Coating of medical and implant biomaterials with an Anti-biofilm agent to prevent and treat infection

Abstract

The present disclosure relates to methods of coating surgical implants with anti-biofilm agents to treat or prevent bacterial infection in patients receiving such implants. The method comprises coating the implant with an anti-DNABII antibody. The method comprises coupling an antibody to an implant, wherein the coupling comprises: treating the implant with aminopropyltriethoxysilane; treating the implant with glutaraldehyde; and incubating the implant with an anti-DNABII antibody.

Description

COATING OF MEDICAL AND IMPLANT BIOMATERIALS WITH AN ANTIBIOFILM AGENT TO PREVENT AND TREAT INFECTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 600,274, filed on November 17, 2023, which is hereby incorporated by reference in its entirety.FELD

[0002] The present disclosure relates to anti-biofilm agent coated implants and methods of coating surgical implants with anti-biofilm agents, to treat or prevent bacterial infection in patients receiving such implants.BACKGROUND

[0003] Infection of orthopedic implants is a common problem for patients. Approximately two-thirds of post-surgical orthopedic implant infections are from Staphylococcus. (See, Ribeiro et al., 2012). Despite their widespread use, the implants provide a surface for the growth of bacteria and the formation of bacterial biofilms.

[0004] Bacteria resident in a biofilm can be 8,000 times more resistant to antibiotics and are resistant to the host immune response as compared to those same bacteria that are planktonic (free living), substantially contributing to the pathogenesis, chronicity, and recurrence of bacterial infections (See, Manning et al., 2020 and Tsai et al., 2019). Bacterial biofilm formation, and correspondingly increased resistance to antibiotics and the immune response, occurs nearly immediately (hours) in host tissue and surfaces (See, Tarabichi et al., 2023 and Kurtz et al., 2012). Within hours to days, bacterial biofilm on foreign implant material can become unresolvable with antibiotics or by the host immune response (See, Premkumar, et al. 2021). The major problem with bacterial biofilms is the inability of the host immune system and / or antibiotics and other antimicrobials to gain access to the bacteria protected within the biofilm. These bacteria may be shed into the body leading to chronic infection, they may be highly resistant to antibiotic treatment, and may result in a chronic inflammatory response at the site of the implant. (See, Ribeiro el al., 2012).

[0005] Thus, patients receiving such implants are at risk for complicated infections since the biofilms formed on the surfaces of these implants serve as a protectant for bacterial growth. Accordingly, a need exists to break through the protective barrier of biofilms to treat, kill, or prevent the associated bacterial infections in patients who surgically receive an orthopedic implant.SUMMARY

[0006] One embodiment described herein is a method of preventing or reducing the likelihood of infection from surgical placement of an implant in a patient in need thereof comprising coating the implant with an anti-DNABII antibody. In one aspect, the coating comprises bonding the anti- DNABII antibody to the surface of the implant. In another aspect, the bonding is covalent or non- covalent. In one aspect, the surface is a solid interface of the implant with tissues and fluids. In another aspect the bonding comprises treating the implant with aminopropyltriethoxysilane and glutaraldehyde to make a treated implant. In another aspect, the method comprises incubating the treated implant with the anti-DNABII antibody. In another aspect, the anti-DNABII antibody is a monoclonal antibody. In another aspect, the implant is made of titanium. In another aspect, the implant is made of polyethylene. In yet another aspect, the implant is an orthopedic implant. In another aspect, the orthopedic implant is a screw, plate, nail, or wires. In another aspect, the orthopedic implant is placed in a knee, shoulder, hip, spine, finger, neck, elbow, or skeletal joint. In one aspect, the infection is a Staphylococcus aureus infection. In one aspect, the infection is reduced by greater than 90%. In another aspect, the method further comprises administering an antibiotic.

[0007] In another embodiment is a method of preventing the formation of or disrupting a biofilm on an implant, the method comprising coupling an antibody to an implant wherein the coupling comprises: (a) treating the implant with aminopropyltriethoxysilane; (b) treating the implant with glutaraldehyde; and (c) incubating the implant with an anti-DNABII antibody. In another aspect the aminopropyltriethoxysilane is a 10% (v / v) solution. In another aspect, the glutaraldehyde is a 4% (v / v) aqueous solution. In another aspect, the antibody is at a concentration of about 0.1 to about 2 mg / mL. In another aspect the antibody is a monoclonal antibody. In another aspect the implant is made out of titanium. In yet another aspect, the implant is made out of ultrahigh molecular weight polyethylene. In another aspect the antibody is coated at a surface density of about 0.01 mg / cm2and 1.0 mg / cm2.

[0008] In another embodiment, is a method of reducing infection from surgical placement of an implant comprising coating the implant with an antibody. In another aspect, the coating the implant with an antibody comprises: treating the implant with aminopropyltriethoxysilane; treating the implant with glutaraldehyde; and incubating the implant with an antibody. In another aspect, the aminopropyltriethoxysilane is a 10% (v / v) solution. In another aspect, the glutaraldehyde is a 4% (v / v) aqueous solution. In another aspect, the antibody is at a concentration of 1 mg / mL. In another aspect the method further comprises administering an antibiotic. In another aspect, the infection is reduced by the disrupting biofilm. The infection is caused by Staphylococcus aureus.

[0009] Another embodiment described herein is an implant comprising an anti-DNABII antibody coating on a surface. In another aspect, the coating comprises a surface density of about 0.01 mg / cm2and 1.0 mg / cm2. In some aspect, the surface is a solid interface of the implant with tissues and fluids. In another aspect, the implant is made out of titanium.BRIEF DESCRIPTION OF THE FIGURES

[0010] Figure 1 is a schematic of three panels, panel 1 depicting bacteria producing a biofilm matrix, consisting of DNABII protein and eDNA. DNABII crosslinks eDNA, facilitating a biofilm matrix scaffold that protects bacteria against immune cells and antibiotics. DNABII is in equilibrium between free floating DNABII and DNABII within the biofilm matrix. Panel 2 describes the anti-DNABII antibody that binds to DNABII, blocking DNABII’ s ability to support the biofilm scaffold and disrupting the equilibrium of DNABII between the biofilm matrix and surrounding environment. Sequestration of free floating DNABII by anti-DNABII antibody facilitates increased migration of DNABII from the biofilm matrix to the surrounding environment to maintain equilibrium. Panel 3 depicts when DNABII migrates from the biofilm matrix, the DNABII-eDNA scaffolding collapses, and biofilm forming bacteria are susceptible to immune cells and antibiotics.

[0011] Figure 2 is a schematic of two panels, panel 1 depicts that the biofilm on the implant prevents immune or antibiotic mediated killing of bacteria on the implant surface. Panel 2 depicts, the antibody binds to the DNABII protein binding domain for eDNA, blocking the eDNA-DNABII scaffolding. Lack of biofilm scaffolding makes bacteria vulnerable to immune cells and antibiotics.Not shown, the antibody also stimulates an equilibrium shift of DNABII from biofilm in surrounding tissue, disrupting biofilm in peripheral tissues.

[0012] Figure 3 is an experimental outline. Panel A describes the anti-DNABII antibody coated Ti wires, or control antibody wires, were placed in a dual transchamber assay. The lower chamber was incubated with .S'. aureus for 24 hours to form biofilm. Titanium (Ti) wires (control or anti-DNABII coated) were then placed in upper chamber. Panel B describes following incubation of implants for two hours in the upper chamber, biofilms in bottom chambers were stained with fluorescent bacterial membrane stain and visualized via confocal laser scanning microscopy (CLSM). Panel C depicts the quantification of biofilm biomass in the lower chamber following incubation of wires for two hours in the upper chamber. Images were analyzed by COMSTAT to calculate biomass.

[0013] Figure 4 depict in panel A, a schematic image of a 12- week-old C57BL / 6 mouse used in the study; (n=l) per group. Groups were: n=l femoral titanium implant coated with anti- DNABII antibody (covalent binding of antibody to implant); n=l femoral titanium implant (uncoated control). Surgical implant placement was performed followed by inoculation at the surgical site with 2 pL of 5xl05CFU / mL Xen36 .S. aureus in PBS (IxlO3CFU of Xen365. aureus total). Additionally, the aminopropyltriethoxy silane coating technique is shown. Panel B depicts a diagram of femoral implant coated with anti-DNABII antibody. Panel C describes a light microscopic image of a control implant and an antibody implant. Panel D describes X-ray images performed following surgical placement of a control implant and an antibody implant. Panel E describes bioluminescent imaging for bioluminescent signal of Xen36 .S'. aureus at the surgical site / knee joint over time in living mice; orange rectangles represent the surgery / inoculation site; green dashed ovals represent the region of interest used for quantification in panel F. Panel F is a plot of the quantification of bioluminescent signal (average radiance over 5 minutes) of Xen36 .S'. aureus at the surgical site / knee joint in living mice. On the final day of bioluminescence imaging, implants were harvested for CFU analysis (CFUs / Implant). Panel G is a plot of the CFUs per implant in the control compared with the anti-DNABII antibody implant.DETAILED DESCRIPTION OF THE DISCLOSURE

[0014] Coating of anti-biofilm molecules to a medical or implant material may reduce infection and subsequent morbidity and mortality of patients receiving such implants. While thiscoating may be applied to a wide-variety of medical or implant material, the present disclosure focuses on orthopedic applications. Thus, the present disclosure relates to a novel method of reducing the likelihood of infection from surgical placement of an implant in a patient comprising coating the implant with an anti-biofilm agent.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0016] As used herein, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" can mean one element or more than one element.

[0017] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some embodiments, the terms "about" or "approximately" when preceding a numerical value indicates the value plus or minus a range of 10%, 5%, or 1%.

[0018] As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the intended use. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions disclosed herein. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0019] “Administration” can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosageof administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include oral administration, nasal administration, injection, and topical application.

[0020] As used herein, the terms “antibody,” “antibodies” and “immunoglobulin” includes whole antibodies and any antigen-binding fragment or a single chain thereof. Thus, the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule. The terms “antibody,” “antibodies” and “immunoglobulin” also include immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fab', F(ab)2, Fv, scFv, dsFv, Fd fragments, dAb, VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies and kappa bodies; multispecific antibody fragments formed from antibody fragments and one or more isolated. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region (which is also referred to herein as a variable domain), a heavy chain or light chain constant region (which is also referred to herein as a constant domain), a framework (FR) region, or any portion thereof, at least one portion of a binding protein, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues. The term “anti-” when used before a protein name, anti-DNABII, anti-IHF, anti- HU, anti-OMP P5, for example, refers to a monoclonal or polyclonal antibody that binds and / or has an affinity to a particular’ protein. The specific antibody may have affinity or bind to proteins other than the protein it was raised against.

[0021] A “biofilm” intends an organized community of microorganisms that at times adhere to the surface of a structure, that may be organic or inorganic, together with the polymers such as DNA that they secrete, release and / or become available in the extracellular milieu due to bacterial lysis. The biofilms are very resistant to microbiotics and antimicrobial agents. Biofilms may also form on the surface of implants, stents, catheter lines and contact lenses. They may grow on pacemakers, heart valve replacements, artificial joints and other surgical implants. The Centers for Disease Control) estimate that over 65% of nosocomial (hospital-acquired) infections are caused by biofilms. In one embodiment described herein, the biofilm comprises a polypeptide or protein. In one aspect, the polypeptide or protein is DNABII. A “DNABII polypeptide or protein” intends a DNA-binding protein or polypeptide that is composed of DNA-binding domains and thus have a specific or general affinity for microbial DNA. In one aspect, they bind DNA in the minor grove. Non-limiting examples of DNABII proteins are an integration host factor (IHF) protein and a histone-like protein from E. coli strain U93 (HU). Other DNA binding proteins that may be associated with the biofilm include DPS (Genbank Accession No.: CAA49169), H-NS (Genbank Accession No.: CAA47740), Hfq (Genbank Accession No.: ACE63256), CbpA (Genbank Accession No.: BAA03950) and CbpB (Genbank Accession No.: NP — 418813).

[0022] As used herein, the term "anti-biofilm agent" refers to a molecule or compound directly targeting the secreted biofilm matrix surrounding the bacterial cell. Currently there are no FDA approved agents clinically available directly targeting the biofilm matrix. Directly targeting the biofilm matrix for disruption or elimination will allow antimicrobial compounds, such as antibiotics and immune cells to kill bacteria in the bacterial biofilm. As described herein, the bacterial biofilm matrix may be disrupted with anti-biofilm antibodies. In one aspect described herein, the anti-biofilm agent is an antibody directed to a biofilm matrix protein. In another aspect described herein, the anti-biofilm agent is an anti-DNABII antibody. Other antibodies targeting a biofilm protein are contemplated herein.

[0023] In some embodiments, the term “disrupt” intends a reduction in the formation of the DNA / protein matrix that is a component of a microbial biofilm. Additionally or alternatively, the term “disrupt” intends a reduction of a formed biofilm, such as dispersing the biofilm partially or completely. Such reduction can be shown in various parameters. For example, the biomass or the bacterial load of a biofilm or both can be evaluated prior to and after a treatment, and the reduction thereof after the treatment can be used to show the efficacy of the treatment. Another example ofthe parameter is relative mucosal biofilm score or biomass score given by a blinded evaluator. Other suitable parameters arc shown in the Examples as disclosed herein. In some embodiments, a treatment reduces a biofilm to at least about 90% (including but not limited to at least about 85%, or at least about 80%, or at least about 75%, or at least about 70%, or at least about 65%, or at least about 60%, or at least about 55%, or at least about 50%, or at least about 45%, or at least about 40%, or at least about 35%, or at least about 30%, or at least about 25%, or at least about 20%, or at least about 15%, or at least about 10%, or at least about 9%%, or at least about 8%, or at least about 7%, or at least about 6%, or at least about 5%, or at least about 4%, or at least about 3 %, or at least about 2 %, or at least about 1%, or less than 1%, or about 0%) of the biofilm prior to the treatment. In certain embodiments, disrupting a biofilm refers to dispersing the biofilm (completely or partially), releasing microorganisms from the DNA / protein matrix of the biofilm, and optionally allowing killing the microorganisms by host immune effectors and / or antibiotics.

[0024] As used herein, "an effective amount" refers to an amount that causes relief of symptoms of a disorder or disease as noted through clinical testing and evaluation, patient observation, and / or the like. An "effective amount" may further designate a dose that causes a detectable change in biological or chemical activity. The detectable changes may be detected and / or further quantified by one skilled in the art for the relevant mechanism or process. Moreover, an "effective amount" may designate an amount that maintains a desired physiological state, i.e., reduces or prevents significant decline and / or promotes improvement in the condition of interest. An "effective amount" may further refer to a “therapeutically effective amount”.

[0025] As used herein the term “implant” is a material capable of being surgically inserted, embedded, grafted or otherwise inserted in a biological body (e.g., human body). For example, the implant may be a dental implant (e.g., a crown), an orthopedic implant (e.g., a hip replacement), a cardiac implant (e.g., stent).

[0026] “Inhibiting, preventing or disrupting” a biofilm intends the prophylactic or therapeutic reduction in the structure of a biofilm. “Inhibiting, preventing or reducing the likelihood” intends the prophylactic, therapeutic reduction in or the chances of, infection.

[0027] The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to 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 subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain atleast two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.

[0028] A “subject” of diagnosis or treatment is a cell or an animal such as a mammal, or a human. Non-human animals subject to diagnosis or treatment and are those subject to infections or animal models, for example, simians, murines, such as, rats, mice, chinchilla, canine, such as dogs, leporids, such as rabbits, livestock, sport animals, and pets. The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human.

[0029] As used herein, "treatment", "treat", and "treating" refers to reversing, alleviating, mitigating, or slowing the progression of, or inhibiting the progress of, a disorder or disease or symptoms associated with such disorder or disease, and as described in more detail herein.

[0030] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0031] Bacterial biofilm consists of bacteria and their self-produced extracellular matrix consisting of extracellular’ DNA (eDNA), proteins, lipids, and exopolysaccharides, which provides protection against environmental stresses including antimicrobials and host immune cells. It is reported that i) eDNA within the biofilm forms a lattice structure that is crucial to the structural integrity of biofilms; ii) the stability of this lattice structure is achieved by the binding and stabilization of the eDNA with DNABII proteins; iii) removal of DNABII-eDNA binding through targeted Ab against the DNABII binding domain results in immediate collapse (-minutes) of biofilm with substantial loss of biofilm biomass in vitro (>90%) and resolution of biofilm- mediated infections in animal models of lung infection and middle ear infection See, Davis et al., 2022;Zmistowski et al. 2013; Segawa et al. 1999; Pelt et al., 2014; Shahi et al., 2015, Qasim et al., 2017; Marschall et al., 2013; Svensson et al. 2021) Anti-DNABII Abs arc known (Novotny et al., 2016) and are in Phase I clinical trials to treat bacterial pneumonia (NCT05629741) and periprosthetic joint infection (NCT04763759) (ClinicalTrials.Gov). The present disclosure entails use of anti-DNABII antibody as well as other antibodies developed to target bacterial biofilm matrix.

[0032] Orthopedic implant devices restore the function of load-bearing joints subjected to stress and the gradual damage or deterioration that results from normal use, aging or orthopedic disease or disorders. Implant-associated infections in patients result from bacterial adhesion to an implant surface and subsequent biofilm formation at the implantation site. Thus, the present disclosure contemplates the use of an anti-biofilm agent with an orthopedic implant.

[0033] Accordingly, one embodiment described herein is a method of preventing or reducing the likelihood of infection from surgical placement of an implant in a patient in need thereof comprising coating the implant with an anti-biofilm agent. Another embodiment described herein is a method of preventing or reducing the likelihood of infection from surgical placement of an implant in a patient in need thereof comprising coating the implant with an anti-DNABII antibody. In some aspects, the anti-DNABII antibody is a monoclonal antibody. In other aspects the anti- DNABII antibody targets the DNABII protein in the biofilm.

[0034] Exemplary orthopedic implant devices include, but are not limited to prostheses for hip, knee, ankle, shoulder and elbow joints, and also include fracture fixation devices such as wires, pins, plates, and screws. Such implants may be made from a variety of materials including metals, such as titanium alloy, cobalt chromium molybdenum alloy, and stainless steel; polymers, such as poly(methyl methacrylate) (PMMA) and ultrahigh-molecular-weight polyethylene (UHMWPE); and ceramics (alumina, zirconia and hydroxyapatite). Specific materials can include, but are not limited to: i) metals and metal alloys incorporating cobalt chromium, titanium, and stainless steel; ii) synthetic polymers, such as polyethylene, polyether ketone polycarbonate, polypropylene, acrylonitrile butadiene styrene, polyethylene terephthalate glycol, polymethyl methacrylate, and polyvinyl chloride; iii) Ceramics, such as alumina and zirconia based ceramics; iv) natural polymers, such as collagen and albumin, and polysaccharides such as chitosan, hyaluronic acid, pectin, silk, alginate, and xanthan gum.

[0035] Surfaces to be coated include the entire surface area of the implant, and the interface of the implant adjacent to body tissues and fluids. Surfaces may be coated by molecules or compounds by either covalent or non-covalent attachment. Exemplary coating methodologies include, but are not limited to, electroporation, direct adsorption, coupling through protein carboxyl groups, sulfhydryl groups, hydroxyl groups, amino groups or aldehyde groups of oxidized carbohydrate residues on proteins. Chemical conjugation is also contemplated as a methodology for coating molecules on a surface. In one aspect described herein, the coating comprises bonding the antibody to the surface of the implant. In some aspects, the bonding may be a covalent bonding, a non-covalent bonding or an adsorption directly on to the surface. In other aspects, the bonding is a chemical bonding. Exemplary chemical bonding includes, but is not limited to, pretreating the implant with a chemical composition to facilitate bonding to an implant surface. One exemplary chemical method includes the use of silane to bond to numerous materials. Silane coatings provide functional moieties that facilitate binding of bioactive molecules to the material surface and silane and glutaraldehyde are used together in a variety of applications. Thus, in one aspect, the bonding comprises treating the implant surface with a silane composition, and subsequently incubating the antibody for chemical conjugation of the antibody to the surface. In some aspects the amount of silane used for conjugation may be between about 0.1% (v / v) solution to about 30% (v / v) solution. In other aspects, the amount of silane composition used for conjugation is about 10% (v / v) solution. In some aspects, glutaraldehyde may used for conjugation of the antibody to the implant. In other aspects, the amount of glutaraldehyde may be between about 0.1% (v / v) solution to about 10% (v / v) solution. In other aspects the amount of glutaraldehyde used for conjugation is about 4% (v / v) aqueous solution. In some aspects, the amount of antibody used for conjugation may be between about 0.1 mg / mL to about 2 mg / mL. In other aspects the amount of antibody used for conjugation is about 1 mg / mL.

[0036] The amount of anti-biofilm agent coating an implant may have vary and may be dependent on the chemical nature of the implant, surface, topography, bonding method, and it’s biological location in the subject.

[0037] Another embodiment described herein is an implant comprising a coating of an antibiofilm agent. In one aspect the anti-biofilm agent is an anti-DNABII antibody. Thus, another embodiment described herein is an implant comprising an anti-DNABII antibody coating. In one aspect, the antibody coated at a surface density of about 0.01 mg / cm2to about 1.0 mg / cm2. Inanother aspect, the antibody is coated at a surface density of about 0. 1 mg / cm2to about 1 .0 mg / cm2. In yet another aspect, the antibody is coated at a surface density of 0.1 mg / cm2to about 0.8 mg / cm2. In yet another aspect antibody is coated at a surface density of about 0.2 mg / cm2. In another aspect the antibody is coated at surface density of 0.7 mg / cm2. In another aspect the antibody is coupled to the implant a surface density of 0.8 mg / cm2.

[0038] Medical and orthopedic implants use occurs in a wide variety of medical areas, including but not limited: neuro surgery, cardiovascular surgery, orthopedic surgery, plastic surgery, ophthalmology urology, gynecology, and dentistry and oral and maxillofacial surgery. Common medical and implant devices include, but are not limited to: spinal and neurological stents, cardiovascular stents, catheters, pacemakers, and heart valves, prosthetic joints, and artificial ligaments, breast implants, intra-ocular lenses, penile implants, urogynecological mesh surgical implants, neural electrodes dental implants and filling materials, sutures used in essentially every surgical procedure, as well as additional orthopedic hardware such as screw, plates, rods, nails, wires, collagen scaffolds, and intra-medullary nails. Such orthopedic devices may be placed in joints, knees, shoulders, hips, spine, fingers, neck, elbow or other skeletal bones or joint. Accordingly, in one aspect of the present disclosure, the implant is an orthopedic implant. In some aspects, the orthopedic implant is a screw, plate, nail, or wires. In other aspects, the orthopedic implant is placed in a knee, shoulder, hip, spine, finger, neck, elbow, or any skeletal joint of bone.

[0039] Medical and implant material may result in an increased risk for infection or persistence of infection by greater than 1,000,000 times (See, Macias -Valcayo A, et al. 2022) and bacterial biofilm on implant material results in 8,000 times greater resistance to resolution of infection by antibiotics (3, 4). Mature bacterial biofilm on foreign implant material may make it difficult to eradicate infection. Infection on and around medical and implant is associated with elevated morbidity and mortality. Exemplary infections in patients from such implants include but are not limited to bacterial infections from, for example, Staphylococcus aureus, Staphylococcus epide rmidis, Pseudomonas aeruginosa, and Escherichia coli.', bone tissue infections such as osteomyelitis, septic arthritis and prosthetic joint infections. In one aspect of the method described herein, the infection is a Staphylococcus aureus infection. In one aspect the infection is reduced by about 10% to about 100%, about 20% to about 90%, about 30% to about 80%, about 40% toabout 70%, about 50% to about 60%. Reduction in infection may be measured by any known mean in the art, including a reduction in colony forming units (CFU).

[0040] In addition to the use of the coated implants described herein, patients may also be administered antibiotics to address infections. Exemplary antibiotics include but are not limited to, nafcillin, ciprofloxacin, vancomycin, cephalosporin, amoxicillin, clavulanic acid, cephalexin, or doxycycline. In some aspects, the method described herein further includes administering an antibiotic in combination with the use of the coated implants described herein.

[0041] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.EXAMPLESExample 1: Titanium(Ti) Coated with Anti-DNABII Monoclonal Antibody Disrupts Bacterial Biofilm

[0042] Ti mAb coating: Ti wires (0.6 mm diameter x 7 mm length) were treated with 10% (v / v) aminopropyltriethoxysilane followed by 4% glutaraldehyde. Wires were then incubated with 1 mg / ml anti-DNABII mAb overnight at room temperature. Residual free aldehydes were blocked with 0.15 M Tris buffer, pH 7.5. In vitro biofilm disruption assay (transwell chamber assay). Xen36 Staphylococcus aureus was incubated for 24 hr in an 8-well chambered coverglass (lower chamber). Ti wires with anti-tip anti-DNABII mAb coating (n=3) and negative control mAb coated wires (n=3) were then placed in upper chambers, separated from the lower chambers, with a semipermeable membrane for 2 hr (Figure 3A). In the lower chamber, biofilms were then stained with FM1-43FX and fixed. Quantification of biofilm biomass was performed using a Zeiss 800 scanning confocal laser microscope and COMSTAT2 software. Statistical analysis: Difference between groups was evaluated with unpaired student’s t-test; p < 0.05 considered significant. Data represented by mean + SD. Biofilm biomass was significantly reduced in the anti-DNABII mAb coated Ti wire group as compared to the negative control mAb Ti wire group (Figure 3B). Quantification of biofilm biomass revealed a >90% reduction in biofilm biomass in the anti-DNABII mAh coated Ti wire group (1 .57 + 0.29 pm3 / pm2 ) vs. the negative control mAb coated Ti wire group (17.0 + 3.91 pm3 / pm2); p = 0.002 (Figure 3C). Anti-DNABII mAb was covalently bound to the Ti wire surface through silane based coating methodology. The mAb coating reduced bacterial biofilm mass >90% over 2 hr. This disruption and collapse of biofilm was mediated through blocking the DNABII protein binding site to eDNA and promoting an equilibrium shift of DNABII from biofilm.

[0043] Further testing was done in mice using 12-week-old C57BL / 6 mouse; (n=l) per group. Groups were: n=l femoral titanium implant coated with anti-DNABII antibody (covalent binding of antibody to implant); n= 1 femoral titanium implant (uncoated control). Surgical implant placement was performed followed by inoculation at the surgical site with 2 pL of 5xl05CFU / mL Xen36 S', aureus in PBS (1x103 CFU of Xen36 S. aureus total) (Figure 4B). Light microscopy images of control implant and antibody implant as well as X-ray images performed following surgical placement of control implant and antibody implant were taken (Figure 4C and D). Bioluminescent images were also taken for biolumine scent signal of Xen36 S. aureus at the surgical site / knee joint over time in living mice (Figure 4E). Quantification of bioluminescent signal (average radiance over 5 minutes) of Xen36 S. aureus was done at the surgical site / knee joint in living mice. On the final day of bioluminescence imaging, implants were harvested for CFU analysis (CFUs / Implant) (Figure 4F and G).Example 2: Covalent coupling of humanized anti-DNABII monoclonal antibody to ultrahigh molecular weight polyethylene (UHMWPE)

[0044] The same method was used to couple the humanized anti-DNABII monoclonal antibody to ultrahigh molecular weight polyethylene as was used to couple the mouse anti- DNABII monoclonal antibody to titanium wires. Discs (3 mm diameter, 3 mm thick) were generated from a UHMWPE sheet (McMaster-Carr no. 8702K196) using a 3 mm inside diameter manual punch (McMaster-Carr no.3424A12). Discs were washed with acetone, then incubated with 10% aminopropyltriethoxysilane (Vectabond, Vector Laboratories) in dry acetone for 12h. Washed 2X with water and incubated with 10% glutaraldehyde in water for 12h. Washed again with water 2X and incubated with 1 mg / ml of humanized anti-DANBII monoclonal antibody (Clarametyx) in PBS for 12h. Residual aldehydes on antibody bound discs were blocked with 0.15 M Tris buffer, 1% bovine serum albumin, pH 7.4 for 12h. All disc incubations were performed in microfuge tubes, with continuous rotation at room temperature.

[0045] An ELISA for human IgG was developed and used to measure the amount of human mAb bound to the surfaces of both UHMWPE disks and Ti wires. A human IgGl isotypc monoclonal antibody (InVivoMAb) was used as a standard in the assay. Duplicate wells were coated overnight with the standard human IgGl in the range of 4 ng to 250 ng per well as two-fold dilutions in 50 mM sodium carbonate buffer, pH 9.5. Plates were washed and BSA 1%, 0.1% Tween 20 in PBS were added to all wells to block any free protein binding sites on the standard coated and blank wells. Antibody-bound materials, either UHMWPE discs or Ti wires, were added to sample wells and sufficient volumes of a secondary antibody, goat anti-human IgG (H+L), horseradish peroxidase conjugate, cross absorbed (for bov, mouse, rabbit)(Invitrogen, cat.no. A18811) was added and the same volume used in standard wells. Secondary antibody-HRP was diluted 1:2000 in PBS, 0.2% BSA, 0.05% Tween 20 and incubated in the wells for Ih at room temperature with shaking. Wells were washed and antibody bound peroxidase activity was detected using a chemiluminescent substrate (SuperSignal ELISA Femto Substrate, ThermoFisher no. 37075) and measured using a multilabel plate reader (Wallac Victor).

[0046] Using this ELISA the mean value for antibody coupled to two UHMWPE discs was 340 ng and on two Ti wires was 97 ng.

[0047] Various aspects of the present disclosure may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is, therefore, not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

Claims

CLAIMS1. A method of preventing or reducing the likelihood of infection from surgical placement of an implant in a patient in need thereof comprising: coating the implant with an anti-DNABII antibody.

2. The method of claim 1, wherein the coating comprises bonding the anti-DNABII antibody to the surface of the implant.

3. The method of claim 2, wherein the bonding is covalent or non-covalent.

4. The method of claim 2, wherein the surface is a solid interface of the implant with tissues and fluids.

5. The method of claim 2, wherein the bonding comprises: treating the implant with aminopropyltriethoxysilane and glutaraldehyde to make a treated implant; and incubating the treated implant with the anti-DNABII antibody.

6. The method of claims 1 and 2, wherein the anti-DNABII antibody is a monoclonal antibody.

7. The method of any of claims 1 to 6, wherein the implant is made of titanium.

8. The method of any of claims 1 to 6, wherein the implant is made of polyethylene.

9. The method of any of claims I to 8, wherein the implant is an orthopedic implant.

10. The method of claim 9, wherein the orthopedic implant is a screw, plate, nail, or wires.

11. The method of claim 10, wherein the orthopedic implant is placed in a knee, shoulder, hip, spine, finger, neck, elbow, or skeletal joint.

12. The method of any of claims 1 to 11, wherein the infection is a Staphylococcus aureus infection.

13. The method of any of claims 1 to 12, wherein the infection is reduced by greater than 90%.

14. The method of any of claims 1 to 13, wherein the method further comprises: administering an antibiotic.

15. A method of preventing the formation of or disrupting a biofilm on an implant, the method comprising: coupling an antibody to an implant wherein the coupling comprises:(a) treating the implant with aminopropyltriethoxysilane;(b) treating the implant with glutaraldehyde; and(c) incubating the implant with an anti-DNABII antibody.

16. The method of claim 15, wherein the aminopropyltriethoxysilane is a 10% (v / v) solution.

17. The method of claims 15 and 16, wherein the glutaraldehyde is a 4% (v / v) aqueous solution.

18. The method of any of claims 15 to 17, wherein the antibody is at a concentration of about 0.1 to about 2 mg / mL.

19. The method of any of claims 15 to 18, wherein the antibody is a monoclonal antibody.

20. The method of any of claims 15 to 19, wherein the implant is made out of titanium.

21. The method of any of claims 15 to 19, wherein the implant is made out of ultrahigh molecular weight polyethylene.

22. The method of any of claims 15 to 21, wherein the antibody is coated at a surface density of about 0.01 mg / cm2and 1.0 mg / cm2.

23. A method of reducing infection from surgical placement of an implant comprising: coating the implant with an antibody.

24. The method of claim 23, wherein the coating the implant with an antibody comprises: treating the implant with aminopropyltriethoxysilane; treating the implant with glutaraldehyde; and incubating the implant with an antibody.

25. The method of claim 24, wherein the aminopropyltriethoxysilane is a 10% (v / v) solution.

26. The method of claims 24 and 25, wherein the glutaraldehyde is a 4% (v / v) aqueous solution.

27. The method of any of claims 24 to 26, wherein the antibody is at a concentration of 1 mg / mL.

28. The method of any of claims 23 to 27, wherein the method further comprises: administering an antibiotic.

29. The method of any of claims 23 to 28, wherein the infection is reduced by the disrupting biofilm.

30. The method of any of claims 23 to 30, wherein the infection is caused by Staphylococcus aureus.

31. An implant comprising an anti-DNABII antibody coating on a surface.

32. The implant of claim 31, wherein the coating comprises a surface density of about 0.01 mg / cnr and 1.0 mg / cnr.

33. The implant of claim 31, wherein the surface is a solid interface of the implant with tissues and fluids34. The implant of claim 31, wherein the implant is made out of titanium.

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