HMGB1 Protein Derivatives For Biofilm Removal

Abstract

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] This application is a divisional application of the invention patent application which entered the Chinese national phase on March 29, 2021, with application number 201980063976.4, application date October 4, 2019, and invention name “HMGB1 protein derivatives for removing biofilms”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS 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 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

[0004] The present invention generally relates to methods and compositions for reducing and / or treating clinical or industrial bacterial biofilms. Background Art

[0005] The persistence of bacteria in biofilms in the human body causes about two-thirds of all chronic / recurrent diseases. These biofilms consist of bacteria protected by an outer "slime," which is usually 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. In addition, biofilms can serve as a reservoir for future acute infections, often with fatal consequences.

[0006] 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 cause a strong innate immune response, the host subject fails to naturally produce specific antibodies against family members due to infection. The main problem of bacterial biofilm is that the host immune system and / or antibiotics and other antimicrobial agents cannot enter the protected bacteria in the biofilm.

[0007] Biofilms are also found in industrial settings. For example, biofilms are involved in a wide range of oil processing problems, from production sites to gas station storage tanks. In the field, sulfate-reducing biofilm bacteria produce hydrogen sulfide (sour oil). In process flows, 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 end-product storage tanks.

[0008] 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.

[0009] Biofilms are relevant to a variety of water treatments in homes and industries. They can grow on surfaces of process equipment and impede the performance of the equipment, such as reduced heat transfer or plugging of filters and membranes. Biofilms growing on cooling tower fill can add enough weight 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 final product. Biofilms growing in drinking water distribution systems can harbor potentially pathogenic organisms, corrosive organisms, or bacteria, reducing the aesthetic quality of the water.

[0010] 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.

[0011] Overview Bacterial biofilms are notoriously 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 therapeutic approaches are urgently needed. One of the defining features 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 biofilms. High Mobility Group B1 (HMGB1) protein is a DNA-binding eukaryotic protein that binds to the same DNA structure as DNABII proteins, leading to the disruption of bacterial biofilms. Derivatives of HMGB1 can be engineered to have the same potent antibiofilm activity, but are smaller and do not cause inflammation.

[0012] Applicants disclose herein a new concept for treating bacterial biofilm-mediated infections by repurposing derivatives of innate immune effectors. HMGB1 domains are functionally different. Domain variants with anti-biofilm activity and no pro-inflammatory outcome represent an embodiment of HMGB1 for treating biofilm-mediated infections without the consequence of excessive inflammation. In vivo and ex vivo experiments have shown that antibodies to 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 are exposed to the anti-biofilm properties of HMGB1 and its various truncated domains (A box, B box, B box joint, mutated B box C106S, AB box, and all with joints).

[0013] Compositions and formulations comprising the protein derivatives can be used to treat refractory or 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 those associated with catheters or prostheses, and ear infections and respiratory infections in chronic / recurrent infections (e.g., cystic fibrosis patients). 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 conjunction with established treatments (i.e., antibiotics).

[0014] Thus, in one aspect, the 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, the 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 at one or both ends. 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 amino acids 1 to 70 of a wild-type HMGB1 polypeptide, optionally comprising, consisting essentially of, or consisting of one or more mutations identified above.

[0015] Also provided herein are isolated B Box polypeptides optionally comprising, or consisting essentially of, 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, or consisting essentially of, 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 B Box polypeptide comprises, or 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 a HMGB1 polypeptide. Other positions for modifying a wild-type HMGB1 B Box polypeptide are shown in FIG. 1C .

[0016] 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.

[0017] In another aspect, provided herein is an isolated AB Box polypeptide 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 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 having the proposed amino acid mutations, or consisting essentially of, or consisting of.

[0018] 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 and the C Box polypeptides. A non-limiting example is a polypeptide linker of the sequence PPKGETKKKF. When recombinantly produced, the AB or A, B and C Box polypeptides may 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 may optionally be partially or fully acetylated, oxidized or phosphorylated.

[0019] In one aspect, the isolated polypeptide further comprises a detectable label.

[0020] 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 located at one or both termini.

[0021] The present invention also provides antibodies that bind to or are produced against the mutant polypeptides described herein. The antibodies can be used as diagnostic and prognostic agents. Further provided are one or more isolated polypeptides and / or antibodies described herein and carriers, such as pharmaceutically acceptable carriers.

[0022] The present invention also provides polynucleotides encoding isolated polypeptides or antibodies and their complementary sequences as described herein. On the one hand, the polynucleotides are detectably labeled. The polynucleotides can optionally be operably connected to a promoter and / or enhancer to express the polynucleotides. A method for recombinantly producing polypeptides is also provided, which is produced and separated by expressing the polynucleotides in a suitable expression system (e.g., a host cell) and then producing and separating the recombinantly produced polypeptides.

[0023] Further provided is a vector comprising, consisting essentially of, or consisting of a polynucleotide described herein.

[0024] In another aspect, an isolated host cell is provided herein, comprising one or more of a polypeptide, a polynucleotide, or a vector as described herein. Also provided is a composition comprising a vector and one or more of a polypeptide, a polynucleotide, or a vector as described herein. In one aspect, the vector is a pharmaceutically acceptable carrier.

[0025] Polypeptide and the compositions comprising 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, which method contacts DNABII polypeptide or protein or microbial DNA with polypeptide or composition as herein described. They can also be used to suppress, prevent or destroy the method for microbial biofilm, which method contacts biofilm with polypeptide or composition as herein described.

[0026] 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.

[0027] The polypeptides and compositions can further be used in a method of inhibiting, preventing or treating a biofilm-producing microbial infection in a subject by administering to the subject an effective amount of a composition or polypeptide described herein.

[0028] 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.

[0029] 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 pneumonia, Burkholderia cenocepacia, S. epidermidis, Streptococcus agalactiae, Neisseria meningitidis, Treponema denticola, Treponema pallidum, Burkholderia cepacia, Burkholderia pseudomallei, Haemophilus influenzae (non-typeable) (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, artificial 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.

[0030] The subject may be a mammal, such as a human, or an infant or a teenager.

[0031] 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

[0032] Figure 1A-1D: Structure of HMGB1, DNABII / HMGB1-DNA complex. (Figure 1A) HMGB1 consists of 3 domains: A Box, B Box and acidic C tail. The A+B Box is mainly a DNA binding domain (brackets), while the C tail mediates nuclear function, transcriptional stimulation and antimicrobial activity. The AA of each domain is shown (left side). C23 and C45 can form disulfide bonds, resulting in reduced 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 1B) Left: IHF dimer bound to DNA. The β-ribbon arm of IHF passes through the minor groove to bend the DNA, wrapping the molecule around the N-terminal α-helix from the concave side. Right: 2 HMGB1A Box domains bound to DNA; mimicking A Box and B Box binding in natural HMGB1. The α helix binds to the minor groove, causing the DNA molecule to bend from the convex surface. Modified from PDB 4QR9. The amino acid sequences, truncations, fusions, and mutant forms of the HMGB1 polypeptide are provided in the sequence table below. (Figure 1C) The HMGB1 B Box construct consists of N-terminal and C-terminal linkers and two predicted α-local regions and a flexible linker between them. Multiple amino acids (AA) in the B Box can be post-translationally modified (PTM, acetylation / methylation of Lys, glycosylation of Asn, phosphorylation of Tyr, oxidation of Cys). However, in the peptides observed in rHMGB1 or nHM from LC-MS / MS analysis, >20% have no B Box PTM. (Figure 1D) The truncation removes the flexible linker from the N and C termini and one of the two α helical regions. It is believed that the two smallest helical region truncations (AA 99-133, 138-164) retain anti-biofilm activity.

[0033] Figure 2 : Model of HMGB1-mediated biofilm collapse. DNABII protein stabilizes the eDNA scaffold by binding to a vertex structure similar to a Holliday junction (HJ). Antibodies (Y) can sequester DNABII protein and alter the dynamic equilibrium, leading to collapse. HMGB1 can also disrupt the structure of eDNA by binding to DNA and forming a transient, unstable intermediate (bracket).

[0034] Figure 3: Structure and constructed variants of HMGB1. Full-length recombinant HMGB1 (rHMGB1) expressed in E. coli contains 3 domains, consisting of A Box, B Box and C tail. The HMGB1 AB Box structure consists of the A and B domains and the 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 the 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 (mHMGB1) and the C106S mutation in the B Box (mB Box) were created to reduce the pro-inflammatory activity of HMGB1. See also Figure 1.

[0035] Figure 4 : HMGB1 variants retain DNA binding ability. Increasing concentrations (100 and 250 nM) of commercial 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% non-denaturing polyacrylamide gel. As shown by the difference in HJ migration, HMGB1 and HMGB1 variants retain DNA binding activity.

[0036] Figures 5A-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 respective preformed bacterial biofilms in vitro for 24 h. After incubation for 16 h, the biofilms were Stained and then observed by confocal laser scanning microscopy. Images were analyzed by COMSTAT to calculate the average thickness and plotted against the control. (Fig. 5A) 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-lHF was added. EcIgG was used for E. faecium and incubated for 1 hour (rather than 16 hours) to avoid degradation by E. faecium proteases. (Figure 5B) Representative images of UPEC biofilms incubated with increasing concentrations of rHMGB1. (Figure 5C) The anti-hemoglobin activity of the various domains of HMGB1 (200nM) was tested as described above (dashed lines represent control values). Bars represent SEM. Statistical significance compared to the control group was assessed using an unpaired t-test, *P<0.05. HMGB1 and its variants (A Box cannot) are able to significantly disrupt established biofilms formed by high-priority human pathogens. ( Figure 5D-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 Stained and then observed by confocal laser scanning microscopy. Images were analyzed by COMSTAT to calculate the average thickness and total biomass. Error bars represent SEM. ***P<0.0001; ns=not significant. HMGB1 and its variants (A Box not) significantly disrupted established Klebsiella pneumoniae biofilms.

[0037] Figure 6 : HMGB1 variants disrupt pathogenic biofilms. The anti-biofilm activity of each domain of HMGB1 (200 nM) was tested as described above (dashed line represents control value). Bars represent SEM. *P<0.05. HMGB1 and its variants (but not A Box) are able to significantly disrupt established biofilms formed by high priority human pathogens.

[0038] Figures 7A-7G:HMGB1 promotes the clearance of staphylococci from mice without causing sepsis. C57BL / 6 mice were challenged intratracheally with 107CFU and received 0.2nmol of HMGB1 variants simultaneously (prevention) or 24 hours later (treatment). (FIG. 7A) Aggregates of Burkholderia cenocepacia can be seen in lung sections probed with α-Burkholderia cenocepacia antibodies by fluorescence microscopy. For prevention, (FIG. 7B) CFUs of bronchoalveolar lavage fluid (BAL) collected 18 hours after inoculation (h post-inoculation, hpi) were analyzed. (FIG. 7C) Neutrophils in BAL were quantified by differential cell counting. (FIG. 7D) Lung tissue collected at 72hpi was fixed, embedded, sliced ​​and stained with hematoxylin and eosin (magnification 10 times). For treatment, CFUs were quantified in BAL at 72hpi (FIG. 7E). (FIG. 7F) 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. (FIG. 7G) 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 lung Burkholderia cenocepacia CFU, mutation of HMGB1 from Cys to Ser abolished proinflammatory activity, and none of these HMGB1 variants caused sepsis.

[0039] Figures 8A-8F :In the experimental otitis media model, rHMGB1 and mHMGB1 promote the dissociation of biofilm. On the 4th and 5th day after attacking with NTHI, diluent, 0.2nmol rHMGB1 or 0.2nmol mHMGB1 were directly delivered to the middle ear of yellow mouse. Animals were killed after 24 hours, and the middle ear was imaged (Fig. 8C, representative images), and according to the standards described in (Fig. 8A, biofilm) and (Fig. 8B, inflammation), the presence of biofilm (Fig. 8D) and inflammation (Fig. 8F) was blindly scored. The CFU (Fig. 8E) of NTH1 quantitatively present in mucosal biomass. Mean values ​​and SEM were plotted. **P<0.01, ****P<0.0001. Image scoring and CFU quantitative proof, rHMGB1 and mHMGB1 both promote the removal of established NTHI biofilm in vivo. It is worth noting that mHMGB1 does not induce obvious inflammation.

[0040] Fig. 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. Stained and imaged by CLSM. Live cells are represented in green and dead cells are represented in red. Note that dead cells increase only in the presence of both HMGB1 and minocycline.

[0041] Figures 10A-10B : HMGB1 in specimens of biofilm-related diseases. (Fig. 10A) Sections of OCT-embedded mucosal biomass from the middle ear of NTHI-infected yellow squirrel were co-labeled for HMGB1 and DNABII proteins for immunofluorescence microscopy. Double-stranded cDNA was labeled with DAPI (white). (Fig. 10B) CF sputum was treated with anti-biofilm treatment agents (PBS, 1:10 rabbit α-1HF Ec , serum, 100U / ml (DNase), 1 mM rHMGB1) were incubated at 37°C for 1 hour, and the optical density of the surrounding medium was measured at 0 and 2 hours. DNABII protein and HMGB1 are present in the scalloped mucosal biofilm in vivo, but do not colocalize on eDNA. Exogenous HMGB1 can disrupt the biofilm present in CF sputum. Detailed description Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any method and material similar to or equal to the method and material described herein can be used in the practice or testing of the present disclosure, preferred methods, devices and materials are now described. All technologies and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein shall be construed as admitting that the present disclosure is not entitled to be disclosed prior to existing disclosure and prior to the disclosure.

[0043] 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, e.g., 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.

[0044] All numerical values ​​(including ranges), such as pH, temperature, time, concentration, and molecular mass, are approximate values ​​with variations of (+) or (-) 0.1 in increments, appropriately or by + / -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, although not always explicitly stated, the agents described herein are exemplary only, and equivalents thereof are known in the art.

[0045] 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.

[0046] As used herein, the term "comprising" or "including" is 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 transitional terms are within the scope of the present invention.

[0047] "Biofilm" means an organized community of microorganisms that sometimes attach to the surface of a structure that can be organic or inorganic, together 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 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, cystic fibrosis patients suffer from Pseudomonas infections, which often lead to the formation of antibiotic-resistant biofilms.

[0048] "DNABII polypeptide or protein" means a DNA binding protein or polypeptide composed of a DNA binding domain and therefore having specificity or general affinity to microbial DNA. On the one hand, they bind DNA in the minor groove. The limiting examples of DNABII protein are integration host factor (IHF) protein 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).

[0049] The "integration host factor" of "IHF" protein is a bacterial protein that is used by bacteriophage to incorporate its DNA into host bacteria. These are DNA binding proteins that work in gene recombination and transcription and translation regulation. They also bind extracellular microbial DNA. The genes encoding IHF protein subunits in Escherichia coli are himA (Genbank accession number: POA6X7.1) and himD (POA6Y1.1) genes. The homologues of these genes are found in other organisms, and the peptides corresponding to these genes from other organisms can be found in Table 1.

[0050] "HMGB1" is a high mobility group box (HMGB) 1 protein that is reported to bind 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.

[0051] "A Box" polypeptide means a polypeptide comprising the A box domain of the HMGB1 protein. The A Box polypeptide may be mutated or comprise other sequences, such as a linker sequence, a signal sequence, or a secretory sequence. Non-limiting examples are shown in the accompanying drawings and sequence listing. One or more point mutations in amino acids K12, C23, and C45 may be introduced.

[0052] By "B Box" polypeptide is meant a polypeptide comprising the B Box domain of the HMGB1 protein. The B Box polypeptide may be mutated or comprise other sequences, such as a linker sequence, a signal sequence, or a secretory sequence. Point mutations in amino acids K114 or C106 may be introduced to affect DNA binding, inflammatory properties, and anti-biofilm activity. Non-limiting examples are shown in the accompanying drawings and sequence listing.

[0053] By "AB Box" polypeptide is meant a polypeptide comprising the A and B box domains of the HMGB1 protein fused together but lacking the amino acids corresponding to the full-length wild-type protein. The AB Box polypeptide may be mutated or comprise other sequences, such as a linker sequence, a signal sequence, or a secretory sequence. One or more point mutations in the amino acids described herein (e.g., at amino acids K12, C23, C45, C106, and / or K114) may be introduced to affect DNA binding, inflammatory properties, and anti-biofilm activity. Non-limiting examples are shown in the accompanying drawings and sequence listing.

[0054] "HU" or "histone-like protein of Escherichia coli strain U93" refers to a class of heterodimeric proteins that are 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.

[0055] "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, about 3 to about 5 amino acid residues. Examples of peptide linkers are Gly-Pro-Ser-Leu-Lys-Leu or PPKGETKKKF.

[0056] "Microbial DNA" means single-stranded or double-stranded DNA from biofilm-producing microorganisms.

[0057] As used herein, the term "detectable label" means a directly or indirectly detectable compound or composition that is directly or indirectly conjugated 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 (e.g., antibodies) to generate "labeled" components. The term also includes sequences conjugated to polynucleotides that will provide signals when the inserted sequence is expressed, such as green fluorescent protein (GFP), etc. The label itself can be detectable (e.g., radioisotope labeling or fluorescent labeling), or in the case of enzyme labeling, can catalyze chemical changes in detectable substrate compounds or compositions. Labels may be suitable for small-scale detection or more suitable for high-throughput screening. In this way, 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. A simply detected response typically includes a response that is only confirmed to exist, while a quantified response typically includes a response with a quantifiable (e.g., digitally reportable) value such as intensity, polarization, and / or other properties. In luminescence or fluorescence determination, the luminophore or fluorophore related to the determination component actually involved in the combination can be directly used to directly produce a detectable reaction, or the luminophore or fluorophore related to another (such as a reporter molecule or an indicator) component can be indirectly used to indirectly produce a detectable response. The example of the luminescent labeling that produces the signal includes but is not limited to bioluminescence and chemiluminescence. The detectable luminescent response generally includes a change or the appearance of the luminescent signal. Suitable methods and luminophores for measuring components of luminescent labels are known in the art, such as 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.

[0058] "Gene delivery vector" is defined as any molecule that can carry an inserted polynucleotide into a host cell. Examples of gene delivery vectors are: liposomes, micelles, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria or viruses, such as baculovirus, adenovirus and retrovirus, phage, cosmid, plasmid, fungal vectors and other recombinant vectors commonly used in the art, which have been described for expression in various eukaryotic and prokaryotic hosts and can be used for gene therapy as well as simple protein expression.

[0059] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vectors. As used herein, "gene delivery", "gene transfer", "transduction" and the like refer to terms for introducing exogenous polynucleotides (sometimes referred to as "transgenics") into host cells, regardless of the method of introduction. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes), and techniques that promote the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques for introducing polynucleotides). The introduced polynucleotides can be stably 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 a replicon of the host cell, such as an extrachromosomal replicon (e.g., plasmid) or a 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.

[0060] As used herein, the term "eDNA" refers to extracellular DNA found as a component of pathogenic biofilms.

[0061] As used herein, ESKAPE pathogens include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. These pathogens are the leading causes of nosocomial infections worldwide.

[0062] A "plasmid" is an extrachromosomal DNA molecule that is separate from the chromosomal DNA and is able to replicate independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a microbial population and often provide a selective advantage under given environmental conditions. Plasmids can carry genes that confer resistance to naturally occurring antibiotics in a competitive environment, or in similar situations, the proteins produced can act as toxins.

[0063] "Plasmids" used in genetic engineering are called "plasmid vectors." Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of a plasmid that 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 that allow DNA fragments to be easily inserted at this location. Another major use of plasmids is to make large quantities of a protein. In this case, researchers can grow bacteria that contain a plasmid with the gene of interest. Just as the bacterium produces the protein that confers it 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.

[0064] "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 free 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 YAC is more unstable than BAC and produces chimeric effects.

[0065] "Viral vector" is defined as a recombinant virus or virus particle containing 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, etc. Vectors based on infectious tobacco mosaic virus (TMV) can be used to make proteins, and are 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.

[0066] As used herein, "retroviral-mediated gene transfer" or "retroviral transduction" have the same meaning and refer to the process of stably transferring a gene or nucleic acid sequence into a host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. Viruses can enter host cells through their normal infection mechanisms or they can be modified to bind to different host cell surface receptors or ligands to enter the cell. As used herein, a retroviral vector refers to a viral particle that is capable of introducing exogenous nucleic acid into a cell through a viral or virus-like entry mechanism.

[0067] Retroviruses carry their genetic information in RNA form; however, once the virus infects a cell, the RNA is reverse transcribed into DNA form, which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0068] In the aspect of gene transfer mediated by a DNA viral vector (e.g., 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.

[0069] 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 part of the clone, to eliminate the extra, potential inappropriate alternative translation initiation codon that may interfere or to interfere or reduce other sequences expressed at the transcription or translation level.Or, can be immediately inserted into the common ribosome bind site at the 5' place of the start codon to enhance expression.

[0070] Gene delivery vectors also include DNA / liposome complexes, micelles, and targeted viral protein-DNA complexes. Liposomes that also contain targeted antibodies or fragments thereof can be used in the methods disclosed herein. In addition to delivering polynucleotides to cells or cell populations, the direct introduction of proteins described herein into cells or cell populations can also be accomplished by non-limiting techniques of protein transfection, or by culture conditions to enhance expression and / or or promote the activity of proteins disclosed herein is other non-limiting techniques.

[0071] "Inhibit, prevent or destroy biofilm" refers to the preventive or therapeutic reduction of biofilm structure. On the one hand, the term "inhibit, compete or titrate" refers to the reduction of DNA / protein matrix (e.g. Figure 6 On the one hand, prevention is excluded from treatment.

[0072] "Bent polynucleotide" means a double-stranded polynucleotide that contains a small loop on one strand that is not paired with the other strand and any polynucleotide, wherein the end-to-end distance is reduced to exceed natural thermal fluctuations, i.e., the bend exceeds the persistence length of 150 bp of natural B-form double-stranded DNA. In some embodiments, the length of the loop is 1 base to about 20 bases, or alternatively 2 bases to about 15 bases long, or alternatively about 3 bases to about 12 bases long, or alternatively about 4 bases to about 10 bases long, or alternatively has about 4, 5, or 6, or 7, or 8, 9, or 10 bases.

[0073] 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, rodents (e.g., rats, mice, ground squirrels), canines (e.g., dogs), lipid animals (e.g., rabbits), livestock, sports animals, and pets.

[0074] 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, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can 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 D and L optical isomers, amino acid analogs and peptidomimetics.

[0075] As used herein with respect to nucleic acids (e.g., DNA or RNA), the term "isolated" or "recombinant" refers to molecules that are separated from other DNA or RNA, respectively, which are present in the natural source of macromolecules and polypeptides. The term "isolated or recombinant nucleic acid" refers to nucleic acid fragments that are not naturally present as fragments and are not found in the natural state. The term "isolated" is also used herein to refer to polynucleotides, polypeptides and proteins separated from other cell 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 usually associated in nature. For example, an isolated cell is a cell separated from a tissue or cell having a different phenotype or genotype. The isolated polynucleotide is separated from 3' and 5' consecutive nucleotides, which are usually associated with it in its natural or natural environment (e.g., on a chromosome). It is obvious 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 naturally occurring counterparts.

[0076] It can be inferred that, without explicit 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 "its biological equivalent" is intended to be synonymous with "its equivalent" when referring to a reference protein, antibody, fragment, polypeptide or nucleic acid, meaning those with minimal homology and still maintaining the desired structure or function. Unless specifically described herein, it is expected that any polynucleotide, polypeptide or protein mentioned herein also includes its equivalent. 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 a biological activity substantially equal to a reference protein, polypeptide or nucleic acid. On the other hand, the term means a polynucleotide hybridized with a reference polynucleotide or its complementary sequence under high stringency conditions.

[0077] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" to another sequence, which means that, when aligned, the percentage of bases (or amino acids) is 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 default parameters are used for alignment. 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; chain = both; cutoff = 60; expect = 10; matrix = BLOSUM62; description = 50 sequences; sort = 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.

[0078] "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.

[0079] "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.

[0080] "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. Hybridization reactions 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.

[0081] 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 washing 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 washing solution of about 5×SSC to about 2×SSC. Examples of high stringency 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 washing solution of about 1×SSC to about 0.1×SSC, or deionized water. In general, the hybridization incubation time is 5 minutes to 24 hours, with 1, 2 or more washing steps, and the washing 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 the equivalent of SSC using other buffer systems may be employed.

[0082] As used herein, the terms "treat," "treating," and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may 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.

[0083] "Prevention" is intended to prevent 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 microorganisms known to produce biofilms.

[0084] "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 the standard reference book Remington's Pharmaceutical Sciences, Mack Publishing Company in this field. 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.

[0085] "Administration / dosing" can be performed continuously or intermittently in one dose throughout the treatment process. 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 treated, and the object 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 agents are known in the art. The route of administration can also be determined, and the method for determining the most effective route of administration is known to those skilled in the art and will vary depending on the composition used for treatment, the purpose of treatment, the health condition or disease stage of the object treated, and the target cell or tissue. Non-limiting examples of routes of administration include oral administration, nasal administration, injection, and topical administration.

[0086] 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, body weight and tolerance to the pharmaceutical composition. In the case of immunogenic compositions, in some embodiments, the effective amount is an amount sufficient to cause a protective response to a pathogen. In other embodiments, the effective amount of an immunogenic composition is an amount sufficient to cause the production of antibodies to 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 above factors, 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. The technician will be able to determine the appropriate amount based on these and other factors.

[0087] 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. According to the embodiment, the effective amount can include one or more administrations of the composition.

[0088] The agents and compositions can be used in the manufacture of medicaments and in the treatment of humans and other animals by administration according to conventional methods (eg, active ingredients in pharmaceutical compositions).

[0089] The agents / medicaments of the present invention can be administered by any suitable route of administration for treatment. It should also be understood that the optimal route will vary with the condition and age of the subject and the disease being treated.

[0090] Examples of solid supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro and magnetite. The nature of the carrier can be soluble or insoluble to some extent. The carrier material can have virtually any possible structural configuration, as long as the coupled molecule is capable of binding to a 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.

[0091] As used herein, the term "antibody" includes complete antibodies and any antigen-binding fragments thereof or single chains thereof. Therefore, 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, a heavy chain or light chain or its ligand-binding portion's complementary determining region (CDR), a heavy chain or light chain variable region, a heavy chain 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 an antigen-binding portion of an antibody and a non-antibody protein.

[0092] The antibodies may be polyclonal, monoclonal, and may be isolated from any suitable biological source (eg, murine, rat, sheep, and dog).

[0093] "Immune response" broadly refers to the antigen-specific response of lymphocytes to foreign substances. Any substance that can cause an immune response is said to be "immunogenic" and is called 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).

[0094] 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 to the antigen (or epitope) 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 expressing molecules that specifically bind to the antigen (or epitope) on their surfaces. 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, antibodies in a sample bind to fixed antigens (or epitopes) and are detected with a detectably labeled secondary antibody (e.g., an enzyme-labeled mouse anti-human Ig antibody).

[0095] 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.

[0096] "HMG domain" or "high mobility group (HMG) box domain" refers to an amino acid sequence involved in binding 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-type DNA conformations (kinked or unwound) with high affinity. The HMG-box domain is located in the high mobility group of 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).

[0097] Modes for carrying out the invention Peptide composition 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.

[0098] Thus, in one aspect, the 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.

[0099] 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.

[0100] Examples of A Box polypeptides include the following sequences, or consist essentially of, or consist of: MGKGDPKKPRRKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKKCSERWK TMSAKEKGKF EDMAKADKAR YEREMKTYIPPKGETKKKF (mouse) MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKKCSERWK TMSAKEKGKFEDMAKADKAR YEREMKTYIP PKGETKKKF (human).

[0101] As used herein, an equivalent of a polypeptide refers to a sequence that has 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, an equivalent polypeptide includes a domain that has 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 the HMG-box domain, which, in one aspect, retains the mutated amino acids. In some aspects, such an equivalent domain retains the function and / or structural features of the HMB-box domain, such as, binding to an HMB-box binding target. In one aspect, an equivalent polypeptide is encoded by a polynucleotide that hybridizes to a polynucleotide encoding a polypeptide of the HMB-box domain under stringent conditions.

[0102] The present invention also provides an isolated B Box polypeptide, which optionally comprises 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 comprises 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 consists essentially of or consists of the same. In one aspect, the B Box polypeptide comprises about amino acids 80 to about 176, or about 88 to about 164, or about 89 to about 162, or about 80 to about 164 of the wild-type HMGB1 B Box polypeptide, which has the aforementioned mutation, or consists essentially of or consists of the same. Other positions for modifying the wild-type HMGB1 B Box polypeptide are shown in FIG. 1C. Examples of the B Box polypeptide include the following sequences, or consist essentially of or consist of the same: KDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAEKLKEKYEKDIAAYRAKGKPDAAKKGVV (mouse). KDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVV (human).

[0103] 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 recombinantly produced, the disclosed B 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.

[0104] 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 mutation. In some aspects, the equivalent of a polypeptide retains the expected function and / or structural features of a 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 retains the amino acid of mutation in one aspect. In some aspects, such an equivalent domain retains the function and / or structural features of an HMB-box domain, such as, binding to a target with an HMB-box 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.

[0105] In another aspect, provided herein is an isolated AB Box polypeptide 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 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.

[0106] In another aspect, the AB Box polypeptide can further comprise a linker polypeptide linking the A Box polypeptide and the B Box polypeptide, and in one aspect, a second linker linking the B Box and the C Box polypeptides. 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.

[0107] Examples of AB Box polypeptides include the following sequences having, consisting essentially of, or consisting of the aforementioned mutations: MGKGDPKKPRRKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGK FEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL SIGDVAKKLGEMWNNTAADDKQPYEKKAEKLKEKYEKDIAAYRAKGKPDAAKKGVV (mouse) MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGK FEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL SIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVV(human).

[0108] As used herein, the equivalent of a polypeptide refers to a sequence having a homology of 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 features of a 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 the amino acid of the mutation is retained in one aspect. In some aspects, such an equivalent domain retains the function and / or structural features of an HMB-box domain, such as, but not inducing a proinflammatory response in conjunction with a target in conjunction with an HMB-box. 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.

[0109] In another aspect, the isolated AB Box polypeptide further comprises a linker polypeptide connecting the A Box polypeptide and the B Box polypeptide.

[0110] Also provided is an isolated HMGB1 polypeptide 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 one or more amino acid mutations selected from K12, C23, C45, C106 or K114, or consisting essentially of, or consisting of. As used herein, an equivalent of a polypeptide refers to a sequence having a homology of less than 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 mutated amino acid. In some aspects, an equivalent of a polypeptide retains the expected function and / or structural features of a polypeptide, such as containing an HMG-box domain but not inducing a proinflammatory response. On the one hand, equivalent polypeptides include domains with 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 with HMG-box domains, which retain mutated amino acids in one aspect. In some aspects, such equivalent domains retain the functional and / or structural features of HMB-box domains, for example, binding to HMB-box targets but not inducing proinflammatory responses. On the one hand, equivalent polypeptides are encoded by polynucleotides that can hybridize with polynucleotides encoding HMB-box domain polypeptides under stringent conditions.

[0111] In another aspect, the isolated AB Box polypeptide further comprises a linker polypeptide linking the A Box polypeptide and the B Box polypeptide, and a second linker linking the B Box polypeptide and the C Box polypeptide. An example of a peptide linker is PPKGETKKKF.

[0112] The polypeptide can be detectably labeled and / or combined with a carrier (eg, a pharmaceutically acceptable carrier).

[0113] Antibodies and their derivatives The present disclosure also provides antibodies that bind and / or specifically recognize and bind to isolated polypeptides for use in the 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, double antibodies, 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. A hybridoma cell line producing the monoclonal antibodies disclosed herein is 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.

[0114] Antibodies can be produced using conventional techniques known in the art and are fully described in the literature. There are several methods for producing polyclonal antibodies. For example, polyclonal antibodies are usually produced by immunizing suitable mammals, such as but not limited to chickens, goats, guinea pigs, hamsters, horses, mice, rats and rabbits. The antigen is injected into the mammal to induce B lymphocytes to produce immunoglobulins specific for the antigen. The immunoglobulins can be purified from the serum of the mammal.

[0115] 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 2A, CHO, PerC.6, YB2 / 0), or hybrid myeloma, its fusion product, 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 latest visit time is November 26, 2007), which has antibody-producing cells, such as but not limited to: isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B-cell-containing cells, or cells expressing Any other cell, virus, bacterium, algae, prokaryotic, amphibian, insect, reptile, fish, mammal, rodent, horse, sheep, goat, sheep, primate, eukaryotic organism, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single-stranded, double-stranded or triple-stranded, hybridized, etc. or any combination thereof that reaches heavy chain or light chain constant or variable or framework or CDR sequence (endogenous or heterologous nucleic acid) (recombinant or endogenous). Antibody-producing cells can also be obtained from peripheral blood or spleen or lymph nodes of people or other suitable animals that have been immunized with target antigens and then screened for target activity. Any other suitable host cell can also be used to express heterologous or endogenous nucleic acids encoding antibodies of the present invention, their specified fragments or variants. Fusion cells (hybridomas) or recombinant cells can be separated using selective culture conditions or other suitable known methods and cloned by limiting dilution or cell sorting or other known methods.

[0116] Other suitable methods for producing or isolating antibodies with the necessary specificity may be used, including but not limited to methods of selecting recombinant antibodies from peptide or protein libraries using methods known in the art (e.g., 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. Pat. 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 (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) capable of producing a human antibody repertoire 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).

[0117] Antibody derivatives of the present disclosure can also be prepared by the following method: the polynucleotide encoding the antibody disclosed herein is delivered 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. Pat. Nos. 5,827,690, 5,849,992, 4,873,316, 5,849,992, 5,994,616, 5,565,362 and 5,304,489.

[0118] The term "antibody derivative" includes post-translational modifications to the linear polypeptide sequence of an antibody or fragment. For example, U.S. Pat. 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.

[0119] Antibodies disclosed herein also include derivatives that are 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 by known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, etc. In addition, the derivative may contain one or more non-classical amino acids.

[0120] 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 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 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, transgenic plants can also be used to produce antibodies according to known methods.

[0121] 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 by human or other amino acids or variable or constant regions from other isotypes.

[0122] 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.

[0123] 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, in order 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). In order to produce humanized antibodies, murine 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 No. 5,530,101, 5,585,089, 5,693,762 and 6,180,370). Similarly, in order to produce primatized antibodies, murine CDR regions can be inserted into primate frameworks using methods known in the art (WO 93 / 02108 and WO99 / 55369).

[0124] The technology of preparing partial 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 chain and light chain antibody genes. A variety of strains of this transgenic mouse 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).

[0125] The antibodies disclosed herein can also be modified to produce chimeric antibodies. Chimeric antibodies are antibodies in which each domain of the heavy and light chains of the antibody is encoded by more than one DNA. See, for example, U.S. Patent No. 4,816,567.

[0126] 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 of one species are judiciously replaced or "veneered" to an antibody of a second species so that the antibody of the first species is not immunogenic in the second species, thereby reducing the immunogenic antibody. Since the antigenicity of a protein depends primarily on the properties of its surface, the immunogenicity of an antibody can be reduced by replacing exposed residues that are different from residues commonly found in antibodies of 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 not be affected. This process is called "veneer" because only the outer surface or skin of the antibody is changed, and the supporting residues remain undisturbed.

[0127] 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 acids and proteins). Non-limiting examples of methods for producing veneered antibodies include EP 519596, U.S. Pat. No. 6,797,492, and are described in Padlan et al. (1991) Mol. Immunol. 28 (4-5): 489-498.

[0128] The term "antibody derivative" also includes "diabodies", which are small antibody fragments with two antigen binding sites, wherein the fragments contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain. (See, e.g., 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 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. Pat. 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 a target antigen that is at least about twice as strong as the binding affinity of the parent antibody for the antigen).

[0129] The term "antibody derivative" also includes engineered antibody molecules, fragments and individual 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] If the tested antibody binds to a protein or polypeptide, the tested antibody and the antibody provided by the present disclosure are equivalent. It is also possible to determine whether the antibody has the same specificity as the antibody disclosed herein by determining whether the tested antibody prevents the antibody disclosed herein from binding to a protein or polypeptide that normally reacts with the antibody without too much experimentation. If the tested antibody competes with the antibody disclosed herein, as shown by reduced binding of the monoclonal antibody disclosed herein, 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 tested antibody to bind to the antigen is inhibited. If the tested antibody is inhibited, it is very likely to have the same or closely related epitope specificity as the antibody disclosed herein.

[0134] 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 techniques to separate class-switched variants using 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 prepare indirectly from a parent hybridoma secreting a monoclonal antibody of a different isotype. Alternatively, recombinant DNA techniques can be used.

[0135] 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.

[0136] 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 to these agents are known in the art. For illustrative purposes only, antibodies can be labeled with detectable moieties (e.g., radioactive atoms, chromophores, fluorophores), etc. Such labeled antibodies can be used for diagnostic techniques in vivo or in isolated test samples.

[0137] 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) supra.

[0138] The variable region of the antibody of the present invention can be modified to improve one or more binding properties (e.g., affinity) of the antibody by mutating the amino acid residues in the 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 effects 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.

[0139] 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.

[0140] In addition, the antibodies disclosed herein can be engineered to include modifications in the Fc region to change one or more functional properties of the antibody, such as serum semi-quantitative, 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 the assembly of light and heavy chains or to increase or reduce the stability of the antibody (U.S. Patent No. 5,677,425), or amino acid mutations in the Fc hinge region to reduce the biological half-life of the antibody (U.S. Patent No. 6,165,745).

[0141] 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. Pat. 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 mechanism. (Shields, RL et al. (2002) J. Biol. Chem. 277: 26733-26740; Umana et al. (1999) Nat. Biotech. 17: 176-180).

[0142] 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 the condition that 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 cover any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. 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).

[0143] In addition, antibodies can be chemically modified by conjugating or fusing the antigen binding region of the antibody to a serum protein (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.

[0144] 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 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 coupled to the antibody or fragment thereof, or indirectly coupled or coupled 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, dichlorotriazineamine 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).

[0145] In addition, the antibodies or fragments thereof of the present invention may 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., nitrogen mustard, 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, alpha-sarcin (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.

[0146] Other suitable conjugated molecules include ribonucleases (RNA enzymes), DNA enzymes I, antisense nucleic acids, inhibitory RNA molecules (such as siRNA molecules), immunostimulatory nucleic acids, aptamers, ribozymes, triplex-forming molecules and external guide sequences. An aptamer is a small nucleic acid of 15-50 bases in length, which is folded into a limited secondary and tertiary structure, such as a stem loop or a G quadruplex, and can bind small molecules (such as ATP (U.S. Patent No. 5,631,146) and theophylline (U.S. Patent No. 5,580,737)) and macromolecules (such as reverse transcriptase (U.S. Patent No. 5,786,462) and thrombin (U.S. Patent No. 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 recognition and combination of target substrates and subsequent cutting. A nucleic acid molecule having triplex-forming function can interact with double-stranded or single-stranded nucleic acids by forming triplexes, wherein the triplex DNA forms a complex that depends on Watson-Crick and Hoogsteen base pairing. The triplex molecule can bind to the target region with high affinity and specificity.

[0147] 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.

[0148] The therapeutic agent can be linked to the antibody directly or indirectly using any of a variety of available methods. For example, the agent is attached to the hinge region of a reduced antibody component by disulfide bond formation, using a cross-linking agent such as N-succinyl 3-(2-pyridyldithio) propionate (SPDP), or through 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)).

[0149] 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).

[0150] The antibodies or antigen binding regions thereof disclosed herein can be linked to another functional molecule (e.g., another antibody or a ligand of 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.

[0151] 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.

[0152] 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 where depletion of NK cells is desired.

[0153] 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.

[0154] Antibodies can also be combined with many different carriers. Therefore, the present invention also provides compositions comprising an antibody and another active or inert substance. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and 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 combining monoclonal antibodies, or will be able to determine such carriers using routine experiments.

[0155] In some aspects of the antibodies provided herein, the antibody is a full-length antibody.

[0156] In some aspects of the antibodies provided herein, the antibody is a monoclonal antibody.

[0157] In some aspects of the antibodies provided herein, the antibody is chimeric or humanized.

[0158] In some aspects of the antibodies provided herein, the antibody is selected from Fab, F(ab)'2, Fab', scF v and F v .

[0159] 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.

[0160] In some aspects of the antibodies provided herein, the antibody comprises a human antibody framework region.

[0161] 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.

[0162] 1) Amino acids with basic side chains: lysine, arginine, histidine.

[0163] 2) Amino acids with acidic side chains: aspartic acid, glutamic acid.

[0164] 3) Amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, tyrosine.

[0165] 4) Amino acids with non-polar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine.

[0166] 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.

[0167] 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.

[0168] Polynucleotides, vectors and host cells The present invention also provides the separation or reorganization polynucleotides encoding one or more of the above-mentioned identified polypeptides or antibodies and their complementary chains respectively. Also provided are carriers comprising the polynucleotides of separation or reorganization, examples of which are known in the art and briefly described herein. In one aspect where the polynucleotides of more than one separation or reorganization will be expressed as a single unit, the polynucleotides of separation or reorganization can be contained in a polycistronic vector. The polynucleotides can be DNA, RNA, mRNA or interfering RNA, for example siRNA, miRNA or dsRNA.

[0169] The present invention further provides a polynucleotide that is operably connected to a promoter for RNA transcription and other regulatory sequences for replication and / or transient or stable expression of DNA and / or DNA. As used herein, the term "operably connected" refers to positioning in such a way that the promoter will instruct RNA to transcribe from the DNA molecule. Examples of such promoters are SP6, T4 and T7. In certain embodiments, a cell-specific promoter is used for cell-specific expression of the inserted polynucleotide. Containing a promoter or promoter / enhancer, with a stop codon and a selection marker sequence, and a vector that can operably connect the inserted DNA fragment to the cloning site of the promoter is 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.

[0170] In one embodiment, the 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).

[0171] 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 an episome or as a component 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 may 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) method described therein. Therefore, the present invention also provides a host cell, such as a mammalian cell, an animal cell (rat or mouse), a human cell, or a prokaryotic cell (such as a bacterial cell), comprising a polynucleotide encoding a protein or polypeptide or an antibody or a fragment thereof.

[0172] The polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure modification may be imparted before or after polynucleotide assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide 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 two complementary single-stranded forms known or predicted to constitute a double-stranded form.

[0173] When the vector is used for in vivo or ex vivo gene therapy, a pharmaceutically acceptable carrier is preferred, such as a retrovirus or adenovirus 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 carrier is a "weakly replicable" carrier, which is defined as being unable to produce viral proteins, thereby preventing the propagation of the carrier in infected host cells. An example of a weakly replicable retroviral vector is LNL6 (Miller et al. (1989) BioTechniques 7: 980-990). A method for retroviral-mediated gene marker gene transfer using a weakly replicable 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).

[0174] 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).

[0175] Polynucleotide can be conjugated to detectable label, such as enzyme label 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 ligands that can provide detectable signal, such as avidin / biotin.On the one hand, people may wish to use fluorescent label or enzyme label, such as urease, alkaline phosphatase or peroxidase, to replace radioactivity or other environmentally unfavorable reagents.In the case of enzyme label, calorimetric indicator substrate can be used to provide human eye visible or spectrophotometric means, to identify specific hybridization with samples containing complementary nucleic acids.Therefore, the present invention further provides a method for detecting single-stranded polynucleotide or its complementary sequence, the method by allowing complementary single-stranded polynucleotide hybridization under conditions (preferably medium stringent hybridization conditions), or more preferably under highly stringent hybridization conditions, the target single-stranded polynucleotide is contacted with a labeled single-stranded polynucleotide (probe) as a part of the polynucleotide of the present invention.The hybridized polynucleotide is separated from the unhybridized single-stranded polynucleotide. Hybridized polynucleotide pairs are detected using methods known to those skilled in the art, such as those described in Sambrook et al. (1989) (supra).

[0176] The polynucleotides embodied in the present invention can be obtained using chemical synthesis, recombinant cloning methods, PCR or any combination thereof. The method for chemical polynucleotide synthesis is known in the art and does not need to be described in detail at this time. 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.

[0177] 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 a linear sequence of polynucleotides, nucleotides, suitable primer molecules, chemicals (e.g., enzymes) and replication instructions thereof, and chemically replicates or connects nucleotides in the appropriate direction to obtain 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 so amplified can be separated 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 thus obtained.

[0178] RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell. The DNA can be delivered by any suitable method, such as by using a suitable gene delivery vehicle (e.g., liposomes, plasmids, or vectors) or by electroporation. When the cells replicate and the DNA is transcribed into RNA; RNA can then be isolated using methods known to those skilled in the art, such 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 resins according to the accompanying instructions provided by the manufacturer.

[0179] 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.

[0180] 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 the hybridization specificity. Typically, up to 20% base pair mismatches (when optimally aligned) can be tolerated. Preferably, the probe used to detect the above 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.

[0181] These probes can be used in radioanalysis (such as Southern and Northern blot analysis) to detect, prognose, diagnose or monitor various cells or tissues comprising these cells. The probe can also be connected to a solid support or array (such as a chip) for use in high-throughput screening assays to detect the expression of the gene corresponding to the polynucleotide of the present invention. Therefore, the present invention also provides a probe comprising or corresponding to a polynucleotide of the present invention or its equivalent or its complementary sequence or its fragment, which is connected to a solid support for high-throughput screening.

[0182] 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, wherein the length of the complementary region can vary, e.g., from at least 5 to 10 to about 100 nucleotides, or even the full length, depending on the complementary sequence that is desired to be detected.

[0183] Usually preferably have the nucleotide probe of complementary sequence on the extended sequence greater than 5 to 10 nucleotide in length, to increase the stability and selectivity of hybrid, thereby improve the specificity of the specific hybrid molecule obtained.More preferably, can design to have the polynucleotide of 10 or more or greater than 50 nucleotide or even longer gene complementary sequence of length when needed.Such fragment can be easily prepared in the following manner: for example, directly synthesize this fragment by chemical method, 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 introducing the sequence of selection into recombinant vector for recombinant production.On the one hand, the length of probe is about 50-75 or more nucleotide, alternatively 50-100 nucleotide.

[0184] 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 a target sequence with reasonable fidelity. Amplification can be carried out by natural or recombinant DNA polymerases, such as Klenow fragments of T7 DNA polymerase, Escherichia coli DNA polymerase, and reverse transcriptase. For illustration purposes only, the length of the primer is identical to the length identified by the probe.

[0185] 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.

[0186] Methods for administering an effective amount of gene delivery vectors or vehicles to cells have been developed and are known to those skilled in the art and are described herein. Methods for detecting gene expression in cells are known in the art and include techniques such as hybridization with DNA microarrays, in situ hybridization, PCR, RNase protection assays, and Northern blot analysis. Such methods can be used to detect and quantify gene expression in cells. Alternatively, the expression of the encoded polypeptide can be detected by a variety of methods. In particular, it is useful to prepare polyclonal or monoclonal antibodies that specifically react with the target polypeptide. Such antibodies can be used to visualize cells expressing polypeptides using techniques such as immunohistology, ELISA, and Western blots. These techniques can be used to determine the expression level of the polynucleotides expressed.

[0187] In one aspect, polypeptides comprising an HMG-box domain include wild-type and recombinantly produced polypeptides and proteins from prokaryotic and eukaryotic host cells.

[0188] 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.

[0189] The polypeptides can also be obtained by chemical synthesis using a commercially available automatic peptide synthesizer, such as produced 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). Therefore, 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.

[0190] 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.

[0191] 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 immune responses in vivo, the carrier can also include an adjuvant that can be used to non-specifically 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 solution 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.

[0192] Treatment 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.

[0193] Another embodiment of the present invention provides a method for inhibiting, preventing or destroying microbial biofilm, which comprises 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.

[0194] Another embodiment of the invention provides a method for disrupting biofilm and clearing 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 the biofilm and clearing without enhancing or inducing an inflammatory response. In some aspects, the contacting is in vitro or in vivo.

[0195] Another embodiment of the present invention provides a method for inhibiting, preventing or destroying biofilm in a subject, comprising administering to the subject an effective amount of a polypeptide as described herein, thereby inhibiting, preventing or destroying 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.

[0196] 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.

[0197] In one aspect of any of the above embodiments, the polypeptide comprising, consisting essentially of, or consisting of an HMG-box domain is 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.

[0198] Equivalents thereof include proteins with AB Box, A Box and B Box, as well as mutants, truncations and fusion proteins described herein (see Figure 3 ) is 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, the equivalents retain the altered amino acids in the polypeptide and retain the ability of the parent or reference protein, peptide, fusion or mutant form. In some aspects, the polypeptide comprising the HMG-box domain comprises, consists essentially of, or consists of a biological equivalent to any of the above polypeptides.

[0199] 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. On the other hand, 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.

[0200] Any of the above methods can further comprise one or more antimicrobial agents, antigenic peptides or adjuvants used to the object in an effective amount, or are basically composed of, or are composed of. On the one hand, the object is a non-human animal or a human patient. On the one hand, the patient is a human teenager or a baby.

[0201] The polypeptides are administered by methods including topically, transdermally, sublingually, rectally, vaginally, ophthalmically, subcutaneously, intramuscularly, intraperitoneally, urethrally, intranasally, by inhalation, or orally.

[0202] In some aspects, the subject is a pediatric patient, and the polypeptide is administered in a formulation for use in pediatric patients.

[0203] In any of the above embodiments, the biofilm may comprise microbial DNA from the microorganisms identified in Table 1.

[0204] Table 1. Examples of bacterial strains that can produce biofilms Streptococcus sobrinus Streptococcus pyogenes (S. pyogenes) S.gordonii Challis Streptococcus agalactiae (S. agalactiae) Streptococcus mutans (S. mutans) Streptococcus pneumoniae Streptococcus gallolyticus Staphylococcus aureus (S. aureus) Staphylococcus epidermidis Escherichia coli (E.coli) Haemophilus influenzae (H.influenza) Salmonella enteric serovar typhi Aggregatibacter actinomycetemcomitans YP_003255304 Porphyromonas gingivalis Neisseria gonorrhoeae (N.gonorrhoeae) Neisseria meningitidis NMB_1302 Pseudomonas aeruginosa Helicobacter pylori (H.pylori) Borrelia burgdorferi Moraxella catarrhalis Vibrio cholera El Tor Burkholderia cenocepacia Burkholderia pseudomallei Mycobacterium tuberculosis Mycobacterium smegmatis Treponema denticola Treponema palladium Nichols Prevotella melaninogenica Prevotella intermedia Bordetella pertusis Tohama Enterococcus faecalis In one embodiment, the polypeptide is administered topically to a microbial infection and disrupts the biofilm.

[0205] In one embodiment, the invention provides a method of inducing or providing an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of a polypeptide as described herein, or consisting essentially of it, or consisting of it. In another embodiment, the administration is localized to where the immune response is needed. In one aspect, the method comprises administering an effective amount of a polypeptide, or consisting essentially of it, or consisting of it, which polypeptide comprises, or consists essentially of, or consists 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 a polypeptide, or consisting essentially of it, or consisting of it, which polypeptide comprises, or consists essentially of, or consists of a B Box polypeptide disclosed herein.

[0206] 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.

[0207] 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 infection site.Other non-limiting examples of use include by one or more methods, including percutaneous, sublingual, rectal, vaginal, ocular, subcutaneous, intramuscular, intraperitoneal, intranasal, by suction or oral.

[0208] 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.

[0209] For some of these methods, contact can be carried out in vitro or in vivo. When contacting in vitro, the method provides the means for determining the effect of medicament of the present invention before animal or clinical study, and can be used to determine whether medicament of the present invention acts in synergy with other antimicrobials. When carrying out in vivo in an animal model, the method provides the means for 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 in synergy with other antimicrobials.

[0210] Microbial infections and diseases that can be treated by the methods of the invention include infections of organisms identified in Table 1, for example, Streptococcus agalactiae, Neisseria meningitidis, Treponema denticola, Treponemapallidum, Burkholderia cepacia, or Burkholderia pseudomallei. In one aspect, the microbial infection is one or more of Haemophilus influenzae (non-typeable) (NTHI), Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and ESKAPE pathogens. These microbial infections may be present in 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).

[0211] Infections may also occur in the mouth (caries, periodontitis) and are caused by Streptococcus mutans, Porphyromonas gingivalis, Aggregatibacter actinomvctemcomitans. Infections may 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 usually caused by Salmonella enterica serovar, Vibriocholerae, and Helicobacter pylori. Reproductive tract infections include those usually caused by Neisseria gonorrhoeae. Enterococcus faecalis can cause bladder infections or indwelling device infections. Infections (usually caused by a variety of bacteria) associated with prosthetic implanted devices (e.g., artificial hip or knee replacements or dental implants) or medical devices (e.g., pumps, catheters, stents, or monitoring systems) can be treated by the methods disclosed herein. These devices can be coated or conjugated to agents as described herein.

[0212] 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.

[0213] 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 necessary, routes of administration can be combined, or adjusted according to the medicament and / or the desired effect. The active agent can be administered in single doses or multiple doses. These methods and embodiments of 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.

[0214] Parenteral routes of administration other than administration by inhalation 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 local or mucosal administration of the drug formulation for invasive or systemic absorption.

[0215] 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).

[0216] Methods for administering active substances through the skin or mucous membranes include, but are not limited to, topical application of suitable pharmaceutical preparations, 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 electrical pulses.

[0217] In various embodiments of the methods disclosed herein, the agent is administered continuously on a daily basis at least once a day (QD) (twice a day (BID), three times a day (TID), or even four times in various embodiments) 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.

[0218] Administration according to the methods disclosed herein can be accomplished using capsules, tablets, oral suspensions, suspensions for intramuscular injections, suspensions for intravenous infusions, ointments or creams for topical application, or suspensions for intra-articular injections.

[0219] 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 kills 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 composition exhibits 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.

[0220] 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, which includes an ED50 that is almost non-toxic or non-toxic. Depending on the dosage form used and the route of administration used, the dosage can vary within this range. For any compound used in the method, a preliminary estimate of the therapeutically effective dose can be obtained from the cell culture assay. Dosages can be formulated in animal models to reach a circulating plasma concentration range including the IC50 (i.e., the concentration of the test compound that reaches 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.

[0221] In some embodiments, the effective amount of the composition that is enough to achieve treatment or preventive effect is to use about 0.000001mg per kg of body weight at each time to about 10,000mg per kg of body weight at each time. Suitably, the dosage range is from about 0.0001mg per kg of body weight at each time to about 100mg per kg of body weight at each time. Administration can be provided with a pre-dose, and then provided with one or more "boost" doses. A booster dose can be provided one day, two days, three days, one week, two weeks, three weeks, one, two, three, six or twelve months after the pre-dose. In some embodiments, a booster dose is used after the reaction of the assessment object to the previous application.

[0222] Those skilled in the art will appreciate that certain factors may affect 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 diseases. 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 prevention.

[0223] 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.

[0224] In some embodiments, the method and composition include deoxyribonuclease (DNase) enzymes, such as DNA enzymes, that act synergistically with the composition of the present invention. DNA enzymes are enzymes that catalyze the cleavage of phosphodiester bonds in any catalytic DNA backbone. Three non-limiting examples of DNA enzymes that are known to target not only cruciform structures but also multiple secondary structures of DNA include DNAseI, T4 EndoVII and T7 EndoI. In certain embodiments, when used in combination with DNA enzymes, the effective amount of the anti-DNABII antibodies required for destroying biofilm stability is reduced. When administered in vitro, DNA enzymes can be directly added to the assay or in a suitable buffer known to stabilize the enzyme. The effective unit dose and assay conditions of DNA enzymes can vary, and can be optimized according to methods known in the art.

[0225] In other embodiments, the methods and compositions can be combined with antibiotics and / or antimicrobial agents. Antimicrobial agents are substances that kill or inhibit the growth of microorganisms (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 treating infections. In other embodiments, the use of antibiotics or antimicrobial agents in combination with the methods and compositions described herein can reduce the effective amount of antimicrobial and / or biofilm reducing agents. Some non-limiting examples of antimicrobial agents 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 antimicrobial agents and / or antibiotics and biofilm reducing agents can be easily determined by conventional methods. In some embodiments, the dose of the combination of antimicrobial agents and biofilm reducing agents is an average effective dose, which has been shown to be effective for other bacterial infections, such as bacterial infections in which the etiology of the infection does not include biofilms. 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 the average effective dose.Can add antibiotic or antimicrobial before, simultaneously or afterwards adding anti-DNABII antibody.

[0226] 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). Using 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 a biofilm reducing agent or a method for reducing biofilm include anti-rsPilA, anti-OMP26, anti-OMP P2, and anti-all-OMP preparations.

[0227] The compositions and methods described herein can be used to sensitize bacterial infections involving biofilms to common treatments that are effective for treating bacterial infections without biofilms, but otherwise are ineffective in treating bacterial infections involving biofilms. In other embodiments, the compositions and methods described herein can be used in combination with treatments that are effective for treating bacterial infections involving biofilms, but the combination of such additional therapies and biofilm reducing agents or methods 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 therapies and biofilm reducing agents or methods produces a synergistic effect, thereby enhancing treatment. The enhancement of treatment can be demonstrated by the shorter time required to treat the infection.

[0228] 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.

[0229] Reagent test kit Also claimed are kits comprising the reagents and instructions necessary to carry out the in vitro and in vivo methods described herein. Thus, the invention provides kits for carrying out these methods which may include the biological agents of the invention and instructions for carrying out the methods of the invention, such as collecting tissues and / or performing screening and / or analyzing results and / or administering effective amounts of the biological agents defined herein. These may be used alone or in combination with other suitable antimicrobial agents.

[0230] In one embodiment, the invention provides a kit comprising a polypeptide as described herein and instructions for decomposing biofilm or inhibiting, preventing or treating microbial infections that produce 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.

[0231] The following examples are intended to illustrate but not to limit the present invention.

[0232] Bacterial biofilm-mediated infections account for about 80% of all chronic / recurrent human infections. Biofilms constitute a protected mode of microbial growth. That is, they consist of microbial communities attached to surfaces and embedded in their own synthesized hydrated polymer matrix. 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 defense. 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 nucleoprotein interactions occurring in the extracellular matrix of biofilms. It is known that the extracellular matrix of biofilms consists 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 there is a large network of interlaced eDNA chains in the formed biofilm, which is stabilized by the DNABII protein located at the vertices of the curved cross-chains of the eDNA. Applicants disclose here a polypeptide of eukaryotic origin having one or more HMG-box domains (e.g., HMGB1) that can interfere with the structure of the extracellular DNA scaffold inside the biofilm. By competing with the microbial proteins bound to the DNA scaffold in the biofilm, these polypeptides destabilize the biofilm, which leads to the destruction and removal of the biofilm by the host immune system ( Figure 2 ).

[0233] 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 for bacteria. Furthermore, HMGB1 domains and mutant variants, 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 consequence of excessive inflammation. In addition, since 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.

[0234] 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 properly folded and functional ( Figure 4 To evaluate the effects of HMGB1 and its variants as well as commercially available native 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 stained and analyzed using confocal laser scanning microscopy (CLSM) and COMSTAT analysis to calculate the average thickness and total biomass of the biofilm. 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 that a single dose of these non-antimicrobial compounds was able to disrupt recalcitrant biofilms. Additionally, the HMGB1 variants disrupted every bacterial species tested to date, including pathogenic Escherichia coli, Burkholderia cenocepacia, nontypeable Haemophilus influenzae ( Figure 6 ). A single dose at this concentration releases the bacteria from its protective shield, making it susceptible to clearance by antibiotics and the immune system.

[0235] The applicant's novel approach to treating biofilm-associated infections using non-antimicrobial agents such as HMGB1 proteins and their variants represents a radical departure from classical therapeutic concepts. Full-length HMGB1 and the smallest variant B Box tested to date with the greatest destructive activity found in vitro and a modified B Box (mB Box) (wherein the cysteine ​​at position 106 was mutated to serine to eliminate its inflammation-inducing ability) were tested for biofilm destruction and inflammatory activity in vivo ( FIG. 7 ). Using an aggregated biofilm infection lung model, the applicant showed that HMGB1, B Box, and each modified form were able to prevent biofilm formation in vivo, and that the modified proteins did not induce an inflammatory response ( FIG. 7B and FIG. 7C ). The applicant further demonstrated that none of the HMGB1 variants induced sepsis at a given dose capable of destroying biofilms ( FIG. 7D ).

[0236] method Experiment 1 Klebsiella pneumoniae (KP), a common cause of nosocomial infections, was used for 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 individual domains on established BBs, each protein (200 nM) was added to preformed BBs over a 24-hour period. At 40 hours, the BBs were washed and incubated with stained, observed by confocal laser scanning microscopy and images analyzed by COMSTAT to calculate average thickness and biomass.

[0237] Exogenous rHMGB1 and its individual domains, except A Box, resulted in significant decreases (p < 0.05) in the average thickness (AT) and biomass (BM) of KP biofilms compared to untreated 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).

[0238] Experiment 2 HMGB1 disrupts pathogenic biofilms: To test the effect of HMGB1 on bacterial biofilms ( FIG. 5 ), the applicants cloned ( 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-agarose chromatography to >95% purity) at 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 (formed for 24 h prior to addition) was evaluated. 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 reduction (P < 0.05) in the average thickness and biomass (not shown) of each biofilm (Figure 5A). Only Enterococcus faecalis and Staphylococcus aureus required higher concentrations (although non-bactericidal) to achieve similar results. It was also shown that native HMGB1 purified from calf thymus (nHMGB1; Chondrex, Inc, Redmond, WA) destroyed selected biofilms (UPEC, Bc, NTHI, Klebsiella pneumoniae) equivalently compared to rHMGB1 (Figure 5A), indicating that any potential differences in post-translational modifications (PTMs) between nHMGB1 and rHMGB1 would not significantly affect this anti-biofilm function. Preliminary analysis of PTMs by LC-MS / MS analysis (MS Bioworks, LLC Ann Arbor, MI) indicated that both rHMGB1 and nHMGB1 were minimally modified (<20% of the observed peptides had any given PTM, Fig. 1C). 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; Fig. 5B), i.e., completely abolished the 3D biofilm structure, implying that the bioburden could be reduced, allowing host immune effectors or other antimicrobial compounds to achieve eradication. Thus, despite intrinsic differences in sensitivity between pathogens, biofilms formed by all high-priority pathogens tested were sensitive to a single dose of these non-antimicrobial compounds.

[0239] Experiment 3 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, Figures 1A and 1C). Addition of the A Box to established biofilms (UPEC, Bc, NTHI, and Klebsiella pneumoniae) had no significant effect on the measured biofilm parameters (Figure 5C). In contrast, the AB Box and the 97 amino acid (AA) B Box retained complete anti-biofilm activity (Figure 5C). Since only the 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. It was reported that B Box contains pro-inflammatory activity, mainly 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 (Figures 1A and 1C). Compared with B Box, the mB Box variant equivalently disrupted bacterial biofilms (UPEC, NTHI, Bc, Klebsiella pneumoniae) in vitro (Figure 5C).

[0240] Experiment 4 rHMGB1 and mHMGB1 destroy biofilm in two different animal models, but inflammatory response is greatly weakened by Cys to Ser mutation: Applicants use the mature experimental OM yellow mouse model (wherein the adherent mucosal biofilm formation plays a key role in pathogenesis) caused by NTHI49,67,68 to test the ability of rHMGB1 and mHMGB1 to treat middle ear infection and the corresponding inflammatory response (Fig. 8A). The middle ear of adult yellow mice with mixed sexes was inoculated with 1000CFU NTHI by small gastrocnemius injection. On the 4th and 5th days after attack, there were a large number of biofilms in the middle ear space at this time, and 5 μg (0.2nmol) rHMGB1, mHMGB1 or diluent were directly delivered to the middle ear (2 times of treatment in total). On the 6th day, animals were killed, and the middle ear was imaged and the residual biofilm (Fig. 8A) and mucosal inflammation (Fig. 8B) were blindly scored. Animals treated with diluent had thick mucosal biofilms that obscured the bony septa (Figs. 8C and 8D). In sharp contrast, in animals treated with rHMGB1 or mHMGB1, residual mucosal biofilms were greatly reduced, with CFUs reduced by approximately 1000-fold (Fig. 8E). These results are particularly noteworthy because biofilms formed in vitro by NTHI did not respond as well to HMGB1 addition as other tested bacterial species (Fig. 5). Middle ear fluid (MEF) collected from animals treated with rHMGB1 had increased proinflammatory cytokines (IL-1β (3-fold), IL-17A (2-fold)) compared to animals treated with mHMGB1 and diluent (data not shown), consistent with rHMGB1 enhancing visible inflammation of the middle ear mucosa (Figs. 8C 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 (Figures 8C 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 aggregate biofilm development. For prevention, 10 7Adult C57BL / 6 mice were challenged intratracheally (it) with CFU of Bc and simultaneously added with 0.2 nmol of rHMGB1 or mHMGB1. After 18 h, mice were euthanized and bronchoalveolar lavage (BAL) and lungs were collected. Bc formed aggregates that were easily seen in the lungs probed with Bc antibodies (Fig. 7A). Tissues were homogenized and CFU were enumerated. Bc was significantly reduced in BAL (Fig. 7B) and lung tissues of mice administered rHMGB1 or mHMGB1 compared to control mice (data not shown; P < 0.05), suggesting that HMGB1 inhibits biofilm formation in murine airways and that this process promotes bacterial clearance. Preliminary results from treatment with B Box and mB Box derivatives showed that these 97AA polypeptides inhibited biofilm development in vivo (reduction in CFU in BAL, Fig. 7B) and that the C106S mutation abolished proinflammatory activity (2-fold reduction compared to B Box) (Fig. 7C). The animal model was then used to study lung injury 72h after Bc attack and the administration of rHMGB1 or mHMGB1. The lungs were collected and the tissues were fixed, embedded in paraffin, sliced, and stained with hematoxylin and eosin (H&E). The lungs of mice treated with mHMGB1 were closer to the lungs of uninfected mice, while severe inflammation and enhanced neutrophil response occurred in mice treated with rHMGB1 (Figure 7D), indicating that mHMGB1 retained 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 attacked as described above and 0.2nmol rHMGB1 or mHMGB1 was administered 24 hours later. 48 hours after treatment, the mice were euthanized and BAL and lungs were collected and processed as described above. Mice treated with rHMGB1 or mHMGB1 had significantly fewer Bc in their lungs (Figure 7E), while mHMGB1 treatment induced less recruitment of neutrophils and inflammatory monocytes to the lungs (P < 0.05, data not shown). Together, these data suggest that mHMGB1 and mB Box did not enhance the inflammatory response, but inhibited the formation of aggregated biofilms, the absorption and clearance of bacteria. 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 neutrophils 70, 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). Four hours later, 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, but mHMGB1 did not ( Fig. 7F ).

[0241] Experiment 5 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, but still show effective anti-biofilm activity, applicants have rigorously studied whether our HMGB1 variants can cause sepsis in the presence or absence of LPS. Applicants used 0.2nmol (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 <300pg endotoxin / μg protein) to intraperitoneally inject naive mice or mice sensitized with a non-lethal dose of LPS (5m / 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 induces more than 100pg / ml of TNF, while rHMGB1, B Box or mB Box alone cannot induce detectable TNF, and when administered to LPS-sensitized mice, they do not induce additional pro-inflammatory signaling (Figure 7G). It is reported that compared with our dose, >450 times the B Box alone is required, or 4 doses of 2 times greater rHMGB1 in 40 hours in mice sensitized with lipopolysaccharide (LPS) are required to induce sepsis. In addition, as described above, the anti-biofilm activity of these endotoxin-free polypeptides was tested in vitro, and it was found that they maintained all functions against UPEC and Bc biofilms (data not shown). Therefore, the variants tested here do not induce sepsis at effective therapeutic doses.

[0242] Experiment 6 Disruption of biofilm 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 ( Fig. 9 ), suggesting that HMGB1 treatment could be used in combination therapies to both improve antimicrobial efficacy and reduce the development of antimicrobial resistance.

[0243] Experiment 7 HMGB1 and DNABII proteins are present in biofilms formed in vivo: Applicants have previously demonstrated that bacterial DNABII proteins are located at the apex of eDNA cross-strands in biofilms formed by NTHI in the OM experimental model. In order to determine the relative presence and spatial distribution of HMGB1 in NTHI biofilms formed in vivo, unfixed biofilms recovered from the middle ear of yellow mice 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 very close to the labeled DNABII protein, but not co-localized, and was detected on the cross-strands of eDNA (as expected) (Figure 10A). It is worth noting that HMGB1 was not observed at the apex. These data indicate that HMGB1 can be integrated into EPS, but will not occupy the same eDNA site (apex) as DNABII protein at the same time, which supports the hypothesis that HMGB1 competes with DNABII protein at the eDNA apex to destroy the stability of EPS. These data also demonstrate that DNABII and HMGB1 proteins do not colocalize and therefore do not interact productively, and further suggest that HMGB1 may function solely through DNA binding / bending.

[0244] Experiment 8 HMGB1 disrupts biofilms present in clinical specimens: Biofilms at sites of chronic disease, such as the CF lung, are composed of multiple species and are therefore difficult to eradicate. Anti-DNABII antibodies have been shown to disrupt biofilms present in CF sputum. Suspensions of CF sputum in PBS containing 1 M Bbox at 37°C for 2 h effectively disrupted sputum and achieved similar levels to those of high concentrations of Bbox. (a therapeutic DNA enzyme used as a mucolytic in CF patients) or anti-DNABII to the same extent (Figure 10B). These data indicate that biofilms formed at sites of chronic infection have a conserved eDNA-dependent structure that is susceptible to infection by HMGB1 and demonstrate that HMGB1 is a host defense against biofilms.

[0245] Experiment 9 Many oral bacteria (e.g., Aggregatibacteractinomycetemcomitans, Porphyromonas gingivalis) have been implicated in the pathogenesis of inflammatory diseases (e.g., periodontitis and peri-implantitis) that destroy the alveolar bone and gingiva. The lack of effective animal models has hampered studies of 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 elucidate their pathogenic mechanisms. This embodiment provides a model for testing the disclosed polypeptides and compositions and their efficacy in treating oral diseases.

[0246] The surfaces of machined titanium dental implants (1.2×4.5 mm) were modified by sandblasting with AlO3 (100 μm) and etching with HCl (pH 7.8 at 80°C for 20 min). 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 implant 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 areas of female rats. In order to detect the presence of Aa biofilm on implants placed in vivo, bacterial samples were collected from saliva and the oral surface of the implant after 2 days. Aa can be detected by culture and PCR analysis. Micro-CT and histological analysis of bone and mucosal tissue around the implant can be performed six weeks after implantation. As described herein, polypeptides and compositions are determined as well as surface connection and biofilm and bacterial growth.

[0247] Experiment 10 This experiment 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.

[0248] 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 recovered to assess the pathological conditions of microbial load and assessment tissue and organ.It is expected that the method and composition of the present invention will be developed to reduce and / or eliminate the treatment and prevention strategies of the resulting Borrelia burgdorferi biofilm formed after the attack and considered to contribute to the pathogenesis of the disease and the chronic nature.

[0249] Experiment 11 This experiment provides a porcine model for preclinical testing of the disclosed polypeptides and compositions for treating 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 attributed to mutations in the gene encoding the CF transmembrane conductance regulator (known as CFTR) anion channel. In this model, pigs that have been specifically bred to carry a defect in the gene known as "CFTR" are called CF pigs, which 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 pathologies.

[0250] Experiment 12 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 about 20 cfu of Mycobacterium tuberculosis (M. tuberculosis) strain Erdman K01 bacteria to their lungs. At 25, 50, 75, 100, 125 and 150 days after the challenge, the animals were killed and the bacterial load was determined and the tissue was recovered for histopathological evaluation. Unlike mice that do not produce classic TB symptoms, guinea pigs attacked in this way form well-organized granulomas with central necrosis, which is a hallmark of human disease. In addition, guinea pigs, like humans, develop severe purulent granulomas and necrotizing lymphadenitis of draining lymph nodes, which is 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.

[0251] Experiment 13 A variety of animal models of catheter / indwelling device biofilm infections are known. See Otto (2009) Nature Reviews Microbiology, 7:555. Although generally considered to be 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. First, 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 usually life-threatening, the difficulties associated with the treatment of these biofilm infections and their frequency of occurrence make them a serious 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) the catheter is sonicated and CFUs are counted, (ii) sections of the catheter are cut or simply placed on a plate and scored, or (iii) the biofilm can be stained with crystal violet or another dye and washed off and the OD measured (as a surrogate for CFU).

[0252] in conclusion Full-length recombinant HMGB1 was able to significantly disrupt established biofilms, as were all truncated HMGB1 forms containing the B-box domain.

[0253] Equivalent 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.

[0254] The present technology illustratively described herein can be suitably implemented 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 descriptions rather than limitations, 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 changes are possible within the scope of the present technology.

[0255] 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.

[0256] 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 the present disclosure. This includes the general description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether the deleted material is specifically described herein.

[0257] 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.

[0258] 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 the event of a conflict, the present specification, including definitions, controls.

[0259] Further aspects are set out in the dependent claims.

[0260] Sequence Listing SEQ ID NO: 1 and 2 – Wild-type HMGB1 (mouse and human) (Human, copied from GenBank accession number CAE48262.1) HMGB1 is a small protein (about 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 (acidic C-tail), consisting of approximately 30 consecutive aspartic acid and glutamic acid residues.

[0261] The amino acids in bold (amino acids 1-70) describe the A Box domain. The italicized amino acids (approximately amino acids 88-164) describe the B Box domain.

[0262] The underlined amino acids (amino acids 186-215) depict the C-tail domain.

[0263] The mutant forms of HMGB1 are shown in Figures 1 and Figure 3 , which have amino acid substitutions.

[0264] SEQ ID NO: 3 and 4 Wild-type mouse HMGB1 B Box: MW = 9735.2; 87aa KDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAE KLKEKYEKDIAAYRAKGKPDAAKKGVV Wild-type human HMGB1 B Box: 87aa KDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAA KLKEKYEKDIAAYRAKGKPDAAKKGVV SEQ ID NO: 5 and 6 Mouse mutant HMGB1 B Box: MW = 9735.2; 87aa KDPNAPKRPPSAFFLFSSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAE KLKEKYEKDIAAYRAKGKPDAAKKGVV Human mutant HMGB1 B Box:87aa KDPNAPKRPPSAFFLFSSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAA KLKEKYEKDIAAYRAKGKPDAAKKGVV Cysteine ​​(C) has been mutated to serine (S) (bold text).

[0265] SEQ ID NO: 7 and 8 Wild-type mouse HMGB1 A+B Box: MW = 20261.42; 176aa MGKGDPKKPRRKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGK FEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL SIGDVAKKLGEMWNNTAADDKQPYEKKAEKLKEKYEKDIAAYRAKGKPDAAKKGVV wild type human HMGB1A+B Box:176aa MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGK FEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL SIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVVCysteine ​​(C) has been mutated to serine (S) (bold text).

[0266] SEQ ID NO:9 and 10 Wild-type mouse HMGB1 B Box + N-linker (underlined): MW = 10876.6; 97aa PPKGETKKKF KDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADD KQPYEKKAEKLKEKYEKDIAAYRAKGKPDAAKKGVV Wild-type human HMGB1 B Box + N-linker (underlined): 97aa PPKGETKKKF KDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADD KQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVV SEQ ID NO: 11 and 12 Mutant HMGB1 B Box + N-linker (underlined): MW = 10876.6; 97aa PPKGETKKKF KDPNAPKRPPSAFFLFSSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADD KQPYEKKAEKLKEKYEKDIAAYRAKGKPDAAKKGVV Human HMGB1 B Box + N-linker (underlined): 97aa PPKGETKKKF KDPNAPKRPPSAFFLFSSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADD KQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVV.

Claims

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

12.

2. An isolated B Box polypeptide prepared by growing a prokaryotic cell expressing an isolated polynucleotide encoding the isolated 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 polypeptide of claim 1.

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

6. An antibody that recognizes and binds to the isolated polypeptide according to any one of claims 1 to 5, or an antigen-binding fragment of the antibody. The antibody or antigen-binding fragment thereof according to claim 6 , further comprising a detectable label.

8. A composition comprising a carrier and one or more isolated polypeptides of claim 1.

9. A composition comprising a carrier and the antibody or antigen-binding fragment of claim 6 or 7.

10. The composition according to claim 8 or 9, wherein the carrier is a pharmaceutically acceptable carrier.

11. A polynucleotide encoding the isolated polypeptide of claim 1, or a complementary sequence of the polynucleotide.

12. A polynucleotide encoding the antibody or antigen-binding fragment of claim 6, or a complementary sequence of the polynucleotide.

13. The polynucleotide according to claim 11 or 12, which is operably linked to a promoter and / or an enhancer.

14. The polynucleotide according to claim 11 or 12, further comprising a detectable label. A vector comprising the polynucleotide according to claim 11 or 12.

16. The vector according to claim 15, wherein the vector is a plasmid or a viral vector.

17. An isolated host cell comprising one or more of the following: a polypeptide as described in any one of claims 1 to 5, an antibody or antigen-binding fragment as described in claim 6 or 7, a polynucleotide as described in any one of claims 11-14, or a vector as described in claim 15 or 16.

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

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

20. 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 a polypeptide described in any one of claims 1 to 5, thereby inhibiting, competing for or titrating the binding of the DNABII protein or polypeptide to the microbial DNA, wherein the biofilm is produced by an organism selected from the group consisting of: uropathogenic Escherichia coli (UPEC), Klebsiella pneumonia, Burkholderia cenocepacia, S. epidermidis, Streptococcus agalactiae, Neisseria meningitidis, Treponema denticola, Treponema pallidum, Burkholderia pseudomallei, Haemophilus influenzae, influenzae) (nontypeable) (NTHI), Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Mycobacterium tuberculosis, or ESKAPE pathogens.

21. An in vitro method for inhibiting, preventing or destroying a microbial biofilm, comprising contacting the biofilm with a polypeptide according to any one of claims 1 to 5, wherein the biofilm is produced by an organism selected from the group consisting of: uropathogenic Escherichia coli (UPEC), Klebsiella pneumonia, Burkholderia cenocepacia, S. epidermidis, Streptococcus agalactiae, Neisseria meningitidis, Treponema denticola, Treponema pallidum, Burkholderia pseudomallei, Haemophilus influenzae (non-typeable) (NTHI), Moraxella catarrhalis (Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Mycobacterium tuberculosis or ESKAPE pathogens, thereby inhibiting, preventing or destroying microbial biofilms.

22. Use of an agent in the preparation of a medicament for inhibiting, preventing or destroying a biofilm in a subject, wherein the agent comprises a polypeptide according to any one of claims 1 to 5, and wherein the biofilm is produced by an organism selected from the group consisting of uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae, Burkholderia cenocepacia, S. epidermidis, Streptococcus agalactiae, Neisseria meningitidis, Treponema denticola, Treponema pallidum, Burkholderia pseudomallei, Haemophilus influenzae (non-typeable) (NTHI), Moraxella catarrhalis, Streptococcus pneumoniae pneumoniae), Streptococcus pyogenes, Mycobacterium tuberculosis, or ESKAPE pathogens.

23. Use of an agent in the preparation of a medicament for inhibiting, preventing or treating a microbial infection that produces a biofilm in a subject, wherein the agent comprises a polypeptide as described in any one of claims 1 to 5, wherein the biofilm is produced by an organism selected from the group consisting of uropathogenic Escherichia coli (UPEC), Klebsiella pneumonia, Burkholderia cenocepacia, S. epidermidis, Streptococcus agalactiae, Neisseria meningitidis, Treponema denticola, Treponema pallidum, Burkholderia pseudomallei, Haemophilus influenzae (non-typeable) (NTHI), Moraxella catarrhalis, Streptococcus pneumoniae, pneumoniae), Streptococcus pyogenes, Mycobacterium tuberculosis, or ESKAPE pathogens.

24. Use of an agent in the preparation of a medicament for disrupting and clearing a biofilm in a subject without enhancing or inducing an inflammatory response, wherein the agent comprises a polypeptide as described in any one of claims 1 to 5, wherein the biofilm is produced by an organism selected from the group consisting of uropathogenic Escherichia coli (UPEC), Klebsiella pneumonia, Burkholderia cenocepacia, S. epidermidis, Streptococcus agalactiae, Neisseria meningitidis, Treponema denticola, Treponema pallidum, Burkholderia pseudomallei, Haemophilus influenzae (non-typeable) (NTHI), Moraxella catarrhalis, Streptococcus pneumoniae, pneumoniae), Streptococcus pyogenes, Mycobacterium tuberculosis, or ESKAPE pathogens.

25. Use of an agent in the preparation of a medicament for treating an infection or condition caused by the presence of a biofilm in a subject in need thereof, wherein the agent comprises a polypeptide as claimed in any one of claims 1 to 5, wherein the biofilm is produced by an organism selected from the group consisting of uropathogenic Escherichia coli (UPEC), Klebsiella pneumonia, Burkholderia cenocepacia, S. epidermidis, Streptococcus agalactiae, Neisseria meningitidis, Treponema denticola, Treponema pallidum, Burkholderia pseudomallei, Haemophilus influenzae (non-typeable) (NTHI), Moraxella catarrhalis, Streptococcus pneumoniae, pneumoniae), Streptococcus pyogenes, Mycobacterium tuberculosis, or ESKAPE pathogens.

26. The method or use of any one of claims 20 to 25, wherein the biofilm is produced by an organism selected from the group consisting of uropathogenic Escherichia coli (UPEC), Klebsiella pneumonia, Burkholderia cenocepacia, Haemophilus influenzae (non-typeable) (NTHI), or ESKAPE pathogens.

27. The use according to any one of claims 22 to 25, wherein the subject is a mammal.

28. The use according to claim 27, wherein the mammal is a human.

29. The use according to claim 28, wherein the human is an infant or a teenager.

30. The use according to any one of claims 22 to 25, further comprising administering an antimicrobial agent.

31. A kit comprising one or more of the following and instructions for use: the isolated polypeptide of any one of claims 1 to 5, the antibody of claim 6 or 7, or the polynucleotide of any one of claims 11-14.

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