HMGB1 protein derivatives for biofilm removal

Abstract

The present invention relates to HMGB1 protein derivatives for use in removing biofilms. Provided herein are derivatives of HMGB1 that have been engineered to have the same potent anti-biofilm activity, but are smaller and do not cause inflammation.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 62 / 742,102, filed on October 5, 2018, the entire contents of which are incorporated herein by reference.

[0003] Federal Support Statement

[0004] This invention was made with government support under Grant No. R01DC011818 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention. Technical Field

[0005] The present invention generally relates to methods and compositions for reducing and / or treating clinical or industrial bacterial biofilms. Background Art

[0006] The persistence of bacteria in biofilms within the human body causes approximately two-thirds of all chronic / recurrent diseases. These biofilms consist of bacteria protected by an outer "slime," which is typically composed primarily of DNA that prevents the innate and adaptive immune systems, antibiotics, and other antimicrobial agents from reaching the bacteria inside the biofilm. Biofilms make it extremely difficult to clear infections from the body. Furthermore, biofilms can serve as a reservoir for future acute infections, often with fatal consequences.

[0007] At least one protein from the DNABII protein family has been found in all known true bacteria and is naturally present outside bacterial cells. Although they elicit a strong innate immune response, the host subject fails to naturally produce specific antibodies against family members due to infection. The main problem with bacterial biofilms is that the host immune system and / or antibiotics and other antimicrobial agents cannot enter the protected bacteria within the biofilm.

[0008] Biofilms also exist in industrial settings. For example, biofilms are a widespread problem in oil processing, from production sites to gas station storage tanks. In the field, sulfate-reducing biofilm bacteria produce hydrogen sulfide (sour oil). Within the process flow, biofilm activity forms slime that can clog filters and orifices. Biofilms and biofilm organisms can also cause corrosion in pipelines and oil processing equipment. These problems can manifest throughout an oil or gas production facility, with fouling and corrosive biofilm organisms even being found on the surfaces of final product storage tanks.

[0009] In the home, biofilms are found in or on any surface that supports the growth of microorganisms, such as in drains, on food preparation surfaces, in toilets, and in swimming pools and spas.

[0010] Biofilms are relevant to various water treatment processes in homes and industry. They can grow on surfaces of process equipment and hinder performance, such as reduced heat transfer or clogging of filters and membranes. Biofilms growing on cooling tower fill can add weight sufficient to cause the fill to collapse. Biofilms can even corrode highly specialized stainless steel. Biofilms in water treatment processes can reduce the value of the end product. Biofilms growing in drinking water distribution systems can harbor potentially pathogenic, corrosive, or bacterial organisms, reducing the aesthetic quality of the water.

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

[0012] It is well known that bacterial biofilms are remarkably resistant to existing treatment modalities (e.g., >1000-fold more resistant to antimicrobial agents than their planktonic counterparts). Due to the high prevalence and enormous consequences of biofilm-mediated infections in terms of attributable mortality and economic burden, novel treatments are urgently needed. One of the defining characteristics of biofilms is the extracellular polymeric substance in which the biofilm cells are embedded. Key components of the extracellular polymeric substance are extracellular DNA and the bacterial DNABII family of proteins, which are essential for the structural integrity of the biofilm. Targeting and sequestering DNABII proteins disrupts the biofilm. High Mobility Group B1 (HMGB1) protein is a eukaryotic protein that binds to DNA and binds to the same DNA structure as DNABII proteins, leading to the destruction of bacterial biofilms. Derivatives of HMGB1 can be engineered to have the same potent anti-biofilm activity, but are smaller and do not cause inflammation.

[0013] Applicants disclose herein a new concept for treating bacterial biofilm-mediated infections by repurposing derivatives of innate immune effectors. HMGB1 domains differ in function. Domain variants with anti-biofilm activity and no pro-inflammatory consequences represent an embodiment of HMGB1 for treating biofilm-mediated infections without the consequence of excessive inflammation. In vivo and in vitro experiments have shown that antibodies against the DNABII family of bacterial nucleoside-related proteins (IHF and HU) are very effective against many different bacterial biofilms that cause a variety of stubborn human infections. In contrast, the present invention utilizes the immune response component HMGB1 to treat biofilm-mediated diseases without the consequence of excessive inflammation. In vitro bacterial biofilms were exposed to the anti-biofilm properties of HMGB1 and its various truncated domains (A box, B box, B box linker, mutated B box C106S, AB box, and all with linkers).

[0014] Compositions and formulations containing the protein derivatives can be used to treat persistent, chronic, or recurrent biofilm-mediated infections. The compositions can be used to treat drug-resistant nosocomial infections, including infections associated with indwelling medical devices, such as catheters or prostheses, as well as ear and respiratory infections in chronic / recurrent infections (e.g., patients with cystic fibrosis). In addition, since many bacteria released from biofilms have been shown to be more sensitive to both host defenses and antimicrobial agents, they can be used in combination with established treatments (i.e., antibiotics).

[0015] Thus, in one aspect, the present invention provides an isolated A Box polypeptide, optionally comprising one or more amino acid mutations selected from K12, C23 and C45 (e.g., native K or C is modified to an amino acid selected from serine, glycine, alanine, valine, isoleucine or threonine) or an equivalent thereof, said equivalent comprising one or more amino acid mutations selected from K12, C23 and C45 (e.g., native K or C is modified to an amino acid selected from serine, glycine, alanine, valine, isoleucine or threonine), or consisting essentially of, or consisting of. In one aspect, the mutation is a C45S mutation. The A Box polypeptide may further comprise a linker or peptide sequence located at one or both termini. A non-limiting example is a polypeptide linker of the sequence PPKGETKKKF. When recombinantly produced, the A Box polypeptide may be partially or fully acetylated, oxidized or phosphorylated. In one aspect, the A Box polypeptide comprises, consists essentially of, or consists of amino acids 1 to 70 of a wild-type HMGB1 polypeptide, optionally comprising one or more mutations identified above.

[0016] Also provided herein are isolated B Box polypeptides that optionally comprise, consist essentially of, or consist of a mutation at amino acid C106 (e.g., a native cysteine ​​to an amino acid selected from serine, glycine, alanine, valine, isoleucine, or threonine), or an equivalent thereof. In one aspect, the B Box polypeptide comprises, consists essentially of, or consists of amino acids about 80 to about 176, or about 88 to about 164, or about 89 to about 162, or about 80 to about 164 of an HMGB1 polypeptide. Other positions for modifying wild-type HMGB1 B Box polypeptides are Figure 1 Shown in C.

[0017] The B Box polypeptide may further comprise a linker or peptide sequence at one or both termini. A non-limiting example is a polypeptide linker of the sequence PPKGETKKKF. When recombinantly produced, the disclosed B Box polypeptides may be partially or fully acetylated, oxidized, or phosphorylated.

[0018] In another aspect, provided herein are isolated AB Box polypeptides optionally comprising, consisting essentially of, or consisting of one or more amino acid mutations selected from K12, C23, C45, or C106 (e.g., native K or C is modified to an amino acid selected from serine, glycine, alanine, valine, isoleucine, or threonine), or an equivalent thereof, the equivalent comprising, consisting essentially of, or consisting of one or more amino acid mutations selected from K12, C23, and C45 (e.g., native K or C is modified to an amino acid selected from serine, glycine, alanine, valine, isoleucine, or threonine). In one aspect, the mutation is a C45S mutation. In another aspect, the polypeptide comprises a mutation at amino acid C106 (e.g., a native cysteine ​​is changed to an amino acid selected from serine, glycine, alanine, valine, isoleucine, or threonine), or an equivalent thereof, the equivalent comprising one or more amino acid mutations selected from K12, C23, C45, and a mutation at amino acid C106 (e.g., a native cysteine ​​is changed to an amino acid selected from serine, glycine, alanine, valine, isoleucine, or threonine). In one aspect, the AB Box polypeptide and equivalents comprise C45S and C106S mutations. In one aspect, the AB Box polypeptide or its equivalent comprises amino acids 1 to 176, or 1 to 162, or 1 to 164 of a wild-type HMGB1 polypeptide with the proposed amino acid mutations, or consists essentially of, or consists of.

[0019] In another aspect, the isolated AB Box polypeptide further comprises a linker polypeptide connecting the A Box polypeptide and the B Box polypeptide, and in one aspect, further comprises a second linker connecting the B Box polypeptide and the C Box polypeptide. A non-limiting example is a polypeptide linker of the sequence PPKGETKKKF. When recombinantly produced, the AB or A, B, and C Box polypeptides can be partially or fully acetylated, oxidized, or phosphorylated. In one aspect, the isolated mutant HMGB1 polypeptide has one or more amino acid substitutions as described herein in the A and / or B Box domains, which can optionally be partially or fully acetylated, oxidized, or phosphorylated.

[0020] In one aspect, the isolated polypeptide further comprises a detectable label.

[0021] Also provided herein is a recombinant polypeptide comprising, consisting essentially of, or consisting of one or more isolated polypeptides as described herein, and further comprising at least one additional amino acid at one or both termini.

[0022] The present invention also provides antibodies that bind to or are generated against the mutant polypeptides described herein. Such antibodies can be used as diagnostic and prognostic agents. Further provided are one or more isolated polypeptides and / or antibodies described herein and a carrier, such as a pharmaceutically acceptable carrier.

[0023] The present invention also provides polynucleotides encoding isolated polypeptides or antibodies as described herein and their complementary sequences. In one aspect, the polynucleotides are detectably labeled. The polynucleotides can optionally be operably linked to a promoter and / or enhancer to express the polynucleotides. A method for recombinantly producing a polypeptide is also provided, which is produced and isolated by expressing the polynucleotides in a suitable expression system (e.g., a host cell) and then producing and isolating the recombinantly produced polypeptide.

[0024] Further provided is a vector comprising, consisting essentially of, or consisting of a polynucleotide described herein.

[0025] In another aspect, the present invention provides an isolated host cell comprising one or more of the polypeptides, polynucleotides, or vectors described herein. Also provided are compositions comprising a vector and one or more of the polypeptides, polynucleotides, or vectors described herein. In one aspect, the vector is a pharmaceutically acceptable carrier.

[0026] Polypeptide and the compositions that comprise them have multiple uses.For example, they can be used to suppress, compete or titrate the method for the combination of DNABII polypeptide or protein and microbial DNA, and this method contacts DNABII polypeptide or protein or microbial DNA with polypeptide as herein described or composition.They can also be used to suppress, prevent or destroy the method for microbial biofilm, and this method contacts biofilm with polypeptide as herein described or composition.

[0027] The polypeptides and compositions can also be used in methods of inhibiting, preventing, or destroying biofilms in a subject or treating infections or diseases associated with biofilms by administering to the subject an effective amount of the composition or polypeptide described herein.

[0028] The polypeptides and compositions can further be used in methods of inhibiting, preventing, or treating biofilm-producing microbial infection in a subject by administering to the subject an effective amount of a composition or polypeptide described herein.

[0029] The method may further comprise contacting or administering an effective amount of an additional agent, such as an antimicrobial agent, to treat the underlying infection.

[0030] Biofilms and infections that can be treated by these methods can be caused by bacterial infections, such as infections caused by ESKAPE pathogens, uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae, Burkholderia cenocepacia, S. epidermidis, Streptococcus agalactiae, Neisseria meningitidis, Treponema denticola, Treponemapallidum, Burkholderia cepacia, Burkholderia pseudomallei, Haemophilus influenzae (nontypeable) (NTHI), Moraxella catarrhalis, and catarrhalis), Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, or Mycobacterium tuberculosis. Device-associated infections caused by biofilms include, for example, ventricular-derived, contact lenses, endotracheal tubes, prosthetic heart valves, pacemakers, as well as vascular grafts, tissue fillers and breast implants, peripheral vascular catheters, urinary catheters, orthopedic implants, and prosthetic joints. Tissue-associated infections that can be treated by the compositions and methods include, for example, chronic otitis media, chronic sinusitis, chronic tonsillitis, dental plaque, chronic laryngitis, endocarditis, lung infections (upper, middle, and lower airway infections (otitis media, sinusitis, bronchitis, and exacerbations of chronic obstructive pulmonary disease (COPD))), chronic cough, complications and / or primary causes of cystic fibrosis (CF) and community-acquired pneumonia (CAP), kidney stones, biliary tract infections, urinary tract infections, Burkholderia infections, osteomyelitis, superficial wounds, and chronic wounds.

[0031] The subject may be a mammal, such as a human, or an infant or a teenager.

[0032] Also provided is a kit comprising, consisting of, or consisting of an isolated polypeptide, antibody, polynucleotide, vector, host cell, or composition described herein and instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A-1D: Structure of HMGB1, DNABII / HMGB1-DNA complex. ( Figure 1 A) HMGB1 consists of three domains: the A Box, the B Box, and the acidic C Tail. The A+B Boxes are primarily DNA binding domains (brackets), while the C Tail mediates nuclear function, transcriptional stimulation, and antimicrobial activity. The AA of each domain is shown (left). C23 and C45 can form disulfide bonds, resulting in decreased DNA binding affinity and increased proinflammatory activity. C106 mediates proinflammatory activity through RLR4-MD2 binding. The C106S mutation has been shown to reduce proinflammatory responses without loss of binding. ( Figure 1 B) Left: IHF dimer bound to DNA. The β-ribbon arms of IHF cross the minor groove to bend the DNA, wrapping the molecule around the N-terminal α-helix from the concave side. Right: Two HMGB1A box domains bound to DNA; mimicking the binding of the A and B boxes in native HMGB1. The α-helix binds to the minor groove, bending the DNA molecule from the convex side. Modified from PDB 4QR9. The amino acid sequences of HMGB1 polypeptides, truncations, fusions, and mutants are provided in the sequence listing below. ( Figure 1 C) The HMGB1 B Box construct consists of N- and C-terminal linkers, two predicted α-localized regions, and a flexible linker between them. Several amino acids (AA) within the B Box can undergo post-translational modifications (PTMs, including acetylation / methylation of Lys, glycosylation of Asn, phosphorylation of Tyr, and oxidation of Cys). However, >20% of the peptides observed from LC-MS / MS analysis of rHMGB1 or nHM showed no B Box PTMs. Figure 1 D) Truncations remove the flexible linker from the N- and C-termini and one of the two alpha helical regions. The two smallest helical region truncations (AA 99-133, 138-164) are believed to retain antibiofilm activity.

[0034] Figure 2 Model of HMGB1-mediated biomembrane collapse. DNABII stabilizes the eDNA scaffold by binding to a vertices structure similar to a Holliday junction (HJ). Antibodies (Y) can sequester DNABII and alter the homeostasis, leading to collapse. HMGB1 can also disrupt the eDNA structure by binding to DNA and forming a transient, unstable intermediate (bracket).

[0035] Figure 3: Structure and constructed variants of HMGB1. Full-length recombinant HMGB1 (rHMGB1) expressed in Escherichia coli contains three domains, consisting of the A Box, B Box, and C Tail. The HMGB1 AB Box structure consists of the A and B domains and a C-terminal linker between the B box and the C tail, but lacks the C tail. The A box construct contains the A box and a C-terminal linker between the A box and the B box, but lacks the B-box and the C tail. The B box construct contains the N- and C-terminal linkers, but lacks the A Box and the C tail. The C45S mutation in rHMGB1 (mH MGB1) and the C106S mutation in the B Box (mB Box) were created to reduce the pro-inflammatory activity of HMGB1. See also Figure 1 .

[0036] Figure 4 HMGB1 variants retain DNA binding ability. Increasing concentrations (100 and 250 nM) of commercially available bovine HMGB1 (bHMGB1), B Box C106S (mB Box), rHMGB1, HMGB1 C45S (mHMGB1), A Box, B Box, and A+B Box were incubated with 5'-end labeled 6-carboxyfluorescein-labeled Holliday junction (HJ) DNA (20 nM) and then separated on a 6% native polyacrylamide gel. As demonstrated by differences in HJ migration, HMGB1 and HMGB1 variants retained DNA binding activity.

[0037] Figure 5 A-5E: Anti-spot effects of HMGB1 variants against high-priority bacterial pathogens. The indicated HMGB1 variants or antibodies against DNABII protein (α-1HF Ec IgG, 1 μM) was added to the preformed bacterial biofilms in vitro for 24 h. The cells were stained and then observed by confocal laser scanning microscopy. The images were analyzed by COMSTAT to calculate the average thickness and plotted against the control. Figure 5 A) Full-length HMGB1 isoforms (200 nM unless otherwise indicated) were added to UPEC, Burkholderia cenocepacia, NTHI, or ESKAPE pathogens. 800 nM rHMGB1 and 200 nM mHMGB1 were added for Staphylococcus aureus (ESKAPE). 800 nM rHMGB1, 800 nM mHMGB1, or 3.3 μM α-1HF were added. Ec IgG was used for E. faecium and incubated for 1 hour (instead of 16 hours) to avoid degradation by E. faecium proteases. Figure 5B) Representative images of UPEC biofilms incubated with increasing concentrations of rHMGB1. ( Figure 5 C) The anti-hemoglobin activity of each domain of HMGB1 (200 nM) was tested as described above (dashed line represents control value). Bars represent SEM. Statistical significance compared to the control group was assessed using an unpaired t-test, *P<0.05. HMGB1 and its variants (Box A cannot) are able to significantly disrupt established biofilms formed by high-priority human pathogens. ( Figure 5 D-5E) HMGB1 and variants disrupted preformed K. pneumoniae biofilms. rHMGB1, HMGB1 C45S (mHMGB1), bovine HMGB1 (bHMGB1), B box, B Box C106S (mB Box), A Box, and A+B Box (200 nM) were added to preformed K. pneumoniae biofilms at 24 h. After 16 h of incubation, the biofilms were incubated with The cells were stained and observed by confocal laser scanning microscopy. Images were analyzed by COMSTAT to calculate mean thickness and total biomass. Error bars represent SEM. ***P < 0.0001; ns = not significant. HMGB1 and its variants (Box A) significantly disrupted established K. pneumoniae biofilms.

[0038] Figure 6 HMGB1 variants disrupt pathogenic biofilms. Antibiofilm activity of individual domains of HMGB1 (200 nM) was tested as described above (dashed line represents control value). Bars represent mean squared error (SEM). *P < 0.05. HMGB1 and its variants (but not A Box) were able to significantly disrupt established biofilms formed by high-priority human pathogens.

[0039] Figure 7 A-7G: HMGB1 promotes the clearance of Staphylococci from mice without causing sepsis. C57BL / 6 mice were challenged intratracheally with 107 CFU and received 0.2 nmol of HMGB1 variants simultaneously (preventive) or 24 hours later (therapeutic). Figure 7 A) Aggregates of Burkholderia cenocepacia are seen by fluorescence microscopy in lung sections probed with α-B. cenocepacia antibodies. Figure 7 B) Bronchoalveolar lavage (BAL) fluid collected 18 hours post-inoculation (hpi) was analyzed for CFU. Figure 7 C) Quantification of neutrophils in BAL by differential cell counting. Figure 7 D) Lung tissue collected at 72 hpi was fixed, embedded, sectioned, and stained with hematoxylin and eosin (magnification 10 times). Figure 7 E) Quantification of CFU in BAL. Figure 7 F) Neutrophil recruitment was analyzed 24 hours after intraperitoneal (ip) administration of HMGB1 derivatives by fluorescence-activated cell sorting of peritoneal lavage fluid stained with α-CD45, α-CD11b, and αLy-6G. Figure 7 G) Serum TNF-α was measured by ELISA in mice injected intraperitoneally with 0.2 nmol HMGB1 variants, 5 mg / kg LPS, or both. LoD: limit of detection. Bars represent SD. *P < 0.05. HMGB1 treatment significantly reduced the CFU of Burkholderia cenocepacia in the lungs. The Cys to Ser mutation of HMGB1 abolished its proinflammatory activity, and none of these HMGB1 variants caused sepsis.

[0040] Figure 8 A-8F: rHMGB1 and mHMGB1 promote biofilm dissociation in an experimental otitis media model. Diluent, 0.2 nmol rHMGB1, or 0.2 nmol mHMGB1 were delivered directly to the middle ear of ground squirrels on days 4 and 5 after challenge with NTHI. Animals were sacrificed 24 hours later, and the middle ear was imaged ( Figure 8 C, representative images), and according to ( Figure 8 A, biofilm) and ( Figure 8 B, inflammation) of the biofilm ( Figure 8 D) and inflammation ( Figure 8 The presence of NTH1 in mucosal biomass was quantified by blind scoring. Figure 8 E). Means and SEM are plotted. **P < 0.01, ****P < 0.0001. Image scoring and CFU quantification demonstrated that both rHMGB1 and mHMGB1 promoted the clearance of established NTHI biofilms in vivo. Notably, mHMGB1 did not induce significant inflammation.

[0041] Figure 9 : HMGB1 enhances antibiotic-mediated killing. Biofilms of Burkholderia cenocepacia were formed for 24 h, and then minocycline (1 μg / ml), rHMGB1 (200 nM), or rHMGB1 (200 nM) + minocycline (1 μg / ml) were added for another 16 h. The cells were stained and imaged by CLSM. Live cells are shown in green, and dead cells in red. Note that the increase in dead cells was only observed in the presence of both HMGB1 and minocycline.

[0042] Figure 10 A-10B: HMGB1 in specimens of biofilm-related diseases. Figure 10 A) Sections of OCT-embedded mucosal biopsy from the middle ear of NTHI-infected ground squirrels were co-labeled for HMGB1 and DNABII proteins for immunofluorescence microscopy. Double-stranded cDNA was labeled with DAPI (white). Figure 10 B) CF sputum was treated with anti-biofilm treatment (PBS, 1:10 rabbit α-1HF Ec , serum, 100U / ml The cells were incubated with DNase (DNase), 1 mM rHMGB1, and 37°C for 1 hour. The optical density of the surrounding culture medium was measured at 0 and 2 hours. DNABII protein and HMGB1 are present in scalloped mucosal biofilms in vivo but do not colocalize on eDNA. Exogenous HMGB1 can disrupt biofilms present in CF sputum. DETAILED DESCRIPTION

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present disclosure, preferred methods, devices, and materials are now described. All techniques and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein shall be construed as an admission that the present disclosure is not entitled to preempt prior disclosure.

[0044] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, for example, Sambrook and Russelleds. (2001) Molecular Cloning: A Laboratory Manual, 3 rdedition; Ausubel etal. eds. (2007) Current Protocols in Molecular Biology series; Methods in Enzymology (Academic Press, Inc., N.Y.) series; MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5 thedition; Gait ed. (1984) Oligonucleotide Synthesis; USPatent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic AcidHybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL) Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) GeneTransfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology(Academic Press, London); and Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology.

[0045] All numerical values ​​(including ranges), such as pH, temperature, time, concentration, and molecular mass, are approximate values ​​with variations of (+) or (-) 0.1, as appropriate, or with variations of + / - 15%, or 10%, or 5%, or 2%. It should be understood that all numerical values ​​are preceded by the term "about", although not always explicitly stated. It should also be understood that the agents described herein are exemplary only, and that equivalents thereof are known in the art.

[0046] As used in the specification and claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a polypeptide" includes a plurality of polypeptides, including mixtures thereof.

[0047] As used herein, the terms "comprising" or "including" are intended to indicate compositions and methods that include the listed elements but do not exclude other elements. When used to define compositions and methods, "consisting essentially of shall mean excluding other elements that have any substantial significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein will not exclude trace contaminants from separation and purification methods and pharmaceutically acceptable carriers (e.g., phosphate buffered saline, preservatives, etc.). "Consisting of" refers to excluding trace elements of other ingredients and substantial method steps for administering the compositions disclosed herein. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0048] "Biofilm" refers to an organized community of microorganisms that sometimes attach to the surface of a structure, which can be organic or inorganic, along with polymers such as DNA that they secrete and / or release. Biofilms are highly resistant to microorganisms and antimicrobial agents. They live on gum tissue, teeth, and restorations, causing caries and periodontal disease (also known as periodontal plaque disease). They can also cause chronic middle ear infections. Biofilms can also form on the surfaces of dental implants, stents, catheters, and contact lenses. They grow on pacemakers, heart valve replacements, artificial joints, and other surgical implants. The Centers for Disease Control estimates that more than 65% of nosocomial (hospital-acquired) infections are caused by biofilms. Fungal biofilms also frequently contaminate medical devices. They can cause chronic vaginal infections and lead to life-threatening systemic infections in people with weakened immune systems. Biofilms are also involved in a variety of diseases. For example, patients with cystic fibrosis suffer from Pseudomonas infections, which often lead to the formation of antibiotic-resistant biofilms.

[0049] " DNABII polypeptide or protein " means to be made up of DNA binding domain and therefore have DNA binding protein or polypeptide of specificity or general affinity to microbial DNA.On the one hand, they combine DNA in minor groove.The limiting examples of DNABII protein are integration host factor (IHF) albumen and histone-like protein (HU) from Escherichia coli strain U93.Other DNA binding proteins that may be relevant to biofilm 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).

[0050] " IHF " protein " integration host factor " is such bacterial protein, and it is used for its DNA to be incorporated in host bacteria by phage.These are DNA binding proteins that work in gene recombination and transcription and translation regulation.They also combine extracellular microbial DNA.The gene of IHF protein subunit in coding Escherichia coli is himA (Genbank accession number: POA6X7.1) and himD (POA6Y1.1) gene.The homologue of these genes is found in other organisms, and can find in table 1 with the peptide corresponding to these genes from other organisms.

[0051] "HMGB1" is a high mobility group box (HMGB) 1 protein that is reported to bind to and distort the minor groove of DNA and is an example of an interfering agent. Recombinant or isolated proteins and polypeptides are commercially available from Atgenglobal, ProSpecBio, Protein1, and Abnova.

[0052] An "A Box" polypeptide is a polypeptide comprising the A Box domain of the HMGB1 protein. A Box polypeptides can be mutated or include other sequences, such as linker sequences, signal sequences, or secretory sequences. Non-limiting examples are shown in the accompanying figures and sequence listing. One or more point mutations can be introduced in amino acids K12, C23, and C45.

[0053] A "B Box" polypeptide is a polypeptide comprising the B Box domain of the HMGB1 protein. B Box polypeptides can be mutated or include other sequences, such as linker sequences, signal sequences, or secretion sequences. Point mutations in amino acids K114 or C106 can be introduced to affect DNA binding, inflammatory properties, and anti-biofilm activity. Non-limiting examples are shown in the accompanying figures and sequence listing.

[0054] An "AB Box" polypeptide is a polypeptide comprising the A and B box domains of an HMGB1 protein fused together but lacking the amino acids corresponding to the full-length wild-type protein. AB Box polypeptides can be mutated or comprise other sequences, such as linker sequences, signal sequences, or secretory sequences. One or more point mutations in the amino acids described herein (e.g., at amino acids K12, C23, C45, C106, and / or K114) can be introduced to affect DNA binding, inflammatory properties, and anti-biofilm activity. Non-limiting examples are shown in the accompanying drawings and sequence listing.

[0055] "HU" or "histone-like protein of Escherichia coli strain U93" refers to a class of heterodimeric proteins normally 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 to HU proteins are commercially available from Abeam.

[0056] "Linker" or "peptide linker" refers to a peptide sequence connected to the N-terminus or C-terminus of a polypeptide sequence. In one aspect, the length of the linker is from about 1 to about 20 amino acid residues, or from 2 to about 10, or from about 3 to about 5 amino acid residues. Examples of peptide linkers are Gly-Pro-Ser-Leu-Lys-Leu or PPKGETKKKF.

[0057] "Microbial DNA" means single-stranded or double-stranded DNA from biofilm-producing microorganisms.

[0058] As used herein, the term "detectable label" means a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, such as an N-terminal histidine tag (N-His), a magnetically active isotope (e.g. 115 Sn, 117 Sn and 119 Sn), non-radioactive isotopes (e.g. 13 C and 15N), polynucleotides or proteins (such as antibodies), to generate "labeled" components. The term also includes sequences conjugated to polynucleotides, which will provide signals when the inserted sequence is expressed, such as green fluorescent protein (GFP) etc. The label itself can be detectable (for example, radioisotope labels or fluorescent labels), or in the case of enzyme labels, can catalyze the chemical changes of detectable substrate compounds or compositions. Labels may be suitable for small-scale detection, or are more suitable for high-throughput screening. Like this, suitable labels include but are not limited to magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes and proteins (including enzymes). Labels can be simply detected or can be quantified. The response that is simply detected generally includes the response that is only confirmed to exist, and the response that is quantified generally includes the response with a quantifiable (for example, digitally reportable) value such as intensity, polarization and / or other properties. In luminescence or fluorescence determination, the luminophore or fluorophore relevant to the actual determination component of participation in combination can be directly used to directly produce a detectable reaction, or the luminophore or fluorophore relevant to another (such as reporter molecule or indicator) component can be indirectly used to indirectly produce a detectable response. The example of the luminescent labeling that produces a signal includes but is not limited to bioluminescence and chemiluminescence. Detectable luminescent response generally includes a change or the occurrence of a luminescent signal. Suitable methods and luminophores for measuring components of luminescent labels are known in the art, for example, described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (sixth edition). The example of luminescent probes includes but is not limited to aequorin and luciferase.

[0059] A "gene delivery vector" is defined as any molecule that can carry an inserted polynucleotide into a host cell. Examples of gene delivery vectors are: liposomes, micelles, biocompatible polymers, including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria or viruses, such as baculoviruses, adenoviruses, and retroviruses, phages, cosmids, plasmids, fungal vectors, and other recombinant vectors commonly used in the art, which have been described for expression in a variety of eukaryotic and prokaryotic hosts and can be used for gene therapy as well as simple protein expression.

[0060] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vectors. As used herein, "gene delivery," "gene transfer," "transduction," etc. refer to terms for introducing exogenous polynucleotides (sometimes referred to as "transgenes") into host cells, regardless of the method of introduction. Such methods include a variety of well-known technologies, such as vector-mediated gene transfer (e.g., by viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes), and technologies that promote the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other technologies for introducing polynucleotides). The introduced polynucleotides can be stable or transiently maintained in the host cell. Stable maintenance generally requires that the introduced polynucleotides include a replication origin compatible with the host cell or are integrated into the replicon of the host cell, such as an extrachromosomal replicon (e.g., plasmid) or nuclear or mitochondrial chromosome. As known in the art and described herein, many vectors are known to be able to mediate the transfer of genes to mammalian cells.

[0061] As used herein, the term "cDNA" refers to extracellular DNA found as a component of pathogenic biofilms.

[0062] As used herein, ESKAPE pathogens include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. These pathogens are the leading cause of nosocomial infections worldwide.

[0063] A "plasmid" is an extrachromosomal DNA molecule that is separate from chromosomal DNA and is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within microbial populations and often provide a selective advantage under given environmental conditions. Plasmids can carry genes that confer resistance to naturally occurring antibiotics in competitive environments, or, in similar situations, produce proteins that can act as toxins.

[0064] The "plasmids" used in genetic engineering are called "plasmid vectors." Many plasmids are commercially available for this purpose. The gene to be replicated is inserted into a copy of the plasmid, which contains a gene that makes the cell resistant to a specific antibiotic and a multiple cloning site (MCS or polylinker), which is a short region containing several commonly used restriction enzyme sites where DNA fragments can be easily inserted. Another major use of plasmids is to produce large quantities of proteins. In this case, researchers can grow bacteria that contain a plasmid carrying the gene of interest. Just as the bacterium produces a protein that confers antibiotic resistance, it can also be induced to produce large quantities of the protein from the inserted gene. This is a cheap and easy way to produce large quantities of a gene or the protein it subsequently encodes.

[0065] "Yeast artificial chromosome" or "YAC" refers to a vector used to clone large DNA fragments (greater than 100kb and up to 3000kb). It is an artificially constructed chromosome that contains the telomere, centromere and replication origin sequences required for replication and maintenance in yeast cells. It is constructed using an initial circular plasmid, which is then linearized using restriction enzymes and then DNA ligase can add the target sequence or gene within the linear molecule by using sticky ends. Yeast expression vectors, such as YAC, YIp (yeast integrating plasmid) and YEp (yeast episomal plasmid), are very useful because eukaryotic protein products with post-translational modifications can be obtained because yeast itself is a eukaryotic cell, but it has been found that YACs are more unstable than BACs and produce chimeric effects.

[0066] "Viral vector" is defined as a recombinantly produced virus or viral particle comprising a polynucleotide to be delivered to a host cell in vivo, in vitro or in vitro. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, alphaviral vectors, and the like. Vectors based on the infectious tobacco mosaic virus (TMV) can be used to produce proteins and have been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15): 6099-6104). Alphaviral vectors, such as vectors based on Semliki Forest virus and vectors based on Sindbis virus, have also been developed for gene therapy and immunotherapy. See Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5: 434-439 and Ying et al. (1999) Nat. Med. 5(7): 823-827. In the context of retroviral vector-mediated gene transfer, the vector construct refers to a polynucleotide comprising the retroviral genome or a portion thereof and the therapeutic gene.

[0067] As used herein, "retroviral-mediated gene transfer" or "retroviral transduction" have synonymous meanings and refer to the process of stably transferring a gene or nucleic acid sequence into a host cell by virtue of viral entry and integration of its genome into the host cell genome. Viruses can enter host cells through their normal infection mechanisms or can be modified to bind to different host cell surface receptors or ligands for cell entry. As used herein, a retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or virus-like entry mechanism.

[0068] Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into DNA, which integrates into the infected cell's genomic DNA. The integrated DNA form is called a provirus.

[0069] In terms of gene transfer mediated by a DNA viral vector, such as adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to a polynucleotide comprising a viral genome or a portion thereof and a transgene. Adenovirus (Ad) is a relatively well-characterized homogeneous virus, including more than 50 serotypes. See, for example, PCT International Application Publication No. WO 95 / 27071. Ad does not need to be integrated into the host cell genome. Recombinant Ad-derived vectors have also been constructed, particularly vectors that reduce the likelihood of recombination and produce wild-type viruses. Referring to PCT International Application Publication Nos. WO 95 / 00655 and WO 95 / 11984, wild-type AAV has high infectivity and specificity for integration into the host cell genome. See Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81: 6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8: 3988-3996.

[0070] The carrier that comprises both promoter and cloning site that polynucleotide can be operably connected to is well known in the art.Such carrier can transcribe RNA in vitro or in vivo, and can be commercially available from sources such as Stratagene (LaJolla, CA) and Promega Biotech (Madison, WI).In order to optimize expression and / or in vitro transcription, it may be necessary to remove, add or change the 5 ' and / or 3 ' non-translated portion of clone to eliminate the extra, potential inappropriate alternative translation initiation codon that may interfere or to interfere or reduce other sequences of expression at transcription or translation level.Or, can be immediately inserted into the consensus ribosome bind site to enhance expression at 5 ' of the start codon.

[0071] Gene delivery vectors also include DNA / liposome complexes, micelles, and targeted viral protein-DNA complexes. Liposomes containing targeting antibodies or fragments thereof can also be used in the methods disclosed herein. In addition to delivering polynucleotides to cells or cell populations, direct introduction of the proteins described herein into cells or cell populations can also be accomplished by the non-limiting technique of protein transfection, or by enhancing expression and / or promoting the activity of the proteins disclosed herein by regulating culture conditions, which are other non-limiting techniques.

[0072] "Inhibit, prevent or disrupt biofilm" refers to the preventive or therapeutic reduction of the structure of the biofilm. In one aspect, the term "inhibit, compete or titrate" refers to the reduction of the DNA / protein matrix (e.g. Figure 6 On the one hand, prevention is excluded from treatment.

[0073] "Bent polynucleotide" means a double-stranded polynucleotide comprising a small loop on one strand that is not paired with the other strand or any polynucleotide, wherein the end-to-end distance is reduced to exceed natural thermal fluctuations, i.e., the bend exceeds the persistent length of natural B-form double-stranded DNA of 150 bp. In some embodiments, the loop is 1 base to about 20 bases in length, or alternatively 2 bases to about 15 bases in length, or alternatively about 3 bases to about 12 bases in length, or alternatively about 4 bases to about 10 bases in length, or alternatively has about 4, 5, or 6, or 7, or 8, 9, or 10 bases.

[0074] The "subject" of diagnosis or treatment is a cell or an animal, such as a mammal or a human. Non-human animals to be diagnosed or treated are animals that undergo infection or animal models, such as monkeys, murines (e.g., rats, mice, ground squirrels), canines (e.g., dogs), lipid animals (e.g., rabbits), livestock, sports animals, and pets.

[0075] The terms "protein," "peptide," and "polypeptide" are used interchangeably and, in their broadest sense, refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds, such as esters, ethers, and the like. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that can constitute a protein or peptide sequence. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and its D and L optical isomers, amino acid analogs, and peptidomimetics.

[0076] As used herein with respect to nucleic acids (e.g., DNA or RNA), the terms "isolated" or "recombinant" refer to molecules that are separated from other DNA or RNA, respectively, that are present in the natural sources of macromolecules and polypeptides. The term "isolated or recombinant nucleic acid" is meant to include nucleic acid fragments that do not naturally exist as fragments and are not found in their natural state. The term "isolated" is also used herein to refer to polynucleotides, polypeptides, and proteins that are separated from other cellular proteins, and is intended to include purified and recombinant polypeptides. In other embodiments, the term "isolated or recombinant" refers to separation from components, cells, or other, wherein cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof are normally associated in nature. For example, an isolated cell is a cell that is separated from a tissue or cell having a different phenotype or genotype. An isolated polynucleotide is separated from 3' and 5' consecutive nucleotides, with which it is normally associated in its natural or native environment (e.g., on a chromosome). It will be apparent to those skilled in the art that non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof do not need to be "isolated" to distinguish them from their naturally occurring counterparts.

[0077] It can be inferred that, without clear narration, unless otherwise indicated, when the present disclosure relates to polypeptides, proteins, polynucleotides or antibodies, their equivalents or biological equivalents should be within the scope of the present disclosure. As used herein, the term "biological equivalents thereof" is intended to be synonymous with "equivalents thereof" when referring to a reference protein, antibody, fragment, polypeptide or nucleic acid, meaning those with minimal homology and still retaining the desired structure or function. Unless specifically stated herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. For example, an equivalent means at least about 70% homology or identity, at least about 80% homology or identity, at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or 98% percent homology or identity and exhibits the same biological activity as the reference protein, polypeptide or nucleic acid. On the other hand, the term refers to a polynucleotide that hybridizes to a reference polynucleotide or its complementary sequence under high stringency conditions.

[0078] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same when comparing the two sequences. Alignments and homology or sequence identity percentages can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, the alignment is performed using default parameters. A preferred alignment program is BLAST, using default parameters. In particular, exemplary programs include BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; description = 50 sequences; rank = HIGH SCORE; database = unique, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following website: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0079] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for the purpose of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity, or alternatively, less than 25% identity with one of the sequences of the present disclosure.

[0080] "Homology" or "identity" or "similarity" may also refer to two nucleic acid molecules that hybridize under stringent conditions to a reference polynucleotide or its complement.

[0081] "Hybridization" refers to the reaction of one or more polynucleotide reactions to form a complex that is stabilized by hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex can include two chains that form a duplex structure, three or more chains that form a multi-chain complex, a single self-hybridizing chain, or any combination thereof. A hybridization reaction can constitute a step in a broader process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0082] Examples of stringent hybridization conditions include: an incubation temperature of about 25°C to about 37°C; a hybridization buffer concentration of about 6×SSC to about 10×SSC; a formamide concentration of about 0% to about 25%; and a wash solution of about 4×SSC to about 8×SSC. Examples of moderate hybridization conditions include: an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9×SSC to about 2×SSC; a formamide concentration of about 30% to about 50%; and a wash solution of about 5×SSC to about 2×SSC. Examples of highly stringent hybridization conditions include: an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1×SSC to about 0.1×SSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1×SSC to about 0.1×SSC, or deionized water. Generally, the hybridization incubation time is 5 minutes to 24 hours, with one, two, or more wash steps, and the wash incubation time is about 1, 2, or 15 minutes. SSC is a 0.15 M NaCl and 15 mM citrate buffer. It will be appreciated that equivalents to SSC using other buffer systems may be employed.

[0083] As used herein, the terms "treating" and "treating" and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic, in terms of complete or partial prevention of a disease or its signs or symptoms, and / or therapeutic, in terms of partial or complete cure of a disease and / or adverse effects attributable to the disease. In one aspect, "treating" does not include prevention.

[0084] "Prevention" refers to the prevention of a disease or effect in or outside a system or subject susceptible to the disease or effect. An example of this is preventing the formation of a biofilm in a system infected with a microorganism known to produce a biofilm.

[0085] "Pharmaceutically acceptable carrier" refers to any diluent, excipient or carrier that can be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (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 silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, a standard reference book in this field, Mack Publishing Company. They can be selected according to the intended form of administration, i.e., oral tablets, capsules, elixirs, syrups, etc., and are consistent with conventional pharmaceutical practice.

[0086] "Administration / dosing" can be performed continuously or intermittently in one dose throughout the course of treatment. Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary depending on the composition used for treatment, the purpose of treatment, the target cell being treated, and the object being treated. Single or multiple administrations can be performed by selecting the dosage level and mode by the treating physician. Suitable dosage forms and methods for administering the agents are known in the art. Route of administration can also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and will vary depending on the composition used for treatment, the purpose of treatment, the health condition or disease stage of the object being treated, and the target cell or tissue. Non-limiting examples of route of administration include oral administration, nasal administration, injection, and topical administration.

[0087] The term "effective amount" refers to an amount sufficient to achieve the desired effect. In the case of therapeutic or preventive applications, the effective amount will depend on the type and severity of the disorder in question and the characteristics of the individual subject, such as overall health, age, sex, weight, and tolerance to the pharmaceutical composition. In the case of immunogenic compositions, in some embodiments, the effective amount is an amount sufficient to elicit a protective response against pathogens. In other embodiments, the effective amount of an immunogenic composition is an amount sufficient to cause the production of antibodies against an antigen. In some embodiments, the effective amount is the amount required to confer passive immunity to a subject in need. With regard to immunogenic compositions, in some embodiments, in addition to the factors mentioned above, the effective amount will depend on the intended use, the degree of immunogenicity of the specific antigenic compound, and the health / responsiveness of the subject's immune system. A skilled person will be able to determine the appropriate amount based on these and other factors.

[0088] In the case of in vitro applications, in some embodiments, the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the in vitro target and the method used. Those skilled in the art will be able to determine the effective amount based on these and other considerations. Depending on the embodiment, the effective amount may include one or more administrations of the composition.

[0089] The agents and compositions can be used in the manufacture of medicaments and in the treatment of humans and other animals by administering them according to conventional methods (eg, as active ingredients in pharmaceutical compositions).

[0090] The agents / medicaments of the present invention may be administered by any suitable route of administration for treatment. It will also be understood that the optimal route will vary with the condition and age of the subject and the disease being treated.

[0091] The example of solid support comprises glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro and magnetite.The property of carrier can be soluble or insoluble to some extent.The carrier material can have any possible structural configuration, as long as the coupled molecule can bind polynucleotide, polypeptide or antibody.Thus, the carrier structure can be spherical, such as beaded, or cylindrical, such as on the inner surface of a test tube or the outer surface of a rod.Alternatively, the surface can be flat, such as a sheet, test strips, etc., or alternatively polystyrene beads.Those skilled in the art will know many other suitable carriers for binding antibodies or antigens, or will be able to determine them by using routine experiments.

[0092] As used herein, the term "antibody" includes complete antibodies and any antigen-binding fragments thereof or single chains thereof. Thus, the term "antibody" includes any protein or peptide molecule comprising at least a portion of an immunoglobulin molecule. Such examples include, but are not limited to, the complementarity determining regions (CDRs) of a heavy or light chain or its ligand-binding portion, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, a binding protein, a chimeric antibody, a humanized antibody, a single-chain antibody, and at least a portion of a fusion protein comprising the antigen-binding portion of an antibody and a non-antibody protein.

[0093] Antibodies can be polyclonal, monoclonal, and can be isolated from any suitable biological source (eg, murine, rat, ovine, and canine).

[0094] "Immune response" broadly refers to the antigen-specific response of lymphocytes to a foreign substance. Any substance that can elicit an immune response is said to be "immunogenic" and is referred to as an "immunogen." All immunogens are antigens, but not all antigens are immunogenic. The immune response disclosed herein can be humoral (through antibody activity) or cell-mediated (through T cell activation).

[0095] As used herein, the term "inducing an immune response in a subject" is a well-known term in the art, and refers to an increase in the immune response (if any) to the antigen (or epitope) after the antigen (or epitope) is introduced into the subject by at least about 2 times, at least about 5 times, at least about 10 times, at least about 100 times, at least about 500 times, or at least about 1000 times or more relative to the immune response (if any) before the antigen (or epitope) is introduced into the subject. The immune response to the 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 that specifically bind to the antigen (or epitope) on their surface. Methods for determining whether an immune response has been induced for a given antigen (or epitope) are well-known in the art. For example, antigen-specific antibodies can be detected using any of a variety of immunoassays known in the art, including but not limited to ELISA, wherein, for example, the antibody in the sample is combined with the fixed antigen (or epitope) and detected with a detectably labeled secondary antibody (e.g., enzyme-labeled mouse anti-human Ig antibody).

[0096] The term "modulating an immune response" includes inducing (increasing, eliciting) an immune response; and decreasing (suppressing) an immune response. An immunomodulatory method (or regimen) is a method of modulating an immune response in a subject.

[0097] "HMG domain" or "high mobility group (HMG) box domain" refers to an amino acid sequence involved in binding to DNA (Stros et al., Cell Mol Life Sci. 64 (19-20): 2590-606 (2007)). In one embodiment, the structure of the HMG-box domain consists of three helices in an irregular array. In another embodiment, the HMG-box domain enables the protein to bind to non-B-form DNA conformations (kinked or unwound) with high affinity. HMG-box domains are located in high mobility group proteins, which are involved in the regulation of DNA-dependent processes such as transcription, replication, and DNA repair, all of which require changes in the conformation of chromatin (Thomas (2001) Biochem. Soc. Trans. 29 (Pt 4): 395-401).

[0098] Modes for carrying out the present invention

[0099] Peptide composition

[0100] Provided herein are polypeptides comprising HMG-box domain truncations and / or mutants described herein, as well as proteins, fragments of these proteins comprising one or more HMG-box domains, truncations, mutants, or equivalents of these proteins or fragments having the disclosed amino acid substitutions.

[0101] Thus, in one aspect, the present invention provides an isolated A Box polypeptide, optionally comprising one or more amino acid mutations selected from K12, C23 and C45 (e.g., native K or C is modified to an amino acid selected from serine, glycine, alanine, valine, isoleucine or threonine) or an equivalent thereof, said equivalent comprising one or more amino acid mutations selected from K12, C23 and C45 (e.g., native K or C is modified to an amino acid selected from serine, glycine, alanine, valine, isoleucine or threonine), or consisting essentially of, or consisting of. In one aspect, the mutation is a C45S mutation. The A Box polypeptide may further comprise a linker or peptide sequence located at one or both termini. An example of a peptide linker is PPKGETKKKF.

[0102] When recombinantly produced, A Box polypeptides can be partially or fully acetylated, oxidized, or phosphorylated using methods known in the art, such as Olia AS, et al. (2015) ACS chemical biology. 10(9): 2034-47. doi: 10.1021 / acschembio.5b00342, PubMed PMID: 26083674; PubMed Central PMCID: PMC4610810; Ugrinova I, et al. (2102) Molecular Biology Reports, 2012; 39(11): 9947-53. Epub 2012 / 06 / 29. doi: 10.1007 / s11033-012-1863-x. PubMed PMID: 22740141; and Ito T, et al. al. (2007) JTH, 5(1): 109-16. doi: 10.1111 / j.1538-7836.2006.02255.x. PubMed PMID: 17239166. In one aspect, the A Box polypeptide comprises, consists essentially of, or consists of amino acids 1 to 70 of a wild-type HMGB1 polypeptide having the aforementioned mutations.

[0103] Examples of A Box polypeptides include the following sequences, or consist essentially of, or consist of: MGKGDPKKPRRKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKKCSERWK TMSAKEKGKF EDMAKADKARYEREMKTYIPPKGETKKKF (mouse)

[0104] MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKKCSERWK TMSAKEKGKFEDMAKADKARYEREMKTYIP PKGETKKKF (human).

[0105] As used herein, an equivalent of a polypeptide refers to a sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% identity to a reference polypeptide, which, in one aspect, retains the mutated amino acids. In some aspects, an equivalent of a polypeptide retains the expected function and / or structural features of the polypeptide, such as containing an HMG-box domain. In one aspect, equivalent polypeptides include domains having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% identity to an HMG-box domain, which, in one aspect, retains the mutated amino acids. In some aspects, such equivalent domains retain the function and / or structural features of the HMB-box domain, such as, binding to an HMB-box binding target. In one aspect, equivalent polypeptides are encoded by polynucleotides that hybridize to a polynucleotide encoding a polypeptide of an HMB-box domain under stringent conditions.

[0106] Also provided herein are isolated B Box polypeptides, which optionally contain a mutation at amino acid C106 or K114 (e.g., the native cysteine is changed to an amino acid selected from serine, glycine, alanine, valine, isoleucine or threonine) or an equivalent thereof, which equivalent contains a mutation at amino acid C106 or K114 (e.g., the native cysteine is changed to an amino acid selected from serine, glycine, alanine, valine, isoleucine or threonine), or consist essentially of, or consist of. In one aspect, the B Box polypeptide contains from about 80 to about 176, or about 88 to about 164, or about 89 to about 162, or about 80 to about 164 amino acids of the wild-type HMGB1 B Box polypeptide, which has the aforementioned mutation, or consist essentially of, or consist of. Other positions for modifying the wild-type HMGB1 B Box polypeptide are in Figure 1shown in C. Examples of B Box polypeptides include the following sequences, or consist essentially of, or consist of: KDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAEKLKEKYEKDIAAYRAKGKPDAAKKGVV (mouse)

[0107] KDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVV (human).

[0108] The B Box polypeptide may further comprise a linker or peptide sequence located at one or both termini. An example of a peptide linker is PPKGETKKKF. When produced recombinantly, the disclosed B Box polypeptides may be partially or fully acetylated, oxidized, or phosphorylated using methods known in the art, such as Olia AS, et al. (2015) ACS chemical biology. 10(9):2034 - 47. doi:10.1021 / acschembio.5b00342, PubMed PMID: 26083674; PubMed Central PMCID: PMC4610810; Ugrinova I, et al. (2102) Molecular Biology Reports, 2012; 39(11):9947 - 53. Epub 2012 / 06 / 29. doi:10.1007 / s11033 - 012 - 1863 - x. PubMed PMID: 22740141; and Ito T, et al. (2007) JTH, 5(1):109 - 16. doi:10.1111 / j.1538 - 7836.2006.02255.x. PubMed PMID: 17239166.

[0109] As used herein, the equivalent of a polypeptide refers to a sequence having a homology of 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% with a reference polypeptide, which, in one aspect, retains the amino acid of the mutation. In some aspects, the equivalent of a polypeptide retains the expected function and / or structural characteristics of the polypeptide, such as containing an HMG-box domain. On the one hand, an equivalent polypeptide includes a domain having a homology of at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% with an HMG-box domain, which, in one aspect, retains the amino acid of the mutation. In some aspects, such an equivalent domain retains the function and / or structural characteristics of the HMB-box domain, such as, binding to the HMB-box binding target and optionally losing its proinflammatory response. In one aspect, the equivalent polypeptide is encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide encoding an HMB-box domain polypeptide.

[0110] In another aspect, provided herein are isolated AB Box polypeptides optionally comprising, consisting essentially of, or consisting of one or more amino acid mutations selected from K12, C23, C45, and C106 or K114 (e.g., native K or C is modified to an amino acid selected from serine, glycine, alanine, valine, isoleucine, or threonine), or an equivalent thereof, the equivalent comprising, consisting essentially of, or consisting of one or more amino acid mutations selected from K12, C23, and C45, and C106 or K114 (e.g., native K or C is modified to an amino acid selected from serine, glycine, alanine, valine, isoleucine, or threonine). In one aspect, the mutation is a C45S mutation. In another aspect, the polypeptide comprises a mutation at amino acid C106 (e.g., a native cysteine ​​is changed to an amino acid selected from serine, glycine, alanine, valine, isoleucine, or threonine) or an equivalent thereof, the equivalent comprising one or more amino acid mutations selected from K12, C23, C45 and a mutation at amino acid C106 (e.g., a native cysteine ​​is changed to an amino acid selected from serine, glycine, alanine, valine, isoleucine, or threonine). In one aspect, the AB Box polypeptide comprises C45S and C106S mutations and the equivalent retains these mutations. In one aspect, the AB Box polypeptide comprises amino acids 1 to 176, or 1 to 162, or 1 to 164 of a wild-type HMGB1 polypeptide having the aforementioned mutations, or consisting essentially of, or consisting of.

[0111] In another aspect, the AB Box polypeptide can further comprise a linker polypeptide connecting the A Box polypeptide and the B Box polypeptide, and in one aspect, a second linker connecting the B Box polypeptide and the C Box polypeptide. When recombinantly produced, the AB or A, B, and C Box polypeptides can be partially or fully acetylated, oxidized, or phosphorylated. An example of a peptide linker is PPKGETKKKF. In one aspect, the isolated mutant HMGB1 polypeptide has one or more amino acid substitutions as described herein in the A and / or B Box domains, which may optionally be partially or fully acetylated, oxidized or phosphorylated using methods known in the art, such as Olia AS, et al. (2015) ACS chemical biology. 10(9): 2034-47. doi: 10.1021 / acschembio.5b00342, PubMed PMID: 26083674; PubMed Central PMCID: PMC4610810; Ugrinova I, et al. (2102) Molecular Biology Reports, 2012; 39(11): 9947-53. Epub 2012 / 06 / 29. doi: 10.1007 / s11033-012-1863-x. PubMed PMID: 22740141; and Ito T, et al. (2007) JTH, 5(1): 109-16. doi: 10.1111 / j.1538-7836.2006.02255.x. PubMed PMID: 17239166.

[0112] Examples of AB Box polypeptides include, have, consist essentially of, or consist of the following sequences, with the aforementioned mutations:

[0113] MGKGDPKKPRRKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGK

[0114] FEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL

[0115] SIGDVAKKLGEMWNNTAADDKQPYEKKAEKLKEKYEKDIAAYRAKGKPDAAKKGVV (mouse)

[0116] MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGK

[0117] FEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL

[0118] SIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVV(human).

[0119] As used herein, the equivalent of a polypeptide refers to a sequence having a homology of 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% with a reference polypeptide, and in one aspect, the amino acid of the mutation is retained. In some aspects, the equivalent of a polypeptide retains the expected function and / or structural characteristics of the polypeptide, such as containing an HMG-box domain. On the one hand, an equivalent polypeptide includes a domain having a homology of at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% with an HMG-box domain, and in one aspect, the amino acid of the mutation is retained. In some aspects, such an equivalent domain retains the function and / or structural characteristics of the HMB-box domain, such as, binding to the HMB-box target but not inducing a proinflammatory response. In one aspect, the equivalent polypeptide is encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide encoding an HMB-box domain polypeptide.

[0120] In another aspect, the isolated AB Box polypeptide further comprises a linker polypeptide connecting the A Box polypeptide and the B Box polypeptide.

[0121] Also provided are isolated HMGB1 polypeptides comprising A, B, and C domains, wherein the polypeptide comprises one or more amino acid mutations selected from K12, C23, C45, C106, or K114, or an equivalent thereof, the equivalent comprising, consisting essentially of, or consisting of one or more amino acid mutations selected from K12, C23, C45, C106, or K114. As used herein, an equivalent of a polypeptide refers to a sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% identity to a reference polypeptide, which, in one aspect, retains the mutated amino acids. In some aspects, an equivalent of a polypeptide retains the intended functional and / or structural features of the polypeptide, such as containing an HMG-box domain but not inducing a proinflammatory response. In one aspect, an equivalent polypeptide comprises a domain having at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% identity to an HMG-box domain, which in one aspect retains the mutated amino acid. In some aspects, such an equivalent domain retains the functional and / or structural features of the HMB-box domain, for example, binding to an HMB-box-bound target without inducing a proinflammatory response. In one aspect, an equivalent polypeptide is encoded by a polynucleotide that can hybridize under stringent conditions to a polynucleotide encoding an HMB-box domain polypeptide.

[0122] In another aspect, the isolated AB Box polypeptide further comprises a linker polypeptide connecting the A Box polypeptide and the B Box polypeptide, and a second linker connecting the B Box polypeptide and the C Box polypeptide. An example of a peptide linker is PPKGETKKKF.

[0123] The polypeptide can be detectably labeled and / or combined with a carrier (eg, a pharmaceutically acceptable carrier).

[0124] Antibodies and their derivatives

[0125] The present disclosure also provides antibodies that bind and / or specifically recognize and bind to isolated polypeptides for use in methods disclosed herein. The antibody can be any of the various antibodies described herein, and such non-limiting examples include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, human antibodies, veneered antibodies, diabodies, humanized antibodies, antibody derivatives, recombinant humanized antibodies, or their respective derivatives or fragments. On the one hand, the fragment comprises the CDR of the antibody, or consists essentially of it, or consists of it. On the one hand, the antibody is detectably labeled or further comprises a detectable label coupled thereto. Hybridoma cell lines producing monoclonal antibodies disclosed herein are also provided. Compositions are further provided herein, comprising one or more of the above embodiments, or consisting essentially of it, or consisting of it. Polynucleotides encoding the amino acid sequences of antibodies and fragments are also provided, as well as methods for producing recombinant or chemically synthesized antibody polypeptides and fragments thereof. Antibody polypeptides can be produced in eukaryotic or prokaryotic cells, or produced by other methods known in the art and described herein.

[0126] Antibodies can be produced using conventional techniques known in the art and are fully described in the literature. Several methods exist for producing polyclonal antibodies. For example, polyclonal antibodies are typically produced by immunizing suitable mammals, such as, but not limited to, chickens, goats, guinea pigs, hamsters, horses, mice, rats, and rabbits. Antigens are injected into mammals to induce B lymphocytes to produce immunoglobulins specific for the antigen. Immunoglobulins can be purified from mammalian serum.

[0127] Monoclonal antibodies can be produced using conventional hybridoma techniques known in the art and fully described in the literature. For example, hybridomas are produced by fusing suitable immortalized cell lines (e.g., myeloma cell lines such as, but not limited to, Sp2 / 0, Sp2 / 0-AG14, NSO, NS1, NS2, AE-1, L.5, P3X63Ag8, 653, Sp2, SA3, Sp2MAI, Sp2 SS1, Sp2SA5, U397, MIA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 313, HL-60, MLA 144, NAMAIWA, NEURO, FLASH ... 2A, CHO, PerC.6, YB2 / 0), etc., or a hybrid myeloma, a fusion product thereof, or any cell or fusion cell derived therefrom, or any other suitable cell line known in the art (see the technology of the following websites, such as atcc.org, lifetech.com, the last access time is November 26, 2007), which has antibody-producing cells, such as but not limited to: isolated or cloned spleen, peripheral blood, lymphocytes, tonsils, or other immune or B cell-containing cells, or cells expressing The antibody of the present invention can be expressed in any other cell, virus, bacterium, algae, protokaryon, amphibian, insect, reptile, fish, mammal, rodent, horse, sheep, goat, sheep, primate, eukaryote, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single-stranded, double-stranded or triple-stranded, hybridization etc. or its any combination. The cell that produces antibody can also be obtained from peripheral blood or spleen or lymph node of people or other suitable animals that have been screened for purpose activity with target antigen immunity. Any other suitable host cell can also be used for expressing the heterologous or endogenous nucleic acid of antibody of the present invention, its specified fragment or variant. Fusion cell (hybridoma) or recombinant cell can use selective culture condition or other suitable known methods to separate, and by limiting dilution or cell sorting or other known methods cloning.

[0128] Other suitable methods for producing or isolating antibodies with the necessary specificity may be used, including but not limited to methods for selecting recombinant antibodies from peptide or protein libraries using methods known in the art (such as, but not limited to, phage, ribosome, oligonucleotide, cDNA, etc. display libraries; such as those available from various commercial suppliers, such as MorphoSys (Martinsreid / Planegg, Del.), BioInvent (Lund, Sweden), Affitech (Oslo, Norway)). Methods known in the art are described in the patent literature, 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. Alternative methods rely on immunization of transgenic animals capable of producing a repertoire of human antibodies (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), as known in the art and / or as described herein. Such techniques include, but are not limited to, ribosome display (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., the selected lymphocyte antibody method ("SLAM") (U.S. Pat. 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 droplet and flow cytometry (Powell et al. (1990) Biotechnol. 8:333-337; One 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).

[0129] Antibody derivatives of the present disclosure can also be prepared by the following method: delivering a polynucleotide encoding an antibody disclosed herein to a suitable host to provide a transgenic animal or mammal (e.g., goats, cattle, horses, sheep, etc.) that produces the antibody in milk. These methods are known in the art and are described, for example, in U.S. Patent Nos. 5,827,690, 5,849,992, 4,873,316, 5,849,992, 5,994,616, 5,565,362, and 5,304,489.

[0130] The term "antibody derivative" includes post-translational modifications to the linear polypeptide sequence of an antibody or fragment. For example, U.S. Patent No. 6,602,684B1 describes a method for producing a modified glycol form of an antibody, including a complete antibody molecule, an antibody fragment, or a fusion protein, which contains a region equivalent to the Fc region of an immunoglobulin, has enhanced iron-mediated cytotoxicity, and thus produces a glycoprotein.

[0131] The antibodies disclosed herein also include derivatives that have been 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-idiotypic response. Antibody derivatives include, but are not limited to, antibodies that have been modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. In addition, derivatives may contain one or more non-classical amino acids.

[0132] Antibody derivatives can also be prepared by the following method: delivering polynucleotides disclosed herein to provide transgenic plants and cultured plant cells (such as, but not limited to, tobacco, corn, and duckweed) that produce such antibodies, specific parts, or variants in plant parts or in cells cultured therefrom. For example, Cramer et al. (1999) Curr. Top. Microbol. Immunol. 240: 95-118 and the references cited therein describe the production of transgenic tobacco leaves expressing large amounts of recombinant proteins, such as using inducible promoters. Transgenic corn has been used to express mammalian proteins at commercial production levels, and its biological activity is equivalent to that produced in other recombinant systems or purified from natural sources. See, for example, Hood et al. (1999) Adv. Exp. Med. Biol. 464: 127-147 and the references cited therein. Antibody derivatives, such as single-chain antibodies (scFv), have also been produced in large quantities from transgenic plant seeds comprising antibody fragments, including tobacco seeds and potato tubers. See, for example, Conrad et al. (1998) Plant Mol. Biol. 38: 101-109 and references cited therein. Thus, according to known methods, transgenic plants can also be used to produce antibodies.

[0133] Antibody derivatives can also be produced by, for example, adding exogenous sequences to modify immunogenicity or to reduce, enhance, or modify binding, affinity, on-rate, off-rate, activity, specificity, half-life, or any other suitable property. Typically, some or all of the non-human or human CDR sequences are retained, while the non-human sequences of the variable and constant regions are replaced with human or other amino acids or with variable or constant regions from other isotypes.

[0134] Typically, the CDR residues are directly and most substantially involved in influencing antigen binding. Humanization or engineering of antibodies can be performed using any known method, such as, but not limited to, the methods described in 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.

[0135] The chimeric, humanized or primatized antibodies of the present disclosure can be prepared based on the sequence of the reference monoclonal antibody prepared using standard molecular biology techniques. DNA encoding heavy and light chain immunoglobulins can be obtained from the hybridoma of interest and transformed using standard molecular biology techniques to include non-reference (e.g., human) immunoglobulin sequences. For example, to produce chimeric antibodies, murine variable regions can be connected to human constant regions using methods known in the art (U.S. Patent No. 4,816,567). To produce humanized antibodies, mouse CDR regions can be inserted into human frameworks using methods known in the art (U.S. Patent No. 5,225,539 and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370). Similarly, to produce primatized antibodies, mouse CDR regions can be inserted into primate frameworks using methods known in the art (WO 93 / 02108 and WO99 / 55369).

[0136] The technology of preparing part to fully human antibodies is known in the art, and any such technology can be used. According to one embodiment, fully human antibody sequences are prepared in transgenic mice that have been engineered to express human heavy and light chain antibody genes. A variety of strains of such transgenic mice that can produce different classes of antibodies have been prepared. The B cells of the transgenic mice that produce the desired antibodies can be fused to prepare hybridoma cell lines to continuously produce the desired antibodies.(See, for example, Russel 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. (1993) Proc. Natl. Acad. Sci. USA 90(6):2551-2555; and U.S. Patent No. 6,075,181).

[0137] The antibodies disclosed herein can also be modified to produce chimeric antibodies. A chimeric antibody is an antibody in which each domain of the heavy and light chains of the antibody is encoded by more than one DNA sequence. See, for example, U.S. Patent No. 4,816,567.

[0138] Alternatively, the antibodies disclosed herein can also be modified to produce veneered antibodies. Veneered antibodies are antibodies in which the external amino acid residues of an antibody from one species are judiciously replaced or "veneered" with an antibody from a second species so that the antibody from the first species is not 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 the antibody can be reduced by replacing exposed residues that are different from the residues typically found in antibodies from another mammalian species. Judicious replacement of external residues should have little or no effect on internal domains or interdomain contacts. Therefore, due to changes limited to variable region framework residues, ligand binding properties should be unaffected. This process is called "veneer" because only the outer surface or skin of the antibody is changed and the supporting residues remain undisturbed.

[0139] The process of "veneer" utilizes the available sequence data of human antibody variable domains, which are compiled by Kabat et al. (1987) Sequences of Proteins of Immunological Interest, 4th ed., Bethesda, Md., National Institutes of Health (an update of this database), as well as other accessible U.S. and foreign databases (nucleic acid and protein). Non-limiting examples of methods for producing veneered antibodies include EP 519596, U.S. Patent No. 6,797,492, and are described in Padlan et al. (1991) Mol. Immunol. 28 (4-5): 489-498.

[0140] The term "antibody derivative" also includes "diabodies", which are small antibody fragments with two antigen-binding sites, wherein the fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain. (See, for example, EP404,097, 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, these domains are forced to pair with the complementary domains of another chain and produce two antigen-binding sites. (See also U.S. Patent No. 6,632,926 to Chen et al., which discloses antibody variants having one or more amino acids inserted into the hypervariable region of a parent antibody and having a binding affinity for the target antigen that is at least about twice as strong as the binding affinity of the parent antibody for the antigen).

[0141] The term "antibody derivative" also includes engineered antibody molecules, fragments and single domains, such as scFv, dAb, nanobodies, minibodies, single bodies and Affibodies & Hudson (2005) Nature Biotech 23(9): 1126-36, U.S. Patent Application Publication No. 2006 / 0211088, PCT International Application Publication No. WO 2007 / 059782, U.S. Patent No. 5,831,012.

[0142] The term "antibody derivative" also includes "linear antibodies". Methods for preparing linear antibodies are known in the art and are described in Zapata et al. (1995) Protein Eng. 8(10): 1057-1062. Briefly, these antibodies contain a pair of tandem Ed segments (V H -C H 1-VH-C H 1), which form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0143] The antibodies disclosed herein can 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") can also be used for purification.

[0144] Antibodies of the present disclosure include naturally purified products, products of chemical synthesis procedures, and products produced by recombinant technology from eukaryotic hosts (including, for example, yeast, higher plants, insects, and mammalian cells) or alternatively from prokaryotic hosts as described above. Many antibody production systems are described in Birch & Radner (2006) Adv. Drug Delivery Rev. 58: 671-685.

[0145] If the antibody being tested binds to a protein or polypeptide, then the antibody being tested and the antibodies provided by the present disclosure are equivalent. It is also possible to determine whether the antibody has the same specificity as the antibodies disclosed herein by determining whether the antibody being tested prevents the antibodies disclosed herein from binding to proteins or polypeptides that normally react with the antibodies without excessive experimentation. If the antibody being tested competes with the antibodies disclosed herein, as shown by reduced binding of the monoclonal antibodies disclosed herein, then the two antibodies are likely to bind to the same or closely related epitopes. Alternatively, the antibodies disclosed herein can be pre-incubated with proteins that normally react with them, and it is determined whether the ability of the antibody being tested to bind to the antigen is inhibited. If the antibody being tested is inhibited, it is very likely to have the same or closely related epitope specificity as the antibodies disclosed herein.

[0146] The term "antibody" is also intended to include antibodies of all immunoglobulin isotypes and subclasses. A specific isotype of a monoclonal antibody can be prepared directly by selection from an initial fusion, or by using sib selection technology to isolate class-switched variants using the methods described in Steplewski et al. (1985) Proc. Natl. Acad. Sci. USA 82:8653 or Spira et al. (1984) J. Immunol. Methods 74:307 to indirectly prepare from parent hybridomas that secrete monoclonal antibodies of different isotypes. Alternatively, recombinant DNA technology can be used.

[0147] Isolation of other monoclonal antibodies having the specificity of the monoclonal antibodies described herein can also be accomplished by one of ordinary skill in the art by generating anti-idiotypic antibodies. Herlyn et al. (1986) Science 232: 100. Anti-idiotypic antibodies are antibodies that recognize unique determinants present on the monoclonal antibody of interest.

[0148] In some aspects disclosed herein, it is useful to detectably or therapeutically label antibodies. Suitable labels are as described above. Methods for coupling antibodies directed against these reagents are known in the art. For illustrative purposes only, antibodies can be labeled with detectable moieties (e.g., radioactive atoms, chromophores, fluorophores). Such labeled antibodies can be used for diagnostic techniques in vivo or in isolated test samples.

[0149] Coupling of an antibody to a low molecular weight hapten can increase the sensitivity of the antibody in the assay. The hapten can then be specifically detected by a second reaction. For example, haptens such as biotin (which can react with avidin) or dinitrophenol, pyridoxal, and fluorescein (which can react with specific anti-hapten antibodies) are commonly used. See Harlow and Lane (1988) above.

[0150] The variable region of the antibody of the present invention can be modified to improve one or more binding properties (such as affinity) of the antibody by mutating the amino acid residues in VH and / or VL CDR 1, CDR 2 and / or CDR 3 regions. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and the impact on antibody binding or other functional properties of interest can be assessed in appropriate in vitro or in vivo assays. In certain embodiments, conservative modifications are introduced and generally no more than 1, 2, 3, 4 or 5 residues are changed in the CDR regions. Mutations can be amino acid substitutions, additions or deletions.

[0151] Framework modifications can be made to antibodies to reduce immunogenicity, for example, by mutating one or more framework residues "back" to the corresponding germline sequence.

[0152] In addition, the antibodies disclosed herein can be engineered to include modifications within the Fc region to alter one or more functional properties of the antibody, such as serum semiquantitation, complement fixation, Fc receptor binding and / or antigen-dependent cellular toxicity. Such modifications include, but are not limited to, changes in the number of cysteine ​​residues in the hinge region to promote assembly of light and heavy chains or to increase or reduce the stability of the antibody (U.S. Patent number 5,677,425), or amino acid mutations in the Fc hinge region to reduce the biological half-life of the antibody (U.S. Patent number 6,165,745).

[0153] In addition, the antibodies disclosed herein can be chemically modified. The glycosylation of the antibody can be changed, for example, by modifying one or more glycosylation sites within the antibody sequence to increase the affinity of the antibody for the antigen (U.S. Patent Nos. 5,714,350 and 6,350,861). Alternatively, in order to increase antibody-dependent cell-mediated cytotoxicity, a hypofucosylated antibody with a reduced amount of fucosyl residues or an antibody with an increased bisecting GlcNac structure can be obtained by expressing the antibody in a host cell with an altered glycosylation machinery. (Shields, R. Let al. (2002) J. Biol. Chem. 277: 26733-26740; Umana et al. (1999) Nat. Biotech. 17: 176-180).

[0154] Antibodies disclosed herein can be pegylated to increase biological half-life by reacting the antibody or its fragment with polyethylene glycol (PEG) or the reactive ester or aldehyde derivative of PEG under conditions where one or more PEG groups become connected to the antibody or antibody fragment. Antibody pegylation can be carried out by acylation reaction or alkylation reaction with reactive PEG molecules (or similar reactive water-soluble polymers). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as single (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. Antibodies to be pegylated can be glycosylated antibodies. Methods for pegylated proteins are known in the art and can be applied to antibodies disclosed herein (EP 0154316 and EP 0401384).

[0155] In addition, antibodies can be chemically modified by conjugating or fusing their antigen-binding regions to serum proteins (e.g., human serum albumin) to increase the half-life of the resulting molecule. Such methods are described, for example, in EP 0322094 and EP 0486525.

[0156] The antibodies or fragments thereof disclosed herein can be conjugated to diagnostic agents and can be used diagnostically, for example, to monitor the development or progression of a disease and to determine the efficacy of a given treatment regimen. Examples of diagnostic agents include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomography techniques, and non-radioactive paramagnetic metal ions. The detectable substance can be directly coupled or conjugated to the antibody or fragment thereof, or indirectly coupled or conjugated through a linker using techniques known in the art. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable repair group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent materials include luciferase, luciferin and aequorin. Examples of suitable radioactive substances include 125 I. 131I, indium 111, lutetium 171, bismuth 212, bismuth 213, astatine 211, copper 62, copper 64, copper 67, yttrium 90, iodine 125, iodine 131, phosphorus 32, phosphorus 33, scandium 47, silver 111, gallium 67, praseodymium 142, samarium 153, terbium 161, dysprosium 166, holmium 166, rhenium 186, rhenium 188, rhenium 189, lead 212, radium 223, actinium 225, iron 59, selenium 75, arsenic 77, strontium 89, molybdenum 99, rhodium 1105, palladium 109, praseodymium 143, promethium 149, erbium 169, iridium 194, gold 198, gold 199, and lead 211. Monoclonal antibodies can be indirectly coupled to radioactive metal ions by using bifunctional chelators covalently linked to the antibody. Chelators can be attached via friendly binding (Meares et al. (1984) Anal. Biochem. 142: 68-78), sulfhydryl groups of amino acid residues (Koyama (1994) Chem. Abstr. 120: 217-262), and carbohydrate groups (Rodwell et al. (1986) PNAS USA 83: 2632-2636; Quadri et al. (1993) Nucl. Med. Biol. 20: 559-570).

[0157] In addition, the antibodies or fragments thereof of the present invention can be conjugated to therapeutic agents. Suitable therapeutic agents include: paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenotoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone dione, mitoxantrone, mithramycin, 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., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, Cisplatin and other platinum derivatives (e.g., carboplatin), antibiotics (e.g., actinomycin, bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), diphtheria toxin and related molecules (e.g., diphtheria A chain and active fragments and hybrid molecules thereof), ricin toxins (e.g., ricin A or deglycosylated ricin A chain toxin), cholera toxin, Shiga-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, aloin, saponins, modeccin, gelanin, abrin A chain, modeccin A chain, α-sarcin, aleurites fordii protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitory factor, curcin, crotin, Sapaonaria officinalis inhibitory factor, gelonin, mitogellin, restrietocin, phenomycin, enomycin toxin and mixed toxins.

[0158] Other suitable conjugated molecules include ribonuclease (RNA enzyme), DNA enzyme I, antisense nucleic acid, inhibitory RNA molecules (such as siRNA molecules), immunostimulatory nucleic acids, aptamers, ribozymes, triplex formation molecules and external guide sequences. Fitamers are small nucleic acids with a length of 15-50 bases, which are folded into the secondary and tertiary structures defined, such as stem loops or G quadruplexes, and can be combined with small molecules (such as ATP (U.S. Patent number 5,631,146) and theophylline (U.S. Patent number 5,580,737)) and macromolecules (such as reverse transcriptase (U.S. Patent number 5,786,462) and thrombin (U.S. Patent number 5,543,293)). Ribozymes are nucleic acid molecules that can catalyze chemical reactions intramolecularly or intermolecularly. Ribozymes are usually cut nucleic acid substrates by the identification and combination of target substrates and subsequent cutting. Nucleic acid molecules with triplex-forming function can interact with double-stranded or single-stranded nucleic acids by forming triplexes, where three strands of DNA form complexes that rely on Watson-Crick and Hoogsteen base pairing. Triplex molecules can bind to target regions with high affinity and specificity.

[0159] A functional nucleic acid molecule can act as an effector, inhibitor, modulator, or stimulator of a specific activity possessed by a target molecule, or a functional nucleic acid molecule can have a de novo activity independent of any other molecule.

[0160] Therapeutic agents can be linked directly or indirectly to antibodies using any of a variety of available methods, for example, by attachment of the agent to the hinge region of a reduced antibody component via disulfide bond formation, using a cross-linking agent such as N-succinyl 3-(2-pyridyldithio) propionate (SPDP), or via a carbohydrate moiety in the Fc region of the antibody. (Yu etal. 1994 Int. J. Cancer 56:244; Upeslacis et al., "Modification of Antibodies by Chemical Methods," in Monoclonal antibodies: principles and applications, Birchet al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies,” in Monoclonalantibodies: Production, engineering and clinical application, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995)).

[0161] Techniques for conjugating therapeutic agents with antibodies are well known (Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy; Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2nd Ed.); Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody in Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,” (1982) Immunol. Rev. 62:119-58).

[0162] The antibodies disclosed herein, or their antigen-binding regions, can be linked to another functional molecule (e.g., another antibody or a ligand for a receptor) to produce a bispecific or multispecific molecule that binds to at least two or more different binding sites or target molecules. The connection of an antibody to one or more other binding molecules (e.g., another antibody, antibody fragment, peptide, or binding mimetic) can be accomplished, for example, by chemical coupling, genetic fusion, or non-covalent binding. In addition to the first and second target epitopes, the multispecific molecule can also include a third binding specificity.

[0163] Bispecific and multispecific molecules can be prepared using methods known in the art. For example, each binding unit of a highly specific molecule can be produced separately and then conjugated to each other. When the binding molecule is a protein or peptide, a variety of coupling agents or cross-linking agents can be used for covalent binding. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phthalimide (oPDM), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8648). When the binding molecules are antibodies, they can be bound through sulfhydryl bonds in the C-terminal hinge region of the two heavy chains.

[0164] The antibodies or fragments thereof of the present disclosure can be linked to moieties that are toxic to cells to which the antibodies bind to form "depleting" antibodies. These antibodies are particularly useful in applications requiring the depletion of NK cells.

[0165] The antibodies disclosed herein can also be attached to a solid support, which is particularly useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0166] Antibodies can also be bound to many different carriers. Thus, the present invention also provides compositions comprising an antibody and another active or inert substance. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. For the purposes disclosed herein, the nature of the carrier can be soluble or insoluble. Those skilled in the art will know other suitable carriers for binding monoclonal antibodies or will be able to determine such carriers using routine experimentation.

[0167] In some aspects of the antibodies provided herein, the antibody is a full-length antibody.

[0168] In some aspects of the antibodies provided herein, the antibody is a monoclonal antibody.

[0169] In some aspects of the antibodies provided herein, the antibody is chimeric or humanized.

[0170] In some aspects of the antibodies provided herein, the antibody is selected from Fab, F(ab)'2, Fab', scF v and F v .

[0171] In some aspects of the antibodies provided herein, the antibody comprises an Fc domain. In some aspects of the antibodies provided herein, the antibody is a non-human animal (e.g., rat, sheep, cattle, dog, cat, or rabbit) antibody. In some aspects of the antibodies provided herein, the antibody is a human or humanized antibody or is non-immunogenic in humans.

[0172] In some aspects of the antibodies provided herein, the antibody comprises human antibody framework regions.

[0173] In other aspects, one or more amino acid residues in the CDR of the antibody provided herein are replaced by another amino acid. With respect to replacement within the same amino acid family, the replacement can be "conservative". Naturally occurring amino acids can be divided into the following four families, and conservative replacements will be performed in these families.

[0174] 1) Amino acids with basic side chains: lysine, arginine, histidine.

[0175] 2) Amino acids with acidic side chains: aspartic acid, glutamic acid.

[0176] 3) Amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, tyrosine.

[0177] 4) Amino acids with non-polar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine.

[0178] In another aspect, one or more amino acid residues are added to or deleted from one or more CDRs of an antibody. Such additions or deletions occur at the N or C termini of a CDR or at a position within a CDR.

[0179] By changing the amino acid sequence of the CDR of an antibody by addition, deletion, or substitution of amino acids, various effects such as increased binding affinity to the target antigen can be obtained.

[0180] Polynucleotides, vectors and host cells

[0181] The present invention also provides polypeptide or antibody encoding one or more of the above-mentioned identifications and their respective complementary chains of separation or reorganization polynucleotides. Also provided are carriers comprising the polynucleotide of separation or reorganization, examples of which are known in the art and briefly described herein. In one aspect in which the polynucleotide of more than one separation or reorganization will be expressed as a single unit, the polynucleotide of separation or reorganization can be contained in a polycistronic vector. The polynucleotide can be DNA, RNA, mRNA or interfering RNA, for example siRNA, miRNA or dsRNA.

[0182] The present invention further provides isolated or recombinant polynucleotides operably linked to promoters for RNA transcription and other regulatory sequences for DNA and / or DNA replication and / or transient or stable expression. As used herein, the term "operably linked" refers to positioning in such a manner that the promoter will direct RNA transcription from the DNA molecule. Examples of such promoters are SP6, T4, and T7. In certain embodiments, cell-specific promoters are used for cell-specific expression of inserted polynucleotides. Vectors containing promoters or promoter / enhancers, with stop codons and selectable marker sequences, and cloning sites to which inserted DNA fragments can be operably linked are known in the art and commercially available. For general methods and cloning strategies, see Gene Expression Technology (Goeddeled., Academic Press, Inc. (1991)) and references cited therein, and Vectors: Essential Data Series (Gacesa and Ramji, eds., John Wiley & Sons, NY (1994)), which contains maps, functional properties, commercial suppliers, and references to GenEMBL accession numbers for various suitable vectors.

[0183] In one embodiment, polynucleotides derived from the polynucleotides of the present invention encode polypeptides, proteins, antibodies, or fragments thereof having diagnostic and therapeutic utility as described herein, as well as probes for identifying protein transcripts that may or may not be present. These nucleic acid fragments can be prepared, for example, by restriction enzyme digestion of larger polynucleotides and then labeled with a detectable marker. Alternatively, nick translation of the molecules can be used to generate random fragments. For methods of preparing and labeling such fragments, see Sambrook, et al. (1989) (supra).

[0184] Expression vectors containing these nucleic acids can be used to obtain host-vector systems to produce proteins and polypeptides. It is implied that these expression vectors must be replicable in the host organism as episomes or as components of chromosomal DNA. Non-limiting examples of suitable expression vectors include plasmids, yeast vectors, viral vectors, and liposomes. Adenovirus vectors are particularly suitable for introducing genes into tissues in vivo due to their high level of expression and efficient transformation of cells in vitro and in vivo. When the nucleic acid is inserted into a suitable host cell (e.g., a prokaryotic or eukaryotic cell) and the host cell replicates, the protein can be recombinantly produced. Suitable host cells will depend on the vector and can include prokaryotic and eukaryotic cells, such as mammalian cells, animal cells, human cells, monkey cells, insect cells, yeast cells, and bacterial cells constructed using known methods. See Sambrook, et al. (1989), supra. In addition to using viral vectors to insert exogenous nucleic acids into cells, nucleic acids can also be inserted into host cells by methods known in the art, such as: transforming bacterial cells; transfecting mammalian cells using calcium phosphate precipitation; or DEAE dextran; electroporation; or microinjection. See, Sambrook et al. (1989) (supra) for the methods described therein. Thus, the present invention also provides host cells, such as mammalian cells, animal cells (rat or mouse), human cells, or prokaryotic cells (such as bacterial cells), comprising a polynucleotide encoding a protein or polypeptide or antibody or a fragment thereof.

[0185] Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, nucleotide structural modifications may be imparted before or after polynucleotide assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment disclosed herein as a polynucleotide includes both a double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0186] When the vector is used for in vivo or ex vivo gene therapy, a pharmaceutically acceptable carrier is preferred, such as a retroviral or adenoviral vector without replication ability. The pharmaceutically acceptable carrier comprising the nucleic acid of the present invention can be further modified to transiently or stably express the inserted polynucleotide. As used herein, the term "pharmaceutically acceptable carrier" includes but is not limited to a carrier or delivery vector with the ability to selectively target nucleic acid and introduce nucleic acid into dividing cells. An example of such a vector is a "weakly replicating" vector, which is defined as being unable to produce viral proteins, thereby preventing the propagation of the vector in infected host cells. An example of a weakly replicating retroviral vector is LNL6 (Miller et al. (1989) BioTechniques 7:980-990). A method for retroviral-mediated gene marker gene transfer using a weakly replicating retrovirus has been established. (Bordignon (1989) PNAS USA 86:8912-8952; Culver (1991) PNAS USA 88:3155; and Rill (1991) Blood 79(10):2694-2700).

[0187] The present invention also provides genetically modified cells comprising and / or expressing the polynucleotides of the present invention.Genetically modified cells can be generated by inserting upstream regulatory sequences (eg, promoters or gene activators) (see, US Pat. No. 5,733,761).

[0188] Polynucleotide can be conjugated to detectable labeling, such as enzyme labeling or radioisotope, to detect nucleic acid and / or gene expression in cell.A variety of suitable detectable labels are known in the art, including fluorescence, radioactivity, enzymatic or other parts that can provide detectable signal, such as avidin / biotin.On the one hand, people may wish to use fluorescent labeling or enzyme labeling, such as urease, alkaline phosphatase or peroxidase, to replace radioactivity or other reagents that are unfavorable to the environment.In the case of enzyme labeling, calorimetric indicator substrates can be used to provide the means of human eye visible or spectrophotometry, to identify the specific hybridization with the sample containing complementary nucleic acid.Therefore, the present invention further provides a method for detecting single-stranded polynucleotide or its complementary sequence, the method is by (preferably medium stringent hybridization conditions) under the conditions allowing complementary single-stranded polynucleotide hybridization, or more preferably under high stringent hybridization conditions, the target single-stranded polynucleotide is contacted with the labeled single-stranded polynucleotide (probe) as a part for polynucleotide of the present invention.The polynucleotide of hybridization is separated from the single-stranded polynucleotide that is not hybridized. Hybridized polynucleotide pairs are detected using methods known to those skilled in the art, such as those described in Sambrook et al. (1989) (supra).

[0189] The polynucleotides embodied in the present invention can be obtained using chemical synthesis, recombinant cloning methods, PCR or any combination thereof. Methods for chemical polynucleotide synthesis are known in the art and do not need to be described in detail herein. Those skilled in the art can use the sequence data provided herein to obtain the required polynucleotides by adopting a DNA synthesizer or from a commercial service.

[0190] The polynucleotides disclosed herein can be separated or replicated using PCR. PCR technology is the subject of U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, and is described in PCR: The Polymerase Chain Reaction (Mullis et al. eds., Birkhauser Press, Boston (1994)) or MacPherson et al. (1991) and (1995), and references cited therein. Alternatively, those skilled in the art can use the sequence provided herein and a commercial DNA synthesizer to replicate DNA. Therefore, the present invention also provides a method for obtaining the polynucleotides of the present invention, which provides the linear sequence of the polynucleotides, nucleotides, suitable primer molecules, chemicals (such as enzymes) and replication instructions thereof, and chemically replicates or connects nucleotides in the appropriate direction to obtain the polynucleotides. In a separate embodiment, these polynucleotides are further separated. Further, those skilled in the art can insert the polynucleotides into a suitable replication vector, and insert the vector into a suitable host cell (prokaryotic or eukaryotic) to replicate and amplify. The DNA amplified in this manner can be isolated from cells by methods known to those skilled in the art. Further provided herein are methods for obtaining polynucleotides by this method and the polynucleotides so obtained.

[0191] RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell. The DNA can be delivered by any suitable method, for example, by using a suitable gene delivery vehicle (e.g., liposomes, plasmids, or vectors) or by electroporation. When the cells replicate and the DNA is transcribed into RNA; the RNA can then be isolated using methods known to those skilled in the art, for example, as described in Sambrook et al. (1989) (supra). For example, mRNA can be isolated using various lytic enzymes or chemical solutions according to the methods described in Sambrook et al. (1989) (supra), or extracted by nucleic acid binding resin according to the accompanying instructions provided by the manufacturer.

[0192] Polynucleotides that exhibit sequence complementarity or homology with the polynucleotides of the present invention can be used as hybridization probes or as equivalents of the specific polynucleotides identified herein. Since the complete coding sequence of the transcript is known, any portion of that sequence or homologous sequence can be used in the methods of the present invention.

[0193] It is known in the art that specific hybridization does not require a "perfect match" probe. Minor changes in the probe sequence achieved by substitution, deletion or insertion of a small number of bases do not affect hybridization specificity. Generally, up to 20% base pair mismatches can be tolerated (when optimally aligned). Preferably, the probe used to detect the above-mentioned mRNA is at least about 80% identical to the homologous region. More preferably, after alignment of the homologous regions, the probe has 85% identity with the corresponding gene sequence; even more preferably, it exhibits 90% identity.

[0194] These probes can be used in radioanalysis (such as Southern and Northern blotting analysis), to detect, prognose, diagnose or monitor various cells or the tissue comprising these cells.Probe can also be connected on solid support or array (such as chip), for high throughput screening and measuring, to detect the expression of the gene corresponding to polynucleotide of the present invention.Therefore, the present invention also provides probe, it comprises or corresponds to polynucleotide of the present invention or its Equivalent or its complementary sequence or its fragment, it is connected to solid support for high throughput screening.

[0195] The total size of the fragments, as well as the size of the complementary fragments, will depend on the intended use or application of the particular nucleic acid fragment. Smaller fragments are generally useful in hybridization embodiments where the length of the complementary region can vary, for example, from at least 5 to 10 to about 100 nucleotides, or even the full length, depending on the complementary sequence that one wishes to detect.

[0196] In some embodiments, the present invention provides the nucleotide probe of the present invention.Usually preferably have the nucleotide probe of the complementary sequence on the extended sequence greater than 5 to 10 Nucleotide in length, to increase the stability and the selectivity of hybrid, thereby improve the specificity of the specific hybrid molecule obtained.More preferably, can design to have a length of 10 or more or greater than 50 Nucleotide or even longer gene complementary sequence when needed.Such fragment can be easily prepared in the following manner: for example, by chemical method directly synthesizing this fragment, by using nucleic acid replication technology, for example, as described in United States Patent (USP) 4,603,102, there is the PCR technology of two primer oligonucleotides, or by being incorporated into the recombinant vector for recombinant production by the sequence of selection.On the one hand, the length of probe is about 50-75 or more Nucleotide, alternatively 50-100 Nucleotide.

[0197] Polynucleotides of the present invention can be used as primers for detecting genes or gene transcripts expressed in cells as described herein. In this article, amplification refers to any method using a primer-dependent polymerase that can replicate the target sequence with reasonable fidelity. Amplification can be carried out by natural or recombinant DNA polymerases, such as T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. For illustration purposes only, the length of the primer is identical to the length identified by the probe.

[0198] A method for amplifying polynucleotides is PCR, and kits for PCR amplification are commercially available. After amplification, the resulting DNA fragments can be detected by any suitable method known in the art, such as by agarose gel electrophoresis, followed by visualization with ethidium bromide staining and ultraviolet irradiation.

[0199] Methods for administering an effective amount of gene delivery vectors or carriers to cells have been developed and are well known to those skilled in the art and are described herein. Methods for detecting gene expression in cells are known in the art and include techniques such as DNA microarray hybridization, in situ hybridization, PCR, RNase protection assays, and Northern blot analysis. Such methods can be used for detecting and quantifying gene expression in cells. Alternatively, the expression of the encoded polypeptide can be detected by a variety of methods. In particular, it is useful to prepare polyclonal or monoclonal antibodies that specifically react with the target polypeptide. Such antibodies can be used to visualize cells expressing polypeptides using techniques such as immunohistology, ELISA, and Western blotting. These techniques can be used to determine the expression level of the polynucleotides expressed.

[0200] In one aspect, polypeptides comprising an HMG-box domain include wild-type and recombinantly produced polypeptides and proteins from prokaryotic and eukaryotic host cells.

[0201] Proteins and polypeptides can be obtained by many methods known to those skilled in the art, including purification, chemical synthesis, and recombinant methods. Polypeptides can be isolated from preparations such as host cell systems by methods such as immunoprecipitation with antibodies and standard techniques such as gel filtration, ion exchange, reverse phase, and affinity chromatography. For such methods, see, for example, Deutscher et al. (1999) Guide To Protein Purification: Methods In Enzymology (Vol. 182, Academic Press). Therefore, the present invention also provides methods for obtaining these polypeptides and products obtainable and obtained by these methods.

[0202] Polypeptides can also be obtained by chemical synthesis using commercially available automated peptide synthesizers, such as those manufactured by Perkin / Elmer / Applied Biosystems, Inc., Model 430A or 431A, Foster City, Calif., 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 method for chemically synthesizing a protein of the present invention by providing the sequence of the protein and reagents (e.g., amino acids and enzymes) and linking the amino acids together in the correct orientation and linear sequence.

[0203] Alternatively, proteins and polypeptides may be obtained by well-known recombinant methods, such as described in Sambrook et al. (1989) (supra), using the host cells and vector systems described herein.

[0204] The polypeptide 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, pharmaceutically acceptable polymers, liposomes, micelles, suspensions, and emulsions). Examples of non-aqueous solvents include propyl glycol, polyethylene glycol, and vegetable oils. When used to prepare antibodies or induce an immune response in vivo, the carrier can also include an adjuvant that can be used to nonspecifically enhance the specific immune response. The technician can easily determine whether an adjuvant is needed and select one. However, for illustrative purposes only, suitable adjuvants include, but are not limited to, Freund's complete and incomplete solutions, mineral salts, and polynucleotides. Other suitable adjuvants include monophosphoryl lipid A (MPL), mutant derivatives of heat-labile enterotoxins of Escherichia coli, mutant derivatives of cholera toxin, CPG oligonucleotides, and adjuvants derived from squalene.

[0205] Treatment

[0206] One embodiment of the present invention provides a kind of method for suppressing, competing or titrating DNABII polypeptide or albumen and microorganism DNA combination, it comprises making described DNABII polypeptide or albumen or microorganism DNA contact with polypeptide as herein described, thereby suppress, compete or titrate described DNABII albumen or polypeptide and microorganism DNA combination.In some respects, described contact is in vitro or in vivo.

[0207] Another embodiment of the present invention provides a method for inhibiting, preventing or destroying microbial biofilm, comprising contacting the biofilm with a polypeptide as described herein, thereby inhibiting, preventing or destroying the microbial biofilm. In some aspects, the contact is in vitro or in vivo.

[0208] Another embodiment of the present invention provides a method for disrupting and clearing a biofilm without enhancing or inducing an inflammatory response, comprising contacting the biofilm with a polypeptide comprising, consisting essentially of, or consisting of a B Box polypeptide as described herein, thereby disrupting and clearing the biofilm without enhancing or inducing an inflammatory response. In some aspects, the contacting is in vitro or in vivo.

[0209] Another embodiment of the present invention provides a method for inhibiting, preventing or destroying a biofilm in a subject, comprising administering to the subject an effective amount of a polypeptide as described herein, thereby inhibiting, preventing or destroying a microbial biofilm. In one aspect, the method comprises administering an effective amount of a polypeptide comprising, consisting essentially of, or consisting of a B Box polypeptide as described herein, or consisting essentially of, or consisting of.

[0210] In another embodiment, a method for inhibiting, preventing, or treating a microbial infection that produces a biofilm in a subject is provided, comprising administering to the subject an effective amount of a polypeptide as described herein, thereby inhibiting, preventing, or treating a microbial infection that produces a biofilm in the subject. In one aspect, the method comprises administering an effective amount of a polypeptide comprising, consisting essentially of, or consisting of a B Box polypeptide as described herein, or consisting essentially of, or consisting of.

[0211] In one aspect of any of the above embodiments, the polypeptides comprising, consisting essentially of, or consisting of an HMG-box domain are described as AB Box, A Box, and B Box, as well as the mutants, truncations, and fusion proteins described herein (see Figure 3 ) and their equivalents.

[0212] Equivalents thereof include proteins that are compatible with AB Box, A Box and B Box, as well as mutants, truncations and fusion proteins described herein (see Figure 3 ) polypeptides that are at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% identical to, or consisting essentially of, or consisting of. In some aspects, equivalents retain the altered amino acids in the polypeptide and retain the abilities of the parent or reference protein, peptide, fusion, or mutant form. In some aspects, polypeptides comprising an HMG-box domain comprise, consist essentially of, or consist of biological equivalents to any of the polypeptides described above.

[0213] In some aspects, the isolated or recombinant protein is a mammalian protein. In a specific aspect, the mammalian protein is a mouse or human protein. In another aspect, the protein is a mammalian protein produced in a eukaryotic or prokaryotic cell. They can be post-translationally modified using methods known in the art.

[0214] Any of the above methods can further comprise one or more antimicrobial agents, antigenic peptides or adjuvants used to object in effective dose, or basically consist of it, or consist of it.On the one hand, this object is non-human animal or human patient.On the one hand, the patient is human teenager or baby.

[0215] The polypeptide is administered by methods including topical, transdermal, sublingual, rectal, vaginal, ophthalmic, subcutaneous, intramuscular, intraperitoneal, urethral, ​​intranasal, by inhalation, or oral.

[0216] In some aspects, the subject is a pediatric patient, and the polypeptide is administered in a formulation for pediatric patients.

[0217] In any of the above embodiments, the biofilm may comprise microbial DNA from the microorganisms identified in Table 1.

[0218] Table 1. Examples of bacterial strains that can produce biofilms

[0219]

[0220]

[0221] In one embodiment, the polypeptide is applied topically to the microbial infection and disrupts the biofilm.

[0222] In one embodiment, the present invention provides a method of inducing or providing an immune response in a subject in need thereof, comprising administering to the subject an effective amount of, consisting essentially of, or consisting of a polypeptide as described herein. In another embodiment, the administration is localized to the site where the immune response is desired. In one aspect, the method comprises administering an effective amount of, consisting essentially of, or consisting of a polypeptide comprising, consisting essentially of, or consisting of a B Box polypeptide disclosed herein. Examples of polypeptides comprising an HMG-box domain are described herein. In one aspect, the method comprises administering an effective amount of, consisting essentially of, or consisting of a polypeptide comprising, consisting essentially of, or consisting of a B Box polypeptide disclosed herein.

[0223] The isolated or recombinant protein can be a mammalian protein, or in particular aspects, a human protein.In certain aspects, the subject is a non-human animal or a human patient.

[0224] Reagent and composition of the present invention can be used simultaneously or successively with other antimicrobial agents and / or surface antigens.In a specific aspect, use is localized to the site of infection.Other non-limiting examples of use comprise by one or more methods, comprise transdermal, sublingual, rectal, vaginal, ophthalmic, subcutaneous, intramuscular, intraperitoneal, intranasal, by inhalation or oral.

[0225] In one embodiment, there is also provided the use of any of the above polypeptides in the preparation of a medicament for disrupting a biofilm or inhibiting, preventing or treating an infection by a biofilm-producing microorganism and providing the medical benefits described herein.

[0226] For some of these methods, contact can be carried out in vitro or in vivo.When contacting in vitro, the method provides the means of determining the effect of medicament of the present invention before animal or clinical studies, and can be used to determine whether medicament of the present invention acts synergistically with other antimicrobials.When carrying out in vivo in an animal model, the method provides the means of determining the effect of medicament of the present invention before studying in human patients, and can be used to determine whether medicament of the present invention acts synergistically with other antimicrobials.

[0227] Infections with microorganisms and diseases that can be treated by the methods of the present invention include infections with organisms identified in Table 1, for example, Streptococcus agalactiae, Neisseria meningitidis, Treponema denticola, Treponemapallidum, Burkholderia cepacia, or Burkholderia pseudomallei. In one aspect, the infection with microorganisms is one or more of Haemophilus influenzae (non-typeable) (NTHI), Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and the ESKAPE pathogen. These microorganisms may infect the upper, middle, and lower airways (otitis, sinusitis, bronchitis) and are complications and / or primary causes of exacerbations of chronic obstructive pulmonary disease (COPD), chronic cough, cystic fibrosis (CF), or community-acquired pneumonia (CAP).

[0228] Infections can also occur in the mouth (dental caries, periodontitis) and are caused by Streptococcus mutans, Porphyromonas gingivalis, and Aggregatibacter actinomucosus. Infections can also be localized to the skin (abscesses, "staph" infections, impetigo, secondary burns, Lyme disease) and are caused by Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Borrelia burdorferi. Urinary tract (UTI) infections can also be treated and are usually caused by Escherichia coli. Gastrointestinal (GI) infections (diarrhea, cholera, gallstones, gastric ulcers) are typically caused by Salmonella enterica serovars, Vibrio cholerae, and Helicobacter pylori. Reproductive tract infections include those typically caused by Neisseria gonorrhoeae. Enterococcus faecalis can cause bladder infections or indwelling device infections. Infections associated with prosthetic implants (e.g., artificial hip or knee replacements or dental implants) or medical devices (e.g., pumps, catheters, stents, or monitoring systems) (typically caused by various bacteria) can be treated by the methods disclosed herein. These devices can be coated or conjugated to reagents as described herein.

[0229] Infections caused by Streptococcus agalactiae can also be treated by the methods disclosed herein and are a leading cause of bacterial sepsis in neonates. Infections caused by Neisseria meningitidis, which can cause meningitis, can also be treated.

[0230] Therefore, routes of administration suitable for methods disclosed herein include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical, intravenous, rectal, nasal, oral and other enteral and parenteral routes of administration. If desired, routes of administration can be combined, or adjusted according to the medicament and / or desired effect. The active agent can be administered in single doses or multiple doses. These methods and embodiments of the routes suitable for delivery include systemic or local routes. Typically, routes of administration suitable for methods disclosed herein include, but are not limited to, enteral, parenteral or inhalation routes.

[0231] Parenteral routes of administration other than inhalation administration include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the digestive tract. Parenteral administration can be performed to achieve systemic or local delivery of the inhibitor. Where systemic delivery is desired, administration typically involves topical or mucosal administration of the pharmaceutical formulation for invasive or systemic absorption.

[0232] The agents disclosed herein can also be delivered to a subject via enteral administration. Enteral routes of administration include, but are not limited to, oral and rectal administration (eg, using suppositories).

[0233] Methods for administering active substances through the skin or mucous membranes include, but are not limited to, topical application of suitable pharmaceutical formulations, transdermal delivery, transdermal delivery, injection, and epidermal administration. For transdermal delivery, absorption enhancers or iontophoresis are suitable methods. Iontophoresis can be achieved using commercially available "patches" that continuously deliver their product over several days or more by means of electrical pulses.

[0234] In various embodiments of the methods disclosed herein, the agent is administered continuously on a daily basis at least once a day (QD) (in various embodiments twice a day (BID), three times a day (TID), or even four times a day) by inhalation, injection, or oral administration. Typically, the therapeutically effective daily dose can be 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.

[0235] Administration according to the methods disclosed herein can be accomplished using capsules, tablets, oral suspensions, suspensions for intramuscular injection, suspensions for intravenous infusion, ointments or creams for topical application, or suspensions for intra-articular injection.

[0236] The dosage, toxicity and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical methods in cell culture or experimental animals, for example, to determine the LD50 (the dose that is lethal to 50% of the population) and the ED50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio of LD50 / ED50. In certain embodiments, the compositions exhibit a high therapeutic index. Although compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the affected tissue site to minimize potential damage to uninfected cells, thereby reducing side effects.

[0237] The data obtained from cell culture assays and animal studies can be used to formulate a dosage range for humans. In certain embodiments, the dosage of such compounds is within a circulating concentration range that includes an ED50 with little or no toxicity. Depending on the dosage form used and the route of administration used, the dosage can vary within this range. For any compound used in this method, a preliminary estimate of the therapeutically effective dose can be obtained from the cell culture assay. Dosage can be formulated in an animal model to reach a circulating plasma concentration range including the IC50 (i.e., the concentration of the test compound that achieves half the maximum inhibition of symptoms) determined in cell culture. This information can be used to more accurately determine a dosage useful to the human body. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0238] In some embodiments, the effective amount of the composition that is enough to realize treatment or preventive effect is to use about 0.000001mg per kilogram of body weight at each time to about 10,000mg per kilogram of body weight at each time. Suitably, dosage range is from about 0.0001mg per kilogram of body weight at each time to about 100mg per kilogram of body weight at each time. Administration can be provided with pre-dose, then provided with one or more " booster " dosages. One day, two days, three days, one week, two weeks, three weeks, one, two, three, six or twelve months after the pre-dose can provide booster dose. In some embodiments, booster dose is used after the reaction previously used by the assessment subject.

[0239] Those skilled in the art will appreciate 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 condition, previous treatments, the patient's overall health and / or age, and other conditions. In addition, treating a subject with a therapeutically effective amount of a therapeutic composition described herein may include a single treatment or a series of treatments. In one aspect, the term "treatment" does not include prophylaxis.

[0240] Compositions of the present invention and related methods can be used in combination with the use of other therapies, or use in the absence of such therapies. These include but are not limited to the use of DNase enzymes, antibiotics, antimicrobials or other antibodies. On the one hand, the polypeptide is used together with the DNase enzyme to treat microbial infection and the biofilm relevant to cystic fibrosis.

[0241] In some embodiments, described method and composition include deoxyribonuclease (DNase) enzyme, such as DNA enzyme, that acts synergistically with the composition of the present invention. DNA enzyme is the enzyme that breaks the phosphodiester bond in any catalysis DNA backbone. Three non-limiting examples of the DNA enzyme of the multiple secondary structure of known not only target cruciform structure but also target DNA include DNAseI, T4 EndoVII and T7 EndoI. In certain embodiments, when used in combination with DNA enzyme, the effective amount of the anti-DNABII antibody required for destroying biofilm stability is reduced. When administered in vitro, DNA enzyme can be directly added in the assay or in the suitable buffer of known stabilizing enzyme. The effective unit dose and assay conditions of DNA enzyme can change, and can be optimized according to methods known in the art.

[0242] In other embodiments, the methods and compositions can be combined with antibiotics and / or antimicrobials. Antimicrobials are substances that kill or inhibit the growth of microorganisms (e.g., bacteria, fungi, or protozoa). Although biofilms are generally resistant to the effects of antibiotics, the compositions and methods described herein can be used to sensitize infections involving biofilms to conventional treatments for infections. In other embodiments, the use of antibiotics or antimicrobials in combination with the methods and compositions described herein can reduce the effective amount of the antimicrobial and / or biofilm reducing agent. Some non-limiting examples of antimicrobials and antibiotics used 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 and / or antibiotic and biofilm reducing agent can be easily determined by conventional methods. In some embodiments, the dose of the antimicrobial and biofilm reducing agent combination is an average effective dose that has been shown to be effective against other bacterial infections, such as bacterial infections where the etiology of the infection does not include biofilm. In other embodiments, dosage is 0.1,0.15,0.2,0.25,0.30,0.35,0.40,0.45,0.50,0.55,0.60,0.65,0.70,0.75,0.8,0.85,0.9,0.95,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,2.5,3.0 or 5 times of average effective dose.Can before adding anti-DNABII antibody, simultaneously or afterwards add antibiotic or antimicrobial.

[0243] In other embodiments, the method and composition can be combined with antibodies for treating bacterial infections. An example of an antibody used in combination with the method and composition described herein is an antibody against an unrelated outer membrane protein (i.e., OMP P5). Use of the antibody alone for treatment will not thin the biofilm in vitro. The effect produced by the combined treatment with the antibody and biofilm reducing agent is greater than the effect that can be achieved by using any one of the agents of the same concentration alone. Other antibodies that may produce synergistic effects when used in combination with biofilm reducing agents or methods for reducing biofilm include anti-rsPilA, anti-OMP26, anti-OMP P2, and anti-all-OMP preparations.

[0244] The compositions and methods described herein can be used to sensitize bacterial infections involving biofilms to common treatment modalities that are effective for treating bacterial infections without biofilms, but that are otherwise ineffective in treating bacterial infections involving biofilms. In other embodiments, the compositions and methods described herein can be used in combination with treatment modalities that are effective for treating bacterial infections involving biofilms, but the combination of such additional therapy and biofilm reducing agent or method produces a synergistic effect, such that the effective dose of the biofilm reducing agent or other therapeutic agent can be reduced. In other cases, the combination of such additional therapy and biofilm reducing agent or method produces a synergistic effect, thereby enhancing treatment. The enhancement of treatment can be demonstrated by a shorter time required to treat the infection.

[0245] Additional treatments may be added before, simultaneously with, or after the method or composition for reducing biofilm and may be contained in the same structure or as separate formulations.

[0246] Reagent test kit

[0247] Also claimed are kits comprising the reagents and instructions necessary to perform the in vitro and in vivo methods described herein. Thus, the present invention provides kits for performing these methods, which may include the biological agents of the present invention and instructions for performing the methods of the present invention, such as collecting tissue and / or performing screening and / or analyzing results and / or administering an effective amount of a biological agent as defined herein. These may be used alone or in combination with other suitable antimicrobial agents.

[0248] In one embodiment, the present invention provides a kit comprising a polypeptide as described herein and instructions for use in decomposing a biofilm or inhibiting, preventing, or treating an infection by a microorganism that produces a biofilm. In one embodiment, the kit further comprises one or more of an adjuvant, an antigenic peptide, or an antimicrobial agent. In another embodiment, the kit further comprises a carrier selected from a liquid carrier, a pharmaceutically acceptable carrier, a solid phase carrier, a pharmaceutically acceptable carrier, an implant, a stent, a paste, a gel, a dental implant, or a medical implant.

[0249] The following examples are intended to illustrate but not to limit the present invention.

[0250] Bacterial biofilm-mediated infections account for approximately 80% of all chronic / recurrent human infections. Biofilms constitute a protected mode of microbial growth. That is, they are composed of microbial communities attached to surfaces and embedded in a hydrated polymer matrix synthesized by themselves. The formation of these sessile communities allows bacteria to survive in adverse environments, making them inherently resistant to conventional treatments including antimicrobial agents and host defenses. Biofilm-related infections are very common and are associated with notorious consequences in terms of attributable mortality and economic burden, so novel treatments are urgently needed. In this regard, the applicant has developed a new immunotherapy for the treatment of stubborn bacterial biofilm-mediated infections. This new approach is based on the nucleoprotein interaction that occurs in the extracellular matrix of biofilms. It is known that the extracellular matrix of biofilms is composed of a variable mixture of proteins, lipids, polysaccharides and extracellular DNA (eDNA). The key components of the extracellular matrix that are critical to the structural integrity of bacterial biofilms are eDNA and proteins of the bacterial DNABII family (IHF and HU). DNABII proteins bind to and bend double-stranded DNA (dsDNA) with high affinity to pre-bend DNA ( Figure 1 ). In vivo, it has been shown that within formed biofilms, there is a vast network of intertwined eDNA chains, which is stabilized by the DNABII protein located at the vertices of the curved cross-strands of the eDNA. Applicants disclose herein eukaryotic polypeptides (e.g., HMGB1) having one or more HMG-box domains that can interfere with the structure of the extracellular DNA scaffold within the biofilm. By competing with microbial proteins bound to the DNA scaffold in the biofilm, these polypeptides destabilize the biofilm, which leads to destruction and removal of the biofilm by the host immune system ( Figure 2 ).

[0251] This new therapeutic approach is innovative because it is the first time that HMGB1 and its variants have been tested for their potential as anti-biofilm therapeutics. Furthermore, HMGB1 domains and mutant variants, including A-box, B-box, C-tail, and A+B-box ( Figure 3 ) to identify the optimal protein fragment with the best antimicrobial film, less anti-inflammatory activity, and the smallest protein fragment size. In this regard, this protein fragment will be able to treat bacterial biofilm diseases without the consequences of excessive inflammation. In addition, because the release of bacteria from biofilms makes these bacteria more susceptible to antibiotic-mediated killing, this suggests that HMGB1 treatment can potentially be combined with conventional treatments (such as antibiotics), which will improve anti-biofilm efficacy and reduce the development of antimicrobial resistance.

[0252] An in vitro biofilm assay was used to test the effects of HMGB1 and its variants on established bacterial biofilms. Applicants expressed (in E. coli) and purified >95% of human recombinant full-length HMGB1 (rHMGB1; 1-215), C45S mutant variant (mHMGB1), and HMGB1 domains A Box (1-89), A+B Box (1-176), B Box (80-179), and B Box C106S (mB Box) ( Figure 3 All full-length and HMGB1 variants retained DNA binding activity, indicating that these domains are correctly folded and functional ( Figure 4 To evaluate the effects of HMGB1 and its variants, as well as commercially available natural bovine HMGB1 (used as a control), on established bacterial biofilms, each protein (200 nM) was added to preformed K. pneumoniae biofilms after 24 h of growth. After 16 h of incubation (40 h of total biofilm growth), the biofilms were incubated with The cells were stained and analyzed using confocal laser scanning microscopy (CLSM) and COMSTAT analysis to calculate the average biofilm thickness and total biomass. Full-length recombinant HMGB1 was able to significantly disrupt established K. pneumoniae biofilms, as were all truncated HMGB1 forms containing the BBox domain ( Figure 5 The results of this study raise a noteworthy observation: a single dose of these non-antimicrobial compounds was able to disrupt recalcitrant biofilms. In addition, the HMGB1 variants destroyed every bacterial species tested to date, including pathogenic Escherichia coli, Burkholderia cenocepacia, non-typeable Haemophilus influenzae ( Figure 6 A single dose at this concentration releases the bacteria from their protective shield, making them susceptible to clearance by antibiotics and the immune system.

[0253] Applicants' novel approach to treating biofilm-associated infections using non-antimicrobial agents such as HMGB1 protein and its variants represents a radical departure from classical therapeutic concepts. Full-length HMGB1, the smallest variant tested to date with the greatest in vitro disrupting activity, and a modified B Box (mB Box) in which the cysteine ​​at position 106 was mutated to serine to eliminate its inflammation-inducing ability, were tested for their biofilm disrupting and inflammatory activities in vivo. Figure 7 Using an aggregated biofilm infection lung model, the applicants showed that HMGB1, B Box, and their respective modified forms were able to prevent biofilm formation in vivo and that the modified proteins did not induce an inflammatory response ( Figure 7 B and Figure 7 C). Applicants further demonstrated that none of the HMGB1 variants induced sepsis at a given dose capable of disrupting biofilms ( Figure 7D).

[0254] method

[0255] Experiment 1

[0256] Klebsiella pneumoniae (KP), a common cause of nosocomial infections, was used in all BB disruption assays. Human recombinant full-length HMGB1 (rHMGB1; 1-215), C45S mutant variant (mHMGB1), and HMGB1 domains A Box (1-89), B Box (90-176), AB Box (1-176), B-Linker Box (80-179), and B-Linker Box C106S were expressed in E. coli and purified to >95%. To evaluate the effects of rHMGB1 and the individual domains on established BBs, each protein (200 nM) was added to preformed BBs within 24 hours. At 40 hours, the BBs were washed and incubated with stained, observed by confocal laser scanning microscopy and images analyzed by COMSTAT to calculate the average thickness and biomass.

[0257] Compared with untreated KP biofilms, exogenous rHMGB1 and its individual domains, except A-Box, resulted in significant (p < 0.05) reductions in the average thickness (AT) and biomass (BM) of KP biofilms (% reduction mean ± SE in AT: 44% ± 0.33, 75% ± 0.04, 63% ± 0.1, 77% ± 0.03, 64% ± 0.08, 54% ± 0.15, and in BM: 61% ± 0.01, 80% ± 0.01, 68% ± 0.02, 67% ± 0.01, 73% ± 0.02, 56% ± 0.02, induced by rHMGB1, mHMGB1, B-Box, B-Linker Box, AB Box, and B-Linker Box C106S, respectively).

[0258] Experiment 2

[0259] HMGB1 disrupts pathogenic biofilms: To test the effect of HMGB1 on bacterial biofilms ( Figure 5 ), the applicant clones ( Untagged human recombinant HMGB1 (rHMGB1) and an engineered C45S variant (mHMGB1), which mimics the reduced form of HMGB1, were expressed (in Escherichia coli) and purified (heparin sepharose chromatography to >95% purity) at 47°C (NEB Ipswich, MA). rHMGB1 readily forms an intramolecular disulfide bond between C23 and C45, contributing to proinflammatory activity, whereas mHMGB1 does not. The ability of these HMGB1 isoforms to disrupt established biofilms (established for 24 h prior to addition) was assessed. After 16 h of exposure to a single dose of rHMGB1 (200 nM; 25-fold higher than typical sepsis serum concentrations, but not capable of directly inducing sepsis), the cells were treated with Biofilms formed by multiple high-priority species were stained, analyzed using confocal laser scanning microscopy (CLSM) and COMSTAT analysis, and compared to control biofilms. Applicants observed a significant decrease (P < 0.05) in the average thickness and biomass (not shown) of each biofilm ( Figure 5 A). Only E. faecalis and S. aureus required higher concentrations (although non-bactericidal) to achieve similar results. Native HMGB1 purified from calf thymus (nHMGB1; Chondrex, Inc, Redmond, WA) was also shown to disrupt selected biofilms (UPEC, Bc, NTHI, K. pneumoniae) equivalently compared to rHMGB1 ( Figure 5 A), suggesting that any potential differences in post-translational modifications (PTMs) between nHMGB1 and rHMGB1 do not significantly affect this anti-biofilm function. Preliminary analysis of PTMs by LC-MS / MS analysis (MS Bioworks, LLC Ann Arbor, MI) showed that both rHMGB1 and nHMGB1 were minimally modified (<20% of the observed peptides had any given PTM, Figure 1 C). Increasing concentrations of rHMGB1 (up to 400 nM) disrupted UPEC biofilms in a dose-dependent manner, down to a monolayer (average thickness of approximately 1 μm; Figure 5 B), i.e., complete elimination of the 3D biofilm structure, implies a reduction in bioburden, allowing host immune effectors or other antimicrobial compounds to complete eradication. Thus, despite inherent differences in sensitivity between pathogens, biofilms formed by all tested high-priority pathogens were sensitive to a single dose of these non-antimicrobial compounds.

[0260] Experiment 3

[0261] HMGB1 domain structure and anti-biofilm activity: Applicants subsequently generated recombinant HMGB1 truncated variants, which are 1) A Box, which is a self-contained DNA binding domain (residues 1-89); 2) AB Box construct (lacking the C-tail; residues 1-185); and 3) B Box (residues 80 to 176, Figure 1 A and 1C). Addition of A Box to established biofilms (UPEC, Bc, NTHI, and K. pneumoniae) had no significant effect on the measured biofilm parameters ( Figure 5 C). In contrast, the AB Box and the 97-amino acid (AA) B Box retained complete antibiofilm activity ( Figure 5 C). Since only B Box can regulate DNA bending, without being bound by theory, it can be speculated that HMGB1 disrupts biofilms at least in part through DNA binding / bending. B Box has been reported to contain pro-inflammatory activity, primarily mediated by interaction with TLR4-MD2 dependent on residue C106, so the applicant created a modified B Box variant (mB Box) with a C106S mutation ( Figure 1 A and 1C). Compared to B Box, mB Box variants equivalently disrupted bacterial biofilms (UPEC, NTHI, Bc, Klebsiella pneumoniae) in vitro ( Figure 5 C).

[0262] Experiment 4

[0263] rHMGB1 and mHMGB1 disrupt biofilms in two different animal models, but the inflammatory response was greatly attenuated by a Cys to Ser mutation: Applicants tested the ability of rHMGB1 and mHMGB1 to treat middle ear infections and the corresponding inflammatory response using a well-established experimental OM model in ground rats due to NTHI49,67,68, in which adherent mucosal biofilm formation plays a key role in pathogenesis ( Figure 8 A). The middle ears of adult ground squirrels of mixed sexes were inoculated with 1000 CFU of NTHI via injection into the gastrocnemius muscle. On days 4 and 5 after challenge, when a large amount of biofilm was present in the middle ear space, 5 μg (0.2 nmol) of rHMGB1, mHMGB1, or diluent was delivered directly to the middle ear (2 treatments in total). On day 6, the animals were sacrificed, the middle ear was imaged, and the residual biofilm ( Figure 8 A) and mucosal inflammation ( Figure 8 B) Blind scoring was performed. Animals treated with diluent had a thick mucosal biofilm that obscured the bony septa ( Figure 8 C and 8D). In stark contrast, in animals treated with rHMGB1 or mHMGB1, residual mucosal biofilm was greatly reduced, with CFU reduced approximately 1000-fold ( Figure 8 E). These results are particularly noteworthy because biofilms formed in vitro by NTHI were less responsive to HMGB1 addition than those of the other tested bacterial species ( Figure 5 Middle ear fluid (MEF) collected from rHMGB1-treated animals had increased proinflammatory cytokines (IL-1β (3-fold), IL-17A (2-fold)) compared to mHMGB1- and diluent-treated animals (data not shown), consistent with rHMGB1 enhancing visible inflammation of the middle ear mucosa ( Figure 8 C and 8F). In contrast, MEFs from mHMGB1-treated animals had increased anti-inflammatory cytokines (IL-4 (2-fold), IL-10 (5-fold)) (data not shown), which corresponded to reduced mucosal inflammation ( Figure 8 C and 8F). Thus, mHMGB1 effectively promoted the clearance of NTHI biofilms in vivo without triggering pro-inflammatory signals. Next, the applicants determined whether rHMGB1 or mHMGB1 could prevent the development of aggregated biofilms. For prevention, 10 7 Adult C57BL / 6 mice were challenged intratracheally (it) with CFU of Bc and 0.2 nmol of rHMGB1 or mHMGB1 was added. 18 hours later, mice were euthanized, and bronchoalveolar lavage (BAL) and lungs were collected. Bc formed aggregates that were readily visible in the lungs probed with Bc antibodies ( Figure 7 A). Tissues were homogenized and CFU were counted. BAL of mice administered with rHMGB1 or mHMGB1 ( Figure 7 B) and Bc in lung tissue were significantly reduced (data not shown; P < 0.05), indicating that HMGB1 inhibits biofilm formation in the murine airways and that this process promotes bacterial clearance. Preliminary results with B Box and mB Box derivatives indicate that these 97AA peptides inhibit biofilm development in vivo (reduction in CFU in BAL, Figure 7 B), and the C106S mutation abolished the pro-inflammatory activity (2-fold reduction compared to B Box) ( Figure 7 C). This animal model was then used to study lung injury 72 hours after Bc challenge and the administration of rHMGB1 or mHMGB1. Lungs were collected and the tissues fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The lungs of mice treated with mHMGB1 more closely resembled those of uninfected mice, whereas severe inflammation and an enhanced neutrophil response occurred in mice treated with rHMGB1 ( Figure 7D), which shows that mHMGB1 retains anti-biofilm activity without the pro-inflammatory activity of rHMGB1. Next, the applicant evaluated the ability of rHMGB1 and mHMGB1 to treat established Bc infection. Mice were challenged as described above and administered 0.2 nmol rHMGB1 or mHMGB1 24 hours later. 48 hours after treatment, mice were euthanized and BAL and lungs were collected and processed as described above. Mice treated with rHMGB1 or mHMGB1 had significantly less Bc in their lungs ( Figure 7 E), while mHMGB1 treatment induced fewer neutrophils and inflammatory monocytes to be recruited to the lungs (P < 0.05, data not shown). Together, these data indicate that mHMGB1 and mB Box did not enhance the inflammatory response, but inhibited the formation of aggregated biofilms, bacterial absorption and clearance. To further confirm the reduced pro-inflammatory activity of mHMGB1, the applicant used an in vivo model of chemotaxis to determine the relative ability to recruit neutrophils70,71. C57BL / 6 mice were injected intraperitoneally (ip) with 0.2 nmol of mHMGB1 or rHMGB1 or 1 ml of 4% thioglycolate (positive control; inducer of neutrophil recruitment). After 4 hours, the mice were euthanized and peritoneal lavage was performed. Cells were stained with anti-CD45, anti-CD11b and anti-Ly6G antibodies, and total neutrophils were quantified by fluorescence-activated cell sorting (FACS). rHMGB1 induced neutrophil recruitment to the peritoneal cavity, whereas mHMGB1 did not ( Figure 7 F).

[0264] Experiment 5

[0265] HMGB1 variants do not induce sepsis at doses required to treat biofilm disease: Although HMGB1 variants with Cys to Ser mutations (mHMGB1, mB Box) do not induce proinflammatory sequelae like single doses of HMGB1 and B Box, they still show effective anti-biofilm activity. Despite this, the applicants rigorously investigated whether our HMGB1 variants can induce sepsis in the presence or absence of LPS. The applicants used 0.2 nmol (the same amount that showed therapeutic effects in in vivo biofilm treatment) of endotoxin-free rHMGB1, B Box, or mB Box (purified with high-capacity endotoxin removal resin (Pierce, Inc) and verified by endotoxin quantification (Genscript) to contain <300 pg endotoxin / μg protein) to intraperitoneally inject naive mice or mice sensitized with a non-lethal dose of LPS (5 m / kg). Mice were monitored for signs of sepsis for 24 hours, and serum TNF-α (TNF-α) levels were measured by ELISA as a surrogate for sepsis induction. No mice showed signs of sepsis requiring euthanasia before the study endpoint. LPS alone induced over 100 pg / ml of TNF, whereas rHMGB1, B Box, or mB Box alone were unable to elicit detectable TNF, and none induced additional pro-inflammatory signaling when administered to LPS-sensitized mice. Figure 7 G). It has been reported that >450-fold more B Box alone is required than our dose, or four doses of twice as much rHMGB1 are required to induce sepsis in mice sensitized with lipopolysaccharide (LPS) over 40 hours. In addition, as described above, these endotoxin-free peptides were tested for anti-biofilm activity in vitro and found to retain full functionality against UPEC and Bc biofilms (data not shown). Therefore, the variants tested here do not induce sepsis at effective therapeutic doses.

[0266] Experiment 6

[0267] Disruption of biofilms by HMGB1 sensitizes released bacteria to antibiotics: 200 nM HMGB1, 1 μg / ml minocycline, or both were added to Bc biofilms as described above. The use of dyes showed synergistic bacterial killing ( Figure 9 ), suggesting that HMGB1 treatment could be used in combination therapies to both improve antimicrobial efficacy and reduce the development of antimicrobial resistance.

[0268] Experiment 7

[0269] HMGB1 and DNABII proteins are present in biofilms formed in vivo: Applicants have previously demonstrated that in biofilms formed by NTHI in the OM experimental model, the bacterial DNABII protein is located at the apex of the eDNA cross-strands. To determine the relative presence and spatial distribution of HMGB1 in NTHI biofilms formed in vivo, unfixed biofilms recovered from the middle ear of ground squirrels were probed with HMGB1 and DNABII antibodies. eDNA was stained with DAPI (white). HMGB1, which was labeled with different periodicities along the length of the dsDNA chain, was in close proximity to the labeled DNABII protein, but did not co-localize, and was detected on the cross-strands of the eDNA (as expected) ( Figure 10 A). Notably, HMGB1 was not observed at the apex. These data suggest that HMGB1 can integrate into the EPS but does not simultaneously occupy the same eDNA site (apex) as DNABII, supporting the hypothesis that HMGB1 competes with DNABII at the eDNA apex to destabilize the EPS. These data also demonstrate that DNABII and HMGB1 proteins do not colocalize and therefore do not interact effectively, further suggesting that HMGB1 may act solely through DNA binding / bending.

[0270] Experiment 8

[0271] HMGB1 disrupts biofilms present in clinical specimens: Biofilms in chronic disease sites (e.g., CF lungs) are composed of multiple species and are therefore difficult to eradicate. Anti-DNABII antibodies have been shown to disrupt biofilms present in CF sputum. Incubation of CF sputum suspensions in PBS containing 1M Bbox at 37°C for 2 h effectively disrupts sputum and achieves the same level of biofilms as those obtained with the addition of high concentrations of Bbox. (a therapeutic DNA enzyme used as a mucolytic in CF patients) or to the same extent as anti-DNABII ( Figure 10 B) These data indicate that biofilms formed at sites of chronic infection possess a conserved eDNA-dependent structure that is susceptible to HMGB1 infection and demonstrate that HMGB1 is a host defense against biofilms.

[0272] Experiment 9

[0273] Many oral bacteria (e.g., Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis) have been implicated in the pathogenesis of inflammatory diseases such as periodontitis and peri-implantitis, which destroy the alveolar bone and gums. The lack of effective animal models has hampered research into the pathogenesis of these bacteria. One of the challenges in studying the pathogenicity of specific bacteria is the difficulty in establishing biofilms when exogenous bacteria are introduced into the oral cavity of an animal. Although animal models of periodontitis have been developed, culturable bacteria are rarely recovered from the oral cavity of the inoculated animal. Establishing an effective animal model that can assess the pathogenicity of specific bacteria would greatly help in elucidating their pathogenic mechanisms. This embodiment provides a model for testing the disclosed polypeptides and compositions and their efficacy in treating oral diseases.

[0274] The surfaces of machined titanium dental implants (1.2×4.5 mm) were modified by sandblasting with AlO 3 (100 μm) and etching with HCl (pH 7.8 at 80° C. for 20 minutes). The machined and nanotextured implants were incubated in TSB medium inoculated with the D7S clinical strain of Actinobacillus actinomycetemcomitans (Aa) at 37° C. for 1 to 3 days. After staining, the bacterial biofilm on the implants was analyzed by SEM and confocal laser scanning microscopy. Implants with or without established Aa biofilm were placed transmucosally in the alveolar bone between the maxillary premolar and incisor regions of female rats. To detect the presence of Aa biofilm on implants placed in vivo, bacterial samples were collected from saliva and the oral surface of the implants after two days. Aa can be detected by culture and PCR analysis. Micro-CT and histological analysis of the bone and mucosal tissue surrounding the implants were performed six weeks after implantation. As described herein, peptides and compositions were tested for surface binding, biofilm, and bacterial growth.

[0275] Experiment 10

[0276] This study provides a mouse model for preclinical testing of interferon for the treatment of Lyme disease. See Dresser et al. Pathogens 5(12)e1000680, Epub 2009 Dec 4. Lyme disease is the most common tick-borne disease in the United States. By definition, these endemic areas are expanding as the human population continues to move from cities to suburban and rural areas and white-tailed deer (which carry the tick species Ixodes) increasingly roam these areas. Lyme disease is caused by the microorganism Borrelia burgdorferi (a spirochete). Borrelia burgdorferi is transmitted through the bite of Ixodes ticks and then spreads to other tissues and organs through the blood.

[0277] In this animal model, by dorsal subcutaneous and intraperitoneal injection, or by intravenous injection, to C3H / HeN mice injection spirochetes.About 7 days after infection, blood and biopsy specimens are reclaimed to assess the pathological condition of microbial load and assessment tissue and organ. Expected method and composition of the present invention will be developed to reduce and / or eliminate the treatment and prevention strategy of the resulting Borrelia burgdorferi biofilm that forms and is considered to the pathogenesis of the disease and chronic nature that all contribute to after attack.

[0278] Experiment 11

[0279] This experiment provides a pig model for preclinical testing of the disclosed polypeptides and compositions for the treatment of lung diseases such as COPD and cystic fibrosis. See Stoltz et al. (2010) Science Translational Medicine 2(29):29ra31. Cystic fibrosis is an autosomal recessive disease due to mutations in the gene encoding the CF transmembrane conductance regulator (called CFTR) anion channel. In this model, pigs that have been specifically bred to carry a defect in the gene called "CFTR" are called CF pigs, and they naturally develop the hallmark features of CF lung disease, including lower respiratory tract infections caused by a variety of bacteria. The composition can be administered to the pigs to deliver the polypeptides to the lungs of these animals by aerosolization to evaluate the improvement of disease signs and related pathology.

[0280] Experiment 12

[0281] The applicant also provides a preclinical model of tuberculosis (TB). See Ordway et al. (2010) Anti.Agents and Chemotherapy 54: 1820. In this animal model, SPF guinea pigs are housed in barrier colonies and infected by aerosol spray to deliver approximately 20 cfu of Mycobacterium tuberculosis (M. tuberculosis) strain Erdman KO1 bacteria to their lungs. At 25, 50, 75, 100, 125 and 150 days after challenge, the animals are sacrificed and the bacterial load is determined and tissues are recovered for histopathological evaluation. Unlike mice that do not develop classic TB symptoms, guinea pigs challenged in this manner develop well-organized granulomas with central necrosis, a hallmark of human disease. In addition, guinea pigs, like humans, develop severe pyogranulomas and necrotizing lymphadenitis of the draining lymph nodes, which are part of the primary lesion complex. The use of this model will provide preclinical screening to confirm and identify therapeutic and preventive strategies to reduce and / or eliminate the production of M. tuberculosis biofilms, which have been observed to form in the lungs of these animals following challenge and are thought to contribute to the pathogenesis and chronicity of the disease.

[0282] Experiment 13

[0283] A variety of animal models of catheter / indwelling device biofilm infections are known. See Otto (2009) Nature Reviews Microbiology, 7:555. Although generally considered a normal skin flora, the microorganism Staphylococcus epidermidis has become considered by many to be a key opportunistic pathogen, ranking first among the pathogens of nosocomial infections. Primarily, this bacterium causes the majority of infections that occur on indwelling medical devices, which are contaminated by this common skin colonizer during device insertion. Although not typically life-threatening, the difficulties associated with the treatment of these biofilm infections and their frequency make them a significant public health burden. There are several animal models of catheter-associated Staphylococcus epidermidis infections, including rabbits, mice, guinea pigs, and rats, all of which are used to study the molecular mechanisms of pathogenesis and to aid in prevention and / or treatment. Rat jugular vein catheters have been used to evaluate therapies that interfere with biofilm formation of Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis. Biofilm reduction is typically measured by one of three methods: (i) sonicating the catheter and counting the CFU, (ii) sectioning the catheter or simply placing it on a plate and scoring it, or (iii) the biofilm can be stained with crystal violet or another dye, washed off, and the OD measured (as a surrogate for CFU).

[0284] in conclusion

[0285] Full-length recombinant HMGB1 was able to significantly disrupt established biofilms, as were all truncated HMGB1 forms containing the B-box domain.

[0286] Equivalent

[0287] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0288] The present technology illustratively described herein may suitably be practiced in the absence of any element or limitation not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like should be understood broadly and without limitation. Furthermore, the terms and expressions employed herein have been used as descriptive and not limiting, and in the use of these terms and expressions, there is no intention to exclude any equivalents of the features shown and described or portions thereof, but it should be recognized that various variations are possible within the scope of the present technology.

[0289] Therefore, it should be understood that the materials, methods, and examples provided herein represent preferred aspects, are exemplary, and are not intended to limit the scope of the present technology.

[0290] The present technology is described broadly and generally herein. Each of the narrower species and subgeneric groupings falling within the general disclosure also forms a part of this disclosure. This includes the general description of the present technology with a proviso or negative limitation that removes any subject matter from that genus, regardless of whether the deleted material is specifically described herein.

[0291] In addition, where features or aspects of the technology are described in terms of Markush groups, those skilled in the art will recognize that embodiments of the disclosure are also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0292] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0293] Further aspects are set out in the accompanying claims.

[0294] Sequence Listing

[0295] SEQ ID NO: 1 and 2—Wild-type HMGB1 (mouse and human)

[0296]

[0297] (Human, copied from GenBank accession number CAE48262.1)

[0298] HMGB1 is a small protein (approximately 30 kDa) consisting of 215 amino acids and composed of three domains: two positively charged domains, the A and B boxes, each consisting of 80 amino acids; and a negatively charged carbonyl terminus (the acidic C-tail), consisting of approximately 30 consecutive aspartic acid and glutamic acid residues.

[0299] The bold amino acids (amino acids 1-70) describe the A Box domain.

[0300] Italicized amino acids (approximately amino acids 88-164) describe the B Box domain.

[0301] The underlined amino acids (amino acids 186-215) depict the C-tail domain.

[0302] Mutated forms of HMGB1 are shown in Figure 1 and Figure 3 , which have amino acid substitutions.

[0303] SEQ ID NO: 3 and 4

[0304] Wild-type mouse HMGB1 B Box: MW = 9735.2; 87aa

[0305] KDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAE

[0306] KLKEKYEKDIAAYRAKGKPDAAKKGVV

[0307] Wild-type human HMGB1 B Box: 87aa

[0308] KDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAA

[0309] KLKEKYEKDIAAYRAKGKPDAAKKGVV

[0310] SEQ ID NO: 5 and 6

[0311] Mouse mutant HMGB1 B Box: MW = 9735.2; 87aa

[0312] KDPNAPKRPPSAFFLFSSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAE

[0313] KLKEKYEKDIAAYRAKGKPDAAKKGVV

[0314] Human mutant HMGB1 B Box:87aa

[0315] KDPNAPKRPPSAFFLFSSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAA

[0316] KLKEKYEKDIAAYRAKGKPDAAKKGVV

[0317] Cysteine ​​(C) has been mutated to serine (S) (bold text).

[0318] SEQ ID NO: 7 and 8

[0319] Wild-type mouse HMGB1 A+B Box: MW = 20261.42; 176aa

[0320] MGKGDPKKPRRKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGK

[0321] FEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL

[0322] SIGDVAKKLGEMWNNTAADDKQPYEKKAEKLKEKYEKDIAAYRAKGKPDAAKKGVV

[0323] Wild-type human HMGB1 A+B Box: 176aa

[0324] MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGK

[0325] FEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL

[0326] SIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVV Cysteine ​​(C) has been mutated to serine (S) (bold text).

[0327] SEQ ID NO: 9 and 10

[0328] Wild-type mouse HMGB1 B Box + N-linker (underlined): MW = 10876.6; 97 aa

[0329] PPKGETKKKF KDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADD

[0330] KQPYEKKAEKLKEKYEKDIAAYRAKGKPDAAKKGVV

[0331] Wild-type human HMGB1 B Box + N-linker (underlined): 97aa

[0332] PPKGETKKKF KDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADD

[0333] KQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVV

[0334] SEQ ID NO: 11 and 12

[0335] Mutant HMGB1 B Box + N-linker (underlined): MW = 10876.6; 97aa

[0336] PPKGETKKKF KDPNAPKRPPSAFFLFSSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADD

[0337] KQPYEKKAEKLKEKYEKDIAAYRAKGKPDAAKKGVV

[0338] Human HMGB1 B Box + N-linker (underlined): 97aa

[0339] PPKGETKKKF KDPNAPKRPPSAFFLFSSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADD

[0340] KQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVV. Sequence Listing <110> Children's National Hospital Research Institute <120> HMGB1 protein derivatives for biofilm removal <130> 106887-7910 <140> PCT / US2019 / 054851 <141> 2019-10-04 <150> 62 / 742,102 <151> 2018-10-05 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 215 <212> PRT <213> Mus sp. <400> 1 Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Arg Lys Met Ser Ser Tyr 1 5 10 15 Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys His Pro 20 25 30 Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg 35 40 45 Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala 50 55 60 Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro 65 70 75 80 Pro Lys Gly Glu Thr Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys 85 90 95 Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys 100 105 110 Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys 115 120 125 Leu Gly Glu Met Trp Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr 130 135 140 Glu Lys Ala Glu Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala 145 150 155 160 Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val 165 170 175 Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Gly Glu Glu 180 185 190 Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu 195 200 205 Glu Glu Asp Asp Asp Asp Glu 210 215 <210> 2 <211> 215 <212> PRT <213> Homo sapiens <400> 2 Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr 1 5 10 15 Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro 20 25 30 Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg 35 40 45 Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala 50 55 60 Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro 65 70 75 80 Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys 85 90 95 Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys 100 105 110 Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys 115 120 125 Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr 130 135 140 Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala 145 150 155 160 Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val 165 170 175 Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu 180 185 190 Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu 195 200 205 Glu Glu Asp Asp Asp Asp Glu 210 215 <210> 3 <211> 87 <212> PRT <213> Mus sp. <400> 3 Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe 1 5 10 15 Cys Ser Glu Tyr Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu Ser 20 25 30 Ile Gly Asp Val Ala Lys Lys Leu Gly Glu Met Trp Asn Asn Thr Ala 35 40 45 Ala Asp Asp Lys Gln Pro Tyr Glu Lys Lys Ala Glu Lys Leu Lys Glu 50 55 60 Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg Ala Lys Gly Lys Pro Asp 65 70 75 80 Ala Ala Lys Lys Gly Val Val 85 <210> 4 <211> 87 <212> PRT <213> Homo sapiens <400> 4 Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe 1 5 10 15 Cys Ser Glu Tyr Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu Ser 20 25 30 Ile Gly Asp Val Ala Lys Lys Leu Gly Glu Met Trp Asn Asn Thr Ala 35 40 45 Ala Asp Asp Lys Gln Pro Tyr Glu Lys Lys Ala Ala Lys Leu Lys Glu 50 55 60 Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg Ala Lys Gly Lys Pro Asp 65 70 75 80 Ala Ala Lys Lys Gly Val Val 85 <210> 5 <211> 87 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 5 Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe 1 5 10 15 Ser Ser Glu Tyr Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu Ser 20 25 30 Ile Gly Asp Val Ala Lys Lys Leu Gly Glu Met Trp Asn Asn Thr Ala 35 40 45 Ala Asp Asp Lys Gln Pro Tyr Glu Lys Lys Ala Glu Lys Leu Lys Glu 50 55 60 Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg Ala Lys Gly Lys Pro Asp 65 70 75 80 Ala Ala Lys Lys Gly Val Val 85 <210> 6 <211> 87 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 6 Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe 1 5 10 15 Ser Ser Glu Tyr Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu Ser 20 25 30 Ile Gly Asp Val Ala Lys Lys Leu Gly Glu Met Trp Asn Asn Thr Ala 35 40 45 Ala Asp Asp Lys Gln Pro Tyr Glu Lys Lys Ala Ala Lys Leu Lys Glu 50 55 60 Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg Ala Lys Gly Lys Pro Asp 65 70 75 80 Ala Ala Lys Lys Gly Val Val 85 <210> 7 <211> 176 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 7 Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Arg Lys Met Ser Ser Tyr 1 5 10 15 Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys His Pro 20 25 30 Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg 35 40 45 Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala 50 55 60 Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro 65 70 75 80 Pro Lys Gly Glu Thr Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys 85 90 95 Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys 100 105 110 Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys 115 120 125 Leu Gly Glu Met Trp Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr 130 135 140 Glu Lys Ala Glu Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala 145 150 155 160 Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val 165 170 175 <210> 8 <211> 176 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 8 Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr 1 5 10 15 Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro 20 25 30 Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg 35 40 45 Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala 50 55 60 Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro 65 70 75 80 Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys 85 90 95 Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys 100 105 110 Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys 115 120 125 Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr 130 135 140 Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala 145 150 155 160 Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val 165 170 175 <210> 9 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 9 Pro Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro 1 5 10 15 Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro 20 25 30 Lys Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys 35 40 45 Lys Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro 50 55 60 Tyr Glu Lys Lys Ala Glu Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile 65 70 75 80 Ala Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val 85 90 95 Val <210> 10 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 10 Pro Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro 1 5 10 15 Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro 20 25 30 Lys Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys 35 40 45 Lys Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro 50 55 60 Tyr Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile 65 70 75 80 Ala Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val 85 90 95 Val <210> 11 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 11 Pro Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro 1 5 10 15 Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Ser Ser Glu Tyr Arg Pro 20 25 30 Lys Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys 35 40 45 Lys Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro 50 55 60 Tyr Glu Lys Lys Ala Glu Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile 65 70 75 80 Ala Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val 85 90 95 Val <210> 12 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 12 Pro Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro 1 5 10 15 Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Ser Ser Glu Tyr Arg Pro 20 25 30 Lys Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys 35 40 45 Lys Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro 50 55 60 Tyr Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile 65 70 75 80 Ala Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val 85 90 95 Val <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 13 Pro Pro Lys Gly Glu Thr Lys Lys Lys Phe 1 5 10 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 14 Gly Pro Ser Leu Lys Leu 1 5 <210> 15 <211> 89 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 15 Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Arg Lys Met Ser Ser Tyr 1 5 10 15 Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro 20 25 30 Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg 35 40 45 Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala 50 55 60 Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro 65 70 75 80 Pro Lys Gly Glu Thr Lys Lys Lys Phe 85 <210> 16 <211> 89 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 16 Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr 1 5 10 15 Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro 20 25 30 Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg 35 40 45 Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala 50 55 60 Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro 65 70 75 80 Pro Lys Gly Glu Thr Lys Lys Lys Phe 85

Claims

1. An isolated B Box polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

12.

2. An isolated B Box polypeptide produced by growing a prokaryotic cell expressing an isolated polynucleotide encoding the isolated B Box polypeptide of claim 1 under conditions for expressing the polynucleotide, and isolating the polypeptide.

3. The isolated B Box polypeptide of claim 2, wherein the prokaryotic cell is an Escherichia coli cell.

4. An isolated B Box polypeptide prepared by chemically synthesizing the isolated B Box polypeptide of claim 1.

5. The isolated B Box polypeptide of any one of claims 1 to 4, further comprising a detectable label.

6. A composition comprising a vector and the isolated B Box polypeptide of any one of claims 1 to 4. The composition according to claim 6 , wherein the carrier is a pharmaceutically acceptable carrier.

8. A polynucleotide encoding the isolated B Box polypeptide of any one of claims 1 to 4, or a complementary sequence of the polynucleotide.

9. The polynucleotide according to claim 8, which is operably linked to a promoter and / or enhancer.

10. The polynucleotide of claim 8, further comprising a detectable label. A vector comprising the polynucleotide according to claim 8.

12. The vector according to claim 11, wherein the vector is a plasmid or a viral vector.

13. An isolated host cell comprising the isolated B Box polypeptide of any one of claims 1 to 4.

14. An isolated host cell comprising the isolated B Box polypeptide of claim 5.

15. An isolated host cell comprising the polynucleotide of claim 8.

16. An isolated host cell comprising the vector of claim 11.

17. An isolated host cell comprising the vector of claim 12.

18. A composition comprising the host cell of claim 13 and a vector.

19. The composition of claim 18, wherein the carrier is a pharmaceutically acceptable carrier.

20. A composition comprising the host cell of claim 14 and a vector.

21. The composition of claim 20, wherein the carrier is a pharmaceutically acceptable carrier.

22. A composition comprising the host cell of claim 15 and a vector.

23. The composition of claim 22, wherein the carrier is a pharmaceutically acceptable carrier.

24. A composition comprising the host cell of claim 16 and a vector.

25. The composition of claim 24, wherein the carrier is a pharmaceutically acceptable carrier.

26. A composition comprising the host cell of claim 17 and a vector.

27. The composition of claim 26, wherein the carrier is a pharmaceutically acceptable carrier.

28. An in vitro method for inhibiting, competing for or titrating the binding of a DNABII polypeptide or protein to a microbial DNA in a biofilm, comprising contacting the DNABII polypeptide or protein or microbial DNA with the isolated BBox polypeptide of any one of claims 1-4, thereby inhibiting, competing for or titrating the binding of the DNABII polypeptide or protein to the microbial DNA, wherein the biofilm is produced by an organism selected from uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae, Burkholderia cenocepacia, or non-typeable Haemophilus influenzae (NTHI).

29. An in vitro method for inhibiting, competing for or titrating the binding of a DNABII polypeptide or protein to microbial DNA in a biofilm, comprising contacting the DNABII polypeptide or protein or microbial DNA with the isolated B Box polypeptide of claim 5 to inhibit, compete for or titrate the binding of the DNABII polypeptide or protein to microbial DNA, wherein the biofilm is produced by an organism selected from uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae, Burkholderia cenocepacia, or non-typeable Haemophilus influenzae (NTHI).

30. An in vitro method for inhibiting, preventing or destroying a microbial biofilm, comprising contacting the biofilm with the isolated B Box polypeptide of any one of claims 1 to 4, wherein the biofilm is produced by an organism selected from the group consisting of uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae, Burkholderia cenocepacia, or non-typeable Haemophilus influenzae (NTHI), thereby inhibiting, preventing or destroying the microbial biofilm.

31. An in vitro method for inhibiting, preventing, or disrupting a microbial biofilm, comprising contacting the biofilm with the isolated B Box polypeptide of claim 5, wherein the biofilm is produced by an organism selected from the group consisting of uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae, Burkholderia cenocepacia, or non-typeable Haemophilus influenzae (NTHI), thereby inhibiting, preventing, or disrupting the microbial biofilm.

32. A kit comprising the isolated B Box polypeptide of any one of claims 1 to 4.

33. A kit comprising the isolated B Box polypeptide of claim 5.

34. A kit comprising the polynucleotide of claim 8 and instructions for use.

35. A kit comprising the polynucleotide of claim 9 and instructions for use.

36. A kit comprising the polynucleotide of claim 10 and instructions for use.

Images