RU2807688C2 - Identification of lysines and their derivatives with antibacterial activity against pseudomonas aeruginosa - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: pharmaceutical composition containing an isolated modified lysine polypeptide of the sequence SEQ ID NO: 3 selected from the group consisting of one or more of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, or a fragment thereof having lysine activity, or a variant thereof having lytic activity, wherein these fragments and variants have at least 80% sequence identity with this modified lysine polypeptide, wherein the modified lysine polypeptide contains amino acid substitutions K100D and R116H relative to SEQ ID NO: 3 and/or contains the peptide sequence SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30 at the N- or C-terminus SEQ ID NO: 3, and a pharmaceutically acceptable carrier, wherein this polypeptide or lysine fragment or variant is present in an amount effective to inhibit the growth or depopulation of, or destruction of gram-negative bacteria.

EFFECT: effective treatment of a gram-negative bacterial infection caused by gram-negative bacteria.

12 cl, 10 tbl, 9 ex

Description

BACKGROUND OF THE ART

[001] Gram-negative bacteria, particularly members of the genus Pseudomonas , are an important cause of serious and potentially life-threatening invasive infections. Pseudomonas infection is a major problem in burn wounds, chronic wounds, chronic obstructive pulmonary disease (COPD) and other structural lung diseases, cystic fibrosis, superficial growth on implanted biomaterials, and on surfaces in hospitals and water supplies, where it poses multiple threats for vulnerable patients such as immunosuppressed patients and intensive care unit (ICU) patients.

[002] Once a patient develops a P. aeruginosa infection, it can be particularly difficult to treat. The genome encodes multiple resistance genes, including multidrug pumps and enzymes that confer resistance to beta-lactam and aminoglycoside antibiotics, making therapy against this Gram-negative pathogen particularly challenging due to the lack of new antimicrobial therapeutics. This problem is compounded by the ability of P. aeruginosa to grow in biofilm, which may enhance its ability to cause infections by shielding the bacteria from host defenses and conventional antimicrobial chemotherapy.

[003] The incidence of drug-resistant strains of Pseudomonas aeruginosa in health care settings is increasing. P. aeruginosa was estimated to cause 7% of all hospital-acquired infections (HAIs) in a multi-country point-in-time prevalence survey (1). More than 6,000 (13%) of the 51,000 cases of HAI caused by P. aeruginosa each year are multidrug resistant (MDR), with approximately 400 deaths per year (2). Extensively drug-resistant (XDR) and all drug-resistant (PDR) strains are emerging threats for which there are limited or no available treatments (3). Invasive P. aeruginosa infections, including bloodstream infections (BSIs), which are among the most lethal HAIs—for example, P. aeruginosa is responsible for 3 to 7% of all BSIs, with mortality rates ranging from 27 to 48% (4). The incidence of invasive bloodstream infections, including those caused by P. aeruginosa, may be underestimated because most health care services in the United States are provided in small, non-clinical community hospitals. In an observational study of BSI in community hospitals, P. aeruginosa was one of the 4 most significant MDR pathogens (5) and the overall in-hospital mortality rate was 18%. In addition, outbreaks of MDR P. aeruginosa have been well described ( 6 ). Poor outcomes are associated with MDR strains of P. aeruginosa, which often require treatment with drugs of last resort such as colistin (7). Clearly, there is an unmet medical need for various antimicrobial agents with novel mechanisms to target MDR P. aeruginosa for the treatment of invasive infections, including, but not limited to, BSI.

[004] An innovative approach to treating bacterial infections focuses on a family of bacteriophage-encoded peptidoglycan (PG) cell wall hydrolases called lysines (8). Currently, lysine-based technology relies on the use of purified recombinant lysine proteins that target a range of Gram-positive (GP) pathogens externally, resulting in bacterial cell lysis on contact with kill rates of several orders of magnitude. Lysines act as “molecular scissors,” thereby destroying the peptidoglycan (PG) network structure responsible for maintaining cell shape and counteracting internal osmotic pressure. The destruction of PG leads to osmotic lysis. In addition to rapid killing and a novel mode of action compared to antibiotics, other hallmarks of lysine activity include activity against biofilms, lack of pre-existing resistance, strong synergy with antibiotics (at concentrations below the minimum inhibitory concentration (MIC)) and inhibition of antibiotic resistance when use of antibiotics in addition to lysines. Importantly, several groups have demonstrated in several animal models the ability to control antibiotic-resistant Gram-positive bacterial pathogens using topical, intranasal, and parenteral administration of lysines (9–11).

[005] Lysine-based technology was originally developed for the treatment of gram-positive pathogens. To date, the development of lysines for targeting Gram-negative (GN) bacteria has been limited. The outer membrane (OM) of Gram-negative bacteria plays a critical role as a barrier to extracellular macromolecules and limits access to the underlying peptidoglycan ( 12 – 14 ).

[006] EM is a hallmark of Gram-negative bacteria and contains a lipid bilayer with an inner phospholipid leaflet and an outer amphiphilic leaflet composed primarily of lipopolysaccharides (LPS) (15). LPS has three main parts: a hexaacylated glucosamine-based phospholipid called lipid A, a polysaccharide core, and an extended outer polysaccharide chain called the O-antigen. VM is a non-liquid continuum stabilized by three main interactions, including: i) avid binding of LPS molecules to each other, particularly if cations are present to neutralize phosphate groups; ii) close packing of mostly saturated acyl chains; and iii) hydrophobic stacking of lipid A groups. The resulting structure is a barrier to both hydrophobic and hydrophilic molecules. PG forms a thin layer under the outer membrane, which is very sensitive to hydrolytic cleavage, unlike the PG of gram-positive bacteria, which has a thickness of 30-100 nm and contains up to 40 layers, the PG of gram-negative bacteria has a thickness of only 2-3 nm and consists of only 1 -3 layers. Potent antimicrobial activity can be achieved if lysins targeting Gram-negative bacteria are designed to penetrate the VM, either alone or in combination with VM-destabilizing agents and/or antibiotics.

[007] Accordingly, the discovery and development of GN-lysines that cross the VM is an important goal and will fill an important, but still unmet, need for the development of effective therapies to treat or prevent infections caused by Gram-negative bacteria. Several agents have been previously described with activities aimed at permeabilizing VMs and destroying VMs. For example, polycationic compounds, including polymyxin antibiotics and aminoglycosides, compete with stabilizing divalent cations in EO for interactions with phospholipids in LPS, resulting in disruption of EO structure (16). Similarly, EDTA and weak acids chelate divalent cations, leading to disruption of the structure of the BM (17). It is also known that a large group of natural antimicrobial peptides and their synthetic peptidomimetics (referred to herein as AMPs) penetrate the VM by self-stimulated uptake (18-20). Translocation of both polycationic and amphipathic AMPs occurs through a primary electrostatic interaction with LPS, followed by displacement of cations, disruption of membrane structure and formation of transient pores, and in some cases due to AMP internalization. The membrane-based antimicrobial activity of many AMPs can be “activated” in the blood by rationally designing amphipathic domains, either by changing the hydrophobicity, net charge and arrangement of polar residues in the hydrophobic surface, or by inserting D, L residues in place of all L- analogues (18, 19, 21, 22).

[008] The present inventors have improved lysine-based technology to combat gram-negative pathogens using various techniques to achieve penetration through the VM as set forth herein. Indeed, the inventors of the present invention previously filed International Patent Application PCT/US2016/052338, filed on September 16, 2016 and published as WO/2017/049233. This prior PCT application is incorporated herein by reference in its entirety for all purposes. For example, the lysines GN2, GN4, GN14, GN43 and GN37 were first disclosed in the aforementioned PCT application.

[009] Recent studies have identified lysines with intrinsic antimicrobial activity against Gram-negative bacteria (12, 13, 17). The antimicrobial effect in some cases is due to N- or C-terminal amphipathic or polycationic α-helical domains, which mediate penetration through LPS and translocation through the EV, leading to PG degradation and osmotic lysis. It is interesting to note that the access of such lysines to PGs can be facilitated by compounds that destabilize VMs, including EDTA and weak organic acids. Although combinations with EDTA and weak organic acids are not used in practice as drugs, the results illustrate the concept of facilitating the activity of GN-lysine.

[0010] A more recent approach uses GN-lysines hybridized to specific α-helical domains that have polycationic, amphipathic, and hydrophobic features to promote translocation through the EM. These results led to the development of GN-lysines, called “artilisins,” which are highly active in vitro and intended for topical application (17). However, low activity of artilisins has been reported in vivo . Accordingly, artilisin GN126, listed as a control in the present invention (see Table 4), also showed low activity.

[0011] Despite the in vitro efficacy of artilisins and lysines, including GN-lysines, with intrinsic antimicrobial activity, a major limitation remains regarding the apparent lack of activity in human blood matrices, making systemic therapy problematic (13, 14). Physiological salt and divalent cations are believed to compete for LPS binding sites and interfere with the α-helical translocation domains of lysines, including GN-lysines, which limits activity in the blood and, more specifically, in the presence of serum, thus limiting the utility of lysines for treatment of invasive infections (23). A similar lack of activity in the blood has been reported for several different AMPs that penetrate and destabilize the IM (18–20, 22).

SUMMARY OF THE INVENTION

[0012] The major design challenge facing the development of GN-lysine for the treatment of invasive infections by systemic administration is the need to attenuate inactivation in blood (or eg human serum).

[0013] Native GN-lysines with intrinsic activity (i.e., high activity in HEPES buffer and low activity in human serum) were first identified and then modified by replacing charged amino acids with uncharged amino acids and/or by hybridization with alpha -helical antimicrobial peptide to improve activity and improve serum potency.

[0014] Based on this work, putative native lysines were identified and their activity was assessed. Lysines are listed in Table 3 and described by their sequence. Unmodified lysines exhibit varying levels of activity in the presence of human serum.

[0015] Modifications of lysine proteins have been based on the following: i) insertion of amino acid substitutions into the lysine protein to change the overall pI of the molecule to facilitate penetration through the IM or reduce sensitivity to human serum or both; and/or ii) hybridizing an antimicrobial peptide sequence (preferably one known to be active in serum) to the N- or C-terminus of a lysine to form a hybrid polypeptide to facilitate penetration and translocation across the outer membrane.

[0016] Modified GN-lysines were prepared by modifying lysine proteins as described herein. Modified GN-lysines have been shown to exhibit improved activity in human serum compared to the activity of the original (unmodified) lysines. Charged amino acid residues of native lysine proteins were subjected to random mutagenesis to replace them with uncharged amino acid residues, and the activity of the resulting polypeptides was tested, including activity in the presence of human serum. Active modified polypeptides, as a rule, differed from the original polypeptides in 1-3 amino acid residues. Alternatively or additionally, antimicrobial peptide (AMP) sequences were hybridized to native or modified GN-lysine sequences with or without the use of a linker. Antimicrobial peptides are characterized by an alpha-helical domain that mediates outer membrane disruption and lysine translocation. Linkers are short peptide sequences, 5 to 20 amino acids in length, that are flexible (eg, rich in serine and/or glycine residues) and are designed to allow free movement of both the AMP and the lysine portion of the hybrid polypeptide without disrupting their structure.

[0017] Each of the putative lysines and modified GN-lysines described herein have been or can be purified to >90% homogeneity and tested in a variety of assays to assess in vitro activity.

[0018] The present invention includes lysine polypeptides and modified lysine polypeptides that are produced synthetically and/or recombinantly. The present invention includes new lysine polypeptides and modified lysine polypeptides, as well as the use of these polypeptides for the treatment of infections caused by gram-negative bacteria, and, in particular, in the presence of blood matrices, for example, human serum.

[0019] Moreover, the present invention includes the use of lysine polypeptides and modified lysine polypeptides for the destruction of biofilms containing gram-negative microorganisms, for example, in prostheses or other medical devices, in vivo , ex vivo or in vitro . Gram-negative biofilm microorganisms include Pseudomonas species, such as Pseudomonas aeruginosa .

[0020] In one aspect, the present invention provides a pharmaceutical composition or dosage formulation comprising an effective amount of an isolated lysine polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 5 - 9 and SEQ ID NO: 13 - 27, or a peptide that is at least 80% identical to the specified sequences, and the specified peptide has lytic activity, while the specified lysine polypeptide inhibits the growth of or reduces the population of, or destroys at least one species of gram-negative bacteria; and a pharmaceutically acceptable carrier.

[0021] In one embodiment, the pharmaceutical composition contains an effective amount of at least one lysine polypeptide selected from the group consisting of peptides GN3, CN147, GN146, GN156, GN54, GN92, GN121, GN94, GN9, GN10, GN13, GN17, GN105, GN108, GN123, GN150, GN200, GN201, GN203, GN204 and GN205, or a fragment thereof retaining lytic activity, wherein said lysine polypeptide or fragment inhibits the growth or reduces the population of at least one species of gram-negative bacteria; and a pharmaceutically acceptable carrier.

[0022] The pharmaceutical compositions/drug formulations of the present invention, in one embodiment, contain an effective amount of at least one lysine polypeptide and at least one antibiotic suitable for treating gram-negative bacteria. According to some embodiments of the present invention, the composition is a combination of two components, which must be administered in combination or separately, one of which contains a lysine in accordance with the present invention, and the other contains an antibiotic. In some embodiments of the present invention, the antibiotic is provided at a suboptimal dose. In some embodiments, the antibiotic may be an antibiotic to which gram-negative bacteria have developed resistance, and lysine is used to overcome this resistance.

[0023] In some embodiments, the compositions (with or without an antibiotic) or combinations (with lysine and an antibiotic) of the present invention are adapted for oral, topical, parenteral, or inhalation administration. According to some embodiments of the present invention, one component of the combination may be adapted to be administered by a different route than the other component. For example, in the experiments detailed below, antibiotics are administered subcutaneously (SC) while lysines are administered intravenously (IV).

[0024] In one embodiment of the present invention, the antibiotic may be selected from the list of antibiotics suitable for gram-negative bacteria presented below, and combinations thereof. In a more specific embodiment, the antibiotic may be selected from amikacin, azithromycin, aztreonam, ciprofloxacin, colistin, rifampicin, carbapenems and tobramycin and combinations of two or more of the above.

[0025] Certain embodiments of the present invention provide a sterile container that contains one of the above-mentioned pharmaceutical compositions containing a lysine polypeptide and optionally one or more additional components. By way of example, but not limitation, the sterile container is one component of the kit; the kit may also contain, for example, a second sterile container that contains at least one additional therapeutic agent. Thus, one of the combinations of GN-antibiotic and GN-lysine disclosed herein may not necessarily be provided in such a kit.

[0026] In one aspect, the present invention includes a vector comprising a nucleic acid molecule that encodes a lysine peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5 - 9 and SEQ ID NOs: 13 - 27 , or a peptide that is at least 80% identical to said sequences, wherein said peptide has lytic activity, wherein said encoded lysine polypeptide inhibits the growth of or reduces the population of or kills at least one species of gram-negative bacteria in the absence or presence of human serum .

[0027] According to another embodiment of the present invention, the vector is a recombinant expression vector containing a nucleic acid encoding one of the above-mentioned lysine polypeptides, including variants thereof that have at least 80% sequence identity, wherein said encoded lysine peptide has growth inhibitory property or reduce the population of at least one species of gram-negative bacteria in the absence and/or presence of human serum, wherein said nucleic acid is operably linked to a heterologous promoter.

[0028] A host cell containing the above vectors is also provided. In some embodiments of the present invention, the nucleic acid sequence is a cDNA sequence.

[0029] In another aspect, the present invention provides an isolated, purified nucleic acid encoding a lysine polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5 - 9 and SEQ ID NOs: 13 - 27. According to In an alternative embodiment of the present invention, the isolated purified nucleic acid contains a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO: 54, their degenerate code and their transcripts. In accordance with embodiments presented herein, a defined nucleic acid includes not only an identical nucleic acid, but also any minor base changes, including, but not limited to, substitutions resulting in a synonymous codon (a different codon specifying the same amino acid residue) . Thus, the claims relating to a nucleic acid are intended to include a sequence complementary to any specified single-stranded sequence. Optionally, the nucleic acid is cDNA.

[0030] In other aspects, the present invention relates to various methods/applications. One is a method/use for inhibiting the growth or population reduction or destruction of at least one species of gram-negative bacteria, the method comprising contacting the bacteria with a composition containing an effective amount of a GN-lysine polypeptide containing a sequence selected from the group, consisting of SEQ ID NOs: 2, 4, 5 - 9 and SEQ ID NOs: 13 - 27, or a peptide that is at least 80% identical to the specified sequences, and the specified peptide has lytic activity for a period of time sufficient to to inhibit said growth or reduce said population, or cause the destruction of said at least one species of gram-negative bacteria in the absence and/or presence of human serum.

[0031] Another such method/use is for inhibiting the growth or population reduction of, or killing, at least one species of gram-negative bacteria, the method comprising contacting the bacteria with a composition containing an effective amount of at least one GN- polypeptide lysine selected from the group consisting of GN-lysines described in SEQ ID NO: 2, 4, 5 - 9 and SEQ ID NO: 13 - 27, or active fragments thereof, wherein said polypeptide or active fragment has the property of inhibiting growth or reduce the population of, or result in the destruction of, P. aeruginosa and optionally at least one other species of gram-negative bacteria in the absence and/or presence of human serum.

[0032] Another method/medical use is for the treatment of a bacterial infection caused by a gram-negative bacterium, such as P. aeruginosa or A. baumannii , and involves administering one or more of the above compositions to a subject diagnosed with, who is at risk for, or who has symptoms of a bacterial infection.

[0033] In any of the above methods/medical uses, the gram-negative bacterium is at least one selected from the group consisting of Acinetobacter baumannii , Pseudomonas aeruginosa , E. coli , Klebsiella pneumoniae , Enterobacter cloacae , Salmonella spp., N. gonorrhoeae and Shigella spp. In another embodiment, the gram-negative bacterium is Pseudomonas aeruginosa .

[0034] Another method/medical use is for the treatment or prevention of local or systemic pathogenic bacterial infection caused by gram-negative bacteria and involves administering to a subject in need of treatment one of the above-mentioned compositions. Local infections include infections that can be treated by topical or topical application of an antibacterial agent. Examples of local infections include infections limited to a specific location, such as an organ or tissue, or an implanted prosthesis or other medical device. Examples include skin infections, gum infections, infected wounds, ear infections, etc., infections in the area where the catheter is inserted, etc.

[0035] Another such method/medicinal use is for the prevention or treatment of a bacterial infection and involves co-administrating to a subject diagnosed with, at risk of, or experiencing symptoms of a bacterial infection, a combination of a first effective amount of one of the above compositions and a second an effective amount of an antibiotic suitable for treating an infection caused by gram-negative bacteria.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0036] As used herein, the following terms and related words shall have the meanings given to them below unless the context clearly indicates otherwise.

[0037] "Carrier", as used in pharmaceutical compositions, refers to the diluent, excipient, additive or carrier with which the active compound is administered. Such pharmaceutical carriers may be sterile liquids such as water, physiological saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, as well as oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil , sesame oil and the like. Other examples include dispersion media, solubilizing agents, coatings, preservatives, isotonic and absorption retarding agents, surfactants, propellants and the like. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, ed. E.W. Martin, 18th ed.

[0038] A “pharmaceutically acceptable carrier” includes any of the above-mentioned carriers that are physiologically compatible. The carrier(s) must be "acceptable" in the sense of not being harmful to the subject being treated in quantities typically used in medicinal products. Pharmaceutically acceptable carriers are compatible with the other ingredients of the composition and do not render the composition unsuitable for its intended purpose. In addition, pharmaceutically acceptable carriers are suitable for use in subjects as provided herein without undue unwanted side effects (such as toxicity, irritation and allergic reaction). Side effects are “unreasonable” when their risk outweighs the benefit provided by the composition.

[0039] “Bactericidal,” when referring to an agent, generally means having the ability to kill bacteria or the ability to kill bacteria to an extent that corresponds to a reduction of at least 3-log10 (99.9%) or more of the original bacterial population over 18- 24 hour period.

[0040] "Bacteriostatic" generally means having the ability to inhibit bacterial growth, including inhibiting the growth of bacterial cells, thereby causing a reduction of 2-log (99%) or more to slightly less than 3-log of the original bacterial population within 18-24- hour period.

[0041] "Antibacterial", when referring to an agent, is generally used to include both bacteriostatic and bactericidal agents.

[0042] "Antibiotic" refers to an antibiotic compound, which may be either a compound that affects the biosynthesis of cell wall peptidoglycan, a compound that affects the integrity of the cell membrane, or a compound that affects DNA or protein synthesis in bacteria. Non-limiting examples of antibiotics active against gram-negative bacteria include cephalosporins such as ceftriaxone-cefotaxime, ceftazidime, cefepime, cefoperazone, ceftobiprole, fluoroquinolones such as ciprofloxacin, levofloxacin, aminoglycosides such as gentamicin, tobramycin, amikacin, piperacil lin, ticarcillin, carbapenems, such as imipenem, meropenem, doripenem, other beta-lactam antibiotics active against gram-negative bacteria such as broad-spectrum penicillins with or without beta-lactamase inhibitors, ansamycins such as rifampicin, and bactericidal polypeptides such as polymyxin B and colistin .

[0043] “Drug-resistant,” when referring to a pathogen and, more specifically, a bacterium, generally refers to a bacterium that is resistant to the antibacterial activity of a drug. In more specific usage, drug resistance specifically refers to resistance to antibiotics. In some cases, a bacterium that is normally susceptible to a particular antibiotic can develop resistance to the antibiotic, causing it to become a drug-resistant microorganism or strain. A “multidrug-resistant” (“MDR”) pathogen is a pathogen that has developed resistance to at least two classes of antimicrobial drugs, each used as monotherapy. For example, certain strains of P. aeruginosa have been found to be resistant to several antibiotics, including, but not limited to, ceftolozane-tazobactam, ceftazidime, cefepime, piperacillin-tazobactam, aztreonam, imipenem, meropenem, ciprofloxacin, ticarcillin, tobramycin, amikacin, and colistin. One skilled in the art can readily determine whether a bacterium is drug resistant using routine laboratory techniques that can determine the susceptibility or resistance of a bacterium to a drug or antibiotic. See, for example, Cabot, G. et al, 2016, Antimicrob. Agents and Chemother. 60(3):1767, DOI: 10.1128/AAC.02676-15; and (Antibiotic Resistant Threats in the United States, 2013, US Department of Health and Human Services, Centers for Disease Control and Prevention).

[0044] "Effective amount" refers to an amount that, when used or administered at an appropriate frequency or dosage schedule, is sufficient to prevent, reduce, inhibit or eliminate bacterial growth or bacterial load or prevent, reduce or improve the onset, severity, duration or progression of the disorder being treated (in this application, growth of or infection with a gram-negative bacterial pathogen), preventing progression of the disorder being treated, causing regression of the disorder being treated, or enhancing or improving the prophylactic(s) or therapeutic(s) effect(s). ov) other therapy, such as antibiotic or bacteriostatic therapy. A suitable effective amount range for the polypeptides of the present invention will be from about 0.01 mg/kg to about 50 mg/kg, with a typical range being from about 0.01 to 25 mg/kg and a typical range being from about 0. 01 to approximately 10 mg/kg. There are adjustments to increase the lower limit depending on the effectiveness of the particular lysine; adjustments to reduce the upper limit are also provided, depending primarily on the toxicity of the particular lysine. Such adjustments are within the skill of the art. In addition, if lysine is administered concomitantly with an antibiotic, the amount of lysine can be adjusted based on the amount needed to re-sensitize the target bacteria to the antibiotic administered simultaneously.

[0045] "Co-administration" is meant to include the separate administration of a lysine polypeptide and an antibiotic or any other antibacterial agent in a sequential manner, as well as the administration of these agents substantially simultaneously, for example, in a single mixture/composition or in doses administered separately, but however, administered to a subject substantially simultaneously, for example, at different times on the same day or over a 24-hour period. Such co-administration of lysine polypeptides with one or more additional antibacterial agents can be provided as a continuous treatment over a period of up to days, weeks or months. In addition, depending on the application, coadministration need not be continuous or uniform in duration. For example, if use as a topical antibacterial agent was used to treat, for example, a bacterial ulcer or an infected diabetic ulcer, lysine could only be administered initially within 24 hours of the first application of the antibiotic, and then the antibiotic could be continued without further administration of lysine.

[0046] "Subject" refers to the subject being treated and includes, but is not limited to, a mammal, plant, lower animal, single-celled organism, or cell culture. For example, the term “subject” is intended to include organisms, such as prokaryotes and eukaryotes, that are susceptible to or affected by bacterial infections, such as infections caused by gram-negative bacteria. Examples of subjects include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and non-human transgenic animals. In certain embodiments of the present invention, the subject is a human, for example, a human suffering from, at risk of, or susceptible to an infection caused by a gram-negative bacteria against which wild-type (parent) lysine is effective, whether such infection is systemic, local or otherwise localized or limited to a specific organ or tissue.

[0047] As used herein, "polypeptide" is used interchangeably with the terms "protein" and "peptide" and refers to a polymer derived from amino acid residues and typically having at least about 30 amino acid residues. The term includes not only polypeptides in isolated form, but also their active fragments and derivatives. The term "polypeptide" also includes fusion proteins or fusion polypeptides containing a lysine polypeptide described below and retaining the function of lysine. Depending on the context, the polypeptide or protein or peptide may be a naturally occurring polypeptide or a recombinant, engineered or synthetically produced polypeptide. A particular lysine polypeptide, for example, may be derived from or isolated from a native protein (i.e., a protein with an amino acid sequence identical to that isolated for that protein from natural sources) by enzymatic or chemical digestion or may be prepared using conventional techniques peptide synthesis (eg, solid phase synthesis) or molecular biology techniques (such as those disclosed in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), or can be rationally truncated or segmented into active fragments while maintaining lysine activity against the same or at least one common target bacterium.

[0048] "Hybrid polypeptide" refers to an expression product resulting from the hybridization of two or more nucleic acid segments to produce a hybrid expression product that typically has two domains or segments with different properties or functionality. More specifically, the term "hybrid polypeptide" also refers to a polypeptide or peptide containing two or more heterologous polypeptides or peptides covalently linked either directly or through an amino acid or peptide linker. Polypeptides forming a fusion polypeptide are typically C-terminal to N-terminal linked, although they may also be C-terminal to C-terminal, N-terminal to N-terminal, or N-terminal to C-terminal linked. the end. The term "fusion polypeptide" may be used interchangeably with the term "fusion protein". Thus, the non-limiting expression “polypeptide comprising” a specific structure includes molecules larger than the specified structure, such as hybrid polypeptides.

[0049] "Heterologous" refers to nucleotide, peptide or polypeptide sequences that are not contiguous in nature. For example, in connection with the present invention, the term “heterologous” can be used to describe a combination or hybrid of two or more peptides and/or polypeptides, wherein said hybrid peptide or polypeptide is not typically found in nature, for example, a lysine polypeptide or an active fragment thereof and a cationic and /or polycationic peptide, amphipathic peptide, sushi peptide (Ding et al. Cell Mol Life Sci., 65(7-8):1202-19 (2008)), defensin peptide (Ganz, T. Nature Reviews Immunology 3, 710 -720 (2003)), a hydrophobic peptide and/or antimicrobial peptide that may have increased lysine activity. Included in this definition are two or more lysine polypeptides or active fragments thereof. They can be used to obtain a hybrid polypeptide with lysine activity.

[0050] "Active fragment" refers to a portion of a full-length polypeptide disclosed herein that retains one or more functions or biological activities of the isolated polypeptide from which the fragment was derived, for example, bactericidal activity against one or more gram-negative bacteria, or, more specifically, lytic activity, regardless of whether it retains the ability to bind to the outer membrane.

[0051] "Amphipathic peptide" refers to a peptide having both hydrophilic and hydrophobic functional groups. Preferably, the secondary structure places hydrophobic and hydrophilic amino acid residues on opposite sides (eg, inner side versus outer side) of the amphipathic peptide. These peptides often adopt a helical secondary structure.

[0052] "Cationic peptide" refers to a peptide having a high percentage of positively charged amino acid residues. Preferably the cationic peptide has a pKa value of 8.0 or higher. The term "cationic peptide" as used herein also includes polycationic peptides, which are synthetically produced peptides consisting primarily of positively charged amino acid residues, in particular lysine and/or arginine residues. Amino acid residues that are not positively charged may be neutrally charged amino acid residues and/or negatively charged amino acid residues and/or hydrophobic amino acid residues.

[0053] “Hydrophobic group” refers to a chemical group, such as an amino acid side chain, that has low or no affinity for water molecules but a higher affinity for oil molecules. Hydrophobic substances tend to have low or no solubility in water or aqueous phases and are generally non-polar, but tend to have higher solubility in oil phases. Examples of hydrophobic amino acids include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met) and tryptophan (Trp).

[0054] "Increase", as used herein, means that the degree of antimicrobial activity is greater than it would otherwise be. “Boost” includes additive as well as synergistic (super additive) effects. For example, structural modifications of native lysines in accordance with the present invention serve to increase the activity of lysine in the presence of serum.

[0055] "Synergistic" or "superadditive" in effect means a beneficial effect due to two active agents in combination that is greater, preferably significantly, than the sum of the effects of the two agents acting independently. One or both active ingredients can be used at a subthreshold level, i.e. a level at which the active substance, if used alone, causes no or very little effect (or at least at a suboptimal level, i.e. , in which the active substance produces an effect that is significantly less than its maximum effect). In another embodiment, the effect can be measured using assays such as the checkerboard assay described herein.

[0056] “Treatment” refers to any method, act, application, therapy, or the like in which medical care is provided to a subject, including a human, for the purpose of curing a disorder or eliminating a pathogen, or improving the condition of the subject, directly or indirectly. Treatment also refers to reducing morbidity or alleviating symptoms, eliminating relapse, preventing relapse, preventing morbidity or reducing the risk of morbidity, improving symptoms, improving prognosis, or combinations thereof. “Treatment” also includes reducing the population, growth rate or virulence of bacteria in a subject and thereby controlling or reducing bacterial infection in the subject or bacterial contamination of an organ or tissue or the environment. Thus, a “treatment” that results in a reduction in disease is effective in inhibiting the growth of at least one Gram-positive bacterium in a particular environment, whether the subject or the environment. On the other hand, “treating” an established infection refers to reducing the population or destroying, inhibiting the growth, including even eliminating, the gram-positive bacteria causing the infection or contamination.

[0057] The term "prevention" includes preventing the incidence, recurrence, spread, onset or development of a disorder, such as a bacterial infection. It is not intended that the present invention be limited to completely preventing or preventing the development of infection. According to some embodiments of the present invention, the onset is delayed, or the severity of a subsequently acquired disease or the likelihood of acquiring a disease is reduced, and these constitute examples of prevention.

[0058] Acquired diseases, in relation to the present invention, include those that manifest clinical or subclinical symptoms, such as the detection of fever, sepsis or bacteremia (BSI), as well as the detection of growth of a bacterial pathogen (for example, in culture), when the symptoms associated with such pathology have not yet manifested themselves.

[0059] The term "derivative", when applied to a peptide or polypeptide (which, as defined herein, includes an active moiety) is intended to include, for example, a polypeptide modified to contain one or more chemical groups other than an amino acid, which do not have a significant undesirable effect on or interfere with the activity of lysine. The chemical group may be covalently linked to the peptide, for example, at the amino-terminal residue of an amino acid, the carboxy-terminal residue of an amino acid, or at an internal residue of an amino acid. Such modifications include the addition of a protecting or capping group on the reactive group, the addition of a detectable label such as an antibody and/or fluorescent label, the addition or modification of glycosylation or the addition of a bulky group such as PEG (PEGylation), and other changes that do not have a significant undesirable effect. influence on the activity of the lysine polypeptide or do not disrupt it. Commonly used protecting groups that can be added to lysine polypeptides include, but are not limited to, t-Boc and Fmoc. Commonly used fluorescent tag proteins, such as, but not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and mCherry, are compact proteins that can be linked covalently or non-covalently to a lysine polypeptide or hybridized to a lysine polypeptide without interfering with the normal functions of cellular proteins. Typically, a polynucleotide encoding a fluorescent protein is inserted in the 5' direction or 3' direction relative to the lysine polynucleotide sequence. This allows for the production of a fusion protein (eg, lysine polypeptide::GFP) that does not disrupt cellular function or the function of the lysine polypeptide to which it is attached. Conjugation of polyethylene glycol (PEG) to proteins has been used as a method to increase the circulating half-life of many pharmaceutical proteins. Thus, when referring to lysine polypeptide derivatives, the term “derivative” includes lysine polypeptides chemically modified by the covalent attachment of one or more PEG molecules. PEGylated lysine polypeptides are expected to exhibit an extended half-life in the bloodstream compared to non-PEGylated lysine polypeptides while maintaining biological and therapeutic activity. Another example is the use of “artilisins,” where short polycationic and amphipathic alpha helices are attached to the N- or C-termini of streptococcal lysines to improve antistreptococcal activity in vitro (Rodriguez-Rubio et al., 2016).

[0060] "Percent amino acid sequence identity" with respect to lysine polypeptide sequences is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular lysine polypeptide sequence after sequence alignment and the introduction of gaps, if necessary, to achieve the maximum percentage sequence identity and without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill in the art, for example, using publicly available software such as BLAST or commercially available software such as from DNASTAR. Two or more polypeptide sequences may be 0-100% identical at any location or by any integer value within the specified values. For purposes of the present invention, two polypeptides are “substantially identical” when at least 80% of the amino acid residues (preferably at least about 85%, at least about 90%, and preferably at least about 95%) are identical.

[0061] The term "percentage (%) amino acid sequence identity" described herein also applies to lysine peptides. Thus, the term “substantially identical” will include mutated, truncated, hybrid, or otherwise sequence-modified variants of the isolated lysine polypeptides and peptides described herein and active fragments thereof, as well as polypeptides with substantial sequence identity (e.g. at least 80%, at least 85%, at least 90%, or at least 95% identity, as measured, for example, by one or more of the methods listed above) compared to a reference (wild type or other intact ) polypeptide. Two amino acid sequences are "substantially homologous" when at least about 80% of the amino acid residues (preferably at least about 85%, at least about 90%, and preferably at least about 95% or 98%) are identical or represent are conservative substitutions. The lysine polypeptide sequences of the present invention are substantially homologous when one or more, or up to 10%, or up to 15%, or up to 20% of the amino acids of the lysine polypeptide are replaced by a similar or conservative amino acid substitution, and the resulting lysine profile types of activity, antibacterial effects and/or types of specificity against bacteria as in the lysine polypeptides disclosed in this application. The meaning of “essentially homologous” as described herein also applies to lysine peptides.

[0062] "Inhalable composition" refers to pharmaceutical compositions of the present invention that are formulated for direct delivery to the respiratory tract during or in conjunction with conventional or artificial respiration (e.g., via intratracheobronchial, pulmonary and/or nasal administration), including, but not limited to, fine, atomized, dry powder and/or aerosol formulations.

[0063] “Biofilm” refers to bacteria that adhere to surfaces and aggregate in a hydrated polymer matrix, which may be composed of bacterial and/or host-derived components. A biofilm is an aggregate of microorganisms in which cells adhere to each other on a biotic or abiotic surface. These adherent cells are often embedded in, but are not limited to, a matrix composed of extracellular polymeric substance (EPS). EPS biofilm, also called mucus (even though not everything described as mucus is biofilm) or plaque, is a polymeric conglomerate typically composed of extracellular DNA, proteins, and polysaccharides.

[0064] “Suitable,” when referring to an antibiotic suitable for use against certain bacteria, refers to an antibiotic that has been found to be effective against those bacteria, even if resistance subsequently develops.

[0065] Identification of lysines with bactericidal activity against P. aeruginosa in human serum. The present invention is based on the identification of five lysines with strong antibacterial activity against Pseudomonas aeruginosa strain PAOl in the exponential growth phase (Examples 1 and 2). This strain is typical of P. aeruginosa strains. To identify the lysine polypeptides of the present invention, the present inventors used a bioinformatics-based approach in combination with antibacterial screening. Putative lysines and lysine-like molecules (see Table 1) were identified in the GenBank database. GenBank sequences were annotated as hypothetical or predicted proteins, and in some cases were listed as putative phage proteins and/or putative lysins. The present inventors were not aware of any reports of activity of these polypeptides. Their activity also could not be predicted based on their sequence, much less their activity in the presence of human serum.

Table 1

Lysine pI GenBank accession number GN3 9.98 WP_012273008.1 GN13 9.47 YP_00638255.1 GN17 7.85 ACD38663.1 GN9 8.85 ECJ78460.1 GN10 9.70 YP_002600773.1 GN105 9.01 WP_016046696.1 GN108 9.28 YP_009288673.1 GN123 9.30 YP_009217242.1 GN150 9.30 WP_034684053.1 GN203 7.87 YP_024745.1

[0066] Identification of modified lysines with improved bactericidal activity against P. aeruginosa in human serum. Five lysines, GN3, GN150, GN203, GN4 and GN37, were used to prepare 12 new GN-lysine derivatives. See Table 2. It is contemplated that modifications (amino acid substitutions or hybridization of the N- or C-terminal peptide with or without the use of a linker) can be applied individually or simultaneously to the native lysine or modified lysine. Thus, for example, the addition of an N- and/or C-terminal peptide disclosed in Table 2 is provided to modify lysine polypeptides. As a more specific example, a peptide that is part of GN156 or GN92 is provided for GN147, even if such a construct was not exemplified in Table 2. And such a peptide could be added, for example, to either GN4 or GN146. In other words, the antimicrobial peptide can be hybridized to the N- or C-terminus of a native lysine or a lysine modified by replacing charged amino acid residues with uncharged amino acid residues. In addition, N-terminal and/or C-terminal peptides and/or antimicrobial peptides can be linked to a lysine polypeptide via a linker domain, for example, the linker domain defined in Table 2, or other suitable linker described in this section above.

Table 2.

Lysine pI Native lysine (accession number/class)* Modification GN147 9.39 GN3
(WP_012273008.1/lysozyme) Amino acid substitutions (R101D, R117H) GN146 8.01 GN4
(YP_002284361.1/lysozyme) Amino acid substitutions (K99D, R115H) GN156 10.51 GN4
(YP_002284361.1/lysozyme) Addition of N-terminal peptide
(GPRRPRRPGRRAPV; SEQ ID NO:28) GN92 9.93 GN4
(YP_002284361.1/lysozyme) Addition of N-terminal peptide
(KFFKFFKFFK; SEQ ID NO:29) with a linker (AGAGAGAGAGAGAGAGAS; SEQ ID NO:31) GN54 10.34 GN4
(YP_002284361.1/lysozyme) Addition of N-terminal peptide
(KRKKRKKRK; SEQ ID NO:30) with a linker (AGAGAGAGAGAGAGAGAS; SEQ ID NO:31) GN201 10.47 GN3
(WP_012273008.1/lysozyme) Addition of C-terminal peptide (GPRRPRRPGRRAPV; SEQ ID NO:28); amino acid substitutions (R101D, R117H) GN202 10.13 GN4
(YP_002284361.1/lysozyme) Addition of C-terminal peptide (GPRRPRRPGRRAPV; SEQ ID NO:28); amino acid substitutions (K99D, R115H) GN121 10.13 GN37
(WP_014102102.1/VanY) Addition of C-terminal peptide (RKKTRKRLKKIGKVLKWI; SEQ ID NO:32) GN94 9.77 GN37
(WP_014102102.1/VanY) Addition of N-terminal peptide
(KFFKFFKFFK; SEQ ID NO:29) with a linker (AGAGAGAGAGAGAGAGAS; SEQ ID NO:31) GN200 9.97 GN150
(WP_034684053.1/VanY) Addition of C-terminal peptide (RKKTRKRLKKIGKVLKWI; SEQ ID NO:32) GN204 9.88 GN203
(YP_024745.1/VanY) Addition of C-terminal peptide (RKKTRKRLKKIGKVLKWI; SEQ ID NO:32) GN205 11.02 GN3
(WP_012273008.1/lysozyme) Addition of N-terminal peptide
(GPRRPRRPGRRAPV; SEQ ID NO:28)

[0067] Lysines and modified GN-lysines, as well as their amino acid sequences according to the present invention are summarized in Table 3. Also included in Table 3 are the unmodified lysines disclosed in WO/2017/049233, as stated above.

Table 3

Lysine Amino acid sequence GN2 MKISLEGLLSLIKKFEGCKLEAYKCSAGVWTIGYGHTAGVKEGDVCTQEEAEKLLRGDIFKFEEYVQDSVKVDLDQSQFDALVAWTFNLGPGNLRSSTMLKKLNNGEYESVPFEMRRWNKAGGKTLDGLIRRRQAESLLFESKEWHQV (SEQ ID NO:1) GN3 mrtsqrglsliksfeglrlqayqdsvgvwtigygttrgvkagmkiskdqaermllndvqrfepeverlikvplnqdqwdalmsftynlgaanlesstlrrllnagnyaaaaeqfprwnkaggqvlagltrrraaerelflgaa (SEQ ID NO:2) GN4 (SEQ ID NO:3) GN146 (SEQ ID NO:4) GN147 mrtsqrglsliksfeglrlqayqdsvgvwtigygttrgvkagmkiskdqaermllndvqrfepeverlikvplnqdqwdalmsftynlgaanlesstlrDllnagnyaaaaeqfpHwnkaggqvlagltrrraaerelflgaa (SEQ ID NO: 5) GN156 GPRRPRRPGRRAPVMRTSQRGIDLIKSFEGLRLSAYQDSVGVWTIGYGTTRGVTRYMTITVEQAERMLSNDIQRFEPELDRLAKVPLNQNQWDALMSFVYNLGAANLASSTLLKLLNKGDYQGAADQFPRWVNAGGKRLDGLVKRRAAERALFLEPLS (SEQ ID NO:6) GN92 KFFKFFKFFKAGAGAGAGAGAGAGAGASMRTSQRGIDLIKSFEGLRLSAYQDSVGVWTIGYGTTRGVTRYMTITVEQAERMLSNDIQRFEPELDRLAKVPLNQNQWDALMSFVYNLGAANLASSTLLKLLNKGDYQGAADQFPRWVNAGGKRLDGLVKRRAAERALFLEPLS (SEQ ID NO:7) GN54 KRKKRKKRKAGAGAGAGAGAGAGASMRTSQRGIDLIKSFEGLRLSAYQDSVGVWTIGYGTTRGVTRYMTITVEQAERMLSNDIQRFEPELDRLAKVPLNQNQWDALMSFVYNLGAANLASSTLLKLLNKGDYQGAADQFPRWVNAGGKRLDGLVKRRAAERALFLEPLS (SEQ ID NO:8) GN202 (SEQ ID NO:9) GN14 MNNELPWVAEARKYIGLREDTSKTSHNPKLLAMLDRMGEFSNESRAWWHDDETPWCGLFVGYCLGVAGRYVVREWYRARAWEAPQLTKLDRPAYGALVTFTRSGGGHVGFIVGKDARGNLMVLGGNQSNAVSIAPFAVSRVTGYFWPSFWRNKTAVKSVPFEERYSLPLLKSNGELSTNEA (SEQ ID NO:10) GN43 MKRTTLNLELESNTDRLLQEKDDLLPQSVTNSSDEGTPFAQVEGASDDNTAEQDSDKPGASVADADTKPVDPEWKTITVASGDTLSTVFTKAGLSTSAMHDMLTSSKDAKRFTHLKVGQEVKLKLDPKGELQALRVKQSELETIGLDKTDKGYSFKREKAQIDLHTAYAHGRITSSLFVAGRNAGLPYNLVTSLSNIFG YDIDFALDLREGDEFDVIYEQHKVNGKQVATGNILAARFVNRGKTYTAVRYTNKQGNTSYYRADGSSMRKAFIRTPVDFARISSRFSLGRRHPILNKIRAHKGVDYAAPIGTPIKATGDGKILEAGRKGGYGNAVVIQHGQRYRTIYGHMSRFAKGIRAGTSVKQGQIIGYVGMTGLATGPHLHYEFQING RHVDPLSAKLPMADPLGGADRKRFMAQTQPMIARMDQEKKTLLALNKQR (SEQ ID NO:11) GN37 MTYTLSKRSLDNLKGVHPDLVAVVHRAIQLTPVDFAVIEGLRSVSRQKELVAAGASKTMNSRHLTGHAVDLAAYVNGIRWDWPLYDAIAVAVKAAAKELGVAIVWGGDWTTFKDGPHFELDRSKYR (SEQ ID NO:12) GN121 MTYTLSKRSLDNLKGVHPDLVAVVHRAIQLTPVDFAVIEGLRSVSRQKEL VAAGASKTMNSRHLTGHAVDLAAYVNGIRWDWPLYDAIAVAVKAAAKELGVAIVWGGDWTTFKDGPHFELDRSKYRRKKTRKRLKKIGKVLKWI (SEQ ID NO:13) GN94 KFFKFFKFFKAGAGAGAGAGAGAGAGASMTYTLSKRSLDNLKGVHPDL VAVVHRAIQLTPVDFAVIEGLRSVSRQKELVAAGASKTMNSRHLTGHAVDLAAYVNGIRWDWPLYDAIAVAVKAAAKELGVAIVWGGDWTTFKDGPHFELDRSKYR (SEQ ID NO:14) GN201 mrtsqrglsliksfeglrlqayqdsvgvwtigygttrgvkagmkiskdqaermllndvqrfepeverlikvplnqdqwdalmsftynlgaanlesstlrDllnagnyaaaaeqfpHwnkaggqvlagltrrraaerelflgaaGPRRPRRPGRRAPV (SEQ ID NO:15) GN205 GPRRPRRPGRRAPVmrtsqrglsliksfeglrlqayqdsvgvwtigygtrgvkagmkiskdqaermllndvqrfepeverlikvplnqdqwdalmsftynlgaanlesstlrrllnagnyaaaaeqfprwnkaggqvlagltrrraaerelflgaa (SEQ ID NO:16) GN200 msfklgkrslsnlegvhpdlikvvkraieltecdftvteglrskerqaql lkekktttsnsrhltghavdlaawvnntvswdwkyyyqiadamkkaaselnvsidwggdwkkfkdgphfeltwskypikgasRKKTRKRLKKIGKVLKWI (SEQ ID NO:17) GN204 mklsekralftqllaqlilwagtqdrvsvaldqvkrtqaeadanaksgagirnslhllglagdlilykdgkymdksedykflgdywkslhplcrwggdfksrpdgnhfslehegvqRKKTRKRLKKIGKVLKWI (SEQ ID NO:18) GN150 msfklgkrslsnlegvhpdlikvvkraieltecdftvteglrskerqaqllkekktttsnsrhltghavdlaawvnntvswdwkyyyqiadamkkaaselnvsidwggdwkkfkdgphfeltwskypikgas (SEQ ID NO:19) GN203 mklsekralftqllaqlilwagtqdrvsvaldqvkrtqaeadanaksgagirnslhllglagdlilykdgkymdksedykflgdywkslhplcrwggdfk srpdgnhfslehegvq (SEQ ID NO:20) GN9 mknfneiiehvlkheggyvndpkdlggetkygitkrfypdldiknltieqateiykkdywdknkveslpqnlwhiyfdmcvnmgkrtavkvlqraavnrgrdievdgglgpatigalkgveldrvrafrv kyyvdlitar peqekfylgw frratev (SEQ ID NO:21) GN10 mskqggvkvaqavaalsspglkidgivgkatraavssmpssqkaatdkilqsagigsldsllaepaaats dtfrevvlav arearkrgln pafyvahial etgwgrsvpklpdgrssynyaglkyaavktqvkgktetntleyikslpkt vrdsfavfasagdfsrvyfwylldspsayrypglknakta qefgdilqkggyatdpayaakvasiastavarygsdvssva (SEQ ID NO:22) GN13 msdkrveitgnvsgffesggrgvktvstgkgdnggvsygkhqlasnngsmalflespfgapyraqfaglkpgtaaftsvynkianetptaferdqfqyiaashydpqaaklkaeginvddrhvavrecvfsvavqygrntsiiikalgsnfrgsdkdfiekvqdyrgatvntyfksssqqt rdsvknrsqqekqmllkllns (SEQ ID No:23) GN17 mtlrygdrsqevrqlqrrlntwaganlyedghfgaatedavrafqrshglvadgiagpktlaalggadcshllqnadlvaaatrlglplatiyavnqvesngqgflgngkpailferhimyrrlaahdqvtadqlaaqfpalvnprpggyaggtaehqrlanarqiddtaalesaswgafqimgfhwqrlgy isvqafaeamgrsesaqfeafvrfidtdpalhkalkarkwadfarlyngpdykrnlydnklarayeqhancaeasa (SEQ ID NO:24) GN105 mavvsektaggrnvlafldmlawsegtstirgsdngynvvvggglfngyadhprlkvylprykvystaagryqllsrywdayreslalkggftpsnqdlvalqqikerrsladiqagrladavqkcsniwaslpgagygqrehslddltahylaaggvls (SEQ ID NO:25) GN108 MILTKDGFSIIRNELFEGKLDQTQVDainfiveKATYGLTYAYLATIYHETGLPSGYRTMQPYAGSYPYYPYGYGYGYGYGYGYGYGYGIGIGIGIGIGIGIGIGIGIGIGIDLVKNEPEKNEPEKALIAIIIIKIIKGMLNGWWF TGVGVGFRKRPVSKYNKQYVARNIINGKALIAIIIIIIFERALRSL (Seq ID NO: 26) GN123 mtllkkgdkgdavkqlqqklkdlgytlgvdgnfgngtdtvvrsfqtkmklsvdgvvgngtmstidstlagikawktsvpfpatnksramamptlteigrltnvdpkllatfcsiesafdytakpykpdgtvyssaegwfqfldatwddevrkhgkqysfpvdpgrslrkdpr anglmgaeflkgnaailrpvlghepsdtdlylahfmgaggakqflmadqnklaaelfpgpakanpnifyksgniartlaevyavldakvakhra (SEQ ID NO:27)

[0068] For GN3 and GN4 (each a member of the lysozyme-like superfamily), modified derivatives GN147 and GN146, respectively, were prepared based on the introduction of two amino acid substitutions at positions equivalent to those shown (31-33) to improve the antibacterial activity of lysozyme humans both in vitro (in buffer and/or media) and in vivo (in animal models of infection).

[0069] The GN3 lysine polypeptide has been modified to include amino acid substitutions, specifically the amino acid substitutions R101D and R117H. This resulted in a modified polypeptide, GN147. These amino acid changes resulted in a decrease in pI from 9.98 in the GN3 polypeptide to 9.39 in the GN147 polypeptide.

[0070] GN4 lysine has been modified to include amino acid substitutions, specifically K99D, R115H. As a result, a modified lysine polypeptide GN146 was obtained. These amino acid changes resulted in a decrease in pI from 9.58 in the GN4 polypeptide to 8.01 in the GN146 polypeptide.

[0071] The positions for each mutation in GN3 and GN4 were calculated based on rough comparison with mutations in human lysozyme (HuLYZ), since HuLYZ does not have significant homology to either GN3 or GN4 at the amino acid sequence level.

[0072] Although the GN3 and GN4 lysines are not similar to T4 lysozyme at the amino acid level, they are similar in size. A number of their structures revealed charged residues. Therefore, equivalence was judged solely by the presence of a charged residue in GN3 and GN4 at approximately the same location as in the primary sequence of lysozyme T4. Similarly, as described above, generally charged amino acids were replaced by those that had no charge, and the mutants were screened for activity.

[0073] Additional modifications to both GN3 and GN4 polypeptides have also been introduced, including the addition of an N-terminal peptide sequence (GPRRPRRPGRRAPV - SEQ ID NO:28) derived from the much larger antimicrobial peptide (AMP) described by Daniels and Schepartz, 2007 (34) , producing GN205 and GN156, respectively.

[0074] GN-lysine polypeptides can be further modified by adding pI modifying mutations. In one embodiment of the present invention, amino acid substitutions (R101D) and (R117H) were introduced into the GN3 lysine to produce the GN147 lysine. In another embodiment of the present invention, amino acid substitutions (K99D) and (R115H) were introduced into the GN4 lysine to produce the GN146 lysine.

[0075] The GN4 polypeptide was also modified by the addition of two different previously described N-terminal cationic AMPs, either KFFKFFKFFK (SEQ ID NO:29) or KRKKRKKRK (SEQ ID NO:30) (35, 36), coupled to GN4 due to the linker domain AGAGAGAGAGAGAGAGAS (SEQ ID NO:31), previously described by Briers et al. 2014 (36), producing modified lysines GN92 and GN54, respectively.

[0076] Modifications of the lysines GN37, GN150 and GN203 (each a member of the VanY superfamily) were made by adding a C-terminal AMP, RKKTRKRLKKIGKVLKWI (SEQ ID NO:32), previously developed as a derivative of porcine myeloid antimicrobial peptide 36 (PMAP-36) (22). Modification of GN37, GN150, and GN203 by addition of the C-terminal peptide sequence of RI18 resulted in the modified derivatives GN121, GN200, and GN204, respectively. An additional modification was also included whereby the AMP (KFFKFFKFFK - SEQ ID NO:29) (35) and linker domain (AGAGAGAGAGAGAGAGAS - SEQ ID NO:31) (36) described above were added to the N-terminus of GN37 to produce a modified lysine GN94.

[0077] It is believed that the peptides used to produce GN121, GN156, GN200, GN201, GN202, GN204 and GN205 have not previously been used to modify lysines. Their use was justified as follows: 1) when added to a given lysine, the predicted secondary structure of both AMP and lysine does not change significantly or at all (as determined using a well-known protein structure prediction program), 2) these peptides have previously been described in literature, as having strong activity; and 3) the present inventors tested these AMPs in serum and found strong activity. The same applies to the peptide used in GN92 and GN9. However, these constructs also used a linker sequence to connect AMP to lysine, producing the corresponding AMP secondary structure (very similar to that of free AMP) when the AMP is hybridized to lysine.

[0078] In the case of GN54, both AMP and linker have previously been used to modify lysines, but there have been no reports of activity in serum in the literature. GN54 is indeed active in serum.

[0079] Lysines GN3, GN9, GN10, GN13, GN17, GN105, GN108, GN123 and GN150 were synthesized and/or produced recombinantly, purified to (>90%) homogeneity and tested in a number of activity assays. MIC analysis was performed using Pseudomonas aeruginosa cultured in two types of media, CAA and CAA supplemented with 25% human serum (“CAA/HuS”). The activity of many GN-lysines (including the control lysozyme T4 in Table 4) is suppressed in both CAA and CAA/HuS.

For the set of 9 novel GN-lysines studied here (i.e., GN3, GN9, GN10, GN13, GN17, GN105, GN108, GN123, and GN150), we observed an MIC range of 2->128 in both CAA and and in CAA/HuS.

[0081] Each of the modified lysines GN54, GN92, GN94, GN121, GN146 and GN147 were purified to (>90%) homogeneity and tested in a series of in vitro activity assays. The MIC value (in µg/ml) for each of the GN-lysines in CAA/HuS is as follows: GN54, 2; GN92, 4; GN94, 2; GN121, 0.5; GN146, 2; GN147, 4, as shown in Table 4.

Table 4: Minimum inhibitory concentration (MIC) analysis of purified GN-lysines.

Lysine Lysine type MIC (µg/ml) in CAA MIC (µg/ml) in CAA/HuS GN3 Native 16 16 GN147 Modified GN3 2 4 GN4 Native 64 16 GN146 Modified GN4 2 2 GN156 Modified GN4 32 2 GN54 Modified GN4 64 2 GN92 Modified GN4 32 4 GN37 Native >128 32 GN121 Modified GN37 0.5 0.5 GN94 Modified GN37 16 2 GN9 Native 8 2 GN10 Native 8 16 GN13 Native 8 >128 GN17 Native 32 16 GN105 Native >128 32 GN108 Native 8 8 GN123 Native 2 128 GN150 Native 2 32 GN126 Native (control) 2 128 T4 LYZ Native (control) >128 >128

[0082] Importantly, the MIC values (in μg/ml) determined using CAA/HuS for each of the parent lysine molecules GN3, GN4 and GN37 are 16, 16 and 32, respectively; therefore, modification of each agent resulted in improved activity in human serum. Lysozyme T4 (MIC => 128 μg/ml) was included as a control standard for GN-lysines, which are inactive in human serum. GN126 (MIC = 128 μg/ml) was also included as a control and corresponds to Art-175 ( 37 ); Art-175 is an artilisin described in the literature, consisting of a hybrid of the AMP SMAP-29 with the GN-lysine KZ144.

[0083] In addition to MIC analysis, modified GN-lysines (GN54, GN92, GN94, GN121, GN146, and GN147) have been shown to have potent anti-biofilm activity, with minimum biofilm elimination concentration (MBEC) values ranging from 0.25 to 2 µg/ml, see table 5.

[0084] GN3, GN9, GN10, GN13, GN17, GN105, GN108 and GN123 have each been shown to have strong anti-biofilm activity with MBEC values ranging from 0.125 to 4 μg/ml (Table 5) and no hemolytic activity (Table 6). The remaining modified lysines are expected to exhibit improved anti-biofilm activity compared to the original lysines and will also have reduced or no hemolytic properties, as well as increased activity in the presence of blood matrices, including human serum.

Table 5: Minimum Biofilm Elimination Concentration (MBEC)

Lysine Lysine type MBEC (µg/ml) GN3 Native 0.25 GN147 Modified GN3 0.25 GN4 Native 1 GN146 Modified GN4 2 GN156 Modified GN4 0.5 GN54 Modified GN4 No data GN92 Modified GN4 0.5 GN37 Native 0.25 GN121 Modified GN37 0.25 GN94 Modified GN37 2 GN9 Native 0.125 GN10 Native 0.5 GN13 Native 0.125 GN17 Native 0.125 GN105 Native 4 GN108 Native 0.125 GN123 Native 4 GN150 Native 0.25

[0085] The modified GN-lysines (GN54, GN92, GN94, GN121, GN146 and GN147) have also been shown to have no hemolytic activity (MHA values >128 μg/ml), see Table 6.

Table 6: Minimum hemolytic concentration (MHC) analysis of purified GN-lysines

Lysine Lysine type MHA (µg/ml) GN3 Native >128 GN147 Modified GN3 >128 GN4 Native >128 GN146 Modified GN4 >128 GN156 Modified GN4 >128 GN54 Modified GN4 >128 GN92 Modified GN4 >128 GN37 Native >128 GN121 Modified GN37 >128 GN94 Modified GN37 >128 GN9 Native >128 GN10 Native >128 GN13 Native >128 GN17 Native >128 GN105 Native >128 GN108 Native >128 GN123 Native >128 GN150 Native >128

[0086] Modified GN-lysines (GN54, GN92, GN94, GN121, GN146, and GN147) have also been shown to have bactericidal activity in a residual microbiological load versus time assay format, as determined by a decrease in CFU ≥3-Log ₁₀ after 3 hours after adding lysine. See table 7 and table 8.

[0087] In a residual microbial load versus time assay format, GN3, GN17, GN108, GN123, and GN150 each demonstrated bactericidal activity at the 3-hour time point after addition at a concentration of 10 μg/mL in both CAA/HuS and HEPES -buffer (Tables 7 and 8, respectively).

Table 7. Analysis of activity of purified GN-lysine in CAA/HuS in relation to residual microbiological load as a function of time

Lysine Lysine type Log ₁₀ CFU/ml T= 0 T = 1 h T = 3 h* No Buffer control 7.8 7.7 7.2 GN3 Native 7.8 5.8 <3.7 GN147 Modified GN3 7.8 6.5 4.2 GN4 Native 7.8 6.0 <3.7 GN146 Modified GN4 7.8 5.9 4.0 GN156 Modified GN4 7.8 5.7 <3.7 GN54 Modified GN4 7.8 No data No data GN92 Modified GN4 7.8 6.2 <3.7 GN37 Native 7.8 6.2 <3.7 GN121 Modified GN37 7.8 7.4 <3.7 GN94 Modified GN37 7.8 6.4 <3.7 GN9 Native 7.8 6.8 7.3 GN10 Native 7.8 7.4 7.4 GN13 Native 7.8 No data No data GN17 Native 7.8 6.4 4.2 GN105 Native 7.8 7.0 6.3 GN108 Native 7.8 5.7 <3.7 GN123 Native 7.8 6.7 <3.7 GN150 Native 7.8 6.0 <3.7

*The detection limit is 3.7 Log ₁₀ CFU/ml. Indicates bactericidal activity.

Table 8: Analysis of activity of purified GN-lysine in HEPES buffer in relation to residual microbiological load as a function of time

Lysine Lysine type Log ₁₀ CFU/ml T= 0 T = 1 h T = 3 h* No Buffer control 7.8 7.7 7.2 GN3 Native 7.8 <3.7 <3.7 GN147 Modified GN3 7.8 <3.7 <3.7 GN4 Native 7.8 5.7 <3.7 GN146 Modified GN4 7.8 6.7 <3.7 GN156 Modified GN4 7.8 5.7 <3.7 GN54 Modified GN4 7.8 No data No data GN92 Modified GN4 7.8 5.7 <3.7 GN37 Native 7.8 6.3 <3.7 GN121 Modified GN37 7.8 <3.7 <3.7 GN94 Modified GN37 7.8 6.0 <3.7 GN9 Native 7.8 6.7 5.7 GN10 Native 7.8 5.7 <3.7 GN13 Native 7.8 No data No data GN17 Native 7.8 5.4 <3.7 GN105 Native 7.8 6.6 <3.7 GN108 Native 7.8 6.4 <3.7 GN123 Native 7.8 5.6 <3.7 GN150 Native 7.8 5.7 <3.7

*The detection limit is 3.7 Log ₁₀ CFU/ml. Indicates bactericidal activity.

[0088] A subset of GN-lysines (GN4, GN37, GN108 and GN150) have been tested in a checkerboard assay using CAA/HuS and have been shown to act synergistically with a number of antibiotics including amikacin, azithromycin, aztreonam, ciprofloxacin, colistin , rifampicin and tobramycin (Table 9).

[0089] It is important to note that each of the modified lysines GN92, GN121 and GN147 have been shown to act synergistically with a number of antibiotics with activity against gram-negative bacteria (amikacin, azithromycin, aztreonam, ciprofloxacin, colistin, rifampicin and tobramycin) in CAA/ HuS as shown in Table 9. These data indicate that the synergy will be maintained in vivo in the presence of human serum.

Table 9: Checkerboard analysis of purified GN-lysines with antibiotics

Amikacin Azithromycin Aztreons Ciprofloxacin Colistin Rifampicin Tobramycin GN4 0.531 0.094 0.156 0.250 0.156 0.156 0.375 GN92 0.375 0.063 0.188 0.281 0.094 0.094 0.500 GN147 0.375 0.250 0.188 0.281 0.188 0.281 0.5 GN37 0.125 0.188 0.531 0.281 0.156 0.281 0.156 GN121 0.375 0.188 0.625 0.313 0.375 0.313 0.188 GN108 0.156 0.060 0.250 0.281 0.133 0.125 0.188 GN150 0.313 0.125 0.188 0.250 0.094 0.094 0.500

[0090] Based on the activity of specific GN-lysines in the presence of human serum (and the nature of their amino acid sequence and homology with other lysines, as well as protein expression and purification profiles), the lysines GN3, GN9, GN10, GN13, GN17, GN105, GN108, GN123, GN150 and 203 are the best candidates for further development, either in the native lysine form or after further modification in the manner described in this application, i.e., using the replacement of typically 1 to 3 charged amino acid residues with uncharged ones residues (and retain activity in the absence and presence of human serum) and/or by hybridization at the N- or C-terminus with an AMP peptide having an alpha-helical structure.

[0091] Modified lysines corresponding to GN200-GN205 are still under analysis and may have activities similar to GN54, GN92, GN94, GN121, GN146 and GN147.

[0092] The present inventors have identified GN-lysines with varying levels of activity in the presence of human serum. In addition, modified GN-lysines have been prepared and demonstrated to exhibit improved activity in the presence of human serum compared to the activity of the parent lysines or the known lysozyme (T4) or the known artilisin (GN126).

[0093] Specific embodiments disclosed in this application may also be limited in the claims using the expression "consisting of" and/or "essentially consisting of." When used in a claim, whether as filed or added by amendment, the transitional term "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional term "essentially consisting of" limits the scope of the claims to the specified materials or steps and those that do not significantly affect the main and novel characteristic(s). Embodiments of the present invention so claimed are certainly or expressly described and included herein. The inventors of the present invention reserve the right to disclaim any embodiment or feature described in this application.

EXAMPLES

[0094] Example 1 Bacterial strains and growth conditions. Antibacterial screening was performed using a clinical isolate of P. aeruginosa (CFS-1292) from human blood obtained from the Hospital for Special Surgery in New York (provided by Dr. Lars Westblade, professor of pathology and laboratory medicine). Strain CFS-1292 was cultured in either lysogeny broth (LB; Sigma-Aldrich), casamino acid (CAA) media (5 g/L casamino acids, Ameresco/VWR; 5.2 mM K ₂ HPO ₄ , Sigma-Aldrich; 1 mM MgSO ₄ , Sigma-Aldrich) or CAA supplemented with 25% human serum (type AB, male, pooled; Sigma-Aldrich). For the purposes of the present invention, the specific isolate of P. aeruginosa is not important, and a commercially available isolate could have been used in these experiments.

[0095] Example 2: Gene synthesis and cloning. All lysines and modified lysines were synthesized as gBlocks (IDT Technologies) and cloned into the arabinose-inducible expression vector pBAD24 ( 24 ) by overlap extension PCR or by ligation of compatible overhangs. All constructs were transformed into E. coli strain TOP10 (Thermo Fisher Scientific). Other commercially available expression vectors and systems could be used.

[0096] Example 3: Identification of lysines with intrinsic activity. A set of up to 250 putative lysines and lysine-like enzymes was identified in the GenBank database of P. aeruginosa genomic sequences. Three search methods were used: i) a targeted BLASTp screen of all P. aeruginosa genomes using sequences of known lysines for queries, ii) a keyword search of all annotated P. aeruginosa genomes, focusing on all categories of superfamilies associated with catalytic and lysine binding domains (and cell wall hydrolase); and iii) visual search of unannotated genomes for lysine-like genes among phage sequences. Once identified, lysine sequences were synthesized as gBlocks, cloned into pBAD24, and transformed into E. coli TOP10 cells. E. coli clones were then tested in a primary screen for antibacterial activity (against live P. aeruginosa) using an agar-on-agar plate method (11, 13) modified to allow detection of GN-lysine activity in overlays consisting of soft agar suspended in 50 mM Tris buffer, pH=7.5. A set of 109 lytic clones were identified and selected for expression and purification.

[0097] Example 4: Expression and purification of lysines and modified lysines.

[0098] A wide variety of host/expression vector combinations can be used to express the polynucleotide sequences encoding the lysine polypeptides of the present invention. Those skilled in the art are aware of a large number of suitable vectors that are commercially available. Examples of suitable vectors are given in Sambrook et al, eds., Molecular Cloning: A Laboratory Manual (3rd ed.), Vols. 1-3, Cold Spring Harbor Laboratory (2001). Such vectors include, but are not limited to, vectors of chromosomal, episomal and viral origin, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papovaviruses such as SV40, vaccinia viruses, adenoviruses, fowlpox viruses, pseudorabies viruses and retroviruses, as well as vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. In addition, these vectors can provide constitutive or inducible expression of the lysine polypeptides of the present invention. More specifically, suitable vectors include, but are not limited to, derivatives of SV40 and known bacterial plasmids, for example, E. coli plasmids colEl, pCRl, pBR322, pMB9 and their derivatives, plasmids such as RP4, pBAD24 and pBAD-TOPO; Phage DNA, for example, multiple derivatives of phage λ, for example, NM989, and other phage DNA, for example, M13, and single-stranded filamentous phage DNA; yeast plasmids such as plasmid 2D or derivatives thereof; vectors that can be used in eukaryotic cells, such as vectors that can be used in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNA, such as plasmids that have been modified to employ phage DNA or other expression control sequences; etc. Many of the vectors mentioned above are commercially available from suppliers such as New England Biolabs, Addgene, Clontech, Life Technologies, etc., many of which also supply suitable host cells.

[0099] In addition, vectors may contain various regulatory elements (including a promoter, ribosome binding site, terminator, enhancer, various cis-elements to control the level of expression), and the vector is designed in accordance with the host cell. Any of a wide variety of expression control sequences (sequences that control the expression of a polynucleotide sequence operably linked to them) can be used in these vectors to express polynucleotide sequences encoding lysine polypeptides. Suitable control sequences include, but are not limited to: SV40, CMV, vaccinia, polyoma or adenovirus early or late promoters, lac system, trp system, TAC system, TRC system, LTR system, phage λ operator and promoter core regions, protein control regions fd shells, promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, acid phosphatase promoters (e.g., Pho5), yeast mating factor promoters, E. coli promoter for expression in bacteria, and other promoter sequences known to control the expression of prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof.

[00100] When expressing lysine polypeptides according to the present invention, a wide range of host cells can be used. Non-limiting examples of host cells suitable for expressing lysine polypeptides of the present invention include well-known eukaryotic and prokaryotic hosts such as E. coli , Pseudomonas , Bacillus , Streptomyces strains, fungi such as yeast, and animal cells such as CHO cells , Rl.l, BW and LM, African green monkey kidney cells (eg COS 1, COS 7, BSC1, BSC40 and BMT10), insect cells (eg Sf9), and human cells and plant cells in tissue culture. Although the expression host can be any known expression host cell, in a preferred embodiment, the expression host is one of the E. coli strains. These include, but are not limited to, commercially available E. coli strains such as Top 10 (Thermo Fisher Scientific), DH5oc (Thermo Fisher Scientific), XLl-Blue (Agilent Technologies), SCS110 (Stratagene), JM109 (Promega), LMG194 (ATCC) and BL21 (Thermo Fisher Scientific). There are several advantages to using E. coli as a host system, including: rapid growth kinetics, with a doubling time of approximately 20 minutes under optimal environmental conditions (Sezonov et al., J. Bacteriol. 189 8746-8749 (2007) ), easy production of high density cultures, easy and rapid transformation of exogenous DNA, etc. Details regarding protein expression in E. coli , including plasmid selection as well as strain selection, are discussed in detail by Rosano, G. and Ceccarelli, E. , Front Microbiol., 5: 172 (2014).

[00101] Efficient expression of lysine polypeptides and their vectors depends on multiple factors, such as optimal expression signals (both at the transcriptional and translational levels), proper protein folding, and cell growth characteristics. With regard to methods for constructing a vector and methods for transducing the constructed recombinant vector into a host cell, conventional methods known in the art can be used. It will be appreciated that while not all vectors, expression control sequences, and hosts will function equally well for the expression of polynucleotide sequences encoding the lysine peptides of the present invention, one skilled in the art will be able to select suitable vectors, expression control sequences, and hosts without unnecessary experiments to achieve target expression without departing from the scope of the present invention. In some embodiments, the present inventors have discovered a correlation between the level of expression and the activity of the expressed polypeptide; in particular, E. coli expression systems at moderate expression levels (e.g., approximately 1 to 10 mg/liter) produce lysine polypeptides with higher levels of activity than those expressed at higher levels in E. coli (e.g. from about 20 to about 100 mg/l), with the latter case sometimes producing completely inactive polypeptides.

[00102] Lysine polypeptides of the present invention can be isolated and purified from recombinant cell cultures using well known methods, including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion exchange or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography , affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography can also be used to purify the lysine polypeptide.

[00103] In another embodiment, the vector system used to produce the lysine polypeptides of the present invention may be a cell-free expression system. Various cell-free expression systems are available commercially, including, but not limited to, systems available from Promega, LifeTechnologies, Clonetech, etc.

[00104] Protein solubilization and purification (using one or more chromatographic techniques) is performed in a well-buffered solution having a suitable monovalent salt ionic strength, for example, an ionic strength equivalent to 300-500 mM NaCl.

[00105] Immobilized metal affinity chromatography (IMAC) is preferably used as an initial purification step. If further purification is required, size exclusion chromatography (gel filtration) can be used as a next step. If necessary, ion exchange chromatography can be used as a final step.

[00106] A range of induction times and temperatures were used to determine optimal conditions for protein expression and purification. The basic methodologies are described in previous studies (11, 13, 25). In general, replicates of each expressed clone were induced in both LB and RM media (Thermo Fisher Scientific) over a 2-24 hour period at 24°C-37°C. Induced cultures were then pelleted and disrupted using a BugBuster (Millipore Sigma) before assessing soluble protein expression by SDS-PAGE with Coomassie staining. The optimal condition for expression of each lysine was then used to increase production. Purifications were performed using anion exchange (HiTrap DEAE FF), cation exchange (HiTrap Capto MMC), hydrophobic interaction columns (HiTrap Phenyl FF) and/or gel filtration columns (HiLoad 16/600 SuperDex) using the Akta™ Pure system FPLC controlled by Unicorn 6.3 software. To improve solubility and increase the ability to bind to chromatographic resins, the addition of Mg ²⁺ was sometimes used. During purification, the target GN-lysine was identified by molecular weight using reducing SDS-PAGE. After the last purification step, fractions containing GN-lysine of interest were pooled, buffer exchanged with 25 mM Tris, 150 mM sodium chloride with pH ranging from 7.2 to 9.0 (depending on protein pI), and concentrated to ca. 2 mg/ml. The concentration was measured using NanoDrop and the protein was stored at -80°C in 500 μL aliquots.

[00107] Example 5: Determination of minimum inhibitory concentration (MIC). The minimum inhibitory concentration of each GN-lysine against P. aeruginosa was determined using a modification of the standard reference broth microdilution method defined by the Clinical and Laboratory Standards Institute (CLSI) ( 26 ). The modification was based on replacing Mueller-Hinton broth with either CAA or CAA media supplemented with 25% human serum.

[00108] Example 6: Determination of Minimum Biofilm Elimination Concentration (MBEC). MBEC CF-301 was determined using a variant of the broth microdilution method for MIC estimation with modifications ( 27 , 28 ). In this application, fresh colonies of P. aeruginosa strain ATCC 17647 were suspended in phosphate-buffered saline (PBS) (0.5 McFarland units), diluted in TSBg (tryptic soy broth with the addition of 0.2% glucose) in a ratio of 1:100, added aliquot 0.15 ml into the Calgary Biofilm Device (96-well plate with a lid carrying 96 polycarbonate pegs; Innovotech) and incubated for 24 hours at 37°C. Biofilms were washed and treated with 2-fold serial dilutions of CF-301 in TSBg at 37°C for 24 hours. All samples were tested in triplicate. After treatment, the wells were washed, air dried at 37°C and stained with 0.05% crystal violet for 10 minutes. After staining, biofilms were decolorized in 33% acetic acid and the OD ₆₀₀ of the extracted crystal violet was determined. The MBEC value of each sample represented the minimum drug concentration required to remove >95% of the biofilm biomass, as assessed by crystal violet quantification.

[00109] Example 7: Checkerboard Assay to Study Antibiotic Synergy. The checkerboard assay is based on a modification of the CLSI method for determining MICs using broth microdilution (26, 29). “Checkerboards” were constructed by first preparing the columns of a 96-well polypropylene microtiter plate in which each well along the horizontal axis contained the same amount of antibiotic diluted 2-fold. Comparable rows were prepared in a separate plate, in which each well along the vertical axis contained the same amount of GN-lysine, diluted 2-fold. Dilutions of GN-lysine and antibiotic were then combined such that each column contained a constant amount of antibiotic and double dilutions of GN-lysine, while each row contained a constant amount of GN-lysine and double dilutions of antibiotic. Thus, each well had a unique combination of GN-lysine and antibiotic. Bacteria were added to drug combinations at concentrations of 1×10 ⁵ CFU/ml in CAA with 25% human serum. The MICs of each drug, alone and in combination, were then recorded after 16 hours at 37°C in ambient air. Total fractional inhibitory concentrations (ΣFIC) were calculated for each drug, and the minimum ΣFIC value (ΣFICmin) was used to determine synergy. ΣFIC was calculated as follows: ΣFIC = FIC A + FIC B, where FIC A is the MIC of each antibiotic in combination/MIC of each antibiotic alone, and FIC B is the MIC of each GN-lysine in combination/MIC of each GN-lysine alone . The combination is considered synergistic if ΣFIC is ≤0.5, highly additive if ΣFIC is >0.5 to <1, additive with ΣFIC 1-<2, and antagonistic if ΣFIC is ≥2.

[00110] Example 8 GN-Lysine Hemolytic Activity Assay. The hemolytic activity of GN-lysines was measured as the amount of hemoglobin released by lysis of human erythrocytes (30). In general, 3 ml of fresh human blood cells (hRBCs) obtained from pooled samples of healthy donors (BioreclamationIVT) in a polycarbonate tube containing heparin were centrifuged at 1000 × g for 5 min at 4°C. The resulting erythrocytes were washed three times with phosphate-buffered saline (PBS) (pH=7.2) and resuspended in 30 PBS. 50 μl of erythrocyte solution was incubated with 50 μl of each GN-lysine (in PBS) at a 2-fold dilution range (128 μg/ml to 0.25 μg/ml) for 1 h at 37°C. Intact erythrocytes were pelleted by centrifugation at 1000 × g for 5 min at 4°C, and the supernatant was transferred to a new 96-well plate. Hemoglobin release was monitored by measuring absorbance at 570 nm. As a negative control, hRBC in PBS were treated as above using 0.1% Triton X-100.

[00111] Example 9: Analysis of GN-lysine activity in relation to residual microbiological load as a function of time. An overnight culture of P. aeruginosa was diluted 1:50 in fresh CAA media and grown for 2.5 h at 37°C with agitation. Exponential growth phase bacteria were then pelleted and resuspended in 1/5 culture volume of 25 mM HEPES, pH=7.4 before final adjustment to an optical density corresponding to a McFarland value of 0.5. The adjusted culture was then diluted 1:50 in either 25 mM HEPES, pH=7.4, or CAA/HuS, and GN-lysines were added at a final concentration of 10 μg/ml. Control cultures without added lysine (i.e., buffer control) were included. All treatments were incubated at 37°C with aeration. Culture samples were collected for quantitative plating on CAA agar plates at time points before addition of lysine (or buffer control) and at 1-h and 3-h intervals thereafter.

[00112] Planned in vivo experiments

[00113] One or more experiments to test the in vivo activity of the polypeptides of the present invention are currently being conducted as follows:

A. Preliminary screening for pharmacokinetics (PK) and efficacy in a mouse model of acute lethal bacteremia

[00114] To identify GN-lysines that have systemic effects, PK screening will be performed on CD1 mice treated with GN-lysine administered as a single IV injection. Blood PK profiles will be analyzed for up to 10 GN-lysines using a research grade bioanalytical assay certified for mouse serum. It is expected that several GN-lysines will be identified that have a corresponding PK profile. Candidates demonstrating blood effects that achieve an AUC/MIC greater than 1 will be tested in a systemic infection model. For this model, P. aeruginosa strains (PAO1 and other clinical isolates) will be resuspended in porcine gastric mucin and administered by intraperitoneal injection to CD1 mice in an inoculum that causes complete morbidity and mortality within 24-48 hours in control mice. Mice will be treated with vehicle or GN-lysine by intravenous (IV) injection into the lateral tail vein 2 hours after lethal challenge. Morbidity and mortality will be assessed within 72 hours. At appropriate dose concentrations, GN-lysine-treated mice are expected to experience reduced morbidity and mortality compared to vehicle-treated controls. Studies may include co-administration of antibiotics. A. Survival data will be analyzed by Kaplan-Meier survival analysis using GraphPad prism and the 50% effective concentration ( _EC50 ) will be calculated for each molecule. It is expected that several GN-lysines with in vivo activity will be identified.

B. Efficacy of GN-lysine in established mouse models of invasive infection

[00115] The purpose of this experiment and other mouse models of infection, such as lung and kidney, is to obtain data on efficacy in these models. Models to evaluate efficacy proposed for this additional purpose typically use bacterial infections of mice in tissues (hips, lungs, or kidneys). Following lysine treatment, tissue bacterial load will be quantified (CFU/gram tissue) at the end of the experiment to assess treatment persistence. In addition, each of these models can be used to determine PK/PD indices and magnitudes to evaluate efficacy, such as AUC/MIC. Therefore, these models will be used to evaluate the efficacy of typical GN-lysines against Pseudomonas in a mouse model of pulmonary infection, alone or in combination with an antibiotic, to identify the best GN-lysine candidates for further in vivo testing and development. Similar models can be created using Acinetobacter baumannii and used to test the effectiveness of lysines according to the present invention.

B.1 Mouse models of neutropenic thigh infection and lung infection

Thigh infection model

[00116] To establish the first model, CD-1 mice (n = 12) were induced to develop neutropenia by administering 20 mg/ml cyclophosphamide administered intraperitoneally according to a dosing regimen that resulted in a greater than 99% reduction in neutrophil counts (150 mg/mL). kg on day -4 and 100 mg/kg on day -1). A thigh infection is established by intramuscular (IM) injection into both lateral thigh muscles of an appropriate P. aeruginosa inoculum (3 x 10 ⁴ or 1 x 10 ⁵ or 3 x 10 ⁵ or 1 x 10 ⁶ CFU/thigh) after 24 hours. after administration of the second dose of the immunosuppressive agent. Mice under inhalational anesthesia are infected by intramuscular injection into both lateral thigh muscles. Each thigh can be inoculated with approximately 1.4 x 10 ⁵ CFU of A. baumannii NCTC 13301 (and/or an equivalent amount of P. aeruginosa strain). However, the amount of inoculum may be adjusted if testing shows that it is appropriate.

[00117] Groups of 4 mice (6, vehicle only (final group)) were administered test GN-lysine or vehicle or control lysine by intravenous (IV) injection at 2, 6 and 10 hours post-infection. At 2 h postinfection, a control group of 4 animals is humanely euthanized using an overdose of pentobarbitone to provide an untreated control group (start). 16 hours after infection, all other groups were humanely euthanized using pentobarbitone. Both femurs from each animal are removed and weighed separately (considered as two independent assessments). The lysines that will be tested will include native and modified lysines that have promising properties, i.e., significant lytic activity and retention of significant activity in the presence of blood matrices. The dosage range tested in this and other experiments detailed herein will be within the broad range of 0.01 to 500 mg/kg, but the upper limit may depend on toxicity and the lower limit may be higher depending on intrinsic activity. Examples of lysines to be tested include GN108, GN121, GN123 and GN156.

[00118] Individual thigh tissue samples are homogenized in ice-cold sterile phosphate-buffered saline. Thigh homogenates were then quantitatively cultured on CLED agar and incubated at 37°C for 24 hours before colony counting. Lysines are expected to significantly reduce or eliminate bacterial colonies.

Mouse model of lung infection

[00119] Groups of up to 8 anesthetized mice (IP injection of 100 mg/kg ketamine/6 mg/kg xylazine) per treatment are infected by intranasal instillation of 20 μl of inoculum into each nostril (5 min between injections into the other nostril) and kept upright for ~10 min after infection. The concentration and amount of the appropriate inoculum are pre-determined as described above.

[00120] The inoculum concentration is ~2.5 x 10 ⁶ CFU/mL (1.0 x 10 ⁵ CFU/lung) for P. aeruginosa ATCC 27853 or ~8.8 x 10 ⁸ CFU/mL (3.5 x 10 ⁷ CFU/lung) for A. baumannii NCTC 13301. Lysines (e.g., lysines identified in the previous experiment) are dosed using the same route of administration and basic dosing principles, and lungs are removed and prepared for counting as for the hip model . Colonies were counted after incubation at 37°C for 24 hours. Efficacy will be assessed based on the weight of the mice and the bacterial load of the lung homogenates. Lysines are expected to perform satisfactorily in reducing infection, as measured by significant reduction or elimination of bacterial colonies.

B.2 Mouse model of neutropenic lung infection

[00121] Neutropenic BALB/c mice will be inoculated with P. aeruginosa bacteria containing an inoculum sufficient to create a lung infection by intranasal instillation under anesthesia. Groups of 4 mice (6, vehicle only (final group)) were treated with GN-lysine, vehicle, or control lysine by subcutaneous (SC) injection at 2, 6, and 10 h postinfection. At 2 h postinfection, a control group of 4 animals is humanely euthanized using an overdose of pentabarbitone to provide an untreated control group (start). 16 hours after infection, all other groups were humanely euthanized using pentabarbitone. Animals are weighed and both lungs from each animal are removed and weighed separately. Lysines and dosage may be similar to those described above.

[00122] Individual lung tissue samples are homogenized in ice-cold sterile phosphate-buffered saline. Thigh homogenates were then quantitatively cultured on CLED agar and incubated at 37°C for 24 hours before colony counting. The effectiveness of treatment is assessed based on the mass and bacterial load.

[00123] Groups of up to 8 anesthetized mice (IP injection of 100 mg/kg ketamine/6 mg/kg xylazine) per treatment are infected by intranasal instillation of P. aeruginosa inoculum into each nostril (5 min between each nostril injection). ) and kept upright for ~10 minutes after infection. Mice were previously immunosuppressed with cyclophosphamide administered subcutaneously at 200 mg/kg on day -4 and 150 mg/kg on day -1. Infection occurs 24 hours after the second dose of the immunosuppressive agent is administered.

[00124] The initial inoculum concentration can be ~2.5 x 10 ⁶ CFU/ml (1.0 x 10 ⁵ CFU/lung) for P. aeruginosa ATCC 27853. The inoculum can be adjusted to increase the bacterial load in untreated mice by approximately 1 log 10 CFU/g lungs. For the survival studies described below, an inoculum will be selected that will result in death within 24-72 hours.

[00125] Lysines are then dosed intranasally, mice are euthanized, weighed, lungs are separated and weighed, and lungs are removed and prepared for counting as in the femoral model. Colonies were counted after incubation at 37°C for 24 hours. The same lysines and dosage can be used as described above. Lysines will be administered intravenously at 5 ml/kg.

[00126] In a related experiment, a suboptimal dose of an antibiotic having activity against gram-negative bacteria will be selected and used at a subthreshold level along with lysine. An appropriate subthreshold level can be established by treating infected mice with varying doses of the antibiotic at doses below, equal to, or above the minimum effective dose. Control mice will receive varying doses of vehicle alone. One vehicle will be used as a replacement for the lysine treatment, and the other vehicle will be used as a replacement for the antibiotic. For each lysine tested, 40 mice (5 per group) will be used.

[00127] If the antibiotic is imipenem, a suitable subthreshold dose will likely be 10 to 100 mg/kg (more generally, a subthreshold or suboptimal dose may be a dose that causes a 1 or 2 log reduction in the bacterial load) and will administered, for example, at 5 ml/kg subcutaneously or intravenously for this experiment and experiments with the combination (antibiotic + lysine).

[00128] It is intended that for a combination experiment, the dose of the antibiotic (eg, imipenem) will be the maximum subthreshold dose tested. The appropriate dose of lysine will be determined by testing different doses of lysine in combination treatments to see where a synergistic effect develops. The optimal set of amounts of lysine and antibiotic will be selected. Lysine and the first antibiotic treatment will be administered 2 hours after infection; a second antibiotic treatment will be administered 6 hours after infection. Tissue will be collected 9 hours after infection.

[00129] A similar study will be conducted using a similar mouse model of pulmonary infection, but only mouse survival will be assessed. Infected mice will be given lysine (or vehicle or control lysine) 24 hours after infection. The use of three different doses of each lysine is provided. Imipenem (or vehicle) will be administered 6 hours after lysine dosing. The experiment will end 72 hours after infection. It is envisaged that 7 mice per group will be used in the survival experiment, i.e. 63 mice for each lysine tested. The survival rate is expected to be higher with the combination.

C. PK/PD analysis in a mouse model of infection

[00130] Animal experiments with anti-infectives that measure PK/PD variables (e.g., Cmax/MIC, AUC/MIC, or % Time/MIC) most closely associated with efficacy are highly predictive of clinical success (31) . Dose fractionation is used to determine the PK/PD parameter associated with efficacy. By fractionating a single total dose into once daily (q24h), twice daily (q12h) or four times daily (q6h) dosing, multiple Cmax values and the time at which free drug concentration >MIC can be obtained (Time >MIC, fT>MIC), while maintaining a constant AUC. Multiple dose fractionation creates unique exposure profiles that, when compared to efficacy endpoints, differentiate Cmax/MIC, fT>MIC, and AUC/MIC as an index and PK value required for efficacy.

[00131] GN-lysines with consistent activity in one or more mouse models of infection previously identified will be subjected to PK/PD analysis. PK studies will be conducted to obtain multiple PK profiles and modeled to include Cmax/MIC, AUC/MIC, and fT>MIC ranges. Dose fractionation studies will be conducted in a pulmonary model to evaluate efficacy as described above (mouse, rat or rabbit). Tissue bacterial load will be used as a PD endpoint and data will be analyzed by plotting CFU/g tissue as a function of various PK/PD parameters. The significance of any PK/PD parameter in relation to efficacy will be determined using nonlinear regression analysis. These data will be used to provide dosing information for preclinical activities.

[00132] One method of conducting a PK study includes the following:

Table 10

Dose level (mg/kg) Route of administration Sample collection time point (h) Number of animals/time point Total number of mice 10 IV 0.083, 0.25, 0.5, 1, 2,
4, 8, 24 3 24 thirty IV 0.083, 0.25, 0.5, 1, 2,
4, 8, 24 3 24 100 IV 0.083, 0.25, 0.5, 1, 2,
4, 8, 24 3 24

[00133] At the time points indicated in Table 10, mice will be euthanized, and in groups of three mice per time point, blood samples will be collected by cardiac puncture. The separated plasma samples will be divided into two aliquots. After blood collection, three bronchoalveolar lavage fluid (BAS) samples will be collected from a narrow transverse hole made in the trachea. The three BAL samples will be pooled and centrifuged to remove any debris from the destroyed cells. The BAL supernatant will be divided into two aliquots. One plasma and BAL sample will be further tested to determine lysine content and bacterial load. The second sample will be analyzed for urea content to calculate epithelial lining fluid (ELF) dilution at the time of BAL collection.

D. Monitoring the development of resistance in vivo

[00134] To identify the potential for development of resistance, in vivo homogenates from mouse efficacy studies will be subjected to MIC analysis. If there is more than a twofold increase in the MIC, the bacteria will be plated and colonies will be isolated for whole genome sequencing.

IMPLEMENTATION OPTIONS

[00135] A. A pharmaceutical composition comprising: an isolated lysine polypeptide selected from the group consisting of one or more of GN147, GN146, GN156, GN92, GN54, GN201, GN202, GN121, GN94, GN200, GN204, GN205, or thereof a fragment having lysine activity, or a variant thereof, having lytic activity and having at least 80% sequence identity with said lysine polypeptide, and a pharmaceutically acceptable carrier, wherein said lysine polypeptide or fragment or variant is present in an amount effective to inhibit the growth or reducing the population of, or eliminating, P. aeruginosa and optionally at least one other species of gram-negative bacteria.

[00136] B. A pharmaceutical composition containing an effective amount of an isolated lysine polypeptide selected from the group consisting of one or more of GN3, GN13, GN17, GN9, GN10, GN105, GN108, GN123, GN150, GN203, or a fragment thereof having activity of lysine, or a variant thereof, having lytic activity and having at least 80% sequence identity with the specified lysine polypeptide, and a pharmaceutically acceptable carrier, and the specified lysine polypeptide is present in an amount effective to inhibit the growth of or reduce the population of or kill P. aeruginosa and optionally at least one other species of gram-negative bacteria; and a pharmaceutically acceptable carrier.

[00137] C. The pharmaceutical composition of Embodiment A or B, which is a solution, suspension, emulsion, inhalable powder, aerosol, or spray.

[00138] D. The pharmaceutical composition of Embodiment B further comprising one or more antibiotics suitable for treating gram-negative bacteria.

[00139] E. A vector containing an isolated polynucleotide containing a nucleic acid molecule that encodes a lysine polypeptide according to embodiment A or B, wherein the encoded lysine polypeptide inhibits the growth of or reduces the population of or kills P. aeruginosa and optionally at least one other a species of gram-negative bacteria, or the complementary sequence of the specified polynucleotide.

[00140] F. A recombinant expression vector comprising a nucleic acid encoding a lysine polypeptide comprising the amino acid sequence of the polypeptide of Embodiment A or B, wherein the encoded lysine polypeptide has the property of inhibiting the growth of or depopulating or killing P. aeruginosa and optionally at least one other species of gram-negative bacteria, wherein said nucleic acid is operably linked to a heterologous promoter.

[00141] G. A host cell containing the vector of embodiment E or F.

[00142] H. The recombinant vector of embodiment E or F, wherein said nucleic acid sequence is a cDNA sequence.

[00143] I. An isolated polynucleotide containing a nucleic acid molecule that encodes a lysine polypeptide selected from the group consisting of GN147, GN146, GN156, GN92, GN54, GN201, GN202, GN121, GN94, GN200, GN204, GN205, or thereof a fragment having lysine activity, or a variant thereof, having lytic activity and having at least 80% sequence identity with said lysine polypeptide, wherein said lysine polypeptide inhibits the growth of or reduces the population of or kills P. aeruginosa and optionally at least one other species gram-negative bacteria.

J. The polynucleotide of Embodiment I, which is a cDNA.

[00145] K. A method of inhibiting the growth or population reduction of, or killing at least one species of gram-negative bacteria, the method comprising contacting said bacteria with a pharmaceutical composition containing a lysine polypeptide selected from the group consisting of one or more GN147 , GN146, GN156, GN92, GN54, GN201, GN202, GN121, GN94, GN200, GN204, GN205, GN3, GN13, GN17, GN9, GN10, GN105, GN108, GN123, GN150, GN203, or a fragment thereof, lytic , or a variant thereof, having lytic activity and having at least 80% sequence identity with the specified lysine polypeptide, in an amount effective to inhibit the growth or population reduction or destruction of P. aeruginosa and optionally at least one other species of gram-negative bacteria.

[00146] L. A method of treating a bacterial infection caused by gram-negative bacteria selected from the group consisting of P. aeruginosa and optionally one or more additional species of gram-negative bacteria, comprising administering to a subject diagnosed with, at risk of, or experiencing symptoms of a bacterial infection, a composition containing a lysine polypeptide selected from the group consisting of one or more of GN147, GN146, GN156, GN92, GN54, GN201, GN202, GN121, GN94, GN200, GN204, GN 205, GN3, GN13, GN17, GN9 , GN10, GN105, GN108, GN123, GN150, GN203, or a fragment thereof having lysine activity, or a variant thereof having lytic activity and having at least 80% sequence identity with said lysine polypeptide, in an amount effective to inhibit the growth or reducing the population of, or eliminating, P. aeruginosa and optionally at least one other species of gram-negative bacteria.

[00147] M. The method of embodiment L, wherein at least one species of gram-negative bacteria is selected from the group consisting of Pseudomonas aeruginosa , Klebsiella spp., Enterobacter spp., Escherichia coli , Citrobacter freundii , Salmonella typhimurium , Yersinia pestis , and Franciscella tulerensis .

[00148] N. The method of embodiment L, wherein said gram-negative bacterial infection is an infection caused by Pseudomonas aeruginosa .

[00149] O. A method of treating a local or systemic pathogenic bacterial infection caused by gram-negative bacteria selected from the group consisting of P. aeruginosa and optionally one or more additional species of gram-negative bacteria in a subject, comprising administering to the subject a composition containing a lysine polypeptide selected from the group consisting of one or more of GN147, GN146, GN156, GN92, GN54, GN201, GN202, GN121, GN94, GN200, GN204, GN 205, GN3, GN13, GN17, GN9, GN10, GN105, GN108, GN123, GN150, GN203, or fragments thereof having lysine activity, or variants thereof, having lytic activity and having at least 80% sequence identity with the specified lysine polypeptide, in an amount effective to inhibit the growth or population reduction or destruction of P. aeruginosa and optionally at least one other gram-negative bacterium.

[00150] P. A method of preventing or treating a bacterial infection, comprising co-administering to a subject diagnosed with, at risk of, or experiencing symptoms of a bacterial infection, a combination of a first effective amount of said composition comprising an effective amount selected from the group consisting of one or more from GN147, GN146, GN156, GN92, GN54, GN201, GN202, GN121, GN94, GN200, GN204, GN 205, GN3, GN13, GN17, GN9, GN10, GN105, GN108, GN123, GN150, GN2 03, or its fragment having lytic activity, or a variant thereof having lytic activity and having at least 80% sequence identity with the specified lysine polypeptide, and a second effective amount of an antibiotic suitable for treating an infection caused by gram-negative bacteria.

[00151] Q. The method of embodiment P, wherein said antibiotic is selected from one or more of ceftazidime, cefepime, cefoperazone, ceftobiprole, ciprofloxacin, levofloxacin, aminoglycosides, imipenem, meropenem, doripenem, gentamicin, tobramycin, amikacin, piperacillin, ticarcillin , penicillin, rifampicin, polymyxin B and colistin.

[00152] R. A method of increasing the effectiveness of an antibiotic suitable for treating an infection caused by gram-negative bacteria, comprising co-administration of said antibiotic in combination with one or more lysine polypeptides selected from the group consisting of one or more of GN147, GN146, GN156, GN92 , GN54, GN201, GN202, GN121, GN94, GN200, GN204, GN205, GN3, GN13, GN17, GN9, GN10, GN105, GN108, GN123, GN150, GN203, or a fragment thereof having lytic activity, or a variant thereof having lytic activity and having at least 80% sequence identity with the specified lysine polypeptide, and the administration of the specified combination more effectively inhibits the growth or reduces the population of, or destroys the specified gram-negative bacteria than the administration of either the specified antibiotic or the specified lysine polypeptide or an active fragment thereof separately.

[00153] S. An isolated lysine polypeptide selected from the group consisting of GN147, GN146, GN156, GN92, GN54, GN201, GN202, GN121, GN94, GN200, GN204, GN205, or a fragment thereof having lysine activity, or a variant thereof having lytic activity and having at least 80% sequence identity with said lysine polypeptide, wherein said lysine polypeptide inhibits the growth of or reduces the population of or kills P. aeruginosa and optionally at least one other species of gram-negative bacteria.

[00154] T. A lysine polypeptide containing native lysine against gram-negative bacteria selected from the group consisting of GN3, GN9, GN10, GN13, GN17, GN105, GN108, GN123, GN150 and GN203, or a fragment thereof having lytic activity, or a variant thereof having lytic activity and having at least 80% sequence identity with said lysine polypeptide, wherein said native lysine or fragment is optionally modified by replacing 1-3 charged amino acid residues with uncharged amino acid residues, wherein said modified native lysine or fragment retains lytic activity.

[00155] U. A lysine polypeptide containing native lysine against gram-negative bacteria selected from the group consisting of GN2, GN4, GN14, GN43 and GN37, or a fragment thereof having lytic activity, or a variant thereof having lytic activity and having at least at least 80% sequence identity with the specified lysine polypeptide, and the specified native lysine or variant, or fragment is modified by replacing 1-3 charged amino acid residues with uncharged amino acid residues, while the specified modified native lysine or fragment retains lytic activity.

[00156] V. The pharmaceutical composition according to embodiment A or B, wherein said lysine polypeptide is selected from the group consisting of one or more of GN156, GN121, GN108 and GN123 or active fragments thereof, or variants thereof having lytic activity and having at least 80% sequence identity with the specified lysine polypeptide.

[00157] W. The method of embodiment K, wherein said bacteria are contained in a biofilm, wherein said method results in destruction of the biofilm.

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Claims (12)

1. A pharmaceutical composition containing: an isolated modified lysine polypeptide of the sequence SEQ ID NO: 3, selected from the group consisting of one or more of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, or a fragment thereof having lysine activity, or a variant thereof having lytic activity, wherein said fragments and variants have at least 80% sequence identity with said modified lysine polypeptide, wherein said modified lysine polypeptide comprises amino acids substitutions K100D and R116H relative to SEQ ID NO: 3 and/or contains the peptide sequence of SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30 at the N- or C-terminus of SEQ ID NO: 3, and a pharmaceutically acceptable carrier wherein said polypeptide or lysine fragment or variant is present in an amount effective to inhibit the growth or population reduction or destruction of gram-negative bacteria. 2. The pharmaceutical composition according to claim 1, which is a solution, suspension, emulsion, inhalable powder, aerosol or spray. 3. The pharmaceutical composition according to claim 1, further containing one or more antibiotics suitable for the treatment of gram-negative bacteria. 4. A method of treating a gram-negative bacterial infection caused by gram-negative bacteria, comprising administering to a subject diagnosed with, at risk for, or exhibiting symptoms of a bacterial infection, a composition comprising a modified lysine polypeptide of the sequence SEQ ID NO: 3 selected from the group consisting of one or more of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, or a fragment thereof having lysine activity, or a variant thereof having lytic activity, wherein said fragments and variants have at least 80% sequence identity with said modified lysine polypeptide, wherein said modified lysine contains amino acid substitutions K100D and R116H relative to SEQ ID NO: 3 and/or contains the peptide sequence SEQ ID NO: 28, SEQ ID NO : 29 or SEQ ID NO: 30 at the N- or C-terminus of SEQ ID NO: 3, in an amount effective to inhibit the growth or population reduction of, or kill, at least one species of gram-negative bacteria. 5. The method according to claim 4, characterized in that at least one type of gram-negative bacteria is selected from the group consisting of Pseudomonas aeruginosa , Klebsiella spp., Enterobacter spp., Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Yersinia pestis and Franciscella tulerensis . 6. The method according to claim 4, characterized in that said gram-negative bacterial infection is an infection caused by Pseudomonas aeruginosa . 7. The method according to claim 4, characterized in that the gram-negative bacterial infection is a local or systemic pathogenic bacterial infection. 8. The method of claim 4, further comprising co-administering to a subject diagnosed with, at risk of, or exhibiting symptoms of a bacterial infection, an effective amount of an antibiotic suitable for treating the infection caused by gram-negative bacteria. 9. The method according to claim 8, characterized in that said antibiotic is selected from one or more of ceftazidime, cefepime, cefoperazone, ceftobiprole, ciprofloxacin, levofloxacin, aminoglycosides, imipenem, meropenem, doripenem, gentamicin, tobramycin, amikacin, piperacillin, ti carcillina, penicillin, rifampicin, polymyxin B and colistin. 10. The method of claim 8, characterized in that the method increases the effectiveness of an antibiotic suitable for treating an infection caused by gram-negative bacteria, and wherein administration of said combination is more effective in inhibiting the growth of or reducing the population of or destroying gram-negative bacteria than administration of either said antibiotic , either the specified lysine polypeptide or its active fragment separately. 11. The method according to claim 8, characterized in that said bacteria are in a biofilm, and this method leads to the destruction of the biofilm. 12. An isolated modified lysine polypeptide of the sequence SEQ ID NO: 3 selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, or a fragment thereof having lysine activity, or a variant thereof having lytic activity, wherein said fragments and variants have at least 80% sequence identity with said modified lysine polypeptide, wherein said modified lysine polypeptide contains amino acid substitutions K100D and R116H relative to SEQ ID NO: 3 and/or contains a peptide sequence of SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30 at the N- or C-terminus of SEQ ID NO: 3, wherein said lysine polypeptide inhibits growth or reduces the population of or destroys gram-negative bacteria.