Compositions for treating intracellular bacterial infections

Abstract

The present disclosure features compositions and methods for the treatment of bacterial infections, such as those caused by bacterial cells residing within host cells (e.g., mammalian cells, e.g., immune cells, e.g., macrophages or dendritic cells). The compositions and methods involve delivering an antimicrobial agent to specifically target the intracellular compartment (endosome, phagosome, lysosome, or cytosol) where the bacterial cells reside.

Description

[Background technology]

[0001] Bacterial pathogens are a major cause of infectious diseases. Many bacteria are successfully detected by the human immune system and quickly eliminated before infection can begin. However, many pathogens evade the host immune system by living within host cells. These intracellular bacteria have developed diverse immune evasion techniques by living and replicating within host cells, such as immune cells (e.g., macrophages or dendritic cells), as well as within the correct intracellular compartments (e.g., endosomes, phagosomes, lysosomes, or cytosol) within the host cells. Bacterial infections that spread within host cells often present challenging treatment barriers due to a lack of accessibility to the intracellular site of infection. While certain antibacterial compositions can treat infections (e.g., in vitro), delivering treatment to the correct intracellular location where the bacteria reside has proven to be a challenging endeavor.

[0002] One group of challenging intracellular bacterial infections is caused by mycobacteria. Mycobacteria are actinomycetes characterized by a thick cell wall rich in mycolic acids. Mycobacteria contain an envelope consisting of a cell membrane composed of a lipid bilayer, a cell wall containing layers of peptidoglycan and arabinogalactan, and an outer membrane containing a hydrophobic mycolic acid layer. Many mycobacteria also contain an outer capsule composed of polysaccharides such as D-glucan, D-arabino-D-mannan, and D-mannan. This complex cell envelope contributes to the resistance of mycobacteria and makes them particularly difficult to penetrate and destroy. Pathogenic mycobacteria are often divided into two groups: M. tuberculosis and nontuberculous mycobacteria (NTM). In contrast to tuberculosis, person-to-person transmission of NTM is rare. Nevertheless, the number of NTM infections is an increasing health concern, especially among individuals with pulmonary disease.

[0003] There is a need for improved compositions and methods for targeting and treating intracellular bacterial infections, such as those caused by mycobacteria. Summary of the Invention

[0004] In one aspect, the invention features a method for delivering an antimicrobial lytic protein to a target intracellular compartment within a subject's professional antigen-presenting cells (e.g., macrophages or dendritic cells). The target intracellular compartment can contain bacterial cells (e.g., mycobacterial cells) therein. The method includes administering to the subject a composition comprising a supramolecular structure containing the antimicrobial lytic protein. After the administering step, the antimicrobial lytic protein is delivered to the target intracellular compartment. The supramolecular structure can further include a targeting moiety. Preferably, the antimicrobial lytic protein is an antimicrobial bacteriophage protein.

[0005] In another aspect, the invention features a method for delivering an antimicrobial lytic protein to a target intracellular compartment within a subject's professional antigen-presenting cells (e.g., macrophages or dendritic cells). The target intracellular compartment can contain a bacterial cell (e.g., a mycobacterial cell) therein. The method includes administering to the subject a composition including a supramolecular structure that includes a targeting moiety and a cargo that includes the antimicrobial lytic protein. After the administration step, the antimicrobial lytic protein is delivered to the target intracellular compartment.

[0006] In another aspect, the invention features a method for treating an intracellular bacterial infection in a subject caused by a bacterial cell. The method includes administering a composition comprising a supramolecular structure and a cargo comprising an antimicrobial lysis protein. The composition can be administered to the subject in an amount and for a duration sufficient to treat the bacterial infection. The supramolecular structure can further comprise a targeting moiety.

[0007] In another aspect, the invention features a method for treating an intracellular bacterial infection in a subject caused by a bacterial cell. The method includes administering a composition comprising a supramolecular structure comprising a targeting moiety and a cargo comprising an antimicrobial lysis protein. The composition can be administered to the subject in an amount and for a duration sufficient to treat the bacterial infection.

[0008] In another aspect, the invention features a composition including a supramolecular structure and a cargo including an antimicrobial lytic protein. The supramolecular structure can further include a targeting moiety. In another aspect, the invention features a composition including a supramolecular structure having a targeting moiety and a cargo comprising an antimicrobial lytic protein.

[0009] In some embodiments according to any of the above aspects, the supramolecular structure has a Z-average mean particle size of about 75 nm to about 750 nm (e.g., about 250 nm to about 750 nm, or about 75 nm to about 250 nm). Preferably, when the supramolecular structure is an LNP or a micelle, the Z-average mean particle size is about 75 nm to about 250 nm. Preferably, when the supramolecular structure is a vesicle (e.g., a liposome), the Z-average mean particle size is about 250 nm to about 750 nm. Non-limiting examples of Z average mean particle size include, for example, about 75 nm to about 100 nm, for example, 75 nm to about 85 nm, for example, about 80 nm, for example, about 80 nm to about 140 nm, about 90 nm to about 130 nm, or about 110 nm to about 130 nm, for example, about 120 nm, for example, about 200 nm to about 300 nm, for example, about 250 nm to about 300 nm, about 260 nm to about 290 nm, about 260 nm to about 280 nm, and about 265 nm to about 275 nm. m, for example, about 270 nm, for example, about 300 nm to about 400 nm, about 400 nm to about 600 nm, for example, about 450 nm to about 550 nm, about 475 nm to about 525 nm, about 480 nm to about 520 nm, about 490 nm to about 510 nm, about 495 nm to about 505 nm, for example, about 500 nm, for example, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm m, approx. 125nm, approx. 130nm, approx. 135nm, approx. 140nm, approx. 145nm, approx. 150nm, approx. 155nm, approx. 160nm, approx. m, approximately 195nm, approximately 200nm, approximately 205nm, approximately 210nm, approximately 215nm, approximately 220nm, approximately 225nm, approximately 230nm, approximately 235nm, approximately 240nm, approximately 245nm, approximately 250nm, approximately 255nm, approximately 260nm , about 265nm, about 270nm, about 275nm, about 280nm, about 285nm, about 290nm, about 295nm, about 300nm, about 305nm, about 310nm, about 315nm, about 320nm, about 325nm, about 330nm , about 335nm, about 340nm, about 345nm, about 350nm, about 355nm, about 360nm, about 365nm, about 370nm, about 375nm, about 380nm, about 385nm, about 390nm, about 395nm, about 400nm,Approx. 405nm, approx. 410nm, approx. 415nm, approx. 420nm, approx. 425nm, approx. 430nm, approx. 435nm, approx. 440nm, approx. 445nm , about 450nm, about 455nm, about 460nm, about 465nm, about 470nm, about 475nm, about 480nm, about 485nm, about 490n m, approx. 495nm, approx. 500nm, approx. 505nm, approx. 510nm, approx. 515nm, approx. 520nm, approx. 525nm, approx. 530nm, approx. 535 nm, approximately 540nm, approximately 545nm, approximately 550nm, approximately 555nm, approximately 560nm, approximately 565nm, approximately 570nm, approximately 575nm, approximately 580 nm, about 585 nm, about 590 nm, about 595 nm, about 600 nm, about 605 nm, about 610 nm, about 615 nm, about 620 nm, about 625 nm, about 630 nm, about 635 nm, about 640 nm, about 645 nm, about 650 nm, about 655 nm, about 660 nm, about 665 nm, about 670 nm, about 675 nm, about 680 nm, about 685 nm, about 690 nm, about 695 nm, about 700 nm, about 705 nm, about 710 nm, about 715 nm, about 720 nm, about 725 nm, about 730 nm, about 735 nm, about 740 nm, about 745 nm, or about 750 nm. In some embodiments, the Z average mean particle size of the supramolecular structure is about 80 nm, about 270 nm, or about 500 nm.

[0010] In some embodiments of any of the above aspects, the bacterial cell is or the antimicrobial lytic protein is capable of killing a Mycobacterium, Salmonella, Neisseria, Brucella, Escherichia, Listeria, Frankincella, Legionella, Yersinia, Staphylococcus, Clostridium, Shigella, or Streptococcus species.

[0011] In some embodiments, the Mycobacterium species is M. tuberculosis, M. leprae, M. lepromatosis, M. avium, M. kansasii, M. fortuitum, M. chelonae, M. marinum, or M. abscessus; the Salmonella species is S. enterica, S. typhimurium, or S. bongori; the Neisseria species is N. gonorrhoeae or N. meningitidis; the Brucella species is B. melitensis, B. abortus, B. suis, or B. canis; the Escherichia species is E. coli; the Listeria species is L. monocytogenes; and the Frankicella species is F. tularensis, F. nov. the Legionella species is L. pneumophila; the Yersinia species is Y. pestis or Y. enterocolitica; the Staphylococcus species is S. aureus; the Clostridium species is C. botulinum, C. perfringens, C. tetani, or C. sordellii; the Shigella species is S. dysenteriae, S. flexneri, S. boydii, or S. sonnei; or the Streptococcus species is S. pyogenes, S. agalactiae, S. dysgalactiae, S. bovis, S. anginosus, S. sanguinis, S. mitis, S. mutans, or S. pneumoniae.

[0012] In some embodiments, the supramolecular structure can have a polydispersity index (PDI) of about 0.05 to about 0.3. In some embodiments, the supramolecular structure can further comprise one or more lipids, such as ionizable lipids. In some embodiments, the supramolecular structure can further comprise at least one targeting moiety.

[0013] In some embodiments, the targeting moiety is an extracellular targeting moiety that targets professional antigen-presenting cells (eg, macrophages or dendritic cells). In some embodiments, the targeting moiety comprises phosphatidylserine.

[0014] In some embodiments, the targeting moiety comprises an antibody or antigen-binding fragment thereof. Thus, the antibody or antigen-binding fragment can be selected from the group consisting of anti-CD163, anti-CD40, anti-CD74, anti-CD206, and anti-CD123 antibodies, and antigen-binding fragments thereof. The antibody or antigen-binding fragment can be selected from the group consisting of anti-DEC205, anti-CD304, anti-CD303, anti-CD40, anti-CD74, anti-BDCA2, and anti-CD123 antibodies, and antigen-binding fragments thereof.

[0015] In some embodiments, the targeting moiety comprises a pathogen-associated molecular pattern (PAMP). In some embodiments, the targeting moiety is a mannose cluster or a folate. In some embodiments, the targeting moiety is a TLR2 agonist. For example, the TLR2 agonist can be selected from the group consisting of MALP-2 lipoprotein, MALP-404 lipoprotein, outer surface lipoprotein A (OspA), porin, LcrV, Hsp60, glycoprotein gH / gL, or glycoprotein gB.

[0016] In some embodiments, the supramolecular structure is a lipid nanoparticle. In some embodiments, the supramolecular structure is a micelle. In some embodiments, the supramolecular structure is a liposome. The liposome can be unilamellar or multilamellar (e.g., 2, 3, 4, 5, or more lamellae). The supramolecular structure can have a polydispersity index of about 0.05 to about 0.3.

[0017] The supramolecular structure can further comprise one or more lipids (eg, ionizable lipids). In some embodiments, the method further comprises administering an antibiotic. In some embodiments, the antibiotic is selected from the group consisting of cephalosporins, carbapenems, penicillins, and fluoroquinolones. In some embodiments, the antibiotic is selected from the group consisting of thiacetazone, sq-109, bedaquiline, delamanid, pyrazinamide, and isoniazid. For example, the antibiotic can be azithromycin, clarithromycin, ethambutol, rifampin, or amikacin, e.g., amikacin.

[0018] In some embodiments, the antimicrobial lytic protein is capable of killing bacterial cells (eg, mycobacterial cells, eg, NTM cells). In some embodiments, the antimicrobial lytic protein is a capsule depolymerase, amylase, or lysin. For example, the capsule depolymerase can be, for example, a hydrolase, metallohydrolase, epoxide hydrolase, peptidoglycan hydrolase, polysaccharase, polysaccharide lyase, endosialidase, hyaluronan lyase, or alginate lyase. The lysin can be, for example, lysin A or lysin B. The amylase can be, for example, an isoamylase or α-amylase. The antimicrobial lytic protein can be an antimicrobial mycobacteriophage protein.

[0019] In some embodiments, the compositions are administered intravenously, orally, topically, or by inhalation. definition As used herein, the term "about" means ±10% of the recited value.

[0020] As used herein, "combination therapy" or "administered in combination" means that two (or more) agents or treatments are administered to a subject as part of a defined therapeutic regimen for a particular disease or condition. The therapeutic regimen defines the dosage and periodicity of administration of each agent so that the effects of the separate agents on the subject overlap and / or synergize. In some embodiments, delivery of two or more agents is simultaneous or concurrent, and the agents may be co-formulated. In some embodiments, two or more agents are not co-formulated but are administered sequentially as part of a prescribed regimen. In some embodiments, administration of two or more agents or treatments in combination is such that the reduction in symptoms or other parameters associated with the disease is greater than that observed with one agent or treatment delivered alone or in the absence of another. The effect of the two treatments may be partially additive, fully additive, or greater than additive, e.g., synergistic. The sequential or substantially simultaneous administration of each therapeutic agent can be by any suitable route, including, but not limited to, oral, intravenous, intramuscular, topical, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes, for example, a first therapeutic agent of the combination may be administered by intravenous injection, while a second therapeutic agent of the combination may be administered orally.

[0021] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of an agent to produce a therapeutic effect described herein, e.g., in a cell, sample, or subject, refer to an amount sufficient to produce a beneficial or desired result, including preclinical or clinical results, when administered to a cell, sample, or subject, including a human; therefore, such an "effective amount," or its equivalents, depends on the context in which the term is used. For example, in the context of treating a disorder, the term refers to an amount of agent sufficient to produce a therapeutic response compared to the response obtained without administration of the agent. The amount of a given agent will vary depending on various factors, such as the given agent, pharmaceutical formulation, route of administration, severity of the bacterial infection, biomarkers, e.g., age, sex, and / or weight of the subject, sample, or host cell, e.g., mammalian immune cell, being treated, etc., but can nevertheless be routinely determined by one of skill in the art. Also, as used herein, the term "therapeutically effective amount" of an agent refers to an amount that produces a beneficial or desired result in a cell or subject compared to a control. As defined herein, a therapeutically effective amount of an agent can be readily determined by one of ordinary skill in the art using routine methods known in the art. Dosage regimens may be adjusted to provide the optimum therapeutic response.

[0022] As used herein, the term "antimicrobial lytic protein" refers to a protein associated with or secreted from a bacteriophage that has bactericidal and / or lytic activity against bacteria. Non-limiting examples of antimicrobial lytic proteins include holins, lysins (e.g., lysin A and / or lysin B), amylases (e.g., isoamylase or α-amylase), and capsule depolymerases (e.g., hydrolases, metallohydrolases, epoxide hydrolases, peptidoglycan hydrolases, polysaccharases, polysaccharide lyases, endosialidases, hyaluronan lyases, or alginate lyases).

[0023] As used herein, the term "endosomal escape moiety" refers to a moiety that enhances the release of endosomal contents or promotes the escape of a molecule from an internal cellular compartment (e.g., an endosome, a phagosome, or a lysosome) relative to a reference molecule that differs only in that it lacks an endosomal escape moiety.

[0024] As used herein, "lipid nanoparticles" or "LNPs" are vesicles comprising a lipid layer encapsulating a substantially solid lipid core, which may contain a pharmaceutically active molecule. LNPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent aggregation of the particles (e.g., PEG-lipid conjugates).

[0025] As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer, or multiple bilayers. Liposomes include unilamellar and multilamellar (e.g., 2, 3, 4, 5, or more lamellae) with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains an antimicrobial lytic protein or a mixture of an antimicrobial lytic protein and other components. The lipophilic material separates the aqueous exterior from the aqueous interior and typically does not contain phage proteins, although in some cases it may. Liposomes also include "sterically stabilized" liposomes, which, as used herein, refers to liposomes containing one or more specialized lipids that, when incorporated into the liposome, result in improved circulation life compared to liposomes lacking such specialized lipids.

[0026] "Micelle" is defined herein as a specific type of substantially spherical supramolecular structure in which amphiphilic molecules, e.g., lipids, are arranged so that the hydrophobic portions of the molecules face inward toward the core, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The reverse arrangement exists when the surrounding environment is hydrophobic. The micelle core can contain an antimicrobial lytic protein or a mixture of proteins.

[0027] As used herein, the term "targeting moiety" refers to a moiety (e.g., a small molecule, e.g., a carbohydrate) that specifically binds to or reactively associates with or forms a complex with a receptor or other receptive moiety associated with a given target cell population (e.g., professional antigen-presenting cells (e.g., macrophages or dendritic cells)). Thus, a targeting moiety can be used to target the supramolecular structures described herein to, for example, professional antigen-presenting cells (e.g., macrophages or dendritic cells).

[0028] As used herein, the term "subject" refers to any organism to which a composition according to the present invention can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal, e.g., mammals such as mice, rats, rabbits, non-human primates, and humans. A subject can be a human or animal seeking or in need of treatment, in need of treatment, undergoing treatment, about to undergo treatment, or being treated by a trained professional for a particular disease or condition.

[0029] As used herein, the term "supramolecular structure" refers to a complex of molecules that are bound to each other by non-covalent bonds, such as hydrogen bonds, van der Waals forces, electrostatic interactions, hydrophobic effects, and π-π interactions. Supramolecular structures can include, for example, large complexes of molecules that form sphere-like structures. Examples of supramolecular structures include lipid-based supramolecular structures, such as liposomes and lipid nanoparticles (e.g., micelles).

[0030] As used herein, the term "target intracellular compartment" means an endosome, phagosome, lysosome, or cytosol. As used herein, the term "targeting moiety" refers to a moiety (e.g., a small molecule, e.g., a carbohydrate) that specifically binds to or reactively associates with or forms a complex with a receptor or other receptive moiety associated with a given target cell population (e.g., professional antigen-presenting cells (e.g., macrophages or dendritic cells)). Thus, a targeting moiety can be used to target the supramolecular structures described herein to, for example, professional antigen-presenting cells (e.g., macrophages or dendritic cells).

[0031] A "vesicle" is defined herein as a type of supramolecular structure in which amphiphilic molecules (e.g., lipids) assemble to define a volume, e.g., a substantially spherical volume. The amphiphilic molecules (e.g., lipids) typically constitute at least one shell of the vesicle. Within this shell, the amphiphilic molecules are arranged within a bilayer membrane, with the hydrophilic portions of the amphiphilic molecules facing outward relative to the plane of the bilayer membrane, and the hydrophobic portions of the amphiphilic molecules being primarily arranged within the bilayer membrane. The reverse arrangement exists when the surrounding medium is hydrophobic. [Brief explanation of the drawings]

[0032] [Figure 1] Schematic diagram of the experimental design to screen and quantify the level of enzybiotic response in grow-out serial dilution plates (GOSD) of Mycobacterium cells, or macrophages infected with Mycobacterium and treated with antimicrobial lytic protein, or cells containing bacteriophage containing a therapeutic payload, respectively. For example, the inset shows a GOSD plate of ECL55 cells infected with Mycobacterium and treated with antimicrobial lytic protein (ABIα) or dilution buffer (DB). [Figure 2]A is a series of graphs showing the dose-dependent response of GOSD-1, -2, -3, -4, -5, -6, -7, and -8 cells to treatment with one (10 μL), two (20 μL), or three (30 μL) doses of an enzyme cocktail of lysin A (A), lysin B (B), isoamylase (I), and α-amylase (α) (ABIα). Quantification of the dose-dependent effect of ABIα treatment on culture optical density (OD590) / lower limit of detection. B is a series of graphs showing the dose-dependent response of M. abscessus cells GOSD-1, -2, -3, -4, -5, -6, -7, and -8 to treatment with one (10 μL), two (20 μL), or three (30 μL) doses of an enzyme cocktail of lysin A (A), lysin B (B), isoamylase (I), and α-amylase (α) (ABIα). Quantification of the effect of treatment on the number of M. abscessus cells. [Figure 3] Graph showing the number of M. abscessus cells after treatment with 3.2 μg, 1.6 μg, 0.8 μg, 0.4 μg, 0.2 μg, and 0.1 μg of ABIα / well, respectively, or no ABIα. [Figure 4] A is a graph showing the OD590 / LLoD of GOSD (-1, -2, -3, -4, -5, -6, -7, and -8) of M. intracellulare cells treated with or without a single dose of ABIα and cultured for 3 or 6 days. B is an image corresponding to the graph showing the OD590 / LLoD of GOSD (-1, -2, -3, -4, -5, -6, -7, and -8) of M. intracellulare cells treated with or without a single dose of ABIα and cultured for 3 or 6 days. [Figure 5] 1 is a tabular collection of data showing the fractional inhibitory concentration index (FICI) of the growth of M. abscessus cells treated with ABIα and the antibiotics amikacin, biapenem, cefoxitin, ethambutol, moxifloxacin, rifampicin, or clarithromycin, respectively. [Figure 6]FIG. 1 is a schematic diagram of the experimental design to evaluate the differential effects of treatment with non-encapsulated (free enzyme) or encapsulated antimicrobial lytic protein (encapsulated enzyme) on attenuation of mycobacterial growth in infected macrophages. [Figure 7] Graph showing OD590 / LLoD of M. abscessus GOSD (-1, -2, -3, -4, -5, -6, -7, and -8) after extraction from infected macrophages treated with non-encapsulated (free) or encapsulated (encapsulated) A, B, I, and combinations of (ABIα, AB, Iα, Biα, encABIα, encAB, encIα, and encBiα), respectively. [Figure 8] 8 is a table showing quantification of the number of M. abscessus cells in the experiment described in FIG. 7. [Figure 9] FIG. 1 is a schematic diagram of the experimental design to evaluate the differential effects of attenuation of mycobacterial growth in infected macrophages treated with non-encapsulated (free bacteriophage) or encapsulated bacteriophage. [Figure 10] Graph showing OD590 / LLoD of S. flexneri GOSD (-1, -2, -3, -4, -5, and -6) after extraction from infected macrophages treated with unencapsulated (phage) or encapsulated (encapsulated phage) Shigella phage EPH34, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0033] Bacteriophages are viruses that infect and replicate within bacteria. Lytic phages infect host bacteria and use the host's machinery to replicate virions. After replication, the phage lyse the host cell, releasing phage progeny to find new bacterial hosts for infection. Bacteriophages are programmed with all the essential machinery to kill targeted cells. Therefore, the protein components of bacteriophages represent attractive antibacterial therapies due to their ability to specifically target and destroy bacterial host cells.

[0034] Some pathogenic bacteria are difficult to target because they reside within host cells. These intracellular bacteria reside and grow within host cells to evade detection by the immune system. To effectively target intracellular bacteria, therapeutic payloads must not only target the bacteria but also the correct intracellular location where the bacteria reside. The present invention solves these problems by providing compositions and methods for targeting antibacterial therapies to treat intracellular bacterial infections. Generally, the compositions feature supramolecular structures, such as lipid-based supramolecular structures, e.g., liposomes, micelles, and lipid nanoparticles (LNPs), that target host cells, e.g., macrophages or dendritic cells, and the correct target intracellular compartment (endosome, phagosome, lysosome, or cytosol). Meanwhile, the supramolecular structures are preloaded with at least one antimicrobial lytic protein, e.g., an antimicrobial mycobacteriophage protein, primed to kill bacterial cells. The supramolecular structure targets the payload to the correct cell type and intracellular compartment, while the antimicrobial lytic protein is able to bind directly to bacteria due to its unique surface recognition properties.

[0035] Antibacterial lytic proteins Lytic bacteriophages contain the machinery necessary to recognize and lyse target bacteria as a result of the phage infection cycle. 30 It has been suggested that there are more than 100 bacteriophages, indicating a wide diversity that could lead to the discovery of antibacterial agents for the development of therapeutics.

[0036] Mycobacteriophages are double-stranded DNA viruses that specifically infect mycobacteria, ultimately leading to the death of the mycobacterial cell at the end of a lytic infection cycle. Mycobacteriophages have evolved specific lytic systems containing antimicrobial proteins, e.g., lytic enzymes, e.g., lipolytic enzymes, that are directed at targeting and cleaving specific types of bonds within specific layers of the highly hydrophobic and complex mycobacterial cell envelope, which comprises a core cell wall containing a covalently bound mAGP complex and an outer layer, e.g., a cell or mucoid capsule, containing non-covalently bound polysaccharides and proteins.

[0037] Antimicrobial lytic proteins, e.g., lytic bacteriophage proteins, can be loaded into supramolecular structures to form supramolecular complexes, which can be administered to cells, samples, or subjects. The supramolecular complexes are endocytosed by mammalian immune cells, e.g., macrophages or dendritic cells, and the antimicrobial lytic proteins are delivered to one or more target intracellular compartments (endosomes, phagosomes, lysosomes, or cytosol) where the bacteria reside. In some embodiments, the antimicrobial lytic proteins attach to the bacterial surface. The antimicrobial lytic proteins cleave specific bonds to porate and / or disrupt components of the bacterial cell envelope, resulting in osmotic destruction and bacterial death. Mycobacteriophage proteins include lysins, e.g., lysin A and lysin B, amylases, e.g., isoamylase and α-amylase, and capsule depolymerase. The lysins and capsule depolymerases are responsible for cleaving different bonds in the mycobacterial mAGP complex-containing cell wall of the cell envelope and the outer cell capsule, respectively. Mycobacteriophage proteins also include holins, which are membrane-associated proteins that oligomerize and pore the mycobacterial cell membrane, allowing non-pore-forming lysins to access their substrates and lyse the cell wall.

[0038] In some embodiments, the antimicrobial lytic protein is, for example, a lytic bacteriophage protein, e.g., a mycobacteriophage protein, hi some embodiments, the bacteriophage protein is derived from a phage that can kill a cell or infect a cell selected from the group consisting of Mycobacterium, Salmonella, Neisseria, Brucella, Escherichia, Listeria, Frankincella, Legionella, Yersinia, Staphylococcus, Clostridium, Shigella, or Streptococcus. In some embodiments, the Mycobacterium species is a species selected from the group consisting of M. tuberculosis, M. leprae, M. lepromatosis, M. avium, M. kansasii, M. fortuitum, M. chelonae, M. marinum, and M. abscessus; the Salmonella species is S. enterica, S. typhimurium, or S. bongori; the Neisseria species is N. gonorrhoeae or N. meningitidis; the Brucella species is B. melitensis, B. abortus, B. suis, or B. canis; the Escherichia species is E. coli; the Listeria species is L. monocytogenes; and the Frankicella species is F. tularensis. , F. novicida, or F. philomiragia; the Legionella species is L. pneumophila; the Yersinia species is Y. pestis or Y. enterocolitica; the Staphylococcus species is S. aureus; the Clostridium species is C. botulinum, C. perfringens, C. tetani, or C. sordellii; the Shigella species is S. dysenteriae, S. flexneri, S. boydii, or S. sonnei; or the Streptococcus species is S. pyogenes, S. agalactiae, S. dysgalactiae, S. bovis, S. anginosus, S. sanguinis, S. mitis, S. mutans, or S. pneumoniae.In some embodiments, the mycobacteriophage protein is a lysin or capsule depolymerase, e.g., capsulase. In some embodiments, the lysin is lysin A or lysin B. Capsule depolymerases are enzymes that destroy the outer cell capsule of mycobacterial cells, e.g., the mucoid capsule. The outer capsule is typically composed of polysaccharides and proteins, plus small amounts of lipids. In some embodiments, the capsule depolymerase is a hydrolase, metallohydrolase, epoxide hydrolase, peptidoglycan hydrolase, polysaccharase, polysaccharide lyase, endosialidase, hyaluronan lyase, or alginate lyase. Capsular polysaccharides within the outer capsule include, for example, D-glucan, D-arabino-D-mannan, and D-mannan. Furthermore, capsule depolymerases are enzymes that destroy these and other capsule polysaccharides.

[0039] In some embodiments, the antimicrobial lytic protein is a naturally occurring protein. In other embodiments, the antimicrobial lytic protein is a recombinant protein. In some embodiments, the compositions described herein comprise two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) different proteins, e.g., that kill two or more bacterial species or strains.

[0040] Non-limiting examples of antimicrobial lytic proteins are shown in Table 1.

[0041] [Table 1]

[0042] intracellular bacteria Intracellular bacteria live within host cells, where they reproduce and cause infection. Intracellular bacteria can reside within immune cells, such as professional antigen-presenting cells (APCs). Professional antigen-presenting cells (APCs) include macrophages, B cells, and dendritic cells. APCs process and present antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells recognize these antigen-presenting complexes using T cell receptors, a process critical for an effective adaptive immune response. Certain bacteria evade this immune response by hiding within immune cells.

[0043] The compositions and methods described herein can be used to treat any intracellular bacteria, including those that reside in professional antigen-presenting cells (e.g., macrophages or dendritic cells). In some embodiments, the bacterial cells are Mycobacterium, Salmonella, Neisseria, Brucella, Escherichia, Listeria, Frankincella, Legionella, Yersinia, Staphylococcus, Clostridium, Shigella, or Streptococcus species. Examples of Mycobacterium species include M. tuberculosis, M. leprae, M. lepromatosis, M. avium, M. kansasii, M. fortuitum, M. chelonae, M. marinum, or M. abscessus. In certain embodiments, the mycobacterium is an NTM. In some embodiments, the NTM is M. avium or M. abscessus. In some embodiments, the infection is caused by a combination of NTM, such as M. avium or M. abscessus.

[0044] Other intracellular bacteria are known in the art. Salmonella species can be, for example, S. enterica, S. typhimurium, or S. bongori. Neisseria species can be, for example, N. gonorrhoeae or N. meningitidis, E. coli. Brucella species can be, for example, B. melitensis, B. abortus, B. suis, or B. canis. Escherichia species can be, for example, E. coli. Listeria species can be, for example, L. monocytogenes. Frankicella species can be, for example, F. tularensis, F. novicida, or F. philomiragia. Legionella species can be, for example, L. pneumophila. Yersinia species can be, for example, Y. pestis or Y. enterocolitica. Staphylococcus species can be, for example, S. aureus. The Clostridium species can be, for example, C. botulinum, C. perfringens, C. tetani, or C. sordellii. The Shigella species can be, for example, S. dysenteriae, S. flexneri, S. boydii, or S. sonnei. The Streptococcus species can be, for example, S. pyogenes, S. agalactiae, S. dysgalactiae, S. bovis, S. anginosus, S. sanguinis, S. mitis, S. mutans, or S. pneumoniae.

[0045] supramolecular structure Supramolecular structures can be used to formulate antimicrobial agents, such as antimicrobial lytic proteins, e.g., mycobacteriophage proteins, for delivery. Supramolecular structures include complexes, e.g., defined complexes of lipids, that are bound to each other by non-covalent bonds, e.g., hydrogen bonds, van der Waals forces, electrostatic interactions, ion-dipole forces, hydrophobic effects, and π-π interactions. Supramolecular structures can include large complexes of molecules that form spheres, helices, or sheet-like structures. Examples of supramolecular structures include lipid-based supramolecular structures, such as micelles, liposomes, and LNPs. Supramolecular structures can have a predetermined size. The size of the structure can vary depending on the size of the components, e.g., proteins, packaged within the structure. The supramolecular complex is endocytosed by cells, e.g., professional antigen-presenting cells such as macrophages or dendritic cells, and the antimicrobial lytic protein is delivered to the target intracellular compartment (endosome, phagosome, lysosome, or cytosol) where the bacteria reside.

[0046] In some embodiments, specific particle sizes are used to guide the structures into specific endocytic pathways that direct them to appropriate target intracellular compartments. The supramolecular structures undergo endocytosis and can be delivered to target intracellular compartments, for example, via clathrin-mediated endocytosis or caveolin-mediated endocytosis. The particle size, e.g., Z-average mean particle size, of the supramolecular structures can vary from about 75 nm to about 750 nm (e.g., about 250 nm to about 750 nm, or about 75 nm to about 250 nm). Preferably, when the supramolecular structures are LNPs or micelles, the Z-average mean particle size is about 75 nm to about 250 nm. Preferably, when the supramolecular structures are vesicles (e.g., liposomes), the Z-average mean particle size is about 250 nm to about 750 nm. Non-limiting examples of the Z average mean particle size include, for example, about 75 nm to about 100 nm, for example, 75 nm to about 85 nm, for example, about 80 nm, for example, about 80 nm to about 140 nm, about 90 nm to about 130 nm, or about 110 nm to about 130 nm, for example, about 120 nm, for example, about 200 nm to about 300 nm, for example, about 250 nm to about 300 nm, about 26 0 nm to about 290 nm, about 260 nm to about 280 nm, about 265 nm to about 275 nm, for example, about 270 nm, for example, about 300 nm to about 400 nm, about 400 nm to about 600 nm, for example, about 450 nm to about 550 nm, about 475 nm to about 525 nm, about 480 nm to about 520 nm, about 490 nm to about 510 nm, about 495 nm to about 505 nm, for example, About 500 nm, for example, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm, about 170 nm, about 175 nm, about 180 nm, Approximately 185nm, approximately 190nm, approximately 195nm, approximately 200nm, approximately 205nm, approximately 210nm, approximately 215nm, approximately 220nm, approximately 225nm, approximately 230nm, approximately 235nm, approximately 24 0nm, about 245nm, about 250nm, about 255nm, about 260nm, about 265nm, about 270nm, about 275nm, about 280nm, about 285nm, about 290nm, about 295nm,Approximately 300nm, approximately 305nm, approximately 310nm, approximately 315nm, approximately 320nm, approximately 325nm, approximately 330nm, approximately 335nm, approximately 340nm, approximately 345nm, approximately 350nm, approximately 35 5nm, about 360nm, about 365nm, about 370nm, about 375nm, about 380nm, about 385nm, about 390nm, about 395nm, about 400nm, about 405nm, about 410nm, Approximately 415nm, approximately 420nm, approximately 425nm, approximately 430nm, approximately 435nm, approximately 440nm, approximately 445nm, approximately 450nm, approximately 455nm, approximately 460nm, approximately 465nm, approximately 470 nm, approximately 475nm, approximately 480nm, approximately 485nm, approximately 490nm, approximately 495nm, approximately 500nm, approximately 505nm, approximately 510nm, approximately 515nm, approximately 520nm, approximately 525nm, approximately 530nm, about 535nm, about 540nm, about 545nm, about 550nm, about 555nm, about 560nm, about 565nm, about 570nm, about 575nm, about 580nm, about 585 nm, approximately 590nm, approximately 595nm, approximately 600nm, approximately 605nm, approximately 610nm, approximately 615nm, approximately 620nm, approximately 625nm, approximately 630nm, approximately 635nm, approximately 640nm, approximately Examples of such diameters include about 645 nm, about 650 nm, about 655 nm, about 660 nm, about 665 nm, about 670 nm, about 675 nm, about 680 nm, about 685 nm, about 690 nm, about 695 nm, about 700 nm, about 705 nm, about 710 nm, about 715 nm, about 720 nm, about 725 nm, about 730 nm, about 735 nm, about 740 nm, about 745 nm, or about 750 nm. In certain embodiments, the Z-average average particle size of the supramolecular structure can be about 75 nm to about 250 nm. In some embodiments, the Z-average average particle size of the supramolecular structure is about 80 nm, about 270 nm, or about 500 nm.

[0047] The average particle size can be measured by zeta potential, dynamic light scattering (DLS), electrophoretic light scattering (ELS), static light scattering (SLS), molecular weight, electrophoretic mobility, size exclusion chromatography (SEC), field-flow fractionation, or other methods known in the art. In certain embodiments, the average particle size is measured by: In certain embodiments, the supramolecular structure contains a Z-average average particle size of about 75 nm to about 250 nm. In certain embodiments, the supramolecular structure contains a Z-average average particle size of about 250 nm to about 750 nm. In certain embodiments, the supramolecular structure contains a Z-average average particle size of about 500 nm. In certain embodiments, the supramolecular structure contains a Z-average average particle size of about 270 nm. In certain embodiments, the supramolecular structure contains a Z-average average particle size of about 80 nm. Those skilled in the art will understand that a collection of supramolecular structures (e.g., liposomes, LNPs, or micelles) can have a range of Z-average mean particle sizes within the collection. Thus, the collection can be polydisperse. The collection can have a polydispersity index of 0.3 or less (e.g., 0.05 to 0.3). The polydispersity index can be measured using DLS (see, e.g., ISO 22412:2017).

[0048] The supramolecular structures can be loaded with a predetermined number or an average number of antimicrobial lytic proteins per supramolecular structure. For example, the supramolecular structures can be loaded with between about 1 protein and about 10 proteins. 6 proteins (e.g., about 1 to about 10 5 , about 1 to about 10 4 , about 1 to about 10 3 , about 1 to about 10 2 , about 1 to about 10, about 10 to about 10 6 , about 10 to about 10 5 , about 10 to about 10 4 , about 10 to about 10 3 , about 10 to about 10 3 , about 10 to about 10 2 , about 10 3 ~about 10 6 , about 10 3 ~about 10 5, about 10 3 ~about 10 4 The number of proteins per structure may depend on the size of the protein and the size of the structure.

[0049] The supramolecular structure can include an endosomal escape moiety. The supramolecular structure including the endosomal escape moiety can provide improved cytosolic delivery of the cargo (e.g., therapeutic agent) contained in the supramolecular structure. Endosome escape moieties are known in the art. The endosomal escape moiety is preferably an ionizable lipid. Ionizable lipids are typical. Ionizable lipids can also serve as supramolecular structure-layer-forming lipids. Non-limiting examples of ionizable lipids include those described in, for example, WO2019 / 067875; WO2018 / 191750; and US9,999,671. Other exemplary endosomal escape moieties include fusogenic lipids (e.g., dioleoylphosphatidyl-ethanolamine (DOPE)); and polymers such as polyethyleneimine (PEI); poly(β-amino esters); polypeptides such as polyarginine (e.g., octaarginine) and polylysine (e.g., octalysine); proton sponges, viral capsids, and peptide transduction domains described herein. For example, fusogenic peptides can be derived from the influenza A virus M2 protein, peptide analogs of influenza virus hemagglutinin, influenza C virus HEF protein, filovirus transmembrane glycoproteins, rabies virus transmembrane glycoproteins, vesicular stomatitis virus transmembrane glycoprotein (G), Sendai virus fusion protein, Semliki Forest virus transmembrane glycoprotein, human respiratory syncytial virus (RSV) fusion protein, measles virus fusion protein, Newcastle disease virus fusion protein, Visna virus fusion protein, murine leukemia virus fusion protein, HTL virus fusion protein, and simian immunodeficiency virus (SIV) fusion protein. Other moieties that can be used to facilitate endosomal escape are described in Dominska et al., Journal of Cell Science, 123(8):1183-1189, 2010.Specific examples of endosomal escape moieties, including moieties suitable for inclusion in or conjugation to the supramolecular structures disclosed herein, are set forth, for example, in WO2015 / 188197, the disclosure of which is incorporated herein by reference.

[0050] Liposomes Liposomes are useful for transporting and delivering antimicrobial proteins to the site of action. Because liposome membranes are structurally similar to biological membranes, when liposomes are applied to tissues, the liposome bilayer fuses with the cell membrane bilayer. When the liposome and cell membrane combine, the internal aqueous contents, including the antimicrobial protein, are delivered to the cell and the antimicrobial lytic protein is delivered intracellularly, where the antimicrobial protein can target and lyse bacterial cells (e.g., mycobacterial cells, e.g., NTM cells) residing within mammalian immune cells. In some cases, liposomes are also specifically targeted, for example, to direct proteins to specific mammalian immune cells and / or to specific intracellular compartments (endosomes, phagosomes, lysosomes, or cytosol) that normally harbor bacteria (e.g., mycobacteria) during infection. The composition of liposomes is typically a combination of phospholipids, usually combined with a steroid such as cholesterol. Other phospholipids or other lipids can also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0051] Preferably, the liposomes described herein comprise a phospholipid, more preferably a glycerophospholipid, such as phosphatidylserine. Phosphatidylserine is a glycerol molecule with two hydroxyl groups replaced with fatty acid ester moieties and one hydroxyl group replaced with a phosphodiester moiety covalently attached to a serine side chain. A typical structure of phosphatidylserine is RO-CH2-CH(OR)-CH2-OP(O)(OH)-OCH2CH(COOH)NH2, or a salt thereof, where each R is independently a fatty acid acyl. Additionally or alternatively, the liposomes described herein can comprise, for example, a lysophospholipid, such as lysophosphatidylserine. Lysophosphatidylserine is phosphatidylserine lacking one of its two fatty acid ester moieties. A typical structure of lysophosphatidylserine is RO-CH2-CH(OR)-CH2-OP(O)(OH)-OCH2CH(COOH)NH2, or a salt thereof, where one R is fatty acyl and the other R is H. Thus, in certain preferred embodiments, the liposomes described herein comprise RO-CH2-CH(OR)-CH2-OP(O)(OH)-OCH2CH(COOH)NH2, or a salt thereof, where each R is H or fatty acyl, provided that at least one R is fatty acyl.

[0052] One of the major types of liposome compositions includes phospholipids other than naturally occurring phosphatidylcholine.Neutral liposome compositions can be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Cationic liposomes have the advantage of being able to fuse with cell membranes. Non-limiting examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleyl kaolin, ... 1,2-Dilinoleyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-s-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyl-3-(2N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or similar. analogues, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienietetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-ieethylazanediedidodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can account for, for example, about 20 mol % to about 50 mol %, or about 40 mol %, of the total lipid present in the particle.

[0053] Non-cationic liposomes, although less efficient at fusing with the plasma membrane, can be taken up by macrophages in vivo and deliver antimicrobial proteins to macrophages. Anionic liposome compositions are typically formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoyl phosphatidylethanolamine (DOPE). Ionizable / non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-ol, and 4-(N-maleimidomethyl)-cyclohexane-1-ol. The lipids can be anionic or neutral lipids, including, but not limited to, carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans-PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt, DOPS), or mixtures thereof. When cholesterol is included, the non-cationic lipid can comprise, for example, about 5 mol% to about 90 mol%, or about 10 mol% to about 58 mol% of the total lipid present in the particle. In some embodiments, the ionizable / non-cationic lipid can be a combination of the lipids described above, for example, a combination of lipids including DOPC, DOPS, Chol, and DOPE.

[0054] Conjugated lipids that inhibit aggregation of liposome particles can be, for example, polyethylene glycol (PEG)-lipids, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. PEG-DAA conjugates can be, for example, PEG-dilauryloxypropyl (C 12 ), PEG-dimyristyloxypropyl (C 14 ), PEG-dipalmityloxypropyl (C 16 ), or PEG-distearyloxypropyl (C 18 The complex lipid that prevents particle aggregation can be, for example, 0 mol % to about 20 mol %, or about 2 mol % of the total lipid present in the particle. In some embodiments, the liposome composition further comprises cholesterol, for example, at about 10 mol % to about 60 mol %, or about 50 mol % of the total lipid present in the particle.

[0055] Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol. Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Feigner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.

[0056] Liposome targeting can also be based on, for example, organ specificity, cell specificity, and organelle specificity, and is known in the art.For liposome-targeted delivery systems, lipid groups can be incorporated into the lipid bilayer of liposome to maintain the targeting of ligand while stably associating with the liposome bilayer.Various linking groups can be used to connect lipid chains to targeting ligands.Further methods are known in the art, and are described, for example, in US Patent Publication No. 20060058255, and the linking groups are incorporated herein by reference.

[0057] Cleavable linking groups are susceptible to cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are more effective or found at higher levels or activity within cells than in serum or blood. Examples of such degradable agents include oxidizing or reducing enzymes or reducing agents present in cells, such as mercaptans, that can degrade redox cleavable linking groups by reduction; esterases; endosomes or agents that can create an acidic environment, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade cleavable acid linking groups by acting as general acids; peptidases (which can be substrate specific); and redox agents that are selective for specific substrates or do not have substrate specificity, including phosphatases.

[0058] Cleavable linking groups, such as disulfide bonds, can be sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH, around 5.0. Some linkers have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand into the cell or into a desired compartment of the cell.

[0059] The linker can include a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the target cell. In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It may also be desirable to test the ability of the candidate cleavable linking group to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative susceptibility to cleavage under a first condition and a second condition can be measured, where the first condition is selected to indicate cleavage in target cells and the second condition is selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, cells, cell culture, organ or tissue culture, or whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm them with further evaluations in whole animals. In preferred embodiments, useful candidate linkers are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0060] lipid nanoparticles The antibacterial agents of the present invention can be fully encapsulated in lipid formulations, such as lipid nanoparticles (LNPs). LNPs exhibit extended circulatory life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically separated from the administration site), making them highly useful for systemic administration. LNPs include "pSPLPs," which contain encapsulated condensing agent-nucleic acid complexes as described in PCT Publication No. WO 2000 / 003683. The particles of the present invention typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially nontoxic. Furthermore, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010 / 0324120; and PCT Publication No. WO 96 / 40964.

[0061] In one embodiment, the lipid:drug ratio (mass / mass ratio) (e.g., lipid:oligonucleotide ratio) ranges from about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above-listed ranges are also contemplated as part of the invention.

[0062] Non-limiting examples of cationic lipids include DODAC, DDAB, DOTAP, DOTMA, DODMA, DLinDMA, DLenDMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLinDAP, DLin-s-DMA, DLin-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl, 1DLin-MPZ, DLinAP, DOAP, DLin-EG-DMA, DLin-K-DMA or analogs thereof, ALN100, MC3, Tech G1, or mixtures thereof. The cationic lipid can comprise, for example, about 20 mol% to about 50 mol%, or about 40 mol% of the total lipid present in the particle.

[0063] The ionizable / non-cationic lipid can be an anionic or neutral lipid, including, but not limited to, DSPC, DOPC, DOPS, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, SOPE, cholesterol, or a mixture thereof. When cholesterol is included, the non-cationic lipid can be, for example, about 5 mol% to about 90 mol%, or about 10 mol% to about 60 mol% of the total lipid present in the particle.

[0064] Conjugated lipids that inhibit particle aggregation can be, for example, polyethylene glycol (PEG)-lipids, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. PEG-DAA conjugates can be, for example, PEG-dilauryloxypropyl (C 12 ), PEG-dimyristyloxypropyl (C 14 ), PEG-dipalmityloxypropyl (C 16 ), or PEG-distearyloxypropyl (C 18The complex lipid that prevents particle aggregation can be, for example, 0 mol % to about 20 mol %, or about 2 mol % of the total lipids present in the particle.

[0065] In some embodiments, the LNPs further comprise cholesterol, for example, at about 10 mol % to about 60 mol %, or about 50 mol % of the total lipids present in the particle. Micelle Micelles are a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure so that the hydrophobic portions of the molecules all face inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. Micelles can be made from lipids. The micellar phase is caused by the confining behavior of single-tailed lipids in a bilayer membrane. The difficulty of filling the entire interior volume of a bilayer membrane while accommodating the area for the head groups acting on the molecules due to hydration of the lipid head groups leads to the formation of micelles. This type of micelle is known as a normal-phase micelle (oil-in-water micelle). Reverse micelles have head groups in the center and tails extending outward (water-in-oil micelle).

[0066] Micelles are approximately spherical in shape. Other shapes are also possible, including ellipsoids, cylinders, and bilayers. The shape and size of a micelle is a function of the molecular geometry of the surfactant molecule and solution conditions such as surfactant concentration, temperature, pH, and ionic strength. The process of micelle formation is known as micellization, and due to its polymorphic nature, it forms part of the phase behavior of many lipids.

[0067] target area The supramolecular structures described herein can include, for example, a targeting moiety. The targeting moiety can be used to target the supramolecular structure to a specific cell type (e.g., professional antigen-presenting cells (e.g., macrophages or dendritic cells)). Specific lipids (e.g., phosphatidylserine) can be used within the supramolecular structure (e.g., vesicles) both as supramolecular structure phase-forming lipids and as targeting moieties. The targeting moiety can be, for example, an antibody or antigen-binding fragment or an engineered derivative thereof (e.g., Fcab or fusion protein (e.g., scFv)). The targeting moiety can be, for example, a polypeptide. Alternatively, the targeting moiety can be, for example, a small molecule (e.g., mannose or folate) or a cluster of small molecules (e.g., a cluster of mannose). The targeting moiety can be covalently or non-covalently associated with the supramolecular structure.

[0068] low molecule The targeting moiety can be a small molecule capable of complexing a receptor expressed on the surface of a target cell. Non-limiting examples of small molecules that can be used as targeting moieties in the supramolecular structures described herein are phosphatidylserine, folate lysophosphatidylserine, mannose, and mannose clusters.

[0069] Preferably, the targeting moiety is phosphatidylserine or lysophosphatidylserine. More preferably, the targeting moiety is phosphatidylserine. The phosphatidylserine and / or lysophosphatidylserine can be present as a supramolecular structure phase-forming lipid, non-covalently bound to the rest of the supramolecular structure.

[0070] Folate can be used as a targeting moiety. In the supramolecular structures described herein, folate can have the following structure:

[0071] [ka]

[0072] Mannose or mannose clusters can be used to target the supramolecular structures described herein to dendritic cells and macrophages. Mannose clusters are known in the art.

[0073] Folate, mannose, and mannose clusters can be covalently bound to supramolecular structures. Conjugation techniques for binding folate, mannose, and mannose clusters are known in the art, for example, as described in US2014 / 0045919, US9,725,479, US8,758,810, US8,450,467, US6,525,031, US6,335,434, and US5,759,572.

[0074] antigen binding part The antigen-binding moiety in the supramolecular structures described herein can be an antibody or an antigen-binding fragment thereof, such as F(ab)2 or Fab, or a recombinant derivative thereof (e.g., Fcab or a fusion protein, such as scFv). Human or chimeric, e.g., humanized, antibodies can be used as antibodies in the supramolecular structures described herein.

[0075] The antigen-binding portion targets APCs bearing the surface antigen recognized by the antigen-binding portion. Dendritic cells can be targeted with anti-DEC205, anti-CD304, anti-CD303, anti-CD40, anti-CD74, anti-BDCA2, or anti-CD123 antibodies, or antigen-binding fragments or recombinant derivatives thereof. Macrophages can be targeted with anti-CD163, anti-CD40, anti-CD74, anti-CD206, or anti-CD123 antibodies, or antigen-binding fragments or recombinant derivatives thereof.

[0076] Non-limiting examples of anti-CD38 antibodies include daratumumab, SAR650984, MOR202, or any one of antibodies Ab79, Ab19, Ab43, Ab72, and Ab110 disclosed in WO2012 / 092616, the disclosures of which are incorporated herein by reference. A non-limiting example of an anti-CD79b antibody is huMA79b v28 disclosed in WO2014 / 011521. A non-limiting example of an anti-CD22 antibody is 10F4 disclosed in US2014 / 0127197. A non-limiting example of an anti-CD20 antibody is rituximab. A non-limiting example of an anti-DEC205 antibody is disclosed in US2010 / 0098704, the disclosures of which are incorporated herein by reference. Non-limiting examples of anti-CD40 antibodies are lucatumumab and dacetuzumab. A non-limiting example of an anti-CD304 antibody is besencumab.

[0077] Conjugation techniques for linking antigen-binding moieties are known in the art, for example, as described in Ansell et al., Methods Mol. Med., 25:51-68, 2000; US 2002 / 0025313; US 6,379,699; and US 5,059,421.

[0078] Polypeptides The targeting moiety can be a polypeptide having affinity for cells (e.g., having affinity for a cell type, e.g., dendritic cells). Non-limiting examples of polypeptides are RGD peptide, rabies virus glycoprotein (RVG), and DC3 peptide. Alternatively, the polypeptide can be a TLR2 agonist, such as MALP-2 lipoprotein, MALP-404 lipoprotein, OspA, porin, LcrV, Hsp60, glycoprotein gH / gL, or glycoprotein gB.

[0079] Conjugation techniques for linking peptides are known in the art, for example, as described in Ansell et al., Methods Mol. Med., 25:51-68, 2000; US 2002 / 0025313; US 6,379,699; and US 5,059,421.

[0080] PAMP The target moiety can be a PAMP. PAMPs are known in the art, for example, CpG ODN. CpG ODNs are generally divided into three classes: class A, class B, and class C. Class A CpG ODNs typically contain poly-G tails with phosphorothioate backbones at the 3' and 5' ends, and a central palindromic sequence containing a phosphate backbone. Class A CpG ODNs typically contain CpG within the central palindromic sequence. Class B CpG ODNs typically contain a fully phosphorothioate backbone, and the sequence at the 5' end of class B CpG ODNs is often important for TLR9 activation. Class C CpG ODNs typically contain a fully phosphorothioate backbone, and the 3' end sequence is capable of duplex formation. PAMPs can be covalently bound to supramolecular structures using techniques and methods known in the art.

[0081] Treatment method The antimicrobial lytic proteins described herein are preferably formulated into pharmaceutical compositions for administration to human subjects to treat diseases or conditions, such as bacterial infections (e.g., intracellular bacterial infections, e.g., mycobacterial infections, e.g., NTM infections). Bacterial infections may occur in otherwise healthy subjects. Alternatively, bacterial infections may occur in subjects with other coexisting diseases or diseases. For example, subjects with weakened immune systems may be more susceptible to bacterial infections.

[0082] Mycobacterial infections caused by NTM bacteria are bacteria normally present in the environment. Inhalation of these bacteria can cause disease in both healthy and immune-compromised patients. NTM disease most commonly affects the lungs in adults, but can affect any body part. Some subjects are at increased risk for developing NTM infection and progression of the disease. Individuals with pre-existing lung diseases, such as bronchiectasis (widened airways), chronic obstructive pulmonary disease (COPD), cystic fibrosis, and alpha-1 antitrypsin deficiency, or those with previous infections, such as tuberculosis, are at increased risk for pulmonary NTM disease. In subjects with advanced HIV infection (CD4 < 50) or immune-related genetic diseases (e.g., interferon-gamma or receptor deficiency, interleukin-12 deficiency), lung disease can develop as part of a disseminated (e.g., widespread) NTM infection. The subject being treated may have any of the aforementioned indications in addition to a bacterial infection, for example.

[0083] The method compositions and methods described herein can be used to reduce the level of infection. For example, the method can reduce the level of infection (e.g., the number of bacteria or the size of the infection) compared to a reference. For example, the infection can be reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0084] Pharmaceutical Composition The antibacterial agents described herein are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.

[0085] As will be appreciated by those skilled in the art, the compositions described herein can be administered to a subject in a variety of forms depending on the selected route of administration. The compositions described herein can be administered, for example, by any route that allows the composition (e.g., a supramolecular structure, e.g., a liposome, micelle, or LNP) to reach target cells. The compositions can be administered, for example, orally, parenterally, intrathecally, intracerebroventricularly, intraparenchymal, buccal, sublingually, nasally, rectally, via patch, pump, or transdermal administration, with the pharmaceutical composition appropriately formulated. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, intranasal, intrapulmonary, intrathecal, intracerebroventricular, intraparenchymal, rectal, and topical administration. In one embodiment, a composition administered parenterally by air can be administered by continuous infusion over a selected period of time. In some preferred embodiments, the compositions described herein are administered by inhalation.

[0086] Administration of two or more antibacterial agents may be by the same route or by different routes, and may occur sequentially or near simultaneously, for example, a first antibacterial agent of the combination may be administered by intravenous injection, while a second therapeutic agent of the combination may be administered orally.

[0087] Certain compositions described herein can be administered, for example, by inhalation. Inhalation can be oral or nasal. The inhalable compositions described herein can be provided in liquid or dry powder form. Dry powder compositions can be administered, for example, by inhalation, either directly or after reconstitution with a vehicle (e.g., saline (e.g., isotonic saline), phosphate buffered saline, or water).

[0088] Inhalable dry powder formulations can be prepared from the liquid compositions described herein by drying (e.g., lyophilization, spray drying, spray freeze drying, or supercritical fluid technology). The inhalable dry powder formulations described herein can include a carrier (e.g., lactose, sucrose, mannitol, etc.), a cryoprotectant (e.g., trehalose, mannitol, etc.), and / or an anti-adhesive agent (e.g., glycine, L-leucine, serine, etc.). The inhalable dry powder formulations described herein can be administered using a dry powder inhaler. Dry powder inhalers are known in the art and may or may not contain a propellant. Non-limiting examples of dry powder inhalers can be found in Newman, Expert Opin. Biol. Ther., 4:23-33, 2004, the entire disclosure of which is incorporated herein by reference.

[0089] The inhalable liquid dosage forms (e.g., aerosol formulations) described herein can be prepared using techniques and methods useful for preparing liquid compositions containing supramolecular structures. Inhalable liquid dosage forms typically comprise a suspension of the supramolecular structures described herein in a physiologically acceptable aqueous or non-aqueous solvent, typically present in a sealed container in a sterile form, in single or multiple doses, which can take the form of a cartridge or refill for use with a spray device. Alternatively, the sealed container can be an integrated dispenser, such as a single-dose nasal inhaler, or an aerosol dispenser fitted with a metering valve for disposal after use. When the dosage form contains an aerosol dispenser, it contains a propellant, which can be a compressed gas, such as compressed air, or an organic propellant, such as a hydrofluoroalkane. The inhalable liquid dosage form can be administered using a nebulizer. The process of converting a bulk liquid into small droplets using compressed air is called atomization. The operation of a gas nebulizer requires a propellant as a driving force for liquid atomization. Various types of nebulizers are described in Respiratory Care, 45:609-622, 2000, the entire disclosure of which is incorporated herein by reference. Alternatively, the inhalable liquid dosage forms described herein can be administered using a metered dose inhaler. Metered dose inhalers are known in the art and typically include a canister, an actuator, and a metering valve.

[0090] The compositions described herein can be orally administered, for example, with an inert diluent or an assimilable edible carrier, or enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into dietary foods. For oral therapeutic administration, the compositions described herein can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. The compositions described herein can also be administered parenterally. Solutions of the compositions described herein can be prepared in water suitably mixed with a surfactant, such as hydroxypropyl cellulose. Dispersions can be prepared in glycerol, liquid polyethylene glycols, DMSO, and mixtures thereof, with or without alcohol, and in oils. These formulations may contain preservatives to prevent the growth of microorganisms under ordinary conditions of storage and use. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2012, 22nd ed.) and The United States Pharmacopeia: The National Formulary (USP 41 NF 36), published in 2018. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and sufficiently fluid for easy administration by syringe. Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, in which the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, gelatin, and glycerin. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base, such as cocoa butter.

[0091] The compositions described herein can be administered to animals, e.g., humans, as described herein, alone or in combination with a pharmaceutically acceptable carrier, the proportions of which will be determined by the solubility and chemical properties of the composition, the chosen route of administration, and standard pharmaceutical practice.

[0092] The dosage of a composition, e.g., a composition comprising an antimicrobial bacteriophage protein described herein, can depend on many factors, such as the pharmacodynamic properties of the antimicrobial lytic protein, the mode of administration, the age, health, and weight of the subject to be treated, the nature and extent of symptoms, the frequency of treatment, and the type of concurrent treatment (if any), as well as the clearance rate of the composition in the treated animal. The compositions described herein can be initially administered at a suitable dosage, which can be adjusted as needed depending on the clinical response. In some embodiments, the dose of a composition, e.g., a composition comprising a bacteriophage, is a prophylactically or therapeutically effective amount. Furthermore, it is understood that the dose can be given continuously or divided into doses given per a given time frame. The composition can be administered, for example, hourly, daily, weekly, monthly, or yearly. In some embodiments, the composition is administered continuously or systemically.

[0093] Combination therapy The pharmaceutical compositions described herein can be administered as part of a combination therapy. Combination therapy refers to the administration of two (or more) different agents or treatments to a subject as part of a defined therapeutic regimen for a particular disease or condition. The therapeutic regimen defines the dosage and periodicity of administration of each agent so that the effects of the separate agents on the subject overlap. In some embodiments, delivery of two or more agents is simultaneous or concurrent, and the agents may be co-formulated. In some embodiments, two or more agents are not co-formulated but are administered sequentially as part of a prescribed regimen. The sequential or substantially simultaneous administration of each therapeutic agent can be by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination may be administered by intravenous injection or by aerosolization, while a second therapeutic agent of the combination may be administered orally.

[0094] In any of the combination embodiments described herein, the first and second therapeutic agents can be administered simultaneously or sequentially in any order. The first therapeutic agent can be administered immediately, up to 15 minutes, up to 30 minutes, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, up to 14 hours, up to 16 hours, up to 17 hours, up to 18 hours, up to 19 hours, up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours, or 1 to 7, 1 to 14, 1 to 21, or 1 to 30 days before or after the second therapeutic agent.

[0095] The pharmaceutical compositions described herein may further comprise an additional antibacterial agent administered with the supramolecular structure comprising the antimicrobial lytic protein. The compositions and methods described herein can further include treatment of underlying pulmonary conditions, e.g., that may be exacerbated by a bacterial infection, e.g., an NTM infection. Suitable pulmonary therapies include, but are not limited to, airway clearance, nebulizers, respiratory masks, and inhalers, e.g., steroid inhalers.

[0096] antibiotics The additional antibacterial agent can be an antibiotic. Suitable antibiotics include penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, azlocillin, temocillin, cephalothin, cephapirin, cephradine, cephaloridine, cefazolin, cefamandole, cefuroxime, cephalexin, cefprozil, cefaclor, loracarbef, cefoxitin, cefmetazole, cefotaxime, ceftizoxime, ceftriaxone, ceftriaxone, cefethamin ... Foperazone, ceftazidime, cefixime, cefpodoxime, ceftibuten, cefdinir, cefpirome, cefepime, chlorhexidine, BAL5788, BAL9141, imipenem, ertapenem, meropenem, aztreonam, clavulanic acid, sulbactam, tazobactam, streptomycin, neomycin, kanamycin, puromycin, gentamicin, tobramycin, amikacin, netilmicin, spectinomycin, sisomicin, isepamicin, tetracycline, chlortetracycline, demeclocycline Clin, minocycline, oxytetracycline, methacycline, doxycycline, erythromycin, azithromycin, clarithromycin, telithromycin, ABT-773, lincomycin, clindamycin, vancomycin, oritavancin, dalbavancin, teicoplanin, quinupristin and dalfopristin, sulfanilamide, p-aminobenzoic acid, sulfadiazine, sulfisoxazole, sulfamethoxazole, sulfatalidine, linezolid, nalidixic acid, oxolinic acid, norfloxacin , pefloxacin, enoxacin, ofloxacin, ciprofloxacin, temafloxacin, lomefloxacin, fleroxacin, grepafloxacin, sparfloxacin, trovafloxacin, clinafloxacin, gatifloxacin, moxifloxacin, gemifloxacin, sitafloxacin, metronidazole, daptomycin, garenoxacin, ramoplanin, faropenem, polymyxin, tigecycline, AZD2563, trimethoprim, ethambutol, and rifampin.In some embodiments, multiple antibiotics are administered with the compositions described herein. In some embodiments, the antibiotics are selected from the group consisting of cephalosporins, carbapenems, penicillins, and fluoroquinolones. In some embodiments, the antibiotics are selected from the group consisting of thiacetazone, sq-109, bedaquiline, delamanid, pyrazinamide, and isoniazid.

[0097] Advantageously, in some embodiments, synergistic interactions with co-administered therapeutic agents may allow for the administration of sub-therapeutic doses of antibiotics when administered without other therapeutic agents.

[0098] The antibiotic can be formulated with a supramolecular structure containing an antibacterial lytic protein. The antibiotic can be administered as a separate pharmaceutical composition. The antibiotic can be administered at a different time from the pharmaceutical composition containing the supramolecular structure with the phage. In some preferred embodiments, the additional antibiotic is amikacin. The amikacin can be, for example, liposomal amikacin formulated for inhalation.

[0099] The following examples are intended to illustrate the invention, but are not intended to limit the invention in any way. Example The following examples are presented to provide one of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.

[0100] Example 1: Materials and Methods Cloning I. Cloning of Lysin A The open reading frame of bacteriophage Halo LysA (gp 10 (accession number NC_001900)) without a stop codon was inserted into the NdeI to XhoI sites of the pet21a plasmid by Genscript.

[0101] II. Cloning of Lysin B The open reading frame of bD29 LysB (gp12 accession number NC_001900) was synthesized by Genscript with adapters and cloned by Gibson-based homology cloning into the pet21 plasmid at position 5237 downstream of the T7 tag.

[0102] Protein preparation I. Expression E. coli Bl21(de3) cells were transduced with the pET21a plasmid containing the therapeutic payload and cultured in tryptic soy broth (TSB) medium. All cultures were grown at 37°C for approximately 3.5 hours or until they reached an optical density (OD) of 0.4. Grown cultures were incubated on ice for 30 minutes before being induced with 40 mM IPTG. Cultures were incubated overnight at low temperature for expression. After removal from the incubator, the cultures were centrifuged in a swinging bucket rotor for 20 minutes at 4000 revolutions per minute (RPM) to separate the cells from the supernatant.

[0103] To purify therapeutic proteins, lysis was performed with a solution containing bacterial protein extraction reagent (B-PER), benzonaceme, and lysozyme. The fast protein liquid chromatography (FPLC) running buffer consisted of 50 mM Tris (pH 8), 250 mM NaCl, 50 mM imidazole (pH 8), 0.5 mM MgCl2, and 10% glycine. The elution buffer consisted of 50 mM Tris (pH 8), 250 mM NaCl, 700 mM imidazole (pH 8), 0.5 mM MgCl2, and 10% glycerol. The eluted fraction was dialyzed overnight at 4°C using 5 mL of Cytiva HisTrap FF 5 with an Äkta Purifier using a 10 kDa dialysis bag. The dialysis buffer consisted of 50 mM Tris (pH 8), 250 mM NaCl, 0.5 mM MgCl2, and 20% glycerol.

[0104] II. Liposome Encapsulation of Payloads To assemble liposomes using the Nanoassembler Ignite (Precision Nanosystems), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt; DOPS):cholesterol (Chol):1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at a total lipid concentration of 1 mg / mL was combined with organic solvent and aqueous buffer (1 M Tris, pH 7.4) in a ratio of 1:0.3:0.4:1.

[0105] To mediate the loading of the payload into the liposomes, 2-4 mg / mL of protein payload was added to an aqueous buffer, followed by 1 x 10 8 ~1×10 10 Phage were added at pfu / mL to aqueous buffer at a total flow rate of 15 mL / min, a 1:1 flow rate ratio, and a total volume of 1 mL. To purify liposomes by two-step dialysis, 30-minute dialysis was performed on a 10 kDa slide-a-lyzer in 100 mM or 10 mM Tris (pH 7.4), 5% glycerol at room temperature. Sample size was analyzed using a dynamic light scattering (DLS) instrument to measure the size and polydispersity of the encapsulated samples. Samples yielding sizes between 0.5 and 1.5 micrometers were selected for assays on cells.

[0106] Therefore, ABIα (lysin A, lysin B, isoamylase, and α-amylase) and BIα (lysin B, isoamylase, and α-amylase) were prepared. Killing of nontuberculous mycobacteria by administration of ABIα On day 0, Middlebrook 7H9 broth + TWEEN (approximately 1 × 10 8A suspension of M. abscessus bacterial cells was prepared by adjusting the cell logarithmic growth to 0.5 using the McFarland standard (cfu / mL) and diluting the prepared suspension 1:5 with 7H9 broth + TWEEN. The assay media solution contained 30 mL of Middlebrook 7H9, ADC, and TWEEN; 3.3 mL of 12X PM additive (Biolog, Inc.); 3.3 mL of IF-01a fluid (Biolog, Inc.); and 400 μL of 100X dye G (Biolog, Inc.). Assay plates were prepared by pipetting 90 μL of the prepared media solution into wells of a 96-well plate, adding 5 μL of the prepared cell suspension, and adding 10 μL of enzyme to a final concentration of 3.2 μg each. Controls included cells alone, with or without dialysis buffer. After the preliminary steps, the assay plates were incubated by tightening the lids and wrapping them in parafilm, then placing them in a static incubator at 37°C.

[0107] On day 1, dosing assay plates were prepared by adding 10 μL of enzyme cocktail to wells designated as doses 2x and 3x. On day 2, 10 μL of ABIα (lysin A, lysin B, isoamylase, and α-amylase) or BIα (lysin B, isoamylase, and α-amylase) cocktail was added to wells designated as dose 3x. On day 3, dye medium for serial dilutions was prepared with 50 mL of Middlebrook 7H9, ADC, and TWEEN, and 100× dye G. Amplified serial dilutions (GOSDs, e.g., -1, -2, -3, -4, -5, -6, -7, and -8) were prepared by aliquoting 90 μL of medium into wells of a 96-well plate, removing the assay plate from the incubator, and pipetting 10 μL of the reaction mixture into the top row of the 96-well plate. Ten-fold serial dilutions were performed in rows A through H by pipetting 10 μL (e.g., pipetting the well and mixing before diluting in the next row). The plates were then covered with lids, wrapped in parafilm, and placed in a 37°C, CO2 incubator for 72 hours. After incubation, the optical density (OD590) of the cultures was read. Experimental data are presented as histograms, with the Y-axis representing the optical density (OD590) of the culture divided by the lower limit of detection (LLoD) (OD590 / LLoD). The LLoD is calculated without a cell control for background measurement, and any value above 1 is considered significant for proliferation at that dilution.

[0108] Killing of the slow-growing mycobacterium M. intracellulare A medium solution was prepared using 5 mL of 12X PM additive solution (Biolog, Inc.), 45 mL of Middlebrook 7H9 (containing ADC supplement), and 500 μL of 100X dye G (Biolog, Inc.). M. intracellulare was treated as follows: 90 μL of the prepared medium solution was added to row A of a 96-well plate. The cell density of the M. intracellulare growth stock was determined using a 0.5 McFarland standard (approximately 1 × 10 8cfu / mL), and 10 μL of cells were added to a 96-well plate to give a 1 × 10 5 An input cell concentration of 100 cfu / well was targeted. As controls, some wells were left untouched and / or 10 μL of dialysis buffer (DB) was added. 10 μL of ABIα cocktail was added to a final concentration of 3.3 μg / well, and the plates were incubated at 37°C for 3 days. OD590 was read and the plates were returned to the incubator as described above. On day 6, the plates were incubated at 37°C and OD590 was determined. In the final step, GOSD reactions were performed with cells only, cells + DB, and cells + ABIα. To do so, 90 μL of medium solution was added to a new 96-well plate, followed by 10 μL of the reaction mixture in the top row, followed by 10-fold serial dilutions from row A to row H. The plates were incubated at 37°C for 6 days, and OD590 was read.

[0109] MIC determination of ABIα cocktail in M. Abscessus Dilute the tube of M. abscessus to Mcfarland standard 0.5 and 1 × 10 5 The antibody was diluted to 100 μL and added to the wells according to the plate map. Next, 10 μL of ABIα cocktail (3.2 μg) was added to each well. For untreated controls, an equal volume of DB was added. Next, biapenem was added to the wells in a 2-fold dilution series. All wells were brought to the appropriate volume and buffer conditions with 1x dye medium. Specifically, in a 50 mL Falcon tube, 30 mL of Middlebrook 7H9, ADC, and TWEEN; 3.3 mL of 12X PM additive; 3.3 mL of IF-01a fluid; and 400 μL of 100X dye G were added.

[0110] Plates were incubated for 24 hours at 37°C with the lid closed and Parafilm wrapped around the sides. Before every serial dilution, the static plate was shaken for at least 10 minutes and pipetted up and down to break up aggregates. Replicate GOSD experiments were performed for 96 hours at 37°C at Omnilog Biolog, Inc. In these experiments, dilutions were 1 x 10-6 It was carried out at.

[0111] Mycobacterial uptake into macrophages On day 1, macrophages were prepared as follows: confluent cultures of macrophages in T-75 flasks were decanted from the medium and replenished with fresh C-DMEM (10 mL). The cells were cultured at approximately 8 × 10 6 At cell confluency, scrape the cells from the bottom of the flask using a cell scraper and culture 1 x 10 cells in C-DMEM. 5 The diluted cells were diluted to a concentration of 1 × 10 cells / mL. 100 μL of diluted cells were added to the wells. 4 The cells were added to the wells of a 96-well tissue culture plate (for a seeding density of 100 cells / well) and the plates were incubated overnight at 37° C. in 5% CO 2 .

[0112] On day 2, macrophage uptake of M. abscessus was performed as follows: growth medium of M. abscessus strains was adjusted to a 0.5 McFarland standard and diluted 1:5 in Middlebrook 7H9, and a 96-well tissue culture plate was removed and wells were diluted three times with 100 μL of phosphate-buffered saline (PBS), 100 μL of fresh, prewarmed C-DMEM, 5 μL of 1 × 10 5M. abscessus cells were added to the wells. The plates were then incubated for 3 hours at 37°C in 5% CO2. After incubation, the cells were washed three times with 100 μL of PBS. Next, 100 μL of C-DMEM containing 250 μg / mL amikacin was added, and the plates were incubated for 1 hour. The cells were washed three times with 100 μL of PBS and supplemented with 85 μL of C-DMEM containing 50 μg / mL amikacin. For enzyme treatment, 15 μL of the prepared enzyme cocktail was added to the designated wells of the tissue culture plate. Free enzyme treatments were performed with 3.2 μg of each component of the ABIα cocktail (e.g., lysin A only, lysin B only, isoamylase only, and α-amylase only). A similar amount of encapsulated ABIα was added at an average payload loading of 50%. The plates were incubated for 72 hours at 37°C in 5% CO2.

[0113] On day 5, macrophage extraction was performed as follows: After incubation, the medium was removed and the cells were washed three times with PBS. 100 μL of 0.5% SDS was added and mixed by pipetting up and down. The plate was incubated at 37°C for 10 minutes and removed from the incubator. The wells were mixed by pipetting up and down, and the medium was transferred to a new 96-well plate. The plate was added to a Biotek shaking incubator to disaggregate the cells, and GOSD was performed on all reaction wells for CFU quantification.

[0114] Shigella phage hunt protocol and phage preparation The Shigella phage hunt protocol and phage preparation were performed as follows: A 500 mL activated sludge sample was collected from the Deerlands Wastewater Treatment Plant. Upon receipt, the sample was treated with TRITON X-100 to a final concentration of 0.1%. The bottle was inverted five times and allowed to stand for 5 minutes. The sample was aliquoted into 50 mL Falcon tubes and centrifuged at 4000 x g for 10 minutes. The supernatant was collected in a clean bottle and treated again with TRITON X-100 to a final concentration of 0.1%. For storage, 40% glycerol was added to the supernatant to a final concentration of 20%. The supernatant was aliquoted into 40 mL volumes in 50 mL Falcon tubes and stored in a -80°C freezer until use.

[0115] Forty mL of TRITON-treated, soiled supernatant stored at -80°C in 20% glycerol was thawed. To a 250 mL flask, 50 mL of TSB, 1 mM magnesium chloride, and 500 μL of overnight Shigella flexneri strain (ATCC 29903) were added. The soiled supernatant was concentrated with an Innovaprep hollow fiber pipette. The flask containing the TSB and cells was then eluted with one pump of elution fluid (0.075% TWEEN 20 + 25 mM Tris (pH 8.0)). The flask was incubated overnight at 37°C with shaking. After incubation, the cells were centrifuged at 4000 x g for 10 minutes. The supernatant was filtered through a 0.2 μm filter. To perform the plaque assay, 100 μL of cells were added to 100 μL of the concentrate and serial dilutions of the concentrate, mixed with 3 mL of 0.5% TSB top agar, and plated onto TSA plates. The plates were incubated overnight at 37°C. Plaques were then grown in 5 mL of TSB containing 1 mM CaCl2 for 24 hours. Cells and debris were pipetted for 10 minutes at 4000 × g, and the supernatant was purified with Innovaprep as before and placed in 1.5 mL of phage buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 10 mM MgCl2, 2 mM CaCl2, and 0.1% gelatin.

[0116] Killing of intracellular Shigella by Shigella phages Killing of intracellular Shigella by Shigella phages was performed as follows: On day 1, macrophage preparation was initiated by culturing confluent macrophages in a T-75 flask, which was decanted from the medium and replenished with fresh C-DMEM (10 mL). Cells were scraped from the bottom of the flask using a cell scraper (approximately 8 × 10 6 cell confluency), 1 × 10 5 The diluted cells were diluted to a concentration of 1 × 10 cells / mL. 100 μL of diluted cells were added to the wells. 4 The liposomes were added to wells of a 96-well tissue culture plate (for a seeding density of 100 cells / well), and the plate was incubated overnight at 37°C in 5% CO2. To prepare liposomes for the ABIα and Shigella experiments, a standard lipid formulation was used for all liposomes, including DOPC, DOPS, DOPE, and Chol. This formulation was generated at a 1:1 flow rate and a flow rate of 15 mL / min. The lipid solvent used included EtOH and aqueous buffer (1 M Tris, pH 8). All liposomes were dialyzed for 1 hour using a 10 kDa membrane. Specifically, a 30-minute dialysis was performed against 100 mM Tris (pH 8, 5% glycerol) and a 30-minute dialysis was performed against 10 mM Tris (pH 8, 5% glycerol). After measuring the protein concentration, a sample was taken, added to cells, diluted 1:1 in 50% IPA·50% PBS, and the absorbance was read at 280 nm on a Nanodrop.

[0117] On day 2, bacterial uptake into macrophages was determined by adjusting the culture to a 0.5 McFarland standard (confirmed by turbidity meter) using a growing culture of S. flexneri. The culture was diluted 1:5 in Middlebrook 7H9 and transferred to a 96-well tissue culture plate. The wells were then washed with 100 μL of PBS. 100 μL of fresh, prewarmed C-DMEM and 5 μL of the prepared culture were added to the wells. The plate was incubated at 37°C in 5% CO2 for 3 hours. After incubation, the cells were washed three times with 100 μL of PBS. 100 μL of C-DMEM containing 250 μg / mL amikacin was added and incubated for 1 hour. The cells were then washed with 100 μL of PBS and replenished with 85 μL of C-DMEM containing 50 μg / mL amikacin. Controls were left without amikacin. For post-treatment with unencapsulated (eg, free) or encapsulated payload, 10 μL of prepared payload was added to designated wells of the tissue culture plate.

[0118] Example 2: Delivery of antimicrobial lytic proteins mediates effective killing of mycobacterial cells This example describes the demonstration of a dose-dependent enzyme cocktail capable of attenuating mycobacterial cell replication.

[0119] Materials and Methods Materials and methods and cell lines are described in Example 1. result Screening of bacterial cell growth in a growth serial dilution series (GOSD) (Figure 1) revealed that Mycobacterium abscessus (M. abscessus) was killed in a dose-dependent manner when treated with single or multiple doses of the enzyme cocktail lysin A (A), lysin B (B), isoamylase (I), and α-amylase (α) (ABIα), as assessed by optical density (OD; Figure 2A) and cell counts (Figure 2B), compared with untreated controls. A dose-dependent experiment was performed to determine whether different doses of ABIα treatment caused M. abscessus to become reproductively incompetent. Figure 3 shows the dose-dependent experiment, in which treatment with 3.2 μg of ABIα was observed to eliminate M. abscessus. Similar experiments were performed with the slow-growing mycobacterium species M. intracellulare, and cell growth was monitored over several days (Figure 4B), revealing that ABIα treatment resulted in sustained attenuation of cell growth (Figure 4A). In similar studies, the ABIα cocktail was found to effectively attenuate cell growth of M. avium, M. fortuitum, M. goodii, M. masiliense, M. boletti, M. chimera, and M. smegmatis (data not shown). Together, these results demonstrate dose-dependent sterilization of mycobacteria by ABIα in an in vitro death / growth assay.

[0120] Example 3: Delivery of antimicrobial lytic proteins and antibiotics mediates synergistic killing of mycobacterial cells This example describes the synergistic effect of the combination of ABIα enzyme cocktail and antibiotics in attenuating mycobacterial production.

[0121] Materials and Methods Materials and methods and cell lines are described in Example 1. result Figure 5 shows the tabular quantification of M. abscessus cell proliferation assays after treatment with ABIα and the antibiotics amikacin, biapenem, cefoxitin, ethambutol, moxifloxacin, rifampicin, or clarithromycin, respectively. Using the fractional inhibitory concentration (FIC) index for quantification, it was revealed that ABIα in combination with amikacin, biapenem, cefoxitin, or moxifloxacin caused a synergistic effect in attenuating M. abscessus growth. Together, these results demonstrate that the ABIα enzyme cocktail in combination with various chemical antibiotics causes a synergistic effect in killing mycobacterial cells.

[0122] Example 4: Delivery of encapsulated antimicrobial lytic proteins mediates highly effective killing of mycobacteria in infected macrophages This example describes the improved effectiveness of encapsulated enzyme cocktails in attenuating the replication of intracellularly localized mycobacteria within infected macrophages.

[0123] Materials and Methods Materials and methods and cell lines are described in Example 1. result To identify the most effective combinations of enzymes (lysin A (A), lysin B (B), isoamylase (I), or α-amylase (α)) and whether their encapsulation improved their effectiveness in attenuating mycobacterial replication (Fig. 6), we measured the optical density of M. abscessus after extraction from infected macrophages treated with unencapsulated ABIα, AB, Iα, or BIα, or encapsulated ABIα, ABIα, Iα, or BIα (encABIα, encAB, encIα, and encBIα, respectively; Fig. 7). We observed that the encapsulated ABIα cocktail showed improved killing of M. abscessus compared with unencapsulated ABIα and all other partial cocktails (Fig. 8). Together, these results demonstrate that a single dose of encapsulated ABIα cocktail killed 99% of intracellular M. abscessus compared to 90% killing with unencapsulated ABIα.

[0124] Example 5: Delivery of encapsulated bacteriophages mediates highly effective killing of mycobacteria in infected macrophages This example describes the effectiveness of encapsulated Shigella bacteriophage in attenuating the replication of intracellularly localized S. flexneri within infected macrophages.

[0125] Materials and Methods Materials and methods and cell lines are described in Example 1. result To determine whether Shigella bacteriophage could be used to attenuate S. flexneri replication in infected macrophages, experiments were performed in which macrophages were treated with S. flexneri and unencapsulated or encapsulated Shigella phage encoding a therapeutic payload (EPH34) (Figure 9). After a 48-hour incubation period, cells were extracted, and GOSD was quantified overnight. It was observed that encapsulated Shigella phage EPH34 (encPhage) showed no growth after extraction from macrophages, whereas unencapsulated phage EPH34 showed growth at a -1 dilution (Figure 10). Together, these results demonstrated that encPhage provided the most effective reduction of S. flexneri in infected macrophages, as indicated by a 10-fold decrease in optical density.

[0126] Other embodiments All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that a term in this application is found to be defined differently than in a document incorporated herein by reference, the definition provided herein shall serve as the definition of that term.

[0127] While the invention has been described in connection with particular embodiments thereof, it will be understood that the invention is capable of further modifications, and that this application is intended to cover generally any variations, uses, or adaptations of the invention in accordance with the principles of the invention and to include departures from the present disclosure which become known or customarily practiced in the art to which this invention pertains, as applicable to the essential features described above, and which comply with the scope of the appended claims.

Claims

1. 1. A composition comprising a supramolecular structure comprising two or more antimicrobial lytic proteins, wherein the supramolecular structure has a Z average mean particle size of about 75 nm to about 750 nm, the supramolecular structure is a lipid nanoparticle, a micelle, or a liposome, and the two or more antimicrobial lytic proteins comprise a lysin and an amylase.

2. The composition of claim 1 further comprising a targeting moiety.

3. A composition comprising a supramolecular structure comprising a targeting moiety and a cargo comprising two or more antimicrobial lytic proteins, wherein the supramolecular structure is a lipid nanoparticle, a micelle, or a liposome, and the two or more antimicrobial lytic proteins comprise a lysin and an amylase.

4. (a) the amylase is an α-amylase or an isoamylase; and (b) the lysin is lysin A or lysin B; The composition according to any one of claims 1 to 3, which is at least one of the following:

5. The composition of any one of claims 2 to 4 (provided that claim 4 is limited to any portion that cites claim 2 or 3), wherein the targeting moiety is an extracellular targeting moiety that targets professional antigen-presenting cells.

6. The composition of claim 5 , wherein the professional antigen-presenting cells are macrophages or dendritic cells.

7. the targeting moiety is (a) phosphatidylserine, (b) an antibody or antigen-binding fragment thereof; (c) pathogen-associated molecular patterns (PAMPs); (d) a mannose cluster or a folate, or (e) TLR2 agonist The composition according to any one of claims 2 to 6 (however, claim 4 is limited to the part that recites claim 2 or 3), comprising:

8. (a) the antibody or antigen-binding fragment thereof is selected from the group consisting of anti-CD163, anti-CD40, anti-CD74, anti-CD206, anti-CD123, anti-DEC205, anti-CD304, anti-CD303, and anti-BDCA2 antibodies, and antigen-binding fragments thereof; or 8. The composition of claim 7, wherein (b) the TLR2 agonist is selected from the group consisting of MALP-2 lipoprotein, MALP-404 lipoprotein, OspA, porin, LcrV, Hsp60, glycoprotein gH / gL, or glycoprotein gB.

9. The composition according to any one of claims 1 to 8, wherein the supramolecular structure is a liposome, and the liposome is unilamellar or multilamellar.

10. The composition according to any one of claims 1 to 9, wherein the supramolecular structure comprises one or more lipids.

11. 11. The composition of claim 10, wherein at least one of the one or more lipids is an ionizable lipid.

12. 12. The composition of any one of claims 1 to 11, wherein the two or more antimicrobial lytic proteins are capable of killing Mycobacterium, Salmonella, Neisseria, Brucella, Escherichia, Listeria, Frankicella, Legionella, Yersinia, Staphylococcus, Clostridium, Shigella, or Streptococcus species.

13. (a) the Mycobacterium species is M. tuberculosis, M. leprae, M. lepromatosis, M. avium, M. kansasii, M. fortuitum, M. chelonae, M. marinum, or M. abscessus; (b) the Salmonella species is S. enterica, S. typhimurium, or S. bongori; (c) the Neisseria species is N. gonorrhoeae or N. meningitidis; (d) the Brucella species is B. melitensis, B. abortus, B. suis, or B. canis; (e) the Escherichia species is E. coli; (f) the Listeria species is L. monocytogenes; (g) the Frankicera species is F. tularensis, F. novicida, or F. philomiragia; (h) the Legionella species is L. pneumophila; (i) the Yersinia species is Y. pestis or Y. enterocolitica; (j) the Staphylococcus species is S. aureus; (k) the Clostridium species is C. botulinum, C. perfringens, C. tetani, or C. sordellii; (l) the Shigella spp. is S. dysenteriae, S. flexneri, S. boydii, or S. sonnei; or (m) The composition of claim 12, wherein the Streptococcus species is S. pyogenes, S. agalactiae, S. dysgalactiae, S. bovis, S. anginosus, S. sanguinis, S. mitis, S. mutans, or S. pneumoniae.

14. 14. A composition according to any one of claims 1 to 13 for use in delivering the two or more antimicrobial lytic proteins to a target intracellular compartment comprising a bacterial cell within a professional antigen-presenting cell of a subject.

15. A composition according to any one of claims 1 to 13 for use in the treatment of an intracellular bacterial infection caused by bacterial cells.

16. 16. The composition of claim 14 or 15, wherein the bacterial cells are Mycobacterium, Salmonella, Neisseria, Brucella, Escherichia, Listeria, Frankincella, Legionella, Yersinia, Staphylococcus, Clostridium, Shigella, or Streptococcus species.

17. (a) the Mycobacterium species is M. tuberculosis, M. leprae, M. lepromatosis, M. avium, M. kansasii, M. fortuitum, M. chelonae, M. marinum, or M. abscessus; (b) the Salmonella species is S. enterica, S. typhimurium, or S. bongori; (c) the Neisseria species is N. gonorrhoeae or N. meningitidis; (d) the Brucella species is B. melitensis, B. abortus, B. suis, or B. canis; (e) the Escherichia species is E. coli; (f) the Listeria species is L. monocytogenes; (g) the Frankicera species is F. tularensis, F. novicida, or F. philomiragia; (h) the Legionella species is L. pneumophila; (i) the Yersinia species is Y. pestis or Y. enterocolitica; (j) the Staphylococcus species is S. aureus; (k) the Clostridium species is C. botulinum, C. perfringens, C. tetani, or C. sordellii; (l) the Shigella spp. is S. dysenteriae, S. flexneri, S. boydii, or S. sonnei; or (m) The composition of claim 16, wherein the Streptococcus species is S. pyogenes, S. agalactiae, S. dysgalactiae, S. bovis, S. anginosus, S. sanguinis, S. mitis, S. mutans, or S. pneumoniae.

18. The composition of any one of claims 14 to 17, wherein the use further comprises administering an antibiotic.

19. 19. The composition of claim 18, wherein the antibiotic is selected from the group consisting of cephalosporins, carbapenems, penicillins, fluoroquinolones, thiacetazone, sq-109, bedaquiline, delamanid, pyrazinamide, isoniazid, azithromycin, clarithromycin, ethambutol, rifampin, and amikacin.

20. The composition of any one of claims 14 to 19, wherein the composition is formulated for intravenous administration, oral administration, or inhalation.

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