Compositions and methods for treating actinomycete infections

Abstract

The present disclosure features compositions and methods for the treatment of actinomycete (e.g., Corynebacterium) infections, e.g., caused by Mycobacterium cells. In one aspect, the present disclosure features a composition comprising an unencapsulated protein comprising one or more of: (a) lysin A; (b) lysin B; (c) an isoamylase; and (d) an alpha-amylase. The composition optionally further comprises a supramolecular structure comprising one or more of the following: (a) lysin A; (b) lysin B; (c) an isoamylase; and (d) an alpha-amylase. In another aspect, the invention features a method of treating a bacterial infection in a subject by administering to the subject a composition of an unencapsulated cleavage protein as described herein, optionally in combination with an encapsulated cleavage protein, in an amount and duration sufficient to treat the bacterial infection. The encapsulated cleavage proteins and the unencapsulated cleavage proteins can be in the same or different compositions, and can be administered simultaneously or sequentially.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy created on April 13, 2023 is named 51486-007WO4_Sequence_Listing_4_13_23.xml and is 660,323 bytes in size. Background Art

[0003] Bacterial pathogens are the main causes of infectious diseases. Many bacteria are successfully detected by the human immune system and are rapidly removed before the onset of infection. However, some bacterial pathogens escape the host immune system by residing in host cells. These intracellular bacteria evolve multiple immune evasion techniques by residing and breeding in correct intracellular compartments (e.g., endosomes, phagosomes, lysosomes or cytosol) in host cells such as immune cells (e.g., macrophages or dendritic cells) and host cells. Due to the lack of subcellular localization accessibility of infection, bacterial infections bred in host cells usually present difficult treatment obstacles. Although some antibacterial compositions can treat infection (e.g., in vitro), it has been proven to be challenging work to deliver treatment to the correct subcellular localization where bacteria reside.

[0004] A challenging group of intracellular bacterial infections is caused by mycobacteria. Mycobacteria are actinomycetia (e.g., corynebacteriales or ropionibacteriales) represented by a thick capsule rich in mycolic acids. Mycobacteria contain an envelope containing a cell membrane composed of lipids, a cell wall composed of peptidoglycan, an arabinogalactan layer, and an outer membrane called a mycomembrane, which is rich in mycolic acids. Many mycobacterial envelopes also contain an outer capsule layer composed of polysaccharides such as D-glucan, D-arabino-D-mannan, and D-mannan. This complex cell envelope contributes to the cold resistance of mycobacteria and is particularly difficult to penetrate and destroy mycobacterial cells.

[0005] These bacteria also contain a complex life cycle in which the bacteria reside in the cytoplasm or other subcellular compartments or outside the host cell. Mycobacteria are endocytosed by host cells and these endocytosed vesicles can fuse with intracellular organelles such as endosomes, phagosomes, or lysosomes. Once inside these intracellular compartments, the bacteria are able to replicate and grow. Subsequently, the membrane dissolves and releases the bacteria into the cytoplasm, where growth continues. Subsequently, the bacteria lyse the host cell and spread as free bacteria. This free form of bacteria may appear in the spleen and liver after being released from lung phagocytes, etc., leading to the expansion of the infection and ultimately death.

[0006] Due to the complex life cycle of bacteria, it is difficult to target bacteria to the appropriate location in time and space to effectively treat infection. For example, when in a host cell, it is necessary to target the correct intracellular compartment at the correct life cycle stage, or target an extracellular location after host cell lysis. Therefore, there is a need for improved compositions and methods for targeting and treating bacterial infections (such as those caused by mycobacteria). Patients may have mycobacteria at various stages of the life cycle simultaneously. Summary of the invention

[0007] In one aspect, the invention features a composition containing unencapsulated proteins, the composition comprising one or more (e.g., two or more, three or more, or all four) of the following: (a) lysin A; (b) lysin B; (c) isoamylase; and (d) alpha-amylase.

[0008] In some embodiments, the composition comprises lysin A and lysin B.

[0009] In some embodiments, the composition comprises lysin A and isoamylase.

[0010] In some embodiments, the composition comprises lysin A and alpha-amylase.

[0011] In some embodiments, the composition comprises lysin B and isoamylase.

[0012] In some embodiments, the composition comprises lysin B and alpha-amylase.

[0013] In some embodiments, the composition comprises an isoamylase and an alpha-amylase.

[0014] In some embodiments, the composition comprises lysin A, lysin B, and isoamylase.

[0015] In some embodiments, the composition comprises lysin A, lysin B, and alpha-amylase.

[0016] In some embodiments, the composition comprises lysin A, isoamylase, and alpha-amylase.

[0017] In some embodiments, the composition comprises lysin B, isoamylase, and alpha-amylase.

[0018] In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and alpha-amylase.

[0019] In some embodiments, the invention features a composition containing an unencapsulated protein, the composition comprising one or more (e.g., two or more, three or more, or all four) of the following: (a) 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; (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 any one of SEQ ID NOs: 183-241; (c) 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; and (d) an alpha-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: Any of NOs: 393-445 has an amino acid sequence with at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity.

[0020] In some embodiments, the invention features a composition containing an unencapsulated protein, the composition comprising one or more (e.g., two or more, three or more, or all four) of the following: (a) 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; (c) an isoamylase 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 alpha-amylase 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: 244 or SEQ ID NO: 245. Any of NOs: 393-398 has an amino acid sequence with at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity.

[0021] In some embodiments, the composition comprises lysin A and lysin B, and 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.

[0022] In some embodiments, the composition comprises lysin A and lysin B, and 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.

[0023] In some embodiments, the composition comprises lysin A and isoamylase, and 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 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.

[0024] In some embodiments, the composition comprises lysozyme A and isoamylase, and lysozyme 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 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.

[0025] In some embodiments, the composition comprises lysin A and an alpha-amylase, and the 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 the alpha-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.

[0026] In some embodiments, the composition comprises lysin A and an alpha-amylase, and the 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 the alpha-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.

[0027] In some embodiments, the composition comprises lysozyme B and isoamylase, and lysozyme 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 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.

[0028] In some embodiments, the composition comprises lysozyme B and isoamylase, and lysozyme 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 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.

[0029] In some embodiments, the composition comprises lysozyme B and an alpha-amylase, and lysozyme 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 the alpha-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.

[0030] In some embodiments, the composition comprises lysozyme B and an alpha-amylase, and lysozyme 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 alpha-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.

[0031] In some embodiments, the composition comprises an isoamylase and an alpha-amylase, and 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 alpha-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.

[0032] In some embodiments, the composition comprises an isoamylase and an alpha-amylase, and 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 alpha-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.

[0033] In some embodiments, the composition comprises lysin A, lysin B, and isoamylase, and 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 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.

[0034] In some embodiments, the composition comprises lysin A, lysin B, and isoamylase, and 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 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.

[0035] In some embodiments, the composition comprises lysin A, lysin B, and an alpha-amylase, and 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 the alpha-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.

[0036] In some embodiments, the composition comprises lysin A, lysin B, and an alpha-amylase, and 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 the alpha-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.

[0037] In some embodiments, the composition comprises lysin A, isoamylase, and alpha-amylase, and 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; 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 alpha-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.

[0038] In some embodiments, the composition comprises lysin A, isoamylase, and alpha-amylase, and 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; 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 alpha-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.

[0039] In some embodiments, the composition comprises lysin B, isoamylase, and alpha-amylase, 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: 242-392; and the alpha-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.

[0040] In some embodiments, the composition comprises lysin B, isoamylase, and alpha-amylase, 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; 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 alpha-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.

[0041] In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and alpha-amylase, and 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; 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 alpha-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.

[0042] In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and alpha-amylase, and 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; 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 alpha-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. In some embodiments, the composition comprises lysozyme A, lysozyme B, isoamylase and alpha-amylase, and lysozyme A comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; lysozyme B comprises the amino acid sequence of SEQ ID NO: 183 or SEQ ID NO: 184; the isoamylase comprises the amino acid sequence of SEQ ID NO: 242 or SEQ ID NO: 243; and the alpha-amylase comprises the amino acid sequence of any one of SEQ ID NOs: 393-398.

[0043] In some embodiments, the composition comprises lysin A of SEQ ID NO: 1, lysin B of SEQ ID NO: 183, isoamylase of SEQ ID NO: 242, and alpha-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, isoamylase of SEQ ID NO: 243, and alpha-amylase of SEQ ID NO: 394.

[0044] In some embodiments, the composition comprises lysin A of SEQ ID NO: 1, lysin B of SEQ ID NO: 183, isoamylase of SEQ ID NO: 242, and alpha-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, isoamylase of SEQ ID NO: 243, and alpha-amylase of SEQ ID NO: 396.

[0045] In some embodiments, the composition comprises lysin A of SEQ ID NO: 1, lysin B of SEQ ID NO: 183, isoamylase of SEQ ID NO: 242, and alpha-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, isoamylase of SEQ ID NO: 243, and alpha-amylase of SEQ ID NO: 398.

[0046] In some embodiments, the composition comprises a concentration of 0.1 mg / mL to 20 mg / mL (e.g., for example, 0.1 mg / mL to 1 mg / mL, such as 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, such as 1 mg / mL to 10 mg / mL, such as 2 mg / mL, 3 mg / mL, 4 mg / mL, In some embodiments, the composition comprises a protein (e.g., lysozyme A, lysozyme B, isoamylase and / or alpha-amylase) at a concentration of 1 mg / mL to 10 mg / mL, for example, 10 mg / mL to 20 mg / mL, for example, 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 lysozyme A, lysozyme B, isoamylase and / or alpha-amylase at a concentration of 1 mg / mL to 10 mg / mL.

[0047] In some embodiments, the composition further comprises a supramolecular structure comprising one or more (e.g., two or more, three or more, or all four) of the following: (a) lysin A; (b) lysin B; (c) isoamylase; and (d) α-amylase.

[0048] In some embodiments, the composition comprises lysin A and lysin B.

[0049] In some embodiments, the composition comprises lysin A and isoamylase.

[0050] In some embodiments, the composition comprises lysin A and alpha-amylase.

[0051] In some embodiments, the composition comprises lysin B and isoamylase.

[0052] In some embodiments, the composition comprises lysin B and alpha-amylase.

[0053] In some embodiments, the composition comprises an isoamylase and an alpha-amylase.

[0054] In some embodiments, the composition comprises lysin A, lysin B, and isoamylase.

[0055] In some embodiments, the composition comprises lysin A, lysin B, and alpha-amylase.

[0056] In some embodiments, the composition comprises lysin A, isoamylase, and alpha-amylase.

[0057] In some embodiments, the composition comprises lysin B, isoamylase, and alpha-amylase.

[0058] In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and alpha-amylase.

[0059] In some embodiments, the composition further comprises a supramolecular structure comprising one or more (e.g., two or more, three or more, or all four) of the following: (a) 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; (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 any one of SEQ ID NOs: 183-241; (c) 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; and (d) an alpha-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: 243-394. Any of NOs: 393-445 has an amino acid sequence with at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity.

[0060] In some embodiments, the composition further comprises a supramolecular structure comprising one or more (e.g., two or more, three or more, or all four) of the following: (a) 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; (c) an isoamylase 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 alpha-amylase 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: 244 or SEQ ID NO: 245. Any of NOs: 393-398 has an amino acid sequence with at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity.

[0061] In some embodiments, the supramolecular structure comprises lysin A and lysin B, and 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.

[0062] In some embodiments, the supramolecular structure comprises lysin A and lysin B, and 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.

[0063] In some embodiments, the supramolecular structure comprises lysin A and isoamylase, and 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 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.

[0064] In some embodiments, the supramolecular structure comprises lysin A and isoamylase, and 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 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.

[0065] In some embodiments, the supramolecular structure comprises lysin A and α-amylase, and 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 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.

[0066] In some embodiments, the supramolecular structure comprises lysin A and α-amylase, and 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 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.

[0067] In some embodiments, the supramolecular structure comprises lysozyme B and isoamylase, and lysozyme 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 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.

[0068] In some embodiments, the supramolecular structure comprises lysozyme B and isoamylase, and lysozyme 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 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.

[0069] In some embodiments, the supramolecular structure comprises lysin B and alpha-amylase, 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; and the alpha-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.

[0070] In some embodiments, the supramolecular structure comprises lysin B and α-amylase, 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; 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.

[0071] In some embodiments, the supramolecular structure comprises an isoamylase and an alpha-amylase, and 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 alpha-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.

[0072] In some embodiments, the supramolecular structure comprises an isoamylase and an alpha-amylase, and 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 alpha-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.

[0073] In some embodiments, the supramolecular structure comprises lysin A, lysin B, and isoamylase, and 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.

[0074] In some embodiments, the supramolecular structure comprises lysin A, lysin B and isoamylase, and 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.

[0075] In some embodiments, the supramolecular structure comprises lysin A, lysin B and α-amylase, and 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.

[0076] In some embodiments, the supramolecular structure comprises lysin A, lysin B and α-amylase, and 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.

[0077] In some embodiments, the supramolecular structure comprises lysin A, isoamylase and alpha-amylase, and 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; 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 alpha-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.

[0078] In some embodiments, the supramolecular structure comprises lysin A, isoamylase and alpha-amylase, and 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; 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 alpha-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.

[0079] In some embodiments, the supramolecular structure comprises lysin B, isoamylase and alpha-amylase, 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: 242-392; and the alpha-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.

[0080] In some embodiments, the supramolecular structure comprises lysin B, isoamylase and alpha-amylase, 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; 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 alpha-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.

[0081] In some embodiments, the supramolecular structure comprises lysin A, lysin B, isoamylase and alpha-amylase, and 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; 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 alpha-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.

[0082] In some embodiments, the supramolecular structure comprises lysin A, lysin B, isoamylase and alpha-amylase, and 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 alpha-amylase 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: 244; NO:393-398 has an amino acid sequence with at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity. In some embodiments, the supramolecular structure comprises lysin A, lysin B, isoamylase and alpha-amylase, and 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 alpha-amylase comprises the amino acid sequence of any one of SEQ ID NO:393-398.

[0083] In some embodiments, the supramolecular structure comprises lysin A of SEQ ID NO: 1, lysin B of SEQ ID NO: 183, isoamylase of SEQ ID NO: 242, and alpha-amylase of SEQ ID NO: 393. In some supramolecular structures, the composition comprises lysin A of SEQ ID NO: 2, lysin B of SEQ ID NO: 184, isoamylase of SEQ ID NO: 243, and alpha-amylase of SEQ ID NO: 394.

[0084] In some embodiments, the supramolecular structure comprises lysin A of SEQ ID NO: 1, lysin B of SEQ ID NO: 183, isoamylase of SEQ ID NO: 242, and α-amylase of SEQ ID NO: 395. In some embodiments, the supramolecular structure comprises lysin A of SEQ ID NO: 2, lysin B of SEQ ID NO: 184, isoamylase of SEQ ID NO: 243, and α-amylase of SEQ ID NO: 396.

[0085] In some embodiments, the supramolecular structure comprises lysin A of SEQ ID NO: 1, lysin B of SEQ ID NO: 183, isoamylase of SEQ ID NO: 242, and α-amylase of SEQ ID NO: 397. In some embodiments, the supramolecular structure comprises lysin A of SEQ ID NO: 2, lysin B of SEQ ID NO: 184, isoamylase of SEQ ID NO: 243, and α-amylase of SEQ ID NO: 398.

[0086] In some embodiments, the Z-average particle size of the supramolecular structure is 75nm to 5μm, such as 75nm to 2μm, 75nm to 1μm, such as 75nm to 750nm (e.g., 250nm to 750nm or 75nm to 250nm). In some embodiments, when the supramolecular structure is LNP or micelle, the Z-average particle size is 75nm to 250nm. In some embodiments, when the supramolecular structure is a vesicle (e.g., liposome), the Z-average particle size is 250nm to 750nm. Non-limiting examples of Z-average particle size include, for example, 75 nm to 100 nm, for example 75 nm to 85 nm (e.g., 80 nm), for example 80 nm to 140 nm, 90 nm to 130 nm, or 110 nm to 130 nm (e.g., 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 (e.g., 270 nm), for example 300 nm to 400 nm, 400 nm to 600 nm, for example 450 nm to 550 nm. m, 475nm to 525nm, 480nm to 520nm, 490nm to 510nm, 495nm to 505nm (e.g., 500nm), for example, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 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, 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, 62 0nm, 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, 7 85nm, 790nm, 795nm, 800nm, 805nm, 810nm, 815nm, 820nm, 825nm, 830nm, 835nm, 840nm, 845nm, 850nm, 855nm, 860nm, 865n m, 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 In some embodiments, the supramolecular structure has a Z-average particle size of 80 nm, 270 nm, or 500 nm. In some embodiments, the supramolecular structure comprises a Z-average particle size of 75 nm to 750 nm. In some embodiments, the Z-average particle size is 250 nm to 750 nm. In some embodiments, the Z-average particle size is 75 nm to 250 nm.

[0087] In some embodiments, the supramolecular structure is a lipid nanoparticle.

[0088] In some embodiments, the supramolecular structure is a micelle.

[0089] In some embodiments, the supramolecular structure is a liposome. The liposome can be unilamellar. Alternatively, the liposome can be multilamellar.

[0090] In some embodiments, the supramolecular structure comprises a polydispersity index of 0.05 to 0.3.

[0091] In some embodiments, the supramolecular structure comprises one or more lipids.

[0092] In some embodiments, at least one of the one or more lipids can be, for example, an ionizable lipid. 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).

[0093] In some embodiments, the lipid is a sterol, such as cholesterol or a derivative thereof.

[0094] In some embodiments, the supramolecular structure comprises a mixture of lipids. For example, the mixture of lipids may include two or more of DOPC, DOPE, DOPS, and cholesterol.

[0095] 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).

[0096] 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).

[0097] In some embodiments, 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).

[0098] 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).

[0099] 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).

[0100] 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).

[0101] 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.

[0102] In some embodiments, the supramolecular structure comprises a lipid at a concentration of 0.1 mg / mL to 10 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, such as 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, such as 1 mg / mL to 10 mg / mL, such as 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 supramolecular structure comprises a lipid at a concentration of 1 mg / mL to 5 mg / mL.

[0103] In some embodiments, the supramolecular structure comprises a concentration of 0.1 mg / mL to 20 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, such as 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, such as 1 mg / mL to 10 mg / mL, such as 2 mg / mL, 3 mg / mL, In some embodiments, the composition comprises a protein (e.g., lysozyme A, lysozyme B, isoamylase and / or alpha-amylase) at a concentration of 1 mg / mL to 10 mg / mL.

[0104] In some embodiments, supramolecular structures as described herein are formulated with one or more buffers and / or excipients. For example, supramolecular structures (e.g., liposomes containing a mixture of a lyase) can be, for example, encapsulated and / or formulated in a buffer (such as glycine, Tris, sodium citrate, sodium acetate, and MES) at a concentration of 10mM to 200mM, such as 50mm to 150mm, such as 10mM, 20mm, 30mm, 40mm, 50mm, 60mm, 70mm, 80mm, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, or 200mM. The supramolecular structure can be formulated at a pH of 5 to 11 (e.g., 5 to 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 to 11, e.g., a pH of 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11). The supramolecular structure may further comprise one or more excipients, such as CaCl2, arginine, NaCl, sodium citrate, MgCl2, or glycerol. In some embodiments, the supramolecular structure comprises, for example, 5 mM to 10 mM CaCl2, 0 to 50 mM arginine, 0 to 200 mM NaCl, 0 to 1 mM sodium citrate, 0 to 1 mM MgCl2, and / or 10%-30% glycerol. In some embodiments, the supramolecular structure 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 also comprise TWEEN, such as TWEEN-80.

[0105] In some embodiments, the supramolecular structure 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.

[0106] In another aspect, the invention features a method of treating a bacterial infection in a subject. The method includes administering to the subject a composition as described herein (eg, any of the above embodiments) in an amount and for a duration sufficient to treat the bacterial infection.

[0107] In some embodiments, the method further comprises administering a supramolecular structure comprising one or more (e.g., two or more, three or more, or all four) of the following: (a) 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; (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 any one of SEQ ID NOs: 183-241; (c) 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; and (d) an alpha-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: 243-394. Any of NOs: 393-445 has an amino acid sequence with at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity.

[0108] In some embodiments, the method further comprises administering a supramolecular structure comprising one or more (e.g., two or more, three or more, or all four) of the following: (a) 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; (c) an isoamylase 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 alpha-amylase 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: 244 or SEQ ID NO: 245. Any of NOs: 393-398 has an amino acid sequence with at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity.

[0109] In some embodiments, the supramolecular structure comprises lysin A and lysin B, and 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.

[0110] In some embodiments, the supramolecular structure comprises lysin A and lysin B, and 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.

[0111] In some embodiments, the supramolecular structure comprises lysin A and isoamylase, and 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 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.

[0112] In some embodiments, the supramolecular structure comprises lysin A and isoamylase, and 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 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.

[0113] In some embodiments, the supramolecular structure comprises lysin A and α-amylase, and 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 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.

[0114] In some embodiments, the supramolecular structure comprises lysin A and α-amylase, and 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 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.

[0115] In some embodiments, the supramolecular structure comprises lysozyme B and isoamylase, and lysozyme 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 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.

[0116] In some embodiments, the supramolecular structure comprises lysozyme B and isoamylase, and lysozyme 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 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.

[0117] In some embodiments, the supramolecular structure comprises lysin B and alpha-amylase, 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; and the alpha-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.

[0118] In some embodiments, the supramolecular structure comprises lysin B and α-amylase, 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; 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.

[0119] In some embodiments, the supramolecular structure comprises an isoamylase and an alpha-amylase, and 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 alpha-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.

[0120] In some embodiments, the supramolecular structure comprises an isoamylase and an alpha-amylase, and 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 alpha-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.

[0121] In some embodiments, the supramolecular structure comprises lysin A, lysin B, and isoamylase, and 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.

[0122] In some embodiments, the supramolecular structure comprises lysin A, lysin B and isoamylase, and 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.

[0123] In some embodiments, the supramolecular structure comprises lysin A, lysin B and α-amylase, and 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.

[0124] In some embodiments, the supramolecular structure comprises lysin A, lysin B and α-amylase, and 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.

[0125] In some embodiments, the supramolecular structure comprises lysin A, isoamylase and alpha-amylase, and 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; 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 alpha-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.

[0126] In some embodiments, the supramolecular structure comprises lysin A, isoamylase and alpha-amylase, and 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; 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 alpha-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.

[0127] In some embodiments, the supramolecular structure comprises lysin B, isoamylase and alpha-amylase, 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: 242-392; and the alpha-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.

[0128] In some embodiments, the supramolecular structure comprises lysin B, isoamylase and alpha-amylase, 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; 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 alpha-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.

[0129] In some embodiments, the supramolecular structure comprises lysin A, lysin B, isoamylase and alpha-amylase, and 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; 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 alpha-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.

[0130] In some embodiments, the supramolecular structure comprises lysin A, lysin B, isoamylase and alpha-amylase, and 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 alpha-amylase 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: 244; Any of NOs: 393-398 has an amino acid sequence with at least 85% (e.g., at least 90%, 95%, 97%, 99% or 100%) sequence identity.

[0131] In some embodiments, the supramolecular structure comprises lysin A, lysin B, isoamylase and α-amylase, and 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; the isoamylase comprises the amino acid sequence of SEQ ID NO: 242 or SEQ ID NO: 243; and the α-amylase comprises the amino acid sequence of any one of SEQ ID NOs: 393-398.

[0132] In some embodiments, the supramolecular structure comprises lysin A of SEQ ID NO: 1, lysin B of SEQ ID NO: 183, isoamylase of SEQ ID NO: 242, and alpha-amylase of SEQ ID NO: 393. In some supramolecular structures, the composition comprises lysin A of SEQ ID NO: 2, lysin B of SEQ ID NO: 184, isoamylase of SEQ ID NO: 243, and alpha-amylase of SEQ ID NO: 394.

[0133] In some embodiments, the supramolecular structure comprises lysin A of SEQ ID NO: 1, lysin B of SEQ ID NO: 183, isoamylase of SEQ ID NO: 242, and α-amylase of SEQ ID NO: 395. In some embodiments, the supramolecular structure comprises lysin A of SEQ ID NO: 2, lysin B of SEQ ID NO: 184, isoamylase of SEQ ID NO: 243, and α-amylase of SEQ ID NO: 396.

[0134] In some embodiments, the supramolecular structure comprises lysin A of SEQ ID NO: 1, lysin B of SEQ ID NO: 183, isoamylase of SEQ ID NO: 242, and α-amylase of SEQ ID NO: 397. In some embodiments, the supramolecular structure comprises lysin A of SEQ ID NO: 2, lysin B of SEQ ID NO: 184, isoamylase of SEQ ID NO: 243, and α-amylase of SEQ ID NO: 398.

[0135] In some embodiments, the composition is administered prior to the supramolecular structure.

[0136] In some embodiments, the composition is administered after the supramolecular structure.

[0137] In some embodiments, the composition and the supramolecular structure are administered substantially simultaneously.

[0138] In some embodiments, the bacterial infection is caused by an Actinomycetes bacteria. In some embodiments, the Actinomycetes is Corynebacteriales or Propionibacteriales. In some embodiments, the Corynebacteriales is a Mycobacterium species.

[0139] 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.

[0140] In some embodiments, the Corynebacteriales is a Nocardia, Corynebacterium, or Rhodococcus species.

[0141] In some embodiments, the Propionibacteriales is a Cutibacterium species.

[0142] In some embodiments, compositions and methods as described herein can be used for targeting other actinomycetes (e.g., Corynebacterials or Propionibacterials) with an envelope component similar to mycobacterium. For example, compositions and methods can be used for targeting Nocardia, Corynebacterials 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. Corynebacterials species can be, for example, C. glutamicum or C. diphtheriae. Rhodococcus species can be, for example, R. fascians or R. equi.

[0143] In some embodiments, the compositions and methods can be used to target Propionibacterium species. The Propionibacterium species can be, for example, Propionibacterium acnes (C. acnes).

[0144] In some embodiments, the method further comprises administering antibiotics. In some embodiments, the antibiotic is cephalosporin, carbapenem, penicillin, aminoglycoside, cephalosporin, rifamycin, macrolide or fluoroquinolone. In some embodiments, the antibiotic is thiosemicarbazide, sq-109, bedaquiline, delamanid, pyrazinamide or isoniazid. In some embodiments, the antibiotic is azithromycin, clarithromycin, ethambutol, rifampicin, biapenem or amikacin. In some embodiments, the antibiotic is a macrolide (for example, azithromycin, clarithromycin, erythromycin). In some embodiments, the antibiotic is a macrolide (for example, azithromycin, clarithromycin, erythromycin). In some embodiments, the antibiotic is an aminoglycoside (for example, kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin (for example, neomycin B, C or E), streptomycin or pramixin).

[0145] In some embodiments, the composition is administered intravenously, orally, topically, or by inhalation (eg, by aerosol).

[0146] In some embodiments, the compositions and methods described herein target bacteria that reside extracellularly for at least a portion of their life cycle.

[0147] In some embodiments, the compositions and methods described herein target bacteria that reside intracellularly for at least a portion of their life cycle.

[0148] In some embodiments, the compositions and methods described herein target bacteria that reside both extracellularly and intracellularly during at least a portion of their life cycle.

[0149] definition

[0150] As used herein, the term "about" means + / - 10% of the recited value.

[0151] As used herein, "combination therapy" or "combined administration" refers to the administration of two (or more) agents or treatments to a subject as part of a defined treatment regimen for a specific disease or illness. The treatment regimen defines the dosage and administration cycle of each agent so that the effects of the individual agents on the subject overlap and / or allow synergy. In some embodiments, the delivery of two or more agents is simultaneous or parallel, and the agents can be co-formulated. In some embodiments, two or more agents are not co-formulated and are administered in a sequential manner as part of a prescription regimen. In some embodiments, the combined administration of two or more agents or treatments causes a reduction in symptoms or other parameters associated with the disease greater than the reduction observed in a single agent or treatment delivered or in the absence of another. The effects of the two treatments may be partially cumulative, completely cumulative, or greater than cumulative (e.g., synergistic). Sequential or substantially simultaneous administration of each therapeutic agent may be performed by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, topical routes, and direct absorption by mucosal tissue. Therapeutic agents may 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.

[0152] As used herein, the term "effective amount", "therapeutically effective amount" and "sufficient amount" of an agent for producing a therapeutic effect in a cell, sample or subject as described herein, for example, refers to an amount sufficient to produce a beneficial or desired result (including preclinical or clinical results) when applied to a cell, sample or subject including a human, and therefore, "effective amount" or its synonyms depend on the context in which it is applied. For example, in the case of treating a disease, it is the amount of an agent sufficient to achieve a therapeutic response compared to the response obtained when the agent is not administered. The amount of a given agent will vary according to various factors, such as a given agent, a pharmaceutical formulation, a route of administration, the severity of a bacterial infection, a biomarker, such as the age, sex and / or weight of the subject being treated, a sample or a host cell (e.g., a mammalian immune cell), etc., but can still be routinely determined by a person of ordinary skill in the art. Similarly, 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, the therapeutically effective amount of an agent can be easily determined by a person of ordinary skill by conventional methods known in the art. Dosage regimens may be adjusted to provide the optimum therapeutic response.

[0153] As used herein, the term "antimicrobial lytic protein" refers to a protein with bactericidal and / or lytic activity against bacteria. Non-limiting examples of antimicrobial lytic proteins include perforins, lysins (e.g., lysin A and / or lysin B), amylases (e.g., isoamylases or α-amylases), capsule depolymerases (e.g., hydrolases, metallohydrolases, epoxide hydrolases, peptidoglycan hydrolases, polysaccharidases, polysaccharide lyases, endosialidases, hyaluronan lyases or alginate lyases), beta-lactamases and lysozymes.

[0154] As used herein, "lipid nanoparticle" or "LNP" is a vesicle comprising a lipid layer that encapsulates a substantially solid lipid core; the lipid core may contain a pharmaceutically active molecule. LNPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates).

[0155] As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids disposed in at least one bilayer (e.g., one or more bilayers). Liposomes include monolayer and multilayer (e.g., 2, 3, 4, 5 or more layers) vesicles having a membrane and an aqueous interior formed by a lipophilic material. The aqueous portion contains an antimicrobial lytic protein or a mixture of an antimicrobial lytic protein and other components. The lipophilic material separates the aqueous interior from the aqueous exterior that does not generally include bacteriophage proteins (although in some instances, it may include). Liposomes also include "sterically stabilized" liposomes, which are terms used herein to refer to liposomes comprising one or more specialized lipids, which when incorporated into liposomes result in an increased circulation life relative to liposomes lacking such specialized lipids.

[0156] "Micelles" are defined herein as a specific type of essentially spherical supramolecular structure in which amphiphilic molecules (e.g., lipids) are arranged so that the hydrophobic portions of the molecules point inwardly toward the core, leaving the hydrophilic portions in contact with the surrounding aqueous phase. If the surrounding environment is hydrophobic, the opposite arrangement exists. The micelle core may contain an antimicrobial lytic protein or a mixture of proteins.

[0157] As used herein, the term "subject" refers to any organism to which a composition according to the invention may be administered, for example, for experimental, diagnostic, preventive and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject may be seeking or in need of treatment, requiring treatment, being treated, about to be treated, or a person or animal under the care of a trained professional for a particular disease or illness.

[0158] As used herein, the term "supramolecular structure" refers to a molecular complex held together by non-covalent bonds such as hydrogen bonds, van der Waals forces, electrostatic interactions, hydrophobic effects, and π-π interactions. Supramolecular structures can include large molecular complexes that form, for example, spherical structures. Supramolecular structures include, for example, lipid-based supramolecular structures, such as liposomes, lipid nanoparticles, and micelles.

[0159] As used herein, the term "targeted intracellular compartment" refers to an endosome, a phagosome, a lysosome, or the cytosol.

[0160] As used herein, the term "unencapsulated protein" refers to a free protein that is not present in a supramolecular structure. For example, an unencapsulated protein is not formulated within a liposome, lipid nanoparticle, or micelle.

[0161] As used herein, the term "targeting moiety" refers to a moiety (e.g., a small molecule, such as a carbohydrate) that specifically binds or reactively associates or complexes with a receptor or other receptive moiety associated with a given target cell population (e.g., a professional antigen presenting cell, e.g., a macrophage or a dendritic cell). Thus, the targeting moiety can be used to target the supramolecular structures described herein, for example, to professional antigen presenting cells (e.g., macrophages or dendritic cells).

[0162] "Vesicle" is defined herein as a type of supramolecular structure in which amphiphilic molecules (e.g., lipids) together define a volume, e.g., a substantially spherical volume. Amphiphilic molecules (e.g., lipids) typically form at least one shell of a vesicle. In such a shell, the amphiphilic molecules are arranged in a bilayer, wherein the hydrophilic portions of the amphiphilic molecules are oriented outwardly relative to the bilayer plane and the hydrophobic portions of the amphiphilic molecules are primarily disposed within the bilayer. If the surrounding medium is hydrophobic, the opposite arrangement exists. BRIEF DESCRIPTION OF THE DRAWINGS

[0163] Figure 1 is a graph showing serial dilutions of M. abscessus infected macrophages treated with free lysin A, lysin B, isoamylase and alpha-amylase (ABIα) or liposomes containing ABIα. DETAILED DESCRIPTION

[0164] Mycobacteria are actinomycetes (e.g., Corynebacteriales or Propionibacteriales) represented by a thick envelope rich in mycolic acid. Mycobacteria contain, from the outside to the inside, a capsule, a mycolic acid layer, an arabinogalactan (AGL) layer, a peptidoglycan (PG), a plasma membrane, and a cytoplasm. This complex cell envelope contributes to the cold resistance of mycobacteria and is particularly difficult to penetrate and destroy, which is required for effective treatment of mycobacterial infections.

[0165] In addition, these bacteria contain a complex life cycle in which the bacteria reside in the cytoplasm or in other subcellular compartments of the host cell or outside the host cell. Mycobacteria are endocytosed by the host cell and these endocytosed vesicles can fuse with intracellular organelles such as endosomes, phagosomes or lysosomes. Once in these intracellular compartments, the bacteria are able to replicate and grow. Subsequently, the membrane dissolves and releases the bacteria into the cytoplasm, where growth continues. Subsequently, the bacteria lyse the host cell and spread in the form of free bacteria. This free form of bacteria may appear in the spleen and liver after being released from lung phagocytes, etc., leading to the expansion of the infection and ultimately death.

[0166] Due to the complex life cycle of bacteria, it is difficult to target bacteria to the appropriate location in time and space to effectively treat infection. For example, when inside a host cell, it is necessary to target the correct intracellular compartment at the correct life cycle stage, or target an extracellular location after host cell lysis. Therefore, there is a need for improved compositions and methods for targeting and treating bacterial infections (such as those caused by mycobacteria).

[0167] The present invention solves this problem through a material composition and a method of using the same, which is rationally designed to degrade the mycobacterial envelope and specifically target the free form intraphagosome stage and intracellular life cycle stage of the mycobacterial life cycle. The composition comprises a mixture of unencapsulated antimicrobial lytic proteins that are triggered to kill bacterial cells inside and outside the host cell. The composition may also comprise a supramolecular structure (e.g., liposome) that targets the host cell (e.g., macrophage or dendritic cell) and the correct intracellular compartment (endosome, phagosome, lysosome or cytosol) to target the intracellular life cycle stage. The liposome guides the payload to the correct cell type and intracellular compartment, while the mixture of antimicrobial lytic proteins degrades the mycobacterial envelope. The free form enzyme can degrade the envelope from the outside to the inside, while the internalized supramolecular structure can degrade the envelope from the inside to the outside to kill bacteria.

[0168] Compositions as described herein comprise a mixture containing two or more of lysin A, lysin B, isoamylase and alpha-amylase. This combination of lysin is particularly advantageous in killing mycobacterial cells and related actinomycetes. In order to obtain protein components, we first rationally attacked three layers of mycobacterial envelope, i.e., capsule, the junction between mycolic acid and the AGL layer and the peptidoglycan layer. For many actinomycetes, such as Corynebacterales (e.g., mycobacteria) and Propionibacterales (such as cutibacteria), the envelope components under the basic structural level were observed.

[0169] These thermostable complexes exhibit 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.

[0170] Antimicrobial lytic protein

[0171] The invention features compositions containing one or more (e.g., one, two, three, or four) of lysin A, lysin B, isoamylase, and alpha-amylase. The invention also features compositions containing unencapsulated proteins, the compositions containing two or more (e.g., two, three, or four) of lysin A, lysin B, isoamylase, and alpha-amylase. The invention also features compositions, the compositions further comprising supramolecular complexes (e.g., liposomes) containing one or more (e.g., one, two, three, or four) of lysin A, lysin B, isoamylase, and alpha-amylase. Suitable lysate proteins for incorporation into the compositions described herein are shown in Table 1 below. These proteins, for example, exhibit improved expression, thermal stability, and antimicrobial effects compared to other orthologs of these proteins.

[0172] Table 1. Cleavage protein sequences

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] The compositions described herein may include 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. Lysin A may comprise or consist of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0180] The compositions described herein may include 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. Lysin B may comprise or consist of the amino acid sequence of SEQ ID NO: 183 or SEQ ID NO: 184.

[0181] The compositions described herein may include an isoamylase 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. The isoamylase may comprise or consist of the amino acid sequence of SEQ ID NO: 242 or SEQ ID NO: 243.

[0182] The compositions described herein may comprise an alpha-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: 393-398. The alpha-amylase may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 393-398.

[0183] The compositions described herein may comprise an alpha-amylase 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: 393 or SEQ ID NO: 394. The alpha-amylase may comprise or consist of the amino acid sequence of SEQ ID NO: 393 or SEQ ID NO: 394.

[0184] In some embodiments, the composition comprises lysin A of SEQ ID NO: 1, lysin B of SEQ ID NO: 183, isoamylase of SEQ ID NO: 242, and alpha-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, isoamylase of SEQ ID NO: 243, and alpha-amylase of SEQ ID NO: 394.

[0185] In some embodiments, the composition comprises lysin A of SEQ ID NO: 1, lysin B of SEQ ID NO: 183, isoamylase of SEQ ID NO: 242, and alpha-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, isoamylase of SEQ ID NO: 243, and alpha-amylase of SEQ ID NO: 396.

[0186] In some embodiments, the composition comprises lysin A of SEQ ID NO: 1, lysin B of SEQ ID NO: 183, isoamylase of SEQ ID NO: 242, and alpha-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, isoamylase of SEQ ID NO: 243, and alpha-amylase of SEQ ID NO: 398.

[0187] Additional sequences useful in the compositions and methods described herein are identified. In some embodiments, the 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 shown in Table 2 below.

[0188] Table 2. Lysin A sequences

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252] Identify additional sequences that can be used in the compositions and methods described herein. In some embodiments, the composition comprises 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 any one of SEQ ID NOs: 183-241 as shown in Table 3 below.

[0253] Table 3. Lysin B sequences

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269] Identify additional sequences that can be used in the compositions and methods described herein. In some embodiments, the 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 shown in Table 4 below.

[0270] Table 4. Isoamylase sequences

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367] Additional sequences useful in the compositions and methods described herein are identified. In some embodiments, the composition comprises an alpha-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 shown in Table 5 below.

[0368] Table 5. α-Amylase sequences

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391] Those skilled in the art will appreciate that the cleavage proteins described herein can be recombinantly produced. Thus, the protein may contain a suitable purification tag, such as a His tag containing, for example, three, four, five, six, seven, eight, nine, ten or more histidine residues present at the N-terminus or C-terminus of the protein. The protein may also contain a removable signal sequence present at the N-terminus or C-terminus of the protein.

[0392] It will also be understood by those skilled in the art that the cleavage proteins described herein may include biologically active fragments thereof, for example, fragments of the cleavage proteins described herein that may be truncated, for example, 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, but still substantially retain their biological activity.

[0393] In some embodiments, one or more of the cleaved proteins (eg, unencapsulated proteins) are mannosylated. Such mannosylation can allow the protein to be targeted to an intracellular destination, such as within an antigen presenting cell.

[0394] In some embodiments, the composition comprises a concentration of 0.1 mg / mL to 20 mg / mL (e.g., for example, 0.1 mg / mL to 1 mg / mL, such as 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, such as 1 mg / mL to 10 mg / mL, such as 2 mg / mL, 3 mg / mL, 4 mg / mL, In some embodiments, the composition comprises a protein (e.g., lysozyme A, lysozyme B, isoamylase and / or alpha-amylase) at a concentration of 1 mg / mL to 10 mg / mL, for example, 10 mg / mL to 20 mg / mL, for example, 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 lysozyme A, lysozyme B, isoamylase and / or alpha-amylase at a concentration of 1 mg / mL to 10 mg / mL.

[0395] Bacterial infection

[0396] Compositions as described herein can be used to treat bacterial infections. In some embodiments, compositions as described herein target bacteria that reside outside the cell in at least a portion of its life cycle. In some embodiments, compositions as described herein target bacteria that reside inside the cell in at least a portion of its life cycle. Intracellular bacteria reside in host cells, and the intracellular bacteria in the host cells multiply and cause infection. Intracellular bacteria can reside in immune cells (such as professional antigen cells). Professional antigen presenting cells (APC) include macrophages, dendritic cells and phagocytes, such as macrophages. APC processes and displays antigens complexed with major histocompatibility complexes (MHC) on its surface. APC places antigens on MHC 2 classes of bacterial pathogens, and the antigens are recognized by T cells, and then stimulate B cells with antibodies complementary to the antigen. This results in the proliferation of specific B cells encoding specific antibodies to counteract organisms with the antigen. Some bacteria escape this immune response by hiding in immune cells.

[0397] The compositions and methods described herein can be used to target mycobacteria, such as intracellular mycobacteria residing in professional antigen presenting cells (e.g., macrophages or dendritic cells). In some embodiments, the mycobacterium species is Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium leprae diffuse, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium marine, Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium chimera, Mycobacterium bolei, Mycobacterium fortuitum, Mycobacterium goode or Mycobacterium masseille. In specific embodiments, the mycobacterium genus is NTM. In some embodiments, NTM is Mycobacterium abscessus, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium chimera, Mycobacterium bolei, Mycobacterium fortuitum, Mycobacterium goode and Mycobacterium masseille.

[0398] In some embodiments, compositions and methods as described herein can be used for targeting other actinomycetes (e.g., Corynebacteriales or Propionibacteriales) with an envelope component similar to mycobacterium. For example, compositions and methods can be used for targeting Nocardia, Corynebacteriales 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. Corynebacteriales species can be, for example, C. glutamicum or C. diphtheriae. Rhodococcus species can be, for example, Rhodococcus equi or Rhodococcus equi. Compositions and methods can be used for targeting Propionibacteriales, such as Propionibacteriales species. The Propionibacterium species can be, for example, Propionibacterium acnes (C. acnes).

[0399] Supramolecular structure

[0400] Supramolecular structures can be used to prepare mixtures of lytic enzymes for delivery. Supramolecular structures include limited molecular (e.g., lipid) complexes that are held together by non-covalent bonds such as hydrogen bonds, van der Waals forces, electrostatic interactions, ion-dipole forces, hydrophobic effects, and π-π interactions. Supramolecular structures can include large molecular complexes that form spherical, rod-shaped, helical, or sheet-like structures. Supramolecular structures include, for example, lipid-based supramolecular structures, such as micelles, liposomes, and LNPs. Supramolecular structures can have a predetermined size. The size of the structure can vary based on the components (e.g., the size of the protein) packaged within the structure. Supramolecular complexes are endocytosed by cells (e.g., professional antigen presenting cells, such as macrophages or dendritic cells), and antibacterial lytic proteins are delivered to the targeted intracellular compartments (endosomes, phagosomes, lysosomes, or cytosols) where the bacteria reside.

[0401] In some embodiments, a specific particle size is used to approach a certain endocytic pathway to guide the structure to an appropriate targeted intracellular compartment. The supramolecular structure can be endocytosed and delivered to a targeted intracellular compartment, for example, by clathrin-dependent endocytosis or by caveolin-dependent endocytosis. The particle size of the supramolecular structure, such as the Z-average particle size, can vary from 75nm to 5 μm, for example, 75nm to 2 μm, 75nm to 1 μm, for example, 75nm to 750nm (e.g., 250nm to 750nm or 75nm to 250nm). In some embodiments, when the supramolecular structure is an LNP or micelle, the Z-average particle size is 75nm to 250nm. In some embodiments, when the supramolecular structure is a vesicle (e.g., liposome), the Z-average particle size is 250nm to 750nm. Non-limiting examples of Z-average particle size include, for example, 75 nm to 100 nm, for example, 75 nm to 85 nm (e.g., 80 nm), for example, 80 nm to 140 nm, 90 nm to 130 nm, or 110 nm to 130 nm (e.g., 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 (e.g., 270 nm), for example, 300 nm to 400 nm, 400 nm to 500 nm, 5 ... 50nm, for example 450nm to 550nm, 475nm to 525nm, 480nm to 520nm, 490nm to 510nm, 495nm to 505nm (e.g. 500nm), for example 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 150nm, 155nm, 160nm 0nm, 165nm, 170nm, 175nm, 180nm, 185nm, 190nm, 195nm, 200nm, 205nm, 210nm, 215nm, 220nm, 225nm, 230nm, 235 nm, 240nm, 245nm, 250nm, 255nm, 260nm, 265nm, 270nm, 275nm, 280nm, 285nm, 290nm, 295nm, 300nm, 305nm, 310n m, 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, 5 55nm, 560nm, 565nm, 570nm, 575nm, 580nm, 585nm, 590nm, 595nm, 600nm, 605nm, 610nm, 615nm, 620nm, 625nm, 630nm, 635nm, 640nm, 645 nanometer , 740nm, 745nm, 750nm, 755nm, 760nm, 765nm, 770nm, 775nm, 780nm, 785nm, 790nm, 795nm, 800nm, 805nm, 810nm, 815nm, 820nm, 825nm, 8 30nm, 835nm, 840nm, 845nm, 850nm, 855nm, 860nm, 865nm, 870nm, 875nm, 880nm, 885nm, 890nm, 895nm, 900nm, 905nm, 910nm, 915nm, 92 0nm, 925nm, 930nm, 935nm, 940nm, 945nm, 950nm, 955nm, 960nm, 965nm, 970nm, 975nm, 980nm, 985nm, 990nm, 995nm, 1μm, 1.1μm, 1.2μm, 1.3 μm, 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 a specific embodiment, the Z-average particle size of the supramolecular structure can be 75 nm to 250 nm. In some embodiments, the Z-average particle size of the supramolecular structure is 80 nm, 270 nm, or 500 nm.

[0402] 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 a specific embodiment, the average particle size is measured by. In a specific embodiment, the supramolecular structure contains a Z-average particle size of 75nm to 250nm. In a specific embodiment, the supramolecular structure contains a Z-average particle size of 250nm to 750nm. In a specific embodiment, the supramolecular structure contains a Z-average particle size of 500nm. In a specific embodiment, the supramolecular structure contains a Z-average particle size of 270nm. In a specific embodiment, the supramolecular structure contains a Z-average particle size of 80nm. It will be appreciated by those skilled in the art that a supramolecular structure population (e.g., liposomes, LNPs or micelles) may have a range of Z-average particle sizes within a population. Therefore, a population may be polydisperse. A 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 can be determined using DLS (see, e.g., ISO 22412:2017).

[0403] The supramolecular structure may be loaded with a predetermined number of antimicrobial lytic proteins or an average number of antimicrobial lytic proteins per supramolecular structure. For example, the supramolecular structure may contain one protein to 10 6 Proteins (e.g., 1 to 10 5 , 1 to 10 4 , 1 to 10 3 , 1 to 10 2 , 1 to 10, 10 to 10 6 , 10 to 10 5 , 10 to 10 4 , 10 to 10 3 , 10 to 10 2 , 10 3 Up to 10 6 , 10 3 Up to 10 5 , 10 3 Up to 10 4 The number of proteins per structure can depend on the size of the protein and the size of the structure.

[0404] The supramolecular structure may include an endosomal escape portion. A supramolecular structure including an endosomal escape portion may provide improved cytosolic delivery of cargo (e.g., therapeutic agent) contained in the supramolecular structure. Endosomal escape portions are known in the art. In some embodiments, the endosomal escape portion is an ionizable lipid. Ionizable lipids may also serve as lipids that form a supramolecular structure layer. Non-limiting examples of ionizable lipids include, for example, those described in WO 2019 / 067875; WO 2018 / 191750; and US9,999,671. Other exemplary endosomal escape portions include fusogenic lipids (e.g., dioleoylphosphatidylethanolamine (DOPE)); and polymers such as polyethyleneimine (PEI); poly(β-amino esters); polypeptides such as polyarginine (e.g., octa-arginine) and polylysine (e.g., octa-lysine); proton sponges, viral capsids, and peptide transduction domains as described herein. For example, the fusion peptide can be derived from the M2 protein of influenza A virus; a peptide analog of influenza virus hemagglutinin; the HEF protein of influenza C virus; the transmembrane glycoprotein of filovirus; the transmembrane glycoprotein of rabies virus; the transmembrane glycoprotein (G) of vesicular stomatitis virus; the fusion protein of Sendai virus; the transmembrane glycoprotein of Semliki Forest virus; the fusion protein of human respiratory syncytial virus (RSV); the fusion protein of measles virus; the fusion protein of Newcastle disease virus; the fusion protein of sheep myelin shedding virus; the fusion protein of murine leukemia virus; the fusion protein of HTL virus; and the fusion protein of simian immunodeficiency virus (SIV). Other moieties that can be used to promote endosomal escape are described in Dominska et al., Journal of Cell Science, 123(8): 1183-1189, 2010. Specific examples of endosomal escape moieties comprising moieties suitable for inclusion in or conjugation to the supramolecular structures disclosed herein are provided, for example, in WO 2015 / 188197; the disclosure of these endosomal escape moieties is incorporated herein by reference.

[0405] Liposomes

[0406] Liposomes are useful for transferring and delivering antimicrobial proteins to the site of action. Because the liposome membrane is similar in structure to the biomembrane, when the liposome is applied to the tissue, the double layer of the liposome merges with the double layer of the cell membrane (e.g., intracellular membrane). In some cases, before merging with the biomembrane, the liposome is engulfed to form a multilayer vesicle, which can then merge with a phagolysosome (e.g., a phagolysosome containing mycobacteria). As the fusion of liposomes and phagolysosomes is carried out, the internal aqueous content containing the antimicrobial protein is delivered to the phagolysosome, wherein the antimicrobial protein can be specifically targeted and cracked to reside in the bacterial cell (e.g., mycobacterial cell, such as NTM cell) in the mammalian immune cell. In some cases, liposomes are also specifically targeted, for example, to guide proteins to specific mammalian immune cell types and / or to specific intracellular compartments (endosomes, phagosomes, lysosomes or cytosol) that usually contain bacteria (e.g., mycobacteria) during infection. The composition of liposomes is usually a combination of phospholipids, usually in combination with steroids such as cholesterol. Other phospholipids or other lipids may also be used.The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0407] 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 substituted by fatty acid ester moieties and one hydroxyl group substituted by a phosphodiester moiety covalently bonded to the serine side chain. The typical structure of phosphatidylserine is RO-CH2-CH(OR)-CH2-OP(O)(OH)-OCH2CH(COOH)NH2 or a salt thereof, wherein each R is independently a fatty acid acyl group. Additionally or alternatively, the liposomes described herein may include, for example, lysophospholipids, such as lysophosphatidylserine. Lysophosphatidylserine is a phosphatidylserine lacking one of its two fatty acid ester moieties. The typical structure of lysophosphatidylserine is RO-CH2-CH(OR)-CH2-OP(O)(OH)-OCH2CH(COOH)NH2 or a salt thereof, wherein one R is a fatty acid acyl group, 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, wherein each R is H or a fatty acid acyl group, provided that at least one R is a fatty acid acyl group.

[0408] One major type of liposome composition comprises phospholipids other than phosphatidylcholine of natural origin. Neutral liposome compositions can be formed, for example, from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Cationic liposomes have the advantage of being able to fuse to cell membranes.Non-limiting examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearoyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), and N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA). A), 1,2-dilinoleylcarbamoyloxy-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 (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-T MA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazine)propane (DLin-MPZ) or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,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 its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriacontria-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-ylethylazetidinol-2-ol (Tech G1) or mixtures thereof.The cationic lipid may comprise, for example, 20 mol% to 50 mol% or 40 mol% of the total lipid present in the particle.

[0409] Non-cationic liposomes can be taken up by macrophages in vivo and can be used to deliver antimicrobial proteins to macrophages. Anionic liposome compositions can be formed from dimyristoylphosphatidylglycerol, and anionic fusogenic liposomes can be formed primarily from dioleoylphosphatidylethanolamine (DOPE). The lipids can be anionic lipids or neutral lipids, including but not limited to: distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)- Cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (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 a mixture thereof. Non-cationic lipids (if cholesterol is included) may, for example, account for 5mol% to 90mol%, 10mol% or 58mol% of the total lipids present in the particles. In some embodiments, the lipid may be a combination of the above-mentioned lipids, for example, a combination of lipids including DOPC, DOPS, Chol and DOPE.

[0410] In some embodiments, the liposomes comprise a mixture of lipids. For example, the mixture of lipids may include two or more of DOPC, DOPE, DOPS, and cholesterol.

[0411] 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).

[0412] 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).

[0413] In some embodiments, 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).

[0414] 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).

[0415] 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).

[0416] 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).

[0417] 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.

[0418] In some embodiments, liposomes comprise a concentration of 0.03 mg / mL to 10 mg / mL, for example 0.1 mg / mL to 10 mg / mL (e.g. 0.1 mg / mL to 1 mg / mL, for example 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, for example 1 mg / mL to 10 mg / mL, for example 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, liposomes comprise a concentration of 1 mg / mL to 5 mg / mL of lipid.

[0419] Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol. Examples of other methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 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.

[0420] The targeting of liposomes is also possible and is known in the art based on, for example, organ-specific, cell-specific, and organelle-specific. In the case of liposome-targeted delivery systems, lipid groups can be incorporated into the lipid bilayer of the liposome to keep the targeting ligand and the liposome bilayer stably associated. Various linking groups can be used to connect the lipid chain to the targeting ligand. Other methods are known in the art and are, for example, described in U.S. Publication No. 20060058255, whose linking groups are incorporated herein by reference.

[0421] Cleavable linking groups are susceptible to the influence of cleavage factors (e.g., pH, redox potential, or the presence of degradation molecules). Typically, cleavage factors are more common or have higher levels or activity in cells than in serum or blood. Examples of such degradation agents include: redox agents selected for specific substrates or without substrate specificity, including, for example, oxidative or reductive enzymes or reductants such as thiols present in cells that can degrade redox cleavable linking groups by reduction; esterases; endosomes or agents that can produce an acidic environment, such as those that produce 5 or lower pH; enzymes that can hydrolyze or degrade acid cleavable linking groups by acting as generalized acids; peptidases (which can be substrate specific); and phosphatases.

[0422] Cleavable linking groups such as disulfide bonds may be sensitive to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, in the range of 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at about 5.0. Some linkers will have a cleavable linking group that cleaves at a preferred pH, thereby releasing the cationic lipid from the ligand within the cell, or into a desired cellular compartment.

[0423] The joint may include a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the joint may depend on the cell to be targeted. In general, the suitability of a candidate cleavable linking group may be assessed by testing the ability of a degradation agent (or condition) to crack the candidate linking group. It will also be desirable to test the ability of a candidate cleavable linking group to resist cracking in blood or when contacting with other non-target tissues. Therefore, it is possible to determine the relative sensitivity of cracking between the first condition and the second condition, wherein the first condition is selected to indicate the cracking in the target cell and the second condition is selected to indicate the cracking in other tissues or biofluids (such as blood or serum). The assessment may be carried out in a cell-free system, in a cell, in a cell culture, in an organ or tissue culture, or in a whole animal. It may be useful to carry out an initial assessment under cell-free or culture conditions and to confirm by further assessment in the 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 in a cell (or under in vitro conditions selected to mimic conditions inside a cell) than in blood or serum (or under in vitro conditions selected to mimic conditions outside a cell).

[0424] Lipid Nanoparticles

[0425] Antimicrobial agent of the present invention can be fully encapsulated in lipid formulations, for example in lipid nanoparticles (LNP).LNP is extremely useful for synthetic applications, because they show the circulation life extended after intravenous (iv) injection and accumulate at distal sites (for example, at the site physically separated from the site of application).LNP includes "pSPLP", which includes the condensing agent-nucleic acid complex of the encapsulation as cited in PCT Publication No. WO 2000 / 003683.Particle of the present invention generally has 50nm to 150nm, more generally 60nm to 130nm, more generally 70nm to 110nm, the average diameter of the most general 70nm to 90nm, and is substantially nontoxic.In addition, when present in nucleic acid-lipid granules of the present invention, nucleic acid has resistance to the degradation of nuclease in aqueous solution. Nucleic acid-lipid particles and methods of making them are disclosed in, for example, U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010 / 0324120 and PCT Publication No. WO 96 / 40964.

[0426] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to peptide ratio) will be in the range of 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-recited ranges are also contemplated as part of the invention.

[0427] 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 its analogs, ALN100, MC3, Tech G1 or mixtures thereof. The cationic lipid may comprise, for example, 20 mol% to 50 mol% or 40 mol% of the total lipid present in the particle.

[0428] Lipid can be anionic lipid or neutral lipid, including but not limited to DSPC, DOPC, DOPS, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, SOPE, cholesterol or their mixture. Non-cationic lipid (if including cholesterol) can for example account for 5mol% to 90mol%, 10mol% or 60mol% of the total lipid present in the particle.

[0429] The conjugated lipid that inhibits 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. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Cer). 12 ), PEG-dimyristyloxypropyl (C 14 ), PEG-dipalmityloxypropyl (C 16 ) or PEG-distearyloxypropyl (C 18 ). The conjugated lipid that prevents aggregation of the particles may be, for example, 0 mol % to 20 mol % or 2 mol % of the total lipid present in the particles.

[0430] In some embodiments, the LNP further comprises cholesterol, eg, from 10 mol % to 60 mol % or 50 mol % of the total lipids present in the particle.

[0431] In some embodiments, LNP comprises a mixture of lipids. For example, the mixture of lipids may include two or more of DOPC, DOPE, DOPS and cholesterol.

[0432] 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).

[0433] 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).

[0434] In some embodiments, 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).

[0435] 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).

[0436] 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).

[0437] 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).

[0438] 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.

[0439] In some embodiments, LNP comprises a concentration of 0.03 mg / mL to 10 mg / mL, for example 0.1 mg / mL to 10 mg / mL (for example 0.1 mg / mL to 1 mg / mL, for example 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, for example 1 mg / mL to 10 mg / mL, for example 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, LNP comprises a concentration of 1 mg / mL to 5 mg / mL of lipid.

[0440] Micelles

[0441] "Micelles" are a specific type of molecular collection in which amphiphilic molecules are arranged in a spherical structure so that all hydrophobic portions of the molecules are oriented inwardly, thereby allowing the hydrophilic portions to be in contact with the surrounding aqueous phase. Micelles can be made of lipids. The micellar phase is caused by the packing behavior of single-tail lipids in a bilayer. The formation of micelles is caused by the difficulty in filling all the volume inside the bilayer and the area of ​​each head group imposed on the molecule to be accommodated by the hydration of the lipid head groups. This type of micelle is called a normal micelle (oil-in-water micelle). Reverse micelles have a head group in the center and the tails extending outward (water-in-oil micelles).

[0442] Micelles are roughly spherical. Other phases, including shapes such as ellipsoids, cylinders, and bilayers, are also possible. The shape and size of micelles vary with the molecular geometry of their surfactant molecules and solution conditions such as surfactant concentration, temperature, pH, and ionic strength. The process of forming micelles is called micellization and forms part of the phase behavior of many lipids depending on their polymorphism.

[0443] Targeting moiety

[0444] The supramolecular structures described herein may include, for example, targeting moieties. The targeting moieties may be used to direct the supramolecular structures to specific cell types (e.g., professional antigen presenting cells, such as macrophages or dendritic cells). Certain lipids (e.g., phosphatidylserine) may be used in supramolecular structures (e.g., vesicles) as both supramolecular structure layer-forming lipids and as targeting moieties. The targeting moiety may be, for example, an antibody or an antigen-binding fragment thereof or an engineered derivative (e.g., Fcab or fusion protein (e.g., scFv)). The targeting moiety may be, for example, a polypeptide. Alternatively, the targeting moiety may be, for example, a small molecule (e.g., mannose or folic acid) or a small molecule cluster (e.g., a mannose cluster). The targeting moiety may be covalently or non-covalently associated with the supramolecular structure.

[0445] Small molecules

[0446] The targeting moiety may be a small molecule capable of complexing a receptor expressed on the surface of a targeted cell.Non-limiting examples of small molecules that can be used as targeting moieties in the supramolecular structures described herein are phosphatidylserine, lysophosphatidylserine folic acid, mannose, and mannose clusters.

[0447] In some embodiments, the targeting moiety is phosphatidylserine or lysophosphatidylserine. In some embodiments, the targeting moiety is phosphatidylserine. Phosphatidylserine and / or lysophosphatidylserine can exist as supramolecular layer-forming lipids that are non-covalently bonded to the rest of the supramolecular structure.

[0448] Folic acid can be used as a targeting moiety. In the supramolecular structure described herein, folic acid can have the following structure:

[0449]

[0450] 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.

[0451] Folic acid, mannose and mannose clusters can be covalently linked to the supramolecular structure. Conjugation techniques for linking folic acid, mannose and mannose clusters are known in the art, for example, as described in US 2014 / 0045919, US 9,725,479, US 8,758,810, US 8,450,467, US 6,525,031, US 6,335,434 and US 5,759,572.

[0452] Antigen binding part

[0453] The antigen binding portion in the supramolecular structure described herein can be an antibody or an antigen binding fragment thereof (e.g., F(ab)2 or Fab) or an engineered derivative thereof (e.g., Fcab or fusion protein (e.g., scFv)). Human or chimeric (e.g., humanized) antibodies can be used as antibodies in the supramolecular structure described herein.

[0454] The antigen binding portion targets APCs with surface antigens recognized by the antigen binding portion. Dendritic cells can be targeted by anti-DEC205, anti-CD304, anti-CD303, anti-CD40, anti-CD74, anti-BDCA2 or anti-CD123 antibodies or their antigen binding fragments or their engineered derivatives. Macrophages can be targeted by anti-CD163, anti-CD40, anti-CD74, anti-CD206 or anti-CD123 antibodies or their antigen binding fragments or their engineered derivatives.

[0455] A non-limiting example of an anti-CD38 antibody is daratumumab, SAR650984, MOR202, or any of Ab79, Ab19, Ab43, Ab72, and Ab110 disclosed in WO 2012 / 092616, the disclosures of which are incorporated herein by reference. A non-limiting example of an anti-CD79b antibody is huMA79b v28 disclosed in WO 2014 / 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 provided in US2010 / 0098704, the disclosures of which are incorporated herein by reference. Non-limiting examples of anti-CD40 antibodies are lucatumumab and dacetuzumab. Non-limiting examples of anti-CD304 antibodies are vesencumab.

[0456] Conjugation techniques for linking antigen binding moieties are known in the art, for example, as described in Ansell et al., Methods Mol. Med., 25:51-68, 2000; US 2002 / 0025313; US 6,379,699; and US 5,059,421.

[0457] Peptides

[0458] The targeting moiety can be a polypeptide having affinity for a cell (e.g., affinity for a cell type such as a dendritic cell). Non-limiting examples of polypeptides are RGD peptide, rabies virus glycoprotein (RVG), and DC3 peptide. Alternatively, the polypeptide can be a TLR2 agonist, such as MALP-2 lipoprotein, MALP-404 lipoprotein, OspA, porin, LcrV, Hsp60, glycoprotein gH / gL, or glycoprotein gB.

[0459] Conjugation techniques for linking peptides are known in the art, for example, as described in Ansell et al., Methods Mol. Med., 25:51-68, 2000; US 2002 / 0025313; US 6,379,699; and US 5,059,421.

[0460] PAMP

[0461] The targeting moiety can be a PAMP. PAMP is known in the art, such as CpG ODN. CpG ODN is generally divided into three categories: Class A, Class B and Class C. Class A CpG ODN generally contains a poly-G tail with a phosphorothioate backbone at the 3'- and 5'-ends and a central palindrome comprising a phosphate backbone. Class A CpG ODN generally contains CpG in the central palindrome. Class B CpG ODN generally contains a fully phosphorothioated backbone, and the sequence at the 5' end of Class B CpG ODN is generally essential for TLR9 activation. Class C CpG ODN contains a fully phosphorothioated backbone, wherein the 3' end sequence can form a duplex. PAMP can be covalently linked to a supramolecular structure using techniques and methods known in the art.

[0462] Assembly Method

[0463] The present invention is characterized by a supramolecular structure (e.g., a lipid-based supramolecular structure, such as a liposome) comprising a plurality of enzymes packaged therein. Assembly methods are described herein to produce structures (e.g., liposomes) containing enzymes (e.g., lysozyme A, lysozyme B, isoamylase and / or alpha-amylase, e.g., having at least 85% sequence identity with the sequences of Table 1). Individual proteins can be overexpressed in any suitable recombinant expression system (e.g., Escherichia coli) and extracted from cells by lysis. In some embodiments, a crude extract from a cell can be purified, for example, by column chromatography. After purification, the enzyme component concentration can be standardized for subsequent encapsulation, for example, encapsulation into 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, such as 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, such as 1 mg / mL to 10 mg / mL, such as 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.

[0464] Then lipid and enzyme can be mixed to prepare liposome.For example, total concentration can be 0.1mg / mL to 10mg / mL (for example 0.1mg / mL to 1mg / mL, for example 0.2mg / mL, 0.3mg / mL, 0.4mg / mL, 0.5mg / mL, 0.6mg / mL, 0.7mg / mL, 0.8mg / mL, 0.9mg / mL or 1mg / mL, for example 1mg / mL to 10mg / mL, for example 2mg / mL, 3mg / mL, 4mg / mL, 5mg / mL, 6mg / mL, 7mg / mL, 8mg / mL, 9mg / mL or 10mg / mL, for example 1mg / mL to 5mg / mL) lipid and 1mg / mL to 10mg / ml His marked protein mix.Lipid can include one or more in for example DOPC, DOPE, DOPS and cholesterol.In some embodiments, lipid includes DOPC, DOPE, DOPS and cholesterol of 10:10:3:4 ratio. The lipid can be resuspended in a suitable organic solvent (e.g., ethanol) and mixed with the protein (e.g., in a 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 aqueous:organic). A suitable system such as IGNITE TM The system (Precision Nanosystems) mixes the mixture at a flow rate of 1 ml / min to 30 ml / min (e.g., 15 ml / min). The organic layer can be removed by dilution at a suitable temperature (such as room temperature) and, for example, dialysis of a formulation buffer for an excess (e.g., 1000X) volume for at least 30 minutes (e.g., 1 hour). The liposomes can then be analyzed by gel electrophoresis (e.g., SDS-PAGE), dynamic light scattering, intrinsic fluorescence, and / or static light scattering and tested for related activity. Such determinations can help confirm the encapsulation of the enzyme and the purity of the enzyme and liposome.

[0465] The composition containing supramolecular structure (for example liposome) can be prepared together with one or more excipients.For example, composition (for example, supramolecular structure, for example, liposome containing the mixture of lyase) can be for example with 10mM to 200mM, for example 50mm to 150mm, for example 10mM, 20mm, 30mm, 40mm, 50mm, 60mm, 70mm, 80mm, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM or 200mM concentration encapsulation and / or be formulated in buffer (such as glycine, Tris, sodium citrate, sodium acetate and MES). The composition can be formulated at a pH of 5 to 11 (e.g., 5 to 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 to 11, e.g., a pH of 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11). The composition can also include one or more excipients, such as CaCl2, arginine, NaCl, sodium citrate, MgCl2, or glycerol. In some embodiments, the composition includes, e.g., 5 mM to 10 mM CaCl2, 0 to 50 mM arginine, 0 to 200 mM NaCl, 0 mM to 1 mM sodium citrate, 0 to 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 also comprise TWEEN, such as TWEEN-80. Once the compositions are in a preferred storage or activity buffer, they may be used in therapy or for assay or preservation for subsequent use.

[0466] Treatment

[0467] The antibacterial lytic proteins described herein are preferably formulated into pharmaceutical compositions for administration to human subjects to treat diseases or illnesses, such as bacterial infections (e.g., Actinomycetes infections, such as Corynebacteriales or Propionibacteriales), such as mycobacterial infections, such as NTM infections. In particular, the compositions and methods described herein can be used to treat bacterial infections caused by Actinomycetes (e.g., Corynebacteriales and Propionibacteriales, due to their similar envelope structures). Bacterial infections can occur in otherwise healthy subjects. Alternatively, bacterial infections can occur in subjects suffering from another comorbidity or disease. For example, subjects with weakened immune systems may be more susceptible to bacterial infections.

[0468] Mycobacterial infections caused by NTM are bacteria that are commonly found in the environment. Inhaling these bacteria can cause disease in healthy patients and those with compromised immune systems. NTM disease most commonly affects the lungs of adults, but it can also affect any part of the body. Some subjects are at a higher risk of contracting NTM and developing the disease. People with existing lung diseases such as bronchiectasis (enlarged airways), chronic obstructive pulmonary disease (COPD), cystic fibrosis, alpha-1 antitrypsin deficiency, or those with previous infections such as tuberculosis are at increased risk for NTM disease in the lungs. Subjects with advanced HIV infection (CD4<50) or immune-related genetic disorders (e.g., interferon-gamma deficiency 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 to be treated may, for example, suffer from any of the aforementioned indications in addition to a bacterial infection.

[0469] The method compositions and methods described herein can be used to reduce the level of infection. For example, the method can reduce the level of infection (e.g., bacterial count or infection size) 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%.

[0470] Pharmaceutical composition

[0471] The antimicrobial agents described herein are preferably formulated into pharmaceutical compositions for administration to human subjects in a biocompatible form suitable for in vivo administration.

[0472] As will be appreciated by those skilled in the art, compositions as described herein may be applied to subjects in various forms depending on the selected route of administration. Compositions as described herein may be applied, for example, by any approach that allows compositions (e.g., unencapsulated mixtures of enzymes and / or supramolecular structures, such as liposomes, micelles or LNPs) to reach target cells. Compositions may be applied, for example, by oral, parenteral, intrathecal, intraventricular, intracerebral parenchyma, buccal, sublingual, nasal, rectal, patch, pump or transdermal administration, and pharmaceutical compositions are formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, epithelial, nasal, intrapulmonary, intrathecal, intraventricular, intracerebral parenchyma, rectal and topical administration modes. In one embodiment, compositions are applied by aerosol. Parenteral administration may be performed by continuous infusion in a selected time period. In some preferred embodiments, compositions as described herein are applied by inhalation.

[0473] Administration of more than one antibacterial agent can be carried out by the same route or by different routes, and can occur sequentially or substantially simultaneously. For example, a first antibacterial agent of the combination can be administered by intravenous injection, while a second therapeutic agent of the combination can be administered orally.

[0474] Certain compositions described herein can be administered, for example, by inhalation. Inhalation can be oral inhalation or nasal inhalation. The inhalable compositions described herein can be provided in liquid dosage form or dry powder dosage form. Dry powder compositions can be administered, for example, by inhalation as is or after reconstitution in a vehicle (e.g., saline (e.g., isotonic saline), phosphate buffered saline or water).

[0475] Inhalable dry powder dosage forms can be prepared from liquid compositions described herein by drying (e.g., by freeze drying, spray drying, spray-freeze drying or supercritical fluid technology). Inhalable dry powder dosage forms described herein may include carriers (e.g., lactose, sucrose, mannitol, etc.), cryoprotectants (e.g., trehalose, mannitol, etc.) and / or antiadhesive agents (e.g., glycine, L-leucine, serine, etc.). Inhalable dry powder dosage forms 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.iol.Ther., 4:23-33, 2004, the disclosure of which is incorporated herein by reference in its entirety.

[0476] Inhalable liquid dosage forms as described herein (e.g., aerosol preparations) can be prepared using techniques and methods that can be used to prepare liquid compositions containing unencapsulated enzymes and / or supramolecular structures. Inhalable liquid dosage forms generally include suspensions of enzymes as described herein and / or supramolecular structures in physiologically acceptable aqueous or non-aqueous solvents, and are generally present in a sealed container in a single dose or multiple dose form in a sterile form, and the sealed container can be a cartridge or a refilled form used with an atomizing device. Alternatively, the sealed container can be an integrated dispensing device, such as a single dose nasal inhaler or an aerosol dispenser equipped with a metering valve intended to be discarded after use. When the dosage form contains an aerosol dispenser, it will contain a propellant, which can be a compressed gas (e.g., compressed air) or an organic propellant (e.g., a fluoroalkane). Inhalable liquid dosage forms can be applied using an atomizer. The process of pneumatically converting bulk liquid into droplets is referred to as atomization. The operation of a pneumatic atomizer requires a propellant as a driving force for liquid atomization. Various types of nebulizers are described in Respiratory Care, 45:609-622, 2000, the disclosure of which is incorporated herein by reference in its entirety. Alternatively, the inhalable liquid dosage form described herein can be administered using a metered dose inhaler. A metered dose inhaler is known in the art and typically includes a canister, an actuator, and a metering valve.

[0477] The compositions described herein can be orally administered, for example, with an inert diluent or with an absorbable edible carrier, or the compositions can be encapsulated in a hard or soft shell gelatin capsule, or the compositions can be compressed into tablets, or the compositions can be incorporated directly with the food of the diet. For oral therapeutic administration, the compositions described herein can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, and wafers. The compositions described herein can also be administered parenterally. The compositions described herein can also be administered by microneedle injection. Solutions of the compositions described herein can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, DMSO, and mixtures thereof with or without alcohol, and in oils. Under normal storage and use conditions, these preparations can contain preservatives to prevent microbial growth. Conventional procedures and ingredients for selecting and preparing suitable formulations are described in, for example, Remington's Pharmaceutical Sciences (2012, 22nd edition) and The United States Pharmacopeia: The National Formulary (USP41NF 36) published in 2018. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it can be easily applied by a syringe. Compositions suitable for buccal or sublingual administration include tablets, lozenges and candy lozenges, wherein the active ingredient is formulated with carriers such as sugar, gum arabic, tragacanth, gelatin and glycerol. Compositions for rectal administration are conveniently in the form of suppositories containing conventional suppository bases such as cocoa butter.

[0478] In some embodiments, compositions as described herein are prepared together with one or more excipients.For example, compositions (for example, unencapsulated enzyme or supramolecular structure, for example, liposome containing the mixture of lyase) can be for example with 10mM to 200mM, for example 50mm to 150mm, for example 10mM, 20mm, 30mm, 40mm, 50mm, 60mm, 70mm, 80mm, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM or 200mM concentration encapsulation and / or be formulated in buffer (such as glycine, Tris, sodium citrate, sodium acetate and MES). The composition can be formulated at a pH of 5 to 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 may also include one or more excipients, such as CaCl2, arginine, NaCl, sodium citrate, MgCl2, or glycerol. In some embodiments, the composition includes, for example, 5mM to 10mM CaCl2, 0 to 50mM arginine, 0 to 200mM NaCl, 0 to 1mM sodium citrate, 0 to 1mM MgCl2, and / or 10%-30% glycerol. In some embodiments, the composition includes 50mM glycine (pH 8.5), 7.5mM CaCl2, 0.5mM MgCl2, 200mM NaCl, 0.33mM sodium citrate, and 10% glycerol. The formulation may also include TWEEN, such as TWEEN-80.

[0479] As described herein, the compositions described herein can be administered to animals (eg, humans) alone or in combination with a pharmaceutically acceptable carrier in proportions determined by the solubility and chemical properties of the composition, the chosen route of administration, and standard pharmaceutical practice.

[0480] The dosage of the compositions described herein (e.g., compositions comprising cleaved proteins) may vary according to many factors, such as the pharmacodynamic properties of the antibacterial cleaved proteins; the mode of administration; the age, health status and weight of the recipient; the nature and extent of the symptoms; the frequency of treatment and the type of concurrent treatment (if any); and the clearance rate of the composition in the animal to be treated. The compositions described herein may be initially administered at a suitable dose, which may be adjusted as needed based on clinical response. In some embodiments, the dosage of the composition (e.g., a composition comprising cleaved proteins) is a preventive or therapeutically effective amount. In addition, it should be understood that all doses may be administered continuously or divided into doses administered per a given time frame. For example, the composition may be administered hourly, daily, weekly, monthly, or annually. In some embodiments, the composition may be administered continuously or systemically.

[0481] Combination therapy

[0482] Compositions described herein can be used as part of a combination therapy. Combination therapy refers to applying two (or more) different agents or treatments to a subject as part of a limited treatment regimen for a specific disease or illness. For example, a first therapeutic agent may include a mixture of unencapsulated proteins, and a second therapeutic agent may include a supramolecular structure containing encapsulated proteins. The treatment regimen defines the dosage and administration cycle of each agent so that the effects of a single agent on the subject overlap. In some embodiments, the delivery of two or more agents is simultaneous or parallel, and the agents can be co-formulated. In some embodiments, two or more agents are not co-formulated, and are administered in a sequential manner as part of a prescription regimen. Sequential or substantially simultaneous administration of each therapeutic agent can be achieved by any appropriate route including but not limited to oral routes, intravenous routes, intramuscular routes, and direct absorption by mucosal tissues. Therapeutic agents can be administered by the same route or by different routes. For example, the first therapeutic agent of the combination can be administered by intravenous injection or by atomization, while the second therapeutic agent of the combination can be administered orally.

[0483] In some embodiments, lysin A, lysin B, isoamylase and / or alpha-amylase (e.g., in unencapsulated form) are administered together. Alternatively, lysin A, lysin B, isoamylase and / or alpha-amylase may be administered at different times.

[0484] In some embodiments, lysin A, lysin B, isoamylase and / or alpha-amylase (e.g., in unencapsulated form, e.g., having at least 85% sequence identity with a sequence in Table 1) are administered together. Alternatively, lysin A, lysin B, isoamylase and / or alpha-amylase may be administered at different times.

[0485] In some embodiments, a first composition containing lysozyme A, lysozyme B, isoamylase and / or alpha-amylase in unencapsulated form is administered as a combination therapy with a supramolecular structure (e.g., liposome) containing one or more of lysozyme A, lysozyme B, isoamylase and / or alpha-amylase.

[0486] In any combination embodiment described herein, the first therapeutic agent and the second therapeutic agent can be used simultaneously or sequentially in any order. The first therapeutic agent (e.g., a composition containing unencapsulated protein) can be used immediately, at most 15 minutes, at most 30 minutes, at most 1 hour, at most 2 hours, at most 3 hours, at most 4 hours, at most 5 hours, at most 6 hours, at most 7 hours, at most 8 hours, at most 9 hours, at most 10 hours, at most 11 hours, at most 12 hours, at most 13 hours, 14 hours, at most 16 hours, at most 17 hours, at most 18 hours, at most 19 hours, at most 20 hours, at most 21 hours, at most 22 hours, at most 23 hours, at most 24 hours, or at most 1-7, 1-14, 1-21 or 1-30 days before or after the second therapeutic agent (e.g., a supramolecular structure containing an encapsulated protein).

[0487] The pharmaceutical compositions described herein may also comprise an additional antibacterial agent administered in conjunction with the supramolecular structure comprising the antibacterial lytic protein.

[0488] The compositions and methods described herein may also include treatment of underlying pulmonary conditions, for example, pulmonary conditions that may be exacerbated by bacterial infection (e.g., NTM infection). Suitable pulmonary therapies include, but are not limited to, airway clearance, nebulizers, respirators, and inhalers (e.g., steroid inhalers).

[0489] antibiotic

[0490] The additional antimicrobial agent may be an antibiotic. Suitable antibiotics include, but are not limited to, penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, azlocillin, temocillin, cephalothin, cefpirin, cephradine, cephalothin, cefazolin, cefmandole, cefuroxime, cephalexin, cefprozil, cefaclor, loracarbef, cefoxitin, cefmetazole, ole), cefotaxime, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefixime, cefpodoxime, ceftibuten, cefdinir, cefpirome, cefepime, chlorhexidine, BAL5788, BAL9141, imipenem, ertapenem, meropenem, astreonam, clavulanic acid, sulbactam, tazobactam, streptomycin, neomycin, kanamycin, paromycin, gentamicin, tobramycin, amikacin, netilmicin, Spectinomycin, sisomicin, dibekalin, isopamicin, tetracycline, chlortetracycline, demeclocycline, minocycline, oxytetracycline, metacycline, doxycycline, erythromycin, azithromycin, clarithromycin, telithromycin, ABT-773, lincomycin, clindamycin, vancomycin, oritavancin, dalbavancin, teicoplanin, quinupristin and dalfopristin, sulfonamides, para-aminobenzoic acid, sulfadiazine, sulfisoxazole, sulfamethoxazole, sulfamethoxazole Sulfatin (sulfathalidine), linezolid, nalidixic acid, oxazolidinone, norfloxacin, pefloxacin, enoxacin, ofloxacin, ciprofloxacin, temafloxacin, lomefloxacin, fleroxacin, grefloxacin, sparfloxacin, trovafloxacin, clinafloxacin, gatifloxacin, moxifloxacin, gemifloxacin, sitafloxacin, metronidazole, daptomycin, garefloxacin, ramoplanin, faropenem, polymyxin, tigecycline, AZD2563, trimethoprim, ethambutol, rifamycin and rifampicin. In some embodiments, a variety of antibiotics are administered in combination 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 thiosemicarbazide, sq-109, bedaquiline, delamanid, pyrazinamide, and isoniazid.

[0491] 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 pramixin).

[0492] Advantageously, in some embodiments, synergy with a co-administered therapeutic agent may allow administration of an antibiotic at a dose that would be subtherapeutic if administered without the other therapeutic agent.

[0493] The antibiotic can be formulated with the supramolecular structure containing the antimicrobial 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 bacteriophage. In some preferred embodiments, the additional antibiotic is amikacin. The amikacin can be liposomal amikacin formulated for, for example, inhalation.

[0494] Example

[0495] The following examples are intended to illustrate the present invention. They are not intended to limit the present invention in any way.

[0496] Example 1. Treatment of Mycobacterium abscessus infected macrophages

[0497] Figure 1 Is a graph showing serial dilutions of Mycobacterium abscessus derived from infected macrophages treated with free lysin A, lysin B, isoamylase and alpha-amylase (ABIα) or liposomes containing ABIα. J774A.1 mouse macrophages were infected with Mycobacterium abscessus (MOI=10:1). The growth time after extraction from infected macrophages was 120 hours. A single dose can have a greater than 100-fold effect relative to untreated cells.

[0498] Other Implementations

[0499] All publications, patents, and patent applications mentioned in this specification 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 herein by reference in its entirety. When a term in this application is found to be differently defined in a document incorporated herein by reference, the definition provided herein will serve as the definition of the term.

[0500] Although the present invention has been described in conjunction with specific embodiments thereof, it will be appreciated that further modifications are possible and this application is intended to cover any changes, uses or adaptations of the present invention that are basically based on the principles of the present invention, and include changes that are not part of the present invention but are within the known or customary practice in the art to which the present invention belongs and that are the essential features described above and that fall within the scope of the claims.

Claims

1. A composition comprising unencapsulated proteins, wherein the unencapsulated proteins comprise two or more of: (a) Lysin A; (b) lysin B; (c) isoamylase; and (d) α-amylase.

2. The composition of claim 1, wherein the unencapsulated protein comprises: (a) lysin A; and (b) Lysin B.

3. The composition of claim 1, wherein the unencapsulated protein comprises: (a) lysin A; and (b) Isoamylase.

4. The composition of claim 1, wherein the unencapsulated protein comprises: (a) lysin A; and (b) α-amylase.

5. The composition of claim 1, wherein the unencapsulated protein comprises: (a) lysin B; and (b) Isoamylase.

6. The composition of claim 1, wherein the unencapsulated protein comprises: (a) lysin B; and (b) α-amylase.

7. The composition of claim 1, wherein the unencapsulated protein comprises: (a) isoamylase; and (b) α-amylase.

8. The composition of claim 1, wherein the unencapsulated protein comprises three or more of: (a) Lysin A; (b) lysin B; (c) isoamylase; and (d) α-Amylase.

9. The composition of claim 8, wherein the unencapsulated protein comprises: (a) Lysin A; (b) lysin B; and (c) Isoamylase.

10. The composition of claim 8, wherein the unencapsulated protein comprises: (a) Lysin A; (b) lysin B; and (c) α-amylase.

11. The composition of claim 8, wherein the unencapsulated protein comprises: (a) Lysin A; (b) isoamylase; and (c) α-amylase.

12. The composition of claim 8, wherein the unencapsulated protein comprises: (a) Lysin B; (b) isoamylase; and (c) α-amylase.

13. The composition of claim 1, wherein the unencapsulated protein comprises all four of: (a) Lysin A; (b) lysin B; (c) isoamylase; and (d) α-Amylase.

14. The composition of claim 1, wherein the unencapsulated protein comprises two or more of: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392; and (d) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

15. The composition of claim 14, wherein the unencapsulated protein comprises two or more of: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and (d) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

16. The composition of claim 14, comprising: (a) lysin A comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; and (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241.

17. The composition of claim 16, comprising: (a) lysin A comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; and (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO:

184.

18. The composition of claim 14, comprising: (a) lysin A comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; and (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392.

19. The composition of claim 18, comprising: (a) lysin A comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; and (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO:

243.

20. The composition of claim 14, comprising: (a) lysin A comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; and (b) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

21. The composition of claim 20, comprising: (a) lysin A comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; and (b) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

22. The composition of claim 14, comprising: (a) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; and (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392.

23. The composition of claim 22, comprising: (a) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; and (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO:

243.

24. The composition of claim 14, comprising: (a) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; and (b) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

25. The composition of claim 24, comprising: (a) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; and (b) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

26. The composition of claim 14, comprising: (a) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392; and (b) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

27. The composition of claim 26, comprising: (a) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and (b) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

28. The composition of claim 1, comprising three or more of: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392; and (d) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

29. The composition of claim 28, comprising three or more of: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and (d) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

30. The composition of claim 28, comprising: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; and (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392.

31. The composition of claim 30, comprising: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; and (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO:

243.

32. The composition of claim 28, comprising: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; and (c) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

33. The composition of claim 32, comprising: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; and (c) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

34. The composition of claim 28, comprising: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392; and (c) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

35. The composition of claim 34, comprising: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and (c) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

36. The composition of claim 28, comprising: (a) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392; and (c) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

37. The composition of claim 36, comprising: (a) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and (c) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

38. The composition of claim 1, comprising all four of the following: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392; and (d) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

39. The composition of claim 38, comprising: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and (d) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

40. The composition of any one of claims 1 to 39, wherein: (a) the lysin A comprises an amino acid sequence having at least 90%, 95%, 97% or 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) the lysin B comprises an amino acid sequence having at least 90%, 95%, 97% or 99% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; (c) the isoamylase comprises an amino acid sequence having at least 90%, 95%, 97% or 99% sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and / or (d) the alpha-amylase comprises an amino acid sequence having at least 90%, 95%, 97% or 99% sequence identity to any one of SEQ ID NOs: 393-398.

41. The composition of claim 40, wherein: (a) the lysin A comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; (b) the lysin B comprises the amino acid sequence of SEQ ID NO: 183 or SEQ ID NO: 184; (c) the isoamylase comprises the amino acid sequence of SEQ ID NO: 242 or SEQ ID NO: 243; and / or (d) the α-amylase comprises the amino acid sequence of any one of SEQ ID NOs: 393-398.

42. The composition of any one of claims 1 to 41, wherein the composition comprises lysin A, lysin B, isoamylase and / or alpha-amylase at a concentration of 0.1 mg / mL to 20 mg / mL.

43. The composition of claim 42, wherein the composition comprises lysin A, lysin B, isoamylase and / or alpha-amylase at a concentration of 1 mg / mL to 10 mg / mL.

44. A method of treating a bacterial infection, the method comprising administering to a subject the composition of any one of claims 1 to 43 in an amount and for a duration sufficient to treat the bacterial infection.

45. The method of claim 44, wherein the bacterial infection is caused by bacteria of the class Actinomycetes.

46. ​​The method of claim 45, wherein the Actinomycetes bacterium is a Corynebacterales or Propionibacterales bacterium.

47. The method of claim 46, wherein the Corynebacteriales is a Mycobacterium species.

48. The method of claim 47, wherein the Mycobacterium species is Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium leprae disseminated, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium marinum, Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium chimera, Mycobacterium boleyi, Mycobacterium fortuitum, Mycobacterium goodii, or Mycobacterium masseille.

49. The method of claim 46, wherein the Corynebacteriales is Nocardia, Corynebacterium, or Rhodococcus.

50. The method of claim 49, wherein: (a) the Nocardia species is Nocardia brasiliensis, Nocardia gelsenkirchen, Nocardia anguillarum, Nocardia novae, Nocardia asteroides, Nocardia brasiliensis or Nocardia caviae; (b) the Corynebacterium species is Corynebacterium glutamicum or Corynebacterium diphtheriae; or (c) The Rhodococcus species is Rhodococcus equi or Rhodococcus equi.

51. The method of claim 46, wherein the Actinomycetes are Propionibacteriales.

52. The method of claim 51, wherein the Propionibacteriales is a Propionibacterium species.

53. The method of claim 52, wherein the Propionibacterium species is Propionibacterium acnes.

54. The method of any one of claims 44 to 53, further comprising administering a supramolecular structure comprising two or more of: (a) Lysin A; (b) lysin B; (c) isoamylase; and (d) α-amylase.

55. The method of claim 54, wherein the supramolecular structure comprises: (a) lysin A; and (b) Lysin B.

56. The method of claim 54, wherein the supramolecular structure comprises: (a) lysin A; and (b) Isoamylase.

57. The method of claim 54, wherein the supramolecular structure comprises: (a) lysin A; and (b) α-amylase.

58. The method of claim 54, wherein the supramolecular structure comprises: (a) lysin B; and (b) Isoamylase.

59. The method of claim 54, wherein the supramolecular structure comprises: (a) lysin B; and (b) α-amylase.

60. The method of claim 54, wherein the supramolecular structure comprises: (a) isoamylase; and (b) α-amylase.

61. The method of claim 54, wherein the supramolecular structure comprises three or more of: (a) Lysin A; (b) lysin B; (c) isoamylase; and (d) α-Amylase.

62. The method of claim 61, wherein the supramolecular structure comprises: (a) Lysin A; (b) lysin B; and (c) Isoamylase.

63. The method of claim 61, wherein the supramolecular structure comprises: (a) Lysin A; (b) lysin B; and (c) α-amylase.

64. The method of claim 61, wherein the supramolecular structure comprises: (a) Lysin A; (b) isoamylase; and (c) α-amylase.

65. The method of claim 61, wherein the supramolecular structure comprises: (a) Lysin B; (b) isoamylase; and (c) α-amylase.

66. The method of claim 54, wherein the supramolecular structure comprises all four of the following: (a) Lysin A; (b) lysin B; (c) isoamylase; and (d) α-Amylase.

67. The method of claim 54, wherein the supramolecular structure comprises two or more of: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392; and (d) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

68. The method of claim 67, wherein the supramolecular structure comprises two or more of: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and (d) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

69. The method of claim 67, wherein the supramolecular structure comprises: (a) lysin A comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; and (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241.

70. The method of claim 69, wherein the supramolecular structure comprises: (a) lysin A comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; and (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO:

184.

71. The method of claim 67, wherein the supramolecular structure comprises: (a) lysin A comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; and (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392.

72. The method of claim 71, wherein the supramolecular structure comprises: (a) lysin A comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; and (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO:

243.

73. The method of claim 67, wherein the supramolecular structure comprises: (a) lysin A comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; and (b) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

74. The method of claim 73, wherein the supramolecular structure comprises: (a) lysin A comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; and (b) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

75. The method of claim 67, wherein the supramolecular structure comprises: (a) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; and (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392.

76. The method of claim 75, wherein the supramolecular structure comprises: (a) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; and (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO:

243.

77. The method of claim 67, wherein the supramolecular structure comprises: (a) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; and (b) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

78. The method of claim 77, wherein the supramolecular structure comprises: (a) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; and (b) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

79. The method of claim 67, wherein the supramolecular structure comprises: (a) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392; and (b) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

80. The method of claim 79, wherein the supramolecular structure comprises: (a) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and (b) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

81. The method of claim 54, wherein the supramolecular structure comprises three or more of: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392; and (d) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

82. The method of claim 81, wherein the supramolecular structure comprises three or more of: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and (d) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

83. The method of claim 81, wherein the supramolecular structure comprises: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; and (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392.

84. The method of claim 83, wherein the supramolecular structure comprises: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; and (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO:

243.

85. The method of claim 81, wherein the supramolecular structure comprises: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; and (c) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

86. The method of claim 85, wherein the supramolecular structure comprises: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; and (c) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

87. The method of claim 81, wherein the supramolecular structure comprises: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392; and (c) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

88. The method of claim 87, wherein the supramolecular structure comprises: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and (c) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

89. The method of claim 81, wherein the supramolecular structure comprises: (a) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392; and (c) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

90. The method of claim 89, wherein the supramolecular structure comprises: (a) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; (b) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and (c) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

91. The method of claim 54, wherein the supramolecular structure comprises all four of the following: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-182; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 183-241; (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 242-392; and (d) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-445.

92. The method of claim 91, wherein the supramolecular structure comprises: (a) lysin A, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) lysin B comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; (c) an isoamylase comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and (d) an alpha-amylase comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 393-398.

93. The method of any one of claims 54 to 92, wherein the supramolecular structure comprises: (a) the lysin A comprises an amino acid sequence having at least 90%, 95%, 97% or 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) the lysin B comprises an amino acid sequence having at least 90%, 95%, 97% or 99% sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; (c) the isoamylase comprises an amino acid sequence having at least 90%, 95%, 97% or 99% sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and / or (d) the alpha-amylase comprises an amino acid sequence having at least 90%, 95%, 97% or 99% sequence identity to any one of SEQ ID NOs: 393-398.

94. The method of claim 93, wherein the supramolecular structure comprises: (a) the lysin A comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; (b) the lysin B comprises the amino acid sequence of SEQ ID NO: 183 or SEQ ID NO: 184; (c) the isoamylase comprises the amino acid sequence of SEQ ID NO: 242 or SEQ ID NO: 243; and / or (d) the α-amylase comprises the amino acid sequence of any one of SEQ ID NOs: 393-398.

95. The method of any one of claims 54 to 94, wherein the supramolecular structure comprises a Z-average particle size of 75 nm to 750 nm.

96. The method of claim 95, wherein the Z-average particle size is 250 nm to 750 nm.

97. The method of claim 96, wherein the Z-average particle size is 75 nm to 250 nm.

98. The method of any one of claims 54 to 97, wherein the supramolecular structure is a lipid nanoparticle.

99. The method of any one of claims 54 to 97, wherein the supramolecular structure is a micelle.

100. The method of any one of claims 54 to 97, wherein the supramolecular structure is a liposome.

101. The method of claim 100, wherein the liposome is unilamellar.

102. The method of claim 100, wherein the liposome is multilaminar.

103. The method of any one of claims 54 to 102, wherein the supramolecular structure comprises a polydispersity index of 0.05 to 0.

3.

104. The method of any one of claims 54 to 103, wherein the supramolecular structure comprises one or more lipids.

105. The method of claim 104, wherein at least one of the one or more lipids is an ionizable lipid.

106. The method of claim 104 or 105, wherein the lipid comprises 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).

107. The method of claim 105, wherein the lipid is a sterol.

108. The method of claim 107, wherein the sterol is cholesterol.

109. The method of any one of claims 104 to 108, wherein the supramolecular structure comprises a mixture of lipids.

110. The method of claim 109, wherein the lipid mixture comprises DOPC, DOPE, DOPS, and cholesterol.

111. The method of claim 110, wherein the DOPC, DOPE, DOPS, and cholesterol are present in a molar ratio of 1-20: 1-20: 1-5: 1-5.

112. The method of claim 111, wherein the DOPC, DOPE, DOPS, and cholesterol are present in a molar ratio of 10:10:3:

4.

113. The method of any one of claims 54 to 112, wherein the supramolecular structure comprises lipids at a concentration of 0.03 mg / mL to 10 mg / mL.

114. The method of claim 113, wherein the supramolecular structure comprises lipids at a concentration of 1 mg / mL to 5 mg / mL.

115. The method of any one of claims 54 to 114, wherein the supramolecular structure comprises a targeting moiety.

116. The method of claim 115, wherein the targeting moiety is an extracellular targeting moiety that targets professional antigen presenting cells.

117. The method of claim 116, wherein the professional antigen presenting cell is a macrophage or a dendritic cell.

118. The method of any one of claims 115 to 117, wherein the targeting moiety comprises phosphatidylserine.

119. The method of any one of claims 54 to 118, wherein the composition is administered before the supramolecular structure.

120. The method of any one of claims 54 to 118, wherein the composition is administered after the supramolecular structure.

121. The method of any one of claims 54 to 118, wherein the composition and the supramolecular structure are administered substantially simultaneously.

122. The method of any one of claims 44 to 121, further comprising administering an antibiotic.

123. The method of claim 122, wherein the antibiotic is a cephalosporin, a carbapenem, a penicillin, an aminoglycoside, a cephalosporin, a rifamycin, a macrolide, or a fluoroquinolone.

124. The method of claim 122, wherein the antibiotic is thiosemicarbazide, sq-109, bedaquiline, delamanid, pyrazinamide, or isoniazid.

125. The method of claim 122, wherein the antibiotic is azithromycin, clarithromycin, ethambutol, rifampicin, biapenem, or amikacin.

126. The method of any one of claims 44 to 125, wherein the composition is administered intravenously, orally, or by inhalation.

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