Compositions and methods for treating actinomycosis

Abstract

The present disclosure relates to compositions and methods for the treatment of actinomycete (e.g., Corynebacteriales) infections caused by, for example, mycobacterial cells. In one aspect, the present disclosure relates to a composition containing an unencapsulated protein comprising one or more of (a) lysin A; (b) lysin B; (c) isoamylase; and (d) α-amylase. The composition may further comprise a supramolecular structure comprising one or more of (a) lysin A; (b) lysin B; (c) isoamylase; and (d) α-amylase. In another aspect, the present invention is a method for treating a bacterial infection in a subject, the method comprising administering to the subject a composition of the unencapsulated lytic protein described herein, optionally in combination with an encapsulated lytic protein, in an amount and for a time sufficient to treat the bacterial infection. The encapsulated lytic protein and the unencapsulated lytic protein can be the same or different compositions and can be administered simultaneously or sequentially.

Description

Technical Field

[0001] Sequence Listing This application is electronically filed in XML format and includes a sequence listing that is hereby incorporated by reference in its entirety. A copy of the XML created on April 13, 2023, is named 51486-007WO4_Sequence_Listing_4_13_23.xml and is 660,323 bytes in size.

Background Art

[0002] Background Bacterial pathogens are a major cause of infectious diseases. Many bacteria are well detected by the human immune system and are rapidly removed before an infection begins. However, some bacterial pathogens evade the host immune system by living inside host cells. These intracellular bacteria have developed various immune evasion techniques by living and multiplying inside host cells, such as immune cells (e.g., macrophages or dendritic cells), and within the correct intracellular compartments within host cells (e.g., endosomes, phagosomes, lysosomes, or the cytosol). Bacterial infections that expand within host cells often present difficult treatment barriers due to the lack of easy access to the intracellular site of infection. Although certain antibacterial compositions can treat infections (e.g., in vitro), delivering the treatment to the correct intracellular location where the bacteria live has proven to be a difficult endeavor.

[0003] The group of intracellular bacterial infections that are difficult are caused by mycobacteria. Mycobacteria are actinomycetes (e.g., Corynebacteriales or Propionibacteriales), which are characterized by a thick envelope rich in mycolic acid. Mycobacteria contain an envelope that includes a cell membrane composed of lipids, a cell wall containing peptidoglycan, an arabinogalactan layer, and an outer membrane known as the mycomembrane that is rich in mycolic acid. Many mycobacterial envelopes also contain an outer capsule layer composed of polysaccharides such as D-glucan, D-arabinono-D-mannan, and D-mannan. This complex cell envelope contributes to the durability of mycobacteria, and in particular, it is difficult to penetrate and destroy mycobacterial cells.

[0004] These bacteria also contain a complex life cycle in which the bacteria exist within the cytoplasm, or within other intracellular compartments, or outside the host cell. Mycobacteria are endocytosed by host cells, and these endocytosed vesicles can integrate with intracellular organelles such as endosomes, phagosomes, or lysosomes. Once inside these intracellular compartments, the bacteria can replicate and proliferate. This is followed by membrane solubilization and release of the bacteria into the cytoplasm, where the bacteria continue to grow. Subsequently, the bacteria lyse the host cell and spread as a free form of the bacteria. Such free forms of the bacteria can appear in the spleen and liver, for example, after release from lung phagocytes, leading to spread of the infection and death.

[0005] Due to the complex life cycle, it is spatially and temporally difficult to target the bacteria at an appropriate locus and effectively treat the infection. Therefore, there is a need for improved compositions and methods for treating bacterial infections, such as those caused by mycobacteria, that target the intracellular bacterial infections. There is a need for improved compositions and methods for treating intracellular bacterial infections, such as those caused by mycobacteria. Furthermore, a patient can have mycobacteria in various states of the life cycle simultaneously. SUMMARY OF THE INVENTION

[0006] In one aspect, the present invention relates to a composition containing an encapsulated protein comprising one or more (e.g., two or more, three or more, or all four) of (a) lysin A; (b) lysin B; (c) isoamylase; and (d) α - amylase.

[0007] In some embodiments, the composition comprises lysin A and lysin B. In some embodiments, the composition comprises lysin A and isoamylase. In some embodiments, the composition comprises lysin A and α - amylase.

[0008] In some embodiments, the composition comprises lysin B and isoamylase. In some embodiments, the composition comprises lysin B and α - amylase. In some embodiments, the composition comprises isoamylase and α - amylase.

[0009] In some embodiments, the composition comprises lysin A, lysin B, and isoamylase. In some embodiments, the composition comprises lysin A, lysin B, and α - amylase.

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

[0011] In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and α - amylase. In some embodiments, the present invention relates to a composition containing an unfused protein comprising: (a) Lysin A comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 to 182; (b) Lysin B comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183 to 241; (c) 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 to 392; and (d) one or more (e.g., two or more, three or more, or all four) of α-Amylase comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393 to 445.

[0012] In some embodiments, the present invention relates to a composition containing an unfused protein comprising: (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) 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) 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) one or more (e.g., two or more, three or more, or all four) of α-Amylase comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393 to 398.

[0013] In some embodiments, the composition comprises lysin A and lysin B. Lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182. Lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 241.

[0014] In some embodiments, the composition comprises lysin A and lysin B. Lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with SEQ ID NO: 183 or SEQ ID NO: 184.

[0015] In some embodiments, the composition comprises lysin A and isoamylase. Lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182. Isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392.

[0016] In some embodiments, the composition comprises lysin A and isoamylase. Lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. Isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243.

[0017] In some embodiments, the composition comprises lysin A and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 445.

[0018] In some embodiments, the composition comprises lysin A and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 398.

[0019] In some embodiments, the composition comprises lysin B and isoamylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 241, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392.

[0020] In some embodiments, the composition comprises lysin B and isoamylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with SEQ ID NO: 242 or SEQ ID NO: 243.

[0021] In some embodiments, the composition comprises lysin B and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 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 with any one of SEQ ID NOs: 393 to 445.

[0022] In some embodiments, the composition comprises lysin B and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with any one of SEQ ID NOs: 393 to 398.

[0023] In some embodiments, the composition comprises isoamylase and α-amylase, wherein isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 445.

[0024] In some embodiments, the composition comprises isoamylase and α-amylase, wherein isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 398.

[0025] In some embodiments, the composition comprises lysin A, lysin B, and isoamylase. Lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182. Lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 241. Isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392.

[0026] In some embodiments, the composition comprises lysin A, lysin B, and isoamylase. Lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with 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 with SEQ ID NO: 242 or SEQ ID NO: 243.

[0027] In some embodiments, the composition comprises lysin A, lysin B, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182, lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 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 with any one of SEQ ID NOs: 393 to 445.

[0028] In some embodiments, the composition comprises lysin A, lysin B, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with 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 with any one of SEQ ID NOs: 393 to 398.

[0029] In some embodiments, the composition comprises lysin A, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 445.

[0030] In some embodiments, the composition comprises lysin A, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393-398.

[0031] In some embodiments, the composition comprises lysin B, isoamylase, and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183-241, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242-392, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393-445.

[0032] In some embodiments, the composition comprises lysin B, isoamylase, and α - amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with SEQ ID NO: 242 or SEQ ID NO: 243, and α - amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 - 398.

[0033] In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and α - amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with any one of SEQ ID NOs: 183 - 241, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 - 392, and α - amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 - 445.

[0034] In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with 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 with SEQ ID NO: 242 or SEQ ID NO: 243, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393-398.

[0035] In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and α-amylase, wherein lysin A comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, lysin B comprises the amino acid sequence of SEQ ID NO: 183 or SEQ ID NO: 184, isoamylase comprises the amino acid sequence of SEQ ID NO: 242 or SEQ ID NO: 243, and α-amylase comprises the amino acid sequence of any one of SEQ ID NOs: 393-398.

[0036] 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 α-amylase of SEQ ID NO: 393. In some embodiments, the composition comprises lysin A of SEQ ID NO: 2, lysin B of SEQ ID NO: 184, isoamylase of SEQ ID NO: 243, and α-amylase of SEQ ID NO: 394.

[0037] 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 α-amylase of SEQ ID NO: 395. In some embodiments, the composition comprises lysin A of SEQ ID NO: 2, lysin B of SEQ ID NO: 184, isoamylase of SEQ ID NO: 243, and α-amylase of SEQ ID NO: 396.

[0038] 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 α-amylase of SEQ ID NO: 397. In some embodiments, the composition comprises lysin A of SEQ ID NO: 2, lysin B of SEQ ID NO: 184, isoamylase of SEQ ID NO: 243, and α-amylase of SEQ ID NO: 398.

[0039] In some embodiments, the composition comprises a protein (e.g., lysin A, lysin B, isoamylase, and / or α-amylase) at a concentration of 0.1 mg / mL to 20 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, e.g., 10 mg / mL to 20 mg / mL, e.g., 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL). In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and / or α-amylase at a concentration of 1 mg / mL to 10 mg / mL.

[0040] 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 (a) lysin A; (b) lysin B; (c) isoamylase; and (d) α-amylase.

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

[0042] In some embodiments, the composition comprises lysin B and isoamylase. In some embodiments, the composition comprises lysin B and α - amylase. In some embodiments, the composition comprises isoamylase and α - amylase.

[0043] In some embodiments, the composition comprises lysin A, lysin B, and isoamylase. In some embodiments, the composition comprises lysin A, lysin B, and α - amylase.

[0044] In some embodiments, the composition comprises lysin A, isoamylase, and α - amylase. In some embodiments, the composition comprises lysin B, isoamylase, and α - amylase.

[0045] In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and α - amylase. 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: (a) Lysin A comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 to 182; (b) Lysin B comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183 to 241; (c) 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 to 392; and (d) α-Amylase comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393 to 445.

[0046] 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: (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) 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) 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) α-Amylase comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393 to 398.

[0047] In some embodiments, the supramolecular structure comprises lysin A and lysin B. Lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 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 with any one of SEQ ID NOs: 183 to 241.

[0048] In some embodiments, the supramolecular structure comprises lysin A and lysin B. Lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with SEQ ID NO: 183 or SEQ ID NO: 184.

[0049] In some embodiments, the supramolecular structure comprises lysin A and isoamylase. Lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392.

[0050] In some embodiments, the supramolecular structure comprises lysin A and isoamylase. Lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243.

[0051] In some embodiments, the supramolecular structure comprises lysin A and α-amylase, where lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 445.

[0052] In some embodiments, the supramolecular structure comprises lysin A and α-amylase, where lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 398.

[0053] In some embodiments, the supramolecular structure comprises lysin B and isoamylase, where lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 241, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392.

[0054] In some embodiments, the supramolecular structure comprises lysin B and isoamylase, where lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with SEQ ID NO: 242 or SEQ ID NO: 243.

[0055] In some embodiments, the supramolecular structure comprises lysin B and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 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 with any one of SEQ ID NOs: 393 to 445.

[0056] In some embodiments, the supramolecular structure comprises lysin B and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with any one of SEQ ID NOs: 393 to 398.

[0057] In some embodiments, the supramolecular structure comprises isoamylase and α-amylase, wherein isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 445.

[0058] In some embodiments, the supramolecular structure comprises isoamylase and α-amylase, wherein the isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, and the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393-398.

[0059] In some embodiments, the supramolecular structure comprises lysin A, lysin B, and isoamylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with 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 with any one of SEQ ID NOs: 242-392.

[0060] In some embodiments, the supramolecular structure comprises lysin A, lysin B, and isoamylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with 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 with SEQ ID NO: 242 or SEQ ID NO: 243.

[0061] In some embodiments, the supramolecular structure comprises lysin A, lysin B, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182, lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 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 with any one of SEQ ID NOs: 393 to 445.

[0062] In some embodiments, the supramolecular structure comprises lysin A, lysin B, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with 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 with any one of SEQ ID NOs: 393 to 398.

[0063] In some embodiments, the supramolecular structure comprises lysin A, isoamylase, and α-amylase. Lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182. Isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392. α-Amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 445.

[0064] In some embodiments, the supramolecular structure comprises lysin A, isoamylase, and α-amylase. Lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. Isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243. α-Amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 398.

[0065] In some embodiments, the supramolecular structure comprises lysin B, isoamylase, and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 241, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 445.

[0066] In some embodiments, the supramolecular structure comprises lysin B, isoamylase, and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with SEQ ID NO: 242 or SEQ ID NO: 243, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 398.

[0067] In some embodiments, the supramolecular structure comprises lysin A, lysin B, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182, lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 241, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 445.

[0068] In some embodiments, the supramolecular structure comprises lysin A, lysin B, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with 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 with SEQ ID NO: 242 or SEQ ID NO: 243, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 398. In some embodiments, the supramolecular structure comprises lysin A, lysin B, isoamylase, and α-amylase, wherein lysin A comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, lysin B comprises the amino acid sequence of SEQ ID NO: 183 or SEQ ID NO: 184, isoamylase comprises the amino acid sequence of SEQ ID NO: 242 or SEQ ID NO: 243, and α-amylase comprises the amino acid sequence of any one of SEQ ID NOs: 393 to 398.

[0069] 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: 393. 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: 394.

[0070] 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.

[0071] 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.

[0072] In some embodiments, the Z-average mean particle size of the supramolecular structure is from 75 nm to 5 μm, for example, from 75 nm to 2 μm, from 75 nm to 1 μm, for example, from 75 nm to 750 nm (for example, from 250 nm to 750 nm, or from 75 nm to 250 nm). In some embodiments, when the supramolecular structure is an LNP or a micelle, the Z-average mean particle size is from 75 nm to 250 nm. In some embodiments, when the supramolecular structure is a vesicle (e.g., a liposome), the Z-average mean particle size is from 250 nm to 750 nm. Non-limiting examples of the Z-average mean particle size include, for example, from 75 nm to 100 nm, for example, from 75 nm to 85 nm, for example, 80 nm, for example, from 80 nm to 140 nm, from 90 nm to 130 nm, or from 110 nm to 130 nm, for example, 120 nm, for example, from 200 nm to 300 nm, for example, from 250 nm to 300 nm, from 260 nm to 290 nm, from 260 nm to 280 nm, from 265 nm to 275 nm, for example, 270 nm, for example, from 300 nm to 400 nm, from 400 nm to 600 nm, for example, from 450 nm to 550 nm, from 475 nm to 525 nm, from 480 nm to 520 nm, from 490 nm to 510 nm, from 495 nm to 505 nm, for example, 500 nm, for example, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, 250 nm, 255 nm, 260 nm, 265 nm, 270 nm, 275 nm, 280 nm, 285 nm, 290 nm, 295 nm, 300 nm, 305 nm, 310 nm, 315 nm, 320 nm, 325 nm, 330 nm, 335 nm, 340 nm, 345 nm, 350 nm, 355 nm, 360 nm, 365 nm, 370 nm, 375 nm, 380 nm, 385 nm, 390 nm, 395 nm, 400 nm, 405 nm, 410 nm, 415 nm, 420 nm, 425 nm, 430 nm, 435 nm, 440 nm, 445 nm, 450 nm, 455 nm, 460 nm, 465 nm,470 nm, 475 nm, 480 nm, 485 nm, 490 nm, 495 nm, 500 nm, 505 nm, 510 nm, 515 nm, 520 nm, 525 nm, 530 nm, 535 nm, 540 nm, 545 nm, 550 nm, 555 nm, 560 nm, 565 nm, 570 nm, 575 nm, 580 nm, 585 nm, 590 nm, 595 nm, 600 nm, 605 nm, 610 nm, 615 nm, 620 nm, 625 nm, 630 nm, 635 nm, 640 nm, 645 nm, 650 nm, 655 nm, 660 nm, 665 nm, 670 nm, 675 nm, 680 nm, 685 nm, 690 nm, 695 nm, 700 nm, 705 nm, 710 nm, 715 nm, 720 nm, 725 nm, 730 nm, 735 nm, 740 nm, 745 nm, 750 nm, 755 nm, 760 nm, 765 nm, 770 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 845 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm, 900 nm, 905 nm, 910 nm, 915 nm, 920 nm, 925 nm, 930 nm, 935 nm, 940 nm, 945 nm, 950 nm, 955 nm, 960 nm, 965 nm, 970 nm, 975 nm, 980 nm, 985 nm, 990 nm, 995 nm, 1 μm, 1.1 μm, 1.2 μm, 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 some embodiments, the Z-average mean particle size of the supramolecular structure is 80 nm, 270 nm, or 500 nm. In some embodiments, the supramolecular structure has a Z-average mean particle size ranging from 75 nm to 750 nm. In some embodiments,The Z-average mean particle size is from 250 nm to 750 nm. In some embodiments, the Z-average mean particle size is from 75 nm to 250 nm.

[0073] In some embodiments, the supramolecular structure is a lipid nanoparticle. In some embodiments, the supramolecular structure is a micelle. In some embodiments, the supramolecular structure is a liposome. The liposome can be monolayer. Alternatively, the liposome can be multilayer.

[0074] In some embodiments, the supramolecular structure has a polydispersity index of from 0.05 to 0.3. In some embodiments, the supramolecular structure contains one or more lipids. 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).

[0075] In some embodiments, the lipid is a sterol, such as cholesterol or a derivative thereof. In some embodiments, the supramolecular structure contains a mixture of lipids. For example, the mixture of lipids can contain two or more of DOPC, DOPE, DOPS, and cholesterol.

[0076] In some embodiments, DOPC and DOPE are present in a molar ratio of from 10:1 to 1:10 (for example, 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).

[0077] 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).

[0078] 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).

[0079] 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).

[0080] 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).

[0081] 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).

[0082] In some embodiments, DOPC, DOPE, DOPS, and cholesterol are present in a molar ratio of 1 to 20:1 to 20:1 to 5:1 to 5. For example, in some embodiments, DOPC, DOPE, DOPS, and cholesterol are present in a molar ratio of 10:10:3:4.

[0083] In some embodiments, the supramolecular structure comprises lipids at a concentration of 0.1 mg / mL to 10 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL). In some embodiments, the supramolecular structure comprises lipids at a concentration of 1 mg / mL to 5 mg / mL.

[0084] In some embodiments, the supramolecular structure comprises proteins (e.g., lysin A, lysin B, isoamylase, and / or α - amylase) at a concentration of 0.1 mg / mL to 20 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, e.g., 10 mg / mL to 20 mg / mL, e.g., 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL). In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and / or α - amylase at a concentration of 1 mg / mL to 10 mg / mL.

[0085] In some embodiments, the supramolecular structures described herein are formulated with one or more buffers and / or excipients. For example, the supramolecular structures (e.g., liposomes containing a cocktail of lysozymes) can be encapsulated and / or formulated in a buffer such as glycine, Tris, sodium citrate, sodium acetate, and MES at a concentration of, for example, 10 mM to 200 mM, for example, 50 mM to 150 mM, for example, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM. The supramolecular structures 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., 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11). The supramolecular structures can further include one or more excipients such as CaCl 2 , arginine, NaCl, sodium citrate, MgCl 2 , or glycerol. In some embodiments, the supramolecular structures include, for example, 5 mM to 10 mM CaCl 2 , 0 to 50 mM arginine, 0 to 200 mM NaCl, 0 to 1 mM sodium citrate, 0 to 1 mM MgCl 2 , and / or 10 to 30% glycerol. In some embodiments, the supramolecular structures include 50 mM glycine (pH 8.5), 7.5 mM CaCl 2 , 0.5 mM MgCl 2 , 200 mM NaCl, 0.33 mM sodium citrate, and 10% glycerol. The formulation can further include Tween, e.g., Tween-80.

[0086] 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.

[0087] In another aspect, the present invention relates to a method for treating a bacterial infection in a subject. The method comprises administering to the subject a composition described herein, for example, any of the compositions of the above embodiments, in an amount and for a period sufficient to treat the bacterial infection.

[0088] 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) 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) 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) 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) α-Amylase comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

[0089] In some embodiments, the method further comprises administering a supramolecular structure comprising (a) Lysin A comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, (b) Lysin B comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184, (c) Isoamylase comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, and (d) one or more (e.g., two or more, three or more, or all four) of α-Amylases comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 - 398.

[0090] In some embodiments, the supramolecular structure comprises Lysin A and Lysin B, Lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with any one of SEQ ID NOs: 183 - 241.

[0091] In some embodiments, the supramolecular structure comprises Lysin A and Lysin B, Lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with SEQ ID NO: 183 or SEQ ID NO: 184.

[0092] In some embodiments, the supramolecular structure comprises lysin A and isoamylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392.

[0093] In some embodiments, the supramolecular structure comprises lysin A and isoamylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243.

[0094] In some embodiments, the supramolecular structure comprises lysin A and α - amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182, and α - amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 445.

[0095] In some embodiments, the supramolecular structure comprises lysin A and α - amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, and α - amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 398.

[0096] In some embodiments, the supramolecular structure comprises lysin B and isoamylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 241, and isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392.

[0097] In some embodiments, the supramolecular structure comprises lysin B and isoamylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with SEQ ID NO: 242 or SEQ ID NO: 243.

[0098] In some embodiments, the supramolecular structure comprises lysin B and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 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 with any one of SEQ ID NOs: 393 to 445.

[0099] In some embodiments, the supramolecular structure comprises lysin B and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with any one of SEQ ID NOs: 393 to 398.

[0100] In some embodiments, the supramolecular structure comprises isoamylase and α-amylase, the isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392, and the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 445.

[0101] In some embodiments, the supramolecular structure comprises isoamylase and α-amylase, the isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, and the α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 398.

[0102] In some embodiments, the supramolecular structure comprises lysin A, lysin B, and isoamylase, the lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182, the lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 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 with any one of SEQ ID NOs: 242 to 392.

[0103] In some embodiments, the supramolecular structure comprises lysin A, lysin B, and isoamylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with 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 with SEQ ID NO: 242 or SEQ ID NO: 243.

[0104] In some embodiments, the supramolecular structure comprises lysin A, lysin B, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182, lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 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 with any one of SEQ ID NOs: 393 to 445.

[0105] In some embodiments, the supramolecular structure comprises lysin A, lysin B, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with 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 with any one of SEQ ID NOs: 393 to 398.

[0106] In some embodiments, the supramolecular structure comprises lysin A, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 1 to 182, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 445.

[0107] In some embodiments, the supramolecular structure comprises lysin A, isoamylase, and α-amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 398.

[0108] In some embodiments, the supramolecular structure comprises lysin B, isoamylase, and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 183 to 241, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 to 392, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 445.

[0109] In some embodiments, the supramolecular structure comprises lysin B, isoamylase, and α-amylase, wherein lysin B comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with SEQ ID NO: 242 or SEQ ID NO: 243, and α-amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 398.

[0110] In some embodiments, the supramolecular structure comprises lysin A, lysin B, isoamylase, and α - amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with any one of SEQ ID NOs: 183 - 241, isoamylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 242 - 392, and α - amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 - 445.

[0111] In some embodiments, the supramolecular structure comprises lysin A, lysin B, isoamylase, and α - amylase, wherein lysin A comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with 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 with 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 with SEQ ID NO: 242 or SEQ ID NO: 243, and α - amylase comprises an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 - 398.

[0112] In some embodiments, the supramolecular structure comprises lysin A, lysin B, isoamylase, and α-amylase, wherein lysin A comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, lysin B comprises the amino acid sequence of SEQ ID NO: 183 or SEQ ID NO: 184, isoamylase comprises the amino acid sequence of SEQ ID NO: 242 or SEQ ID NO: 243, and α-amylase comprises any one of the amino acid sequences of SEQ ID NOs: 393 to 398.

[0113] 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: 393. 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: 394.

[0114] 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.

[0115] 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.

[0116] In some embodiments, the composition is administered before the supramolecular structure. In some embodiments, the composition is administered after the supramolecular structure. In some embodiments, the composition is administered simultaneously with the supramolecular structure.

[0117] In some embodiments, the bacterial infection is caused by actinomycete bacteria. In some embodiments, the actinomycetes are of the order Corynebacteriales or Propionibacteriales. In some embodiments, the order Corynebacteriales is of the Mycobacterium species.

[0118] In some embodiments, the Mycobacterium species are 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.

[0119] In some embodiments, the order Corynebacteriales is Nocardia, Corynebacterium, or Rhodococcus species. In some embodiments, the order Propionibacteriales is Cutibacterium species.

[0120] In some embodiments, using the compositions and methods described herein, other actinomycetes (e.g., of the order Corynebacteriales or Propionibacteriales) having envelope components similar to Mycobacteria can be targeted. For example, the compositions and methods can be used to target Nocardia, Corynebacterium, or Rhodococcus species. For example, Nocardia species can be, for example, N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. asteroids, N. brasiliensis, and N. caviae. Corynebacterium species can be, for example, C. glutamicum or C. diphtheriae. Rhodococcus species can be, for example, R. fascians or R. equi.

[0121] In some embodiments, the compositions and methods can be used to target Cutibacterium species. The Cutibacterium species can be, for example, C. acnes.

[0122] In some embodiments, the method further comprises administering an antibiotic. In some embodiments, the antibiotic is a cephalosporin, carbapenem, penicillin, aminoglycoside, cephalosporin, rifamycin, macrolide, or fluoroquinolone. In some embodiments, the antibiotic is thiacetazone, sq-109, bedaquiline, delamanid, pyrazinamide, or isoniazid. In some embodiments, the antibiotic is azithromycin, clarithromycin, ethambutol, rifampin, biapenem, or amikacin. In some embodiments, the antibiotic is a macrolide (e.g., azithromycin, clarithromycin, erythromycin). In some embodiments, the antibiotic is a macrolide (e.g., azithromycin, clarithromycin, erythromycin). In some embodiments, the antibiotic is an aminoglycoside (e.g., kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin (e.g., neomycin B, C, or E), streptomycin, or plazomycin).

[0123] In some embodiments, the composition is administered intravenously, orally, or by inhalation (e.g., via an aerosol). In some embodiments, the compositions and methods described herein target bacteria that are extracellular for at least a portion of their life cycle.

[0124] In some embodiments, the compositions and methods described herein target bacteria that are intracellular for at least a portion of their life cycle. In some embodiments, the compositions and methods described herein target bacteria that are both extracellular and intracellular for at least a portion of their life cycle.

[0125] Definition As used herein, the term "about" means ±10% of the recited value. As used herein, "combination therapy" or "co - administration" means that two (or more) agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen defines the dosage and dosing period of each agent such that the effects of the individual agents on the subject overlap and / or result in a synergistic effect. In some embodiments, the delivery of two or more agents is done simultaneously or in parallel, and the agents may be formulated as a mixture. In some embodiments, two or more agents are not co - formulated and are administered sequentially as part of the prescribed regimen. In some embodiments, administering two or more agents or treatments in combination results in a decrease in symptoms or other parameters associated with the disease that is greater than that observed with one agent or treatment delivered alone or in the absence of the other. The effects of the two treatments can be partially additive, fully additive, or supra - additive, e.g., synergistic. The sequential or substantially simultaneous administration of each therapeutic agent can be by any suitable route, including, but not limited to, oral, intravenous, intramuscular, topical, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes. For example, the first therapeutic agent of a combination may be administered by intravenous injection while the second therapeutic agent of the combination may be administered orally.

[0126] As used herein, for example, in reference to a cell, a sample, or a subject, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of an agent that provides a therapeutic effect described herein mean an amount sufficient to provide a beneficial or desired result, including preclinical or clinical outcomes, when administered to a cell, a sample, or a subject, including a human, and thus such "effective amount," or synonyms thereof, are context-dependent. For example, in the context of treating a disorder, the term refers to an amount of an agent sufficient to achieve a therapeutic response as compared to a response obtained without administration of the agent. The amount of a given agent will vary depending on various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the severity of the bacterial infection, the subject, sample, or host cell being treated, e.g., biomarkers of mammalian immune cells, e.g., age, sex, and / or weight, etc., but can nevertheless be routinely determined by one of ordinary skill in the art. Also, as used herein, the term "therapeutically effective amount" of an agent means an amount that provides a beneficial or desired result in a cell or subject as compared to a control. As defined herein, a therapeutically effective amount of an agent can be readily determined by one of ordinary skill in the art by routine methods known in the art. A dosing regimen may be adjusted to provide an optimal therapeutic response.

[0127] As used herein, the term "antibacterial lytic protein" means a protein having bactericidal and / or lytic activity against bacteria. Non-limiting examples of antibacterial lytic proteins include holins, lysins (e.g., lysin A and / or lysin B), amylases (e.g., isoamylase or α-amylase), pseudopolimerases (e.g., hydrolases, metallohydrolases, epoxide hydrolases, peptidoglycan hydrolases, polysaccharases, polysaccharide lyases, endosialidases, hyaluronan lyases, or alginate lyases), beta-lactamases, and lysozyme.

[0128] As used herein, "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer encapsulating a substantially solid lipid core, which can contain a pharmaceutically active molecule. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particles (e.g., a PEG-lipid conjugate).

[0129] As used herein, the term "liposome" means a vesicle composed of amphiphilic lipids disposed within at least one bilayer membrane, e.g., one bilayer membrane or multiple bilayer membranes. Liposomes include unilamellar and multilamellar (e.g., 2, 3, 4, 5, or more lamellae) having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains an antibacterial lytic protein or a mixture of an antibacterial lytic protein and other components. The lipophilic material separates the aqueous exterior from the aqueous interior and usually does not contain phage proteins, but may contain them in some cases. Liposomes also include "sterically stabilized" liposomes, which, as used herein, means liposomes containing one or more dedicated lipids that provide an improved circulation lifetime when incorporated into the liposome as compared to liposomes lacking such dedicated lipids.

[0130] "Micelle" is defined herein as a particular type of substantially spherical supramolecular structure in which amphiphilic molecules, e.g., lipids, are arranged such that the hydrophobic portions of the molecules face inward towards the core and the hydrophilic portions remain in contact with the surrounding aqueous phase. The reverse arrangement exists when the surrounding environment is hydrophobic. The micelle core can contain an antibacterial lytic protein or a mixture of multiple proteins.

[0131] As used herein, the term "subject" refers to any organism to which a composition according to the invention can be administered for, e.g., experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal, such as mammals, e.g., mice, rats, rabbits, non-human primates, and humans. A subject can be a subject seeking or in need of treatment, a subject in need of treatment, a subject receiving treatment, a subject who is going to receive treatment in the future, or a human or animal under the care of a specialist trained in a particular disease or disorder.

[0132] As used herein, the term "supramolecular structure" means a complex of molecules that are bound to each other by non-covalent bonds, such as hydrogen bonds, van der Waals forces, electrostatic interactions, hydrophobic effects, and π-π interactions. Supramolecular structures can include, for example, large complexes of molecules that form a globular-like structure. Examples of supramolecular structures include lipid-based supramolecular structures, such as liposomes, lipid nanoparticles, and micelles.

[0133] As used herein, the term "target intracellular compartment" means an endosome, phagosome, lysosome, or cytosol. As used herein, the term "unencapsulated protein" means a free protein that is not present in a supramolecular structure. For example, unencapsulated proteins are not formulated within liposomes, lipid nanoparticles, or micelles.

[0134] As used herein, the term "target moiety" refers to a moiety (e.g., a small molecule, such as a carbohydrate) that specifically binds to, or reacts associatively with, or forms a complex with a receptor or other receptive moiety that associates with a given target cell population (e.g., professional antigen-presenting cells, such as macrophages or dendritic cells). Thus, a target moiety can be used to target the supramolecular structures described herein to, for example, professional antigen-presenting cells (such as macrophages or dendritic cells).

[0135] In this specification, a "vesicle" is defined as a type of supramolecular structure in which amphiphilic molecules (e.g., lipids) aggregate to define a volume, e.g., a substantially spherical volume. Amphiphilic molecules (e.g., lipids) usually constitute at least one shell of the vesicle. Within this shell, the amphiphilic molecules are arranged in a bilayer, with the hydrophilic portions of the amphiphilic molecules facing outward with respect to the plane of the bilayer and the hydrophobic portions of the amphiphilic molecules being mainly arranged within the bilayer. The reverse arrangement exists when the surrounding medium is hydrophobic. BRIEF DESCRIPTION OF THE DRAWINGS

[0136]

Figure 1

[0137] Mycobacteria are actinomycetes (e.g., Corynebacteriales or Propionibacteriales) characterized 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, peptidoglycan (PG), a plasma membrane, and cytoplasm. This complex cell envelope contributes to the durability of mycobacteria and, in particular, is difficult to penetrate and disrupt, necessitating effective treatment of mycobacterial infections.

[0138] Furthermore, these bacteria also contain a complex life cycle in which the bacteria exist in the cytoplasm, or within other intracellular 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 inside these intracellular compartments, the bacteria can replicate and proliferate. This is followed by membrane solubilization and release of the bacteria into the cytoplasm, where the bacteria continue to grow. Subsequently, the bacteria lyse the host cell and spread as free forms of the bacteria. Such free forms of the bacteria can appear in the spleen and liver, for example, after release from lung phagocytes, leading to spread of the infection and death.

[0139] Due to the complex life cycle, it is spatially and temporally difficult to target the bacteria at the appropriate locus and effectively treat the infection. Accordingly, there is a need for improved compositions and methods for treating bacterial infections, such as those caused by mycobacteria, that target intracellular bacterial infections. There is a need for improved compositions and methods for treating intracellular bacterial infections, such as those caused by mycobacteria.

[0140] The present invention solves this problem by a composition of the subject matter, and a method of using the same, which is rationally designed to target both the free-form phagosomal intracellular stage and the intracellular life cycle stage of the mycobacterial life cycle by degrading the mycobacterial envelope. The composition comprises a cocktail of unencapsulated antibacterial lytic proteins primed to kill bacterial cells both inside and outside of the host cell. The composition can further comprise a supramolecular structure (e.g., liposome) that targets a host cell, such as a macrophage or dendritic cell, and the correct target intracellular compartment (endosome, phagosome, lysosome, or cytosol) that targets the intracellular life cycle stage. The liposome directs the payload to the correct cell type and intracellular compartment, while the cocktail of antibacterial lytic proteins degrades the mycobacterial envelope. The free-form enzymes can degrade the envelope from the outside inwards, while the internalized supramolecular structure can degrade the envelope from the inside outwards and kill the bacteria.

[0141] The compositions described herein comprise a cocktail containing two or more of lysin A, lysin B, isoamylase, and α-amylase. Such combinations of lytic proteins are particularly advantageous in the killing of mycobacterial cells and related actinomycetes. To come up with the protein components, we first rationally attacked the three-layer mycobacterial envelope, the capsule, the junction between mycolic acid and the AGL layer, and the peptidoglycan layer. The components of the envelope at the basic structural level have been observed in many actinomycetes such as the order Corynebacteriales (e.g., mycobacteria), and the order Propionibacteriales such as Cutibacterium.

[0142] These thermostable complexes exhibit a robust anti-mycobacterial effect and can be used to treat infections caused by various mycobacteria and related actinomycetes (e.g., the order Corynebacteriales or Propionibacteriales) having a similar envelope structure.

[0143] Antibacterial lytic protein The present invention relates to a composition containing one or more (e.g., 1, 2, 3, or 4) of lysin A, lysin B, isoamylase, and α-amylase. The present invention also relates to a composition containing an unencapsulated protein containing two or more (e.g., 2, 3, or 4) of lysin A, lysin B, isoamylase, and α-amylase. The present invention also relates to a composition further comprising a supramolecular complex (e.g., liposome) containing one or more (e.g., 1, 2, 3, or 4) of lysin A, lysin B, isoamylase, and α-amylase. Suitable lytic proteins for incorporation into the compositions described herein are shown below in Table 1. These proteins exhibit, for example, improved expression, thermal stability, and antibacterial effects compared to other orthologs of these proteins.

[0144]

Table 1-1

[0145]

Table 1-2

[0146]

Table 1-3

[0147]

Table 1-4

[0148]

Table 1-5

[0149]

Table 1-6

[0150] The compositions described herein can include Lysin A comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. Lysin A can comprise, or consist of, the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0151] The compositions described herein can include Lysin B comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184. Lysin B can comprise, or consist of, the amino acid sequence of SEQ ID NO: 183 or SEQ ID NO: 184.

[0152] The compositions described herein can include isoamylase comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243. Isoamylase can comprise, or consist of, the amino acid sequence of SEQ ID NO: 242 or SEQ ID NO: 243.

[0153] The compositions described herein can include α-amylase comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with any one of SEQ ID NOs: 393 to 398. α-Amylase can comprise, or consist of, the amino acid sequence of any one of SEQ ID NOs: 393 to 398.

[0154] The compositions described herein can include α-amylase comprising an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with SEQ ID NO: 393 or SEQ ID NO: 394. α-Amylase can comprise, or consist of, the amino acid sequence of SEQ ID NO: 393 or SEQ ID NO: 394.

[0155] 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 α-amylase of SEQ ID NO: 393. In some embodiments, the composition comprises lysin A of SEQ ID NO: 2, lysin B of SEQ ID NO: 184, isoamylase of SEQ ID NO: 243, and α-amylase of SEQ ID NO: 394.

[0156] 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 α-amylase of SEQ ID NO: 395. In some embodiments, the composition comprises lysin A of SEQ ID NO: 2, lysin B of SEQ ID NO: 184, isoamylase of SEQ ID NO: 243, and α-amylase of SEQ ID NO: 396.

[0157] 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 α-amylase of SEQ ID NO: 397. In some embodiments, the composition comprises lysin A of SEQ ID NO: 2, lysin B of SEQ ID NO: 184, isoamylase of SEQ ID NO: 243, and α-amylase of SEQ ID NO: 398.

[0158] Additional sequences that may be useful in the compositions and methods described herein have been identified. In some embodiments, the composition comprises, as shown in Table 2 below, lysin A comprising any one of SEQ ID NOs: 1 to 182 and an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity.

[0159]

Table 2-1

[0160]

Table 2-2

[0161]

Table 2-3

[0162]

Table 2-4

[0163]

Table 2-5

[0164]

Table 2-6

[0165]

Table 2-7

[0166]

Table 2-8

[0167]

Table 2-9

[0168]

Table 2-10

[0169]

Table 2-11

[0170]

Table 2-12

[0171]

Table 2-13

[0172]

Table 2-14

[0173]

Table 2-15

[0174]

Table 2-16

[0175]

Table 2-17

[0176]

Table 2-18

[0177]

Table 2-19

[0178]

Table 2-20

[0179]

Table 2-21

[0180]

Table 2-22

[0181]

Table 2-23

[0182]

Table 2-24

[0183]

Table 2-25

[0184]

Table 2-26

[0185]

Table 2-27

[0186]

Table 2-28

[0187]

Table 2-29

[0188]

Table 2-30

[0189]

Table 2-31

[0190]

Table 2-32

[0191]

Table 2-33

[0192]

Table 2-34

[0193]

Table 2-35

[0194]

Table 2-36

[0195]

Table 2-37

[0196]

Table 2-38

[0197]

Table 2-39

[0198]

Table 2-40

[0199]

Table 2-41

[0200]

Table 2-42

[0201]

Table 2-43

[0202]

Table 2-44

[0203]

Table 2-45

[0204]

Table 2-46

[0205]

Table 2-47

[0206]

Table 2-48

[0207]

Table 2-49

[0208]

Table 2-50

[0209]

Table 2-51

[0210]

Table 2-52

[0211]

Table 2-53

[0212]

Table 2-54

[0213]

Table 2-55

[0214]

Table 2-56

[0215]

Table 2-57

[0216]

Table 2-58

[0217]

Table 2-59

[0218]

Table 2-60

[0219]

Table 2-61

[0220]

Table 2-62

[0221]

Table 2-63

[0222]

Table 2-64

[0223]

Table 2-65

[0224]

Table 2-66

[0225] Additional sequences that may be useful in the compositions and methods described herein were identified. In some embodiments, the composition comprises lysin B comprising any one of SEQ ID NOs: 183 to 241 and an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity.

[0226]

Table 3-1

[0227]

Table 3-2

[0228]

Table 3-3

[0229]

Table 3-4

[0230]

Table 3-5

[0231]

Table 3-6

[0232]

Table 3-7

[0233]

Table 3-8

[0234]

Table 3-9

[0235]

Table 3-10

[0236]

Table 3-11

[0237]

Table 3-12

[0238]

Table 3-13

[0239]

Table 3-14

[0240]

Table 3-15

[0241]

Table 3-16

[0242] Additional sequences that may be useful in the compositions and methods described herein have been identified. In some embodiments, the composition comprises an isoamylase comprising any one of SEQ ID NOs: 242 to 392 and an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity, as shown in Table 4 below.

[0243] [Table 4-1]

[0244] [Table 4-2]

[0245] [Table 4-3]

[0246] [Table 4-4]

[0247] [Table 4-5]

[0248] [Table 4-6]

[0249] [Table 4-7]

[0250] [Table 4-8]

[0251]

Table 4-9

[0252]

Table 4-10

[0253]

Table 4-11

[0254]

Table 4-12

[0255]

Table 4-13

[0256]

Table 4-14

[0257]

Table 4-15

[0258]

Table 4-16

[0259]

Table 4-17

[0260]

Table 4-18

[0261]

Table 4-19

[0262]

Table 4-20

[0263]

Table 4-21

[0264]

Table 4-22

[0265]

Table 4-23

[0266]

Table 4-24

[0267]

Table 4-25

[0268]

Table 4-26

[0269]

Table 4-27

[0270]

Table 4-28

[0271]

Table 4-29

[0272]

Table 4-30

[0273]

Table 4-31

[0274]

Table 4-32

[0275]

Table 4-33

[0276]

Table 4-34

[0277]

Table 4-35

[0278]

Table 4-36

[0279]

Table 4-37

[0280]

Table 4-38

[0281]

Table 4-39

[0282]

Table 4-40

[0283]

Table 4-41

[0284]

Table 4-42

[0285]

Table 4-43

[0286]

Table 4-44

[0287]

Table 4-45

[0288]

Table 4-46

[0289]

Table 4-47

[0290]

Table 4-48

[0291]

Table 4-49

[0292]

Table 4-50

[0293]

Table 4-51

[0294]

Table 4-52

[0295]

Table 4-53

[0296]

Table 4-54

[0297]

Table 4-55

[0298]

Table 4-56

[0299]

Table 4-57

[0300]

Table 4-58

[0301]

Table 4-59

[0302]

Table 4-60

[0303]

Table 4-61

[0304]

Table 4-62

[0305]

Table 4-63

[0306]

Table 4-64

[0307]

Table 4-65

[0308]

Table 4-66

[0309]

Table 4-67

[0310]

Table 4-68

[0311]

Table 4-69

[0312]

Table 4-70

[0313]

Table 4-71

[0314]

Table 4-72

[0315]

Table 4-73

[0316]

Table 4-74

[0317]

Table 4-75

[0318]

Table 4-76

[0319]

Table 4-77

[0320]

Table 4-78

[0321]

Table 4-79

[0322]

Table 4-80

[0323]

Table 4-81

[0324]

Table 4-82

[0325]

Table 4-83

[0326]

Table 4-84

[0327]

Table 4-85

[0328]

Table 4-86

[0329]

Table 4-87

[0330]

Table 4-88

[0331]

Table 4-89

[0332]

Table 4-90

[0333]

Table 4-91

[0334]

Table 4-92

[0335]

Table 4-93

[0336]

Table 4-94

[0337]

Table 4-95

[0338]

Table 4-96

[0339]

Table 4-97

[0340]

Table 4-98

[0341]

Table 4-99

[0342] Additional sequences that may be useful in the compositions and methods described herein have been identified. In some embodiments, the composition comprises an α - amylase comprising any one of SEQ ID NOs: 393 - 445 and an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity.

[0343]

Table 5 - 1

[0344]

Table 5 - 2

[0345]

Table 5 - 3

[0346]

Table 5 - 4

[0347]

Table 5 - 5

[0348]

Table 5 - 6

[0349]

Table 5 - 7

[0350]

Table 5 - 8

[0351]

Table 5 - 9

[0352]

Table 5-10

[0353]

Table 5-11

[0354]

Table 5-12

[0355]

Table 5-13

[0356]

Table 5-14

[0357]

Table 5-15

[0358]

Table 5-16

[0359]

Table 5-17

[0360]

Table 5-18

[0361]

Table 5-19

[0362]

Table 5-20

[0363]

Table 5-21

[0364]

Table 5-22

[0365]

Table 5-23

[0366] One skilled in the art will understand that the soluble proteins described herein can be produced recombinantly. Thus, the protein can contain a suitable purification tag, such as a His tag, containing, for example, 3, 4, 5, 6, 7, 8, 9, 10 or more histidine residues present at the N-terminus or C-terminus of the protein. The protein can also contain a removable signal sequence present at the N-terminus or C-terminus of the protein.

[0367] One skilled in the art will also understand that the soluble proteins described herein can be cleaved by, for example, 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 of their biologically active fragments, but still substantially retain their biological activity, and can include, for example, fragments of the soluble proteins described herein.

[0368] In some embodiments, one or more of the lysed proteins (e.g., unencapsulated proteins) are mannosylated. Such mannosylation allows the protein to be targeted to an intracellular destination, for example, within an antigen-presenting cell.

[0369] In some embodiments, the composition comprises a protein (e.g., lysin A, lysin B, isoamylase, and / or α-amylase) at a concentration of 0.1 mg / mL to 20 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, e.g., 10 mg / mL to 20 mg / mL, e.g., 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL). In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and / or α-amylase at a concentration of 1 mg / mL to 10 mg / mL.

[0370] Bacterial infection The compositions described herein are useful for treating bacterial infections. In some embodiments, the compositions described herein target bacteria that are extracellular for at least a portion of their life cycle. In some embodiments, the compositions described herein target bacteria that are intracellular for at least a portion of their life cycle. Intracellular bacteria inhabit host cells where they multiply and cause infection. Intracellular bacteria can inhabit immune cells such as professional antigen-presenting cells. Professional antigen-presenting cells (APCs) include macrophages, dendritic cells, and phagocytic cells such as macrophages. APCs process antigens complexed with major histocompatibility complex (MHC) on their surface and present them on their surface. APCs present antigens to MHC class 2 against bacterial pathogens, which are recognized by T cells, which then stimulate B cells having antibodies complementary to the antigens. This results in the proliferation of specific B cells encoding specific antibodies to fight organisms having the plateau. Certain bacteria avoid this immune response by hiding within immune cells.

[0371] Using the compositions and methods described herein, mycobacteria such as intracellular acid-fast bacteria that inhabit professional antigen-presenting cells (e.g., macrophages or dendritic cells) can be treated. In some embodiments, the mycobacterial species are M. tuberculosis, M. leprae, M. lepromatosis, M. avium, M. kansasii, M. fortuitum, M. chelonae, M. marinum, M. intracellulare, M. abscessus, M. chimera, M. boletti, M. fortuitum, M. goodii, or M. masiliense. In certain embodiments, the mycobacterium is NTM. In some embodiments, the NTM is M. abscessus, M. intracellulare, M. avium, M. chimera, M. boletti, M. fortuitum, M. goodii, and M. masiliense.

[0372] In some embodiments, the compositions and methods described herein can be used to target other actinomycetes (e.g., Corynebacteriales or Propionibacteriales) having envelope components similar to mycobacteria. For example, the compositions and methods can be used to target Nocardia, Corynebacterium, or Rhodococcus species. For example, Nocardia species can be, for example, N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. asteroids, N. brasiliensis, and N. caviae. Corynebacterium species can be, for example, C. glutamicum or C. diphtheriae. Rhodococcus species can be, for example, R. fascians or R. equi. The compositions and methods can be used to target Propionibacteriales such as Cutibacterium species. Cutibacterium species can be, for example, C. acnes.

[0373] Supramolecular structure Supramolecular structures can be used to formulate a cocktail of lytic enzymes for delivery. Supramolecular structures include complexes, such as defined complexes of lipids, that are bound to each other 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 complexes of molecules that form spherical, helical, or sheet-like structures. Examples of supramolecular structures include lipid-based supramolecular structures such as micelles, liposomes, and LNPs. Supramolecular structures can have a predetermined size. The size of the structure can vary depending on the size of the components packed within the structure, such as proteins. The supramolecular complex is endocytosed by cells, such as professional antigen-presenting cells like macrophages or dendritic cells, and the antibacterial lytic protein is delivered to the target intracellular compartment (endosome, phagosome, lysosome, or cytosol), where the bacteria reside.

[0374] In some embodiments, a specific particle size is used to direct the structure to a particular endocytosis pathway that targets an appropriate intracellular compartment of the cell. The supramolecular structure undergoes endocytosis and can be delivered to the target intracellular compartment, for example, via clathrin-dependent endocytosis or via caveolin-dependent endocytosis. The particle size of the supramolecular structure, e.g., the Z-average mean particle size, can vary from 75 nm to 5 μm, e.g., from 75 nm to 2 μm, from 75 nm to 1 μm, e.g., from 75 nm to 750 nm (e.g., from 250 nm to 750 nm, or from 75 nm to 250 nm). In some embodiments, when the supramolecular structure is an LNP or a micelle, the Z-average mean particle size is from 75 nm to 250 nm. In some embodiments, when the supramolecular structure is a vesicle (e.g., a liposome), the Z-average mean particle size is from 250 nm to 750 nm. Non-limiting examples of the Z-average mean particle size include, for example, from 75 nm to 100 nm, e.g., from 75 nm to 85 nm, e.g., 80 nm, e.g., from 80 nm to 140 nm, from 90 nm to 130 nm, or from 110 nm to 130 nm, e.g., 120 nm, e.g., from 200 nm to 300 nm, e.g., from 250 nm to 300 nm, from 260 nm to 290 nm, from 260 nm to 280 nm, from 265 nm to 275 nm, e.g., 270 nm, e.g., from 300 nm to 400 nm, from 400 nm to 600 nm, e.g., from 450 nm to 550 nm, from 475 nm to 525 nm, from 480 nm to 520 nm, from 490 nm to 510 nm, from 495 nm to 505 nm, e.g., 500 nm, e.g., 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, 250 nm, 255 nm, 260 nm, 265 nm, 270 nm, 275 nm, 280 nm, 285 nm, 290 nm, 295 nm, 300 nm, 305 nm, 310 nm, 315 nm, 320 nm, 325 nm, 330 nm, 335 nm, 340 nm,345 nm, 350 nm, 355 nm, 360 nm, 365 nm, 370 nm, 375 nm, 380 nm, 385 nm, 390 nm, 395 nm, 400 nm, 405 nm, 410 nm, 415 nm, 420 nm, 425 nm, 430 nm, 435 nm, 440 nm, 445 nm, 450 nm, 455 nm, 460 nm, 465 nm, 470 nm, 475 nm, 480 nm, 485 nm, 490 nm, 495 nm, 500 nm, 505 nm, 510 nm, 515 nm, 520 nm, 525 nm, 530 nm, 535 nm, 540 nm, 545 nm, 550 nm, 555 nm, 560 nm, 565 nm, 570 nm, 575 nm, 580 nm, 585 nm, 590 nm, 595 nm, 600 nm, 605 nm, 610 nm, 615 nm, 620 nm, 625 nm, 630 nm, 635 nm, 640 nm, 645 nm, 650 nm, 655 nm, 660 nm, 665 nm, 670 nm, 675 nm, 680 nm, 685 nm, 690 nm, 695 nm, 700 nm, 705 nm, 710 nm, 715 nm, 720 nm, 725 nm, 730 nm, 735 nm, 740 nm, 745 nm, 750 nm, 755 nm, 760 nm, 765 nm, 770 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 845 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm, 900 nm, 905 nm, 910 nm, 915 nm, 920 nm, 925 nm, 930 nm, 935 nm, 940 nm, 945 nm, 950 nm, 955 nm, 960 nm, 965 nm, 970 nm, 975 nm, 980 nm, 985 nm, 990 nm, 995 nm, 1 μm, 1.1 μm, 1.2 μm, 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 μmExamples include 4.7 μm, 4.8 μm, 4.9 μm, or 5 μm. In certain embodiments, the Z-average mean particle size of the supramolecular structure can be from about 5 nm to 250 nm. In some embodiments, the Z-average mean particle size of the supramolecular structure is 80 nm, 270 nm, or 500 nm.

[0375] The average particle size can be measured by zeta potential, dynamic light scattering (DLS), electrophoretic light scattering (ELS), static light scattering (SLS), molecular weight, electrophoretic mobility, size exclusion chromatography (SEC), field flow fractionation, or other methods known in the art. In certain embodiments, the average particle size is measured by. In certain embodiments, the supramolecular structure has a Z-average mean particle size of from 75 nm to 250 nm. In certain embodiments, the supramolecular structure has a Z-average mean particle size of from 250 nm to 750 nm. In certain embodiments, the supramolecular structure has a Z-average mean particle size of 500 nm. In certain embodiments, the supramolecular structure has a Z-average mean particle size of 270 nm. In certain embodiments, the supramolecular structure has a Z-average mean particle size of 80 nm. One of ordinary skill in the art will understand that an aggregate of supramolecular structures (e.g., liposomes, LNPs, or micelles) can have a range of Z-average mean particle sizes within the aggregate. Thus, the population can be polydisperse. The aggregate can have a polydispersity index of 0.5 or less, e.g., 0.3 or less (e.g., from 0.05 to 0.3). The polydispersity index can be measured using DLS (see, e.g., ISO 22412:2017).

[0376] The supramolecular structure can be loaded with a predetermined number, or an average number, of antibacterial lytic proteins per supramolecular structure. For example, the supramolecular structure can have from 1 protein to 10 6 proteins (e.g., from 1 to 10 5 proteins, from 1 to 10 4 proteins, from 1 to 10 3 proteins, from 1 to 10 2 proteins, from 1 to 10 proteins, from 10 to 10 6 proteins, from 10 to 105 , 10 to 10 4 pieces, 10 to 10 3 pieces, 10 to 10 2 pieces, 10 3 to 10 6 pieces, 10 3 to 10 5 pieces, 10 3 to 10 4 It can contain (pieces). The number of proteins per structure can depend on the size of the protein and the size of the structure.

[0377] The supramolecular construct can include an endosomal escape moiety. A supramolecular construct that includes an endosomal escape moiety can be provided for improved cytosolic delivery of a cargo (e.g., a therapeutic agent) included in the supramolecular construct. Endosomal escape moieties are known in the art. In some embodiments, the endosomal escape moiety is an ionizable lipid. The ionizable lipid can also serve as a supramolecular construct-forming lipid. Non-limiting examples of ionizable lipids include, for example, those described in WO2019 / 067875; WO2018 / 191750; and US9,999,671. Other exemplary endosomal escape moieties include membrane-fusogenic lipids (e.g., dioleoylphosphatidylethanolamine (DOPE)); and polymers such as polyethyleneimine (PEI); poly(β-amino esters); polypeptides such as polyarginine (e.g., octaarginine) and polylysine (e.g., octalysine); proton sponges, virus capsids, and peptide transduction domains described herein. For example, membrane-fusogenic peptides include the M2 protein of influenza A virus; peptide analogs of influenza virus hemagglutinin; the HEF protein of influenza C virus; transmembrane glycoproteins of filoviruses; transmembrane glycoproteins of rabies virus; transmembrane glycoprotein (G) of vesicular stomatitis virus; the fusion protein of Sendai virus; transmembrane glycoproteins 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 visna 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 facilitate endosomal escape are described in Dominska et al., Journal of Cell Science, 123(8):1183-1189, 2010.Specific examples of endosomal escape moieties that are included in or conjugated to the supramolecular structures disclosed herein and that are suitable for inclusion in or conjugation to such supramolecular structures are shown, for example, in WO 2015 / 188197, the disclosure of which is incorporated herein by reference.

[0378] Liposome Liposomes are useful for transporting and delivering antibacterial proteins to the site of action. Since the liposome membrane is structurally similar to biological membranes, when liposomes are applied to tissues, the liposome bilayer fuses with cell membranes, such as the bilayer of intracellular membranes. In some cases, prior to fusing with the biological membrane, liposomes are phagocytosed to form multilamellar vesicles, which can subsequently fuse with phagolysosomes, such as phagolysosomes containing mycobacteria. When liposome and phagolysosome trafficking coincide, the internal aqueous contents containing the antibacterial protein are delivered into the phagolysosome and the antibacterial lytic protein into the cell, where the antibacterial protein can target and lyse bacterial cells (e.g., mycobacterial cells, e.g., NTM cells) that inhabit mammalian immune cells. In some cases, liposomes can also be specifically targeted, for example, to direct proteins to specific mammalian immune cells and / or to specific intracellular compartments (endosomes, phagosomes, lysosomes, or cytosol) that normally harbor bacteria (e.g., mycobacteria) during infection. Liposome compositions typically are combinations of phospholipids, usually combined with steroids such as cholesterol. Other phospholipids or other lipids can also be used. The physical characteristics of liposomes are influenced by pH, ionic strength, and the presence of divalent cations.

[0379] In some embodiments, the liposomes described herein contain phospholipids. In some embodiments, glycerophospholipids, such as phosphatidylserine. Phosphatidylserine is a glycerol molecule having two hydroxyl groups substituted with fatty acid ester moieties and one hydroxyl group substituted with a phosphodiester moiety covalently bonded to a serine side chain. A typical structure of phosphatidylserine is RO-CH 2 -CH(OR)-CH 2 -OP(O)(OH)-OCH 2 CH(COOH)NH 2 , or a salt thereof [wherein each R is independently a fatty acid acyl]. Additionally, or alternatively, the liposomes described herein can include, for example, lysophospholipids, such as lysophosphatidylserine. Lysophosphatidylserine is phosphatidylserine lacking one of its two fatty acid ester moieties. A typical structure of lysophosphatidylserine is RO-CH 2 -CH(OR)-CH 2 -OP(O)(OH)-OCH 2 CH(COOH)NH 2 , or a salt thereof [wherein one R is a fatty acid acyl and the other R is H]. Thus, in certain preferred embodiments, the liposomes described herein are RO-CH 2 -CH(OR)-CH 2 -OP(O)(OH)-OCH 2 CH(COOH)NH 2 , or a salt thereof [wherein each R is H or a fatty acid acyl, provided that at least one R is a fatty acid acyl].

[0380] One of the main types of liposomal compositions includes phospholipids other than naturally occurring phosphatidylcholine. Neutral liposomal compositions can be formed, for example, from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Cationic liposomes have the advantage that they can fuse with cell membranes. Non-limiting examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-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-dioleyloxypropylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-s-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.(Cl), 1,2-dilinoleoyl-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyl-3-(2N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or an analog thereof, (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)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethane-1,2-diamine dodecane-2-ol (Tech G1), or a mixture thereof. The cationic lipid can account for, for example, 20 mol% to 50 mol%, or about 40 mol% of the total lipid present in the particles.

[0381] Non-cationic liposomes can be taken up in vivo by macrophages and can deliver antibacterial lytic proteins to macrophages. Anionic liposome compositions can be formed from dimyristoyl phosphatidylglycerol, while anionic membrane-fusogenic liposomes can mainly be formed from dioleoyl phosphatidylethanolamine (DOPE). Lipids can be dipalmitoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans-PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt, DOPS), or mixtures thereof, and can be anionic or neutral lipids. When cholesterol is included, the non-cationic lipid can be, for example, 5 mol% to 90 mol%, 10 mol% to 58 mol% of the total lipids present in the particles. In some embodiments, the lipid can be a combination of the lipids described above, for example, a combination of lipids including DOPC, DOPS, Chol, and DOPE.

[0382] In some embodiments, the liposome comprises a mixture of lipids. For example, the mixture of lipids can comprise two or more of DOPC, DOPE, DOPS, and cholesterol.

[0383] 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).

[0384] 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).

[0385] 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).

[0386] 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).

[0387] 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).

[0388] 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).

[0389] 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.

[0390] In some embodiments, the liposome contains lipids at a concentration of 0.03 mg / mL to 10 mg / mL, e.g., 0.1 mg / mL to 10 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL). In some embodiments, the liposome contains lipids at a concentration of 1 mg / mL to 5 mg / mL.

[0391] Another type of liposomal composition is formed from phosphatidylcholine (PC), such as soy PC and egg PC, for example. 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. Patent Nos. 5,283,185; 5,171,678; WO94 / 00569; WO93 / 24640; WO91 / 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.

[0392] Targeting of liposomes is also possible, for example, based on organ specificity, cell specificity, and organelle specificity, and is known in the art. In the case of a delivery system targeted by liposomes, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting of the ligand while stably associating with the liposomal bilayer. Various linking groups can be used to attach the lipid chain to the target ligand. Further methods are known in the art and are described, for example, in U.S. Patent Publication No. 20060058255, the contents of which are incorporated herein by reference.

[0393] Cleavable linking groups can be susceptible to the influence of cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are more effective or found at higher levels or activities intracellularly than in serum or blood. Examples of such degradative agents include, for example, oxidative or reductive enzymes such as mercaptans present in cells that can degrade redox-cleavable linking groups by reduction, or reducing agents; esterases; endosomes or agents that can create an acidic environment, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade cleavable acid linking groups by acting as general acids; peptidases (which can be substrate-specific); and redox agents that are selective for a particular substrate or have no substrate specificity, including phosphatases.

[0394] Cleavable linking groups, such as disulfide bonds, can potentially be susceptible to the influence of 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 of 5.5 - 6.0, and lysosomes have a more acidic pH of approximately 5.0. Some linkers have cleavable linking groups that are cleaved at a preferred pH to release cationic lipids from ligands into the cell or into a desired compartment of the cell.

[0395] The linker can include a cleavable linking group that is cleavable by a specific enzyme. The type of cleavable linking group incorporated into the linker can be influenced by the target cell. Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It may also be desirable to test the ability of a candidate cleavable linking group to resist cleavage when in the blood or when in contact with other non-target tissues. Thus, the relative sensitivity to cleavage under a first condition and a second condition can be measured, where the first condition is selected to indicate cleavage in the target cell and the second condition is selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. The evaluation can be performed in a cell-free system, cells, cell culture, organ or tissue culture, or whole animals. An initial evaluation can be performed under cell-free or culture conditions and can be usefully confirmed by further evaluation in whole animals. In a preferred embodiment, a linker that is a useful candidate is cleaved at least 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0396] Lipid nanoparticles The antibacterial agent of the present invention can be completely encapsulated in a lipid formulation, such as lipid nanoparticles (LNP). LNP shows an extended circulation life after intravenous (i.v.) injection and accumulates at distal sites (e.g., sites physically separated from the administration site), making it extremely useful for systemic administration. LNP contains "pSPLP", which includes an encapsulated condensing agent-nucleic acid complex as described in PCT Publication No. WO2000 / 003683. The particles of the present invention typically have an average diameter of 50 nm to 150 nm, more typically 60 nm to 130 nm, more typically 70 nm to 110 nm, and most typically 70 nm to 90 nm, and are substantially non-toxic. Further, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010 / 0324120, and PCT Publication No. WO 96 / 40964.

[0397] In one embodiment, the lipid:drug ratio (mass / mass ratio) (e.g., lipid:peptide ratio) ranges from 1:1 to 50:1, 1:1 to 25:1, 3:1 to 15:1, 4:1 to 10:1, 5:1 to 9:1, or 6:1 to 9:1. Ranges intermediate to the ranges listed above are also contemplated as part of the present invention.

[0398] Non-limiting examples of cationic lipids include DODAC, DDAB, DOTAP, DOTMA, DODMA, DLinDMA, DLenDMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl, 1DLin-MPZ, DLinAP, DOAP, DLin-EG-DMA, DLin-K-DMA or analogs thereof, ALN100, MC3, Tech G1, or mixtures thereof. The cationic lipid can, for example, account for 20 mol% to 50 mol%, or about 40 mol% of the total lipids present in the particles.

[0399] The lipid can be an anionic lipid or a 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 mixtures thereof. When cholesterol is included, the non-cationic lipid can be, for example, 5 mol% to 90 mol%, or 10 mol% to 60 mol% of the total lipids present in the particle.

[0400] The conjugate lipid that inhibits particle aggregation can be a polyethylene glycol (PEG)-lipid, including but not limited to, for example, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate can be, for example, PEG-dilauroxypropyl (C 12 ), PEG-dimyristyloxypropyl (C 14 ), PEG-dipalmityloxypropyl (C 16 ), or PEG-distearyloxypropyl (C 18 ). The complex lipid that prevents particle aggregation can be, for example, 0 mol% to 20 mol%, or about 2 mol% of the total lipids present in the particle.

[0401] In some embodiments, the LNP further comprises cholesterol, for example, 10 mol% to 60 mol%, or 50 mol% of the total lipids present in the particle. In some embodiments, the LNP comprises a mixture of lipids. For example, the mixture of lipids can comprise two or more of DOPC, DOPE, DOPS, and cholesterol.

[0402] 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).

[0403] 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).

[0404] 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).

[0405] 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).

[0406] 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).

[0407] 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).

[0408] 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.

[0409] In some embodiments, the LNP contains lipids at a concentration of 0.03 mg / mL to 10 mg / mL, such as 0.1 mg / mL to 10 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL). In some embodiments, the LNP contains lipids at a concentration of 1 mg / mL to 5 mg / mL.

[0410] Micelle A micelle is a particular type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that the hydrophobic parts of the molecules all face inward and the hydrophilic parts remain in contact with the surrounding aqueous phase. Micelles can be made from lipids. The micellar phase is caused by confining the behavior of single-tailed lipids in the bilayer phase. The hydration of the lipid headgroups results in the difficulty of filling the entire volume inside the bilayer while accommodating a region for the headgroups acting on the molecule, which leads to the formation of micelles. This type of micelle is known as a normal-phase micelle (oil-in-water micelle). Inverse micelles have the headgroups in the center and the tails extending outward (water-in-oil micelles).

[0411] Micelles are approximately spherical in shape. Other shapes including ellipsoids, cylinders, and bilayers are also possible. The shape and size of micelles are a function of the molecular geometry of the surfactant molecules and the conditions of the solution such as surfactant concentration, temperature, pH, and ionic strength. The process of micelle formation is known as micellization and, due to its polymorphism, forms part of the phase behavior of many lipids.

[0412] Target moiety The supramolecular structures described herein can include, for example, a target moiety. The target moiety can be used to direct the supramolecular structure to a specific cell type (e.g., professional antigen-presenting cells such as macrophages or dendritic cells). A particular lipid (e.g., phosphatidylserine) can be used within the supramolecular structure (e.g., vesicles) both as a supramolecular structure-forming lipid and as a target moiety. The target moiety can be, for example, an antibody or antigen-binding fragment or engineered derivative thereof (e.g., Fcab or fusion protein (e.g., scFv)). The target moiety can be, for example, a polypeptide. Alternatively, the target moiety can be, for example, a small molecule (e.g., mannose or folate), or a cluster of small molecules (e.g., a cluster of mannose). The target moiety can associate with the supramolecular structure either covalently or non-covalently.

[0413] Small molecule The target moiety can be a small molecule capable of complexing with a receptor expressed on the surface of the target cell. Non-limiting examples of small molecules that can be used as a target moiety in the supramolecular structures described herein are phosphatidylserine, folate lysophosphatidylserine, mannose, and mannose clusters.

[0414] In some embodiments, the target moiety is phosphatidylserine or lysophosphatidylserine. In some embodiments, the target moiety is phosphatidylserine. Phosphatidylserine and / or lysophosphatidylserine can be present as a supramolecular structure-forming lipid non-covalently bound to the rest of the supramolecular structure.

[0415] Folate can be used as a target moiety. In the supramolecular structures described herein, folate can assume the following structure:

[0416]

Chemical formula

[0417] Mannose or a mannose cluster can be used to direct the supramolecular structures described herein to dendritic cells and macrophages. Mannose clusters are known in the art.

[0418] Folate, mannose, and mannose clusters can be covalently attached to the supramolecular structure. Conjugation techniques for attaching folate, mannose, and mannose clusters are known in the art, as described, for example, in US2014 / 0045919, US9,725,479, US8,758,810, US8,450,467, US6,525,031, US6,335,434, and US5,759,572.

[0419] Antigen-binding moiety The antigen-binding moiety in the supramolecular structures described herein can be an antibody or an antigen-binding fragment thereof, such as F(ab) 2 or Fab, or a recombinant derivative thereof, such as Fcab or a fusion protein, such as scFv. Human or chimeric, such as humanized, antibodies can be used as antibodies within the supramolecular structures described herein.

[0420] The antigen-binding portion targets APCs having a surface antigen 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 antigen-binding fragments or recombinant derivatives thereof. Macrophages can be targeted by anti-CD163, anti-CD40, anti-CD74, anti-CD206, or anti-CD123, or antigen-binding fragments or recombinant derivatives thereof.

[0421] Non-limiting examples of anti-CD38 antibodies are daratumumab, SAR650984, MOR202, or any one of antibodies Ab79, Ab19, Ab43, Ab72, and Ab110 disclosed in WO2012 / 092616, the disclosures of these antibodies being incorporated herein by reference. A non-limiting example of an anti-CD79b antibody is huMA79b v28 disclosed in WO2014 / 011521. A non-limiting example of an anti-CD22 antibody is 10F4 disclosed in US2014 / 0127197. A non-limiting example of an anti-CD20 antibody is rituximab. A non-limiting example of an anti-DEC205 antibody is shown in US2010 / 0098704, the antibody being incorporated herein by reference. Non-limiting examples of anti-CD40 antibodies are lucatumumab and dacetuzumab. A non-limiting example of an anti-CD304 antibody is besencumab.

[0422] Conjugation techniques for binding the antigen-binding portion are known in the art, for example, as described in Ansell et al., Methods Mol. Med., 25:51-68, 2000; US2002 / 0025313; US6,379,699; and US5,059,421.

[0423] Polypeptide The targeting moiety can be a polypeptide having an affinity for cells (e.g., having an affinity for a cell type, e.g., dendritic cells). Non-limiting examples of polypeptides are RGD peptides, rabies virus, rabies virus glycoprotein (RVG), and DC3 peptides. Alternatively, the polypeptide can be a TLR2 agonist, such as MALP-2 lipoprotein, MALP-404 lipoprotein, OspA, polyinosine, LcrV, Hsp60, glycoprotein gH / gL, or glycoprotein gB.

[0424] Conjugation techniques for binding peptides are known in the art, such as those described in Ansell et al., Methods Mol. Med., 25:51-68, 2000; US2002 / 0025313; US6,379,699; and US5,059,421.

[0425] PAMP The targeting moiety can be a PAMP. PAMPs are known in the art and include, for example, CpG ODNs. CpG ODNs are generally divided into three classes: class A, class B, and class C. Class A CpG ODNs typically contain a poly-G tail with a phosphorothioate backbone at the 3' and 5' ends and a central palindromic sequence containing a phosphate backbone. Class A CpG ODNs usually contain CpG within the central palindromic sequence. Class B CpG ODNs generally contain a fully phosphorothioated backbone, and the sequence at the 5' end of class B CpG ODNs is often important for TLR9 activation. Class C CpG ODNs contain a fully phosphorothioated backbone, and the 3' end sequence allows for duplex formation. PAMPs can be covalently attached to the supramolecular structure using techniques and methods known in the art.

[0426] Assembly method The present invention relates to a supramolecular structure comprising a plurality of enzymes packaged therein, such as a lipid-based supramolecular structure (e.g., liposome). An assembly method for producing a structure (e.g., liposome) containing enzymes (e.g., lysin A, lysin B, isoamylase, and / or α-amylase having at least 85% sequence identity with the sequences in Table 1) is described herein. The individual proteins can be overexpressed in any suitable recombinant expression system (e.g., E. coli) and extracted from the cells by cell lysis. In some embodiments, the crude extract from the cells can be purified, for example, by column chromatography. After purification, the component concentration of the enzyme can be standardized, for example, for subsequent 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, e.g., 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL). In some embodiments, the concentration can be standardized at 0.33 mg / mL.

[0427] Next, the lipid can be mixed with an enzyme for formulating liposomes. For example, a lipid at a total concentration of 0.1 mg / mL to 10 mg / mL (e.g., 0.1 mg / mL to 1 mg / mL, e.g., 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, e.g., 1 mg / mL to 10 mg / mL, e.g., 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL, e.g., 1 mg / mL to 5 mg / mL) can be mixed with a His-tagged protein at 1 mg / mL to 10 mg / mL. The lipid can include, for example, one or more of DOPC, DOPE, DOPS, and cholesterol. In some embodiments, the lipid includes DOPC, DOPE, DOPS, and cholesterol in a ratio of 10:10:3:4. The lipid is resuspended in a suitable organic solvent (e.g., ethanol) and mixed with the protein (e.g., at an aqueous:organic ratio of 20:1 to 1:1, e.g., 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1, e.g., 3:1 to 10:1, e.g., 3:1 to 8:1). The mixture can be mixed at a flow rate of 1 mL / min to 30 mL / min, e.g., 15 mL / min, using a suitable system such as the NANOASSEMBLR® IGNITE™ system (Precision Nanosystems). The organic layer can be removed by diluting and dialyzing, for example, against an excess (e.g., 1000-fold) volume of formulation buffer for at least 30 minutes (e.g., 1 hour) at a suitable temperature such as room temperature. Next, the liposomes can be analyzed by gel electrophoresis (e.g., SDS-PAGE), dynamic light scattering, intrinsic fluorescence, and / or static light scattering, and the relevant activities can be tested. Such assays can help confirm the encapsulation of the enzyme as well as the purity of the enzyme and liposomes.

[0428] A composition containing a supramolecular structure (e.g., liposome) can be formulated with one or more excipients. For example, the composition (e.g., a supramolecular structure, e.g., a liposome containing a cocktail of lysozyme) can be encapsulated and / or formulated in a buffer such as glycine, Tris, sodium citrate, sodium acetate, and MES at a concentration of, for example, 10 mM to 200 mM, for example, 50 mM to 150 mM, for example, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM. The composition can be formulated at a pH of 5 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., 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11). The composition can further contain one or more excipients such as CaCl 2 , arginine, NaCl, sodium citrate, MgCl 2 , or glycerol. In some embodiments, the composition contains, for example, 5 mM to 10 mM CaCl 2 , 0 to 50 mM arginine, 0 to 200 mM NaCl, 0 to 1 mM sodium citrate, 0 nM to 1 mM MgCl 2 , and / or 10 to 30% glycerol. In some embodiments, the composition contains 50 mM glycine (pH 8.5), 7.5 mM CaCl 2 , 0.5 mM MgCl 2 , 200 mM NaCl, 0.33 mM sodium citrate, and 10% glycerol. The formulation can further contain Tween, e.g., Tween-80. If the compositions are placed in a preferred storage or active buffer, they can be used in therapy or for assays, or stored for later use.

[0429] Treatment method The antibacterial lytic proteins described herein are preferably formulated into pharmaceutical compositions for administration to a human subject for treating a disease or condition, such as a bacterial infection (e.g., an actinomycete infection, e.g., an infection by bacteria of the order Corynebacteriales or Propionibacteriales), such as a mycobacterial infection, such as an NTM infection. In particular, the compositions and methods described herein are useful for treating bacterial infections caused by actinomycetes, such as Corynebacterium and Propionibacteriales, due to their similar envelope structures. Bacterial infections can occur in an otherwise healthy subject in other cases. Alternatively, bacterial infections can occur in a subject having another co-existing disease or disorder. For example, a subject with a weakened immune system may be more susceptible to bacterial infections.

[0430] Mycobacterial infections caused by NTM are bacteria that are normally present in the environment. By inhaling these bacteria, disease can be caused in both healthy patients and patients with a compromised immune system. NTM diseases most often affect the lungs in adults, but can also affect any part of the body. Some subjects are at high risk of developing NTM infections and the disease progressing. People with existing lung diseases, such as bronchiectasis (enlargement of the airways), chronic obstructive pulmonary disease (COPD), cystic fibrosis, alpha-1 antitrypsin deficiency, or those who have previously had an infection such as tuberculosis, have an increased risk of pulmonary NTM disease. In subjects with advanced HIV infection (CD4 <50) or an immune-related genetic disease (e.g., interferon-gamma deficiency or receptor deficiency, interleukin-12 deficiency), lung disease can progress as part of a disseminated (e.g., widespread throughout the body) NTM infection. The subject to be treated may have any of the aforementioned indications, in addition to, for example, a bacterial infection.

[0431] Using the methods, compositions, and methods described herein, the levels of infectious diseases can be reduced. For example, the method can reduce the level of an infectious disease (e.g., the number of bacteria or the size of the infection) compared to a reference. For example, the infectious disease 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%.

[0432] Pharmaceutical composition The antibacterial agents described herein are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for in vivo administration.

[0433] As will be understood by those skilled in the art, the compositions described herein can be administered to a subject in various forms depending on the selected route of administration. The compositions described herein can be administered, for example, by any route that allows the composition (e.g., an unencapsulated mixture of enzymes and / or supramolecular structures such as liposomes, micelles, or LNPs) to reach the target cells. The compositions can be administered, for example, orally, parenterally, intrathecally, intraventricularly, intracorporally, buccally, sublingually, nasally, rectally, topically, by pump, or transdermally, and the pharmaceutical compositions are formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, trans-epithelial, nasal, pulmonary, intrathecal, intraventricular, intracorporal, rectal, and topical administration methods. In one embodiment, the composition is administered by aerosol. Parenteral administration can be by continuous infusion over a selected period. In some preferred embodiments, the compositions described herein are administered by inhalation.

[0434] The administration of two or more antibacterial agents can be by the same route or by different routes and can occur continuously or almost simultaneously. For example, the first antibacterial agent in combination can be administered by intravenous injection while the second therapeutic agent in the combination can be administered orally.

[0435] Certain compositions described herein can be administered, for example, by inhalation. The inhalation can be oral inhalation or nasal inhalation. The inhalable compositions described herein can be provided as a liquid dosage form or a dry powder dosage form. The dry powder composition can be administered by inhalation, for example, as is or after reconstitution with a vehicle (e.g., physiological saline (e.g., isotonic saline), phosphate buffered saline, or water).

[0436] The inhalable dry powder dosage form can be prepared from the liquid compositions described herein by drying (e.g., by lyophilization, spray drying, spray freeze drying, or supercritical fluid technology). The inhalable dry powder dosage form described herein can include a carrier (e.g., lactose, sucrose, mannitol, etc.), a cryoprotectant (e.g., trehalose, mannitol, etc.), and / or an anti-adherent agent (e.g., glycine, L-leucine, serine, etc.). The inhalable dry powder dosage form described herein can be administered using a dry powder inhaler. Dry powder inhalers are known in the art and may or may not contain a propellant. Non-limiting examples of dry powder inhalers can be found in Newman, Expert Opin. Biol. Ther., 4:23-33, 2004, the entire disclosure of which is incorporated herein by reference.

[0437] The inhalable liquid dosage forms (e.g., aerosol formulations) described herein can be prepared using techniques and methods useful in the preparation of liquid compositions containing unencapsulated enzymes and / or supramolecular structures. Inhalable liquid dosage forms typically contain a suspension of the enzymes and / or supramolecular structures described herein in a physiologically acceptable aqueous or non-aqueous solvent, and are typically present in a sealed container in a sterile form, in single or multiple doses, which can take the form of a cartridge or refill for use with a nebulizer device. Alternatively, the sealed container can be an integrated dispensing device, such as a single-dose nasal inhaler, or an aerosol dispenser fitted with a throttle valve for purposes of being discarded after use. When the dosage form contains an aerosol dispenser, the dosage form contains a propellant, which can be a compressed gas, such as compressed air, or an organic propellant, such as a hydrofluoroalkane. Inhalable liquid dosage forms can be administered using a nebulizer. The process of converting bulk liquid into droplets by compressed air is called atomization. The operation of a gas nebulizer requires a propellant as a driving force for liquid atomization. Various types of nebulizers are described in Respiratory Care, 45:609-622, 2000, the entire disclosure of which is incorporated herein by reference. Alternatively, the inhalable liquid dosage forms described herein can be administered using a metered-dose inhaler. Metered-dose inhalers are known in the art and typically include a canister, an actuator, and a throttle valve.

[0438] The compositions described in the present invention can be administered orally, for example, with an inert diluent or an assimilable edible carrier, or can be encapsulated in hard or soft shell gelatin capsules, or can be compressed into tablets, or can be incorporated directly into foodstuffs for diet. In the case of oral therapeutic administration, the compositions described herein can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. The compositions described herein can also be administered parenterally. The compositions described herein can also be administered by micro-needle injection. Solutions of the compositions described herein can be prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, DMSO, and their mixtures with or without alcohol, and in oils. These formulations may contain preservatives to prevent the growth of microorganisms under normal storage and use conditions. Conventional procedures and ingredients for selecting and preparing suitable formulations are described, for example, in Remington’s Pharmaceutical Sciences (2012, 22nd ed.), and The United States Pharmacopeia: The National Formulary (USP 41 NF 36) issued in 2018. Pharmaceutical forms suitable for injection use include sterile aqueous solutions or sterile dispersions, and sterile powders for the immediate preparation of injectable sterile solutions or sterile dispersions. In all cases, this form must be sterile and must be fluid to the extent that it can be easily administered by syringe. Compositions suitable for buccal or sublingual administration include tablets, lozenges, and troches, where the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, gelatin, and glycerin. Compositions for rectal administration conveniently take the form of suppositories containing conventional suppository bases such as cocoa butter.

[0439] In some embodiments, the compositions described herein are formulated with one or more excipients. For example, the composition (e.g., an unencapsulated enzyme or supramolecular structure, e.g., a liposome containing a cocktail of lysozymes) can be encapsulated and / or formulated in a buffer such as glycine, tris, sodium citrate, sodium acetate, and MES at a concentration of, for example, 10 mM to 200 mM, for example, 50 mM to 150 mM, for example, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM. The composition can be formulated at a pH of 5 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 can further comprise one or more excipients such as CaCl 2 , arginine, NaCl, sodium citrate, MgCl 2 , or glycerol. In some embodiments, the composition comprises, for example, 5 mM to 10 mM CaCl 2 , 0 to 50 mM arginine, 0 to 200 mM NaCl, 0 to 1 mM sodium citrate, 0 to 1 mM MgCl 2 , and / or 10 to 30% glycerol. In some embodiments, the composition comprises 50 mM glycine (pH 8.5), 7.5 mM CaCl 2 , 0.5 mM MgCl 2 , 200 mM NaCl, 0.33 mM sodium citrate, and 10% glycerol. The formulation can further comprise Tween, e.g., Tween-80.

[0440] The compositions described herein can be administered to an animal, e.g., a human, alone or in combination with a pharmaceutically acceptable carrier, as described herein, in a proportion determined by the solubility and chemical nature of the composition, the selected route of administration, and standard pharmaceutical practice.

[0441] The dosage of a composition, e.g., a composition comprising a lysed protein as described herein, may depend on many factors, such as the pharmacodynamic properties of the antibacterial lysed protein, the mode of administration, the age, health status, and weight of the subject to be treated, the nature and extent of the symptoms, the frequency of treatment, and the type of co - treatment (if any), as well as the clearance rate of the composition in the animal being treated. The compositions described herein can initially be administered at a suitable dosage that can be adjusted as needed according to the clinical response. In some embodiments, the dosage of a composition, e.g., a composition comprising a lysed protein, is an amount effective for prophylaxis or treatment. Further, it is understood that all dosages can be administered continuously or divided into dosages given at intervals within a given time frame. The composition can be administered, for example, hourly, daily, weekly, monthly, or annually. In some embodiments, the composition is administered continuously or systemically.

[0442] Combination therapy The compositions described herein can be administered as part of a combination therapy. Combination therapy means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. For example, the first therapeutic agent can comprise a cocktail of unencapsulated proteins while the second therapeutic agent can comprise a supramolecular structure containing an encapsulated protein. The treatment regimen defines the dosage and dosing period of each agent such that the effects of the individual agents on the subject overlap. In some embodiments, the delivery of two or more agents is made simultaneously or in parallel, and the agents may be prepared as a mixture. In some embodiments, two or more agents are not co - formulated and are administered sequentially as part of the prescribed regimen. The sequential or substantially simultaneous administration of each therapeutic agent can be by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes. For example, the first therapeutic agent in the combination may be administered by intravenous injection or by aerosolization, while the second therapeutic agent in the combination may be administered orally.

[0443] In some embodiments, for example, unencapsulated lysin A, lysin B, isoamylase, and / or α - amylase are administered together. Alternatively, lysin A, lysin B, isoamylase, and / or α - amylase can be administered at different times.

[0444] In some embodiments, for example, unencapsulated lysin A, lysin B, isoamylase, and / or α - amylase having at least 85% sequence identity with the sequences in Table 1 are administered together. Alternatively, lysin A, lysin B, isoamylase, and / or α - amylase can be administered at different times.

[0445] In some embodiments, a first composition containing unencapsulated lysin A, lysin B, isoamylase, and / or α - amylase is administered as combination therapy with a supramolecular structure (e.g., liposome) containing one or more of lysin A, lysin B, isoamylase, and / or α - amylase.

[0446] In any of the combination embodiments described herein, the first therapeutic agent and the second therapeutic agent can be administered sequentially, either simultaneously or in either order. The first therapeutic agent (e.g., a composition containing an unencapsulated protein) can be administered immediately, up to 15 minutes, up to 30 minutes, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, up to 14 hours, up to 16 hours, up to 17 hours, up to 18 hours, up to 19 hours, up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours, or 1 - 7, 1 - 14, 1 - 21, or 1 - 30 days before or after the second therapeutic agent (e.g., a supramolecular structure containing an encapsulated protein).

[0447] The pharmaceutical compositions described herein can further comprise an additional antibacterial agent that is administered with a supramolecular structure comprising an antibacterial lytic protein. The compositions and methods described herein can further comprise treatment of an underlying lung condition that can be exacerbated by, for example, a bacterial infection, such as an NTM infection. Suitable lung therapies include, but are not limited to, airway clearance, nebulizers, breathing masks, and inhalers, such as steroid inhalers.

[0448] Antibiotics The additional antibacterial agent can 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, cephapirin, cephradine, cephaloridine, cefazolin, cefamandole, cefuroxime, cephalexin, cefprozil, cefaclor, loracarbef, cefoxitin, cefmetazole, cefotaxime, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefixime, cefpodoxime, cefibuten, cefdinir, cefepime, chlorhexidine, BAL5788, BAL9141, imipenem, ertapenem, meropenem, aztreonam, clavulanic acid, sulbactam, tazobactam, streptomycin, neomycin, kanamycin, puromycin, gentamicin, tobramycin, amikacin, netilmicin, spectinomycin, sisomicin, dibekacin, isepamicin, tetracycline, chlortetracycline, demeclocycline, minocycline, oxytetracycline, methacycline, doxycycline, erythromycin, azithromycin, clarithromycin, telithromycin, ABT-773, lincomycin, clindamycin, vancomycin, oritavancin, dalbavancin, teicoplanin, quinupristin and dalfopristin, sulfanilamide, p-aminobenzoic acid, sulfadiazine, sulfisoxazole, sulfamethoxazole, sulfathalidine, linezolid, nalidixic acid, oxolinic acid, norfloxacin, pefloxacin, enoxacin, ofloxacin, ciprofloxacin, temafloxacin, lomefloxacin, fleroxacin, grepafloxacin, sparfloxacin, trovafloxacin, clinafloxacin, gatifloxacin, moxifloxacin, gemifloxacin, sitafloxacin, metronidazole, daptomycin, galenoxacin, ramoplanin, faropenem, polymyxin, tigecycline, AZD2563, trimethoprim, ethambutol, rifamycin, and rifampin.In some embodiments, a plurality of antibiotics are administered with the compositions described herein. In some embodiments, the antibiotics are cephalosporin, carbapenem (e.g., biapenem), penicillin, macrolide, aminoglycoside, or fluoroquinolone. In some embodiments, the antibiotics are selected from the group consisting of thiacetazone, sq-109, bedaquiline, delamanid, pyrazinamide, and isoniazid.

[0449] In some embodiments, the antibiotics are macrolides (e.g., azithromycin, clarithromycin, erythromycin). In some embodiments, the antibiotics are aminoglycosides (e.g., kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin (e.g., neomycin B, C, or E), streptomycin, or plazomycin).

[0450] Advantageously, in some embodiments, due to the synergistic effect with co-administered therapeutic agents, administration of the antibiotic at a dose below the therapeutic dose may be possible when administered without other therapeutic agents.

[0451] The antibiotics can be formulated with supramolecular structures containing bacteriophages and antibacterial lytic proteins. The antibiotics can be administered as individual pharmaceutical compositions. The antibiotics can be administered at a different time from the pharmaceutical composition containing the supramolecular structure having phages. In some preferred embodiments, the additional antibiotic is amikacin. Amikacin can be, for example, liposomal amikacin formulated for inhalation.

[0452] Examples The following examples are intended to illustrate the present invention. The examples do not limit the present invention in any way.

[0453] Example 1. Treatment of macrophages infected with M. abscessus FIG. 1 is a graph showing serial dilutions of M. abscessus from infected macrophages treated with either free lysin A, lysin B, isoamylase, and α - amylase (ABIα), or liposomes containing ABIα. J774A.1 mouse macrophages were infected with Mycobacterium abscessus (MOI = 10:1). The growth length after extraction from the infected macrophages was 120 hours. A single administration could have an effect exceeding 100 - fold compared to untreated cells.

[0454] Other embodiments All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. If it is found that the terms of this application are defined differently from the documents incorporated by reference herein, the definitions provided herein shall serve as the definitions of those terms.

[0455] Although the invention has been described in connection with its specific embodiments, the invention is capable of further modification, and this application generally covers any variations, uses, or adaptations of the invention that follow the principles of the invention and include departures from the disclosure that become known in the art to which the invention pertains or are customarily practiced, and are applicable to the essential features described above and are understood to follow the scope of the claims.

Claims

1. A composition containing an unencapsulated protein, wherein the unencapsulated protein is (a)lysin A; (b)lysin B; (c)isoamylase; and (d)α-amylase and contains two or more of the above, said composition.

2. The unencapsulated protein is (a)lysin A; and (b)lysin B and contains the composition according to claim 1.

3. The unencapsulated protein is (a)lysin A; and (b)isoamylase and contains the composition according to claim 1.

4. The unencapsulated protein is (a)lysin A; and (b)α-amylase and contains the composition according to claim 1.

5. The unencapsulated protein is (a)lysin B; and (b)isoamylase and contains the composition according to claim 1.

6. The unencapsulated protein is (a)lysin B; and (b)α-amylase and contains the composition according to claim 1.

7. The unencapsulated protein is (a)isoamylase; and (b)α-amylase and contains the composition according to claim 1.

8. The unencapsulated protein is (a)lysin A; (b)lysin B; (c)isoamylase; and (d)α-amylase and contains three or more of the above, said composition according to claim 1.

9. The unencapsulated protein is (a)lysin A; (b)lysin B; and (c)isoamylase and contains the composition according to claim 8.

10. The unencapsulated protein is (a)lysin A; (b)lysin B; and (c)α-amylase and contains the composition according to claim 8.

11. The unencapsulated protein is (a)lysin A; (b)isoamylase; and (c)α-amylase and contains the composition according to claim 8.

12. The unencapsulated protein is (a)lysin B; (b)isoamylase; and (c)α-amylase and contains the composition according to claim 8.

13. The unencapsulated protein is (a)lysin A; (b)lysin B; (c)isoamylase; and (d)α-amylase and contains all four of the above, said composition according to claim 1.

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

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

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

17. (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, and (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184 The composition according to claim 16, comprising the above.

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

19. (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, and (b) An isoamylase comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243 The composition according to claim 18, comprising the above.

20. (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 182, and An α-amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445 The composition according to claim 14, comprising the same. **Claim 21** (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, and An α-amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 398 The composition according to claim 20, comprising the same. **Claim 22** (a) Lysin B comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 183 to 241, and An isoamylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 242 to 392, the composition according to claim 1. **Claim 23** (a) Lysin B comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184, and An isoamylase comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243 The composition according to claim 22, comprising the same. **Claim 24** (a) Lysin B comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 183 to 241, and An α-amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445 The composition according to claim 14, comprising the same. **Claim 25** (a) Lysin B comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184, and An α-amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 398 The composition according to claim 24, comprising the same. **Claim 26** (a) An isoamylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 242 to 392, and An α-amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445 The composition according to claim 14, comprising the same. **Claim 27** (a) An isoamylase comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, and An α-amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 398 The composition according to claim 26, comprising the same. **Claim 28** (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 182, (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 183 to 241, (c) Isoamylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 242 to 392, and (d) An α-amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445 The composition according to claim 1, comprising all four of the above. **Claim 29** (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184, (c) Isoamylase comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, and (d) An α-amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 398 The composition according to claim 28, comprising the same. **Claim 30** (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 182, (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 183 to 241, and (c) Isoamylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 242 to 392 The composition according to claim 28, comprising the same. **Claim 31** (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184, and (c) Isoamylase comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243 The composition according to claim 30, comprising the same. **Claim 32** Lysin A comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 182, Lysin B comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 183 to 241, and α - amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445 The composition according to claim 28, comprising the same.

33. Lysin A comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, Lysin B comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184, and α - amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 398 The composition according to claim 32, comprising the same.

34. Lysin A comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 182, Isoamylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 242 to 392, and α - amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445 The composition according to claim 28, comprising the same.

35. Lysin A comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, Isoamylase comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, and α - amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 398 The composition according to claim 34, comprising the same.

36. Lysin B comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 183 to 241, Isoamylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 242 to 392, and α - amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445 The composition according to claim 28, comprising the same.

37. (a) Lysin B comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184, (b) Isoamylase comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, and (c) α-Amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 398 The composition according to claim 36, comprising.

38. (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 182, (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 183 to 241, (c) Isoamylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 242 to 392, and (d) α-Amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445 The composition according to claim 1, comprising all four of.

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

40. (a) the lysin A comprises an amino acid sequence having at least 90%, 95%, 97%, or 99% sequence identity with 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 with 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 with SEQ ID NO: 242 or SEQ ID NO: 243, and / or (d) the α-amylase comprises an amino acid sequence having at least 90%, 95%, 97%, or 99% sequence identity with any one of SEQ ID NOs: 393 to 398, The composition according to any one of claims 1 to 39.

41. (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 any one of the amino acid sequences of SEQ ID NOs: 393 to 398, The composition according to claim 40.

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

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

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

45. The method according to claim 44, wherein the bacterial infection is caused by actinomycete bacteria.

46. The method according to claim 45, wherein the actinomycete bacteria are Corynebacteriales or Propionibacteriales bacteria.

47. The method according to claim 46, wherein the Corynebacteriales are Mycobacterium species.

48. The method according to claim 47, wherein the Mycobacterium species are M. tuberculosis, M. leprae, M. lepromatosis, M. avium, M. kansasii, M. fortuitum, M. chelonae, M. marinum, M. intracellulare, M. abscessus, M. chimaera, M. bolletti, M. fortuitum, M. goodii, or M. massiliense.

49. The method according to claim 46, wherein the Corynebacteriales are Nocardia, Corynebacterium, or Rhodococcus.

50. (a) the Nocardia species is N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. asteroids, N. brasiliensis, or N. caviae; (b) the Corynebacterium species is C. glutamicum or C. diphtheriae, or (c) the Rhodococcus species is R. fascians or R. equi; The method according to claim 49.

51. The method according to claim 46, wherein the actinomycete is of the order Propionibacteriales.

52. The method according to claim 51, wherein the order Propionibacteriales is of the genus Cutibacterium.

53. The method according to claim 52, wherein the genus Cutibacterium is C. acnes.

54. (a) Lysin A; (b) Lysin B; (c) Isoamylase; and (d) α-Amylase The method according to any one of claims 44 to 53, further comprising administering a supramolecular structure comprising two or more of the above.

55. The supramolecular structure is (a) Lysin A; and (b) Lysin B The method according to claim 54.

56. The supramolecular structure is (a) Lysin A; and (b) Isoamylase The method according to claim 54.

57. The supramolecular structure is (a) Lysin A; and (b) α-Amylase The method according to claim 54.

58. The supramolecular structure is (a) Lysin B; and (b) Isoamylase The method according to claim 54.

59. The supramolecular structure is (a) Lysin B; and (b) α-Amylase The method according to claim 54.

60. The supramolecular structure is (a) Isoamylase; and (b) α-Amylase The method according to claim 54.

61. The supramolecular structure is (a) Lysin A; (b) Lysin B; (c) Isoamylase; and (d) α-Amylase The method according to claim 54, comprising three or more of the above.

62. The supramolecular structure is (a) Lysin A; (b) Lysin B; and (c) Isoamylase The method according to claim 61.

63. The supramolecular structure is (a) Lysin A; (b) Lysin B; and (c) α-Amylase The method according to claim 61.

64. The supramolecular structure is (a) Lysin A; (b) isoamylase; and (c) α-amylase The method according to claim 61, comprising the same. **Claim 65** The supramolecular structure is (a) Lysin B; (b) isoamylase; and (c) α-amylase The method according to claim 61, comprising the same. **Claim 66** The supramolecular structure is (a) Lysin A; (b) Lysin B; (c) isoamylase; and (d) α-amylase The method according to claim 54, comprising all four of the same. **Claim 67** The supramolecular structure is (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 182, (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 183 to 241, (c) isoamylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 242 to 392, and (d) α-amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445 The method according to claim 54, comprising two or more of the same. **Claim 68** The supramolecular structure is (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184, (c) isoamylase comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, and (d) α-amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 398, the method according to claim 67, comprising two or more of the same. **Claim 69** The supramolecular structure is (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 182, and (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 183 to 241 The method according to claim 67, comprising the same. **Claim 70** The supramolecular structure is (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184 The method according to claim 69, comprising the same. **Claim 71** wherein the supramolecular structure comprises (a) lysin A comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 182, and (b) isoamylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 242 to 392, The method according to claim 67. **Claim 72** wherein the supramolecular structure comprises (a) lysin A comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, and (b) isoamylase comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, The method according to claim 71. **Claim 73** wherein the supramolecular structure comprises (a) lysin A comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 182, and (b) α-amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445, The method according to claim 67. **Claim 74** wherein the supramolecular structure comprises (a) lysin A comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, and (b) α-amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 398, The method according to claim 73. **Claim 75** wherein the supramolecular structure comprises (a) lysin B comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 183 to 241, and (b) isoamylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 242 to 392, The method according to claim 67. **Claim 76** wherein the supramolecular structure comprises (a) lysin B comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184, and (b) isoamylase comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, The method according to claim 75. **Claim 77** wherein the supramolecular structure comprises (a) lysin B comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 183 to 241, and (b) α-amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445 The method according to claim 67, comprising

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

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

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

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

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

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

84. The supramolecular structure is (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184, and (c) Isoamylase comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243 The method according to claim 83, comprising.

85. The supramolecular structure is (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 182, (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 183 to 241, and (c) α - Amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445 The method according to claim 81, comprising.

86. The supramolecular structure is (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184, and (c) α - Amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 398 The method according to claim 85, comprising.

87. The supramolecular structure is (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 182, (b) Isoamylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 242 to 392, and (c) α - Amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445 The method according to claim 81, comprising.

88. The supramolecular structure is (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, (b) Isoamylase comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, and (c) α-Amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 398 The method according to claim 87, comprising the above.

89. The supramolecular structure is (a) Lysin B comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 183 to 241, (b) Isoamylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 242 to 392, and (c) α-Amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445 The method according to claim 81, comprising the above.

90. The supramolecular structure is (a) Lysin B comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 183 or SEQ ID NO: 184, (b) Isoamylase comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 242 or SEQ ID NO: 243, and (c) α-Amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 398 The method according to claim 89, comprising the above.

91. The supramolecular structure is (a) Lysin A comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1 to 182, (b) Lysin B comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 183 to 241, (c) Isoamylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 242 to 392, and (d) α-Amylase comprising an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 393 to 445 The method according to claim 54, comprising all four of the above.

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

93. The supramolecular structure is (a) The lysin A comprises an amino acid sequence having at least 90%, 95%, 97%, or 99% sequence identity with 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 with 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 with SEQ ID NO: 242 or SEQ ID NO: 243, and / or (d) The α-amylase comprises an amino acid sequence having at least 90%, 95%, 97%, or 99% sequence identity with any one of SEQ ID NOs: 393 to 398, The method according to any one of claims 54 to 92, comprising

94. The supramolecular structure is (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 to 398, The method according to claim 93, comprising

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

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

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

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

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

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

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

102. The method according to claim 100, wherein the liposome is multilamellar.

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

3.

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

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

106. The method according to claim 104 or 105, wherein the lipid comprises 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 according to claim 105, wherein the lipid is a sterol.

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

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

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

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

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

4.

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

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

116. The method according to claim 115, wherein the target moiety is an extracellular target moiety that targets professional antigen-presenting cells.

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

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

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

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

121. The method according to any one of claims 54 to 118, wherein the composition is administered simultaneously with the supramolecular structure.

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

123. The method according to claim 122, wherein the antibiotic is cephalosporin, carbapenem, penicillin, aminoglycoside, cephalosporin, rifamycin, macrolide, or fluoroquinolone.

124. The method according to claim 122, wherein the antibiotic is thiacetazone, sq-109, bedaquiline, delamanid, pyrazinamide, or isoniazid.

125. The method according to claim 122, wherein the antibiotic is azithromycin, clarithromycin, ethambutol, rifampin, biapenem, or amikacin.

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

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