-111, Lutetium-171, Bismuth-212, Bismuth-213, Astatine-211, Copper-62, Copper-64, Copper-67, Yttrium-90, Iodine-125, Iodine-131, Phosphorus-32, Phosphorus-33, Scandium-47, Silver-111, Gallium-67, Praseodymium-142, Samarium-153, Terbium-161, Dysprosium-166, Holmium-1 66, rhenium-186, rhenium-188, rhenium-189, lead-212, radium-223, actinium-225, iron-59, selenium-75, arsenic-77, strontium-89, molybdenum-99, rhodium-1105, palladium-109, praseodymium-143, promethium-149, erbium-169, iridium-194, gold-198, gold-199, and lead-211. Monoclonal antibodies can be indirectly conjugated with radioactive metal ions through the use of a bifunctional chelating agent covalently linked to the antibody. Chelating agents can be attached via amino groups (amities) (Meares et al., 1984 Anal. Biochem. 142: 68-78), sulfhydryl groups (Koyama 1994 Chem. Abstr. 120: 217262t), and carbohydrate groups (Rodwell et al. 1986 PNAS USA 83: 2632-2636; Quadri et al. 1993 Nucl. Med. Biol. 20: 559-570) of amino acid residues.

[0307] Furthermore, the antibodies or fragments thereof may be conjugated to therapeutic agents. Suitable therapeutic agents include: Taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, and dihydroxyanthracine dione. Dione), mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (e.g., mechloretamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozosin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives (e.g., carboplatin) )), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mitramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), diphtheria toxin and related molecules (e.g., diphtheria A chain and its active fragments and hybrid molecules), lysine toxin (e.g., lysine A or deglycosylated lysine A chain toxin), cholera toxin, Shiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 allonucleotide, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alloline, saporin, modesin, geranin, abrin A chain, modesin A chain, α-sarcin, Aleurites Fordii protein, dianthin protein, PhytolaccaAmericana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, geronin, mitogellin, restrietocin, phenomycin, enomycin toxin, and mixed toxins.

[0308] Further suitable conjugate molecules include ribonucleases (RNases), DNases, antisense nucleic acids, inhibitory RNA molecules (e.g., siRNA molecules), immunostimulatory nucleic acids, aptamers, ribozymes, triple-helix-forming molecules, and external guide sequences. Aptamers are small nucleic acids ranging from 15 to 50 nucleotides in length that fold into defined secondary and tertiary structures (e.g., stem-loop or G-quartet) and can bind to small molecules (e.g., ATP (US Patent No. 5,631,146) and theophylline (US Patent No. 5,580,737)) and large molecules (e.g., reverse transcriptase (US Patent No. 5,786,462) and thrombin (US Patent No. 5,543,293)). Ribozymes are nucleic acid molecules that can catalyze chemical reactions either intramolecularly or intermolecularly. Ribozymes typically cleave nucleic acid substrates through recognition and binding of their target substrates, which accompanies subsequent cleavage. Triple-helix-forming nucleic acid molecules can interact with double-stranded or single-stranded nucleic acids by forming a triple helix, where the three strands of DNA form a complex dependent on both Watson-Crick and Hoogsteen base pairing. Triple-helix molecules can bind to target regions with high affinity and specificity. Suitable conjugate molecules may further include any protein that binds to DNA, provided it does not form or stabilize a biofilm structure; it is conceivable that at least a partial set of such proteins could facilitate the kinetics of binding to the drugs disclosed herein.

[0309] Functional nucleic acid molecules may act as effectors, inhibitors, regulators, and stimulants of the specific activity of target molecules, or they may possess novel activities independent of any other molecules.

[0310] Therapeutic agents can be linked to their antibodies directly or indirectly using one of a very large number of available methods. For example, the drug can be attached to the hinge region of the reduced antibody component via disulfide bond formation using a crosslinking agent such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP), or via the carbohydrate moiety in the Fc region of the antibody (Yu et al. 1994 Int. J. Cancer). 56: 244; Upeslacis et al., "Modification of Antibodies by Chemical Methods", in Monoclonal antibodies: principles and applications, Birch et al. (eds.), pp. 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. (ed.), pp. 60-84 (Cambridge University Press 1995)).

[0311] 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.). (ed.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd edition), Robinson et al. (ed.), 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. (ed.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of 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).

[0312] The antibodies or antigen-binding domains disclosed herein may be ligated to another functional molecule (e.g., another antibody or ligand) to generate a bispecific or multispecific molecule that binds to at least two or more different binding sites or target molecules. Ligation of the antibody to one or more other binding molecules (e.g., another antibody, antibody fragment, peptide or binding mimetic) may be carried out, for example, by chemical coupling, gene fusion, or non-covalent association. The multispecific molecule may further include a third binding specificity in addition to the first and second target epitopes.

[0313] Bi-specific and multi-specific molecules can be prepared using methods known in the art. For example, each binding unit of the bi-specific molecule can be generated separately and then conjugated together. If the binding molecule is a protein or peptide, various coupling agents or crosslinking agents can be used for covalent conjugation. Examples of crosslinking 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, they can be conjugated by sulfhydryl bonds in the C-terminal hinge regions of their two heavy chains.

[0314] The antibodies disclosed herein may also be attached to solid supports that are particularly useful for immunoassays or purification of their target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0315] The antibody can also be bound to many different carriers. Therefore, this disclosure also provides compositions comprising the antibody and other substances (active or inactive). Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The properties of the carrier may be either soluble or insoluble for the purposes disclosed herein. Those skilled in the art may know of or confirm other suitable carriers for binding monoclonal antibodies using conventional experiments.

[0316] The antibodies disclosed herein may be used to purify the polypeptides disclosed herein and to identify bioequivalent polypeptides and / or polynucleotides. They may also be used to identify agents that modify the function of the polypeptides disclosed herein. These antibodies include polyclonal antisera, monoclonal antibodies, and various reagents familiar to those skilled in the art and derived from these preparations described above.

[0317] Antibodies that neutralize the activity of proteins encoded by identified genes can also be used in vivo and in vitro to demonstrate function by adding such neutralizing antibodies to in vivo and in vitro test systems. They are also useful as agents to modulate the activity of polypeptides disclosed herein.

[0318] Various antibody preparations can also be used in analytical methods such as ELISA assays or Western blotting to demonstrate the expression of proteins encoded by identified genes by testing cells in vitro or in vivo. Fragments of such proteins, produced by protease degradation during metabolism, can also be identified by using appropriate polyclonal antiserum along with samples derived from the experimental sample.

[0319] Furthermore, in some embodiments, the antibodies disclosed herein may be used to visualize and / or detect biofilms. In such embodiments, the antibodies may be detectably labeled with, for example, a radioisotope, an enzyme that produces a detectable product, a fluorescent protein, or conjugated to another part (e.g., a member of a specific binding pair (e.g., biotin (a member of the biotin-avidin specific binding pair))). The detectably labeled antibody may then be introduced into a sample suspected of containing a biofilm colony and visualized and / or detected by microscopy or other methods known to detect the associated label (e.g., spectroscopy, cytometry, or other common techniques). The conjugated antibody or the unlabeled antibody may also be identified by known analytical methods that target the conjugated part or antibody, respectively. For example, in some embodiments, a detectably labeled secondary antibody specific to the isotype of the antibody disclosed herein may be used in the visualization and / or detection of biofilms.

[0320] kit Kits comprising the necessary agents and instructions for carrying out the in vitro and in vivo methods as described herein are also claimed. Accordingly, the present invention provides kits for carrying out these methods, which may include the modified high mobility group-box 1 domain disclosed herein and instructions for carrying out the methods of the present invention (e.g., collecting tissue and / or performing screening and / or analyzing the results and / or administering an effective dose thereof as defined herein). These may be used alone or in combination with other suitable antimicrobial agents.

[0321] In one embodiment, the disclosure provides a polypeptide comprising a modified high mobility group-box 1 domain comprising one or more substitutions selected from the group C23S, C45S, and C106S, and a kit comprising instructions for use in inhibiting, preventing, or treating microbial infections that disrupt or biofilms. Examples of modified high mobility group-box 1 domains comprising one or more substitutions selected from the group C23S, C45S, and C106S are described above. In some embodiments, the modified high mobility group-box 1 domain comprises the substitution C45S. In some embodiments, the modified high mobility group-box 1 domain comprises the substitutions C23S, C45S, and C106S. In one embodiment, the kit further comprises one or more of an adjuvant, an antigenic peptide, or an antimicrobial substance. In yet another embodiment, the kit further comprises a carrier selected from the group consisting of liquid carriers, pharmaceutically acceptable carriers, solid-phase carriers, pharmaceutically acceptable carriers, implants, stents, pastes, gels, dental implants, or medical implants.

[0322] It is recognized that among the equivalents to the modified high mobility group-box 1 domain are modified high mobility group-box 2, modified high mobility group-box 3, and modified high mobility group-box 4, which include cysteine-to-serine substitutions at one or more amino acid positions corresponding to C23S, C45S, and C106S of modified high mobility group box 1. For modified high mobility group-box 2, its corresponding cysteine ​​residues are found at positions 23, 45, and 106; modified high mobility With respect to group-box 3, the corresponding cysteine ​​residues are found at positions 23, 45, and 104; and with respect to modified high mobility group-box 4, the corresponding cysteine ​​residues are found at positions 45, 104, 164, and 178. Accordingly, the applicant states that a kit surface disclosed with respect to modified high mobility group-box 1, which includes one or more substitutions selected from the group C23S, C45S, and C106S, is a modified high mobility group-box species (e.g., modified high mobility group-box 1, which includes one or more substitutions selected from the group C23S, C45S, and C106S) having one or more cysteine-to-serine substitutions at the position mentioned. Mobility group-box 2; Modified high mobility including one or more substitutions selected from the groups C23S, C45S, and C104S. We believe this is equally applicable to group-box 3; and modified high mobility group-box 4), which includes one or more substitutions selected from the groups C45S, C104S, C164S, and C178S.

[0323] The following examples are intended to illustrate but not to limit the invention. [Examples]

[0324] Example 1 - mHMGBI(C45S) test The applicant added 0.1 μg / ml, 1 μg / ml, and 5 μg / ml of HMGB1 to biofilms formed by urinary tract pathogenic E. coli UTI89 at seeding and at 24 hours. Dose-dependent disruption of UPEC biofilms was observed when HMGB1 was added at seeding and at 24 hours. Based on this information, the applicant thereby discloses a method for inhibiting, competing for, or titrating the binding of deoxyribonucleic acid B II (DNABII) polypeptides to microbial DNA in a biofilm, the method comprising, or instead essentially consisting of, or even more than, a step of contacting the microbial DNA in the biofilm with an effective amount of an isolated polypeptide or recombinant polypeptide containing a modified high mobility group-box 1 domain (mHMGB1) with a C45S substitution, thereby inhibiting, competing for, or titrating the binding of the DNABII polypeptide to the microbial DNA. The contacting step can be achieved in vitro or in vivo. Non-limiting examples of organisms that promote or cause such biofilms include one or more of the following: Burkholderia cenocepacia, Enterobacter spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterococcus faecium, urinary tract pathogenic Escherichia coli (UPEC), non-encapsulated Haemophilus influenzae (NTHI), and / or Aggregatibacter actinomycetemcomitans.

[0325] Preliminary results mHMGB1(C45S) retains its antibiofilm function in vitro against numerous human pathogens. To determine the antibiofilm function of the recombinant HMGB1(mHMGB1(C45S)), the applicant expressed and purified human recombinant HMGB1(rHMGB1) and recombinant recombinant HMGB1(mHMGB1(C45S)) in E. coli. In vitro 24-hour biofilms formed by a number of human pathogens, as shown in Table 1, were incubated with 5 μg / ml rHMGB1 or mHMGB1(C45S) for 16 hours. Enterococcus faecium biofilms were incubated with 20 μg / ml rHMGB1 or mHMGB1(C45S) for 16 hours. The biofilms were washed, stained with LIVE / DEAD® stain, and analyzed using confocal laser scanning microscopy and COMSTAT analysis. The applicant observed that mHMGB1(C45S) was effective in disrupting each of the pre-formed biofilms in vitro, as indicated by significant reductions in mean thickness and biomass compared to the control (Table 1). As shown in Table 1, the antibiofilm activity of mHMGB1(C45S) was also comparable to that of rHMGB1. These data suggest that substituting cysteine ​​with serine at position 45 does not qualitatively alter the antibiofilm activity of recombinant human HMGB1.

[0326] mHMGB1(C45S) retains its antibiofilm function in vivo in two different animal models, but its inflammatory response is significantly attenuated. To test the antibiofilm function of mHMGB1(C45S) in vivo, the applicant used two different animal models. In the first model, the applicant tested the ability of rHMGB1 and mHMGB1(C45S) to clear the NTHI biofilm in experimental OM, as performed by the applicant in a well-established chinchilla model (Novotny et al. 2011; 2013b; Novotny et al. 2016). The applicant first established the NTHI strain 86-028NP biofilm in the middle ear of chinchillas over four days. The applicant then treated the four-day-old NTHI biofilm with two doses (days 4 and 5; 5 μg each) of rHMGB1, mHMGB1(C45S), or a diluent. On day 6, the animals were sacrificed and their middle ears were blinded and scored for the presence of any remaining biofilm. Animals treated with the diluent showed a thick mucosal biofilm in the middle ear (Figures 1A-1B). Notably, rHMGB1 and mHMGB1(C45S) were highly effective in clearing the biofilm from the middle ear, as indicated by recognizable bony septa (Figures 1A-1B). In a second model, the applicant tested the ability of rHMGB1 and mHMGB1(C45S) to inhibit the biofilm formation of Burkholderia cenocepacia in the mouse airway. C57BL / 6 mice were given 10 7CFUs of B. cenocepacia were used to infect the respiratory tracts, and 5 μg of rHMGB1 or mHMGB1(C45S) was added simultaneously. After 18 hours, the animals were sacrificed, and bronchoalveolar lavage fluid (BAL) and lungs were collected. CFUs (Figure 2A), total inflammatory infiltration (Figure 2B), and total infiltrating neutrophils (Figure 2C) were counted in the BAL. rHMGB1 and mHMGB1(C45S) were effective in reducing the bacterial load in the BAL compared to the control (Figure 2A). rHMGB1 induced a strong inflammatory response, as evidenced by the increase in total inflammatory infiltration and neutrophils, while mHMGB1(C45S) showed a significantly weaker inflammatory response (Figures 2B and 2C). The applicant also evaluated lung injury 72 hours after infection and treatment, observing that lungs treated with rHMGB1 showed severe inflammation and an increased neutrophil response, while lungs treated with mHMGB1(C45S) more closely resembled uninfected mouse lungs (Figure 2D). Finally, the applicant examined the inflammatory activity of rHMGB1 and mHMGB1(C45S) in an in vivo chemotaxis model to determine their ability to recruit neutrophils (Orlova et al. 2007; Penzo et al. 2010). C57BL / 6 mice were injected with either 5 μg of mHMGB1(C45S) or rHMGB1, or 1 ml of 4% thioglycolate. After 4 hours, the mice were sacrificed, and total neutrophils in the peritoneal lavage fluid were quantified. As is clear from Figure 3, rHMGB1 induced neutrophil recruitment into the peritoneal cavity, whereas mHMGB1(C45S) did not recruit neutrophils. In summary, these results indicate that mHMGB1(C45S) promotes bacterial clearance without the undesirable pro-inflammatory activity of rHMGB1. [Table 1]

[0327] HMGB1 isoform (5 μg / ml) was added to bacterial biofilms prepared in vitro over 24 hours. 16 hours after inoculation (total 40 hours), the biofilms were washed, stained with LIVE / DEAD®, and subsequently visualized using CLSM. The biofilms were analyzed by Comstat to determine the average thickness. a (AT) and biomass a (BM) was calculated.

[0328] Further experiments were conducted to confirm the effects of mHMGB1(C45S) (Figures 4-7).

[0329] Example 2 - Triple mutant HMGB1 (C23S, C45S, and C106S) and other variants The applicant generates triple mutant HMGB1 and other variants containing C23S, C45S, and C106S substitutions (e.g., HMGB1 variants containing C23S alone, C23S and C45S, C23S and C106S, C45S and C106S, and C106S alone). These modified high mobility group-box 1 domains are tested for efficacy in the same manner as in Example 1. The same objective is carried out for the HMGB2, HMGB3, and HMGB4 variants disclosed herein (i.e., single, double, triple, and quadruple mutants whose sequences are provided herein above).

[0330] Equal parts Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains.

[0331] The technologies illustrated herein may be adequately implemented in the presence of any elements or limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be read broadly and without limitation. Furthermore, the terms and expressions used herein are for illustrative purposes only and not limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features or parts thereof shown or described; however, it is acknowledged that various modifications are possible within the scope of the claimed technologies.

[0332] Therefore, it should be understood that the materials, methods, and examples provided herein are representative and illustrative of preferred aspects and are not intended as limitations to the scope of this art.

[0333] This technology is described broadly and comprehensively in this specification. Each of the narrower species and subordinate comprehensive groupings that fall within the scope of the comprehensive disclosure also forms part of this technology. This includes the comprehensive description of this technology, with any subject matter to be removed from its genus, whether or not the material being removed is specifically described in this specification, along with any proviso or negative limitation.

[0334] Furthermore, if any feature or aspect of the present technology is described in terms of the Markush Group, a person skilled in the art will recognize that the present technology is also described in terms of any individual member or subgroup of any member of the Markush Group.

[0335] All publications, patent applications, patents, and other references referred to herein are expressly incorporated herein by reference to the same extent that each is incorporated by reference individually. In the event of any conflict, this specification, including its definitions, shall prevail.

[0336] Other aspects are described within the scope of the following claims. References Abraham, E., Arcaroli, J., Carmody, A., Wang, H., and Tracey, KJ (2000) HMG-1 as a mediator of acute lung inflammation. J Immunol 165: 2950-4. Agnello, D., Wang, H., Yang, H., Tracey, K. J., and Ghezzi, P. (2002) HMGB-1, a DNA-binding protein with cytokine activity, induces brain TNF and IL-6 production, and mediates anorexia and taste aversion. Cytokine 18: 231-6. Brandstetter, KA, Jurcisek, JA, Goodman, SD, Bakaletz, LO, and Das, S. (2013) Antibodies directed against integration host factor mediate biofilm clearance from Nasopore. Laryngoscope 123: 2626-32. Brockson, ME et al. (2014) Evaluation of the kinetics and mechanism of action of anti-integration host factor mediated disruption of bacterial biofilms. Mol Microbiol. Davalos, AR et al. (2013) p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes. J Cell Biol 201: 613-29.Devaraj, A., Justice, S.S., Bakaletz, L.O., and Goodman, S.D. (2015) DNABII proteins play a central role in UPEC biofilm structure. Mol Microbiol. Freire, M.O. et al. (2016) A Bacterial Biofilm Induced Oral Osteolytic Infection Can be Successfully Treated by Immuno-Targeting an Extracellular Nucleoid Associated Protein. Mol Oral Microbiol. Gong, W., Li, Y., Chao, F., Huang, G., and He, F. (2009) Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1. J Biomed Sci 16: 83. Goodman, S.D. et al. (2011) Biofilms can be dispersed by focusing the immune system on a common family of bacterial nucleoid-associated proteins. Mucosal Immunol 4: 625-37. Goodwin, G.H., Sanders, C., and Johns, E.W. (1973) A new group of chromatin-associated proteins with a high content of acidic and basic amino acids. Eur J Biochem 38: 14-9. Gustave, J.E., Jurcisek, J.A., McCoy, K.S., Goodman, S.D., and Bakaletz, L.O. (2013) Targeting bacterial integration host factor to disrupt biofilms associated with cystic fibrosis. J Cyst Fibros 12: 384-9. Idicula, W.A. et al. (2016) Identification of biofilms in post-tympanostomy tube otorrhea. Laryngoscope In Press. Justice, S.S. et al. (2012) Aberrant community architecture and attenuated persistence of uropathogenic Escherichia coli in the absence of individual IHF subunits. PLoS One 7: e48349. Kang, R. et al. (2014) HMGB1 in health and disease. Mol Aspects Med 40: 1-116. Kazama, H., Ricci, J.E., Herndon, J.M., Hoppe, G., Green, D.R., and Ferguson, T.A. (2008) Induction of immunological tolerance by apoptotic cells requires caspase-dependent oxidation of high-mobility group box-1 protein. Immunity 29: 21-32. Lee, H. et al. (2010) Analysis of nuclear high mobility group box 1 (HMGB1)-binding proteins in colon cancer cells: clustering with proteins involved in secretion and extranuclear function. J Proteome Res 9: 4661-70. Mardente, S. et al. (2012) HMGB1 induces the overexpression of miR-222 and miR-221 and increases growth and motility in papillary thyroid cancer cells. Oncol Rep 28: 2285-9. Melloni, E., Sparatore, B., Patrone, M., Pessino, A., Passalacqua, M., and Pontremoli, S. (1995a) Extracellular release of the 'differentiation enhancing factor', a HMG1 protein type, is an early step in murine erythroleukemia cell differentiation. FEBS Lett 368: 466-70. Melloni, E., Sparatore, B., Patrone, M., Pessino, A., Passalacqua, M., and Pontremoli, S. (1995b) Identity in molecular structure between "differentiation enhancing factor" of murine erythroleukemia cells and the 30 kD heparin-binding protein of developing rat brain. Biochem Biophys Res Commun 210: 82-9. Novotny, L.A., Amer, A.O., Brockson, M.E., Goodman, S.D., and Bakaletz, L.O. (2013a) Structural stability of Burkholderia cenocepacia biofilms is reliant on eDNA structure and presence of a bacterial nucleic acid binding protein. PLoS One 8: e67629. Novotny, L.A., Clements, J.D., and Bakaletz, L.O. (2011) Transcutaneous immunization as preventative and therapeutic regimens to protect against experimental otitis media due to nontypeable Haemophilus influenzae. Mucosal Immunol 4: 456-67. Novotny, L.A., Clements, J.D., and Bakaletz, L.O. (2013b) Kinetic analysis and evaluation of the mechanisms involved in the resolution of experimental nontypeable Haemophilus influenzae-induced otitis media after transcutaneous immunization. Vaccine 31: 3417-26. Novotny, L.A., Jurcisek, J.A., Goodman, S.D., and Bakaletz, L.O. (2016) Monoclonal antibodies against DNA-binding tips of DNABII proteins disrupt biofilms in vitro and induce bacterial clearance in vivo. EBioMedicine 10: 33-44. Orlova, V.V. et al. (2007) A novel pathway of HMGB1-mediated inflammatory cell recruitment that requires Mac-1-integrin. EMBO J 26: 1129-39. Paull, T.T., Haykinson, M.J., and Johnson, R.C. (1993) The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures. Genes Dev 7: 1521-34. Penzo, M. et al. (2010) Inhibitor of NF-kappa B kinases alpha and beta are both essential for high mobility group box 1-mediated chemotaxis [corrected]. J Immunol 184: 4497-509. Pistoia, V. and Raffaghello, L. (2011) Damage-associated molecular patterns (DAMPs) and mesenchymal stem cells: a matter of attraction and excitement. Eur J Immunol 41: 1828-31. Ranzato, E., Patrone, M., Pedrazzi, M., and Burlando, B. (2009) HMGb1 promotes scratch wound closure of HaCaT keratinocytes via ERK1 / 2 activation. Mol Cell Biochem 332: 199-205. Rocco, C.J., Davey, M.E., Bakaletz, L.O., and Goodman, S.D. (2016) Natural antigenic differences in the functionally equivalent extracellular DNABII proteins of bacterial biofilms provide a means for targeted biofilm therapeutics. Mol Oral Microbiol. Segall, A.M., Goodman, S.D., and Nash, H.A. (1994) Architectural elements in nucleoprotein complexes: interchangeability of specific and non-specific DNA binding proteins. EMBO J 13: 4536-48. Tang, D., Kang, R., Livesey, K.M., Zeh, H.J., 3rd, and Lotze, M.T. (2011) High mobility group box 1 (HMGB1) activates an autophagic response to oxidative stress. Antioxid Redox Signal 15: 2185-95. Wang, H. et al. (1999) HMG-1 as a late mediator of endotoxin lethality in mice. Science 285: 248-51. Yang, D., Chen, Q., Yang, H., Tracey, K.J., Bustin, M., and Oppenheim, J.J. (2007) High mobility group box-1 protein induces the migration and activation of human dendritic cells and acts as an alarmin. J Leukoc Biol 81: 59-66. Yang, H. et al. (2012) Redox modification of cysteine residues regulates the cytokine activity of high mobility group box-1 (HMGB1). Mol Med 18: 250-9. Zetterstrom, C.K., Strand, M.L., and Soder, O. 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Claims

1. Isolated polynucleotides or recombinant polynucleotides encoding a modified high mobility group-box 1 domain, wherein the modified high mobility group-box 1 domain comprises Sequence ID No. 2 having a C45S substitution or a C23S and / or C106S substitution in addition to C45S.

2. The isolated polynucleotide or recombinant polynucleotide according to claim 1, wherein the modified high mobility group-box 1 domain comprises the substituted C23S, C45S, and C106S.

3. The isolated polynucleotide or recombinant polynucleotide according to claim 1 or 2, wherein the isolated polynucleotide or recombinant polynucleotide comprises DNA or RNA.

4. A vector comprising an isolated polynucleotide or recombinant polynucleotide according to any one of claims 1 to 2.

5. The vector according to claim 4, wherein the vector is selected from plasmids or viral vectors.

6. A composition comprising an isolated polynucleotide or recombinant polynucleotide according to any one of claims 1 to 2.

7. A composition comprising the vector described in Claim 4.

8. A host cell comprising an isolated polynucleotide or recombinant polynucleotide according to any one of claims 1 to 2.

9. A host cell comprising the vector described in Claim 4.

10. A host cell comprising the composition described in Claim 6.

11. A method for producing an isolated polypeptide or recombinant polypeptide comprising a modified high mobility group-box 1 domain, comprising growing the host cells described in claim 8 under conditions that enable the production of the isolated polypeptide or recombinant polypeptide.

12. A method for producing an isolated polypeptide or recombinant polypeptide comprising a modified high mobility group-box 1 domain, comprising growing the host cells described in claim 9 under conditions that enable the production of the isolated polypeptide or recombinant polypeptide.

13. A method for producing an isolated polypeptide or recombinant polypeptide comprising a modified high mobility group-box 1 domain, comprising growing the host cells described in claim 10 under conditions that enable the production of the isolated polypeptide or recombinant polypeptide.

14. The method according to claim 13, further comprising isolating the polypeptide from the host cells or cell culture.

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