Compositions and methods for treating actinomycete infections

Abstract

The present disclosure features compositions and methods for the treatment of actinomycete (e.g., Corynebacteriales) infections caused by mycobacterial cells residing within a host cell (e.g., a mammalian cell, such as an immune cell, e.g., a macrophage or dendritic cell). The compositions and methods involve delivering a mixture of lytic enzymes to specifically target the cell envelope of a group of related bacterial species within intracellular compartments (endosomes, phagosomes, lysosomes, or cytosol) where bacteria of the aforementioned orders infect, grow, hide, and rest.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. A copy of said XML, created on April 5, 2023, is named 51486-005WO3_Sequence_Listing_4_5_23.XML and is 660,322 bytes in size. [Background technology]

[0002] Bacterial pathogens are a major cause of infectious diseases. Many bacteria are successfully detected by the human immune system and quickly removed before infection begins. However, many bacterial pathogens evade the host immune system by living within host cells. These intracellular bacteria have developed diverse immune evasion techniques by living and multiplying 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 propagate within host cells often present a challenging treatment barrier due to a lack of accessibility to the intracellular site of infection. Although 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.

[0003] One group of challenging intracellular bacterial infections is caused by mycobacteria. Mycobacteria are actinomycetes (e.g., Corynebacteriales or Propionibacteriales) that are characterized by a thick cell wall that is rich in mycolic acids. Mycobacteria contain an envelope that contains a cell membrane composed of a lipid bilayer, a cell wall that includes a peptidoglycan layer and an arabinogalactan layer, and an outer membrane that contains 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 durability of mycobacteria and is 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. Nonetheless, the number of NTM infections is a growing health concern, especially among people with lung disease.

[0004] There is a need for improved compositions and methods that target and treat intracellular bacterial infections, such as those caused by mycobacteria. Summary of the Invention

[0005] In one aspect, the present invention relates to (a) lysin A comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 to 182; (b) lysin B comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183 to 241; and (c) lysin B comprising at least one amino acid sequence having at least one of SEQ ID NOs: 242 to 392. and (d) an isoamylase comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393 to 445; and a supramolecular structure comprising one or more (e.g., two or more, three or more, or all four) of α-amylases comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393 to 445. In some embodiments, the invention provides a lysin A comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2; (b) a lysin B comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:183 or SEQ ID NO:184; or (c) a lysin B comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:242 or SEQ ID NO:243. and (d) an isoamylase comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393 to 398; and a supramolecular structure comprising one or more (e.g., two or more, three or more, or all four) of an α-amylase comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393 to 398.

[0006] In some embodiments, the composition comprises lysin A and lysin B, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, and lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241.

[0007] In some embodiments, the composition comprises lysin A and lysin B, where lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, and lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:183 or SEQ ID NO:184.

[0008] In some embodiments, the composition comprises lysin A and isoamylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392.

[0009] In some embodiments, the composition comprises lysin A and isoamylase, where lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:242 or SEQ ID NO:243.

[0010] In some embodiments, the composition comprises lysin A and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

[0011] In some embodiments, the composition comprises lysin A and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs:393-398.

[0012] In some embodiments, the composition comprises lysin B and isoamylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392.

[0013] In some embodiments, the composition comprises lysin B and isoamylase, where lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243.

[0014] In some embodiments, the composition comprises lysin B and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

[0015] In some embodiments, the composition comprises lysin B and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184, and the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-398.

[0016] In some embodiments, the composition comprises an isoamylase and an α-amylase, wherein the isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392, and the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

[0017] In some embodiments, the composition comprises an isoamylase and an α-amylase, wherein the isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:242 or SEQ ID NO:243, and the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs:393-398.

[0018] In some embodiments, the composition comprises lysin A, lysin B, and isoamylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392.

[0019] In some embodiments, the composition comprises lysin A, lysin B, and isoamylase, where lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:183 or SEQ ID NO:184, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:242 or SEQ ID NO:243.

[0020] In some embodiments, the composition comprises lysin A, lysin B, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

[0021] In some embodiments, the composition comprises lysin A, lysin B, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:183 or SEQ ID NO:184, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs:393-398.

[0022] In some embodiments, the composition comprises lysin A, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

[0023] In some embodiments, the composition comprises lysin A, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, wherein the isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:242 or SEQ ID NO:243, and wherein the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs:393-398.

[0024] In some embodiments, the composition comprises lysin B, isoamylase, and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

[0025] In some embodiments, the composition comprises lysin B, isoamylase, and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184, wherein the isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243, and wherein the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-398.

[0026] In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, and lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241. The isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242 to 392, and the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393 to 445.

[0027] In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:183 or SEQ ID NO:184, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:242 or SEQ ID NO:243, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs:393-398.

[0028] In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and α-amylase, wherein lysin A comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, lysin B comprises the amino acid sequence of SEQ ID NO:183 or SEQ ID NO:184, isoamylase comprises the amino acid sequence of SEQ ID NO:242 or SEQ ID NO:243, and α-amylase comprises the amino acid sequence of any one of SEQ ID NOs:393-398.

[0029] In some embodiments, the composition comprises lysin A of SEQ ID NO:1, lysin B of SEQ ID NO:183, an isoamylase of SEQ ID NO:242, and an α-amylase of SEQ ID NO:393. In some embodiments, the composition comprises lysin A of SEQ ID NO:2, lysin B of SEQ ID NO:184, an isoamylase of SEQ ID NO:243, and an α-amylase of SEQ ID NO:394.

[0030] In some embodiments, the composition comprises lysin A of SEQ ID NO:1, lysin B of SEQ ID NO:183, an isoamylase of SEQ ID NO:242, and an α-amylase of SEQ ID NO:395. In some embodiments, the composition comprises lysin A of SEQ ID NO:2, lysin B of SEQ ID NO:184, an isoamylase of SEQ ID NO:243, and an α-amylase of SEQ ID NO:396.

[0031] In some embodiments, the composition comprises lysin A of SEQ ID NO:1, lysin B of SEQ ID NO:183, an isoamylase of SEQ ID NO:242, and an α-amylase of SEQ ID NO:397. In some embodiments, the composition comprises lysin A of SEQ ID NO:2, lysin B of SEQ ID NO:184, an isoamylase of SEQ ID NO:243, and an α-amylase of SEQ ID NO:398.

[0032] In some embodiments according to any of the above aspects, the Z average average particle size of the supramolecular structure is 75 nm to 5 μm, for example, 75 nm to 2 μm, 75 nm to 1 μm, for example, 75 nm to 750 nm (for example, 250 nm to 750 nm, or 75 nm to 250 nm). In some embodiments, when the supramolecular structure is an LNP or a micelle, the Z average average particle size is 75 nm to 250 nm. In some embodiments, when the supramolecular structure is a vesicle (for example, a liposome), the Z average average particle size is 250 nm to 750 nm. Non-limiting examples of the Z average mean particle size include, for example, 75 nm to 100 nm, for example, 75 nm to 85 nm, for example, 80 nm, for example, 80 nm to 140 nm, 90 nm to 130 nm, or 110 nm to 130 nm, for example, 120 nm, for example, 200 nm to 300 nm, for example, 250 nm to 300 nm, 260 nm to 290 nm, 260 nm to 280 nm, 265 nm to 275 nm, for example, 270 nm, For example, 300 nm to 400 nm, 400 nm to 600 nm, for example, 450 nm to 550 nm, 475 nm to 525 nm, 480 nm to 520 nm, 490 nm to 510 nm, 495 nm to 505 nm, for example, 500 nm, for example, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150nm, 155nm, 160nm, 165nm, 170nm, 175nm, 180nm, 185nm, 190nm, 195nm, 200nm, 205nm, 210nm, 215nm, 220nm, 225nm, 230nm, 235nm, 240nm, 245nm, 250nm, 255nm, 260nm, 265nm, 270nm, 275nm, 280nm, 285nm, 290nm, 295nm, 300n m, 305nm, 310nm, 315nm, 320nm, 325nm, 330nm, 335nm, 340nm, 345nm, 350nm, 355nm, 360nm, 365nm, 370nm, 375nm, 38 0nm, 385nm, 390nm, 395nm, 400nm, 405nm, 410nm, 415nm, 420nm, 425nm, 430nm, 435nm, 440nm, 445nm, 450nm, 455nm,460nm, 465nm, 470nm, 475nm, 480nm, 485nm, 490nm, 495nm, 500nm, 505nm, 510nm, 515nm, 520nm, 525nm, 530nm, 535nm, 540nm, 545nm, 550 nm, 555nm, 560nm, 565nm, 570nm, 575nm, 580nm, 585nm, 590nm, 595nm, 600nm, 605nm, 610nm, 615nm, 620nm, 625nm, 630nm, 635nm, 640nm, 6 45nm, 650nm, 655nm, 660nm, 665nm, 670nm, 675nm, 680nm, 685nm, 690nm, 695nm, 700nm, 705nm, 710nm, 715nm, 720nm, 725nm, 730nm, 735n m, 740nm, 745nm, 750nm, 755nm, 760nm, 765nm, 770nm, 775nm, 780nm, 785nm, 790nm, 795nm, 800nm, 805nm, 810nm, 815nm, 820nm, 825nm, 83 0nm, 835nm, 840nm, 845nm, 850nm, 855nm, 860nm, 865nm, 870nm, 875nm, 880nm, 885nm, 890nm, 895nm, 900nm, 905nm, 910nm, 915nm, 920nm , 925nm, 930nm, 935nm, 940nm, 945nm, 950nm, 955nm, 960nm, 965nm, 970nm, 975nm, 980nm, 985nm, 990nm, 995nm, 1μm, 1.1μm, 1.2μm, 1.3μm , 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2 μm, 2.1 μm, 2.2 μm, 2.3 μm, 2.4 μm, 2.5 μm, 2.6 μm, 2.7 μm, 2.8 μm, 2.9 μm, 3 μm, 3.1 μm, 3.2 μm, 3.3 μm, 3.4 μm, 3.5 μm, 3.6 μm, 3.7 μm, 3.8 μm, 3.9 μm, 4 μm, 4.1 μm, 4.2 μm, 4.3 μm, 4.4 μm, 4.5 μm, 4.6 μm, 4.7 μm, 4.8 μm, 4.9 μm, or 5 μm. In some embodiments, the supramolecular structure has a Z average mean particle size of 80 nm, 270 nm, or 500 nm.The Z average average particle size is from 75 nm to 750 nm. In some embodiments, the Z average average particle size is from 250 nm to 750 nm. In some embodiments, the Z average average particle size is from 75 nm to 250 nm.

[0033] 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. Alternatively, the liposome can be multilamellar.

[0034] In some embodiments, the supramolecular structure comprises a polydispersity index of 0.05 to 0.3. In some embodiments, the supramolecular structure comprises one or more lipids. In some embodiments, at least one of the one or more lipids can be, for example, an ionizable lipid.

[0035] In some embodiments, the lipid can be, for example, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS).

[0036] In some embodiments, the lipid is a sterol, such as cholesterol or a derivative thereof. In some embodiments, the composition comprises a mixture of lipids. For example, the mixture of lipids can include two or more of DOPC, DOPE, DOPS, and cholesterol.

[0037] In some embodiments, DOPC and DOPE are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0038] In some embodiments, DOPC and DOPS are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0039] In some embodiments, the DOPC and cholesterol are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0040] In some embodiments, DOPE and DOPS are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0041] In some embodiments, DOPE and cholesterol are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0042] In some embodiments, DOPS and cholesterol are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0043] In some embodiments, DOPC, DOPE, DOPS, and cholesterol are present in a molar ratio of 1-20:1-20:1-5:1-5. For example, in some embodiments, DOPC, DOPE, DOPS, and cholesterol are present in a molar ratio of 10:10:3:4.

[0044] In some embodiments, the composition comprises a lipid concentration of 0.03 mg / mL to 10 mg / mL, e.g., 0.1 mg / mL to 10 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL). In some embodiments, the composition comprises a lipid concentration of 1 mg / mL to 5 mg / mL.

[0045] In some embodiments, the composition comprises a concentration of 0.1 mg / mL to 20 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, In some embodiments, the composition comprises a protein (e.g., lysin A, lysin B, isoamylase, and / or α-amylase) at a concentration of 1 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, e.g., 10 mg / mL to 20 mg / mL, e.g., 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL). In some embodiments, the composition comprises a protein (e.g., lysin A, lysin B, isoamylase, and / or α-amylase at a concentration of 1 mg / mL to 10 mg / mL.

[0046] In some embodiments, the compositions described herein are formulated with one or more buffers and / or excipients. For example, the compositions (e.g., supramolecular structures, e.g., liposomes containing a cocktail of lytic enzymes) can be encapsulated and / or formulated in buffers such as glycine, Tris, sodium citrate, sodium acetate, and MES, for example, at a concentration of 10 mM to 200 mM, e.g., 50 mm to 150 mm, e.g., 10 mM, 20 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM. The composition can be formulated at a pH of 5-11 (e.g., 5-6, e.g., 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6, e.g., 6-11, e.g., 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11). The composition can further comprise one or more excipients, such as CaCl2, arginine, NaCl, sodium citrate, MgCl2, or glycerol. In some embodiments, the composition comprises, for example, 5 mM-10 mM CaCl2, 0-50 mM arginine, 0-200 mM NaCl, 0-1 mM sodium citrate, 0-1 mM MgCl2, and / or 10-30% glycerol. In some embodiments, the composition comprises 50 mM glycine (pH 8.5), 7.5 mM CaCl2, 0.5 mM MgCl2, 200 mM NaCl, 0.33 mM sodium citrate, and 10% glycerol. The formulation may further comprise a Tween, e.g., Tween-80.

[0047] In some embodiments, the composition further comprises a targeting moiety. The targeting moiety can be, for example, an extracellular targeting moiety that targets professional antigen-presenting cells (e.g., macrophages or dendritic cells). In some embodiments, the targeting moiety is phosphatidylserine.

[0048] In another aspect, the invention relates to a method of treating a bacterial infection in a subject, the method comprising administering to the subject a composition described herein, e.g., a composition of any of the above embodiments, in an amount and for a duration sufficient to treat the bacterial infection.

[0049] In some embodiments, the bacterial infection is caused by an Actinomycete bacterium. In some embodiments, the Actinomycete is a Corynebacteriales or Propionibacteriales. In some embodiments, the Corynebacteriales is a Mycobacterium species.

[0050] In some embodiments, the Mycobacterium species is M. tuberculosis, M. leprae, M. lepromatosis, M. avium, M. kansasii, M. fortuitum, M. chelonae, M. marinum, M. intracellulare, M. abscessus, M. chimera, M. boletti, M. fortuitum, M. goodii, or M. masiliense.

[0051] In some embodiments, the Corynebacteriales is a Nocardia, Corynebacterium, or Rhodococcus species. In some embodiments, the Propionibacteriales is a Cutibacterium species.

[0052] For example, in some embodiments, the compositions and methods described herein can be used to target other actinomycetes (e.g., Corynebacteriales or Propionibacteriales) that have similar envelope components as mycobacteria. For example, the compositions and methods can be used to target Nocardia, Corynebacterium, or Rhodococcus species. For example, Nocardia species can be, for example, N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. asteroides, N. brasiliensis, and N. caviae. Corynebacterium species can be, for example, C. glutamicum or C. diphtheriae. Rhodococcus species can be, for example, R. fascians or R. equi.

[0053] In some embodiments, the compositions and methods can be used to target Cutibacterium species, which can be, for example, C. acnes.

[0054] In some embodiments, the method further comprises administering an antibiotic. In some embodiments, the antibiotic is a cephalosporin, a carbapenem, a penicillin, an aminoglycoside, a cephalosporin, a rifamycin, a macrolide, or a fluoroquinolone. In some embodiments, the antibiotic is a thiacetazone, sq-109, bedaquiline, delamanid, pyrazinamide, or isoniazid. In some embodiments, the antibiotic is an azithromycin, clarithromycin, ethambutol, rifampin, biapenem, or amikacin. In some embodiments, the antibiotic is a macrolide (e.g., azithromycin, clarithromycin, erythromycin). In some embodiments, the antibiotic is a macrolide (e.g., azithromycin, clarithromycin, erythromycin). In some embodiments, the antibiotic is an aminoglycoside (eg, kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin (eg, neomycin B, C, or E), streptomycin, or plazomicin).

[0055] In some embodiments, the compositions are administered intravenously, orally, or by inhalation (eg, via aerosol). In another aspect, the invention relates to a method of assembly, the method comprising providing a sample comprising two or more (e.g., two, three, or four) of lysin A, lysin B, isoamylase, and α-amylase, the method further comprising mixing the sample with one or more lipids in the presence of an organic solvent to form liposomes comprising two or more of lysin A, lysin B, isoamylase, and α-amylase.

[0056] In some embodiments, lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182; lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241; isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392; and / or α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

[0057] In some embodiments, lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2; lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:183 or SEQ ID NO:184; isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:242 or SEQ ID NO:243; and / or α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs:393-398.

[0058] In some embodiments, the composition comprises lysin A and lysin B, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, and lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241.

[0059] In some embodiments, the composition comprises lysin A and lysin B, where lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, and lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:183 or SEQ ID NO:184.

[0060] In some embodiments, the composition comprises lysin A and isoamylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392.

[0061] In some embodiments, the composition comprises lysin A and isoamylase, where lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:242 or SEQ ID NO:243.

[0062] In some embodiments, the composition comprises lysin A and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

[0063] In some embodiments, the composition comprises lysin A and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs:393-398.

[0064] In some embodiments, the composition comprises lysin B and isoamylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392.

[0065] In some embodiments, the composition comprises lysin B and isoamylase, where lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243.

[0066] In some embodiments, the composition comprises lysin B and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

[0067] In some embodiments, the composition comprises lysin B and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184, and the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-398.

[0068] In some embodiments, the composition comprises an isoamylase and an α-amylase, wherein the isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392, and the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

[0069] In some embodiments, the composition comprises an isoamylase and an α-amylase, wherein the isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:242 or SEQ ID NO:243, and the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs:393-398.

[0070] In some embodiments, the composition comprises lysin A, lysin B, and isoamylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392.

[0071] In some embodiments, the composition comprises lysin A, lysin B, and isoamylase, where lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:183 or SEQ ID NO:184, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:242 or SEQ ID NO:243.

[0072] In some embodiments, the composition comprises lysin A, lysin B, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

[0073] In some embodiments, the composition comprises lysin A, lysin B, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:183 or SEQ ID NO:184, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs:393-398.

[0074] In some embodiments, the composition comprises lysin A, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

[0075] In some embodiments, the composition comprises lysin A, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, wherein the isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:242 or SEQ ID NO:243, and wherein the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs:393-398.

[0076] In some embodiments, the composition comprises lysin B, isoamylase, and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

[0077] In some embodiments, the composition comprises lysin B, isoamylase, and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184, wherein the isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243, and wherein the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-398.

[0078] In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, and lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241. The isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242 to 392, and the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393 to 445.

[0079] In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:183 or SEQ ID NO:184, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:242 or SEQ ID NO:243, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs:393-398.

[0080] In some embodiments, the organic solvent comprises ethanol. In some embodiments, ethanol is provided to the aqueous portion of the sample in a ratio of 10:1 to 1:1. For example, in some embodiments, the ratio is 8:1 to 3:1.

[0081] In some embodiments, ethanol is provided to the aqueous portion of the sample in a ratio of 1:10 to 1:1. For example, in some embodiments, the ratio is 1:8 to 1:3.

[0082] definition As used herein, the term "about" means ±10% of the recited value.

[0083] As used herein, "combination therapy" or "co-administration" means that two (or more) agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen defines the dosage and dosing period of each agent so that the effects of the individual agents on the subject overlap and / or synergize. In some embodiments, the delivery of two or more agents is simultaneous or parallel, and the agents may be formulated in admixture. In some embodiments, the two or more agents are not co-formulated, but are administered sequentially as part of a prescribed regimen. In some embodiments, the 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 the other. 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.

[0084] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of an agent, e.g., to produce a therapeutic effect as described herein in a cell, sample, or subject, refer to an amount sufficient to produce a beneficial or desired result, including a preclinical or clinical result, when administered to a cell, sample, or subject, including a human, and thus such an "effective amount," or synonyms thereof, depend on the context in which the term is used. For example, in the context of treating a disorder, the term is an amount of an 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 a variety of factors, e.g., the given agent, pharmaceutical formulation, route of administration, severity of bacterial infection, biomarkers of the subject, sample, or host cell, e.g., mammalian immune cell, being treated, e.g., age, sex, and / or weight, 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 is 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.

[0085] As used herein, the term "antimicrobial lytic protein" refers to a protein that has bactericidal and / or bacteriolytic 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), capsule depolymerases (e.g., hydrolases, metallohydrolases, epoxide hydrolases, peptidoglycan hydrolases, polysaccharases, polysaccharide lyases, endosialidases, hyaluronan lyases, or alginate lyases), beta-lactamases, and lysozymes.

[0086] As used herein, a "lipid nanoparticle" or "LNP" is a vesicle that includes a lipid layer that encapsulates a substantially solid lipid core, which may contain a pharma- ceutically active molecule. LNPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent aggregation of the particle (e.g., PEG-lipid conjugates).

[0087] 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 antimicrobial lytic protein and other components. The lipophilic material separates the aqueous exterior from the aqueous interior, and typically does not contain phage proteins, but may in some cases. Liposomes also include "sterically stabilized" liposomes, which term, as used herein, refers to liposomes that contain one or more specialized lipids that, when incorporated into the liposome, result in improved circulation life compared to liposomes lacking such specialized lipids.

[0088] "Micelle" is defined herein as a particular type of substantially spherical supramolecular structure in which amphiphilic molecules, e.g., lipids, are arranged such 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 multiple proteins.

[0089] The term "subject" as used herein refers to any organism to which the compositions according to the present invention may be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Exemplary subjects include any animal, e.g., mammals such as mice, rats, rabbits, non-human primates, and humans. A subject may be a subject seeking or needing treatment, a subject in need of treatment, a subject undergoing treatment, a subject who will undergo treatment in the future, or a human or animal under the care of a trained professional for a particular disease or condition.

[0090] 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 pi-pi interactions. Supramolecular structures can include, for example, large complexes of molecules that form globular-like structures. Supramolecular structures include, for example, lipid-based supramolecular structures, such as liposomes, lipid nanoparticles, and micelles.

[0091] 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, targeting moieties can be used to target the supramolecular structures described herein, for example, to professional antigen-presenting cells (e.g., macrophages or dendritic cells).

[0092] 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) usually constitute at least one shell of the vesicle. In this shell, the amphiphilic molecules are arranged within a bilayer membrane, with the hydrophilic parts of the amphiphilic molecules facing outward relative to the plane of the bilayer membrane, and the hydrophobic parts of the amphiphilic molecules are primarily arranged within the bilayer membrane. The reverse arrangement exists when the surrounding medium is hydrophobic.

[0093] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0094] [Figure 1] A shows the growth curve of mycobacteria over a 5-day period in the presence of ABIα, and B shows the growth curve of mycobacteria over a 5-day period in the absence of ABIα. [Diagram 2] FIG. 2A is a brightfield image of mycobacterial rod-shaped cells that have been shredded by ABIα into intracellular fragments containing stainable lipids that give rise to red chromosomes isolated from genomic DNA. The experiment was performed at pH 6.2. Untreated mycobacterial cells containing fragment ratios are shown. Lipids are shown in red and DNA in green. The green stained DNA indicates that the compromised cell envelope has provided access to the dye. Intact cells do not readily take up the red dye. FIG. 2B is a brightfield image of mycobacterial rod-shaped cells that have been shredded by ABIα into intracellular fragments containing stainable lipids that give rise to red chromosomes isolated from genomic DNA. The experiment was performed at pH 6.2. ABIα treated cells and fragments are shown. Lipids are shown in red and DNA in green. The intracellular fragments glow with the lipophilic red dye. The green DNA is spread out compared to FIG. 2A. [Diagram 3]A shows a bright field image similar to that of Fig. 2A, except that the experiment was performed at pH 5.3. B shows a bright field image similar to that of Fig. 2B, except that the experiment was performed at pH 5.3. [Figure 4] FIG. 4A is a brightfield image showing ABIα mechanism by fragmenting mycobacteria into fragments. Untreated mycobacterial cells containing fragments are shown. FM4-64 is a dye that stains lipids, envelopes, and lipid bodies. FIG. 4B is a brightfield image showing ABIα mechanism by fragmenting mycobacteria into fragments. Treated mycobacterial cells with fragments are shown. Cytox-Green stains DNA in cells with compromised envelopes. [Figure 5A] FIG. 1 is a schematic diagram showing a liposome containing ABIα. [Figure 5B] Dynamic light scattering analysis showed a mean hydrodynamic diameter of 413 nM and a polydispersity index of 0.19, indicating high homogeneity. [Figure 6] 1 is a photograph of a gel showing the ABIα enzyme within liposomes. [Figure 7] 1 is a table showing the activity of ABIα liposome formulations. [Figure 8] Photograph of a gel showing partially purified ABIα liposomes with improved encapsulation. [Figure 9] FIG. 1 is a schematic showing the proposed mechanism of ABIα killing mycobacteria inside macrophages. [Figure 10]Graph showing treatment of J774A.1 murine macrophages (MΦ) uninfected or infected (MOI=10:1) with Mycobacterium abscessus with ABI α-PPL Formulation 8. The amount of necrotic cell death was measured for MΦ to investigate the safety and efficacy of ABI α-PPL treatment for mycobacterial infection. The extent of necrosis for MΦ was quantified as the ratio of the number of cells stained with SYTOX™ Green Nucleic Acid Stain (Invitrogen™) and Hoechst-33342 (Invitrogen™) in the population and counted per FOV (N=2-3 per well) by HCIA platform (BZ-X800; Keyence). A reduction in necrosis was observed in both uninfected (Form8 MΦ only) and infected (Form8-treated IIM) Formulation 8-treated groups compared to untreated control (untreated IIM). IIM: internally infected MΦ; Form8: formulation 8; HCIA: high content imaging analysis; FOV: field of view of imaging. [Figure 11]Graph showing treatment of J774A.1 murine macrophages (MΦ) uninfected or infected (MOI=10:1) with Mycobacterium abscessus with ABI α-PPL formulation 8. The amount of necrotic cell death was measured for MΦ to investigate the safety and efficacy of ABI α-PPL treatment for mycobacterial infection. The extent of necrosis for MΦ was quantified as the ratio of the number of cells stained with SYTOX™ Green Nucleic Acid Stain (Invitrogen™) and Hoechst-33342 (Invitrogen™) in the population and counted per FOV (N=3 per well) by HCIA platform (BZ-X800; Keyence). Compared to the untreated control group (untreated IIM), reduced necrosis was observed in both Formulation 8-treated groups (MΦ only and Form8-treated IIM) and in uninfected MΦ treated with empty liposomes (Lipo MΦ only). Lipo: empty liposomes; IIM: internally infected MΦ; Form8: Formulation 8; HCIA: high content imaging and analysis; FOV: field of view. [Figure 12] Graph showing treatment of J774A.1 murine macrophages (MΦ) uninfected or infected (MOI=10:1) with Mycobacterium abscessus with ABI α-PPL. The extent of necrosis and oxidative stress in MΦ was quantified to investigate the safety and efficacy of ABI α-PPL treatment against mycobacterial infection. The extent of necrosis for MΦ was calculated as the ratio of the number of cells stained with SYTOX™ Green Nucleic Acid Stain (Invitrogen™) and Hoechst-33342 (Invitrogen™) in the population and counted per FOV (N=2-3 per well) by HCIA platform (Keyence). IIM: internally infected MΦ; Form8: Formulation 8; Form8 w / Tw: Formulation 8 with Tween-80; ROS: reactive oxygen species; HCIA: high content imaging analysis; FOV: field of view. [Figure 13]Graph showing the degree of oxidative stress for MΦ quantified as the ratio of cells stained with CellROX™ (Invitrogen™) to the number of cells stained with Hoechst-33342 (Invitrogen™) and counted per FOV (N=2-3 per well) by HCIA platform (BZ-X800; Keyence) in a population. A reduction in necrosis was observed in all treatment groups compared to the untreated control group (untreated IMM). A reduction in oxidative stress was observed in two treatment groups (MΦ only and Form8-treated IIM**) compared to untreated IIM. IIM: internally infected MΦ; Form8: formulation 8; Form8 w / Tw: formulation 8 with Tween-80; ROS: reactive oxygen species; HCIA: high content imaging analysis; FOV: field of view. [Figure 14] 1 is a table showing the various formulations used to assemble the payload liposomes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0095] Mycobacteria are actinomycetes (e.g., Corynebacteriales or Propionibacteriales) characterized by a thick cell wall that is rich in mycolic acids. From the outside to the inside, mycobacteria contain a capsule, a mycolic acid layer, an arabinogalactan (AGL) layer, peptidoglycan (PG), a plasma membrane, and a cytoplasm. This complex cell envelope contributes to the durability of mycobacteria, making them particularly difficult to penetrate and destroy, and there is a need for effective treatment of mycobacterial infections.

[0096] The present invention relates to subject compositions and methods of use that are rationally designed to degrade mycobacterial envelopes. In general, the compositions described herein relate to liposomes that target host cells (e.g., macrophages or dendritic cells) and the correct target intracellular compartment (endosomes, phagosomes, lysosomes, or cytosol) while the liposomes are preloaded with a cocktail of antimicrobial lytic proteins primed to kill bacterial cells. The liposomes target the payload to the correct cell type and intracellular compartment while the cocktail of antimicrobial lytic proteins degrades the mycobacterial envelope and kills the bacteria.

[0097] The composition includes a cocktail containing two or more of lysin A, lysin B, isoamylase, and α-amylase. Such a combination of lytic proteins is particularly advantageous in killing mycobacterial cells. To come up with the protein components, we first rationally attacked the three-layer mycobacterial envelope, the capsule, the junction between the mycolic acid and AGL layers, and the peptidoglycan layer. At the basic structure level, components of the envelope have been observed in many actinomycetes, such as Corynebacteriales (e.g., Mycobacteria) and Propionibacteriales, such as Cutibacteria.

[0098] To identify optimal protein components in the composition, we used bioinformatic analyses to analyze a variety of proteins, screening for important enzymatic properties such as yield, enzymatic activity, purity, thermostability, and antimicrobial activity.

[0099] We further identified improved excipients for the assembly of proteins into liposomes. These excipients improved encapsulation and reduced precipitation, key characteristics for enzyme activity. These thermostable complexes showed robust antimycobacterial effects and can be used to treat infections caused by a variety of mycobacteria and related actinomycetes (e.g., Corynebacteriales or Propionibacteriales) that have similar envelope structures.

[0100] Antibacterial Lytic Proteins The present invention relates to supramolecular complexes (e.g., liposomes) containing one or more (e.g., one, two, three, or four) of lysin A, lysin B, isoamylase, and α-amylase. Additionally, the present invention relates to the discovery of distinct protein sequences that exhibit robust bactericidal effects. These proteins, as shown below in Table 1, exhibit improved expression, thermostability, and antibacterial effects, for example, compared to other orthologs of these proteins.

[0101] [Table 1-1]

[0102] [Table 1-2]

[0103] [Table 1-3]

[0104] [Table 1-4]

[0105] [Table 1-5]

[0106] The compositions described herein can include a lysin A that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2. Lysin A can include or consist of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0107] The compositions described herein can include lysin B comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184. Lysin B can comprise or consist of the amino acid sequence of SEQ ID NO: 183 or SEQ ID NO: 184.

[0108] The compositions described herein can include an isoamylase that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243. The isoamylase can include or consist of the amino acid sequence of SEQ ID NO: 242 or SEQ ID NO: 243.

[0109] The compositions described herein can include an α-amylase that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-398. The α-amylase can include or consist of the amino acid sequence of any one of SEQ ID NOs: 393-398.

[0110] The compositions described herein can include an α-amylase that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 393 or SEQ ID NO: 394. The α-amylase can include or consist of the amino acid sequence of SEQ ID NO: 393 or SEQ ID NO: 394.

[0111] In some embodiments, the composition comprises lysin A of SEQ ID NO:1, lysin B of SEQ ID NO:183, an isoamylase of SEQ ID NO:242, and an α-amylase of SEQ ID NO:393. In some embodiments, the composition comprises lysin A of SEQ ID NO:2, lysin B of SEQ ID NO:184, an isoamylase of SEQ ID NO:243, and an α-amylase of SEQ ID NO:394.

[0112] In some embodiments, the composition comprises lysin A of SEQ ID NO:1, lysin B of SEQ ID NO:183, an isoamylase of SEQ ID NO:242, and an α-amylase of SEQ ID NO:395. In some embodiments, the composition comprises lysin A of SEQ ID NO:2, lysin B of SEQ ID NO:184, an isoamylase of SEQ ID NO:243, and an α-amylase of SEQ ID NO:396.

[0113] In some embodiments, the composition comprises lysin A of SEQ ID NO:1, lysin B of SEQ ID NO:183, an isoamylase of SEQ ID NO:242, and an α-amylase of SEQ ID NO:397. In some embodiments, the composition comprises lysin A of SEQ ID NO:2, lysin B of SEQ ID NO:184, an isoamylase of SEQ ID NO:243, and an α-amylase of SEQ ID NO:398.

[0114] Additional sequences have been identified that may be useful in the compositions and methods described herein. In some embodiments, a composition comprises a lysin A comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-182, as set forth in Table 2 below.

[0115] [Table 2-1]

[0116] [Table 2-2]

[0117] [Table 2-3]

[0118]

Table 2-4

[0119]

Table 2-5

[0120]

Table 2-6

[0121]

Table 2-7

[0122]

Table 2-8

[0123]

Table 2-9

[0124]

Table 2-10

[0125]

Table 2-11

[0126]

Table 2-12

[0127]

Table 2-13

[0128]

Table 2-14

[0129]

Table 2-15

[0130]

Table 2-16

[0131]

Table 2-17

[0132]

Table 2-18

[0133]

Table 2-19

[0134]

Table 2-20

[0135]

Table 2-21

[0136]

Table 2-22

[0137]

Table 2-23

[0138]

Table 2-24

[0139]

Table 2-25

[0140]

Table 2-26

[0141]

Table 2-27

[0142]

Table 2-28

[0143]

Table 2-29

[0144]

Table 2-30

[0145]

Table 2-31

[0146]

Table 2-32

[0147]

Table 2-33

[0148]

Table 2-34

[0149]

Table 2-35

[0150]

Table 2-36

[0151]

Table 2-37

[0152]

Table 2-38

[0153]

Table 2-39

[0154]

Table 2-40

[0155]

Table 2-41

[0156]

Table 2-42

[0157]

Table 2-43

[0158]

Table 2-44

[0159]

Table 2-45

[0160]

Table 2-46

[0161]

Table 2-47

[0162]

Table 2-48

[0163]

Table 2-49

[0164]

Table 2-50

[0165]

Table 2-51

[0166]

Table 2-52

[0167]

Table 2-53

[0168]

Table 2-54

[0169]

Table 2-55

[0170]

Table 2-56

[0171]

Table 2-57

[0172]

Table 2-58

[0173]

Table 2-59

[0174]

Table 2-60

[0175]

Table 2-61

[0176]

Table 2-62

[0177]

Table 2-63

[0178]

Table 2-64

[0179] [Table 2-65]

[0180] [Table 2-66]

[0181] Additional sequences have been identified that may be useful in the compositions and methods described herein. In some embodiments, a composition comprises lysin B comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241, as set forth in Table 3 below.

[0182] [Table 3-1]

[0183] [Table 3-2]

[0184] [Table 3-3]

[0185] [Table 3-4]

[0186] [Table 3-5]

[0187] [Table 3-6]

[0188]

Table 3-7

[0189]

Table 3-8

[0190]

Table 3-9

[0191]

Table 3-10

[0192]

Table 3-11

[0193]

Table 3-12

[0194]

Table 3-13

[0195]

Table 3-14

[0196]

Table 3-15

[0197]

Table 3-16

[0198] [Table 3-17]

[0199] Additional sequences have been identified that may be useful in the compositions and methods described herein. In some embodiments, a composition comprises an isoamylase comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs:242-392, as set forth in Table 4 below.

[0200] [Table 4-1]

[0201] [Table 4-2]

[0202] [Table 4-3]

[0203] [Table 4-4]

[0204] [Table 4-5]

[0205] [Table 4-6]

[0206] [Table 4-7]

[0207]

Table 4-8

[0208]

Table 4-9

[0209]

Table 4-10

[0210]

Table 4-11

[0211]

Table 4-12

[0212]

Table 4-13

[0213]

Table 4-14

[0214]

Table 4-15

[0215]

Table 4-16

[0216]

Table 4-17

[0217]

Table 4-18

[0218]

Table 4-19

[0219]

Table 4-20

[0220]

Table 4-21

[0221]

Table 4-22

[0222]

Table 4-23

[0223]

Table 4-24

[0224]

Table 4-25

[0225]

Table 4-26

[0226]

Table 4-27

[0227]

Table 4-28

[0228]

Table 4-29

[0229]

Table 4-30

[0230]

Table 4-31

[0231]

Table 4-32

[0232]

Table 4-33

[0233]

Table 4-34

[0234]

Table 4-35

[0235]

Table 4-36

[0236]

Table 4-37

[0237]

Table 4-38

[0238]

Table 4-39

[0239]

Table 4-40

[0240]

Table 4-41

[0241]

Table 4-42

[0242]

Table 4-43

[0243]

Table 4-44

[0244]

Table 4-45

[0245]

Table 4-46

[0246]

Table 4-47

[0247]

Table 4-48

[0248]

Table 4-49

[0249]

Table 4-50

[0250]

Table 4-51

[0251]

Table 4-52

[0252]

Table 4-53

[0253]

Table 4-54

[0254]

Table 4-55

[0255]

Table 4-56

[0256]

Table 4-57

[0257]

Table 4-58

[0258]

Table 4-59

[0259]

Table 4-60

[0260]

Table 4-61

[0261]

Table 4-62

[0262]

Table 4-63

[0263]

Table 4-64

[0264]

Table 4-65

[0265]

Table 4-66

[0266]

Table 4-67

[0267]

Table 4-68

[0268]

Table 4-69

[0269]

Table 4-70

[0270]

Table 4-71

[0271]

Table 4-72

[0272]

Table 4-73

[0273]

Table 4-74

[0274]

Table 4-75

[0275]

Table 4-76

[0276]

Table 4-77

[0277]

Table 4-78

[0278]

Table 4-79

[0279]

Table 4-80

[0280]

Table 4-81

[0281]

Table 4-82

[0282]

Table 4-83

[0283]

Table 4-84

[0284]

Table 4-85

[0285]

Table 4-86

[0286]

Table 4-87

[0287]

Table 4-88

[0288]

Table 4-89

[0289]

Table 4-90

[0290]

Table 4-91

[0291]

Table 4-92

[0292]

Table 4-93

[0293]

Table 4-94

[0294]

Table 4-95

[0295]

Table 4-96

[0296]

Table 4-97

[0297]

Table 4-98

[0298]

Table 4-99

[0299] [Table 4-100]

[0300] [Table 4-101]

[0301] [Table 4-102]

[0302] [Table 4-103]

[0303] [Table 4-104]

[0304] [Table 4-105]

[0305] [Table 4-106]

[0306] Additional sequences have been identified that may be useful in the compositions and methods described herein. In some embodiments, a composition comprises an α-amylase comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445, as set forth in Table 5 below.

[0307] [Table 5-1]

[0308]

Table 5-2

[0309]

Table 5-3

[0310]

Table 5-4

[0311]

Table 5-5

[0312]

Table 5-6

[0313]

Table 5-7

[0314]

Table 5-8

[0315]

Table 5-9

[0316]

Table 5-10

[0317]

Table 5-11

[0318]

Table 5-12

[0319]

Table 5-13

[0320]

Table 5-14

[0321]

Table 5-15

[0322]

Table 5-16

[0323]

Table 5-17

[0324]

Table 5-18

[0325]

Table 5-19

[0326]

Table 5-20

[0327]

Table 5-21

[0328] [Table 5-22]

[0329] [Table 5-23]

[0330] [Table 5-24]

[0331] Those skilled in the art will understand that the soluble protein described herein can be produced recombinantly.Therefore, the protein can contain a suitable purification tag, such as His tag, which contains 3, 4, 5, 6, 7, 8, 9, 10 or more histidine residues, present at the N-terminus or C-terminus of the protein.The protein can also contain a removable signal sequence present at the N-terminus or C-terminus of the protein.

[0332] One of skill in the art will also understand that the lysis proteins described herein can include biologically active fragments thereof, e.g., fragments of the lysis proteins described herein that can be truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids, yet still substantially retain their biological activity.

[0333] In some embodiments, the composition comprises a concentration of 0.1 mg / mL to 20 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, In some embodiments, the composition comprises a protein (e.g., lysin A, lysin B, isoamylase, and / or α-amylase) at a concentration of 1 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, e.g., 10 mg / mL to 20 mg / mL, e.g., 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL). In some embodiments, the composition comprises a protein (e.g., lysin A, lysin B, isoamylase, and / or α-amylase at a concentration of 1 mg / mL to 10 mg / mL.

[0334] intracellular bacteria Intracellular bacteria live within host cells, where they reproduce and cause infection. Intracellular bacteria can live within immune cells, such as professional antigen cells. Professional antigen presenting cells (APCs) include macrophages, dendritic cells, and phagocytes, such as macrophages. APCs process antigens complexed with major histocompatibility complexes (MHC) on their surface and present them on their surface. APCs present antigens to bacterial pathogens on MHC class 2, which are recognized by T cells, which then stimulate B cells with antibodies complementary to the antigen. This leads to the proliferation of specific B cells that code for specific antibodies to fight the organisms that carry the antigen. Certain bacteria evade this immune response by hiding within immune cells.

[0335] The compositions and methods described herein can be used to treat mycobacteria, such as intracellular acid-fast bacteria that reside in professional antigen-presenting cells (e.g., macrophages or dendritic cells). In some embodiments, the mycobacterial species is M. tuberculosis, M. leprae, M. lepromatosis, M. avium, M. kansasii, M. fortuitum, M. chelonae, M. marinum, M. intracellulare, M. abscessus, M. chimera, M. boletti, M. fortuitum, M. goodii, or M. masiliense. In certain embodiments, the mycobacterium is NTM. In some embodiments, the NTM is M. abscessus, M. intracellulare, M. avium, M. chimera, M. boletti, M. fortuitum, M. goodii, and M. masiliense.

[0336] In some embodiments, the compositions and methods described herein can be used to target other actinomycetes (e.g., Corynebacteriales or Propionibacteriales) that have similar envelope components as mycobacteria. For example, the compositions and methods can be used to target Nocardia, Corynebacterium, or Rhodococcus species. For example, Nocardia species can be, for example, N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. asteroids, N. brasiliensis, and N. caviae. Corynebacterium species can be, for example, C. glutamicum or C. diphtheriae. Rhodococcus species can be, for example, R. fascians or R. equi. The compositions and methods can be used to target Propionibacteriales, such as Cutibacterium species. Cutibacterium species can be, for example, C. acnes.

[0337] supramolecular structure Supramolecular structures can be used to formulate cocktails of lytic enzymes 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 pi-pi interactions. Supramolecular structures can include large complexes of molecules that form spheres, helices, or sheet-like structures. Supramolecular structures include lipid-based supramolecular structures, e.g., 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, packed into the structure. The supramolecular complexes are endocytosed by cells, e.g., professional antigen-presenting cells, such as macrophages or dendritic cells, and the antibacterial lytic proteins are delivered to the target intracellular compartment (endosomes, phagosomes, lysosomes, or cytosol) where the bacteria reside.

[0338] In some embodiments, a specific particle size is used to guide the structure into a specific endocytic pathway that directs it to the appropriate target intracellular compartment. The supramolecular structure can undergo endocytosis and be delivered to the target intracellular compartment, for example, via clathrin-dependent endocytosis or via caveolin-dependent endocytosis. The particle size, for example, the Z-average average particle size, of the supramolecular structure can vary from 75 nm to 5 μm, for example, 75 nm to 2 μm, 75 nm to 1 μm, for example, 75 nm to 750 nm (for example, 250 nm to 750 nm, or 75 nm to 250 nm). In some embodiments, when the supramolecular structure is a LNP or a micelle, the Z-average average particle size is from 75 nm to 250 nm. In some embodiments, when the supramolecular structure is a vesicle (for example, a liposome), the Z-average average particle size is from 250 nm to 750 nm. Non-limiting examples of the Z average mean particle size include, for example, 75 nm to 100 nm, for example, 75 nm to 85 nm, for example, 80 nm, for example, 80 nm to 140 nm, 90 nm to 130 nm, or 110 nm to 130 nm, for example, 120 nm, for example, 200 nm to 300 nm, for example, 250 nm to 300 nm, 260 nm to 290 nm, 260 nm to 280 nm, 265 nm to 275 nm, for example, 270 nm, for example, 300 nm to 400 nm, 400 nm to 600 nm, for example, 450 nm to 550 nm, 475 nm to 525 nm, 480 nm to 520 nm, 490 nm to 510 nm, 495 nm to 505 nm, for example, 500 nm, for example, 75 nm, 80 nm, 85 nm, m, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 150n m, 155nm, 160nm, 165nm, 170nm, 175nm, 180nm, 185nm, 190nm, 195nm, 200nm, 205nm, 210nm, 215 nm, 220nm, 225nm, 230nm, 235nm, 240nm, 245nm, 250nm, 255nm, 260nm, 265nm, 270nm, 275nm, 2 80nm, 285nm, 290nm, 295nm, 300nm, 305nm, 310nm, 315nm, 320nm, 325nm, 330nm, 335nm, 340nm,345nm、350nm、355nm、360nm、365nm、370nm、375nm、380nm、385nm、390nm、395nm、400nm、405nm、410nm、415nm、420nm、425nm、430nm、435nm、440nm、445nm、450nm、455nm、460nm、465nm、470nm、475nm、480nm、485nm、490nm、495nm、500nm、505nm、510nm、515nm、520nm、525nm、530nm、535nm、540nm、545nm、550nm、555nm、560nm、565nm、570nm、575nm、580nm、585nm、590nm、595nm、600nm、605nm、610nm、615nm、620nm、625nm、630nm、635nm、640nm、645nm、650nm、655nm、660nm、665nm、670nm、675nm、680nm、685nm、690nm、695nm、700nm、705nm、710nm、715nm、720nm、725nm、730nm、735nm、740nm、745nm、750nm、755nm、760nm、765nm、770nm、775nm、780nm、785nm、790nm、795nm、800nm、805nm、810nm、815nm、820nm、825nm、830nm、835nm、840nm、845nm、850nm、855nm、860nm、865nm、870nm、875nm、880nm、885nm、890nm、895nm、900nm、905nm、910nm、915nm、920nm、925nm、930nm、935nm、940nm、945nm、950nm、955nm、960nm、965nm、970nm、975nm、980nm、985nm、990nm、995nm、1μm、1.1μm、1.2μm、1.3μm、1.4μm、1.5μm、1.6μm、1.7μm、1.8μm、1.9μm、2μm、2.1μm、2.2μm、2.3μm、2.4μm、2.5μm、2.6μm、2.7μm、2.8μm、2.9μm、3μm、3.1μm、3.2μm、3.3μm、3.4μm、3.5μm、3.6μm、3.7μm、3.8μm、3.9μm、4μm、4.1μm、4.2μm、4.3μm、4.4μm、4.5μm、4.6μm、In certain embodiments, the Z-average average particle size of the supramolecular structure can be about 5 nm to 250 nm. In some embodiments, the Z-average average particle size of the supramolecular structure is 80 nm, 270 nm, or 500 nm.

[0339] 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 75 nm to 250 nm. In certain embodiments, the supramolecular structure contains a Z average average particle size of 250 nm to 750 nm. In certain embodiments, the supramolecular structure contains a Z average average particle size of 500 nm. In certain embodiments, the supramolecular structure contains a Z average average particle size of 270 nm. In certain embodiments, the supramolecular structure contains a Z average average particle size of 80 nm. One skilled in the art will appreciate that a population of supramolecular structures (e.g., liposomes, LNPs, or micelles) may have a range of Z-average mean particle sizes within the population. Thus, the population may be polydisperse. The population may have a polydispersity index of 0.5 or less, such as 0.3 or less (e.g., 0.05 to 0.3). The polydispersity index may be measured using DLS (see, e.g., ISO 22412:2017).

[0340] The supramolecular structures can be loaded with a given number of antimicrobial lysis proteins or an average number of antimicrobial lysis proteins per supramolecular structure. For example, the supramolecular structures can be loaded with between 1 protein and 10 6 proteins (e.g., 1-10 5 pieces, 1~10 4 pieces, 1~10 3 pieces, 1~10 2 pieces, 1~10 pieces, 10~10 6 pieces, 10~105 ,10~10 4 pieces, 10~10 3 pieces, 10~10 2 pieces, 10 3 ~10 6 pieces, 10 3 ~10 5 pieces, 10 3 ~10 4 The number of proteins per structure may depend on the size of the protein and the size of the structure.

[0341] The supramolecular structure may include an endosomal escape moiety. The supramolecular structure including the endosomal escape moiety may provide for improved cytosolic delivery of the cargo (e.g., therapeutic agent) contained in the supramolecular structure. The endosomal escape moiety is known in the art. In some embodiments, the endosomal escape moiety is an ionizable lipid. The ionizable lipid may also serve as a supramolecular structure-layer forming lipid. 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, the fusogenic peptide can be derived from the M2 protein of influenza A virus; a peptide analogue of influenza virus hemagglutinin; the HEF protein of influenza C virus; a filovirus transmembrane glycoprotein; a rabies virus transmembrane glycoprotein; a vesicular stomatitis virus transmembrane glycoprotein (G); a Sendai virus fusion protein; a Semliki Forest virus transmembrane glycoprotein; a human respiratory syncytial virus (RSV) fusion protein; a measles virus fusion protein; a Newcastle disease virus fusion protein; a Visna virus fusion protein; a murine leukemia virus fusion protein; a HTL virus fusion protein; and a 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 WO 2015 / 188197, the disclosure of which is incorporated herein by reference.

[0342] 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 proceed together, the internal aqueous contents containing the antimicrobial protein are delivered to the cell and the antimicrobial lytic protein is delivered into the cell, where the antimicrobial protein can target and lyse bacterial cells (e.g., mycobacterial cells, e.g., NTM cells) that reside 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 usually a combination of phospholipids, usually combined with steroids 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.

[0343] In some embodiments, the liposomes described herein include phospholipids. In some embodiments, glycerophospholipids, 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 fatty acyl. Additionally or alternatively, the liposomes described herein can include, for example, lysophospholipids, 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, with the proviso that at least one R is fatty acyl.

[0344] One of the major types of liposome composition includes phospholipids other than naturally occurring phosphatidylcholine.Neutral liposome composition can be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Cationic liposome has the advantage that it can fuse with cell membrane. 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 kaline, ... 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-propanediol (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-yeethylazanediedidodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can, for example, comprise 20 mol % to 50 mol %, or about 40 mol % of the total lipid present in the particle.

[0345] Non-cationic liposomes cannot fuse as efficiently with the plasma membrane, but can be taken up by macrophages in vivo and deliver antimicrobial proteins to the macrophages. Anionic liposome compositions are usually formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoyl phosphatidylethanolamine (DOPE). The lipids were distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate ( The lipids may be anionic or neutral, including, but not limited to, DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (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. The non-cationic lipid, when cholesterol is included, may be, for example, 5 mol% to 90 mol%, 10 mol% to 58 mol% of the total lipid present in the particle. In some embodiments, the lipid may be a combination of the lipids described above, for example, a combination of lipids including DOPC, DOPS, Chol, and DOPE.

[0346] In some embodiments, the liposome comprises a mixture of lipids, for example, the mixture of lipids can include two or more of DOPC, DOPE, DOPS, and cholesterol.

[0347] In some embodiments, DOPC and DOPE are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0348] In some embodiments, DOPC and DOPS are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0349] In some embodiments, the DOPC and cholesterol are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0350] In some embodiments, DOPE and DOPS are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0351] In some embodiments, DOPE and cholesterol are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0352] In some embodiments, DOPS and cholesterol are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0353] In some embodiments, DOPC, DOPE, DOPS, and cholesterol are present in a molar ratio of 1-20:1-20:1-5:1-5. For example, in some embodiments, DOPC, DOPE, DOPS, and cholesterol are present in a molar ratio of 10:10:3:4.

[0354] In some embodiments, the liposomes contain lipids at a concentration of 0.03 mg / mL to 10 mg / mL, e.g., 0.1 mg / mL to 10 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL). In some embodiments, the liposomes contain lipids at a concentration of 1 mg / mL to 5 mg / mL.

[0355] Another type of liposomal composition is formed from phosphatidylcholine (PC), such as soybean PC, egg PC, etc. 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.

[0356] 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 system, lipid group can be incorporated into the lipid bilayer of liposome to maintain the targeting of ligand while stably associated with the liposome bilayer.Various linking groups can be used to connect lipid chain to targeting ligand.Additional methods are known in the art, and are described, for example, in US Patent Publication No. 20060058255, whose linking groups are incorporated herein by reference.

[0357] Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. In general, cleaving agents are more effective or found at higher levels or activity in cells than in serum or blood. Examples of such degradable agents include oxidizing or reducing enzymes, such as mercaptans, present in cells, which can degrade redox cleavable linking groups by reduction, or reducing agents; 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 certain substrates or have no substrate specificity, including phosphatases.

[0358] Cleavable linking groups, such as disulfide bonds, can be sensitive to pH. Human serum has a pH of 7.4, while the average intracellular pH is slightly lower, ranging from 7.1 to 7.3. Endosomes have a more acidic pH of 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand into the cell or into a desired compartment of the cell.

[0359] The linker may include a cleavable linking group that is cleavable by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the cells to be targeted. 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 in 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 in a whole animal. It may be useful to perform initial evaluation in a cell-free or culture conditions and confirm with further evaluation in a whole animal. In preferred embodiments, useful candidate linkers are cleaved at least 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster within 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).

[0360] 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 following intravenous (iv) injection and accumulate at distal sites (e.g., sites physically separated from the site of administration), making them extremely useful for systemic administration. LNPs include "pSPLPs," which contain encapsulated condensing agent-nucleic acid complexes as described in PCT Publication No. WO2000 / 003683. The particles of the present invention typically have an average diameter of 50 nm to 150 nm, more typically 60 nm to 130 nm, more typically 70 nm to 110 nm, and most typically 70 nm to 90 nm, and are substantially non-toxic. Furthermore, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is resistant in aqueous solution to degradation by nucleases. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Pat. 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.

[0361] In one embodiment, the lipid:agent ratio (mass / mass ratio) (e.g., lipid:peptide ratio) ranges from 1:1 to 50:1, 1:1 to 25:1, 3:1 to 15:1, 4:1 to 10:1, 5:1 to 9:1, or 6:1 to 9:1. Ranges intermediate to the above-listed ranges are also contemplated as part of the invention.

[0362] 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, for example, comprise 20 mol% to 50 mol%, or about 40 mol% of the total lipid present in the particle.

[0363] The lipids can be anionic or neutral lipids, 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 mixtures thereof. The non-cationic lipid, when cholesterol is included, can be, for example, 5 mol% to 90 mol%, 10 mol% to 60 mol% of the total lipid present in the particle.

[0364] 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-phospholipids, 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 20 mol %, or about 2 mol % of the total lipid present in the particle.

[0365] In some embodiments, the LNPs further comprise cholesterol, for example, at 10 mol % to 60 mol %, or 50 mol % of the total lipid present within the particle. In some embodiments, the LNPs comprise a mixture of lipids, for example, the mixture of lipids can include two or more of DOPC, DOPE, DOPS, and cholesterol.

[0366] In some embodiments, DOPC and DOPE are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0367] In some embodiments, DOPC and DOPS are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0368] In some embodiments, the DOPC and cholesterol are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0369] In some embodiments, DOPE and DOPS are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0370] In some embodiments, DOPE and cholesterol are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0371] In some embodiments, DOPS and cholesterol are present in a molar ratio of 10:1 to 1:10 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10).

[0372] In some embodiments, DOPC, DOPE, DOPS, and cholesterol are present in a molar ratio of 1-20:1-20:1-5:1-5. For example, in some embodiments, DOPC, DOPE, DOPS, and cholesterol are present in a molar ratio of 10:10:3:4.

[0373] In some embodiments, the LNP comprises a lipid at a concentration of 0.03 mg / mL to 10 mg / mL, e.g., 0.1 mg / mL to 10 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL). In some embodiments, the LNP comprises a lipid at a concentration of 1 mg / mL to 5 mg / mL.

[0374] Micellar Micelles are a particular type of molecular assembly in which amphiphilic molecules are arranged in a globular structure such that the hydrophobic parts of the molecules all face inwards, leaving the hydrophilic parts in contact with the surrounding aqueous phase. Micelles can be made from lipids. The micellar phase is caused by the trapping behavior of single-tailed lipids in the bilayer membrane phase. The difficulty of filling the entire inner volume of the bilayer membrane while accommodating areas for the head groups to act 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 middle and tails extending outwards (water-in-oil micelle).

[0375] 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 the solution conditions, such as surfactant concentration, temperature, pH, and ionic strength. The process of micelle formation is known as micellization, and due to their polymorphic nature, forms part of the phase behavior of many lipids.

[0376] target area The supramolecular structures described herein can include, for example, targeting moieties. Targeting moieties can be used to target the supramolecular structures to specific cell types (e.g., professional antigen-presenting cells, such as macrophages or dendritic cells). Specific lipids (e.g., phosphatidylserine) can be used in the supramolecular structures (e.g., vesicles) both as supramolecular structure phase-forming lipids and as targeting moieties. The targeting moiety can be, for example, an antibody or an antigen-binding fragment or an engineered derivative thereof (e.g., Fcab or a 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.

[0377] 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.

[0378] In some embodiments, the targeting moiety is phosphatidylserine or lysophosphatidylserine. In some embodiments, the targeting moiety is phosphatidylserine. Phosphatidylserine and / or lysophosphatidylserine can be present as supramolecular structure phase-forming lipids non-covalently bound to the remainder of the supramolecular structure.

[0379] Folate can be used as a targeting moiety. In the supramolecular structures described herein, folate can have the following structure:

[0380] [ka]

[0381] 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.

[0382] 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.

[0383] 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, such as 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.

[0384] The antigen-binding portion targets APCs that have a 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, or antigen-binding fragments or recombinant derivatives thereof.

[0385] Non-limiting examples of anti-CD38 antibodies are daratumumab, SAR650984, MOR202, or any one of the 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.

[0386] Conjugation techniques for linking antigen-binding moieties are known in the art, e.g., as described in Ansell et al., Methods Mol. Med., 25:51-68, 2000; US2002 / 0025313; US6,379,699; and US5,059,421.

[0387] 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, e.g., MALP-2 lipoprotein, MALP-404 lipoprotein, OspA, porin, LcrV, Hsp60, glycoprotein gH / gL, or glycoprotein gB.

[0388] 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; US2002 / 0025313; US6,379,699; and US5,059,421.

[0389] 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 usually contain a poly-G tail with a phosphorothioate backbone at the 3' and 5' ends, and a central palindromic sequence with a phosphate backbone. Class A CpG ODNs usually contain CpG in the central palindromic sequence. Class B CpG ODNs usually 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 contain a fully phosphorothioate backbone, and the 3' end sequence is capable of forming a duplex. PAMPs can be covalently attached to supramolecular structures using techniques and methods known in the art.

[0390] Assembly Method The present invention relates to a supramolecular structure (e.g., a lipid-based supramolecular structure, such as a liposome), which comprises a plurality of enzymes packaged therein. Described herein are assembly methods for producing structures (e.g., liposomes) containing enzymes (e.g., lysin A, lysin B, isoamylase, and / or α-amylase having at least 85% sequence identity with the sequences in Table 1). Individual proteins can be overexpressed in any suitable recombinant expression system (e.g., E. coli) and extracted from cells by cell lysis. In some embodiments, crude extracts from cells can be purified, for example, by column chromatography. After purification, the component concentrations of the enzymes can be standardized for subsequent encapsulation, for example, in liposomes. The concentration can be standardized, for example, at 0.1 mg / mL to 10 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, e.g., 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL). In some embodiments, the concentration can be standardized at 0.33 mg / mL.

[0391] The lipids can then be mixed with an enzyme to formulate liposomes. For example, a total concentration of lipids of 0.1 mg / mL to 10 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, e.g., 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL, e.g., 1 mg / mL to 5 mg / mL) can be mixed with 1 mg / mL to 10 mg / mL of His-tagged protein. The lipids can include, for example, one or more of DOPC, DOPE, DOPS, and cholesterol. In some embodiments, the lipids include DOPC, DOPE, DOPS, and cholesterol in a ratio of 10:10:3:4. The lipids can be resuspended in a suitable organic solvent (e.g., ethanol) and mixed with the protein (e.g., in an aqueous:organic ratio of 20:1 to 1:1, e.g., 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1, e.g., 3:1 to 10:1, e.g., 3:1 to 8:1). The mixture can be mixed using a suitable system, e.g., the NANOASSEMBLR® IGNITE™ system (Precision Nanosystems), at a flow rate of 1 ml / min to 30 mL / min, e.g., mL / min. The organic layer can be removed, for example, by dialysis against an excess (e.g., 1000-fold) volume of formulation buffer for at least 30 minutes (e.g., 1 hour) at a suitable temperature, such as room temperature. The liposomes can then be analyzed and tested for associated activity by gel electrophoresis (e.g., SDS-PAGE), dynamic light scattering, intrinsic fluorescence, and / or static light scattering. Such assays can help confirm enzyme encapsulation and purity of the enzyme and liposomes.

[0392] The composition containing the supramolecular structure (e.g., liposome) can be formulated with one or more excipients. For example, the composition (e.g., the supramolecular structure, e.g., liposome containing a cocktail of lytic enzymes) can be encapsulated and / or formulated in a buffer solution such as glycine, Tris, sodium citrate, sodium acetate, and MES, for example, at a concentration of 10 mM to 200 mM, for example, 50 mm to 150 mm, for example, 10 mM, 20 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM. The composition can be formulated at a pH of 5-11 (e.g., 5-6, e.g., 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6, e.g., 6-11, e.g., 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11). The composition can further comprise one or more excipients, such as CaCl2, arginine, NaCl, sodium citrate, MgCl2, or glycerol. In some embodiments, the composition comprises, for example, 5 mM-10 mM CaCl2, 0-50 mM arginine, 0-200 mM NaCl, 0-1 mM sodium citrate, 0 nM-1 mM MgCl2, and / or 10-30% glycerol. In some embodiments, the compositions comprise 50 mM glycine (pH 8.5), 7.5 mM CaCl2, 0.5 mM MgCl2, 200 mM NaCl, 0.33 mM sodium citrate, and 10% glycerol. The formulations can further comprise Tween, e.g., Tween-80. Once the compositions are in the preferred storage or activity buffer, they can be used in therapy or for assays, or can be stored for future use.

[0393] 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., actinomycete infections, e.g., Corynebacteriales or Propionibacteriales), intracellular bacteriosis (e.g., mycobacteria infections, e.g., NTM infections). In particular, the compositions and methods described herein are useful for treating bacterial infections caused by actinomycetes, e.g., Corynebacteriales and Propionibacteriales, due to their similar envelope structures. Bacterial infections may occur in otherwise healthy subjects. Alternatively, bacterial infections may occur in subjects with other comorbidities or diseases. For example, subjects with weakened immune systems may be more susceptible to bacterial infections.

[0394] Mycobacterial infections caused by NTM are bacteria that are usually found in the environment. Inhalation of these bacteria can cause disease in both healthy and immune-compromised patients. NTM disease most often affects the lungs in adults, but can affect any body site. Some subjects are at high risk of acquiring and progressing to NTM infection. People with pre-existing lung disease, such as bronchiectasis (widening of airways), chronic obstructive pulmonary disease (COPD), cystic fibrosis, alpha-1 antitrypsin deficiency, or people with previous infections, such as tuberculosis, are at increased risk of pulmonary NTM disease. Subjects with advanced HIV infection (CD4<50) or immune-related genetic diseases (e.g., interferon-gamma or receptor deficiency, interleukin-12 deficiency) may develop lung disease as part of a disseminated (e.g., widespread in the body) NTM infection. The subject being treated may have, for example, any of the aforementioned indications in addition to a bacterial infection.

[0395] The method compositions and methods described herein can be used to reduce the level of infection. For example, the methods 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 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0396] Pharmaceutical Compositions 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.

[0397] 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 route of administration selected. The compositions described herein can be administered, for example, by any route that allows the composition (e.g., supramolecular structures, such as liposomes, micelles, or LNPs) to reach the target cells. The compositions can be administered, for example, orally, parenterally, intrathecally, intracerebroventricularly, intraparenchymal, buccal, sublingual, nasal, rectal, patch, pump, or transdermal, with the pharmaceutical composition being formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, pulmonary, intrathecal, intracerebroventricular, intraparenchymal, rectal, and topical methods of administration. In one embodiment, the compositions are administered by aerosol. Parenteral administration can be by continuous infusion over a selected period of time. In some preferred embodiments, the compositions described herein are administered by inhalation.

[0398] Administration of the two or more antibacterial agents may be by the same route or by different routes and may occur sequentially or nearly 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.

[0399] 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 as is or after reconstitution with a vehicle (e.g., saline (e.g., isotonic saline), phosphate buffered saline, or water).

[0400] 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). Inhalable dry powder formulations described herein can include carriers (e.g., lactose, sucrose, mannitol, etc.), cryoprotectants (e.g., trehalose, mannitol, etc.), and / or anti-adhesive agents (e.g., glycine, L-leucine, serine, etc.). 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 include 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.

[0401] The inhalable liquid dosage forms (e.g., aerosol formulations) described herein can be prepared using techniques and methods useful in the preparation of liquid compositions containing supramolecular structures. Inhalable liquid dosage forms usually comprise a suspension of the supramolecular structures described herein in a physiologically acceptable aqueous or non-aqueous solvent, usually present in a sterile form in a sealed container, in single or multiple doses, which can take the form of a cartridge or refill for use with a nebulizer. 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, intended to be discarded 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 forms can be administered using a nebulizer. The process of converting a bulk liquid into small droplets by 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.

[0402] The compositions described herein may be administered orally, for example, with an inert diluent or an assimilable edible carrier, or may be enclosed in hard or soft shell gelatin capsules, or may be compressed into tablets, or may be incorporated directly into dietary foods. For oral therapeutic administration, the compositions described herein may 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 may also be administered parenterally. The compositions described herein may also be microneedle injected. Solutions of the compositions described herein may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, DMSO, and mixtures thereof with or without alcohol, and in oils. These preparations may contain a preservative 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 injection 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 must be sufficiently fluid that it may be easily administered by syringe. Compositions suitable for buccal or sublingual administration include tablets, lozenges, and troches, 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.

[0403] In some embodiments, the compositions described herein are formulated with one or more excipients. For example, the compositions (e.g., supramolecular structures, e.g., liposomes containing a cocktail of lytic enzymes) can be encapsulated and / or formulated in buffers such as glycine, Tris, sodium citrate, sodium acetate, and MES, for example, at a concentration of 10 mM to 200 mM, for example, 50 mm to 150 mm, for example, 10 mM, 20 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM. The composition can be formulated at a pH of 5-11 (e.g., a pH of 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11). The composition can further include one or more excipients, such as CaCl2, arginine, NaCl, sodium citrate, MgCl2, or glycerol. In some embodiments, the composition includes, for example, 5 mM-10 mM CaCl2, 0-50 mM arginine, 0-200 mM NaCl, 0-1 mM sodium citrate, 0-1 mM MgCl2, and / or 10-30% glycerol. In some embodiments, the composition comprises 50 mM glycine (pH 8.5), 7.5 mM CaCl2, 0.5 mM MgCl2, 200 mM NaCl, 0.33 mM sodium citrate, and 10% glycerol. The formulation may further comprise a Tween, e.g., Tween-80.

[0404] The compositions described herein, as described herein, can be administered to animals, e.g., humans, alone or in combination with pharma- ceutically acceptable carriers, 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.

[0405] The dose of the composition, e.g., a composition comprising a lytic protein as described herein, may 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 may be initially administered in a suitable dosage, which may be adjusted as necessary depending on the clinical response. In some embodiments, the dose of the composition, e.g., a composition comprising a lytic protein, is a prophylactically or therapeutically effective amount. Furthermore, it is understood that all doses may be given continuously or divided into doses given per given time frame. The composition may be administered, for example, hourly, daily, weekly, monthly, or yearly. In some embodiments, the composition is administered continuously or systemically.

[0406] Combination therapy The pharmaceutical compositions described herein can be administered as part of a combination therapy. Combination therapy means that two (or more) different 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 dose and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, the delivery of two or more agents is simultaneous or parallel, and the agents may be prepared in admixture. In some embodiments, the 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.

[0407] In any of the combination embodiments described herein, the first and second therapeutic agents can be administered simultaneously or sequentially in either 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 1-7, 1-14, 1-21, or 1-30 days before or after the second therapeutic agent.

[0408] The pharmaceutical compositions described herein may further comprise an additional antibacterial agent administered together with the supramolecular structure comprising the antimicrobial lysis protein. The compositions and methods described herein can further include treatment of underlying pulmonary conditions, e.g., that may be exacerbated by bacterial infections, e.g., NTM infections. Suitable pulmonary therapies include, but are not limited to, airway clearance, nebulizers, respiratory masks, and inhalers, e.g., steroid inhalers.

[0409] antibiotics The additional antimicrobial 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, cefopera Zon, 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, dibekacin, isepamicin, tetracycline, chlortetracycline, demeclocycline cyclosporine, 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, pefloromycin, cyclosporine ... These include, but are not limited to, oxacin, enoxacin, ofloxacin, ciprofloxacin, temafloxacin, lomefloxacin, fleroxacin, grepafloxacin, sparfloxacin, trovafloxacin, clinafloxacin, gatifloxacin, moxifloxacin, gemifloxacin, sitafloxacin, metronidazole, daptomycin, garenoxacin, ramoplanin, faropenem, polymyxin, tigecycline, AZD2563, trimethoprim, ethambutol, rifamycin, and rifampin.In some embodiments, multiple antibiotics are administered with the compositions described herein. In some embodiments, the antibiotic is a cephalosporin, a carbapenem (e.g., biapenem), a penicillin, a macrolide, an aminoglycoside, or a fluoroquinolone. In some embodiments, the antibiotic is selected from the group consisting of thiacetazone, sq-109, bedaquiline, delamanid, pyrazinamide, and isoniazid.

[0410] In some embodiments, the antibiotic is a macrolide (e.g., azithromycin, clarithromycin, erythromycin). In some embodiments, the antibiotic is an aminoglycoside (e.g., kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin (e.g., neomycin B, C, or E), streptomycin, or plazomicin).

[0411] Advantageously, in some embodiments, synergy with co-administered therapeutic agents may allow for the administration of sub-therapeutic doses of the antibiotic when administered without other therapeutic agents.

[0412] The antibiotic can be formulated with a supramolecular structure containing a bacteriophage and an antibacterial lytic protein. The antibiotic can be administered as a separate pharmaceutical composition. The antibiotic can be administered at a different time than 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, a liposomal amikacin formulated for inhalation.

[0413] Working Example The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be purely illustrative of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.

[0414] Example 1 background Mycobacteria have inherent resistance to antibiotics due in part to their complex cell surface envelope, which contains, from the inside out, a multilayer of the plasma membrane PM, peptidoglycan (PG), arabinogalactan (AGL), mycolic acid layer (MA), and capsule layer (CL).

[0415] Endolytix Cocktail 1 (EC1) was built on four enzymes that catalytically attack three layers within the mycobacterial envelope. The first enzyme is lysin A, a phage-encoded protein that represents a conserved family of proteins that catalyze the lysis of the PG layer for the release of bacteriophage progeny. Mycobacteria also display a diversity of chemical bonds in their PG layer, and can control the ratio of 3,3 and 4,3 linked stem peptides that crosslink the many glycan layers that form the PG layer.

[0416] The second enzyme, lysin B, is exclusive to actinomycete phages, including, for example, mycobacteriophages and related host species mentioned above, where the host contains an MA layer. Lysin B is an ester that hydrolyzes the bond between arabinogalactan and mycolic acid.

[0417] The mechanism of action for lysin A and lysin B naturally attacks the mycobacterial host from the inside out. The problem of providing exogenous access to substrates for lysin A and lysin B was addressed by adding enzymes that degrade the M. tuberculosis capsule. Capsulase hydrolyzes α-1,4 and α-1,6 linked glucose in the capsular polysaccharide. The structure of the polysaccharide in the mycobacterial envelope CL consists of heptameric α-1,4-glycan chains linked by two α-1,6-glycosidic bonds, creating a meshwork of cross-linked heptamers. Cleavage of the capsular polysaccharide exposes the mycolic acid and arabinogalactan layers, making the target layer (i.e., substrate) accessible to both lysin A and lysin B. α-Amylase (EC 3.2.1.1) is a hydrolase that cleaves α-1,4-glycan chains. Isoamylase (EC 3.2.1.68) dissolves α-1,6-glycosidic chains which are linked together with α-1,4-glycan chains.

[0418] This unique combination of enzymes allows the mycobacterial envelope to be degraded from the outside in, reversing traditional phage-based approaches and also avoiding the inactivation of many genome surveillance pathways that would cleave the bacteriophage genome and kill the bacteriophage.

[0419] Another problem for mycobacterial therapeutics is that they can kill intracellular bacteria. Because phosphatidylserine (PS) is an indicator of apoptosis when it appears on the outer leaflet of mammalian cells, we addressed the intracellular delivery of enzymes by placing them in liposomes containing the phospholipid PS, which stimulates macrophages to engulf such cells.

[0420] We demonstrated the disruption of the mycobacterial envelope by an enzyme cocktail designed to attack the capsule, the bond between MA and AGL, and the PG. We also demonstrated the mechanism of action of EC1, which results in mycobacterial cell death by fragmentation into intracellular fragments. We also demonstrated the ability of EC1 to rescue macrophages from the apoptotic phenotype associated with mycobacterial-infected macrophages (MIMs). Here, we show the development and performance of EC1 in nontuberculous mycobacterial (NTM) species, in vitro and ex vivo (Figure 9).

[0421] In silico and biochemical identification of lysin A Lysin A genes are modular and typically contain two chains: an N-terminal catalytic domain and a C-terminal protein-binding domain. Lysin A normally catalyzes the degradation of the peptidoglycan layer of the mycobacterial envelope. To select lysin A for EC1, we used computational screening to identify unique functional domains and validated the identified genes by biophysical performance testing.

[0422] We collected all phage genes from PhageDB (371,279 genes; deposited on April 21, 2021) and performed bioinformatics analysis using BLAST. Genes annotated as lysin A were a subset from the gene set and used to generate a protein homology database. The remaining genes were compared to the lysin A protein database for homology using BLASTp. Queries with at least 90% sequence identity to the target lysin A were kept, resulting in the addition of 78 unlabeled genes to the 1753 lysin A genes already labeled. Additional quality control tests led us to discover two lysin A genes that were incorrectly labeled as lysin B, bringing the total number of lysin A targets to 1833. The identified domain sequences within each lysin A were extracted, resulting in 27 unique domain architectures. These unique domain architectures were cloned into a modified pET21a vector and screened for yield. Nine of the plasmids were high yielding and all nine high yielding enzymes were capable of hydrolyzing peptidoglycan. The thermostability and aggregation of the nine enzymes were tested using Unchained Labs' UNICLE® device. Three enzymes had aggregation and melting temperatures above 40° C. The enzyme with the highest aggregation and melting temperature was selected for the lysin A component of EC1. The biophysical properties of lysin A in EC1 were screened and experimentally verified from 371,279 genes down to just three candidates.

[0423] Sources of additional components in EC1 The pipeline applied to discover lysin A peptides was applied to the lysin B peptide sequence, identifying 15 unique lysin B architectures. Among these unique architectures were candidates from the preliminary screen. This lysin B gene showed both catalytic activity and excellent yield. The aggregation and melting temperatures were 52.3°C and 58.2°C, respectively.

[0424] α-Amylase was obtained commercially and a fungal isoamylase with a known crystal structure was expressed to investigate the hypothesis that EC1 passes through the outer leaflet, allowing lysin A and lysin B access to their target substrates.

[0425] Liposomal packaging of EC1 The enzyme component (i.e., SEQ ID NOs: 2, 184, 392, and 394) concentrations were standardized at 0.33 mg / mL. The protein solutions were packaged into liposomes or related lipid drug delivery vehicles using a Precision Nanosystems NANOASSEMBLR® IGNITE™.

[0426] Lipids (DO-phosphatidylcholine, DO-phosphatidylethanolamine, DO-phosphatidylserine, and cholesterol in a ratio of 1:1:0.3:0.4) resuspended in EtOH at a total concentration of 1-5 mg / mL were mixed with 1-10 mg / mL of his-tagged protein (capsules, which may contain lysin) in a 3:1 aqueous:organic ratio at a flow rate of 15 mL / min using a NANOASSEMBLR® IGNITE™ system (Precision Nanosystems). Liposomes were then analyzed by SDS-PAGE (8-16% Gradient Mini-Protean Pre-cast gels; Bio-Rad), dynamic light scattering (Zetasizer Ultra; Malvern), intrinsic fluorescence (Uncle; Unchained Labs), and static light scattering (Uncle) to test for associated antibiotic activity (Figures 5, 6, and 8). Dynamic light scattering revealed a mean hydrodynamic diameter of 413 nm and a PDI of 0.19, indicating high homogeneity (Figures 5A and 5B).

[0427] The formulation buffer consisted of at least one buffer component from glycine, Tris, sodium citrate, sodium acetate, and MES, with one or more excipients (5-10 mM CaCl2, 0-50 mM arginine, 0-200 nM NaCl, 0.33-1 mM sodium citrate, 0.5 mM MgCl2, and 10-30% glycerol) at concentrations of 50-150 mM and pHs of 5-11. One formulation contained 50 mM glycine (pH 8.5), 7.5 nM CaCl2, 0.5 mM MgCl2, 200 mM NaCl, 0.33 mM sodium citrate, and 10% glycerol (Figure 14). This formulation (formulation 8) showed improved killing compared to other conditions (Figure 7).

[0428] Growth curve identification algorithm To identify growth of NTM in the presence of our enzymes, reaction optical density information was collected using Biolog's OMNILOG®. Observations were collected from the 96-well plate every 30 minutes for 121 hours, resulting in 242 observations per well. To identify growth from the kinetic data, the observation values ​​were smoothed and the second derivative was calculated to determine the inflection point. A tangent line was calculated at the observed inflection point, and the x location where the tangent line intersects the y-intercept was identified. The y value at the x location of the tangent line was used as the inflection onset. A threshold was calculated by dividing the y value of the inflection point by the inflection onset. If the measured threshold was greater than 1.4, the well was labeled as proliferating NTM. An increase in the inflection onset over time also indicated a growth inhibitory effect. Furthermore, since NTM grows slower than other bacteria, an inflection point that showed growth within 24 hours identified a possible contamination (Figures 1A and 1B).

[0429] Mycobacterial cells clump together, making traditional methods inaccurate for quantification. We introduced enzyme and sonication treatments to optimize time and frequency and demonstrate more ideal dilution behavior in the absence of clumping, a major problem in the field.

[0430] Calculation of influence factors A dilution series was created and kinetic growth was measured to calculate the log growth change upon application of EC1. Binary growth calls across dilutions in the initial inoculum, untreated control, and treated samples were compared. An impact factor was calculated by the binary dilution factor of untreated growth versus treated growth (Figure 6).

[0431] In vitro component analysis The effect of individual enzymes and all possible component combinations on the initiation of NTM growth was analyzed. The concentrations of the tested combinations ranged from 0.0125 μg to 5 μg.

[0432] Imaging EC1 in macrophages Figures 2A and 2B are brightfield images of mycobacterial rod-shaped cells that were sheared by ABIα into intracellular fragments containing stainable lipids that gave rise to red chromosomes isolated from genomic DNA. The experiment was performed at pH 6.2. Figure 2A shows untreated mycobacterial cells containing fragment ratios. Lipids are shown in red and DNA in green. The green stained DNA indicates that the compromised cell envelope has provided access to the dye. Intact cells do not readily take up the red dye. Figure 2B shows cells and fragments treated with ABIα. Lipids are shown in red and DNA in green. The intracellular fragments glow with the lipophilic red dye. The green DNA is spread out compared to Figure 2A. Figures 3A and 3B show a similar experiment, where shearing was observed at pH 5.3. Figures 4A and 4B are brightfield images showing the ABIα mechanism by which mycobacteria are sheared into fragments. Figure 4A shows an untreated mycobacterial cell containing fragments. FM4-64 is a dye that stains lipids, envelopes, and lipid bodies. Figure 4B shows a treated mycobacterial cell with fragments. Cytox-Green stains DNA in cells with compromised envelopes.

[0433] Cell killing assay FIG. 10 is a graph showing treatment of J774A.1 murine macrophages (MΦ) uninfected or infected (MOI=10:1) with Mycobacterium abscessus with ABI α-PPL Formulation 8. The amount of necrotic cell death was measured for MΦ to investigate the safety and efficacy of ABI α-PPL treatment for mycobacterial infection. The extent of necrosis for MΦ was quantified as the ratio of the number of cells stained with SYTOX™ Green Nucleic Acid Stain (Invitrogen™) and Hoechst-33342 (Invitrogen™) in the population and counted per FOV (N=2-3 per well) by HCIA platform (BZ-X800; Keyence). A reduction in necrosis was observed in both uninfected (Form8 MΦ only) and infected (Form8-treated IIM) Formulation 8-treated groups compared to the untreated control group (untreated IIM).

[0434] FIG. 11 is a graph showing treatment of J774A.1 murine macrophages (MΦ) uninfected or infected (MOI=10:1) with Mycobacterium abscessus with ABI α-PPL formulation 8. The amount of necrotic cell death was measured for MΦ to investigate the safety and efficacy of ABI α-PPL treatment for mycobacterial infection. The extent of necrosis for MΦ was quantified as the ratio of the number of cells stained with SYTOX™ Green Nucleic Acid Stain (Invitrogen™) and Hoechst-33342 (Invitrogen™) in the population, and counted per FOV (N=3 per well) by HCIA platform (BZ-X800; Keyence). Compared to the untreated control group (untreated IIM), reduced necrosis was observed in both Formulation 8-treated groups (MΦ only and Form8-treated IIM) and in uninfected MΦ treated with empty liposomes (Lipo MΦ only).

[0435] FIG. 12 is a graph showing treatment of J774A.1 murine macrophages (MΦ) uninfected or infected (MOI=10:1) with Mycobacterium abscessus with ABI α-PPL. The extent of necrosis and oxidative stress in MΦ was quantified to investigate the safety and efficacy of ABI α-PPL treatment against mycobacterial infection. The extent of necrosis for MΦ was calculated as the ratio of the number of cells stained with SYTOX™ Green Nucleic Acid Stain (Invitrogen™) and Hoechst-33342 (Invitrogen™) in the population, counted per FOV (N=2-3 per well) by HCIA platform (Keyence).

[0436] FIG. 13 is a graph showing the degree of oxidative stress for MΦ quantified as the ratio of cells stained with CellROX™ (Invitrogen™) to the number of cells stained with Hoechst-33342 (Invitrogen™) and counted per FOV (N=2-3 per well) by HCIA platform (BZ-X800; Keyence) within a population. A reduction in necrosis was observed in all treatment groups compared to the untreated control group (untreated IMM). A reduction in oxidative stress was observed in two treatment groups (MΦ only and Form8-treated IIM**) compared to untreated IIM.

[0437] conclusion In this study, we show that EC1 can degrade the mycobacterial envelope and kill NTM both in vitro and inside macrophages. EC1 differs from small antibiotic molecules in its destruction by degrading the mycobacterial envelope regardless of the microbial replication state. EC1 destroys multiple cleavage sites in the mycobacterial envelope at different levels, destabilizing the envelope and sterilizing the bacteria.

[0438] Other embodiments All publications, patents, and patent applications mentioned herein are incorporated herein 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 in a document incorporated herein by reference, the definition provided herein serves as the definition of that term.

[0439] 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 any variations, uses, or adaptations of the invention in accordance with the principles of the invention in general, including departures from the present disclosure which have become known or are customarily practiced in the art to which the invention pertains, as applicable to the essential features described above, and in accordance with the scope of the appended claims.

Claims

1. (a) A lysant A containing an amino acid sequence having at least 85% sequence identity with one of sequence numbers 1 to 182, (b) A lysin B containing an amino acid sequence having at least 85% sequence identity with any one of sequence numbers 183 to 241, (c) an isoamylase containing an amino acid sequence having at least 85% sequence identity with any one of sequence numbers 242 to 392, and (d) α-amylase containing an amino acid sequence having at least 85% sequence identity with one of sequence numbers 393 to 445 A composition comprising a supramolecular structure containing one or more of the following.

2. (a) Lysine B containing an amino acid sequence having at least 85% sequence identity with any one of sequence numbers 183 to 241, and / or (b) Isoamylase containing an amino acid sequence having at least 85% sequence identity with any one of sequence numbers 242 to 392. The composition according to claim 1, comprising:

3. (a) Lysine B containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184, and / or (b) Isoamylase containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243 The composition according to claim 2, comprising:

4. (a) Lysine B comprising an amino acid sequence having at least 85% sequence identity with Sequence ID No. 184, and / or (b) Isoamylase containing an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 243 The composition according to claim 3, comprising:

5. (a) The supramolecular structure comprises a Z-average particle size of 75 nm to 750 nm, (b) The supramolecular structure is a lipid nanoparticle, a micelle, or a liposome, (c) The composition according to claim 1, wherein the composition further comprises a target portion.

6. (a) The Z-average average particle size is 75 nm to 250 nm, (b) The supramolecular structure contains one or more lipids including 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or 1,2-dioleyl-sn-glycero-3-phospho-L-serine (DOPS), (c) The composition according to claim 5, wherein the target portion is an extracellular target portion that targets professional antigen-presenting cells, and / or the target portion comprises phosphatidylserine.

7. (a) The supramolecular structure comprises a mixture of lipids including DOPC, DOPE, DOPS, and cholesterol. (b) The composition contains a sterol, and the sterol is cholesterol, or (c) The composition according to claim 6, wherein the professional antigen-presenting cell is a macrophage or a dendritic cell.

8. The composition according to claim 7, wherein the DOPC, DOPE, DOPS, and cholesterol are present in a molar ratio of 1-20:1-20:1-5:1-5.

9. The composition according to claim 8, wherein the DOPC, DOPE, DOPS, and cholesterol are present in a molar ratio of 10:10:3:

4.

10. The composition according to claim 1, for use in treating a bacterial infection in a subject.

11. (a) The bacterial infection is caused by actinomycetes, (b) The treatment further comprises administering an antibiotic, and / or (c) The composition according to claim 10, wherein the composition is administered intravenously, orally, or by inhalation.

12. (a) The actinomycete bacteria are bacteria of the orders Corynebacteriales or Propionibacteriales, and / or (b) The antibiotic is (i) Cephalosporins, carbapenems, penicillins, aminoglycosides, cephalosporins, rifamycin, macrolides, or fluoroquinolones, (ii) Thiacetazone, SQ-109, bedaquiline, delamanid, pyrazinamide, or isoniazid, (iii) The composition according to claim 11, wherein the composition is azithromycin, clarithromycin, ethambutol, rifampin, biapenem, or amikacin.

13. (a) The order Corynebacteriales is a species of Mycobacterium, Nocardia, Corynebacterium, or Rhodococcus, (b) The composition according to claim 12, wherein the order Propionibacteriales is the species Cutibacterium.

14. (a) The Mycobacterium species is M. tuberculosis, M. leprae, M. lepromatosis, M. avium, M. kansasii, M. fortuitum, M. chelonae, M. marinum, M. intracellulare, M. abscessus, M. chimera, M. boletti, M. fortuitum, M. goodii, or M. Masiliense, (b) The Nocardia species is N. brasilensis, N. cyriacigeorgica, N. farcinica, N. nova, N. asteroids, N. brasilensis, or N. caviae. (c) The Corynebacterium species is C. glutamicum or C. diphtheriae. (d) The Rhodococcus species is R. fascians or R. equii, (e) The composition according to claim 13, wherein the Cutibacterium species is C. acnes.

15. (a) To provide a sample containing one or more of solubilizer A, solubilizer B, isoamylase, and α-amylase, (b) Mixing the sample with one or more lipids in the presence of an organic solvent to form liposomes containing one or more of the solubilizer A, solubilizer B, isoamylase, and α-amylase, An assembly method including

16. (i) The lysant A comprises an amino acid sequence having at least 85% sequence identity with any one of sequence numbers 1 to 182, (ii) The lysant B comprises an amino acid sequence having at least 85% sequence identity with any one of sequence numbers 183 to 241, (iii) The isoamylase comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or 243, and / or (iv) The α-amylase comprises an amino acid sequence having at least 85% sequence identity with any one of sequence numbers 393 to 445. The method according to claim 15.

17. (i) The lysant A contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or 2, (ii) The solubilizer B contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or 184, (iii) The isoamylase comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or 243, and / or (iv) The α-amylase contains an amino acid sequence having at least 85% sequence identity with any one of sequence numbers 393 to 398. The method according to claim 15.

18. The method according to claim 15, wherein the organic solvent contains ethanol.

19. The method according to claim 18, wherein the ethanol is provided in a ratio of 10:1 to 1:1 or 1:10 to 1:1 with respect to the aqueous portion of the sample.

20. The method according to claim 19, wherein the ratio is 8:1 to 3:1 or 1:8 to 1:3.