Modified bacteriophage

Abstract

The present invention provides a bacteriophage having a bacteriolytic activity against Mycobacterium avium and / or Mycobacterium intracellularis, the bacteriophage having a genome containing a nucleic acid sequence represented by the genome of the bacteriophage specified by the preservation number NITE BP-03513 or NITE BP-03514 or the preservation number NITE BP-03918, the bacteriophage having a bacteriolytic activity against Mycobacterium avium and / or Mycobacterium intracellularis, and the bacteriophage having a bacteriolytic activity against Mycobacterium avium and / or Mycobacterium intracellularis. The capsid of the phage is connected with the cell-penetrating peptide through a tag.

Description

Technical Field

[0001] This invention relates to novel bacteriophages and modified bacteriophages, as well as the uses of these bacteriophages. Background Technology

[0002] Non-tuberculous mycobacteria (NTM) diseases are infections caused by mycobacteria other than Mycobacterium tuberculosis and Mycobacterium leprae. NTMs are environmentally native bacteria found in lakes, soil, etc., and cause respiratory symptoms through inhalation of aerosols. Examples of NTMs include: Mycobacterium avium (M. avium), Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium gordonae, Mycobacterium szulgai, Mycobacterium kansasii, and Mycobacterium genavense (Infection. 2004;32:257.). Infections caused by the Mycobacterium avium complex (MAC), which includes Mycobacterium avium and Mycobacterium intracellulare, are called MAC diseases, with lung infections being particularly known as pulmonary MAC diseases.

[0003] Treatment for bacterial infections, including NTM disease, typically involves multidrug therapy with macrolide antibiotics. However, in recent years, phage therapy utilizing the lytic activity of bacteriophages (also known 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 have shown 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. A bacteriophage infects specific bacteria, injecting its genome into the bacteria and using the host bacteria's metabolic mechanisms to reproduce progeny bacteriophages. Subsequently, the lysozyme carried by the bacteriophage genome destroys the bacterial cell wall, releasing the progeny bacteriophages, while the host bacteria die.

[0005] Bacteriophages can be broadly classified into virulent phages (lysogenic phages) and temperate phages (lysogenic phages) (Scientifica (Cairo). 2014; 2014:581639.). Virulent phages replicate without integrating into the host bacterial DNA. They reproduce progeny phages within the bacteria, causing bacterial lysis and releasing mature phage particles. On the other hand, temperate phages integrate into the host bacterial DNA after infecting bacteria and replicate along with it. Integration into the host bacterial DNA is harmless to the host bacteria, maintaining a lysogenic state (the state where phage DNA integrates into the host bacterial DNA and replicates along with it). However, if temperate phages are exposed to external stimuli such as ultraviolet light, they will release lysozyme, similar to virulent phages, causing the lysis of the host bacteria and the release of mature phage particles.

[0006] Phages that delete genes associated with lysogenic elements have also been reported. For example, phages that delete integrase genes have been reported (Nat Med. 2019; 25(5):730.) and phages that delete integrase genes and immunorepressor protein genes have been reported (mBio. 2021; 12(3):e00973.).

[0007] A bacteriophage consists of a head (a capsid containing the bacteriophage genome) and a tail. The bacteriophage capsid is created by synthesizing various capsid proteins within the host bacterium using the bacteriophage genome and assembling these capsid proteins. A bacteriophage with a target peptide or protein attached to its capsid can be created by introducing a plasmid containing a DNA sequence encoding a capsid protein and a DNA sequence encoding a target peptide or protein to the host bacterium. For example, it has been reported that when a recombinant host bacterium carrying a plasmid containing a DNA sequence encoding an S-tag peptide downstream of a DNA sequence encoding a capsid protein of wild-type TM4 bacteriophage is infected with wild-type TM4 bacteriophage, a modified TM4 bacteriophage with a capsid protein fused with an S-tag peptide can be obtained (Non-Patent Literature 1).

[0008] It is known that MAC can also infect cells. If bacteriophages can be effectively delivered into cells, a more potent therapeutic effect can be expected, as the bacteriophages can lyse intracellular MAC. As one of the effective means of delivering bacteriophages into cells, the utilization of cell penetrating peptides (CPP) has been explored (Non-Patent Literature 2).

[0009] As a CPP, there are many known sequences. For example, the peptide Inv3 contained in Mycobacterium cell entry protein (Mce1A), a protein derived from Mycobacterium tuberculosis, has been reported to have CPP activity (Non-Patent Literature 3). Existing technical documents Non-patent literature

[0010] Non-patent literature 1: Appl Environ Microbiol. 2013; 79(18):5608 Non-patent literature 2: Adv Drug Deliv Rev. 2021; 176:113864 Non-patent literature 3: Anal. Biochem. 2006; 353(1):7 Summary of the Invention The technical problem that the invention aims to solve

[0011] The technical problem of this invention is to provide a bacteriophage with lytic activity against Mycobacterium avium and / or intracellular Mycobacteria. Another technical problem of this invention is to provide means and methods, particularly bacteriophages and pharmaceutical compositions, for the prevention or treatment of nontuberculous mycobacterial diseases such as pulmonary MAC. Technical means to solve technical problems

[0012] Through persistent innovation and research in the preparation of bacteriophages, the inventors obtained a novel bacteriophage (Example 1) and discovered that this bacteriophage is resistant to freeze-thaw cycles (Example 2). Furthermore, a bacteriophage with a modified capsid was also obtained (Example 3). The novel and modified bacteriophages were found to have lytic activity against Mycobacterium avium and Mycobacterium intracellularis (Examples 4-5). Further, the inventors discovered that pharmaceutical compositions containing combinations of specific bacteriophage strains exhibit excellent bactericidal activity (Example 6). Based on the above insights, the present invention was completed.

[0013] That is, the present invention may include the following inventions as substances or methods that are useful in medicine or industry. [1] A bacteriophage, wherein the bacteriophage is a bacteriophage with lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) contains a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacks an integrase gene and / or an immunorepressor protein gene, the capsid of the bacteriophage being linked to an S tag. [2] The phage as described in [1], wherein the genome of the phage contains the nucleic acid sequence of the genome of the phage specified by the accession number NITE BP-03513. [3] The phage as described in [1] or [2], wherein the genome of the phage is composed of the nucleic acid sequence of the genome of the phage specified by the accession number NITE BP-03513. [4] A bacteriophage, wherein the bacteriophage is a bacteriophage with lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03513, and the capsid of the bacteriophage or its passage being attached to an S tag. [5] The phage as described in any one of [1] to [4], wherein the capsid is further linked to a cell-penetrating peptide via an S-tag. [6] The phage as described in [5], wherein the cell-penetrating peptide comprises Inv3. [7] A pharmaceutical composition comprising any one of [1] to [6] a bacteriophage and a pharmaceutically acceptable excipient. [8] The pharmaceutical composition as described in [7], wherein the pharmaceutical composition further comprises one or more strains of bacteriophages having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare.

[0014] [9] A bacteriophage, wherein the bacteriophage is a bacteriophage with lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) contains a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene.

[10] The phage as described in [9], wherein the phage is resistant to freeze-thaw cycles.

[11] The phage as described in [9], wherein the genome of the phage contains the nucleic acid sequence of the genome of the phage specified by the accession number NITE BP-03918.

[12] The phage as described in [9] or

[11] , wherein the genome of the phage is composed of the nucleic acid sequence of the genome of the phage specified by the accession number NITE BP-03918.

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

[14] The phage as described in any one of [9] to

[13] , wherein the capsid of the phage is attached to a tag.

[15] The phage as described in any one of [9] to

[13] , wherein the capsid of the phage is linked to a cell-penetrating peptide via a tag.

[16] The phage as described in

[14] or

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

[17] The phage as described in

[15] or

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

[18] A pharmaceutical composition comprising any one of the bacteriophages in [9] to

[17] and a pharmaceutically acceptable excipient.

[19] The pharmaceutical composition as described in

[18] , wherein the pharmaceutical composition further comprises one or more strains of bacteriophages having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare.

[0015]

[20] The pharmaceutical composition as described in [7], wherein the pharmaceutical composition further comprises any one of [1] to [6] and one or more phages selected from the group consisting of phages of (1) to (3) below: (1) (A) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514 and (b) lacks an integrase gene and / or an immunorepressor protein gene, or (B) a bacteriophage that is the bacteriophage of (A), the capsid of which is linked to a cell-penetrating peptide via a tag; (2) (C) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene; or (D) a bacteriophage that is (C) the bacteriophage, the capsid of which is linked to a cell-penetrating peptide via a tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage contains a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

[0016]

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

[0017]

[22] The pharmaceutical composition as described in [7], wherein the pharmaceutical composition further comprises any one of [1] to [6] and one or more phages selected from the group consisting of phages of (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 specified by accession number NITE BP-03514, or (B) a bacteriophage that is the bacteriophage of (A), the capsid of which is linked to a cell-penetrating 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 accession number NITE BP-03918; or (D) a bacteriophage that is (C) and whose capsid is linked to a cell-penetrating 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.

[0018]

[23] The pharmaceutical composition as described in [7], wherein the pharmaceutical composition further comprises any one of [1] to [6] and one or more phages selected from the group consisting of phages of (1) to (3) below: (1) (A) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03514, or (B) a bacteriophage being the bacteriophage of (A), the capsid of the bacteriophage or its passage being linked to a cell-penetrating peptide via a tag; (2) (C) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage or its passage specified in accession number NITE BP-03918, or (D) a bacteriophage being the bacteriophage of (C), the capsid of the bacteriophage or its passage being linked to a cell-penetrating peptide via a tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is the bacteriophage or its passage specified by accession number NITE BP-03519.

[0019]

[24] A pharmaceutical composition comprising the bacteriophages 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 the bacteriophage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacking an integrase gene and / or an immunorepressor protein gene, 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 which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514, and (b) lacks an integrase gene and / or an immunorepressor protein gene; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacking an integrase gene and / or an immunorepressor protein gene, the capsid of the bacteriophage being linked to Inv3 via an S tag.

[0020]

[25] The pharmaceutical composition as described in

[24] , wherein the pharmaceutical composition comprises the following (1) to (3): (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of the bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage containing 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, the genome of the bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, the capsid of the bacteriophage being linked to Inv3 via an S tag.

[0021]

[26] The pharmaceutical composition as described in

[24] or

[25] , wherein the pharmaceutical composition comprises the following (1) to (3): (1) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of said bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting 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, the genome of the bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03918, the capsid of the bacteriophage being linked to Inv3 via an S tag.

[0022]

[27] A pharmaceutical composition comprising the bacteriophages 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 the bacteriophage or its passage specified by accession number NITE BP-03513, the capsid of the bacteriophage or its passage being connected to Inv3 via an S tag. (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03918, the capsid of the bacteriophage or its passage being linked to Inv3 via an S tag.

[0023]

[28] A pharmaceutical composition comprising the bacteriophages 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 the bacteriophage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacking an integrase gene and / or an immunorepressor protein gene, 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 which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene, the capsid of which 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 contains a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

[0024]

[29] The pharmaceutical composition as described in

[28] , wherein the pharmaceutical composition comprises the following (1) to (3): (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of the bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, the capsid of said bacteriophage being 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 contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

[0025]

[30] The pharmaceutical composition as described in

[28] or

[29] , wherein the pharmaceutical composition comprises the following (1) to (3): (1) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of said bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, the capsid of said bacteriophage being 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 the accession number NITE BP-03519.

[0026]

[31] A pharmaceutical composition comprising the bacteriophages 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 the bacteriophage or its passage specified by accession number NITE BP-03513, the capsid of the bacteriophage or its passage being connected to Inv3 via an S tag. (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03918, the capsid of said bacteriophage or its passage being linked to Inv3 via an S-tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is the bacteriophage or its passage specified by accession number NITE BP-03519.

[0027]

[32] A pharmaceutical composition comprising the bacteriophages 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 the bacteriophage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacking an integrase gene and / or an immunorepressor protein gene, 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 which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514, and (b) lacks an integrase gene and / or an immunorepressor protein gene; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage contains a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

[0028]

[33] The pharmaceutical composition as described in

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

[0029]

[34] The pharmaceutical composition as described in

[32] or

[33] , wherein the pharmaceutical composition comprises the bacteriophages described in (1) to (3) below: (1) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of said bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting 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, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03519.

[0030]

[35] A pharmaceutical composition comprising the bacteriophages 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 the bacteriophage or its passage specified by accession number NITE BP-03513, the capsid of the bacteriophage or its passage being connected to Inv3 via an S tag. (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is the bacteriophage or its passage specified by accession number NITE BP-03519.

[0031]

[36] A pharmaceutical composition as described in any one of [7], [8],

[18] ,

[19] and

[20] to

[35] , wherein the pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of nontuberculous mycobacterial diseases.

[37] The pharmaceutical composition as described in

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

[0032] The bacteriophages and modified bacteriophages of the present invention, as well as the pharmaceutical compositions thereof, exhibit lytic activity against Mycobacterium avium and / or intracellular mycobacteria. The bacteriophages and modified bacteriophages of the present invention, as well as the pharmaceutical compositions thereof, are expected to be used for the prevention or treatment of nontuberculous mycobacterial diseases such as pulmonary MAC. Attached Figure Description

[0033] Figure 1 This is a photograph showing the results of a plaque formation assay comparing the effect of physical damage caused by freeze-thaw operations on the number of bacteriophages for strain #63 with a lysogen gene deletion (No.63Δrep,int) and strain #63 with freeze-thaw resistance (No.63Δrep,int_R12-23). Figure 2 The images show the lysogenic activity of the following strains against Mycobacterium avium KCH-ASGF-MA-05 (A) or clarithromycin-resistant Mycobacterium intracellularis KCH-ASGF-MAC-475 (B): 1. S-tag and Inv3 fusion D29 lysogen deletion strain (D29Δint-Stag-Inv3), 2. S-tag and Inv3 fusion B1 lysogen deletion strain (B1Δrep,int-Stag-Inv3), 3. S-tag and Inv3 fusion #63 freeze-thaw resistant strain (No.63Δrep,int_R12-23-Stag-Inv3), and 4. #63 freeze-thaw resistant strain (No.63Δrep,int_R12-23). Figure 3 Figure A shows the bactericidal activity of phages of the S-tag fusion D29 lysogen gene deletion strain (N), the S-tag fusion D29 lysogen gene deletion strain (C), and the D29 lysogen gene deletion strain against intracellularly infected Mycobacterium avium KCH-ASGF-MA-05. Figure 3 B is a graph showing the bactericidal activity of S-tag fusion #63 freeze-thaw resistant strain and #63 freeze-thaw resistant strain bacteriophages against intracellularly infected Mycobacterium avium KCH-ASGF-MA-12. The vertical axis represents the colony-forming unit concentration (CFU / mL). The horizontal bar represents the geometric mean. Figure 4 This is a graph showing the bactericidal activity of various bacteriophage mixtures against intracellularly infected Mycobacterium avium KCH-ASGF-MA-05. The vertical axis represents the colony-forming unit concentration (CFU / mL). The horizontal bar represents the geometric mean. Figure 5This graph shows the bactericidal activity of various bacteriophages against intracellularly infected Mycobacterium avium KCH-ASGF-MA-05. The vertical axis represents the colony-forming unit concentration (CFU / mL). The horizontal bar represents the geometric mean. This indicates that when the Tukey multiple comparison test is performed at a significance level of less than 1%, a significant difference (p<0.01) is confirmed between the groups. Detailed Implementation

[0034] This application claims priority to 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 now be described in detail. The following embodiments are illustrative examples and are not intended to limit the invention to these embodiments only. The invention can be practiced in various ways without departing from its spirit.

[0036] <1. The bacteriophage of the present invention> In one embodiment, the present invention provides a novel bacteriophage (hereinafter sometimes referred to as "the bacteriophage of the present invention"). The bacteriophage involved in the present invention is a bacteriophage with lytic activity against nontuberculous mycobacteria (NTM), particularly avian mycobacteria and / or intracellular mycobacteria that cause pulmonary MAC disease.

[0037] The freeze-thaw resistant strain #63, which is the bacteriophage of the present invention, was deposited by the applicant on June 22, 2023, with the Patent Microbial Collection Center of the Technical Base for Product Evaluation (Room 2-5-8122, Kazusa Kamata, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan) under the provisions of the Budapest Treaty on the Preservation of Patent Microorganisms (Accession No. NITE BP-03918).

[0038] In one embodiment, the bacteriophage of the present invention includes the following bacteriophages: A bacteriophage having lysing activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks a lysogenic gene.

[0039] In this specification, "identity" means the identity of a base sequence or amino acid sequence as determined by known techniques in the art. As a method for sequence alignment, generally disclosed 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), and the BLAST program (including BLASTP, BLASTN, BLASTX, TBLASTN, TBLASTX, etc.) (Journal of Molecular Biology. 1990; 215(3):403-10) can be used, but it is not limited to these. As a specific sequence alignment method, for example, it refers to the value of Identity obtained by searching using the NEEDLE program (Journal of Molecular Biology. 1970; 48(3):443-53) with default parameters. The parameters are shown below. Gap penalty=10 Extend penalty = 0.5 Matrix=EBLOSUM62

[0040] In this specification, "lysogenic gene" refers to a gene associated with the induction and / or maintenance of lysogenic state. Examples of "lysogenic genes" include integrase genes, immunorepressor protein genes, Cro genes, etc. Specific lysogenic genes and their sequence information are also well known (Nat Med. 2019; 25(5):730., mBio. 2021; 12(3):e00973., Cell. 2018; 172(6):1260.).

[0041] In this specification, "deleted lysogen" means: 1) the full-length deletion of at least one lysogen in the genome sequence of a bacteriophage; or 2-1) modification of a portion of at least one lysogen region in the genome sequence of a bacteriophage by deletion, substitution, insertion, appending, or a combination thereof, thereby making the bacteriophage a lysogenic bacteriophage, or 2-2) loss of function of the lysogen. The deletion of the lysogen can be performed by methods known in the art, depending on the type of lysogen to be deleted (e.g., Nat Med. 2019; 25(5):730., mBio. 2021; 12(3):e00973., PLoS One. 2008; 3(12):e3957.).

[0042] In this specification, "having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare" means causing lytic plaques to appear in at least one strain of bacteria belonging to Mycobacterium avium or Mycobacterium intracellulare. The lytic activity of these bacteria can be confirmed using methods and techniques known in the art. For example, the method described in Example 4 of this specification can be used to confirm approximately 10 10 pfu / mL ~10 5 Whether a bacteriophage (or a mixture of multiple bacteriophages) at pfu / mL induces lytic plaques in Mycobacterium avium or Mycobacterium intracellulare can be used to confirm whether the bacteriophage has lytic activity.

[0043] In one embodiment, the phage of the present invention is resistant to freeze-thaw cycles. For example, the phage of the present invention includes the following phages: A bacteriophage having lysing activity against Mycobacterium avium and / or Mycobacterium intracellulare, the phage's genome (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the phage specified by accession number NITE BP-03918, and (b) lacking a lysogenic gene, the phage being resistant to freeze-thaw cycles.

[0044] In this specification, "tolerance to freeze-thaw" means that although freeze-thaw treatment is known to kill or damage a portion of the phage, the phage remains at the desired level after freeze-thaw treatment. For example, the number of phages after freeze-thaw treatment remains approximately the same as before freeze-thaw treatment, or remains 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 than before freeze-thaw treatment. Tolerance to freeze-thaw can be confirmed using methods and means known in the art. For example, the method described in Example 2 of this specification can be used to confirm whether there is a reduction in the number of phages before and after freeze-thaw treatment, thereby confirming whether the phage is tolerant to freeze-thaw. Alternatively, tolerance to freeze-thaw can also be confirmed by confirming whether the activity of the phage (e.g., lytic activity against Mycobacterium avium and / or intracellular Mycobacteria) remains at the desired level after freeze-thaw treatment. For example, if a phage after freeze-thaw treatment retains approximately the same activity as before freeze-thaw treatment, or retains 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 its activity compared to before freeze-thaw treatment, then the phage can be said to be resistant to freeze-thaw.

[0045] In one embodiment, the bacteriophage of the present invention includes the following bacteriophages: A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene.

[0046] "Deletion of integrase gene and / or immunorepressor protein gene" means: 1) full-length deletion of integrase gene and / or immunorepressor protein gene in the genome sequence of a bacteriophage; or 2-1) modification of a portion of integrase gene and / or immunorepressor protein gene in the genome sequence of a bacteriophage by deletion, substitution, insertion, appending or a combination thereof, thereby making the bacteriophage a lysogenic bacteriophage, or 2-2) loss of function of integrase gene and / or immunorepressor protein gene. As mentioned above, the deletion of lysogenic genes such as integrase gene and immunorepressor protein 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 comprises the following bacteriophage: a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) contains a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or more, or 99% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene.

[0048] The bacteriophages of the present invention include the following bacteriophages: A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918.

[0049] The bacteriophages of the present invention include the following bacteriophages: A bacteriophage whose genome is composed of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918.

[0050] The bacteriophages of the present invention also include the following bacteriophages: The phage specified by accession number NITE BP-03918.

[0051] The bacteriophages of the present invention also include the following bacteriophages: 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 5000 (e.g., 1 to 1000, 1 to 500, or 1 to 100) bases are modified by deletion, substitution, insertion, or addition, or combinations thereof, of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene.

[0052] Regarding the aforementioned modifications such as deletion, substitution, insertion, or addition of bases, multiple modifications may be present consecutively, or multiple modifications may exist at different positions. Such modified forms are included in the bacteriophages of the present invention as long as they possess lytic activity against Mycobacterium avium and / or intracellular Mycobacteria (and optionally, resistance to freeze-thaw cycles).

[0053] During the passage, production, and / or replication of bacteriophages, it is possible to generate bacteriophage variants in which a portion of the nucleic acid sequence of the genome contained in the bacteriophage is deleted, substituted, inserted, and / or added. Such variants are also included in the bacteriophages of the present invention, provided that they have lytic activity against Mycobacterium avium and / or Mycobacterium intracellularis (and optionally, tolerance to freeze-thaw cycles).

[0054] The passages of the phage specified in the above accession number NITE BP-03918 are also included in the phages of the present invention, provided that the phages have lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare (and optionally tolerance to freeze-thaw cycles).

[0055] In this specification, "subcultured strain" means a bacteriophage obtained by subculturing distributed bacteriophages.

[0056] The present invention also includes polynucleotides comprising the genome contained in the phage of the present invention (hereinafter sometimes referred to as "the genome of the present invention"). Therefore, the present invention provides: polynucleotides comprising a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the phage specified in accession number NITE BP-03918, and lacking an integrase gene and / or an immunorepressor protein gene; and polynucleotides comprising a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the phage specified in accession number NITE BP-03918. In one embodiment, the polynucleotide is a polynucleotide encoding a phage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare.

[0057] In one embodiment, the genome of the present invention comprises the following genome: a genome that (a) contains a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or more, or 99% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene.

[0058] The genome of this invention includes the following genomes: A genome comprising the nucleic acid sequence of the genome of a bacteriophage specified by accession number NITE BP-03918.

[0059] The genome of this invention includes the following genomes: A genome comprising the nucleic acid sequence of the genome of a bacteriophage specified by accession number NITE BP-03918.

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

[0061] The bacteriophage of the present invention can be obtained by submitting a distribution application to the aforementioned depository center.

[0062] Furthermore, the phage of the present invention can be prepared by analyzing the nucleic acid sequence of the genome of the distributed phage using general techniques known in the art, based on that sequence information. For example, the genome of the present invention prepared by the above method can be introduced into host bacteria (for example, if the purpose is to obtain a phage that shows lysing activity against Mycobacterium spp., Mycobacterium spp. is used as the host bacteria) using electroporation. Then, bacteria with the introduced genome are added to a plate covered with multiple layers of soft agar and cultured. Afterward, a single lysing plaque is obtained by phage plaque assay. The single lysing plaque is added to the host bacterial culture medium and cultured. The culture supernatant obtained by standing or centrifugation is filtered, thereby preparing the phage of the present invention. In addition, to prepare the phage of the present invention, the lysogen gene can be deleted from the phage genome using Bacteriophage Recombineering of Electroporated DNA (BRED) (PLoSOne. 2008; 3(12):e3957.).

[0063] The bacteriophages of the present invention can be prepared using general culturing, isolation, and purification methods known in the art. For example, host bacteria (Mycobacterium smegmatis, Mycobacterium avium, or Mycobacterium intracellularis, etc.) are pre-cultured, and the bacteriophages of the present invention are infecting these host bacteria and cultured at 37°C. After culturing, the culture supernatant obtained by standing or centrifugation is filtered to obtain purified bacteriophages. The culture medium can be appropriately selected according to the bacteria used; for example, 7H9 medium can be used when culturing Mycobacterium smegmatis. In the case of preparing multiple bacteriophages, they can be propagated in different host bacteria or in the same host bacteria.

[0064] Furthermore, the bacteriophages of the present invention can be preserved in various forms (liquid, freeze-dried, etc.) by implementing appropriate methods known in the art.

[0065] <2. The modified phage of the present invention> In another embodiment, the present invention provides a modified bacteriophage (hereinafter sometimes referred to as "the modified bacteriophage of the present invention"). The modified bacteriophage of the present invention is a bacteriophage with a modified head (also called a capsid) and / or tail. There are no particular limitations on the bacteriophage to be modified, as long as it has lytic activity against nontuberculous mycobacteria (NTM), especially avian mycobacteria and / or intracellular mycobacteria that cause pulmonary MAC. For example, any of the following bacteriophages (1) to (3) can be modified: (1) A bacteriophage having lysing activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacking a lysogenic gene; (2) A bacteriophage having lysing activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) containing a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacking a lysogenic gene; (3) A phage having lysing activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the phage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the phage specified by accession number NITE BP-03514, and (b) lacking a lysogen gene.

[0066] In one embodiment, the bacteriophage to be modified includes any one of the following (1) to (3): (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacking an integrase gene and / or an immunorepressor protein gene; (2) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) containing a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacking an integrase gene and / or an immunorepressor protein gene; (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514, and (b) lacks an integrase gene and / or an immunorepressor protein gene.

[0067] In one embodiment, the bacteriophage to be modified includes any one of the following (1) to (3): (1) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918; (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514.

[0068] In one embodiment, the bacteriophage to be modified includes any one of the following (1) to (3): (1) A bacteriophage whose genome is composed of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03513; (2) A bacteriophage, wherein the genome of the bacteriophage is composed of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03918; (3) A bacteriophage whose genome is composed of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03514.

[0069] If the phage has lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the passages of the phages specified by the above accession numbers NITE BP-03513, NITE BP-03918 or NITE BP-03514 are also included in the phage to be modified.

[0070] In one embodiment, the bacteriophage to be modified includes any one of the following (1) to (3): (1) The phage or its passages specified by the accession number NITE BP-03513; (2) The phage or its passages specified by the accession number NITE BP-03918; (3) The phage or its passages specified by the accession number NITE BP-03514.

[0071] In addition, the bacteriophage (#63 freeze-thaw resistant strain #63ΔR12-23) designated by accession number NITE BP-03918 has been internationally deposited by the applicant on June 22, 2023, with the Patent Microbiology Collection Center of the Technical Base for Product Evaluation (Room 122, 2-5-8 Kazusa Kamata, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan) under the provisions of the Budapest Treaty on the Preservation of Patent Microorganisms (accession number NITE BP-03918).

[0072] The phage (D29 lysogen gene deletion strain D29Δ) designated by accession number NITE BP-03513 has been internationally deposited by the applicant on August 26, 2021, with the Patent Microbiology Collection Center of the Technical Base for Product Evaluation (Room 122, 2-5-8 Kazusa Kamata, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan) under the provisions of the Budapest Treaty on the Preservation of Patent Microorganisms (accession number NITE BP-03513).

[0073] The phage (B1 lysogen gene deletion strain B1Δ) designated by accession number NITE BP-03514 has been internationally deposited by the applicant on August 26, 2021, with the Patent Microbial Collection Center of the Technical Base for Product Evaluation (Room 122, 2-5-8 Kazusa Kamata, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan) (accession number NITEBP-03514).

[0074] Among the phages to be modified, the phages specified by the accession number can be obtained by submitting a distribution application to the aforementioned accession center.

[0075] The preparation and culture of the phage to be modified can be carried out in the same manner as described in the preceding paragraph <1. The phage of the present invention>.

[0076] In one embodiment, the modification includes the connection of the capsid and / or tail to a tag peptide. As the tag peptide, various tag peptides known in the art, such as S-tags, T7 tags, His tags, and FLAG tags, can be used. In one embodiment, the modification includes the connection of the capsid and / or tail to an S-tag. That is, the capsid and / or tail of the modified phage of the present invention are connected to an S-tag. In one embodiment, the modification includes the connection of the capsid to an S-tag.

[0077] The S-tag is one type of tag peptide containing the amino acid sequence WSHPQFEK (sequence number 2), and is well known in the art. The number of S-tags attached to the capsid and / or tail is not particularly limited; for example, it can be one, two, or four relative to any protein.

[0078] As a method for attaching a tag peptide (e.g., an S-tag) to the capsid and / or tail of a bacteriophage, those skilled in the art can perform this using conventional methods. For example, a plasmid containing a DNA sequence encoding an S-tag peptide, linked upstream or downstream of a DNA sequence encoding a capsid constituent protein and / or a tail constituent protein, is introduced into the host bacteria of the bacteriophage. This allows the expression of a fusion protein of the capsid constituent protein and the S-tag in the host bacteria, and the bacteriophage to infect the host bacteria, thereby creating a bacteriophage with an S-tag attached to its capsid and / or tail. In this specification, a bacteriophage with an S-tag attached to its capsid and / or tail is sometimes referred to as an S-tag fusion bacteriophage. As the capsid constituent protein and / or tail constituent protein, proteins belonging to the following protein groups can generally be used: known major capsid proteins constituting the capsid, minor capsid proteins, accessory proteins, neck proteins, and known tail proteins constituting the tail. For example, Non-Patent Document 1 reports a modified bacteriophage with an S-tag attached to its capsid. The connection between the S-tag and the capsid can be a connection between the S-tag peptide and the N-terminal side of the capsid-forming protein, or it can be a connection with the C-terminal side.

[0079] Modified phages with a capsid and / or tail-attached tagged peptide (e.g., an S-tag) exhibit higher lytic activity against Mycobacterium avium and / or intracellular Mycobacteria compared to unmodified phages. For example, the lytic activity of modified phages 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 phages. This lytic activity can be confirmed, for example, by the method described in Example 5.

[0080] 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, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacks an integrase gene and / or an immunorepressor protein gene, the capsid of which is linked to an S tag.

[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, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and the capsid of which is linked to an S-tag.

[0082] 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, 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 which is linked to an S-tag.

[0083] 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, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03513, the capsid of the bacteriophage or its passage being attached to an S-tag.

[0084] 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, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene, the capsid of which 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, the phage's genome (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the phage genome specified by accession number NITE BP-03918, and (b) lacking an integrase gene and / or an immunorepressor protein gene, the phage being resistant to freeze-thaw cycles, and the phage's capsid being linked to an S-tag.

[0086] 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, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and the capsid of which is linked to an S-tag.

[0087] 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, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and the capsid of which is linked to an S-tag.

[0088] 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, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03918, the capsid of the bacteriophage or its passage being attached to an S-tag.

[0089] In one embodiment, the modification includes a direct or indirect connection between the capsid and / or tail of the modified phage and the cell-penetrating peptide. That is, the capsid and / or tail of the modified phage of the present invention are directly or indirectly connected to the cell-penetrating peptide. In the case of indirect connection, a adapter or tag may be used, for example.

[0090] In one embodiment, the modification includes a direct link between the capsid and / or tail and the cell-penetrating peptide.

[0091] In this specification, cell-penetrating peptide (CPP) means a peptide that, when attached to the capsid and / or tail of a bacteriophage, increases the uptake of the bacteriophage into the cell. CPPs are well known in the art, and numerous CPP sequences and their mechanisms have been reported (e.g., Crit. Rev. Microbiol. 2021; 47(4):461). For example, without limitation, peptides such as Inv3 (TKRRITPKDVIDVRSVTTEINT: sequence number 1) at positions 25-46 of the rMceIa sequence, and Inv5 (Anal. Biochem. 2006; 353(1):7), R9, TAT, TP10, and L17E are examples of CPPs.

[0092] In one embodiment, the modification includes the capsid and / or tail being linked to a cell-penetrating peptide via a tag.

[0093] There are no particular limitations on the tag, as long as it enables the capsid and / or tail to be linked to the cell-penetrating peptide. Examples of tags include S-tags, T7 tags, His tags, and FLAG tags. The linkage between the cell-penetrating peptide and the capsid and / or tail via the tag can be a linkage to the N-terminal side of the capsid constituent protein and / or the tail constituent protein, or a linkage to the C-terminal side.

[0094] 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, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacks an integrase gene and / or an immunorepressor protein gene, the capsid of which is linked to a cell-penetrating peptide via an S-tag.

[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, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and the capsid of which is linked to a cell-penetrating peptide via an S-tag.

[0096] 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, 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 which is linked to a cell-penetrating peptide via an S-tag.

[0097] 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, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03513, wherein the capsid of the bacteriophage or its passage is linked to a cell-penetrating peptide via an S-tag.

[0098] 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, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene, the capsid of which is linked to a cell-penetrating peptide.

[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, the phage's genome (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the phage genome specified by accession number NITE BP-03918, and (b) lacking an integrase gene and / or an immunorepressor protein gene, the phage being resistant to freeze-thaw cycles, and the phage's capsid being linked to a cell-penetrating peptide.

[0100] 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, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and the capsid of which is linked to a cell-penetrating peptide.

[0101] 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, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and the capsid of which is linked to a cell-penetrating peptide.

[0102] 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, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03918, wherein the capsid of the bacteriophage or its passage is linked to a cell-penetrating peptide.

[0103] 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, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene, the capsid of which is linked to a cell-penetrating peptide via a tag.

[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, the phage's genome (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the phage genome specified by accession number NITE BP-03918, and (b) lacking an integrase gene and / or an immunorepressor protein gene, the phage being resistant to freeze-thaw cycles, and the phage's capsid being linked to a cell-penetrating 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, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and the capsid of which is linked to a cell-penetrating peptide via a tag.

[0106] 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, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and the capsid of which is linked to a cell-penetrating peptide via a tag.

[0107] 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, the bacteriophage being the bacteriophage or its passage designated by accession number NITE BP-03918, the capsid of the bacteriophage or its passage linked to a cell-penetrating peptide via a tag.

[0108] In one implementation, the tag attached to the modified bacteriophage includes an S-tag.

[0109] In one embodiment, the cell-penetrating peptides modified to link the bacteriophage include Inv3.

[0110] 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, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacks an integrase gene and / or an immunorepressor protein gene, the capsid of which is linked to Inv3 via an S tag.

[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, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and the capsid of which is linked to Inv3 via an S-tag.

[0112] 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, 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 which is linked to Inv3 via an S tag.

[0113] 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, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03513, the capsid of the bacteriophage or its passage being linked to Inv3 via an S-tag.

[0114] 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, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene, the capsid of which 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, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene, the bacteriophage being resistant to freeze-thaw cycles, and the capsid of the bacteriophage being 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, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and the capsid of which is linked to Inv3 via an S-tag.

[0117] 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, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and the capsid of which is linked to Inv3 via an S tag.

[0118] 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, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03918, the capsid of the bacteriophage or its passage being linked to Inv3 via an S-tag.

[0119] The direct or tag-linked connection of the cell-penetrating peptide to the capsid and / or tail can be performed by those skilled in the art using conventional methods (Non-Patent Document 1, Non-Patent Document 2). For example, a plasmid containing a DNA sequence encoding a cell-penetrating peptide or a tag and cell-penetrating peptide encoded by a DNA sequence upstream or downstream of a DNA sequence encoding a capsid constituent protein and / or a tail constituent protein is introduced into the host bacteria of the bacteriophage. This allows the expression of a fusion protein of the capsid constituent protein and / or tail constituent protein with the cell-penetrating peptide, or a fusion protein of the capsid constituent protein and / or tail constituent protein with the tag and cell-penetrating peptide, in the host bacteria, and the bacteriophage infects the host bacteria. This allows the creation of a bacteriophage with a capsid and / or tail linked to a cell-penetrating peptide, or a bacteriophage with a capsid and / or tail linked to a tag and cell-penetrating peptide. In this specification, bacteriophages with a capsid and / or tail linked to a tag and cell-penetrating peptide are sometimes referred to as tag and cell-penetrating peptide fusion bacteriophages. As capsid constituent proteins and / or tail constituent proteins, proteins belonging to the following protein groups can generally be used: known major capsid proteins, minor capsid proteins, accessory proteins, neck proteins, and known tail constituent proteins.

[0120] Modified phages with cell-penetrating peptides directly attached to their capsid and / or tail, or with such peptides attached via a tag, exhibit higher lytic activity against intracellular Mycobacterium avium and / or intracellular Mycobacteria compared to unmodified phages. For example, the lytic activity of modified phages against intracellular Mycobacterium avium and / or intracellular Mycobacteria 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 phages. Regarding the lytic activity against intracellular Mycobacterium avium and / or intracellular Mycobacteria, the methods described in Examples 5-6 can be used, for example, to confirm whether a decrease in the number of Mycobacterium avium or intracellular Mycobacteria occurs before and after treatment with the modified phage (or a mixture of multiple phages), thereby confirming whether the modified phage has lytic activity against intracellular Mycobacterium avium and / or intracellular Mycobacteria.

[0121] <3. Uses of the pharmaceutical compositions and bacteriophages of the present invention> The bacteriophages and / or modified bacteriophages of the present invention exhibit lytic activity against nontuberculous mycobacteria (NTM), particularly Mycobacterium avium and / or intracellular mycobacteria that cause pulmonary MAC. Therefore, the bacteriophages and / or modified bacteriophages of the present invention show promise for the treatment or prevention of nontuberculous mycobacterial diseases (NTM), particularly pulmonary MAC.

[0122] In another embodiment, the present invention provides the use of the bacteriophage of the present invention and / or the modified bacteriophage of the present invention, for example, a pharmaceutical composition comprising the bacteriophage of the present invention and / or the modified bacteriophage of the present invention as an active ingredient (hereinafter sometimes referred to as "the pharmaceutical composition of the present invention").

[0123] The pharmaceutical compositions of the present invention include a pharmaceutical composition comprising a bacteriophage containing at least one of the bacteriophages of the present invention and / or the modified bacteriophages of the present invention, and a pharmaceutically acceptable excipient.

[0124] The pharmaceutical compositions of the present invention may further comprise one or more strains of bacteriophages known to have lytic activity against Mycobacterium avium and / or Mycobacteria intracellularis. Such strains are not particularly limited, and examples include the bacteriophages of the present invention, the modified bacteriophages of the present invention, and the bacteriophages described in International Application No. PCT / JP2023 / 016600.

[0125] In one embodiment, the pharmaceutical composition of the present invention comprises the modified phage of the present invention as follows: A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of said bacteriophage (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacks an integrase gene and / or an immunorepressor protein gene, and the capsid of said bacteriophage is linked to a cell-penetrating peptide via an S-tag; and Selected from one or more bacteriophages from the group consisting of bacteriophages from (1) to (3) below: (1) (A) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514 and (b) lacks an integrase gene and / or an immunorepressor protein gene, or (B) a bacteriophage that is the bacteriophage of (A), the capsid of which is linked to a cell-penetrating peptide via a tag; (2) (C) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene; or (D) a bacteriophage that is (C) the bacteriophage, the capsid of which is linked to a cell-penetrating peptide via a tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage contains a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

[0126] In addition, the bacteriophage (parental strain #123) designated by accession number NITE BP-03519 has been internationally deposited by the applicant on August 26, 2021, with the Patent Microbial Collection Center of the Technical Base for Product Evaluation (Room 122, 2-5-8 Kazusa Kamata, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan) (accession number NITE BP-03519), based on the provisions of the Budapest Treaty on the Preservation of Patent Microorganisms.

[0127] In one embodiment, the pharmaceutical composition of the present invention comprises the modified phage of the present invention as follows: A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of said bacteriophage being linked to an S-tag; and Selected from one or more bacteriophages from the group consisting of bacteriophages from (1) to (3) below: (1) (A) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514, or (B) a bacteriophage that is the bacteriophage of (A), the capsid of which is linked to a cell-penetrating peptide via a tag; (2) (C) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918; or (D) A bacteriophage that is (C) of which the capsid is linked to a cell-penetrating peptide via a tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

[0128] In one embodiment, the pharmaceutical composition of the present invention comprises the modified phage of the present invention as follows: A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of said bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and the capsid of said bacteriophage is linked to an S-tag; and Selected from one or more bacteriophages from the group consisting of bacteriophages from (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 specified by accession number NITE BP-03514, or (B) a bacteriophage that is the bacteriophage of (A), the capsid of which is linked to a cell-penetrating 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 accession number NITE BP-03918; or (D) a bacteriophage that is (C) and whose capsid is linked to a cell-penetrating 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.

[0129] In one embodiment, the pharmaceutical composition of the present invention comprises the modified phage of the present invention as follows: A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03513, wherein the capsid of said bacteriophage or its passage is attached to an S-tag; and Selected from one or more bacteriophages from the group consisting of bacteriophages from (1) to (3) below: (1) (A) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03514, or (B) a bacteriophage being the bacteriophage of (A), the capsid of the bacteriophage or its passage being further linked to a cell-penetrating peptide via a tag; (2) (C) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage or its passage specified in accession number NITE BP-03918, or (D) a bacteriophage being the bacteriophage of (C), the capsid of the bacteriophage or its passage being further linked to a cell-penetrating peptide via a tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is the bacteriophage or its passage specified by accession number NITE BP-03519.

[0130] In one embodiment, the pharmaceutical composition of the present invention comprises the modified phage of the present invention as follows: A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of said bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and the capsid of said bacteriophage is linked to Inv3 via an S-tag; and Selected from one or more bacteriophages from the group consisting of bacteriophages from (1) to (3) below: (1) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, the capsid of said bacteriophage being 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 the accession number NITE BP-03519.

[0131] In one embodiment, the pharmaceutical composition of the present invention comprises the modified phage of the present invention as follows: A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03513, wherein the capsid of said bacteriophage or its passage is linked to Inv3 via an S-tag; and Selected from one or more bacteriophages from the group consisting of bacteriophages from (1) to (3) below: (1) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03514; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03918, the capsid of said bacteriophage or its passage being linked to Inv3 via an S-tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is the bacteriophage or its passage specified by accession number NITE BP-03519.

[0132] In one embodiment, the pharmaceutical composition of the present invention comprises the modified phage of the present invention as follows: A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of said bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and the capsid of said bacteriophage is linked to Inv3 via an S-tag; and Selected from one or more bacteriophages from the group consisting of bacteriophages from (1) to (3) below: (1) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of said bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting 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, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03519.

[0133] In one embodiment, the pharmaceutical composition of the present invention comprises the modified phage of the present invention as follows: A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03918, wherein the capsid of said bacteriophage or its passage is linked to Inv3 via an S-tag; and Selected from one or more bacteriophages from the group consisting of bacteriophages from (1) to (3) below: (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03513, the capsid of the bacteriophage or its passage being connected to Inv3 via an S tag. (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is the bacteriophage or its passage specified by accession number NITE BP-03519.

[0134] The pharmaceutical compositions of the present invention can be prepared using excipients commonly used in the art (i.e., pharmaceutical excipients or pharmaceutical carriers, etc.) and by commonly used methods. Examples of dosage forms for these pharmaceutical compositions include non-oral formulations such as injections, intravenous drips, powder inhalers, and nebulizers, which can be administered intravenously or via the lungs. During formulation, excipients, carriers, or additives suitable for these dosage forms can be used within pharmaceutically acceptable limits. For example, the pharmaceutical compositions of the present invention can be manufactured by mixing bacteriophages with pharmaceutically acceptable excipients or by suspending bacteriophages in pharmaceutically acceptable excipients.

[0135] The pharmaceutical compositions of the present invention include a pharmaceutical composition comprising at least two bacteriophages and a pharmaceutically acceptable excipient.

[0136] The pharmaceutical composition of the present invention includes a pharmaceutical composition comprising three bacteriophages and a pharmaceutically acceptable excipient.

[0137] In one embodiment, the pharmaceutical composition of the present invention comprises a pharmaceutical composition containing the following bacteriophages (1) to (3) and a pharmaceutically acceptable excipient: (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacking an integrase gene and / or an immunorepressor protein gene, 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 which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514, and (b) lacks an integrase gene and / or an immunorepressor protein gene; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacking an integrase gene and / or an immunorepressor protein gene, the capsid of the bacteriophage being linked to Inv3 via an S tag.

[0138] In the above embodiments, the pharmaceutical composition of the present invention comprises the following bacteriophages (1) to (3): (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of the bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage containing 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, the genome of the bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, the capsid of the bacteriophage being linked to Inv3 via an S tag.

[0139] In the above embodiments, the pharmaceutical composition of the present invention comprises the following bacteriophages (1) to (3): (1) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of said bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting 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, the genome of the bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03918, the capsid of the bacteriophage being linked to Inv3 via an S tag.

[0140] In one embodiment, the pharmaceutical composition of the present invention comprises a pharmaceutical composition containing the following bacteriophages (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 or its passage specified by accession number NITE BP-03513, the capsid of the bacteriophage or its passage being connected to Inv3 via an S tag. (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03918, the capsid of the bacteriophage or its passage being linked to Inv3 via an S tag.

[0141] In one embodiment, the pharmaceutical composition of the present invention comprises a pharmaceutical composition containing the following bacteriophages (1) to (3) and a pharmaceutically acceptable excipient: (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacking an integrase gene and / or an immunorepressor protein gene, 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 which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene, the capsid of which 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 contains a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

[0142] In the above embodiments, the pharmaceutical composition of the present invention comprises the following bacteriophages (1) to (3): (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of the bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, the capsid of said bacteriophage being 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 contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

[0143] In the above embodiments, the pharmaceutical composition of the present invention comprises the following bacteriophages (1) to (3): (1) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of said bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, the capsid of said bacteriophage being 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 the accession number NITE BP-03519.

[0144] In one embodiment, the pharmaceutical composition of the present invention comprises a pharmaceutical composition containing the following bacteriophages (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 or its passage specified by accession number NITE BP-03513, the capsid of the bacteriophage or its passage being connected to Inv3 via an S tag. (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03918, the capsid of said bacteriophage or its passage being linked to Inv3 via an S-tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is the bacteriophage or its passage specified by accession number NITE BP-03519.

[0145] In one embodiment, the pharmaceutical composition of the present invention comprises a pharmaceutical composition containing the following bacteriophages (1) to (3) and a pharmaceutically acceptable excipient: (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacking an integrase gene and / or an immunorepressor protein gene, 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 which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514, and (b) lacks an integrase gene and / or an immunorepressor protein gene; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage contains a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

[0146] In the above embodiments, the pharmaceutical composition of the present invention comprises the following bacteriophages (1) to (3): (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of the bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage containing 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, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

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

[0148] In one embodiment, the pharmaceutical composition of the present invention comprises a pharmaceutical composition containing the following bacteriophages (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 or its passage specified by accession number NITE BP-03513, the capsid of the bacteriophage or its passage being connected to Inv3 via an S tag. (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is the bacteriophage or its passage specified by accession number NITE BP-03519.

[0149] Any variant of the phage specified in any of the above accessions NITE BP-03918, NITE BP-03513, NITE BP-03514, or NITE BP-03519 that exhibits lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare is also included in the pharmaceutical composition of the present invention. Specifically, the pharmaceutical composition of the present invention may contain variants of the following phages: (1) (A) A phage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence in which 1 to 5000 (e.g., 1 to 1000, 1 to 500, or 1 to 100) bases are modified by deletion, substitution, insertion, or addition or combinations thereof, and (b) lacks an integrase gene and / or an immunorepressor protein gene, or (B) a phage whose capsid of (A) is further linked to a cell-penetrating peptide via a tag; (2) (C) A phage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) contains a nucleic acid sequence of 1 to 5000 (e.g., 1 to 1000, 1 to 500, or 1 to 100) bases modified by deletion, substitution, insertion, or addition or combinations thereof, and (b) lacks an integrase gene and / or an immunorepressor protein gene, or (D) a phage whose capsid of (C) is further linked to a cell-penetrating peptide via a tag; (3) (E) A phage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence in which 1 to 5000 (e.g., 1 to 1000, 1 to 500, or 1 to 100) bases are modified by deletion, substitution, insertion, or addition, or combinations thereof, and (b) lacks an integrase gene and / or an immunorepressor protein gene; or (F) a phage whose capsid of (E) is further linked to a cell-penetrating peptide via a tag; and / or (4) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which 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 are modified by deletion, substitution, insertion, or addition, or combinations thereof, of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

[0150] Regarding the above-mentioned modifications such as the deletion, substitution, insertion, or addition of bases, multiple modifications can exist consecutively, or multiple modifications can exist in different positions.

[0151] As long as it has lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the passaged strain of any of the bacteriophages specified in the above accession numbers NITE BP-03918, NITE BP-03513, NITE BP-03514 or NITE BP-03519 is also included in the pharmaceutical composition of the present invention.

[0152] Effective dosage, frequency, and duration of administration vary depending on the purpose of administration (therapeutic or prophylactic), the severity and age of the recipient's symptoms, the dosage form of the formulation used, or the titer of the phage. For example, an effective dosage for a single phage or for the combined effective dosage of two or more phages may be approximately 10. 4 ~10 14 Plaque-forming units (PFU). The ratio of dosages of two or more phages can be appropriately adjusted based on the severity of the patient's symptoms and age, the dosage form of the formulation used, or the titer of the phages. For example, in a pharmaceutical composition containing three phages, approximately equal amounts (e.g., 3.3 × 10⁻⁶) can be used. 3 PFU ~ 3.3 × 10 13 The phages in pfu can also contain different ratios of phages.

[0153] The pharmaceutical compositions of the present invention can be used as a preventive or therapeutic agent for NTM diseases (e.g., MAC disease or pulmonary MAC disease).

[0154] In this instruction manual, "treatment" means at least partial improvement of the symptoms of NTM disease (e.g., cough, sputum, hemoptysis, fever, dyspnea, fatigue, pulmonary nodules, bronchiectasis, etc.), cessation of the progression or worsening of NTM disease, including negative sputum culture, or complete cure. In this instruction manual, "prevention" means preventing individuals who do not have NTM disease from developing NTM disease and preventing recurrence of NTM disease.

[0155] The present invention includes a pharmaceutical composition for the prevention or treatment of NTM diseases (e.g., MAC disease or pulmonary MAC disease) comprising the bacteriophage or modified bacteriophage of the present invention or the pharmaceutical composition of the present invention.

[0156] Furthermore, the present invention also includes a method for preventing or treating NTM diseases (e.g., MAC disease or pulmonary MAC disease) in a subject, the method comprising administering a therapeutically effective amount of the phage or modified phage of the present invention or a pharmaceutical composition of the present invention. In one embodiment, at least two (e.g., three) phages or modified phages of the present invention may be administered to a subject, and the multiple phages may be administered simultaneously or separately.

[0157] Furthermore, the present invention includes a phage or modified phage of the present invention or a pharmaceutical composition of the present invention intended for the prevention or treatment of NTM diseases (e.g., MAC disease or pulmonary MAC disease). Furthermore, the present invention includes the use of a phage or modified phage of the present invention or a pharmaceutical composition of the present invention in the manufacture of a pharmaceutical composition for the prevention or treatment of NTM diseases (e.g., MAC disease or pulmonary MAC disease).

[0158] In this invention, the recipient of the bacteriophage is not limited to any mammal, such as mice, rats, dogs, pigs, monkeys, and humans. For example, the phage can be administered to individuals diagnosed with NTM disease or individuals at risk of developing NTM disease.

[0159] Furthermore, the phage or modified phage of the present invention, or the pharmaceutical composition of the present invention, can be used or administered in combination with other ingredients effective for the treatment or prevention of NTM disease, and the phage or modified phage of the present invention, or the pharmaceutical composition of the present invention, can be provided as a combination medicine with other relevant active ingredients.

[0160] Although the invention has been fully described, specific embodiments are provided herein for further understanding. These embodiments are intended to be illustrative and not to limit the invention. Example

[0161] For the use of commercially available kits or reagents, unless otherwise specified, follow the accompanying instructions. Furthermore, for convenience, concentration is expressed as M in mol / L. For example, 1M sodium hydroxide aqueous solution means a 1 mol / L sodium hydroxide aqueous solution.

[0162] <Example 1: Preparation of a Novel Bacteriophage> Using the #63 lysogen gene deletion strain (accession number NITE BP-03517) as the parent phage, the freeze-thaw resistant strain #63 was obtained by the following method.

[0163] Ethyl methanesulfonate (Nacalai Tesque) was added to phage buffer (10mM Tris-HCl (pH 7.5) containing 1mM CaCl2, 10mM MgSO4, and 68.4mM NaCl) to achieve a final concentration of 33mM for strain #63, and the mixture was incubated at 37°C for 1 hour. The resulting solution was then mixed with *Mycobacterium smegmatis* in soft agar and layered onto 7H10 plates (19g / L Middlebrook 7H10 Agar (Difco), 6.3g / L glycerol, 100mL / L Middlebrook OADC Enrichment (Difco)) and incubated overnight at 37°C. Phage buffer was added to the incubated plates, the supernatant was collected, and the solution was filtered using a 0.22μm filter. The filtered solution was frozen at -80°C and then thawed at 4°C. The solution was mixed with Mycobacterium smegmatis in soft agar, and cultured and filtered to obtain the supernatant using the same procedure as described above. This process was considered one cycle, and a total of 12 cycles were performed.

[0164] The supernatant was cultured for 12 cycles using the same procedure as described above, and the #63 freeze-thaw resistant strain was obtained from a single plaque. The preservation number of this #63 freeze-thaw resistant strain is NITE BP-03918.

[0165] <Example 2: Freeze-thaw resistance activity of #63 freeze-thaw resistant strain> The freeze-thaw resistance activity of the #63 freeze-thaw resistant strain obtained in Example 1 was confirmed by the following methods.

[0166] Colonies of *Mycobacterium smegmatis* grown on 7H10 plates were scraped, suspended in LB (Luria-Bertani) medium, and cultured at 37°C. After culturing, either the #63 lysogen deletion strain or the #63 freeze-thaw resistant strain was added, and the culture was continued at 37°C for approximately one day.

[0167] The supernatant after culture was filtered using a 0.22 μm filter to obtain phage lysates. These lysates were then purified by ultracentrifugation to obtain phage solutions.

[0168] Purification by ultracentrifugation: DNase I and RNase A were added to the phage lysates of the #63 lysogen gene deletion strain and the #63 freeze-thaw resistant strain at a final concentration of 1 μg / mL, respectively, and incubated at room temperature for 30 minutes. NaCl was added to dissolve the lysates at a final concentration of 1M, and the solution was incubated on ice or in a refrigerator for 1 hour. Polyethylene glycol 8000 was added to dissolve the lysates at a final concentration of 10% w / v, and the solution was incubated on ice or in a refrigerator for 2 hours. The supernatant was removed by centrifugation at 11000g for 10 minutes at 4°C. Phage buffer was added to suspend the precipitate, and the solution was recovered. 0.5 g of cesium chloride per mL was added to the recovered solution to ensure stable dissolution, and the solution was transferred to an ultracentrifugation container. Cesium chloride solutions of concentrations of 1.45 g / mL, 1.5 g / mL, and 1.7 g / mL were injected sequentially into the bottom of the container using a syringe. Centrifugation was performed at 22000g for 2 hours at 4℃ to recover the phage-containing bands. The recovered phage-containing solution was then dialyzed to replace the phage buffer solution, which was used as the phage solution.

[0169] The phage solutions of strain #63 (lysogen gene deletion) or strain #63 (freeze-thaw resistant) were diluted and adjusted to 10⁻⁶ mcg. 7 Approximately pfu / mL. For each phage solution, samples were prepared and stored at 4℃, as well as samples frozen at -80℃ for 1 hour and then stored at 4℃.

[0170] Colonies of *Mycobacterium smegmatis* grown on 7H10 plates were 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 incubated for 3 days. The culture medium was then washed three times with 7H9 medium without polyoxyethylene sorbitan monooleate to adjust the bacterial suspension.

[0171] Mix 300 μL of bacterial suspension 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 layer it onto a 7H10 plate.

[0172] For the sample groups of #63 lysogen gene deletion strain and #63 freeze-thaw resistant strain prepared as described above, repeated 10-fold dilutions were performed using phage buffer to create a total of 6 concentration dilution series. After confirming that the soft agar was completely solidified, 2.5 μL of each was added dropwise and incubated at 37°C for 24 hours.

[0173] The results are shown in Figure 1 The PFU of the #63 lysogen gene deletion strain (No.63Δrep,int) decreased by approximately 1000-fold due to the freeze-thaw operation, while the PFU of the #63 freeze-thaw resistant strain (No.63Δrep,int_R12-23) remained at roughly the same level.

[0174] <Example 3: Preparation of Capsid-Modified Bacteriophages> Capsid-modified bacteriophages were prepared using the following method.

[0175] For the purpose of expressing S-tag fused capsid constituent proteins or S-tag and Inv3 fused capsid constituent proteins according to promoter activity, plasmids were created by linking genes encoding S-tag peptide sequences (Sequence No. 2) or genes encoding S-tag peptide sequences and Inv3 peptide sequences (Sequence No. 1) upstream or downstream of the genes encoding capsid constituent proteins obtained from the genome sequences of the D29 lysogen gene deletion strain (accession number NITE BP-03513), the B1 lysogen gene deletion strain (accession number NITE BP-03514), and the #63 freeze-thaw resistant strain obtained in Example 1.

[0176] The plasmid was introduced into *Mycobacterium smegmatis* via electroporation, facilitating the expression of various fusion capsid constituent proteins. During fusion capsid protein expression, the phage was infected with the corresponding phage, which served as the source of the capsid protein, to create a capsid-modified phage with a capsid composed of fusion capsid constituent proteins in a specific ratio. The capsid-modified phage was purified using an S-tag-binding resin (Strep-Tactin XT, IBA GmbH).

[0177] Various capsid-modified phages as shown below were obtained using the same method. • S-tag fusion D29 lysogen gene deletion strain (a modified phage with an S-tag fused to the N-terminus or C-terminus of the capsid constituent proteins. These are referred to as S-tag fusion D29 lysogen gene deletion strain (N) and S-tag fusion D29 lysogen gene deletion strain (C), respectively.) • S-tag and Inv3 fusion D29 lysogen gene deletion strain (a modified phage with a capsid fused to the C-terminus of the capsid constituent proteins with an S-tag and Inv3 sequence) • S-tag fusion #63 freeze-thaw resistant strain (a modified phage with an S-tag fused to the C-terminus of the capsid constituent proteins) • S-tag and Inv3 fusion #63 freeze-thaw resistant strain (a modified phage with a capsid fused to the C-terminus of the capsid constituent proteins with an S-tag and Inv3 sequence) • S-tag and Inv3 fusion B1 lysogen gene deletion strain (a modified phage with a capsid fused to the C-terminus of the capsid constituent proteins with an S-tag and Inv3 sequence)

[0178] <Example 4: Lysis Activity of Capsid-Modified Phages> The lysis activity of capsid-modified bacteriophages was evaluated using the following methods.

[0179] Scrape strains of Mycobacterium avium KCH-ASGF-MA-05 or clarithromycin-resistant intracellular Mycobacterium avium KCH-ASGF-MAC-475 (distributed from the Kinki Central Respiratory Center) grown at 37°C for approximately two weeks on 7H10 plates and suspend them in 7H9 medium (4.7 g / L Middlebrook 7H9 Broth (Difco)). After suspension, adjust the bacterial suspension using a spectrophotometer to a final OD600 of 1.5 via 7H9 medium.

[0180] Mix 300 μL of bacterial suspension 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 layer it onto a 7H10 plate.

[0181] After confirming that the soft agar has completely solidified, use phage buffer to become 10 10 pfu / mL to 10 5 The following strains obtained in Example 3 were diluted at six pfu / mL levels: the S-tag and Inv3 fusion D29 lysogen deletion strain (D29Δint-Stag-Inv3), the S-tag and Inv3 fusion B1 lysogen deletion strain (B1Δrep,int-Stag-Inv3), the S-tag and Inv3 fusion #63 freeze-thaw resistant strain (No.63Δrep,int_R12-23-Stag-Inv3), or the #63 freeze-thaw resistant strain obtained in Example 2 (No.63Δrep,int_R12-23). ​​2.5 μL of each strain was added dropwise. The strains were incubated at 37°C for approximately 9 days. Furthermore, preliminary studies confirmed that even adding only phage buffer to solidified soft agar did not result in plaque formation.

[0182] The results are shown in Figure 2 For both *Mycobacterium avium* strain KCH-ASGF-MA-05 (A) and clarithromycin-resistant *Mycobacterium intracellulare* strain KCH-ASGF-MAC-475 (B), it was confirmed that all phages formed lysis plaques. Based on the above, it was confirmed that the S-tag and Inv3 fusion D29 lysogen deletion strain, the S-tag and Inv3 fusion B1 lysogen deletion strain, the S-tag and Inv3 fusion #63 freeze-thaw resistant strain, and the #63 freeze-thaw resistant strain obtained in Example 2 possess lysis activity against *Mycobacterium avium* and *Mycobacterium intracellulare*.

[0183] <Example 5: Bactericidal activity of capsid-modified bacteriophages against intracellular infectious bacteria> Various phage solutions were obtained using the methods described in Example 2 or Example 3.

[0184] Two strains of Mycobacterium avium (KCH-ASGF-MA-05 or KCH-ASGF-MA-12 (distributed by the Kinki Central Respiratory Center)) that had been cultured and grown on 7H10 plates at 37°C for 4 days to approximately 2 weeks were scraped and suspended in cell culture medium. After suspension, the bacterial suspension was adjusted to a final OD600 of 0.07 using a spectrophotometer. Bronchoalveolar lavage fluid (BALF) from mice was collected, and mouse alveolar macrophages were prepared from the BALF solution and cultured at 37°C in the presence of 5% CO2 for approximately one week. The cultured cells were then mixed and seeded at 10 μL per well in a 96-well plate. 4 Cells were cultured at 37°C and 5% CO2 for one day, after which the supernatant was replaced with the bacterial suspension described above. Two hours later, 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 the culture was prepared to a final concentration of 3.0 × 10⁻⁶ cells / day. 8 pfu / mL (KCH-ASGF-MA-05) or 1.4×10 9 Various phage solutions prepared by the above method were added at pfu / mL (KCH-ASGF-MA-12). After culturing for 3 days, the supernatant was removed, cell lysis solution was added, the cells were resuspended, and the solution was recovered to count the CFU.

[0185] The results are shown in Figure 3 All phage strains exhibited bactericidal activity against intracellularly infected Mycobacterium avium KCH-ASGF-MA-05, but the S-tag fusion D29 lysogen deletion strain (N) and the S-tag fusion D29 lysogen deletion strain (C) showed higher bactericidal activity than the D29 lysogen deletion strain. Figure 3A). Furthermore, both phage strains exhibited bactericidal activity against intracellularly infected Mycobacterium avium KCH-ASGF-MA-12, but the S-tag fusion #63 freeze-thaw resistant strain showed higher bactericidal activity than the #63 freeze-thaw resistant strain. Figure 3 B). Based on the above, it was confirmed that the S-tag fusion D29 lysogen gene deletion strain (N), the S-tag fusion D29 lysogen gene deletion strain (C), and the S-tag fusion #63 freeze-thaw resistant strain have high lytic activity against Mycobacterium avium.

[0186] <Example 6: Bactericidal activity of various phage mixtures against intracellular infectious bacteria> The B1 lysogen gene-deleted strain (accession number NITE BP-03514) and parental strain #123 (accession number NITE BP-03519) were purified by ultracentrifugation. The bactericidal activity of various phage mixtures against intracellularly infected Mycobacterium avium KCH-ASGF-MA-05 was evaluated using the method described in Example 5.

[0187] The bacteriophage mixture consisted of the following three types, each with a final concentration of 1.0 × 10⁻⁶. 9 A mixture of equal amounts of three bacteriophages at pfu / mL. Mixture #1: S-tag and Inv3 fusion D29 lysogen deletion strain, B1 lysogen deletion strain, and parental strain #123 Mixture #2: S-tag and Inv3 fusion D29 lysogen deletion strain, S-tag and Inv3 fusion #63 freeze-thaw resistant strain, and B1 lysogen deletion strain Mixture #3: The S-tag and Inv3 fusion D29 lysogen gene deletion strain, the S-tag and Inv3 fusion #63 freeze-thaw resistant strain, and the #123 parental strain.

[0188] The results are shown in Figure 4 The results showed that all phage mixtures tested exhibited bactericidal activity against intracellularly infected Mycobacterium avium KCH-ASGF-MA-05.

[0189] <Example 7: Bactericidal activity of capsid-modified bacteriophages against intracellular infectious bacteria> The D29 lysogen gene deletion strain (accession number NITE BP-03513) and the B1 lysogen gene deletion strain (accession number NITE BP-03514) were purified by ultracentrifugation to obtain their respective phage solutions. The S-tag and Inv3 fusion D29 lysogen gene deletion strain and the S-tag and Inv3 fusion B1 lysogen gene deletion strain were purified using the method described in Example 3 to obtain their respective phage solutions.

[0190] 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 the bacteriophages was 3.0 × 10⁻⁶. 9 Phages were added at a rate of pfu / mL.

[0191] The results are shown in Figure 5 All phage strains exhibited bactericidal activity against intracellularly infected Mycobacterium avium KCH-ASGF-MA-05. The S-tag and Inv3 fusion D29 lysogen deletion strain showed higher bactericidal activity than the D29 lysogen deletion strain. Figure 5 A), the S-tag and Inv3 fusion B1 lysogen deletion strain showed higher bactericidal activity than the B1 lysogen deletion strain. Figure 5 B).

[0192] All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety. Industrial applicability

[0193] The bacteriophages and modified bacteriophages of the present invention, as well as the pharmaceutical compositions thereof, are expected to be effective in the prevention or treatment of nontuberculous mycobacterial diseases (e.g., MAC disease or pulmonary MAC disease).

[0194] [Collection Number] Accession number NITE BP-03513 (phage D29 lysogen gene deletion strain D29Δ, deposited on August 26, 2021) Accession number NITE BP-03514 (phage B1 lysogen gene deletion strain B1Δ, deposited on August 26, 2021) Accession number NITE BP-03519 (parental strain of phage #123, deposited on August 26, 2021) Accession number NITE BP-03918 (phage #63 freeze-thaw resistant strain, deposited on June 22, 2023) [Sequence List Free Text]

[0195] Sequence number 1: Synthesized construct (Inv3) Sequence number 2: Synthetic construct (S tag) PCT / RO / 134 form

Claims

1. A bacteriophage, wherein, The phage is a phage with lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) contains a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the phage specified by accession number NITE BP-03513, and (b) lacks an integrase gene and / or an immunorepressor protein gene, and the capsid of the phage is linked to an S tag.

2. The bacteriophage as described in claim 1, wherein, The genome of the phage contains the nucleic acid sequence of the genome of the phage specified by accession number NITE BP-03513.

3. The bacteriophage as described in claim 1 or 2, wherein, The genome of the phage consists of the nucleic acid sequence of the genome of the phage specified by the accession number NITE BP-03513.

4. A bacteriophage, wherein, The phage is a phage with lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, and is the phage or its passage specified by accession number NITE BP-03513, wherein the capsid of the phage or its passage is attached to an S tag.

5. The phage according to any one of claims 1 to 4, wherein, The capsid is further linked to the cell-penetrating peptide via an S-tag.

6. The bacteriophage as described in claim 5, wherein, The cell-penetrating peptide includes Inv3.

7. A pharmaceutical composition comprising the bacteriophage of any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

8. The pharmaceutical composition of claim 7, wherein, The pharmaceutical composition further comprises one or more strains of bacteriophages that have lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare.

9. A bacteriophage, wherein, The phage is a phage with lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) contains a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the phage specified by accession number NITE BP-03918, and (b) lacks an integrase gene and / or an immunorepressor protein gene.

10. The bacteriophage of claim 9, wherein, The bacteriophage is resistant to freeze-thaw cycles.

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

12. The bacteriophage as claimed in claim 9 or 11, wherein, The genome of the phage consists of the nucleic acid sequence of the genome of the phage specified by the accession number NITE BP-03918.

13. A bacteriophage, wherein, The phage is a phage with lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, and the phage is the phage or its passage specified by the accession number NITE BP-03918.

14. The phage according to any one of claims 9 to 13, wherein, The bacteriophage's capsid is attached to the tag.

15. The phage according to any one of claims 9 to 13, wherein, The phage capsid is linked to a cell-penetrating peptide via a tag.

16. The bacteriophage as claimed in claim 14 or 15, wherein, The label is an S label.

17. The bacteriophage as claimed in claim 15 or 16, wherein, The cell-penetrating peptide includes Inv3.

18. A pharmaceutical composition comprising the bacteriophage of any one of claims 9 to 17 and a pharmaceutically acceptable excipient.

19. The pharmaceutical composition of claim 18, wherein, The pharmaceutical composition further comprises one or more strains of bacteriophages that have lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare.

20. The pharmaceutical composition of claim 7, wherein, The pharmaceutical composition further comprises the phage according to any one of claims 1 to 6 and one or more phages selected from the group consisting of phages from (1) to (3) below: (1) (A) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514 and (b) lacks an integrase gene and / or an immunorepressor protein gene, or (B) a bacteriophage that is the bacteriophage of (A), the capsid of which is linked to a cell-penetrating peptide via a tag; (2) (C) A phage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) contains a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the phage specified by accession number NITE BP-03918 and (b) lacks an integrase gene and / or an immunorepressor protein gene, or (D) a phage that is the phage of (C), the capsid of which is linked to a cell-penetrating peptide via a tag; as well as (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage contains a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

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

22. The pharmaceutical composition of claim 7, wherein, The pharmaceutical composition further comprises the phage according to any one of claims 1 to 6 and one or more phages selected from the group consisting of phages from (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 specified by accession number NITE BP-03514, or (B) a bacteriophage that is the bacteriophage of (A), the capsid of which is linked to a cell-penetrating 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 accession number NITE BP-03918, or (D) A bacteriophage that is (C) of which the capsid is linked to a cell-penetrating peptide via a tag; as well as (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 of claim 7, wherein, The pharmaceutical composition further comprises the phage according to any one of claims 1 to 6 and one or more phages selected from the group consisting of phages from (1) to (3) below: (1) (A) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03514, or (B) a bacteriophage being the bacteriophage of (A), the capsid of the bacteriophage or its passage being linked to a cell-penetrating peptide via a tag; (2) (C) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage or its passage specified in accession number NITE BP-03918, or (D) a bacteriophage being the bacteriophage of (C), the capsid of the bacteriophage or its passage being linked to a cell-penetrating peptide via a tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is the bacteriophage or its passage specified by accession number NITE BP-03519.

24. A pharmaceutical composition, wherein, The pharmaceutical composition comprises the phages described in (1) to (3) below and pharmaceutically acceptable excipients: (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacking an integrase gene and / or an immunorepressor protein gene, 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 (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514, and (b) lacking an integrase gene and / or an immunorepressor protein gene; as well as (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacking an integrase gene and / or an immunorepressor protein gene, the capsid of the bacteriophage being linked to Inv3 via an S tag.

25. The pharmaceutical composition of claim 24, wherein, The pharmaceutical composition comprises the following bacteriophages (1) to (3): (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of the bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage containing 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, the genome of the bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, the capsid of the bacteriophage being linked to Inv3 via an S tag.

26. The pharmaceutical composition of claim 24 or 25, wherein, The pharmaceutical composition comprises the following bacteriophages (1) to (3): (1) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of said bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting 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, the genome of the bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03918, the capsid of the bacteriophage being linked to Inv3 via an S tag.

27. A pharmaceutical composition, wherein, The pharmaceutical composition comprises the phages described in (1) to (3) below and pharmaceutically acceptable excipients: (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03513, the capsid of the bacteriophage or its passage being connected to Inv3 via an S tag. (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03514; as well as (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03918, the capsid of the bacteriophage or its passage being linked to Inv3 via an S tag.

28. A pharmaceutical composition, wherein, The pharmaceutical composition comprises the phages described in (1) to (3) below and pharmaceutically acceptable excipients: (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacking an integrase gene and / or an immunorepressor protein gene, 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 (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, and (b) lacking an integrase gene and / or an immunorepressor protein gene, the capsid of the bacteriophage being linked to Inv3 via an S tag; as well as (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage contains a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

29. The pharmaceutical composition of claim 28, wherein, The pharmaceutical composition comprises the following bacteriophages (1) to (3): (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, 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 containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, the capsid of the bacteriophage being linked to Inv3 via an S tag; as well as (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

30. The pharmaceutical composition of claim 28 or 29, wherein, The pharmaceutical composition comprises the following bacteriophages (1) to (3): (1) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of said bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03918, the capsid of said bacteriophage being linked to Inv3 via an S tag; as well as (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, wherein, The pharmaceutical composition comprises the phages described in (1) to (3) below and pharmaceutically acceptable excipients: (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03513, the capsid of the bacteriophage or its passage being connected to Inv3 via an S tag. (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03918, the capsid of said bacteriophage or its passage being linked to Inv3 via an S-tag; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is the bacteriophage or its passage specified by accession number NITE BP-03519.

32. A pharmaceutical composition, wherein, The pharmaceutical composition comprises the phages described in (1) to (3) below and pharmaceutically acceptable excipients: (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, and (b) lacking an integrase gene and / or an immunorepressor protein gene, 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 (a) containing a nucleic acid sequence having more than 90% identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514, and (b) lacking an integrase gene and / or an immunorepressor protein gene; as well as (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage contains a nucleic acid sequence that is more than 90% identical to the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

33. The pharmaceutical composition of claim 32, wherein, The pharmaceutical composition comprises the following bacteriophages (1) to (3): (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage containing the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of the bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage containing 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, the genome of which contains the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03519.

34. The pharmaceutical composition of claim 32 or 33, wherein, The pharmaceutical composition comprises the following bacteriophages (1) to (3): (1) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513, the capsid of said bacteriophage being linked to Inv3 via an S tag; (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage consisting 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, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-03519.

35. A pharmaceutical composition, wherein, The pharmaceutical composition comprises the phages described in (1) to (3) below and pharmaceutically acceptable excipients: (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03513, the capsid of the bacteriophage or its passage being connected to Inv3 via an S tag. (2) A bacteriophage, said bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, said bacteriophage being the bacteriophage or its passage specified by accession number NITE BP-03514; as well as (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the bacteriophage is the bacteriophage or its passage specified by accession number NITE BP-03519.

36. The pharmaceutical composition according to any one of claims 7, 8, 18, 19 and 20 to 35, wherein, The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of nontuberculous mycobacterial diseases.

37. The pharmaceutical composition of claim 36, wherein, Nontuberculous mycobacterial disease is pulmonary MAC disease.

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