Modified bacteriophage

Abstract

The present invention provides a bacteriophage having bacteriolytic activity against Mycobacterium avium and / or Mycobacterium intracellulare. The genome of said bacteriophage contains a nucleic acid sequence found in the genome of a bacteriophage identified by deposition number NITE BP-03513, deposition number NITE BP-03514, or deposition number NITE BP-03918. The capsid of said bacteriophage is linked to a membrane-permeable peptide via a tag.

Description

Engineered bacteriophage

[0001] The present invention relates to novel and modified bacteriophages, and uses of these bacteriophages.

[0002] Nontuberculous mycobacteria (NTM) disease is an infectious disease caused by acid-fast bacteria other than Mycobacterium tuberculosis and Mycobacterium leprae. NTM bacteria are common in the environment, such as lakes and soil, and can cause respiratory symptoms when inhaled as aerosols. Examples of NTM include Mycobacterium avium (M. avium), Mycobacterium intracellulare (M. intracellulare), Mycobacterium fortuitum (M. fortuitum), Mycobacterium chelonae (M. chelonae), Mycobacterium gordonae (M. gordonae), Mycobacterium szulgai (M. kansasii), and Mycobacterium genavense (M. genavense) (Infection. 2004; 32: 257.). Among these, infections caused by the Mycobacterium avium complex (MAC), which includes Mycobacterium avium and Mycobacterium intracellulare, are called MAC diseases, and among these, pulmonary infections are particularly called pulmonary MAC diseases.

[0003] Treatment methods for bacterial infections, including NTM disease, are generally multidrug combination therapies including macrolide antibiotics. In recent years, phage therapy utilizing the lytic activity of bacteriophages (hereinafter also referred to as phages) has become known (Microorganisms. 2021; 9 (3): 596., International Publication No. 2021 / 092362). For example, D29, TM4, and ZoeJ have been reported as phages that exhibit lytic activity against Mycobacterium avium (Journal of Medical Microbiology 2006; 55 (1): 37., Microorganisms. 2021; 9 (3): 596., Microb. Drug Resist. 2006; 12: 1.).

[0004] Bacteriophages are viruses that use bacteria as their host. When a phage infects a specific bacterium, it injects its genome into the bacterium and uses the host bacterium's metabolic machinery to propagate progeny phages. Subsequently, the bacterial cell wall is destroyed by a lytic enzyme contained in the phage genome, releasing the progeny phages and causing the host bacterium to die.

[0005] Phages can be broadly classified into non-temperate phages (lytic phages) and temperate phages (lysogenic phages) (Scientifica (Cairo) 2014; 2014: 581639). Non-temperate phages replicate without integrating into the host bacterial DNA. They grow progeny phages within the bacteria, lyse the bacteria, and release mature phage particles. On the other hand, temperate phages are integrated into the host bacterial DNA after infecting the bacteria and replicate together with the host bacterial DNA. Simply being integrated into the host bacterial DNA is harmless to the host bacteria, and they are able to maintain a lysogenic state (a state in which the phage DNA is integrated into the host bacterial DNA and replicates together with the host bacterial DNA). However, when temperate phages are exposed to an external stimulus such as ultraviolet light, they release lytic enzymes like non-temperate phages, lyse the host bacteria, and release mature phage particles.

[0006] Phages lacking genes involved in lysogenization have also been reported, such as phages lacking the integrase gene (Nat Med. 2019; 25(5): 730.) and phages lacking the integrase gene and the immunity repressor gene (mBio. 2021; 12(3): e00973.).

[0007] Phages consist of a head (a capsid containing the phage genome) and a tail. Phage capsids are produced by synthesizing multiple proteins that make up the capsid from the phage genome within host bacteria and assembling these proteins. Phages with the desired peptide or protein attached to the capsid are produced by introducing a plasmid containing a DNA sequence encoding a capsid-constituting protein linked to a DNA sequence encoding a target peptide or protein and a phage genome into host bacteria. For example, it has been reported that when a genetically modified host bacterium harboring a plasmid containing a DNA sequence encoding an S-tag peptide linked downstream of the DNA sequence encoding the capsid-constituting protein of wild-type TM4 phage is infected with wild-type TM4 phage, a modified TM4 phage having a capsid protein fused with an S-tag peptide can be obtained (Non-Patent Document 1).

[0008] MAC is known to infect cells. If phages can be efficiently delivered into cells, the phages can lyse the MAC inside the cells, which is expected to have a more potent therapeutic effect. The use of cell-penetrating peptides (CPPs) has been discussed as an effective means of delivering bacteriophages into cells (Non-Patent Document 2).

[0009] Many CPP sequences are known. For example, it has been reported that Inv3, a peptide contained in Mycobacterium cell entry protein (Mce1A), a protein derived from Mycobacterium tuberculosis, has CPP activity (Non-Patent Document 3).

[0010] Appl Environ Microbiol. 2013;79(18):5608Adv Drug Deliv Rev. 2021;176:113864Anal.Biochem. 2006;353(1):7

[0011] An object of the present invention is to provide a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare. Another object of the present invention is to provide means and methods, particularly bacteriophages and pharmaceutical compositions, for preventing or treating nontuberculous mycobacterial diseases such as pulmonary mycobacterial infection (MAC) disease.

[0012] As a result of considerable ingenuity and research into the creation of bacteriophages, the present inventors obtained a novel bacteriophage (Example 1) and discovered that the bacteriophage was resistant to freezing and thawing (Example 2). They also obtained a bacteriophage with a modified capsid (Example 3). They then discovered that the novel bacteriophage and the modified bacteriophage had lytic activity against Mycobacterium avium and Mycobacterium intracellulare (Examples 4 and 5). Furthermore, the present inventors discovered that a pharmaceutical composition containing a combination of specific bacteriophage strains had excellent bactericidal activity (Example 6). Based on these findings, the present invention was completed.

[0013] That is, the present invention may include the following inventions as medically or industrially useful substances or methods: [1] A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to an S tag. [2] The bacteriophage according to [1], wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513. [3] The bacteriophage according to [1] or [2], wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513. [4] A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is the bacteriophage identified by accession number NITE BP-03513 or a subculture strain thereof, and the capsid of the bacteriophage or the subculture strain thereof is linked to an S tag. [5] The bacteriophage according to any of [1] to [4], wherein the capsid is further linked to a cell membrane-penetrating peptide via an S tag. [6] The bacteriophage according to [5], wherein the cell membrane-penetrating peptide includes Inv3. [7] A pharmaceutical composition comprising the bacteriophage according to any one of [1] to [6] and a pharmaceutically acceptable excipient. [8] The pharmaceutical composition according to [7], further comprising one or more strains of bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare.

[0014] [9] A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene.

[10] The bacteriophage according to [9], which is resistant to freezing and thawing.

[11] The bacteriophage according to [9], wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918.

[12] The bacteriophage according to [9] or

[11] , wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918.

[13] A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is the bacteriophage identified by accession number NITE BP-03918 or a passage strain thereof.

[14] The bacteriophage according to any of [9] to

[13] , wherein the capsid of the bacteriophage is linked to a tag.

[15] The bacteriophage according to any of [9] to

[13] , wherein the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via a tag.

[16] The bacteriophage according to

[14] or

[15] , wherein the tag is an S tag.

[17] The bacteriophage according to

[15] or

[16] , wherein the cell membrane-permeable peptide comprises Inv3.

[18] A pharmaceutical composition comprising the bacteriophage according to any of [9] to

[17] and a pharmaceutically acceptable excipient.

[19] The pharmaceutical composition according to

[18] , further comprising one or more strains of bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare.

[0015]

[20] The pharmaceutical composition according to [7], further comprising one or more bacteriophages selected from the group consisting of the bacteriophage according to any one of [1] to [6] and the bacteriophages (1) to (3) below: (1) (A) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, and (b) is deficient in an integrase gene and / or an immunity repressor gene, or (B) the bacteriophage according to (A), the capsid of which is linked to a cell membrane-permeable peptide via a tag; (2) (C) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene, or (D) a bacteriophage according to (C), wherein the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via a tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage is identified by accession number NITE BP-03918. A bacteriophage comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified as BP-03519.

[0016]

[21] The pharmaceutical composition according to [7], further comprising one or more bacteriophages selected from the group consisting of the bacteriophage according to any one of [1] to [6] and the bacteriophages (1) to (3) below: (1) (A) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, or (B) the bacteriophage (A), the capsid of which is linked to a cell membrane-permeable peptide via a tag; (2) (C) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, or (D) a bacteriophage of (C), wherein the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via a tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519.

[0017]

[22] The pharmaceutical composition according to [7], further comprising one or more bacteriophages selected from the group consisting of the bacteriophage according to any one of [1] to [6] and the bacteriophages (1) to (3) below: (1) (A) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, or (B) the bacteriophage (A), the capsid of which is linked to a cell membrane-permeable peptide via a tag; (2) (C) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, or (D) A bacteriophage of (C), wherein the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via a tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519.

[0018]

[23] The pharmaceutical composition according to [7], further comprising one or more bacteriophages selected from the group consisting of the bacteriophage according to any one of [1] to [6] and the bacteriophages (1) to (3) below: (1) (A) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage identified by accession number NITE BP-03514 or a passage strain thereof, or (B) the bacteriophage (A), wherein the capsid of the bacteriophage or a passage strain thereof is linked to a cell membrane-permeable peptide via a tag; (2) (C) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage identified by accession number NITE BP-03918, or a subcultured strain thereof, or (D) a bacteriophage of (C), wherein the capsid of the bacteriophage or a subcultured strain thereof is linked to a cell membrane-permeable peptide via a tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage identified by accession number NITE BP-03519, or a subcultured strain thereof.

[0019]

[24] A pharmaceutical composition comprising a bacteriophage of any one of (1) to (3) below and a pharmaceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) is deficient in the integrase gene and / or the immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag.

[0020]

[25] The pharmaceutical composition according to

[24] , which contains a bacteriophage of any one of the following (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and the capsid of the bacteriophage is linked to Inv3 via an S tag.

[0021]

[26] The pharmaceutical composition according to

[24] or

[25] , which contains a bacteriophage of any one of the following (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and the capsid of the bacteriophage is linked to Inv3 via an S tag.

[0022]

[27] A pharmaceutical composition comprising a bacteriophage of any one of the following (1) to (3) and a pharmaceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage identified by accession number NITE BP-03513 or a passage strain thereof, and the capsid of the bacteriophage or the passage strain thereof being linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage identified by accession number NITE BP-03514 or a passage strain thereof; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage identified by accession number NITE BP-03918 or a subcultured strain thereof, and the capsid of the bacteriophage or the subcultured strain thereof is linked to Inv3 via an S tag.

[0023]

[28] A pharmaceutical composition comprising a bacteriophage of any one of (1) to (3) below and a pharmaceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, (b) a bacteriophage that contains a nucleic acid sequence that is 90% or more identical to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and that is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag; and (3) a bacteriophage that has lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage containing a nucleic acid sequence that is 90% or more identical to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519.

[0024]

[29] The pharmaceutical composition according to

[28] , which contains the bacteriophage of (1) to (3) below: (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage identified by NITE Accession No. BP-03519.

[0025]

[30] The pharmaceutical composition according to

[28] or

[29] , which contains a bacteriophage of any one of the following (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by NITE Accession Number BP-03519.

[0026]

[31] A pharmaceutical composition comprising a bacteriophage of any one of the following (1) to (3) and a pharmaceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage identified by accession number NITE BP-03513 or a subculture strain thereof, and the capsid of the bacteriophage or the subculture strain thereof being linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being identified by accession number NITE BP-03513 or a subculture strain thereof; (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage identified by accession number NITE BP-03519 or a subculture strain thereof, wherein the capsid of the bacteriophage or the subculture strain thereof is linked to Inv3 via an S tag; and

[0027]

[32] A pharmaceutical composition comprising a bacteriophage of any one of (1) to (3) below and a pharmaceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, (b) a bacteriophage comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, and which is deficient in the integrase gene and / or the immunity repressor gene; and (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519.

[0028]

[33] The pharmaceutical composition according to

[32] , which contains the bacteriophage of (1) to (3) below: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519.

[0029]

[34] The pharmaceutical composition according to

[32] or

[33] , which contains a bacteriophage of any one of the following (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519.

[0030]

[35] A pharmaceutical composition comprising a bacteriophage of any one of the following (1) to (3) and a pharmaceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage identified by accession number NITE BP-03513 or a subcultured strain thereof, and the capsid of the bacteriophage or the subcultured strain thereof being linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage identified by accession number NITE BP-03514 or a subcultured strain thereof; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being identified by accession number NITE BP-03519, or a passage strain thereof.

[0031]

[36] The pharmaceutical composition according to any one of [7], [8],

[18] ,

[19] , and

[20] to

[35] , which is a pharmaceutical composition for preventing or treating nontuberculous mycobacterial disease.

[37] The pharmaceutical composition according to

[36] , wherein the nontuberculous mycobacterial disease is pulmonary MAC disease.

[0032] The bacteriophages and modified bacteriophages, as well as the pharmaceutical compositions of the present invention, exhibit lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, and are expected to be useful in the prevention or treatment of nontuberculous mycobacterial diseases such as pulmonary MAC disease.

[0033] Photographs showing the results of a plaque formation assay comparing the effect of physical damage caused by freeze-thawing on the number of phages in the #63 lysogen gene-deficient strain (No. 63Δrep, int) and the #63 freeze-thaw resistant strain (No. 63Δrep, int_R12-23). ​​1. S-tag and Inv3 fused D29 lysogen gene-deficient strain (D29Δint-Stag-Inv3), 2. S-tag and Inv3 fused B1 lysogen gene-deficient strain (B1Δrep, int-Stag-Inv3), 3. S-tag and Inv3 fused B1 lysogen gene-deficient strain (B1Δrep, int-Stag-Inv3), 4. S-tag and Inv3 fused B1 lysogen gene-deficient strain (B1Δrep, int-Stag-Inv3), and 5. S-tag and Inv3 fused B1 lysogen gene-deficient strain (B1Δrep, int-Stag-Inv3) were compared against Mycobacterium avium KCH-ASGF-MA-05 (A) or clarithromycin-resistant Mycobacterium intracellulare KCH-ASGF-MAC-475 strain (B). Photographs showing the lytic activity of the S-tag and Inv3-fused #63 freeze-thaw resistant strain (No. 63Δrep, int_R12-23-Stag-Inv3) and #63 freeze-thaw resistant strain (No. 63Δrep, int_R12-23). ​​A: Graph showing the bactericidal activity of bacteriophages of the S-tag fused D29 lysogen gene-deficient strain (N), the S-tag fused D29 lysogen gene-deficient strain (C), and the D29 lysogen gene-deficient strain against Mycobacterium avium KCH-ASGF-MA-05 cells infected intracellularly. B: Graph showing the bactericidal activity of bacteriophages of the S-tag fused #63 freeze-thaw resistant strain and #63 freeze-thaw resistant strain against Mycobacterium avium KCH-ASGF-MA-12 cells infected intracellularly. The vertical axis represents colony-forming unit concentration (CFU / mL). 4 is a graph showing the bactericidal activity of various bacteriophage cocktails against intracellularly infected Mycobacterium avium KCH-ASGF-MA-05. The vertical axis indicates colony-forming unit concentration (CFU / mL). The bars indicate geometric mean values. 4 is a graph showing the bactericidal activity of various bacteriophages against intracellularly infected Mycobacterium avium KCH-ASGF-MA-05. The vertical axis indicates colony-forming unit concentration (CFU / mL). The bars indicate geometric mean values. ** indicates that a significant difference was observed between groups when tested by Tukey's multiple comparison test at a significance level of less than 1% (p<0.01).

[0034] This application claims priority from Japanese Patent Application No. 2023-116013, filed on July 14, 2023, the entire contents of which are incorporated herein by reference.

[0035] The present invention will be described in detail below. The following embodiments are merely examples for explaining the present invention, and are not intended to limit the present invention to these embodiments. The present invention can be embodied in various forms without departing from the gist of the present invention.

[0036] <1. Bacteriophage of the Present Invention> In one aspect, the present invention provides a novel bacteriophage (hereinafter, may be referred to as "bacteriophage of the present invention"). The bacteriophage of the present invention is a bacteriophage having lytic activity against nontuberculous mycobacteria (NTM), particularly Mycobacterium avium and / or Mycobacterium intracellulare, which cause pulmonary MAC disease.

[0037] The bacteriophage #63 freeze-thaw resistant strain of the present invention has been internationally deposited by the applicant with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan), an international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, on June 22, 2023 (Accession No. NITE BP-03918).

[0038] In one embodiment, the bacteriophage of the present invention includes the following bacteriophage: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) lacks a lysogeny gene.

[0039] As used herein, "identity" refers to the identity of a nucleotide sequence or an amino acid sequence determined by a technique known in the art. As a method for aligning sequences, publicly available alignment software can be used. For example, the CLUSTAL W program (Nucleic Acids Research. 1994; 22(22): 4673-80), the FASTA program (Proceedings of the National Academy of Sciences of the United States of America. 1988; 85(8): 2444-8), the BLAST program (including BLASTP, BLASTN, BLASTX, TBLASTN, TBLASTX, etc.) (Journal of Molecular Biology. 1990; 215(3): 403-10) and the like can be used, but are not limited to these. A specific sequence alignment method is, for example, the value "Identity" obtained by searching with the NEEDLE program (Journal of Molecular Biology. 1970; 48(3): 443-53) using the default parameters. The parameters are as follows: Gap penalty = 10 Extend penalty = 0.5 Matrix = EBLOSUM62

[0040] As used herein, the term "lysogenic gene" refers to a gene involved in inducing and / or maintaining a lysogenic state. Examples of "lysogenic genes" include the integrase gene, the immunity repressor gene, and the Cro gene. Specific lysogenic genes and their sequence information are also publicly known (Nat Med. 2019; 25(5): 730., mBio. 2021; 12(3): e00973., Cell. 2018; 172(6): 1260.).

[0041] As used herein, "lysogenic gene deletion" means 1) the deletion of the entire length of at least one lysogenic gene from the genome sequence of the bacteriophage, or 2-1) the bacteriophage has become a lytic bacteriophage by modifying at least one lysogenic gene region from the genome sequence of the bacteriophage by deletion, substitution, insertion, addition, or a combination thereof, or 2-2) the function of the lysogenic gene is deleted. Lysogenic gene deletion can be performed by methods known in the art depending on the type of lysogenic gene to be deleted (e.g., Nat Med. 2019; 25(5): 730., mBio. 2021; 12(3): e00973., PLoS One. 2008; 3(12): e3957.).

[0042] As used herein, "having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare" means that it shows lytic plaques against at least one strain of bacteria belonging to Mycobacterium avium or Mycobacterium intracellulare. The lytic activity against these bacteria can be confirmed using methods and means known in the art. For example, the method described in Example 4 of the present specification was used to confirm the lytic activity of approximately 10 １０ pfu / mL ~ 10 ５ Whether a bacteriophage has lytic activity can be determined by determining whether pfu / mL of the bacteriophage (or a mixture of bacteriophages) shows lytic plaques against Mycobacterium avium or Mycobacterium intracellulare.

[0043] In one embodiment, the bacteriophage of the present invention is resistant to freezing and thawing. For example, the bacteriophage of the present invention includes the following bacteriophage: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) comprising a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) lacking a lysogeny gene, and the bacteriophage is resistant to freezing and thawing.

[0044] As used herein, "freeze-thaw resistant" means that the bacteriophage is maintained to a desired level even after freeze-thaw treatment, even though it is known that some bacteriophages are killed or damaged by freeze-thaw treatment. For example, the number of bacteriophages after freeze-thaw treatment is approximately the same as that before freeze-thaw treatment, or the number of bacteriophages after freeze-thaw treatment is maintained at at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more compared to that before freeze-thaw treatment. Freeze-thaw resistance can be confirmed using methods and means known in the art. For example, whether a bacteriophage is resistant to freeze-thaw can be confirmed by determining whether the number of bacteriophages before and after freeze-thaw treatment is reduced using the method described in Example 2 of the present specification. Alternatively, resistance to freeze-thawing can be confirmed by determining whether the bacteriophage retains a desired activity (e.g., lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare) after the freeze-thaw treatment. For example, if the bacteriophage after the freeze-thaw treatment retains approximately the same activity as that before the freeze-thaw treatment, or at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of the activity compared to that before the freeze-thaw treatment, the bacteriophage can be said to be resistant to freeze-thawing.

[0045] In one embodiment, the bacteriophage of the present invention includes the following bacteriophage: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene.

[0046] "Deficient in integrase gene and / or immunity repressor gene" means 1) that the entire integrase gene and / or immunity repressor gene is deleted from the genome sequence of the bacteriophage, or 2-1) that the bacteriophage has become a lytic bacteriophage by modifying the genome sequence of the bacteriophage by deletion, substitution, insertion, addition, or a combination thereof of a portion of the integrase gene and / or the immunity repressor gene, or 2-2) that the function of the integrase gene and / or the immunity repressor gene is defective. As described above, deletion of lysogenic genes such as the integrase gene and the immunity repressor gene can be performed by methods known in the art depending on the type of lysogenic gene to be deleted (e.g., Nat Med. 2019; 25(5): 730., mBio. 2021; 12(3): e00973., PLoS One. 2008; 3(12): e3957.).

[0047] In one embodiment, the bacteriophage of the present invention includes a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene.

[0048] The bacteriophage of the present invention includes the following bacteriophage: A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918.

[0049] The bacteriophage of the present invention includes the following bacteriophage: A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918.

[0050] The bacteriophage of the present invention also includes the following bacteriophage: A bacteriophage identified by accession number NITE BP-03918.

[0051] The bacteriophage of the present invention also includes the following bacteriophage: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence modified by deletion, substitution, insertion or addition of 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases, or a combination thereof, in the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene.

[0052] With regard to the above-mentioned modifications such as deletion, substitution, insertion, or addition of bases, multiple modifications may be consecutive or multiple modifications may be present at different positions. Such modifications are included in the bacteriophage of the present invention as long as they have lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare (and, optionally, resistance to freeze-thawing).

[0053] Bacteriophage mutants may arise in the process of subculture, production, and / or replication of bacteriophages, in which the nucleic acid sequences of the genome contained in the bacteriophage have been partially deleted, substituted, inserted, and / or added, and such mutants are also included in the bacteriophages of the present invention, so long as they have lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare (and, optionally, resistance to freeze-thawing).

[0054] The bacteriophage of the present invention also includes passaged strains of the bacteriophage identified by the above-mentioned accession number NITE BP-03918, so long as they have lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare (and, optionally, resistance to freeze-thawing).

[0055] As used herein, the term "passaged strain" refers to a bacteriophage obtained by subculturing a provided bacteriophage.

[0056] The present invention also includes a polynucleotide consisting of a genome contained in the bacteriophage of the present invention (hereinafter, sometimes referred to as the "genome of the present invention"). Accordingly, the present invention provides a polynucleotide of a bacteriophage genome comprising a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918 and lacking an integrase gene and / or an immunity repressor gene, and a polynucleotide of a bacteriophage genome comprising a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918. In one embodiment, the polynucleotide is a polynucleotide encoding a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare.

[0057] In one embodiment, the genome of the present invention includes a genome that (a) comprises a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene.

[0058] The genomes of the present invention include the following genomes: A genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918.

[0059] The genomes of the present invention include the following genomes: A genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918.

[0060] The genomes of the present invention can be produced using common techniques known in the art, such as recombinant DNA techniques (e.g., polymerase chain reaction (PCR) amplification, cloning), enzymatic or chemical synthesis, or a combination thereof. For example, the genomes of the present invention can be produced by linking multiple polynucleotides containing partial base sequences of the genomes of the present invention using genetic engineering techniques. In one embodiment, the full-length or partial sequence of the genomes of the present invention may be contained in a vector known in the art.

[0061] The bacteriophage of the present invention can be obtained by requesting it from the above-mentioned depository center.

[0062] Furthermore, the bacteriophage of the present invention can be produced by deciphering the nucleic acid sequence of the genome of the provided bacteriophage using common techniques known in the art and based on the sequence information. For example, the genome of the present invention produced by the above-described method is introduced into a host bacterium by electroporation (e.g., Mycobacterium is used as the host bacterium to obtain a bacteriophage that exhibits lytic activity against Mycobacterium). The bacteria into which the genome has been introduced are then placed on a plate overlaid with soft agar and cultured. Single plaques are then obtained by plaque assay. The single plaques are added to a host bacterium culture medium and cultured. The culture supernatant obtained by standing or centrifuging is then filtered, whereby the bacteriophage of the present invention can be produced. Furthermore, to prepare the bacteriophage of the present invention, the lysogen gene can be deleted from the phage genome using Bacteriophage Recombineering of Electroporated DNA (BRED) (PLoS One. 2008; 3(12): e3957.).

[0063] The bacteriophage of the present invention can be prepared by common culture, isolation, and purification methods known in the art. For example, host bacteria (e.g., Mycobacterium smegmatis, Mycobacterium avium, or Mycobacterium intracellulare) are cultured in advance, infected with the bacteriophage of the present invention, and cultured at 37°C. After culture, the culture supernatant obtained by standing or centrifugation is filtered to obtain purified bacteriophage. The medium can be selected appropriately depending on the bacterium used. For example, 7H9 medium can be used when culturing Mycobacterium smegmatis. When preparing multiple bacteriophages, they may be grown in different host bacteria or the same host bacteria.

[0064] Furthermore, the bacteriophage of the present invention can be stored in various forms (liquid, lyophilized, etc.) by following appropriate methods known in the art.

[0065] 2. Engineered Bacteriophage of the Present Invention In another aspect, the present invention provides an engineered bacteriophage (hereinafter, sometimes referred to as "engineered bacteriophage of the present invention"). The engineered bacteriophage of the present invention is a bacteriophage whose head (also referred to as capsid) and / or tail have been modified.

[0066] The bacteriophage to be modified is not particularly limited, as long as it has lytic activity against nontuberculous mycobacteria (NTM), particularly Mycobacterium avium and / or Mycobacterium intracellulare, which cause pulmonary MAC disease. For example, any of the following bacteriophages (1) to (3) can be modified: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) contains a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in a lysogeny gene; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) contains a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514, and (b) is deficient in a lysogenic gene; (4) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514, and (b) is deficient in a lysogenic gene.

[0067] In one embodiment, the bacteriophage to be modified includes any of the following (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene; (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514, and (b) is deficient in the integrase gene and / or the immunity repressor gene; (4) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514, and (b) is deficient in the integrase gene and / or the immunity repressor gene.

[0068] In one embodiment, the bacteriophage to be modified includes any of the following (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918; (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514.

[0069] In one embodiment, the bacteriophage to be modified includes any of the following (1) to (3): (1) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513; (2) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918; (3) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514.

[0070] The bacteriophage to be modified also includes passaged strains of the bacteriophage identified by the above-mentioned accession number NITE BP-03513, accession number NITE BP-03918, or accession number NITE BP-03514, so long as it has lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare.

[0071] In one embodiment, the bacteriophage to be modified includes any of the following (1) to (3): (1) a bacteriophage identified by accession number NITE BP-03513 or a passage strain thereof; (2) a bacteriophage identified by accession number NITE BP-03918 or a passage strain thereof; (3) a bacteriophage identified by accession number NITE BP-03514 or a passage strain thereof.

[0072] The bacteriophage identified by accession number NITE BP-03918 (#63 freeze-thaw resistant strain #63ΔR12-23) was deposited by the applicant at the National Institute of Technology and Evaluation, an international depository authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Room 122) on June 22, 2023 (accession number NITE BP-03918).

[0073] The bacteriophage identified by accession number NITE BP-03513 (D29 lysogen gene-deficient strain D29Δ) has been internationally deposited by the applicant with the Patent Microorganisms Deposit Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan), an international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, on August 26, 2021 (accession number NITE BP-03513).

[0074] The bacteriophage identified by accession number NITE BP-03514 (B1 lysogen gene-deficient strain B1Δ) was deposited internationally by the applicant at the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) on August 26, 2021 (accession number NITE BP-03514).

[0075] Among the bacteriophages to be modified, those identified by accession numbers can be obtained by requesting the above-mentioned depository center.

[0076] The bacteriophage to be modified can be prepared and cultured in the same manner as in the previous section <1. Bacteriophage of the present invention>.

[0077] In one embodiment, the modification comprises linking the capsid and / or tail to a tag peptide. Various tag peptides known in the art, such as S tag, T7 tag, His tag, and FLAG tag, can be used as the tag peptide. In one embodiment, the modification comprises linking the capsid and / or tail to an S tag. That is, the capsid and / or tail of the modified bacteriophage of the present invention are linked to an S tag. In one embodiment, the modification comprises linking the capsid to an S tag.

[0078] The S tag is a type of tag peptide and is known in the art, and includes the amino acid sequence WSHPQFEK (SEQ ID NO: 2). The number of S tags linked to the capsid and / or tail is not particularly limited, and may be, for example, one, two, or four for any protein.

[0079] Those skilled in the art can use conventional methods to link a tag peptide (e.g., an S-tag) to the capsid and / or tail of a bacteriophage. For example, a bacteriophage having an S-tag linked to its capsid and / or tail can be produced by introducing a plasmid containing a polynucleotide containing a DNA sequence encoding an S-tag peptide linked upstream or downstream of a DNA sequence encoding a capsid-constituting protein and / or a tail-constituting protein into a bacteriophage host bacterium, expressing a fusion protein of the capsid-constituting protein and an S-tag in the host bacterium, and infecting the bacteriophage with the S-tag. Herein, a bacteriophage having an S-tag linked to its capsid and / or tail may also be referred to as an S-tag fusion bacteriophage. Examples of proteins that can be used as the capsid-constituting protein and / or tail-constituting protein include proteins included in the group of proteins known to generally constitute capsids, such as major capsid proteins, minor capsid proteins, accessory proteins, neck proteins, and tail proteins, which are known to constitute tails. For example, Non-Patent Document 1 reports a modified phage in which an S tag is linked to the capsid. The S tag may be linked to the capsid by linking the S tag peptide to the N-terminus or C-terminus of the capsid-constituting protein.

[0080] Engineered bacteriophages with a tag peptide (e.g., S-tag) linked to the capsid and / or tail have higher lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare than unmodified bacteriophages. For example, the engineered bacteriophages have lytic activity that is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher than that of unmodified bacteriophages. The lytic activity can be confirmed, for example, by the method described in Example 5.

[0081] In one embodiment, the modified bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to an S tag.

[0082] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to an S tag.

[0083] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to an S tag.

[0084] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage identified by accession number NITE BP-03513 or a subcultured strain thereof, and the capsid of the bacteriophage or a subcultured strain thereof is linked to an S tag.

[0085] In one embodiment, the modified bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to an S tag.

[0086] In one embodiment, the modified bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene, the bacteriophage is resistant to freeze-thawing, and the capsid of the bacteriophage is linked to an S tag.

[0087] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and the capsid of the bacteriophage is linked to an S tag.

[0088] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and the capsid of the bacteriophage is linked to an S tag.

[0089] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage identified by accession number NITE BP-03918 or a subcultured strain thereof, and the capsid of the bacteriophage or the subcultured strain thereof is linked to an S tag.

[0090] In one embodiment, the modification includes directly or indirectly linking the capsid and / or tail to a cell membrane-permeable peptide. That is, the capsid and / or tail of the modified bacteriophage of the present invention are directly or indirectly linked to a cell membrane-permeable peptide. When the capsid and / or tail are indirectly linked, for example, a linker or tag can be used.

[0091] In one embodiment, the modification comprises linking the capsid and / or tail directly to a cell membrane-penetrating peptide.

[0092] As used herein, a cell-penetrating peptide (CPP) refers to a peptide that, when linked to the capsid and / or tail of a bacteriophage, enhances the uptake of the bacteriophage into the cell. CPPs are well known in the art, and the sequences and mechanisms of many CPPs have been reported (e.g., Crit. Rev. Microbiol. 2021; 47(4):461). Examples of CPPs include, but are not limited to, peptide Inv3 (TKRRITPKDVIDVRSVTTEINT: SEQ ID NO: 1) from positions 25 to 46 of the rMceIa sequence, peptide Inv5 (Anal. Biochem. 2006; 353(1):7), R9, TAT, TP10, L17E, and the like.

[0093] In one embodiment, the modification comprises linking the capsid and / or tail to a cell membrane-permeable peptide via a tag.

[0094] The tag is not particularly limited as long as it is a tag peptide that can link the capsid and / or tail to the cell membrane-penetrating peptide. Examples of tags include S tags, T7 tags, His tags, and FLAG tags. The cell membrane-penetrating peptide may be linked to the capsid and / or tail via the tag at the N-terminus or C-terminus of the capsid-constituting protein and / or tail-constituting protein.

[0095] In one embodiment, the modified bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via an S tag.

[0096] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via an S tag.

[0097] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via an S tag.

[0098] In one embodiment, the engineered bacteriophage of the present invention includes the following: A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage identified by accession number NITE BP-03513 or a subcultured strain thereof, and the capsid of the bacteriophage or a subcultured strain thereof is linked to a cell membrane-permeable peptide via an S tag.

[0099] In one embodiment, the modified bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to a cell membrane-permeable peptide.

[0100] In one embodiment, the engineered bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene, the bacteriophage is resistant to freezing and thawing, and the capsid of the bacteriophage is linked to a cell membrane-permeable peptide.

[0101] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and the capsid of the bacteriophage is linked to a cell membrane-permeable peptide.

[0102] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and the capsid of the bacteriophage is linked to a cell membrane-permeable peptide.

[0103] In one embodiment, the engineered bacteriophage of the present invention includes the following: A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage identified by accession number NITE BP-03918 or a subcultured strain thereof, and the capsid of the bacteriophage or a subcultured strain thereof is linked to a cell membrane-permeable peptide.

[0104] In one embodiment, the modified bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via a tag.

[0105] In one embodiment, the modified bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deleted in an integrase gene and / or an immunity repressor gene, the bacteriophage is resistant to freezing and thawing, and the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via a tag.

[0106] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via a tag.

[0107] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via a tag.

[0108] In one embodiment, the engineered bacteriophage of the present invention includes the following: A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage identified by accession number NITE BP-03918 or a subcultured strain thereof, and the capsid of the bacteriophage or a subcultured strain thereof is linked to a cell membrane-permeable peptide via a tag.

[0109] In one embodiment, the tag linked to the engineered bacteriophage comprises an S tag.

[0110] In one embodiment, the cell membrane penetrating peptide linked to the engineered bacteriophage comprises Inv3.

[0111] In one embodiment, the modified bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag.

[0112] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag.

[0113] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag.

[0114] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage identified by accession number NITE BP-03513 or a subcultured strain thereof, and the capsid of the bacteriophage or the subcultured strain thereof is linked to Inv3 via an S tag.

[0115] In one embodiment, the modified bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag.

[0116] In one embodiment, the modified bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deleted in the integrase gene and / or the immunity repressor gene, the bacteriophage is resistant to freezing and thawing, and the capsid of the bacteriophage is linked to Inv3 via an S tag.

[0117] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and the capsid of the bacteriophage is linked to Inv3 via an S tag.

[0118] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and the capsid of the bacteriophage has an S tag linked to Inv3.

[0119] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage identified by accession number NITE BP-03918 or a subcultured strain thereof, and the capsid of the bacteriophage or the subcultured strain thereof is linked to Inv3 via an S tag.

[0120] Those skilled in the art can link a cell membrane-permeable peptide to the capsid and / or tail directly or via a tag using conventional methods (see Non-Patent Documents 1 and 2). For example, a plasmid in which a polynucleotide containing a DNA sequence encoding a cell membrane-permeable peptide or a tag and a cell membrane-permeable peptide is linked upstream or downstream of a DNA sequence encoding a capsid-constituting protein and / or a tail-constituting protein is introduced into a bacteriophage host bacterium, a fusion protein of the capsid-constituting protein and / or the tail-constituting protein with the cell membrane-permeable peptide or the tag and the cell membrane-permeable peptide is expressed in the host bacterium, and the bacteriophage is then infected to produce a bacteriophage in which a cell membrane-permeable peptide or a tag and a cell membrane-permeable peptide are linked to the capsid and / or tail. Herein, a bacteriophage in which a tag and a cell membrane-permeable peptide are linked to the capsid and / or tail may also be referred to as a tag and cell membrane-permeable peptide-fused bacteriophage. As the capsid-forming protein and / or tail-forming protein, proteins included in the protein group known as major capsid protein, minor capsid protein, accessory protein, neck protein, and tail protein, which are generally known to constitute capsids, can be used.

[0121] Engineered bacteriophages having a cell membrane-permeable peptide linked to their capsid and / or tail, either directly or via a tag, have higher lytic activity against intracellular Mycobacterium avium and / or Mycobacterium intracellulaire than unmodified bacteriophages. For example, the engineered bacteriophages have at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher lytic activity against intracellular Mycobacterium avium and / or Mycobacterium intracellulaire than unmodified bacteriophages. The lytic activity against intracellular Mycobacterium avium and / or Mycobacterium intracellulare can be determined by, for example, using the methods described in Examples 5 and 6 to determine whether Mycobacterium avium or Mycobacterium intracellulare show a decrease in number before and after treatment with the modified bacteriophage (or a mixture of multiple bacteriophages). This can be done to determine whether the modified bacteriophage has lytic activity against intracellular Mycobacterium avium and / or Mycobacterium intracellulare.

[0122] 3. Uses of the Pharmaceutical Composition and Bacteriophage of the Present Invention The bacteriophage of the present invention and / or the modified bacteriophage of the present invention have lytic activity against nontuberculous mycobacteria (NTM), particularly Mycobacterium avium and / or Mycobacterium intracellulare, which cause pulmonary MAC disease. Therefore, the bacteriophage of the present invention and / or the modified bacteriophage of the present invention are expected to be effective in treating or preventing nontuberculous mycobacterial disease (NTM disease), particularly pulmonary MAC disease.

[0123] In another aspect, the present invention provides uses of the bacteriophage of the present invention and / or the engineered bacteriophage of the present invention, for example, pharmaceutical compositions containing the bacteriophage of the present invention and / or the engineered bacteriophage of the present invention as an active ingredient (hereinafter sometimes referred to as the "pharmaceutical composition of the present invention").

[0124] Pharmaceutical compositions of the present invention include those comprising at least one bacteriophage, including a bacteriophage of the present invention and / or an engineered bacteriophage of the present invention, and a pharmaceutically acceptable excipient.

[0125] The pharmaceutical composition of the present invention may further comprise one or more strains of bacteriophage known to have lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, including, but not limited to, the bacteriophage of the present invention, the modified bacteriophage of the present invention, and the bacteriophage described in International Application No. PCT / JP2023 / 016600.

[0126] In one embodiment, the pharmaceutical composition of the present invention comprises the following modified bacteriophage of the present invention: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via an S tag; and one or more bacteriophages selected from the group consisting of the bacteriophages of (1) to (3) below: (1) (A) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, and (b) is deficient in an integrase gene and / or an immunity repressor gene, or (B) the bacteriophage of (A), wherein the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via a tag; (2) (C) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, and (b) is deficient in an integrase gene and / or an immunity repressor gene; (b) a bacteriophage comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified as BP-03918, and deficient in an integrase gene and / or an immunity repressor gene, or (D) a bacteriophage according to (C), wherein the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via a tag;and (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519;

[0127] The bacteriophage (#123 parent strain) identified by the accession number NITE BP-03519 has been internationally deposited by the applicant with the Patent Microorganisms Deposit Center of the National Institute of Technology and Evaluation (ROI 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818), an international depository authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, on August 26, 2021 (accession number NITE BP-03519).

[0128] In one embodiment, the pharmaceutical composition of the present invention comprises the following modified bacteriophage of the present invention: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage being linked to an S tag, and one or more bacteriophages selected from the group consisting of the bacteriophages (1) to (3) below: (1) (A) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage being linked to an S tag (B) a bacteriophage according to (A), wherein the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via a tag; (2) (C) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage specified by NITE Accession No. BP-03918, or (D) a bacteriophage according to (C), wherein the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via a tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519.

[0129] In one embodiment, the pharmaceutical composition of the present invention comprises the following modified bacteriophage of the present invention: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to an S tag, and one or more bacteriophages selected from the group consisting of the bacteriophages (1) to (3) below: (1) (A) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to an S tag (2) (C) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, or (D) A bacteriophage of (C), the capsid of which is linked to a cell membrane-permeable peptide via a tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519.

[0130] In one embodiment, the pharmaceutical composition of the present invention comprises the following modified bacteriophage of the present invention: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage identified by accession number NITE BP-03513 or a subculture strain thereof, wherein the capsid of the bacteriophage or the subculture strain thereof is linked to an S tag, and one or more bacteriophages selected from the group consisting of the bacteriophages (1) to (3) below: (1) (A) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being identified by accession number NITE BP-03513 or a subculture strain thereof, wherein the capsid of the bacteriophage or the subculture strain thereof is linked to an S tag (B) a bacteriophage specified by (A), wherein the capsid of the bacteriophage or its subculture strain is further linked to a cell membrane-permeable peptide via a tag; (2) (C) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is specified by accession number NITE BP-03918, or its subculture strain, or (D) a bacteriophage specified by (C), wherein the capsid of the bacteriophage or its subculture strain is further linked to a cell membrane-permeable peptide via a tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being identified by accession number NITE BP-03519, or a passage strain thereof.

[0131] In one embodiment, the pharmaceutical composition of the present invention comprises the following modified bacteriophage of the present invention: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag, and one or more bacteriophages selected from the group consisting of the bacteriophages (1) to (3) below: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by NITE Accession No. BP-03918, and the capsid of the bacteriophage is linked to Inv3 via an S tag; and (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by NITE Accession No. BP-03519.

[0132] In one embodiment, the pharmaceutical composition of the present invention comprises the following modified bacteriophage of the present invention: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage identified by accession number NITE BP-03513 or a subculture strain thereof, wherein the capsid of the bacteriophage or the subculture strain thereof is linked to Inv3 via an S tag, and one or more bacteriophages selected from the group consisting of the bacteriophages (1) to (3) below: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage identified by accession number NITE BP-03513 or a subculture strain thereof, wherein the capsid of the bacteriophage or the subculture strain thereof is linked to Inv3 via an S tag, (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage specified by accession number NITE BP-03918 or a passaged strain thereof, and the capsid of the bacteriophage or the passaged strain thereof is linked to Inv3 via an S tag; and (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage specified by accession number NITE BP-03519 or a passaged strain thereof.

[0133] In one embodiment, the pharmaceutical composition of the present invention comprises the following modified bacteriophage of the present invention: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and the capsid of the bacteriophage is linked to Inv3 via an S tag, and one or more bacteriophages selected from the group consisting of the bacteriophages (1) to (3) below: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and the capsid of the bacteriophage is linked to Inv3 via an S tag (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by NITE Accession No. BP-03514; and (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by NITE Accession No. BP-03519.

[0134] In one embodiment, the pharmaceutical composition of the present invention comprises the following modified bacteriophage of the present invention: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage identified by accession number NITE BP-03918 or a subculture strain thereof, wherein the capsid of the bacteriophage or the subculture strain thereof is linked to Inv3 via an S tag, and one or more bacteriophages selected from the group consisting of the following bacteriophages (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being identified by accession number NITE BP-03918 or a subculture strain thereof, wherein the capsid of the bacteriophage or the subculture strain thereof is linked to Inv3 via an S tag, (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage specified by NITE Accession No. BP-03513 or a subculture strain thereof, and wherein the capsid of the bacteriophage or the subculture strain thereof is linked to Inv3 via an S tag; (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage specified by NITE Accession No. BP-03519 or a subculture strain thereof.

[0135] The pharmaceutical compositions of the present invention can be prepared by commonly used methods using excipients commonly used in the art, i.e., pharmaceutical excipients and pharmaceutical carriers. Examples of dosage forms of these pharmaceutical compositions include parenteral preparations such as injections, infusions, powder inhalants, and nebulizers, which can be administered intravenously or via pulmonary administration. When formulating the compositions, excipients, carriers, or additives appropriate for these dosage forms can be used within a pharmaceutically acceptable range. For example, the pharmaceutical compositions of the present invention can be produced by mixing a bacteriophage with a pharmaceutically acceptable excipient, or by suspending a bacteriophage in a pharmaceutically acceptable excipient.

[0136] The pharmaceutical compositions of the present invention include those containing at least two types of bacteriophages and a pharmaceutically acceptable excipient.

[0137] The pharmaceutical compositions of the present invention include those containing three types of bacteriophages and a pharmaceutically acceptable excipient.

[0138] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of (1) to (3) below and a pharmaceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, and (b) is deficient in the integrase gene and / or the immunity repressor gene; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, A bacteriophage comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified as BP-03918, and (b) lacking an integrase gene and / or an immunity repressor gene, wherein the capsid of the bacteriophage is linked to Inv3 via an S tag.

[0139] In the above-mentioned embodiment, the pharmaceutical composition of the present invention includes the following bacteriophages (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and the capsid of the bacteriophage is linked to Inv3 via an S tag.

[0140] In the above-mentioned embodiment, the pharmaceutical composition of the present invention contains the following bacteriophages (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and the capsid of the bacteriophage is linked to Inv3 via an S tag.

[0141] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of the following (1) to (3) and a pharmaceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage identified by accession number NITE BP-03513 or a subcultured strain thereof, and the capsid of the bacteriophage or the subcultured strain thereof is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage identified by accession number NITE BP-03514 or a subcultured strain thereof; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage identified by accession number NITE BP-03918 or a subcultured strain thereof, and the capsid of the bacteriophage or the subcultured strain thereof is linked to Inv3 via an S tag.

[0142] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of (1) to (3) below and a pharmaceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag; A bacteriophage comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified as BP-03519.

[0143] In the above embodiment, the pharmaceutical composition of the present invention includes the following bacteriophages (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage identified by NITE Accession No. BP-03519.

[0144] In the above embodiment, the pharmaceutical composition of the present invention contains the following bacteriophages (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by NITE Accession Number BP-03519.

[0145] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising the bacteriophage of any one of (1) to (3) below and a pharmaceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage identified by accession number NITE BP-03513 or a subculture strain thereof, and the capsid of the bacteriophage or the subculture strain thereof being linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage identified by accession number NITE BP-03513 or a subculture strain thereof, (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage identified by accession number NITE BP-03519 or a subculture strain thereof, wherein the capsid of the bacteriophage or the subculture strain thereof is linked to Inv3 via an S tag; and

[0146] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of (1) to (3) below and a pharmaceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, and (b) is deficient in the integrase gene and / or the immunity repressor gene; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519.

[0147] In the above-mentioned embodiment, the pharmaceutical composition of the present invention includes the following bacteriophages (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519.

[0148] In the above-mentioned embodiment, the pharmaceutical composition of the present invention contains the following bacteriophages (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519.

[0149] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of the following (1) to (3) and a pharmaceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage identified by accession number NITE BP-03513 or a subcultured strain thereof, and the capsid of the bacteriophage or the subcultured strain thereof is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage identified by accession number NITE BP-03514 or a subcultured strain thereof; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being identified by accession number NITE BP-03519, or a passage strain thereof.

[0150] The pharmaceutical composition of the present invention also includes mutant strains of the bacteriophage identified by any of the above-mentioned accession numbers NITE BP-03918, NITE BP-03513, NITE BP-03514, and NITE BP-03519, so long as they have lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare. Specifically, the pharmaceutical composition of the present invention may contain the following mutant strains of bacteriophage: (1) (A) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof has been modified in the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918, and (b) the bacteriophage is deficient in an integrase gene and / or an immunity repressor gene, or (B) the capsid of the bacteriophage (A) is further linked to a cell membrane-permeable peptide via a tag; (2) (C) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof has been modified in the nucleic acid sequence of the genome of the bacteriophage identified by Accession No. NITE BP-03513, and (b) the bacteriophage is deficient in an integrase gene and / or an immunity repressor gene, or (D) the capsid of the bacteriophage (C) is further linked to a cell membrane-permeable peptide via a tag;(3) (E) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof has been modified in the nucleic acid sequence of the genome of the bacteriophage identified by Accession No. NITE BP-03514, and (b) the bacteriophage is deficient in an integrase gene and / or an immunity repressor gene, or (F) the capsid of the bacteriophage (E) is further linked to a cell membrane-permeable peptide via a tag; and / or (4) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof has been modified in the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519;

[0151] With regard to the above-mentioned modifications such as deletion, substitution, insertion or addition of bases, multiple modifications may be consecutive, or multiple modifications may be present at different positions.

[0152] The pharmaceutical composition of the present invention also includes passaged strains of the bacteriophage identified by any of the above-mentioned accession numbers NITE BP-03918, NITE BP-03513, NITE BP-03514, and NITE BP-03519, so long as they have lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare.

[0153] The effective dose, number of doses, and duration of administration vary depending on the purpose of administration (therapeutic or preventive), the severity and age of the subject, the dosage form of the preparation used, the titer of the bacteriophage, etc. For example, the effective dose of a single bacteriophage or the total effective dose of two or more bacteriophages is 10 ４ ~10 １４ The dosage ratio of two or more bacteriophages can be adjusted appropriately depending on the severity of symptoms and age of the patient, the dosage form of the preparation used, the titer of the bacteriophage, etc. For example, when a pharmaceutical composition contains three bacteriophages, each bacteriophage is used in approximately equal amounts (e.g., 3.3 × 10 ３ ~3.3 x 10 １３ pfu), or each bacteriophage may be contained in a different ratio.

[0154] The pharmaceutical composition of the present invention can be used as an agent for preventing or treating NTM diseases, such as MAC disease or pulmonary MAC disease.

[0155] As used herein, "treatment" means at least partial improvement of symptoms of NTM disease (e.g., cough, sputum, bloody sputum, fever, dyspnea, fatigue, pulmonary nodules, bronchiectasis, etc.), arrest of the progression or worsening of NTM disease including negative sputum culture, complete cure, etc. As used herein, "prevention" means preventing a subject not suffering from NTM disease from contracting NTM disease, preventing the recurrence of NTM disease, etc.

[0156] The present invention includes a pharmaceutical composition for preventing or treating NTM diseases, such as MAC disease or pulmonary MAC disease, which comprises the bacteriophage or modified bacteriophage of the present invention or the pharmaceutical composition of the present invention.

[0157] The present invention also includes a method for preventing or treating an NTM disease, e.g., MAC disease or pulmonary MAC disease, in a subject, comprising administering a therapeutically effective amount of a bacteriophage or modified bacteriophage of the present invention or a pharmaceutical composition of the present invention. In one embodiment, at least two, e.g., three, types of bacteriophages or modified bacteriophages of the present invention can be administered to a subject, and the multiple bacteriophages can be administered simultaneously or separately.

[0158] Furthermore, the present invention includes the bacteriophage or modified bacteriophage of the present invention, or the pharmaceutical composition of the present invention for use in the prevention or treatment of NTM diseases, such as MAC disease or pulmonary MAC disease. Furthermore, the present invention includes use of the bacteriophage or modified bacteriophage of the present invention, or the pharmaceutical composition of the present invention in the manufacture of a pharmaceutical composition for the prevention or treatment of NTM diseases, such as MAC disease or pulmonary MAC disease.

[0159] In the present invention, the subject to which the bacteriophage is administered is not limited as long as it is a mammal, and examples thereof include mice, rats, dogs, pigs, monkeys, humans, etc. For example, the bacteriophage is administered to subjects diagnosed with NTM disease or subjects at risk of contracting NTM disease.

[0160] Furthermore, the bacteriophage or modified bacteriophage of the present invention or the pharmaceutical composition of the present invention may be used or administered in combination with other ingredients that are effective in the treatment or prevention of NTM disease, and the bacteriophage or modified bacteriophage of the present invention or the pharmaceutical composition of the present invention may be provided as a combined medicine with such other active ingredients.

[0161] Having generally described the invention, reference is now made to specific examples to provide a further understanding thereof, which are intended for purposes of illustration and not as limitations of the invention.

[0162] Unless otherwise specified, experiments using commercially available kits or reagents were conducted according to the accompanying protocols. For convenience, concentrations are expressed as M (mol / L). For example, a 1M sodium hydroxide solution means a 1 mol / L sodium hydroxide solution.

[0163] Example 1: Preparation of a novel bacteriophage A #63 freeze-thaw resistant strain was obtained by the following method using the #63 lysogen gene-deficient strain (accession number NITE BP-03517) as a parent phage.

[0164] Phage buffer (1 mM CaCl ２ , 10 mM MgSO ４ To the #63 lysogen gene-deficient strain, ethyl methanesulfonate (Nacalai Tesque) was added to a final concentration of 33 mM in 10 mM Tris-HCl (pH 7.5) containing 68.4 mM NaCl, and the mixture was incubated at 37°C for 1 hour. The reaction solution was mixed with Mycobacterium smegmatis in soft agar, layered on a 7H10 plate (19 g / L Middlebrook 7H10 Agar (Difco), 6.3 g / L glycerol, 100 mL / L Middlebrook OADC Enrichment (Difco)), and incubated overnight at 37°C. After incubation, phage buffer was added to the plate, and the supernatant was collected and filtered through a 0.22 μm filter. The filtered solution was frozen in a -80°C freezer and then thawed at 4°C. This solution was mixed with Mycobacterium smegmatis in soft agar and cultured in the same manner as above, and the supernatant was filtered. The above procedure constituted one cycle, and a total of 12 cycles were performed.

[0165] The supernatant after 12 cycles was cultured in the same manner as above, and freeze-thaw resistant strain #63 was isolated from a single plaque. The accession number of this freeze-thaw resistant strain #63 is NITE BP-03918.

[0166] Example 2: Freeze-thaw resistance activity of #63 freeze-thaw resistant strain The freeze-thaw resistance activity of #63 freeze-thaw resistant strain obtained in Example 1 was confirmed by the following method.

[0167] Bacterial colonies of Mycobacterium smegmatis grown on a 7H10 plate were scraped off and suspended in LB (Luria-Bertani) medium, followed by expansion culture at 37° C. After culture, the #63 lysogen gene-deficient strain or the #63 freeze-thaw resistant strain was added, and culture was continued at 37° C. for approximately one day.

[0168] The supernatant after the culture was filtered through a 0.22 μm filter to obtain each phage lysate, which was then purified by ultracentrifugation to obtain each phage solution.

[0169] Purification by ultracentrifugation: Phage lysates from the #63 lysogeny gene-deficient strain and the #63 freeze-thaw-resistant strain were added with DNase I and RNase A to a final concentration of 1 μg / mL, respectively, and incubated at room temperature for 30 minutes. NaCl was added to a final concentration of 1 M, followed by dissolution and incubation on ice or in a refrigerator for 1 hour. Polyethylene glycol 8000 was added to a final concentration of 10% w / v, followed by incubation on ice or in a refrigerator for 2 hours. The supernatant was removed by centrifugation at 11,000 g for 10 minutes at 4°C, and the precipitate was suspended and recovered in phage buffer. The recovered solution was gently dissolved in 0.5 g of cesium chloride per mL and transferred to an ultracentrifugation container. Cesium chloride solutions at concentrations of 1.45 g / mL, 1.5 g / mL, and 1.7 g / mL were sequentially injected into the bottom of the container using a syringe. The band containing the phage was collected by centrifugation at 22,000 g for 2 hours at 4° C. The solution containing the collected phage was replaced with a phage buffer by dialysis to obtain a phage solution.

[0170] The phage solution of the #63 lysogen gene-deficient strain or the #63 freeze-thaw resistant strain was diluted to 10 ７ The concentration was adjusted to about pfu / mL. For each phage solution, a sample stored at 4°C and a sample frozen at -80°C for 1 hour and then stored at 4°C were prepared.

[0171] A bacterial colony of Mycobacterium smegmatis grown on a 7H10 plate was scraped and inoculated into 7H9 medium (4.7 g / L Middlebrook 7H9 Broth (Difco), 100 mL / L Middlebrook OADC Enrichment (Difco), 0.5 g / L polyoxyethylene sorbitan monooleate) and cultured for 3 days. The culture was washed three times with 7H9 medium minus polyoxyethylene sorbitan monooleate to prepare a bacterial suspension.

[0172] 300 μL of the bacterial suspension was mixed with 3 mL of 7H9 medium containing 0.6% soft agar (4.7 g / L Middlebrook 7H9 Broth (Difco), 6 g / L agarose, 100 mL / L Middlebrook OADC Enrichment (Difco)), and the mixture was layered on a 7H10 plate.

[0173] The samples of the #63 lysogen gene-deficient strain and the #63 freeze-thaw resistant strain prepared as described above were repeatedly diluted 10-fold with phage buffer to obtain a dilution series of six concentrations in total. After confirming that the soft agar had completely solidified, 2.5 μL of each sample was added dropwise and cultured at 37°C for 24 hours.

[0174] The results are shown in Figure 1. The pfu of the #63 lysogen gene-deficient strain (No. 63Δrep, int) was reduced by about 1000-fold by the freeze-thaw procedure, while the pfu of the #63 freeze-thaw resistant strain (No. 63Δrep, int_R12-23) remained almost the same.

[0175] Example 3: Preparation of capsid-modified phages Capsid-modified phages were prepared by the following method.

[0176] For the purpose of expressing an S-tag fusion capsid-forming protein or an S-tag and Inv3 fusion capsid-forming protein in response to promoter activity, plasmids were prepared in which a gene encoding each of the capsid-forming proteins obtained from the genomic sequences of the D29 lysogen gene-deficient strain (accession number NITE BP-03513), the B1 lysogen gene-deficient strain (accession number NITE BP-03514), and the #63 freeze-thaw resistant strain obtained in Example 1 was ligated and inserted upstream or downstream of the gene encoding each of the capsid-forming proteins.

[0177] The plasmids were introduced into Mycobacterium smegmatis by electroporation to express various fusion capsid-constituting proteins. During expression of the fusion capsid proteins, the cells were infected with the corresponding phages as a source of capsid proteins, thereby producing capsid-modified phages containing capsids containing a mixture of fusion capsid-constituting proteins at a certain ratio. The capsid-modified phages were purified using a resin that binds to the S tag (Strep-Tactin XT, IBA GmbH).

[0178] Using a similar method, the following various capsid-modified phages were obtained: - S-tag fused D29 lysogen gene-deficient strain (a modified phage having a capsid in which an S-tag is fused to the N-terminus or C-terminus of the capsid-forming protein; these are referred to as the S-tag fused D29 lysogen gene-deficient strain (N) and the S-tag fused D29 lysogen gene-deficient strain (C), respectively) - S-tag and Inv3 fused D29 lysogen gene-deficient strain (a modified phage having a capsid in which an S-tag and an Inv3 sequence are fused to the C-terminus of the capsid-forming protein) - S-tag fused #63 freeze-thaw resistant strain (a modified phage having a capsid in which an S-tag and an Inv3 sequence are fused to the C-terminus of the capsid-forming protein) - S-tag and Inv3 fused #63 freeze-thaw resistant strain (a modified phage having a capsid in which an S-tag and an Inv3 sequence are fused to the C-terminus of the capsid-forming protein) - S-tag and Inv3-fused B1 lysogen gene-deficient strain (a modified phage having a capsid in which an S-tag and Inv3 sequence are fused to the C-terminus of the capsid-constituting protein)

[0179] Example 4: Lytic activity of capsid-modified phages The lytic activity of capsid-modified phages was evaluated by the following method.

[0180] Mycobacterium avium KCH-ASGF-MA-05 or clarithromycin-resistant Mycobacterium intracellulare KCH-ASGF-MAC-475 strain (provided by Kinki Central Respiratory Center) grown on a 7H10 plate at 37°C for approximately two weeks was scraped and suspended in 7H9 medium (4.7 g / L Middlebrook 7H9 Broth (Difco)). After suspension, the bacterial suspension was adjusted in 7H9 medium to a final OD600 of 1.5 using a spectrophotometer.

[0181] 300 μL of the bacterial suspension was mixed with 3 mL of 7H9 medium containing 0.6% soft agar (4.7 g / L Middlebrook 7H9 Broth (Difco), 6 g / L agarose, 100 mL / L Middlebrook OADC Enrichment (Difco)), and the mixture was layered on a 7H10 plate.

[0182] After confirming that the soft agar had completely solidified, the S-tag and Inv3 fused D29 lysogen gene-deficient strain (D29Δint-Stag-Inv3), the S-tag and Inv3 fused B1 lysogen gene-deficient strain (B1Δrep,int-Stag-Inv3), the S-tag and Inv3 fused #63 freeze-thaw resistant strain (No. 63Δrep,int_R12-23-Stag-Inv3) obtained in Example 3, or the #63 freeze-thaw resistant strain (No. 63Δrep,int_R12-23) obtained in Example 2 was diluted with phage buffer for 10 min. １０ pfu / mL to 10 ５ The solution was diluted to six concentrations up to pfu / mL, and 2.5 μL of each was dropped onto the plates. The plates were cultured at 37°C for approximately 9 days. Preliminary studies have confirmed that adding only the phage buffer to solidified soft agar does not result in plaque formation.

[0183] The results are shown in Figure 2. Plaques formed by all phages were observed against both Mycobacterium avium KCH-ASGF-MA-05 (A) and clarithromycin-resistant Mycobacterium intracellulare KCH-ASGF-MAC-475 strain (B). These results confirm that the S-tag and Inv3-fused D29 lysogen gene-deficient strain, the S-tag and Inv3-fused B1 lysogen gene-deficient strain, the S-tag and Inv3-fused #63 freeze-thaw-resistant strain, and the #63 freeze-thaw-resistant strain obtained in Example 2 have lytic activity against Mycobacterium avium and Mycobacterium intracellulare.

[0184] Example 5: Bactericidal activity of capsid-modified phages against intracellularly infected bacteria Various phage solutions were obtained by the method of Example 2 or Example 3.

[0185] Two strains of Mycobacterium avium (KCH-ASGF-MA-05 or KCH-ASGF-MA-12 (provided by the Kinki Central Respiratory Center)) were cultured on a 7H10 plate at 37°C for 4 days to approximately 2 weeks, then scraped and suspended in cell culture medium. After suspension, the bacterial suspension was adjusted using a spectrophotometer to a final OD600 of 0.07. Bronchoalveolar lavage fluid (BALF solution) was collected from the mice, and mouse alveolar macrophages were prepared from the BALF solution and incubated at 37°C in 5% CO ２ After culturing, the cells were pooled and plated in a 96-well plate at 10 cells per well. ４ The cells were seeded at 37°C and 5% CO ２ After culturing in the presence of amikacin, the supernatant was replaced with the bacterial suspension. After 2 hours, the supernatant of the bacterial suspension was removed, and the cells were washed three times with phosphate-buffered saline containing amikacin. Cell culture medium was then added and the cells were cultured for three days. After culturing, the supernatant was removed, and various phage solutions prepared by the above method were added at a final concentration of 3.0 × 10 ８ pfu / mL (KCH-ASGF-MA-05), or 1.4 × 10 ９After culturing for 3 days, the supernatant was removed, the cell disruption solution was added, the cells were suspended, and the solution was recovered and the CFU was counted.

[0186] The results are shown in Figure 3. All bacteriophage strains had bactericidal activity against Mycobacterium avium KCH-ASGF-MA-05 cells, but the S-tag-fused D29 lysogen gene-deficient strain (N) and the S-tag-fused D29 lysogen gene-deficient strain (C) showed higher bactericidal activity than the D29 lysogen gene-deficient strain (Figure 3A). Furthermore, both bacteriophage strains had bactericidal activity against Mycobacterium avium KCH-ASGF-MA-12 cells, but the S-tag-fused #63 freeze-thaw-resistant strain showed higher bactericidal activity than the #63 freeze-thaw-resistant strain (Figure 3B). These results confirm that the S-tag-fused D29 lysogen gene-deficient strain (N), the S-tag-fused D29 lysogen gene-deficient strain (C), and the S-tag-fused #63 freeze-thaw-resistant strain have high lytic activity against Mycobacterium avium.

[0187] Example 6: Bactericidal activity of each bacteriophage cocktail against intracellularly infected bacteria The B1 lysogen gene-deficient strain (accession number NITE BP-03514) and the #123 parent strain (accession number NITE BP-03519) were purified by ultracentrifugation. Using the method described in Example 5, the bactericidal activity of various bacteriophage cocktails against intracellularly infected Mycobacterium avium KCH-ASGF-MA-05 was evaluated.

[0188] The cocktail of bacteriophages consisted of the following three types, each at a final concentration of 1.0 × 10 ９ This cocktail was prepared by mixing equal amounts of three phages (pfu / mL): Cocktail #1: S-tag and Inv3 fusion D29 lysogen gene-deficient strain, B1 lysogen gene-deficient strain, and #123 parent strain Cocktail #2: S-tag and Inv3 fusion D29 lysogen gene-deficient strain, S-tag and Inv3 fusion #63 freeze-thaw resistant strain, and B1 lysogen gene-deficient strain Cocktail #3: S-tag and Inv3 fusion D29 lysogen gene-deficient strain, S-tag and Inv3 fusion #63 freeze-thaw resistant strain, and #123 parent strain

[0189] The results are shown in Figure 4. All of the bacteriophage cocktails tested were shown to have bactericidal activity against intracellularly infected Mycobacterium avium KCH-ASGF-MA-05.

[0190] Example 7: Bactericidal activity of capsid-modified phages against intracellularly infected bacteria The D29 lysogen gene-deficient strain (accession number NITE BP-03513) and the B1 lysogen gene-deficient strain (accession number NITE BP-03514) were purified by ultracentrifugation to obtain respective phage solutions. The S-tag and Inv3-fused D29 lysogen gene-deficient strain and the S-tag and Inv3-fused B1 lysogen gene-deficient strain were purified by the method of Example 3 to obtain respective phage solutions.

[0191] The bactericidal activity of various bacteriophages against intracellularly infected Mycobacterium avium KCH-ASGF-MA-05 was evaluated using the method described in Example 5. The final concentration of bacteriophages was 3.0 × 10 ９ The solution was added so as to give a concentration of pfu / mL.

[0192] The results are shown in Figure 5. All bacteriophage strains had bactericidal activity against intracellularly infected Mycobacterium avium KCH-ASGF-MA-05. The S-tag and Inv3-fused D29 lysogen gene-deficient strain exhibited higher bactericidal activity than the D29 lysogen gene-deficient strain (Figure 5A), and the S-tag and Inv3-fused B1 lysogen gene-deficient strain exhibited higher bactericidal activity than the B1 lysogen gene-deficient strain (Figure 5B).

[0193] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0194] The bacteriophage of the present invention and the modified bacteriophage of the present invention, as well as the pharmaceutical composition, are expected to be useful in the prevention or treatment of nontuberculous mycobacterial diseases, such as MAC disease or pulmonary MAC disease.

[0195] [Accession Number] Accession Number NITE BP-03513 (bacteriophage D29 lysogen gene-deficient strain D29Δ, deposited on August 26, 2021) Accession Number NITE BP-03514 (bacteriophage B1 lysogen gene-deficient strain B1Δ, deposited on August 26, 2021) Accession Number NITE BP-03519 (bacteriophage #123 parent strain, deposited on August 26, 2021) Accession Number NITE BP-03918 (bacteriophage #63 freeze-thaw resistant strain, deposited on June 22, 2023)

[0196] SEQ ID NO: 1: Synthetic construct (Inv3) SEQ ID NO: 2: Synthetic construct (S tag)

Claims

1. A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-03513, and (b) lacking an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to an S tag.

2. The bacteriophage of claim 1, wherein the genome of said bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513.

3. The bacteriophage according to claim 1 or 2, wherein the genome of said bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-03513.

4. A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is a bacteriophage identified by the accession number NITE BP-03513 or a subcultured strain thereof, and the capsid of the bacteriophage or the subcultured strain thereof is linked to an S tag.

5. A bacteriophage according to any one of claims 1 to 4, wherein the capsid is further linked to a cell membrane-permeable peptide via an S tag.

6. The bacteriophage of claim 5, wherein the cell membrane-permeable peptide comprises Inv3.

7. A pharmaceutical composition comprising a bacteriophage according to any one of claims 1 to 6 and a pharma- ceutically acceptable excipient.

8. The pharmaceutical composition according to claim 7, further comprising one or more strains of bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare.

9. A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene.

10. The bacteriophage of claim 9, which is resistant to freezing and thawing.

11. The bacteriophage of claim 9, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03918.

12. The bacteriophage according to claim 9 or claim 11, wherein the genome of said bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-03918.

13. A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage identified by the accession number NITE BP-03918 or a subcultured strain thereof.

14. The bacteriophage of any one of claims 9 to 13, wherein the capsid of the bacteriophage is linked to a tag.

15. A bacteriophage according to any one of claims 9 to 13, wherein the capsid of the bacteriophage is linked to a cell membrane-permeable peptide via a tag.

16. A bacteriophage according to claim 14 or claim 15, wherein the tag is an S tag.

17. The bacteriophage of claim 15 or claim 16, wherein the cell membrane-permeable peptide comprises Inv3.

18. A pharmaceutical composition comprising a bacteriophage according to any one of claims 9 to 17 and a pharma- ceutically acceptable excipient.

19. The pharmaceutical composition of claim 18, further comprising one or more strains of bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare.

20. The pharmaceutical composition according to claim 7, further comprising one or more bacteriophages selected from the group consisting of the bacteriophage according to any one of claims 1 to 6 and the bacteriophages (1) to (3) below: (1) (A) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-03514, and (b) is deficient in an integrase gene and / or an immunity repressor gene, or (B) the bacteriophage according to (A), the capsid of which is linked to a cell membrane-permeable peptide via a tag; (2) (C) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03918, and (b) is deficient in an integrase gene and / or an immunity repressor gene, or (D) a bacteriophage according to (C), the capsid of which is linked to a cell membrane-permeable peptide via a tag; and (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03918, A bacteriophage comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified in BP-03519.

21. The pharmaceutical composition according to claim 7, further comprising one or more bacteriophages selected from the group consisting of the bacteriophage according to any one of claims 1 to 6 and the bacteriophages (1) to (3) below: (1) (A) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-03514, or (B) the bacteriophage (A), the capsid of which is linked to a cell membrane-permeable peptide via a tag; (2) (C) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises a nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03918, or (D) a bacteriophage of (C), the capsid of which is linked to a cell membrane-permeable peptide via a tag; and (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises a nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03519.

22. The pharmaceutical composition according to claim 7, further comprising one or more bacteriophages selected from the group consisting of the bacteriophage according to any one of claims 1 to 6 and the bacteriophages (1) to (3) below: (1) (A) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-03514, or (B) the bacteriophage (A), the capsid of which is linked to a cell membrane-permeable peptide via a tag; (2) (C) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03918, or (D) a bacteriophage of (C), the capsid of which is linked to a cell membrane-permeable peptide via a tag; and (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03519.

23. The pharmaceutical composition according to claim 7, further comprising one or more bacteriophages selected from the group consisting of the bacteriophage according to any one of claims 1 to 6 and the bacteriophages (1) to (3) below: (1) (A) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage identified by the accession number NITE BP-03514 or a subculture strain thereof, or (B) the bacteriophage (A), the capsid of the bacteriophage or a subculture strain thereof being linked to a cell membrane-permeable peptide via a tag; (2) (C) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage identified by accession number NITE BP-03918, or a subcultured strain thereof, or (D) a bacteriophage of (C), the capsid of the bacteriophage or a subcultured strain thereof being linked to a cell membrane-permeable peptide via a tag; and (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage identified by accession number NITE BP-03519, or a subcultured strain thereof.

24. A pharmaceutical composition comprising a bacteriophage of any one of (1) to (3) below and a pharma- ceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage specified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage specified by accession number NITE BP-03513, (a) a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage specified by NITE BP-03514, and (b) a lack of an integrase gene and / or an immunity repressor gene; and (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage specified by NITE BP-03918, and (b) a lack of an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag.

25. The pharmaceutical composition according to claim 24, comprising a bacteriophage of any one of (1) to (3) below: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage comprising a nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage being linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage comprising a nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-03918, and the capsid of the bacteriophage is linked to Inv3 via an S tag.

26. The pharmaceutical composition according to claim 24 or 25, comprising a bacteriophage of any one of the following (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-03918, and the capsid of the bacteriophage is linked to Inv3 via an S tag.

27. A pharmaceutical composition comprising a bacteriophage of any one of (1) to (3) below and a pharma- ceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage specified by accession number NITE BP-03513 or a subcultured strain thereof, and the capsid of the bacteriophage or a subcultured strain thereof is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage specified by accession number NITE BP-03514 or a subcultured strain thereof; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage identified by the accession number NITE BP-03918 or a subcultured strain thereof, and the capsid of the bacteriophage or the subcultured strain thereof is linked to Inv3 via an S tag.

28. A pharmaceutical composition comprising a bacteriophage of any one of (1) to (3) below and a pharma- ceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage specified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage specified by accession number NITE BP-03513, (b) a bacteriophage that is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag; and (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage comprising a nucleic acid sequence that has 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03519.

29. The pharmaceutical composition according to claim 28, comprising a bacteriophage of any one of (1) to (3) below: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage comprising a nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-03513, and the capsid of the bacteriophage being linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage comprising a nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-03513, and the capsid of the bacteriophage being linked to Inv3 via an S tag; (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises a nucleic acid sequence of the genome of a bacteriophage specified by NITE Accession No. BP-03519.

30. The pharmaceutical composition according to claim 28 or 29, comprising a bacteriophage of any one of the following (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of a nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of a nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03519.

31. A pharmaceutical composition comprising a bacteriophage of any one of (1) to (3) below and a pharma- ceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage identified by accession number NITE BP-03513 or a subculture strain thereof, and the capsid of the bacteriophage or the subculture strain thereof being linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage identified by accession number NITE BP-03513 or a subculture strain thereof, (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage specified by accession number NITE BP-03519 or a subculture strain thereof, wherein the capsid of the bacteriophage or the subculture strain thereof is linked to Inv3 via an S tag; and 32. A pharmaceutical composition comprising a bacteriophage of any one of (1) to (3) below and a pharma- ceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage specified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage specified by accession number NITE BP-03513, (b) a bacteriophage having a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by NITE BP-03514, and (b) lacking an integrase gene and / or an immunity repressor gene; and (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by NITE BP-03519.

33. The pharmaceutical composition according to claim 32, comprising a bacteriophage of any one of (1) to (3) below: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage comprising a nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and the capsid of the bacteriophage being linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage comprising a nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises a nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-03519.

34. The pharmaceutical composition according to claim 32 or 33, comprising a bacteriophage of any one of the following (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03513, and the capsid of the bacteriophage is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-03519.

35. A pharmaceutical composition comprising a bacteriophage of any one of (1) to (3) below and a pharma- ceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage specified by accession number NITE BP-03513 or a subcultured strain thereof, and the capsid of the bacteriophage or a subcultured strain thereof is linked to Inv3 via an S tag; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being a bacteriophage specified by accession number NITE BP-03514 or a subcultured strain thereof; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being identified by the accession number NITE BP-03519, or a passage strain thereof.

36. A pharmaceutical composition according to any one of claims 7, 8, 18, 19, and 20 to 35, which is a pharmaceutical composition for preventing or treating nontuberculous mycobacterial disease.

37. The pharmaceutical composition according to claim 36, wherein the nontuberculous mycobacterial disease is pulmonary mycobacterial infection.

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