Cell wall hydrolases that target C. acnes

Abstract

This disclosure relates to novel cell wall-binding domains, enzyme-active domains, and chimeric cell wall hydrolases having anti-Cutibacterium acnes activity. This disclosure also relates to compositions comprising these domains and to the use of these compositions in the treatment of Cutibacterium acnes and related conditions.

Description

Technical Field

[0001] Cross - Reference to Related Applications

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 529,224, filed on July 27, 2023, the content of which is hereby incorporated by reference in its entirety.

[0002] Statement Regarding Federally Sponsored Research

[0002] The present invention was made with government support under Grant No. R43 AR082722 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] Reference to Electronic Sequence Listings

[0003] The content of the electronic sequence listing (TOPB_003_01WO_SeqList_ST26.xml; size: 3,993,956 bytes; and creation date: July 26, 2024) is hereby incorporated by reference in its entirety.

[0004] Field of the Disclosure

[0004] The present disclosure relates to novel enzyme activity domains, cell wall binding domains, and cell wall hydrolases having activity against Cutibacterium acnes. The present disclosure also relates to compositions containing these recombinant domains and proteins, and methods of treating conditions associated with Cutibacterium acnes.

Background Art

[0005] Background

[0005] Acne vulgaris is a chronic inflammatory disease of the follicular sebaceous gland units, affecting up to 50 million people annually in the United States. Acne can have significant adverse effects on psychosocial functioning, such as increased anxiety, decreased self-esteem, depression, academic failure, and suicidal ideation. The anaerobic Gram-positive bacterium Cutibacterium acnes (formerly known as Propionibacterium acnes) plays a significant role in the development of acne. C. acnes is the most common species in the follicular sebaceous gland units, and in contrast to previous models that linked acne to C. acnes proliferation, metagenomic analysis now shows that the relative abundance of C. acnes is similar between healthy skin and acne-prone skin. Instead, it is now understood that acne development is associated with a loss of C. acnes phylogenetic diversity. C. acnes strains are classified into six major phylogenetic types based on their genome sequence: IA1, IA2, IB, IC, II, and III. Numerous studies have linked acne to a higher relative abundance of phylogenetic type IA1. This relative abundance is specifically reduced in non-phylogenetic IA1 strains, which is thought to induce innate immune stimulation and acne development. Antibiotics continue to play a central role in the treatment of acne. First-line combination therapy includes topical antibiotics for mild to moderate acne and systemic oral antibiotics for moderate to severe acne. Although effective, high levels of antibiotic use, especially systemic oral antibiotics, present challenges. Firstly, there may be unintended adverse effects on the gut and skin microbiome, such as changes in microbial composition, reduced microbial diversity, and altered functional attributes. Secondly, the overuse of antibiotics exacerbates antibiotic resistance, which poses an imminent public health threat. Therefore, there is a strong need for novel antimicrobial agents that can target C. acnes and provide microbiome-friendly alternatives to standard antibiotics.

[0006]

[0006] Endorcine is a phage-encoded bacterial cell wall-degrading enzyme that catalyzes the rapid and selective death of bacteria in the skin microbiome. Endorcine is typically expressed in the later stages of the phage lysis cycle. Endorcine binds to the host bacterial cell wall and rapidly degrades it, causing the cell to rupture and releasing mature viral particles. Endorcine can lyse its congeners even within biofilms, and bacteria have difficulty evolving resistance to endolysin because they have difficulty altering their peptidoglycans without harmful effects. However, all previous attempts to develop endolysin targeting C. acnes have faced significant challenges.

[0007]

[0007] Firstly, the diversity of known C. acnes phage / endolysin is extremely low. Through comprehensive efforts to isolate and sequence C. acnes phage diversity, all C. acnes phages isolated to date have been found to be very similar to one another, with little variation in gene content between genomes. The nucleotide sequence identity between any pair of C. acnes phage genomes is in the range of 85-100% (Liu et al., ISME J. 2015 Sep; 9(9): 2078-2093; Marinelli et al., mBio. 2012 Sep-Oct; 3(5): e00279-12). These genomes contain only a single, highly conserved endolysin, which significantly limits the amount of natural diversity available for endolysin development (e.g., the protein sequence with NCBI accession ID: YP_006907103.1, hereafter referred to as "CaLys1"). In contrast, endolysins targeting Staphylococcus sp. species can be classified into at least 27 subgroups (Oliveira et al., BMC Genomics. 2019 May 9; 20(1):357).

[0008]

[0008] The second challenge is that attempts to characterize CaLys1 and its closely related homologs from C. acnes phage face problems involving low solubility and low activity. Several groups have attempted to express CaLys1 and its closely related homologs by recombination and have found that the proteins are insoluble. Resolubilizing the proteins requires labor-intensive methods such as the use of urea gradients, which is not a commercially viable approach. In addition, this endolysin exhibits low activity in activity assays using high concentrations (e.g., 100 mg / mL) of the protein (see, for example, International Publication No. 2021 / 175606; Varotsou et al., Int J Mol Sci. 2023 May 10; 24(10):8523). [Overview of the project] [Problems that the invention aims to solve]

[0009]

[0009] Therefore, there is a strong unmet need for novel endolysins (and, more generally, bacterial cell wall hydrolases) that have improved properties compared to the currently known CaLys1 family and can selectively target C. acnes. [Means for solving the problem]

[0010] overview

[0010] This disclosure teaches chimeric cell wall hydrolases (CWHs) having desirable properties. In some embodiments, the CWHs of this disclosure can selectively treat conditions associated with Cutibacterium acnes.

[0011]

[0011] In one embodiment, the present disclosure provides a chimeric cell wall hydrolase (CWH) comprising a CW_7 cell wall binding domain (CBD) and an enzyme activity domain (EAD) of the CLC1 family.

[0012]

[0012] In another aspect, the present disclosure relates to a chimeric cell wall hydrolase (CWH), a. It is a cell wall binding domain (CBD), i. Sequence ID 47; ii. An amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 46, 48, 49, 50, and 51; iii. A CW_7 sequence consisting of amino acid sequences selected from the group comprising sequence numbers 166 to 223; iv. A CW_7 sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938; or v. CW_7 sequence composed of amino acid sequences selected from Table 7 CBD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to the sequence, or containing a CW_7 amino acid sequence having this sequence; b. Enzyme-active domain (EAD), i. Sequence ID 35; ii. Sequence ID 21; iii. Sequence ID 73; iv. An amino acid sequence selected from the group consisting of SEQ ID NOs. 20-36; v. EAD derived from sequence numbers 1-19; vi. EAD derived from CLC16, CLC2, CLC1, CLC3, CLC4, CLC5, CLC6, CLC7, CLC8, CLC9, CLC10, CLC11, CLC12, CLC13, CLC14, CLC15, CLC17, CLC18, or CLC19; vii. Sequence ID 64; or viii. Sequence ID 63 An EAD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to the sequence, or an EAD containing an amino acid sequence having this sequence. This provides chimeric cell wall hydrolase (CWH) containing the following:

[0013] In another aspect, the present disclosure provides a recombinant protein comprising an enzyme active domain (EAD) of the CLC1 family.

[0014] In another aspect, the present disclosure provides a recombinant protein comprising a CW_7 cell wall binding domain (CBD).

[0015] In another aspect, the present disclosure provides a recombinantly expressed CLC1 family protein or CaLys1 protein that is enzymatically active and has a truncated C-terminus.

[0016] In another aspect, the present disclosure provides a formulation comprising any one of the chimeric CWH, recombinant protein, or truncated protein of the foregoing embodiments, and optionally, the formulation is a topical formulation.

[0017] In another aspect, the present disclosure provides a method of treating a condition associated with Cutibacterium acnes (C. acnes), the method comprising administering a composition comprising any one of the recombinant proteins, chimeric CWH, or truncated proteins of the foregoing embodiments or administering any one of the formulations of the foregoing embodiments.

[0018] In another aspect, the present disclosure provides a method of restoring the phylogenetic diversity of Cutibacterium acnes, the method comprising administering a composition comprising any one of the recombinant proteins, chimeric CWH, or truncated proteins of the foregoing embodiments or administering any one of the formulations of the foregoing embodiments.

[0019]

[0019] In another aspect, the present disclosure also relates to a method for identifying a novel CW_7 cell wall binding domain for use in binding, targeting, and / or lysis of Cutibacterium acnes, a. A step of searching a gene database using a known CW_7 query sequence; b. A step of identifying sequences that exceed the amino acid sequence identity cutoff for this query sequence; c. A step of cloning a CW_7 sequence or a CW_7-containing CBD sequence into a chimeric cell wall hydrolase in combination with an enzyme activity domain (EAD) to form a CW_7 chimera; and d. The process of assaying the CW_7 chimera for its binding activity, targeting activity, and / or lysis activity against Cutibacterium acnes. We also provide methods that include this.

[0020] Brief explanation of each figure in the drawing

[0020] The accompanying drawings incorporated herein and forming part of herein illustrate some exemplary embodiments and / or features, which are neither sole nor exclusive. It is intended that the embodiments and drawings disclosed herein should be considered illustrative rather than restrictive. [Brief explanation of the drawing]

[0021] [Figure 1A]

[0021] Figure 1A shows a phylogenetic tree containing endolysins found in the genomes of C. acnes strains / phages such as CLC1-CLC19 and CaLys1. The CLC1 family proteins form a completely separate group from the CaLys1 family of endolysins. [Figure 1B]

[0021] Figure 1B shows a phylogenetic tree comparing the amidase domains of CLC1-CLC19 with the amidase domains of endolysins found in the genomes of C. acnes strains / phages such as CaLys1. The amidase domains of the CLC1 family proteins form a completely separate group from the amidase domains found in the endolysins of the CaLys1 family. The CLC18 amidase domain is identical to the CLC1 amidase domain, and the CLC19 amidase domain is identical to the CLC13 amidase domain; as a result, these domains are not shown separately in Figure 1B. [Figure 2]

[0022] Figure 2 shows an image illustrating the formation of a zone clearing on a plate embedded with C. acnes, caused by the cell lysate of BL21 cells expressing CLC1. [Figure 3]

[0023] Figure 3 is a chart showing the results of a turbidity reduction assay demonstrating that purified CLC1 exhibits lytic activity against C. acnes. [Figure 4]

[0024] Figure 4 shows the results of a turbidity reduction assay demonstrating that CLC1 truncation exhibits higher C. acnes solubility compared to full-length natural CLC1. [Figure 5A]

[0025] Figure 5 shows the results of a turbidity reduction assay for C. acnes, comparing C-terminal truncation of full-length native CLC1 family proteins with CaLys1. Figure 5A corresponds to the results for CLC2. [Figure 5B]

[0025] Figure 5B corresponds to the results of CLC3. [Figure 5C]

[0025] Figure 5C corresponds to the results of CLC16. [Figure 5D]

[0025] Figure 5D corresponds to the results of CaLys1. [Figure 6]

[0026] Figure 6 shows a diagram of the domain composition of the proteins shown. CLB1, CLB2, CLB3, and CLB4 contain putative cell wall-binding domains consisting of one or two CW-7 domains. In contrast, the C-terminus of CLC1 does not encode any protein domains predicted by calculation. [Figure 7A]

[0027] Figure 7A shows the taxonomic distribution of proteins containing the CW_7 repeat domain, as identified by the protein database search performed herein. [Figure 7B]

[0027] Figure 7B shows the detailed taxonomic distribution of proteins containing the CW_7 repeat domain, as identified by the protein database search performed herein. [Figure 8A]

[0028] Figure 8 shows the results of a lysis activity assay demonstrating that chimeric endolysins targeting C. acnes can be created using CLB1-CBD and CLB2-CBD. Figure 8A shows the results for CLB1-CBD and CLB2-CBD combined with EAD derived from the Clostridium dificile-targeting protein (CD27L). [Figure 8B]

[0028] Figure 8B shows the results for CLB1-CBD and CLB2-CBD combined with EAD derived from a Geobacillus sp. targeting protein (PlyGVE2). [Figure 9A]

[0029] Figure 9A is a chart showing the results of a turbidity reduction assay, demonstrating that a chimeric protein linking CLC1 EAD with CLB1 CBD and CLB2 CBD exhibited significantly higher C. acnes lytic activity compared to the natural CLC1 protein. [Figure 9B]

[0029] Figure 9B is a chart of the results of a turbidity reduction assay showing that the chimeric protein linking CLC1 EAD with CLB3 CBD and CLB4 CBD exhibited significantly higher C. acnes lytic activity compared to the natural CLC1 protein. [Figure 9C]

[0029] Figure 9C is a chart of the results of a turbidity reduction assay showing that the CLC1 EAD+CLB2 CBD chimera exhibited significantly higher C. acnes lytic activity compared to the natural CLC1 protein and CLC1 truncation. [Figure 10A]

[0030] Figure 10A is a chart showing the results of a turbidity reduction assay, demonstrating that a chimeric protein linking CLC2 EAD with CLB1 CBD and CLB2 CBD exhibited significantly higher C. acnes lytic activity compared to the native CLC2 protein. [Figure 10B]

[0030] Figure 10B is a chart of the results of a turbidity reduction assay showing that the CLC2 EAD+CLB2 CBD chimera exhibited significantly higher C. acnes lytic activity compared to the natural CLC2 protein and CLC2 truncation. [Figure 11A]

[0031] Figure 11A is a chart showing the results of a turbidity reduction assay, demonstrating that a chimeric protein linking CLC3 EAD with CLB1 CBD and CLB2 CBD exhibited significantly higher C. acnes lytic activity compared to the natural CLC3 protein. [Figure 11B]

[0031] Figure 11B is a chart of the results of a turbidity reduction assay showing that the CLC3 EAD+CLB2 CBD chimera exhibited significantly higher C. acnes lytic activity compared to the natural CLC3 protein and CLC3 truncation. [Figure 12]

[0032] Figure 12 is a chart showing that the four chimeric proteins are active over a wide range of pH values, with maximum activity at approximately pH 6. [Figure 13A]

[0033] Figure 13A shows the results of a turbidity reduction assay, demonstrating the selectivity of the following chimeric protein of this disclosure for C. acnes over other genera of symbiotic skin bacteria: CLC1-EAD + CLB1-CBD. [Figure 13B]

[0033] Figure 13B shows the results of a turbidity reduction assay, demonstrating the selectivity of the following chimeric protein of this disclosure for C. acnes over other genera of symbiotic skin bacteria: CLC1-EAD + CLB2-CBD. [Figure 13C]

[0033] Figure 13C shows the results of a turbidity reduction assay, demonstrating the selectivity of the following chimeric protein of this disclosure for C. acnes over other genera of symbiotic skin bacteria: CLC3-EAD+CLB1-CBD. [Figure 13D]

[0033] Figure 13D shows the results of a turbidity reduction assay, demonstrating the selectivity of the following chimeric protein of this disclosure for C. acnes over other genera of symbiotic skin bacteria: CLC3-EAD+CLB2-CBD. [Figure 14A]

[0034] Figure 14A shows the results of a thermal stability assay for the following chimeric protein of this disclosure: CLC1-EAD+CLB1-CBD. [Figure 14B]

[0034] Figure 14B shows the results of a thermal stability assay for the following chimeric protein of this disclosure: CLC1-EAD+CLB2-CBD. [Figure 14C]

[0034] Figure 14C shows the results of a thermal stability assay for the following chimeric protein of this disclosure: CLC3-EAD+CLB1-CBD. [Figure 14D]

[0034] Figure 14D shows the results of a thermal stability assay for the following chimeric protein of this disclosure: CLC3-EAD+CLB2-CBD. [Figure 15]

[0035] Figure 15 is a chart showing the CFU of C. acnes cells under specified conditions in a quantitative cell death assay. Six hours of incubation with CLC1-EAD + CLB1-CBD resulted in a more than three-order-of-magnitude reduction in the number of viable C. acnes cells. [Figure 16A]

[0036] Figure 16A shows the results of a turbidity reduction assay for C. acnes with the following chimeric protein of this disclosure: CLC4-EAD+CLB2-CBD. [Figure 16B]

[0036] Figure 16B shows the results of a turbidity reduction assay for C. acnes with the following chimeric protein of this disclosure: CLC5-EAD+CLB2-CBD. [Figure 16C]

[0036] Figure 16C shows the results of a turbidity reduction assay for C. acnes with the following chimeric protein of this disclosure: CLC8-EAD+CLB2-CBD. [Figure 16D]

[0036] Figure 16D shows the results of a turbidity reduction assay for C. acnes with the following chimeric protein of this disclosure: CLC10-EAD+CLB2-CBD. [Figure 16E]

[0036] Figure 16E shows the results of a turbidity reduction assay for C. acnes with the following chimeric protein of this disclosure: CLC14-EAD+CLB2-CBD. [Figure 16F]

[0036] Figure 16F shows the results of a turbidity reduction assay for C. acnes with the following chimeric protein of this disclosure: CLC16-EAD+CLB2-CBD. [Figure 16G]

[0036] Figure 16G shows the results of a turbidity reduction assay for the CLC16-EAD+CLB2-CBD chimera compared to full-length CLC16 and CLC16-truncation. [Figure 17]

[0037] Figure 17 shows the percentage of amino acid sequence identity between EDAs derived from CLC1, CLC5, CLC4, CLC8, CLC3, CLC10, CLC2, CLC14, and CLC16. [Figure 18A]

[0038] Figure 18A shows the results of a thermal stability assay for the following chimeric protein of this disclosure: CLC4-EAD+CLB2-CBD. [Figure 18B]

[0038] Figure 18B shows the results of a thermal stability assay for the following chimeric protein of this disclosure: CLC5-EAD+CLB2-CBD. [Figure 18C]

[0038] Figure 18C shows the results of a thermal stability assay for the following chimeric protein of this disclosure: CLC8-EAD+CLB2-CBD. [Figure 18D]

[0038] Figure 18D shows the results of a thermal stability assay for the following chimeric protein of this disclosure: CLC10-EAD+CLB2-CBD. [Figure 18E]

[0038] Figure 18E shows the results of a thermal stability assay for the following chimeric protein of this disclosure: CLC14-EAD+CLB2-CBD. [Figure 18F]

[0038] Figure 18F shows the results of a thermal stability assay for the following chimeric protein of this disclosure: CLC16-EAD+CLB2-CBD. [Figure 19]

[0039] Figure 19 shows the results of a turbidity reduction assay for the CLC16-EAD+CLB2-CBD chimeric protein in C. acnes and three other symbiotic bacterial strains. [Figure 20A]

[0040] Figure 20 shows the results of turbidity reduction assays for various strains and phyla of C. acnes regarding the two chimeras of this disclosure. Figure 20A shows the results for the CLC1-EAD+CLB2-CBD chimera for strains ATCC 11827, HL013PA1, HL027PA1, HL030PA1, and HL060PA1. [Figure 20B]

[0040] Figure 20B shows the results for CLC1-EAD+CLB2-CBD chimeras for ATCC 11827, HL001PA1, and HL056PA1 strains. [Figure 20C]

[0040] Figure 20C shows the results for CLC16-EAD+CLB2-CBD chimeras for ATCC 11827, HL013PA1, HL027PA1, HL030PA1, and HL060PA1 strains. [Figure 20D]

[0040] Figure 20D shows the results for CLC16-EAD+CLB2-CBD chimeras for ATCC 11827, HL001PA1, and HL056PA1 strains. [Figure 21A]

[0041] Figure 21A shows the results of turbidity reduction assays for full-length CaLys1 and CaLys1-EAD+CLB2-CBD chimeric proteins against C. acnes. [Figure 21B]

[0041] Figure 21B shows the results of a turbidity reduction assay for full-length CaLys1 and CaLys1-EAD+CLB2-CBD chimeric proteins against C. striatum. [Figure 21C]

[0041] Figure 21C shows the results of a turbidity reduction assay for full-length CaLys1 and CaLys1-EAD+CLB2-CBD chimeric protein against S. aureus. [Figure 22]

[0042] Figure 22 shows the results of turbidity reduction assays for C. acnes using full-length CaLys1, CaLys1-truncation, and CaLys1-EAD+CLB2-CBD chimeric protein. [Figure 23A]

[0043] Figure 23A shows the results of screening for various CW_7 CBD whole cell lysates, including chimeric ones, in a turbidity reduction assay. [Figure 23B]

[0043] Figure 23B shows the results of screening for various additional CW_7 CBD whole cell lysates, including chimeras, in the turbidity reduction assay. [Figure 23C]

[0043] Figure 23C visually shows the sequence motif shared by the CW_7 repeat that exhibits anti-C. acnes activity in a chimeric combination with CLC2-EAD. [Figure 24A]

[0044] Figure 24A shows the results of turbidity reduction assays for chimeric proteins containing CPL3, CPL25, and CPL36 CBD. For comparison, results for full-length CLC2, CLC2-truncation, and CLC2-EAD+CLB2-CBD chimeric proteins are also shown. [Figure 24B]

[0044] Figure 24B shows the results of turbidity reduction assays for chimeric proteins containing CPL46, CPL54, and CPL66 CBD. For comparison, results for full-length CLC2, CLC2-truncation, and CLC2-EAD+CLB2-CBD chimeric proteins are also shown. [Figure 25]

[0045] Figure 25 shows the results of turbidity reduction assays for C. acnes using CLC1-EAD+CLB1-CW7-1 and CLC1-EAD+CLB1-CW7-2 chimeras compared to full-length CLC1. [Figure 26]

[0046] Figure 26 shows the results of quantitative killing assays against C. acnes using HPMC-based hydrogel formulations of CLC1-EAD+CLB2-CBD and CLC16-EAD+CLB2-CBD chimeras, compared to an untreated control. [Figure 27]

[0047] Figure 27 shows the motif analysis of the EAD of the CLC1 family. [Modes for carrying out the invention]

[0022] Detailed explanation

[0048] All publications, patents, and patent applications (including all drawings and annexes) are incorporated herein by reference to the same extent as each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0023]

[0049] The following description contains information that may be useful in understanding this disclosure. This does not constitute an endorsement that any information provided herein is prior art, nor does it endorse any publication specifically or implicitly referenced as prior art.

[0024] definition

[0050] The terms “a” or “an” refer to one or more entities, that is, they can refer to multiple entities. Therefore, the terms “a,” “an,” “one or more,” and “at least one” are used synonymously in this specification. In addition, a reference to “one element” using the indefinite article “a” or “an” does not exclude the possibility that multiple elements exist, unless the context explicitly requires that only one element exists.

[0025]

[0051] Throughout this application, the term “approximately” is used to indicate that a value includes inherent variations in the error of the apparatus or method used to determine that value, or variations present between the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “approximately” means within plus or minus 10 percent of the reported value (except where such a value is greater than 100% of the possible value or less than 0%). When used in conjunction with a range or set of values, the term “approximately” applies to each of the endpoints of the range or the values ​​enumerated in the set, unless otherwise indicated. As used in this application, the terms “approximately” and “about” are used interchangeably.

[0026]

[0052] Unless otherwise indicated, all figures used in this specification and in the claims to represent quantities, ratios, and numerical characteristics of components, reaction conditions, etc., should be understood to be intended in all cases to be modifiable with the term "approximately".

[0027]

[0053] As used herein, the term “cell wall hydrolase” or “CWH” refers to a bacterial cell wall hydrolase, which is an enzyme that degrades peptidoglycan in the bacterial cell wall by cleaving the bonds in the peptidoglycan chain and side chain branches. Cell wall hydrolases can have various domain structures. A CWH includes an “enzyme-active domain” or “EAD,” which is a domain involved in the degradation of peptidoglycan. In some embodiments, the EAD has glycosidase activity, amidase activity, and / or peptidase activity. In some embodiments, a CWH includes a “cell wall-binding domain” or “CBD,” which is a domain that binds to the bacterial cell wall.

[0028]

[0054] The term "natural" protein is used to refer to proteins that exist naturally and have not undergone any artificial modification or recombination.

[0029]

[0055] The term “recombinant” is used herein to describe nucleic acids, proteins, vectors, and host cells in configurations that do not exist in nature or are not found in nature in relation to nucleic acids. Thus, “recombinant protein” refers to a protein that does not exist in nature. In some embodiments, recombinant protein refers to a chimeric protein, as used herein. In some embodiments, recombinant protein refers to the expression product of either the EAD or CBD sequence of this disclosure alone, or an expression product within a protein that does not exist in nature. For example, this disclosure assumes a recombinant EAD or CBD sequence of this disclosure fused to any protein tag, such as 6×His.

[0030]

[0056] As used herein, “heterogeneous” refers to any genetic material that is artificially introduced into a non-natural context. For example, a heterogeneous domain refers to a domain (e.g., EAD or CBD) that is artificially introduced into a recombinant protein sequence, and the resulting recombinant protein sequence is non-natural. Two polypeptide sequences or domains are heterogeneous if they originate from different natural polypeptide sequences or proteins. For example, in some embodiments, CBD and EAD are heterogeneous if they originate from different natural proteins.

[0031]

[0057] As used herein, “chimeric protein” is any recombinant protein comprising two or more heterogeneous domains (e.g., EAD and / or CBD).

[0032]

[0058] As used herein, a protein “domain” is a functional and / or structural subunit within a protein. In some embodiments, these are involved in specific functions or interactions and contribute to the overall role of the protein. Protein domains are the fundamental units of protein structure, folding, function, evolution, and design. See, for example, Wang et al., “Protein domain identification methods and online resources,” Comput Struct Biotechnol J 2021; 19:1145-1153 (incorporated herein by reference).

[0033]

[0059] As used herein, “chimeric cell wall hydrolase” or “chimeric CWH” is a chimeric protein that acts as a cell wall hydrolase and contains at least one heterologous domain (e.g., heterologous EAD or CBD) compared to a native CWH sequence. In some embodiments, chimeric CWH refers herein to a recombinant protein containing two heterologous CWH domains (e.g., EAD and CBD).

[0034]

[0060] As used herein, “activity” refers to the ability of a chimeric protein to inhibit the proliferation of cells derived from the species Cutibacterium acnes and / or to lyse these cells. The term “active against” refers, as used herein in relation to the target species of Cutibacterium acnes, to a chimeric protein of the disclosure (e.g., CWH) that is capable of inhibiting the proliferation of cells belonging to this target species of Cutibacterium acnes and / or lysing these cells. Activity can be calculated in various ways depending on the assay performed. In some embodiments, the level of activity is indicated based on the minimum inhibitory concentration ("MIC"), for example, based on the minimum concentration of protein required to prevent the proliferation of the target Cutibacterium acnes species in an MIS assay. In some embodiments, the level of activity is indicated based on turbidity reduction, for example, the activity is -ΔOD 600 It is calculated as / min / (mg of enzyme). In some embodiments, activity is shown based on the reduction of viable bacterial cells in the culture after an incubation period with the protein (e.g., 2 hours).

[0035]

[0061] In relation to anti-Cutibacterium acnes activity, the terms “selective” and “selectivity,” as used herein, refer to the property of a Cutibacterium acnes to exhibit higher activity against one target species compared to another species of Cutibacterium acnes. Selectivity may be calculated by comparing the reciprocal of the MIC of the chimeric protein against the first species with the MIC of the protein against the second species. In some embodiments, selectivity is determined based on relative activity in a turbidity reduction assay.

[0036]

[0062] As used herein, the term “sequence identity” refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences remain invariant throughout the entire window of residue (e.g., nucleotide or amino acid) alignment. The “identity fraction” of the aligned segments of the test sequence and the reference sequence is the number of identical residues shared by the two aligned sequences divided by the total number of residues in the reference sequence segment (i.e., the entire reference sequence or a smaller defined portion of the reference sequence). The “identity percentage” is the identity fraction multiplied by 100. Sequence comparisons for determining the identity percentage can be achieved by many well-known methods, for example, by using mathematical algorithms (e.g., those in the BLAST suite of sequence analysis programs). Unless otherwise stated, the term “sequence identity” in the claims refers to sequence identity as calculated using MUSCLE (www.ebi.ac.uk / Tools / msa / muscle / ) with default parameters.

[0037]

[0063] As used herein, the terms “CW_7 cell wall-binding domain” or “CW_7 CBD” refer to a cell wall-binding domain comprising one or more CW_7 sequences. In some embodiments, the CW_7 CBD comprises, essentially comprises, or consists of CW_7 sequences.

[0038]

[0064] The term "including all ranges and subranges between them" or its equivalent is used herein to indicate the intent that any disclosure of any range or set of possible values ​​also essentially discloses all ranges and subranges encompassed by the disclosed highest and lowest values. This term includes the entire range from the disclosed highest to the lowest values, as well as subranges from any two or more disclosed points. This term is also intended to disclose, to one decimal place, any subranges (including between any two points explicitly listed in the document) that are encompassed within the range of the disclosed highest and lowest values. Thus, the disclosure of the values ​​0, 5, 10, 15, and 20 (including all ranges and subranges between them) should be interpreted as including the ranges 0 to 20, 0 to 5 or 5 to 15, and 2 to 16 or 3.1 to 19.8, etc.

[0039]

[0065] Unless otherwise indicated, all figures used herein to represent quantities, ratios, and numerical characteristics of components, reaction conditions, etc., should be understood to be intended in all cases to be modifiable with the term "including all ranges and partial ranges between them."

[0040] overview

[0066] This disclosure provides novel enzyme-active domains (EADs), truncated enzymes, cell wall-binding domains (CBDs), and chimeric cell wall hydrolases (CWHs) exhibiting binding and / or enzymatic activity to Cutibacterium acnes. Also provided herein are compositions comprising the EADs, truncated enzymes, CBDs, and chimeric CWHs, and the use of these compositions in targeting Cutibacterium acnes and treating conditions associated with Cutibacterium acnes.

[0041]

[0067] CWHs are enzymes that degrade bacterial peptidoglycans by cleaving bonds in the peptidoglycan chain and side chain branches. CWH-mediated degradation of the peptidoglycan cell wall can lead to rapid lysis of bacterial cells due to their inability to resist internal turgor pressure. One advantage of using CWHs to treat conditions associated with Cutibacterium acnes is their high specificity. CWHs (e.g., Gram-positive endolysin) possess one or more cell wall-binding domains that bind to specific epitopes within the target cell wall. Because the composition and structure of peptidoglycan cell walls can vary significantly among bacterial species, CWHs often exhibit lysis specificity down to the genus, species, and even subspecies level. In active acne, C. acnes lineage diversity is reduced, and consequently, extensive removal of endogenous C. acnes can promote the re-establishment of lineage diversity. CWHs also offer a more direct and stable mechanism of action compared to phage therapy, another proposed antibiotic alternative.

[0042]

[0068] As demonstrated in the examples herein, this disclosure provides novel and highly effective EADs, truncated enzymes, CBDs, and chimeric CWHs exhibiting high lytic activity and / or Cutibacterium acnes species specificity. In some embodiments, the chimeric CWHs consist of domains derived from parent proteins and possessing superior properties to any of these parent proteins. In some embodiments, the CWHs herein bind to highly specific epitopes in target cell walls. In some embodiments, the CWHs herein have lytic activity down to a single species or a group of related species. Due to these properties, in some embodiments, the CWHs herein act as highly specific skin microbiome modifiers. For example, in some embodiments, the CWHs herein exhibit remarkably low activity against healthy symbiotic bacteria but are capable of specifically killing or modulating the diversity of Cutibacterium acnes.

[0043] Enzyme active domain (EAD) and truncated enzyme

[0069] In some embodiments, the recombinant protein of this disclosure comprises the EAD, enzyme, or truncated enzyme disclosed herein. In some embodiments, the recombinant protein of this disclosure comprises the EAD disclosed herein. In some embodiments, the recombinant protein of this disclosure is essentially composed of or consists of the EAD disclosed herein. In some embodiments, the recombinant protein is a chimeric protein or chimeric CWH comprising the EAD. In some embodiments, the recombinant protein of this disclosure comprises the EAD disclosed herein in combination with the CBD disclosed herein.

[0044]

[0070] This disclosure provides novel enzymes and enzyme-active domains (EADs). The inventors of this disclosure have discovered a novel class of enzymes having activity against C. acnes, which are referred to herein as the CLC1 family of enzymes. See Figures 1A-1B and Tables 1-2. As shown in the examples herein, these novel enzymes and the EADs contained therein exhibit significant activity against C. acnes even in the absence of CBD.

[0045] [Table 1]

[0046] [Table 2]

[0047] [Table 3]

[0048] [Table 4]

[0049] [Table 5]

[0050] [Table 6]

[0051]

[0071] In some embodiments, the recombinant protein of the Disclosure includes an EAD of the CLC1 family. In some embodiments, the recombinant protein of the Disclosure includes an EAD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to an EAD of the CLC1 family. In some embodiments, the recombinant protein of the Disclosure includes an EAD having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to an EAD of the CLC1 family. In some embodiments, the recombinant proteins of this disclosure include an EAD of the CLC1 family that differs from an EAD by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0052]

[0072] In some embodiments, the recombinant proteins of this disclosure include EADs of the CLC1 family disclosed in Table 2. In some embodiments, the recombinant proteins of this disclosure include EADs having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with respect to the CLC1 family EADs disclosed in Table 2. In some embodiments, the recombinant proteins of this disclosure include EADs having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with respect to the CLC1 family EADs disclosed in Table 2. In some embodiments, the recombinant proteins of this disclosure include EADs of the CLC1 family disclosed in Table 2 that differ from EADs by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0053]

[0073] In some embodiments, the recombinant protein of the Disclosure comprises CLC16 EAD (SEQ ID NO: 35). In some embodiments, the recombinant protein of the Disclosure comprises an EAD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to CLC16 EAD (SEQ ID NO: 35). In some embodiments, the recombinant protein of the Disclosure comprises an EAD having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to CLC16 EAD (SEQ ID NO: 35). In some embodiments, the recombinant protein of the present disclosure comprises CLC16 EAD (SEQ ID NO: 35) and an EAD that differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0054]

[0074] In some embodiments, the recombinant protein of the Disclosure comprises CLC2 EAD (SEQ ID NO: 21). In some embodiments, the recombinant protein of the Disclosure comprises an EAD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to CLC2 EAD (SEQ ID NO: 21). In some embodiments, the recombinant protein of the Disclosure comprises an EAD having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to CLC2 EAD (SEQ ID NO: 21). In some embodiments, the recombinant protein of the present disclosure comprises CLC2 EAD (SEQ ID NO: 21) and an EAD that differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0055]

[0075] The newly discovered EAD of this CLC1 family has the following sequence: [ka] They share the "EAD motif of the CLC1 family," which is a conserved sequence motif having (X represents any amino acid).

[0056]

[0076] In some embodiments, the recombinant proteins of the Disclosure include an EAD comprising the EAD motif of the CLC1 family (SEQ ID NO: 2939). In some embodiments, the recombinant proteins of the Disclosure include an EAD comprising the EAD motif of the CLC1 family (SEQ ID NO: 2939) and having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with respect to the CLC1 family EADs disclosed in Table 2. In some embodiments, the recombinant proteins of this disclosure include an EAD motif of the CLC1 family (SEQ ID NO: 2939) and an EAD having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with respect to the CLC1 family EADs disclosed in Table 2. In some embodiments, the recombinant proteins of this disclosure include an EAD motif of the CLC1 family (SEQ ID NO: 2939) and an EAD that differs from the CLC1 family EADs disclosed in Table 2 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0057]

[0077] In some embodiments, the recombinant protein of the Disclosure includes an EAD comprising a sequence following any one of SEQ ID NOs: 20-36 or an EAD region comprising any one of SEQ ID NOs: 1-19. In some embodiments, the recombinant protein of the Disclosure includes an EAD comprising a sequence following any one of SEQ ID NOs: 20-36 or an EAD region comprising any one of SEQ ID NOs: 1-19. In some embodiments, the recombinant protein of the Disclosure includes an EAD having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with a sequence following any one of SEQ ID NOs: 20-36 or an EAD region comprising any one of SEQ ID NOs: 1-19. In some embodiments, the recombinant protein of the present disclosure comprises an EAD region that follows a sequence according to any one of SEQ ID NOs: 20-36 or an EAD region that follows any one of SEQ ID NOs: 1-19 and an EAD that differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0058] [Table 7]

[0059] [Table 8]

[0060]

[0078] In some embodiments, the recombinant protein of the Disclosure comprises an EAD comprising the sequence of Table 4 or the EAD region of the sequence of Table 3. In some embodiments, the recombinant protein of the Disclosure comprises an EAD comprising the sequence of Table 4 or the EAD region of the sequence of Table 3. In some embodiments, the recombinant protein of the Disclosure comprises an EAD having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the sequence of Table 4 or the EAD region of the sequence of Table 3. In some embodiments, the recombinant protein of the Disclosure comprises an EAD that differs from the sequence of Table 4 or the EAD region of the sequence of Table 3 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0061]

[0079] In some embodiments, the recombinant protein of the Disclosure comprises an EAD derived from CD27L or PlyGVE2. In some embodiments, the recombinant protein of the Disclosure comprises an EAD comprising a sequence following any one of SEQ ID NOs. 63-64 or an EAD region comprising any one of SEQ ID NOs. 60-61. In some embodiments, the recombinant protein of the Disclosure comprises an EAD comprising a sequence following any one of SEQ ID NOs. 63-64 or an EAD region comprising any one of SEQ ID NOs. 60-61. In some embodiments, the recombinant protein of the Disclosure comprises an EAD having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with a sequence following any one of SEQ ID NOs. 63-64 or an EAD region comprising any one of SEQ ID NOs. 60-61. In some embodiments, the recombinant protein of the present disclosure comprises an EAD that differs from either a sequence following any one of SEQ ID NOs. 63-64 or an EAD region of any one of SEQ ID NOs. 60-61 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0062]

[0080] In some embodiments, the recombinant protein of the Disclosure comprises an EAD of CaLys1. In some embodiments, the recombinant protein of the Disclosure comprises an EAD comprising a sequence according to SEQ ID NO: 73 or an EAD region of SEQ ID NO: 72. In some embodiments, the recombinant protein of the Disclosure comprises an EAD comprising a sequence according to SEQ ID NO: 73 or an EAD region of SEQ ID NO: 72. In some embodiments, the recombinant protein of the Disclosure comprises an EAD having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the sequence according to SEQ ID NO: 73 or the EAD region of SEQ ID NO: 72. In some embodiments, the recombinant protein of the Disclosure comprises an EAD that differs from the sequence according to SEQ ID NO: 73 or the EAD region of SEQ ID NO: 72 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0063]

[0081] In some embodiments, the recombinant protein of this disclosure comprises an EAD derived from lysine. In some embodiments, this lysine is endolysine, taillysine, exolysine, bacteriocin, or autolysine. In some embodiments, this EAD is derived from any one of the endolysines listed herein. In some embodiments, this EAD is a glycosidase. In some embodiments, this EAD is an amidase. In some embodiments, this EAD is a peptidase.

[0064]

[0082] In some embodiments, the recombinant protein herein comprises an EAD according to any one of the embodiments described above. In some embodiments, the recombinant protein herein comprises one EAD. In some embodiments, this recombinant protein comprises multiple EADs. In some embodiments, this recombinant protein comprises two EADs. In some embodiments, this recombinant protein comprises three, four, five, six, seven, eight, nine, or ten EADs.

[0065] Truncate enzymes

[0083] In one embodiment, the present disclosure provides a truncated enzyme having lytic activity against C. acnes. As demonstrated in the examples herein, the inventors have surprisingly discovered that truncation of the conserved C-terminal region of CLC1 family enzymes and CaLys1 results in a truncated enzyme with higher lytic activity compared to the corresponding full-length native protein. Table 5 provides exemplary truncated enzymes of the present disclosure.

[0066] [Table 9]

[0067] [Table 10]

[0068] [Table 11]

[0069] [Table 12]

[0070]

[0084] In some embodiments, the recombinant protein of this disclosure comprises a sequence of a truncated enzyme disclosed herein. In some embodiments, the recombinant protein of this disclosure consists of a sequence of a truncated enzyme disclosed herein. In some embodiments, the recombinant protein of this disclosure comprises a truncated enzyme having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the sequence of a truncated enzyme disclosed herein. In some embodiments, the recombinant protein of this disclosure comprises a truncated enzyme that differs from the sequence disclosed herein by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0071]

[0085] In some embodiments, the recombinant protein of this disclosure comprises a truncated enzyme sequence as disclosed in Table 5. In some embodiments, the recombinant protein of this disclosure consists of a truncated enzyme sequence as disclosed in Table 5. In some embodiments, the recombinant protein of this disclosure comprises a truncated enzyme having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the truncated enzyme sequences disclosed in Table 5. In some embodiments, the recombinant protein of this disclosure comprises a truncated enzyme that differs from the sequences disclosed in Table 5 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0072]

[0086] In some embodiments, the recombinant protein of this disclosure comprises a C-terminal truncation of an enzyme sequence disclosed in Table 1 or Table 3. In some embodiments, the recombinant protein of this disclosure consists of a C-terminal truncation of an enzyme sequence disclosed in Table 1 or Table 3. In some embodiments, the recombinant protein of this disclosure comprises a truncated enzyme having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the C-terminal truncation of an enzyme sequence disclosed in Table 1 or Table 3. In some embodiments, the recombinant protein of this disclosure comprises a truncated enzyme that differs from the C-terminal truncation of an enzyme sequence disclosed in Table 1 or Table 3 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0073]

[0087] In some embodiments, the C-terminal truncation is a truncation of the entire C-terminal region following the EAD. In some embodiments, the C-terminal truncation is a truncation of the conserved C-terminal tail. In some embodiments, the C-terminal truncation is a truncation of the C-terminal region of the enzyme that does not have any protein domain annotations. In some embodiments, the C-terminal truncation is a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 from the C-terminus. ,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, In some embodiments, the C-terminal truncation is a truncation of approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 140, 145, or 150 amino acids. In some embodiments, the C-terminal truncation is a truncation of approximately 60 to 90 amino acids from the C-terminus.

[0074]

[0088] In some embodiments, the truncated enzyme is truncated CLC1, and the truncation is a truncation of about 60 to 90 amino acids from the full-length CLC1 enzyme.

[0075]

[0089] In some embodiments, the truncated enzyme is truncated CaLys1, CLC1, CLC2, CLC3, CLC4, CLC5, CLC6, CLC7, CLC8, CLC9, CLC10, CLC11, CLC12, CLC13, CLC14, CLC16, CLC18, or CLC19, and the truncation is a truncation of approximately 50 to 90 amino acids from the C-terminus of the full-length native enzyme. In some embodiments, the truncated enzyme is truncated CaLys1, CLC1, CLC2, CLC3, CLC4, CLC5, CLC6, CLC7, CLC8, CLC9, CLC10, CLC11, CLC12, CLC13, CLC14, CLC16, CLC18, or CLC19, and the truncation is a truncation of approximately 80-85 amino acids from the C-terminus of the full-length native enzyme.

[0076]

[0090] In some embodiments, the truncated enzyme is truncated CLC15, and the truncation is a truncation of approximately 90–130 amino acids from the full-length CLC15 enzyme. In some embodiments, the truncated enzyme is truncated CLC15, and the truncation is a truncation of approximately 120–125 amino acids from the full-length CLC15 enzyme.

[0077]

[0091] In some embodiments, the truncated enzyme is truncated CLC17, and the truncation is a truncation of approximately 30–70 amino acids from the full-length CLC17 enzyme. In some embodiments, the truncated enzyme is truncated CLC17, and the truncation is a truncation of approximately 60–70 amino acids from the full-length CLC17 enzyme.

[0078] Cell wall-binding domain (CBD)

[0092] In some embodiments, the recombinant proteins of this disclosure include a cell wall-binding domain (CBD).

[0079]

[0093] The inventors have discovered a novel class of C. acnes-binding CDRs containing the CW_7 sequence. As shown in the examples of this disclosure, a wide variety of these CW_7 CBDs, when combined with CLC1 family EADs, were able to increase the lytic activity against C. acnes compared to the corresponding full-length CLC1 family enzymes. Information on CLB1-4 is provided in Table 6.

[0080] [Table 13]

[0081]

[0094] In some embodiments, the recombinant protein of the Disclosure comprises a CBD containing the CW_7 sequence. In some embodiments, the recombinant protein of the Disclosure comprises a CBD containing the CW_7 sequence disclosed herein. In some embodiments, the recombinant protein of the Disclosure comprises a CBD containing the CW_7 sequence assigned to Interpro domain entry IPR013168. In some embodiments, the CBD consists of the CW_7 sequence.

[0082]

[0095] In some embodiments, the recombinant protein of the present disclosure comprises one CBD of any one of CLB1 to 4. In some embodiments, this CBD comprises a sequence following any one of SEQ ID NOs: 41 to 44, or a CBD region of any one of SEQ ID NOs: 37 to 40. In some embodiments, this CBD comprises a sequence following any one of SEQ ID NOs: 41 to 44, or a CBD region of any one of SEQ ID NOs: 37 to 40. In some embodiments, this CBD has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with a sequence following any one of SEQ ID NOs: 41 to 44, or with a CBD region of any one of SEQ ID NOs: 37 to 40. In some embodiments, the CBD differs from the sequence following any one of SEQ ID NOs: 41-44 or from the CBD region of any one of SEQ ID NOs: 37-40 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0083]

[0096] In some embodiments, the recombinant protein of the present disclosure contains CBD of CLB2. In some embodiments, this CBD consists of a sequence according to SEQ ID NO: 42 or the CBD region of SEQ ID NO: 38. In some embodiments, this CBD contains a sequence according to SEQ ID NO: 42 or the CBD region of SEQ ID NO: 38. In some embodiments, this CBD has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the sequence according to SEQ ID NO: 42 or the CBD region of SEQ ID NO: 38. In some embodiments, this CBD differs from the sequence according to SEQ ID NO: 42 or the CBD region of SEQ ID NO: 38 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0084]

[0097] In some embodiments, the recombinant protein of the present disclosure comprises a CBD containing a CW_7 sequence derived from CLB1-4. In some embodiments, this CW_7 sequence consists of a sequence following any one of SEQ ID NOs. 45-51. In some embodiments, this CW_7 sequence includes a sequence following any one of SEQ ID NOs. 45-51. In some embodiments, this CW_7 sequence has at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% identity with a sequence following any one of SEQ ID NOs. 45-51. In some embodiments, this CW_7 sequence includes a sequence following any one of SEQ ID NOs. 45-51. In some embodiments, the CW_7 sequence has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the sequence following any one of sequence numbers 45-51. In some embodiments, the CW_7 sequence differs from the sequence following any one of sequence numbers 45-51 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0085]

[0098] In some embodiments, the recombinant protein of the present disclosure comprises a CBD containing the CW_7 sequence of CLB2. In some embodiments, this CW_7 sequence consists of a sequence according to SEQ ID NO: 47. In some embodiments, this CW_7 sequence includes a sequence according to SEQ ID NO: 47. In some embodiments, this CW_7 sequence has at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% identity with the sequence according to SEQ ID NO: 47. In some embodiments, this CW_7 sequence includes a sequence according to SEQ ID NO: 47. In some embodiments, this CW_7 sequence has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the sequence according to SEQ ID NO: 47. In some embodiments, this CW_7 sequence differs from the sequence according to SEQ ID NO: 47 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0086]

[0099] In some embodiments, the recombinant protein of the present disclosure comprises a CBD of any one of the CPL-designated proteins illustrated in the examples herein. In some embodiments, this CBD comprises a sequence following any one of SEQ ID NOs: 166-223 or a CBD region of any one of SEQ ID NOs: 108-165. In some embodiments, this CBD comprises a sequence following any one of SEQ ID NOs: 166-223 or a CBD region of any one of SEQ ID NOs: 108-165. In some embodiments, this CBD has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with a sequence following any one of SEQ ID NOs: 166-223 or a CBD region of any one of SEQ ID NOs: 108-165. In some embodiments, the CBD differs from the sequence following any one of SEQ ID NOs: 166-223 or from the CBD region of any one of SEQ ID NOs: 108-165 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0087]

[0100] In some embodiments, the recombinant protein of this disclosure comprises a CW_7 sequence derived from any one of the CPL-designated proteins illustrated in the examples herein. In some embodiments, the recombinant protein comprises a CW_7 sequence comprising any one of SEQ ID NOs. 166-223 or a CBD region comprising any one of SEQ ID NOs. 108-165. In some embodiments, the recombinant protein comprises a CW_7 sequence contained in any one of SEQ ID NOs. 166-223 or a CBD region comprising any one of SEQ ID NOs. 108-165. In some embodiments, the recombinant protein comprises a CW_7 sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% identity with a CW_7 sequence comprising any one of SEQ ID NOs. 166-223 or a CBD region comprising any one of SEQ ID NOs. 108-165. In some embodiments, the recombinant protein includes a CW_7 sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with a CW_7 sequence comprising any one of SEQ ID NOs. 166-223 or with a CBD region comprising any one of SEQ ID NOs. 108-165. In some embodiments, the CW_7 sequence differs from a CW_7 sequence comprising any one of SEQ ID NOs. 166-223 or with a CBD region comprising any one of SEQ ID NOs. 108-165 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0088]

[0101] In some embodiments, the recombinant protein of this disclosure comprises CW_7 CBD derived from a family of proteins comprising CW_7 CBD disclosed herein. In some embodiments, this recombinant protein comprises CW_7 CBD comprising a sequence following any one of SEQ ID NOs. 282-2938. In some embodiments, this recombinant protein comprises CBD contained in any one of SEQ ID NOs. 282-2938. In some embodiments, this recombinant protein comprises CBD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90%, or 95% identity with CBD comprising a sequence following any one of SEQ ID NOs. 282-2938. In some embodiments, the recombinant protein contains CBD having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with CBD consisting of a sequence following any one of SEQ ID NOs. 282-2938. In some embodiments, the CBD sequence differs from the CBD sequence consisting of a sequence following any one of SEQ ID NOs. 282-2938 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0089]

[0102] In some embodiments, the recombinant protein of this disclosure comprises a CW_7 sequence derived from a family of proteins comprising the CW_7 sequence disclosed herein. In some embodiments, the recombinant protein comprises a CW_7 sequence comprising a sequence following any one of SEQ ID NOs. 282-2938. In some embodiments, the recombinant protein comprises a CW_7 sequence contained in any one of SEQ ID NOs. 282-2938. In some embodiments, the recombinant protein comprises a CW_7 sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% identity with a CW_7 sequence comprising a sequence following any one of SEQ ID NOs. 282-2938. In some embodiments, the recombinant protein includes a CW_7 sequence that has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with a CW_7 sequence consisting of a sequence following any one of SEQ ID NOs. In some embodiments, this CW_7 sequence differs from a CW_7 sequence consisting of a sequence following any one of SEQ ID NOs. by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0090]

[0103] In some embodiments, the recombinant protein of this disclosure comprises CW_7 CBD derived from a family of proteins comprising CW_7 CBD disclosed herein. In some embodiments, this recombinant protein comprises CW_7 CBD comprising the sequences of Table 7. In some embodiments, this recombinant protein comprises CBD comprising any one of the sequences of Table 7. In some embodiments, this recombinant protein comprises CBD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% identity with CBD comprising the sequences of Table 7. In some embodiments, this recombinant protein comprises CBD having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with CBD comprising the sequences of Table 7. In some embodiments, this CBD sequence differs from the CBD sequence consisting of the sequences in Table 7 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0091]

[0104] In some embodiments, the recombinant protein of this disclosure comprises a CW_7 sequence derived from a family of proteins comprising the CW_7 sequence disclosed herein. In some embodiments, the recombinant protein comprises a CW_7 sequence comprising the sequences in Table 7. In some embodiments, the recombinant protein comprises a CW_7 sequence included in the sequences in Table 7. In some embodiments, the recombinant protein comprises a CW_7 sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% identity with the CW_7 sequence comprising the sequences in Table 7. In some embodiments, the recombinant protein comprises a CW_7 sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the CW_7 sequence comprising the sequences in Table 7. In some embodiments, the CW_7 sequence differs from the CW_7 sequence consisting of the sequences in Table 7 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0092]

[0105] In some embodiments, the recombinant protein of the Disclosure contains one CBD. In some embodiments, the recombinant protein of the Disclosure contains multiple CBDs. In some embodiments, the recombinant protein of the Disclosure contains two CBDs. In some embodiments, the recombinant protein of the Disclosure contains three, four, five, six, seven, eight, nine, or ten CBDs.

[0093]

[0106] In some embodiments, the recombinant protein of the Disclosure comprises one CW_7 sequence. In some embodiments, the recombinant protein of the Disclosure comprises multiple CW_7 sequences. In some embodiments, the recombinant protein of the Disclosure comprises two CW_7 sequences. In some embodiments, the recombinant protein of the Disclosure comprises three, four, five, six, seven, eight, nine, or ten CW_7 sequences.

[0094]

[0107] In some embodiments, the recombinant protein of the Disclosure comprises a CBD containing a CW_7 sequence having the CW_7-21 motif (SEQ ID NO: 2940). In some embodiments, the recombinant protein of the Disclosure comprises a CW_7 sequence having the CW_7-21 motif (SEQ ID NO: 2940). In some embodiments, this CW_7 sequence has the CW_7-21 motif (SEQ ID NO: 2940) and has at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to the CLB2 CW_7 repeat. In some embodiments, this CW_7 sequence has the CW_7-21 motif (SEQ ID NO: 2940) and has at least 43% or at least 58% identity to the CLB2 CW_7 repeat. In some embodiments, this CW_7 sequence has a CW_7-21 motif (SEQ ID NO: 2940) and differs from the CLB2 CW_7 repeat by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids.

[0095]

[0108] In some embodiments, the recombinant protein of the Disclosure comprises a CBD containing a CW_7 sequence having the CW_7-19 motif (SEQ ID NO: 2941). In some embodiments, the recombinant protein of the Disclosure comprises a CW_7 sequence having the CW_7-19 motif (SEQ ID NO: 2941). In some embodiments, this CW_7 sequence has the CW_7-19 motif (SEQ ID NO: 2941) and has at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to the CLB2 CW_7 repeat. In some embodiments, this CW_7 sequence has the CW_7-19 motif (SEQ ID NO: 2941) and has at least 43% or at least 58% identity to the CLB2 CW_7 repeat. In some embodiments, this CW_7 sequence has a CW_7-19 motif (sequence number 2941) and differs from the CLB2 CW_7 repeat by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids.

[0096] [Table 14]

[0097] [Table 15]

[0098] [Table 16]

[0099] [Table 17]

[0100] [Table 18]

[0101] [Table 19]

[0102] [Table 20]

[0103] [Table 21]

[0104] [Table 22]

[0105] [Table 23]

[0106] [Table 24]

[0107] [Table 25]

[0108] [Table 26]

[0109] [Table 27]

[0110] [Table 28]

[0111] Chimeric cell wall hydrolase of the present disclosure

[0109] In some embodiments, the recombinant proteins of the Disclosure are chimeric proteins. In some embodiments, the recombinant proteins of the Disclosure are chimeric CWHs. The Disclosure is partly based on the inventors' development of novel, highly active chimeric CWHs. As disclosed in the examples herein, the exemplary CWHs of the Disclosure are remarkable in that they have highly effective anti-Cutibacterium acnes properties resulting from the unique properties of the novel CBD and / or EAD composed of the CWH.

[0112]

[0110] In some embodiments, the Disclosure provides recombinant proteins comprising sequences of EAD and / or CBD according to any one of the embodiments disclosed herein. In some embodiments, the recombinant protein is a chimeric protein. In some embodiments, the chimeric protein is a chimeric cell wall hydrolase (CWH). The chimeric CWHs herein comprise at least one heterologous domain compared to a native CWH sequence, and comprise, for example, heterologous EAD or CBD.

[0113]

[0111] In some embodiments, the chimeric CWH of this specification is a chimeric protein comprising EAD and CBD derived from different natural proteins. In some embodiments, the chimeric CWH of this disclosure comprises EAD and CBD as disclosed herein. In some embodiments, the chimeric CWH of this disclosure comprises EAD having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with EAD as disclosed herein, and CBD having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with CBD as disclosed herein.

[0114]

[0112] In some embodiments, the chimeric CWH of the Disclosure includes an EAD of the CLC1 family. In some embodiments, the chimeric CWH of the Disclosure includes an EAD having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an EAD of the CLC1 family disclosed herein.

[0115]

[0113] In some embodiments, the chimeric CWH of the Disclosure comprises CW_7 CBD. In some embodiments, the chimeric CWH of the Disclosure comprises CBD having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with CW_7 CBD disclosed herein.

[0116]

[0114] In some embodiments, the chimeric CWH of the Disclosure comprises an EAD of the CLC1 family and a CW_7 CBD. In some embodiments, the chimeric CWH of the Disclosure comprises an EAD having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an EAD of the CLC1 family disclosed herein and a CBD having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with a CW_7 CBD disclosed herein.

[0117]

[0115] In some embodiments, the chimeric CWH of the Disclosure includes an EAD (SEQ ID NO: 35) derived from CLC16. In some embodiments, the chimeric CWH of the Disclosure includes an EAD having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with the EAD (SEQ ID NO: 35) derived from CLC16.

[0118]

[0116] In some embodiments, the chimeric CWH of the Disclosure includes an EAD (SEQ ID NO: 21) derived from CLC2. In some embodiments, the chimeric CWH of the Disclosure includes an EAD having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with the EAD (SEQ ID NO: 21) derived from CLC2.

[0119]

[0117] In some embodiments, the chimeric CWH of the Disclosure includes an EAD derived from CaLys1. In some embodiments, the chimeric CWH of the Disclosure includes an EAD having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with the EAD derived from CaLys1.

[0120]

[0118] In some embodiments, the chimeric CWH of the Disclosure comprises CBD derived from CLB2. In some embodiments, the chimeric CWH of the Disclosure comprises CBD having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with CBD derived from CLB2.

[0121]

[0119] In some embodiments, the chimeric CWH of the Disclosure comprises the sequence of Table 8. In some embodiments, the chimeric CWH of the Disclosure consists of the sequence of Table 8. In some embodiments, the chimeric CWH of the Disclosure has at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% identity with the sequence of Table 8. In some embodiments, the chimeric CWH of the Disclosure has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the sequence of Table 8. In some embodiments, the sequence of this chimeric CWH differs from the sequence of Table 8 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0122] [Table 29]

[0123] [Table 30]

[0124] [Table 31]

[0125] Table 32

[0126] Table 33

[0127] Table 34

[0128] Table 35

[0129] Table 36

[0130] Table 37

[0131] Table 38

[0132] Table 39

[0133]

[0120] In some embodiments, the chimeric CWH of the Disclosure comprises the amino acid sequence of SEQ ID NO: 106. In some embodiments, the chimeric CWH of the Disclosure consists of the amino acid sequence of SEQ ID NO: 106. In some embodiments, the chimeric CWH of the Disclosure has at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% identity with the amino acid sequence of SEQ ID NO: 106. In some embodiments, the chimeric CWH of the Disclosure has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the amino acid sequence of SEQ ID NO: 106. In some embodiments, the sequence of this chimeric CWH differs from the amino acid sequence of SEQ ID NO: 106 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0134] Linker

[0121] In some embodiments, the chimeric proteins of this specification comprise multiple domains, which are linked by a linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is an amino acid sequence having a length of 1 to 100 amino acids (including all values ​​and partial ranges between these). In some embodiments, the linker comprises one or more glycines and / or serines. Those skilled in the art will be familiar with other linkers that may be used in the chimeric proteins of this disclosure.

[0135] Protein tags

[0122] In some embodiments, the recombinant proteins of the present disclosure include a protein tag. The protein tag is typically a short sequence of amino acids or protein domains fused to the recombinant protein to facilitate purification and / or visualization. In some embodiments, the protein tag improves the solubility of the protein. In some embodiments, the tag is a His tag, GST tag, MBP tag, Strep tag, FLAG tag, GFP tag, HA tag, V5 tag, Avi tag, CBP tag, ZZ tag, SUMO tag, Fc tag, thioredoxin tag, protein kinase A (PKA) tag, Myc tag, or S tag, or any combination thereof. In some embodiments, the tag is a His tag and contains six histidine residues.

[0136] Nucleic acids, vectors, and host cells of this disclosure

[0123] The Disclosure also provides nucleic acids encoding recombinant proteins of the Disclosure (e.g., CWH). The Disclosure also provides vectors and host cells for the expression of recombinant proteins of the Disclosure. In some embodiments, the vector is a plasmid, cosmid, bacteriophage, or virus containing the nucleic acid of the Disclosure. In some embodiments, the host cell contains the nucleic acid of the Disclosure or the vector of the Disclosure. In some embodiments, the host cell is a bacterial cell, yeast cell, insect cell, mammalian cell, or plant cell.

[0137] Formulations of the Disclosure

[0124] This disclosure provides compositions comprising recombinant proteins disclosed herein. This disclosure provides compositions comprising EAD, enzymes, truncated enzymes, CBD, chimeric CWH, nucleic acids, vectors, and host cells disclosed herein. In some embodiments, the compositions are formulated for delivery to subjects to treat conditions associated with Cutibacterium acnes.

[0138] Topical formulations, parenteral formulations, and enteral formulations

[0125] In some embodiments, the compositions of the present disclosure are formulated for topical, parenteral, or enteral administration.

[0139]

[0126] In some embodiments, the compositions herein are formulated for topical administration. Formulations for topical administration include lotions, hydrogels, creams, ointments, gels, drops, transdermal patches, colloidal patches, powders, suppositories, sprays, liquids, semi-solids, single-phase compositions, multi-phase compositions (e.g., oil-in-water, water-in-oil), foams, microsponges, liposomes, nanoemulsions, aerosol foams, polymers, fullerenes, and powders. In some embodiments, the formulation is supplemented with carriers, bases, thickeners, penetration enhancers, buffers, diluents, emulsifiers, humectants, dispersants, binders, and / or excipients. In some embodiments, the composition is formulated as a hydrogel. In some embodiments, the composition is formulated as a lotion. In some embodiments, the composition is formulated as a cream. In some embodiments, the composition is formulated as a lyophilized powder that can be reconstituted in liquid, for example, before use. In some embodiments, the composition is a colloidal patch. In some embodiments, the composition is formulated as a colloidal patch. In some embodiments, the composition is formulated as a microneedle patch.

[0140]

[0127] In some embodiments, the compositions of the present disclosure are formulated for parenteral administration. As used herein, “parenteral administration” of a composition includes any route of administration characterized by physical perforation of the tissue of interest, and administration of the composition via tissue perforation, and therefore generally direct administration into the bloodstream, muscle, or viscera. Therefore, parenteral administration includes, but is not limited to, administration of the composition by injection, administration of the composition by application of the composition via surgical incision, administration of the composition by application of the composition via a tissue-permeable nonsurgical wound, and the like. In particular, intended parenteral administration includes, but is not limited to, injection or infusion into the subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, urethral, ​​intracranial, intratumoral, and cerebrospinal fluid; and renal dialysis infusion techniques.

[0141]

[0128] In some embodiments, the compositions of this specification are prepared for oral administration. The terms “oral,” “enteral,” “enteral,” “oral,” “non-parenteral,” “non-parenteral,” and similar terms refer to the administration of a compound or composition to a solid by a route or manner along the gastrointestinal tract. Examples of “oral” routes of administration of a composition include swallowing the composition by mouth in liquid or solid form, administration of the composition via nasojejunal or gastrostomy tube, intraduodenal administration of the composition, and rectal administration, including, but not limited to, the use of suppositories for the lower intestinal tract of the gastrointestinal tract. The compositions of this disclosure may be formulated into any of many possible dosage forms (e.g., tablets, capsules, liquid syrups, soft gels, suppositories, aerosols, and enemas). The compositions of this disclosure may also be formulated as suspensions in aqueous, non-aqueous, or mixed media. The aqueous suspension may further contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0142] Pharmaceutical ingredients

[0129] In some embodiments, the composition comprises an emulsifier. In some embodiments, the composition comprises a mixture of emulsifiers. In some embodiments, the composition comprises about 0.5 w / v% to about 5 w / v% of an emulsifier or a mixture of emulsifiers.

[0143]

[0130] Examples of emulsifiers suitable for use in some embodiments of the present disclosure include: xanthan gum, polysorbate 80, oleoyl polyoxyl-6 glyceride, polyoxyl-35 hydrogenated castor oil, sucrose distearate, saponins, sodium alginate, guar gum, tocopherol polyethylene glycol 1000 succinate, lauroyl polyoxyl-32 glyceride, sorbitan monooleate, glyceryl stearate, cetearyl alcohol, sodium stearoyl lactylate, salts thereof, derivatives thereof, and mixtures thereof. In some embodiments, the emulsifier is xanthan gum.

[0144]

[0131] In some embodiments, the emulsifier component is selected from medium to long-chain mono-, di-, and triglyceride polyglycolated glycoglycerides and polyoxyethylene glycerides, for example, almond oil PEG-6 ester, almond oil PEG-60 ester, apricot kernel oil PEG-6 ester (Labrafil® M1944CS), caprylic / capric acid triglyceride PEG-4 ester (Labrafac® Hydro WL 1219), Caprylic / Capric Triglyceride PEG-4 Complex (Labrafac® Hydrodrophile), Caprylic / Capric Glyceride PEG-6 Ester (Softigen® 767), Caprylic / Capric Glyceride PEG-8 Ester (Labrasol®), Castor Oil PEG-50 Ester, Hydrogenated Castor Oil PEG-5 Ester, Hydrogenated Castor Oil PEG-7 Ester, Hydrogenated Castor Oil PEG-9 Ester, Corn Oil PEG-6 Ester (Labrafil® M 2125 CS), Corn Oil PEG-8 Ester (Labrafil® WL 2609 BS), Corn Glyceride PEG-60 Ester, Olive Oil PEG-6 Ester (Labrafil® M1980 CS), Hydrogenated Palm / Palm Kernel Oil PEG-6 Ester (Labrafil® M 2130 BS), Hydrogenated palm / palm kernel oil containing palm kernel oil, PEG-6, palm oil (Labrafil® M 2130 CS), palm kernel oil PEG-40 ester, peanut oil PEG-6 ester (Labrafil® M 1969CS), glycerol esters of saturated C8-C18 fatty acids (Gelucire® 33 / 01), glyceryl esters of saturated C12-C18 fatty acids (Gelucire® 39 / 01 and 43 / 01), glyceryl laurate / PEG-32 laurate (Gelucire® 44 / 14), glyceryl laurate / PEG-20 laurate, glyceryl laurate / PEG-32 laurate, glyceryl, glyceryl laurate / PEG-40 laurate, glyceryl oleate / PEG-20 glyceryl, glyceryl oleate / PEG-30 oleate, glyceryl palmitostearate / PEG-32 palmitostearate (Geluc Selected from: Gelucire® 50 / 13), glyceryl stearate / PEG stearate, glyceryl stearate / PEG-32 stearate (Gelucire® 53 / 10), saturated polyglycolized glycerides (Gelucire® 37 / 02 and Gelucire® 50 / 02), triisostearin PEG-6 ester (i.e., Labrafil® Isostearique), triolein PEG-6 ester, trioleate PEG-25 ester, polyoxyl 35 castor oil (Cremophor® EL or Kolliphor® EL), polyoxyl 40 hydrogenated castor oil (Cremophor® RH 40 or Kolliphor® RH40), polyoxyl 60 hydrogenated castor oil (Cremophor® RH60), lecithin, phospholipids, and mixtures thereof.

[0145]

[0132] In some embodiments, the emulsifier may be polyglycolated derivatives of medium to long chain fatty acids and polyoxyethylene esters or other derivatives, various surfactants trade names Brij and Myrj, and propylene glycol esters of medium to long chain fatty acids, such as caprylate / caprate diglyceride, glyceryl monooleate, glyceryl ricinoleate, glyceryl laurate, glyceryl dilaurate, glyceryl dioleate, mono / dioleate glyceride, caprylic / caprate glyceride, medium chain (C8 / C10) mono- and diglycerides (Capmul® MCM, Capmul Examples include MCM(L), mono- and diacetylated monoglycerides, polyglyceryl oleate, polyglyceryl-2 dioleate, polyglyceryl-10 trioleate, polyglyceryl-10 laurate, polyglyceryl-10 oleate, and polyglyceryl-10 monodioleate, propylene glycol caprylate / caplate (Labrafac(PC)), propylene glycol dicaprylate / dicaplate (Miglyol(840)), propylene glycol monolaurate, propylene glycol ricinolate, propylene glycol monooleate, propylene glycol dicaprylate / dicaplate, propylene glycol dioctanoate, and mixtures thereof.

[0146]

[0133] In some embodiments, the composition comprises a humectant. In some embodiments, the composition is a topical formulation comprising a humectant, which may be referred to as a sedative, smoothing agent, moisturizing agent, or protective agent. The humectants of the Disclosure function to stabilize the water content of the tissue to which the humectant is applied in the presence of fluctuating humidity.

[0147]

[0134] In some embodiments, the humectants are polyglycols (as defined below), propylene glycol, sorbitol, lactic acid, sodium lactate, glycerol, glycerin, ethoxylated castor oil, calamine, dodecyl sulfate, sodium lauryl sulfate (SLS); polyoxyethylene esters of polysorbitan, such as monooleate, monolaurate, monopalmitate, monostearate; esters of sorbitan, polyoxyethylene ethers, dioctyl sulfate Selected from sodium phosuccinate (DOSS), lecithin, sodium doxate, hexylene glycol, butylene glycol, aloe vera gel, aloe vera powder, hyaluronic acid, alpha-hydroxy acids, such as lactic acid, egg yolk, egg white, glyceryl triacetate, honey, molasses, high molecular weight polyols, such as polydextrose, quillaja, sodium hexametaphosphate e452i; sugar alcohols (sugar polyols), such as glycerol, sorbitol, xylitol, maltitol; urea, and castor oil.

[0148]

[0135] In some embodiments, the composition comprises a humectant selected from the list consisting of aloe vera, betaine, butylene glycol, caprylyl glycol, dimethicone, fructose, glucomannan, glucose, glycerin, glyceryl glucoside, honey, hyaluronic acid, lactic acid, panthenol, polyethylene glycol, propylene glycol, propanediol, sodium hyaluronate, sodium lactate, sodium pyrrolidone carboxylate, sorbitol, and urea. In some embodiments, the composition comprises 0.1 to 50 w / v% of the humectant (including all values ​​and partial ranges between these). In some embodiments, the composition comprises 0.5 to 10 w / v% of the humectant.

[0149]

[0136] In some embodiments, the composition comprises hyaluronic acid. In some embodiments, the composition is a hyaluronic acid-based hydrogel for topical application. In some embodiments, the composition comprises 0.1 to 10 w / v% hyaluronic acid. In some embodiments, the composition comprises 0.5 to 5.0 w / v% hyaluronic acid. In some embodiments, the composition comprises 1 to 2 w / v% hyaluronic acid. In some embodiments, the composition comprises a hydrogel. In some embodiments, the hydrogel comprises a cellulose polymer. In some embodiments, the hydrogel comprises hydroxypropyl methylcellulose.

[0150]

[0137] In some embodiments, the composition comprises a cellulose polymer. In some embodiments, the cellulose polymer is hydroxyethylcellulose, methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, microcrystalline cellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, or cellulose acetate. In some embodiments, the composition comprises 0.1 to 20 w / v% of the cellulose polymer (including all values ​​and partial ranges between these). In some embodiments, the composition comprises 0.5 to 10 w / v% of the cellulose polymer. In some embodiments, the composition comprises 1 to 5 w / v% of the cellulose polymer. In some embodiments, the composition comprises hydroxypropyl methylcellulose (HPMC). In some embodiments, the composition comprises 0.5 to 10 w / v% of HPMC. In some embodiments, the composition comprises 1 to 5 w / v% of HPMC.

[0151]

[0138] In some embodiments, the composition comprises a thickener, a gelling agent, and / or a polymer. In some embodiments, the composition comprises an acrylate. In some embodiments, the composition comprises a carbomer.

[0152]

[0139] In some embodiments, the composition contains salt. In some embodiments, the composition contains salt selected from the list consisting of calcium chloride, Dead Sea salt, Epsom salt, Himalayan pink salt, magnesium chloride, sea salt, and sodium chloride. In some embodiments, the composition contains salt in concentrations of 10 to 500 mM (including all values ​​and partial ranges between these). In some embodiments, the composition contains salt in concentrations of 50 to 250 mM.

[0153]

[0140] In some embodiments, the composition comprises a buffer. In some embodiments, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, acetic acid, ammonium acetate, boric acid, citric acid, glycine, phosphoric acid, potassium hydroxide, potassium phosphate, sodium acetate, sodium bicarbonate, sodium borate, sodium carbonate, sodium citrate, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium hydroxide, sodium phosphate, sodium tetraborate, tris(hydroxymethyl)aminomethane, or trisodium phosphate. In some embodiments, the composition comprises a buffer of 1 to 250 mM (including all values ​​and partial ranges between these). In some embodiments, the composition comprises a buffer of 5 to 50 mM.

[0154]

[0141] In some embodiments, the composition comprises a surfactant. In some embodiments, the composition comprises a surfactant selected from the list consisting of ceteareth-20, cocamidopropyl betaine, coco glucoside, decyl glucoside, decyl polyglucose, disodium laureth sulfosuccinate, glycereth-26, lauryl glucoside, lauryl polyglucose, sodium cocoyl glutamate, sodium cocoyl isethionate, sodium laureth sulfate, and sodium lauryl sulfate. In some embodiments, the composition comprises 0.1 to 20 w / v% of the surfactant (including all values ​​and partial ranges between these). In some embodiments, the composition comprises 1 to 10 w / v% of the surfactant.

[0155]

[0142] In some embodiments, the composition comprises oil. In some embodiments, the composition comprises oil selected from the list consisting of argan oil, avocado oil, baobab oil, camellia oil, carrot seed oil, coconut oil, evening primrose oil, grape seed oil, hemp seed oil, jojoba oil, macadamia nut oil, marula oil, mineral oil, olive oil, pomegranate seed oil, raspberry seed oil, rosehip seed oil, squalane oil, sunflower seed oil, sweet almond oil, and tamanu oil. In some embodiments, the composition comprises oil in an amount of 0.1 to 20 w / v% (including all values ​​and partial ranges between these).

[0156]

[0143] In some embodiments, the composition contains an alcohol. In some embodiments, the composition contains an alcohol selected from the list consisting of cetyl alcohol, ethyl alcohol, isopropyl alcohol, and stearyl alcohol. In some embodiments, the composition contains an alcohol of 0.1 to 20 w / v% (including all values ​​and partial ranges between these). In some embodiments, the composition contains an alcohol of 1 to 10 w / v%.

[0157]

[0144] In some embodiments, the composition comprises free amino acids. In some embodiments, the composition comprises alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, the composition comprises amino acids selected from the list consisting of alanine, arginine, cysteine, glutamine, glycine, histidine, lysine, methionine, proline, serine, and threonine. In some embodiments, the composition comprises amino acids in concentrations of 10 to 250 mM (including all values ​​and partial ranges between these). In some embodiments, the composition comprises amino acids in concentrations of 25 to 150 mM.

[0158]

[0145] In some embodiments, the composition contains glycerol. In some embodiments, the composition contains 0.5 to 50 w / v% glycerol (including all values ​​and partial ranges between these). In some embodiments, the composition contains 1 to 30 w / v% glycerol. In some embodiments, the composition contains 1 to 5 w / v% glycerol.

[0159]

[0146] In some embodiments, the composition contains petrolatum. In some embodiments, the composition contains 0.1 to 20 w / v% petrolatum (including all values ​​and partial ranges between these).

[0160]

[0147] The compositions of the present disclosure may contain one or more additional agents, whether active or inactive. Examples of such agents include sweeteners, flavoring agents, colorants, fillers, binders, lubricants, excipients, preservatives, emollients, hydrates, smoothing agents, or manufacturing agents. Additional excipients or additives may be added to the compositions. For example, any commonly accepted soluble or insoluble inactive filler (diluent) material may be included in the final product (e.g., solid dosage form) as needed. Such inactive fillers may include monosaccharides, disaccharides, polyhydric alcohols, inorganic phosphates, sulfates or carbonates, and combinations thereof. Examples of suitable inactive fillers include sucrose, dextrose, lactose, xylitol, fructose, sorbitol, calcium phosphate, calcium sulfate, calcium carbonate, microcrystalline cellulose, and combinations thereof. An effective amount of any commonly accepted lubricant (e.g., calcium or magnesium soap) may be added.

[0161]

[0148] Depending on the dosage form, optional additives and modifiers may further include one or more of the following: acids, bases, acidity adjusters, alcohols, anti-caking agents, defoaming agents, antioxidants, bulking agents, coagulants, color retainers, emulsifiers, flavor enhancers, flour treatment agents, gelling agents, glazing agents, humectants, leavening agents, tracer gases, preservatives, stabilizers, sweeteners, softeners, and thickeners.

[0162]

[0149] The compositions of the present disclosure may further include other conventional auxiliary components. For example, the compositions may include additional suitable active substances, such as antipruritic agents, astringents, topical anesthetics, or anti-inflammatory agents, or additional substances useful for physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavorings, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, such substances, if added, should not excessively interfere with the biological activity of the components of the compositions of the present disclosure. Such formulations may be sterilized and, if necessary, mixed with auxiliary agents that do not have harmful interactions with nucleic acids in the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, colorants, flavorings, and / or fragrances, and the like.

[0163]

[0150] In some embodiments, the composition comprises a skin protectant. In some embodiments, the composition comprises an FDA-approved skin protectant. In some embodiments, the composition comprises colloidal oatmeal. In some embodiments, the composition comprises a skin protectant selected from the list consisting of allantoin, aluminum hydroxide gel, calamine, cocoa butter, cod liver oil, colloidal oatmeal, dimethicone, glycerin, hard fat, kaolin, lanolin, mineral oil, petrolatum, sodium bicarbonate, topical starch, white petrolatum, zinc acetate, zinc carbonate, and zinc oxide. In some embodiments, the composition comprises one of the following skin protectants in the following ranges: allantoin, 0.5-2%; aluminum hydroxide gel, 0.15-5%; calamine, 1-25%; cocoa butter, 50-100%; cod liver oil, 5-13.56%; colloidal oatmeal, at least 0.007%, or at least 0.003% in combination with mineral oil; dimethicone, 1-30%; glycerin. Serine, 20-45%; hard fat, 50-100%; kaolin, 4-20%; lanolin, 12.5-50%; mineral oil, 50-100%, or 30-35% in combination with colloidal oatmeal; petrolatum, 30-100%; sodium bicarbonate; topical starch, 10-98%; white petrolatum, 30-100%; zinc acetate, 0.1-2%; zinc carbonate, 0.2-2%; zinc oxide, 1-25%. See, for example, Sec. 347.10 of CFR Title 21, Volume 5, “Skin protectant active ingredients,” (this entire section is incorporated herein by reference).

[0164]

[0151] In some embodiments, the composition contains ingredients approved by the FDA for the treatment of acne. In some embodiments, the composition contains Aklief (triphalotene), Arazlo (tazarotene), Avita (tretinoin) Gel and Cream, Benzamycin (erythromycin 3%-benzoyl peroxide 5% topical gel), Cabtreo (clindamycin phosphate, adapalene, and benzoyl peroxide) topical gel, Estrostep Fe (norethisterone acetate and ethinylestradiol, USP, and ferrous fumarate), Retin-A Micro (tretinoin gel) 0.06% and 0.08%, Seysara (thalecycline), Tazorac (tazarotene) gel and cream 0.05% and 0.1%, Veltin (clindamycin phosphate and tretinoin), or Winlevi (crascoterone). In some embodiments, the composition comprises triphalotene, tazarotene, tretinoin, erythromycin, benzoyl peroxide, clindamycin phosphate, adapalene, norethisterone acetate, ethinylestradiol, ferrous fumarate, salecycline, and / or clascoterone.

[0165]

[0152] In some embodiments, the composition also contains other components, such as antibiotics; preservatives; antifungal agents; corticosteroids; sedatives; anti-aging agents; smoothing agents; moisturizers; and protective agents. In some embodiments, the composition contains an antibiotic.

[0166] Characteristics of the compositions disclosed herein

[0153] This disclosure provides chimeric proteins and compositions comprising these chimeric proteins. These compositions have properties beneficial for therapeutic use against the target Cutibacterium acnes.

[0167]

[0154] In addition to selectivity issues, previous CWHs and endolysins known in the art have properties that make them unsuitable for therapeutic indications (e.g., topical application). For example, previously characterized CWHs have problems with weak activity, low thermal stability, low solubility, and / or narrow or inappropriate pH ranges.

[0168]

[0155] This disclosure provides chimeric CWH having beneficial characteristics (e.g., high anti-Cutibacterium acnes activity, Cutibacterium acnes species specificity, thermal stability, solubility, and a broader or more preferred pH range).

[0169] Anti-Cutibacterium acnes activity

[0156] In some embodiments, the compositions of the present disclosure are active against the species Cutibacterium acnes. In some embodiments, the compositions of the present disclosure have activity against Cutibacterium acnes, and the degree of this activity is determined based on the minimum inhibitory concentration (MIC) for this target species. In some embodiments, the MIC is 100, 90, 80, 70, 60, 50, 40, 30, 20, or less than 10 μg / mL. In some embodiments, the MIC is 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 μg / mL. In some embodiments, the MIC is less than 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, or 0.5 μg / mL. In some embodiments, the MIC is less than 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 μg / mL.

[0170]

[0157] In some embodiments, the compositions of the present disclosure have activity against Cutibacterium acnes at a concentration of less than 50 μg / mL. In some embodiments, the compositions of the present disclosure have activity against Cutibacterium acnes at concentrations of less than 40, 30, 20, or 10 μg / mL. In some embodiments, the compositions of the present disclosure have activity against Cutibacterium acnes at a concentration of 50 μg / mL. In some embodiments, the compositions of the present disclosure have activity against Cutibacterium acnes at a concentration of 24 μg / mL. In some embodiments, the compositions of the present disclosure have activity against Cutibacterium acnes at a concentration of 12 μg / mL. In some embodiments, the compositions of the present disclosure have activity against Cutibacterium acnes at a concentration of 6 μg / mL.

[0171]

[0158] In some embodiments, the compositions of the present disclosure are active against one or more different lineages of Cutibacterium acnes. In some embodiments, the compositions of the present disclosure are active against lineage IA1. In some embodiments, the compositions of the present disclosure are active against lineage IA2. In some embodiments, the compositions of the present disclosure are active against lineages IA1, IA2, IB, II, and / or III. In some embodiments, the compositions of the present disclosure are active against lineages IA1, IA2, IB, and II.

[0172] Selectivity

[0159] The inventors have found that the exemplary novel chimeric organisms disclosed herein exhibit remarkable selective activity against Cutibacterium acnes more than other bacterial species. As is known in the art, the only CWH (CaLys1) previously known in the art to exhibit lytic activity against C. acnes also exhibits similar lytic activity against other bacterial strains, which is highly undesirable for topical skin microbiome applications. In contrast, the exemplary chimeric CWH of this disclosure is capable of distinguishing C. acnes from symbiotic skin bacteria.

[0173]

[0160] In some embodiments, the recombinant proteins of the Disclosure exhibit specificity to Cutibacterium acnes more than other species. In some embodiments, the recombinant proteins of the Disclosure exhibit specificity to Cutibacterium acnes more than symbiotic bacterial species. In some embodiments, the recombinant proteins of the Disclosure exhibit specificity to Cutibacterium acnes more than Corynebacterium xerosis, Corynebacterium striatum, and / or Staphylococcus epidermidis.

[0174]

[0161] In some embodiments, the recombinant proteins of the Disclosure have a selectivity of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the recombinant proteins of the Disclosure have a selectivity of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times. In some embodiments, the recombinant proteins of the Disclosure have substantially no activity (or no measurable activity) against a certain species, but have measurable activity against C. acnes.

[0175]

[0162] In some embodiments, the compositions of the present disclosure have broad anti-Cutibacterium acnes activity. In some embodiments, the compositions have high activity against multiple strains of Cutibacterium acnes. In some embodiments, the compositions have high activity against a group of related Cutibacterium acnes strains. In some embodiments, the compositions have high activity against a diverse group of Cutibacterium acnes strains. In some embodiments, the compositions are suitable for use as a broad-spectrum therapeutic agent.

[0176] thermal stability

[0163] The inventors of this disclosure have also found, surprisingly, that some of the novel chimeric CWHs of this disclosure exhibit desirable high thermal stability.

[0177]

[0164] In some embodiments, the compositions of the present disclosure exhibit thermal stability at temperatures up to 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60°C (including all values ​​and ranges between these). In some embodiments, the compositions of the present disclosure exhibit thermal stability at temperatures of at least 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60°C (including all values ​​and ranges between these).

[0178]

[0165] As used herein, thermal stability at a given temperature refers to the ability to maintain an activity level at that temperature or the ability to maintain an activity level after exposure of a protein to that temperature. In some embodiments, thermal stability at a given temperature is measured after exposure to that temperature over a period of time. In some embodiments, thermal stability is determined based on experiments that test activity at a temperature or after exposure to a given temperature over a period of time (e.g., showing a measurable reduction in target bacterial density at or after exposure to that temperature). Thus, in some embodiments, an EAD, CBD, or chimeric protein is considered thermally stable at a temperature if it still exhibits measurable activity at a temperature or after exposure to a temperature. In some embodiments, an EAD, CBD, or recombinant chimeric protein is considered thermally stable at a critical temperature if it still exhibits measurable activity at its intended use temperature after exposure to the critical temperature (e.g., activity tested after the protein is exposed to this critical temperature for 30 minutes). In some embodiments, thermal stability after exposure to a given temperature is determined based on an assay performed at room temperature. In some embodiments, thermal stability after exposure to a given temperature is determined based on an assay that measures activity. In some embodiments, this assay is a turbidity reduction assay.

[0179]

[0166] In the context of comparing two proteins (e.g., two EADs, two CBDs, or two chimeric CWHs), one protein may be considered more thermally stable at a given temperature than the other if it exhibits higher absolute activity at that temperature or after exposure to that temperature (e.g., resulting in a greater reduction in microbial density over a measured period of time). In other embodiments, one protein may be considered more thermally stable at a given temperature than the other if it exhibits higher relative activity at that temperature or after exposure to that temperature (e.g., if this protein exhibits a smaller reduction in activity after exposure to that temperature compared to a relative reduction in activity of another protein after exposure to that temperature).

[0180]

[0167] In some embodiments, thermal stability at a given temperature is determined based on the ability to maintain activity after exposure to this temperature for 10, 20, 30, 40, 50, or 60 minutes (including all values ​​and ranges in between). In some embodiments, thermal stability at a given temperature is determined after exposure to this temperature for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours (including all values ​​and ranges in between). In some embodiments, thermal stability at a given temperature is determined after exposure to this temperature for 1, 2, 3, 4, 5, 6, or 7 days (including all values ​​and ranges in between). In some embodiments, thermal stability at a given temperature is determined after exposure to this temperature for 1, 2, 3, or 4 weeks (including all values ​​and ranges in between). In some embodiments, thermal stability at a given temperature is determined after exposure to this temperature for 1, 2, 3, 4, 5, or 6 months (including all values ​​and ranges in between).

[0181]

[0168] In some embodiments, the thermal stability is measured based on a test of the activity after maintaining the composition at a given temperature for a period of time (e.g., several weeks or months). In some embodiments, the thermal stability is determined based on the activity retained after two months at a given temperature.

[0182]

[0169] In some embodiments, the compositions herein are thermally stable at 45 °C for at least four weeks. In some embodiments, the compositions herein are thermally stable at 45 °C for at least two months. In some embodiments, the compositions herein are thermally stable at 50 °C for at least two months.

[0183]

[0170] In some embodiments, the compositions herein are considered to be thermally stable or storable at room temperature if they retain at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (including all values and ranges therebetween) of their original activity at room temperature after exposure to a temperature of 45 °C for four weeks.

[0184] pH range

[0171] In some embodiments, the compositions herein are stable within a range of pH values. A composition (e.g., a chimeric protein) is considered stable at a given pH level if it exhibits activity at that pH level. In some embodiments, pH stability at various pH values ​​is determined based on activity assays performed at various pH values. For example, in some embodiments, pH stability is determined by incubating the composition of the Disclosure (e.g., a chimeric protein) with target bacterial cells at various pH levels (e.g., in a turbidity reduction assay). The results of this assay provide the activity levels (e.g., maximum activity levels) of the composition at various pH values. In some embodiments, a composition is stable at a certain pH level if, at that pH, it exhibits the same activity as the maximum activity level of the composition. In some embodiments, a composition is stable at a certain pH level if, at that pH, it exhibits at least 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the maximum activity of the composition. In some embodiments, the composition is stable at a given pH level if, at that pH, it exhibits approximately 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the composition's maximum activity.

[0185]

[0172] In some embodiments, pH stability is determined based on an activity assay. In some embodiments, this assay is a turbidity reduction assay.

[0186]

[0173] In some embodiments, the compositions herein are stable at a pH of 3, 4, 5, 6, 7, 8, 9, or 10, or any range in between. In some embodiments, the compositions herein are stable in a pH range of 6 to 8. In some embodiments, the compositions herein are stable in a pH range of 5 to 8. In some embodiments, the compositions herein are stable at a pH value most suitable for topical skin application. For example, in some embodiments, the compositions herein are stable at a pH of 4, 5, or 6.

[0187] Synergistic effect

[0174] In some embodiments, the Disclosure provides a combination composition comprising at least two compositions of the Disclosure (e.g., chimeric proteins). In some embodiments, at least two compositions of the Disclosure are administered together. In some embodiments, two compositions of the Disclosure are administered sequentially or simultaneously. In some embodiments, the combination composition of the Disclosure exhibits synergistic results compared to the individual constituent compositions.

[0188]

[0175] As used herein, the term “synergistic” means, when the term refers to a composition of the present disclosure, a composition that exhibits an effect (y) that exceeds the predicted effect of the composition calculated by the reference model. In some embodiments, “synergistic” means an effect that is greater than a simple additive effect. In some embodiments, a synergistic combination is one in which the MIC of the combination is lower than the MIC of the individual components of the combination. In some embodiments, a synergistic combination is one in which the MIC of the combination is lower than the MIC of the individual components of the combination when calculated by compositional percentages. In some embodiments, a composition is said to have a synergistic effect if it exhibits properties that exceed the additive properties of its individual components.

[0189] How to treat Cutibacterium acnes

[0176] The present disclosure provides a method for treating a condition associated with Cutibacterium acnes, comprising administering a composition of the present disclosure.

[0190]

[0177] The compositions disclosed herein may be used to treat subjects suffering from conditions associated with the species of Cutibacterium acnes as defined herein. In some embodiments, the subject is an animal. In some embodiments, the animal is a mammal. In some embodiments, the subject is a human.

[0191] situation

[0178] In some embodiments, the compositions of the present disclosure are used in the treatment of a condition associated with Cutibacterium acnes. In some embodiments, this condition is acne vulgaris.

[0192]

[0179] In some embodiments, this condition is related to the skin. In some embodiments, this condition is a skin infection. In some embodiments, this condition is impetigo, cellulitis, folliculitis, atopic dermatitis, acute radiation dermatitis, acne, or abscess.

[0193]

[0180] In some embodiments, this condition is a wound infection, pneumonia, food poisoning, toxic shock syndrome, bloodstream infection, pneumonia, urinary tract infection, bone or joint infection (e.g., osteomyelitis, septic arthritis), endocarditis, meningitis, sepsis, ear infection (e.g., otitis externa), eye infection (e.g., conjunctivitis, keratitis), sinus infection, gastroenteritis, mastitis, peritonitis, prosthesis infection, sternal wound infection, catheter-related infection, or tonsillitis. In some embodiments, this condition is a skin infection. In some embodiments, this condition is a soft tissue infection. In some embodiments, this condition is an opportunistic infection. In some embodiments, this condition is a wound infection. In some embodiments, this condition is a chronic wound. In some embodiments, this condition is acne vulgaris, skin infection, cellulitis, orthopedic infection, endophthalmitis, prosthetic valve endocarditis, otitis media, or osteomyelitis. In some embodiments, this condition is an inflammatory lesion. In some embodiments, this condition is characterized by comedones (pimples), papules, pustules, nodules, or cysts. In some embodiments, this condition is characterized by redness, swelling, pain, tenderness, or abnormal texture of the skin. In some embodiments, this condition is characterized by psychosocial conditions associated with acne vulgaris.

[0194]

[0181] In some embodiments, this condition is the presence of Cutibacterium acnes species (in particular, an excess abundance of phylogenetic type IA1) on the skin.

[0195] Dosage

[0182] In some embodiments, the composition of the Disclosure comprises 0.1 to 100 μg / mL of the recombinant protein disclosed herein (e.g., a chimeric protein). In some embodiments, the composition comprises 0.5 to 50 μg / mL of the recombinant protein disclosed. In some embodiments, the composition comprises 1 to 25 μg / mL of the recombinant protein disclosed. In some embodiments, the composition comprises 2 to 15 μg / mL of the recombinant protein disclosed.

[0196]

[0183] In some embodiments, the compositions of the present disclosure are about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 5 The protein comprises 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μg / mL of the protein of this disclosure.

[0197] Administration

[0184] The composition may be formulated in various forms for administration purposes. The term “dosage form” refers to any form of formulation containing an amount sufficient to achieve at least partial therapeutic effect with a single or repeated dose. In some embodiments, the dosage form is a topical dosage form. In some embodiments, the dosage form is a lotion, oil, gel, ointment, or body balm. In some embodiments, the dosage form is a lotion.

[0198]

[0185] The compositions can be formulated in forms including, but not limited to, liquids, gels, semi-solids, and solids. The compositions disclosed herein can be further processed into forms including, but not limited to, solids, liquids, suspensions, gels, lotions, balms, and other forms considered herein.

[0199]

[0186] In some embodiments, an effective amount of the composition is administered to the subject. The terms “effective amount” or “therapeutically effective amount” refer to an amount of the composition described herein sufficient to achieve an intended use, including but not limited to the reduction of the Cutibacterium acnes population. The therapeutically effective amount may vary depending on the subject and condition being treated (e.g., the subject’s weight and age, the severity of the disease condition, the mode of administration, and likewise), which can be readily determined by those skilled in the art. The term also applies to a dose that elicits a specific response at the target site (e.g., reduction of inflammation, pain, acne, fever, etc.). The specific dose will vary depending on the specific formulation of the composition, the administration regimen followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, the route of administration, and the physical delivery system to which it is carried.

[0200]

[0187] In some embodiments, a composition disclosed herein is said to be active, functionally or therapeutically active, or treatable, preventable, and / or delayable if it reduces or alleviates one or more symptoms associated with a condition associated with Cutibacterium acnes. In some embodiments, a composition is considered therapeutically active if, after treatment, it reduces the severity of the symptoms by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% compared to the severity of the symptoms before treatment. In some embodiments, the symptoms include pain, fever, swelling, redness, dry skin, lesion count, lesion size, rash, warmth, drainage, secretion, cough, shortness of breath, rapid heart rate, hypotension or hypertension, chills, nausea, vomiting, diarrhea, stomach cramps, chest pain, or organ failure.

[0201]

[0188] In some embodiments, the compositions of the present invention are therapeutically active in reducing the amount of target Cutibacterium acnes species present in the subject or in the in vitro system, which preferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less of the initial amount of Cutibacterium acnes species are still detectable after treatment. In some embodiments, Cutibacterium acnes species are undetectable after treatment. In this specification, the expression “amount of Cutibacterium acnes species” refers to living Cutibacterium acnes species. In some embodiments, Cutibacterium acnes species are detected using sequencing techniques such as 16S sequencing or shotgun sequencing to quantify the amount of various Cutibacterium acnes species present in the sample and to further evaluate the species present in the entire microbiome of interest. Live Cutibacterium acnes species can be detected using standard techniques known to those skilled in the art, such as microbiological bacterial culture techniques and / or real-time quantitative reverse transcription polymerase chain reaction for assaying bacterial mRNA. In some embodiments, the reduction is evaluated in the tissue or cells of the individual or patient by comparing it to the amount present in the individual or patient before treatment with the composition disclosed herein. In some embodiments, if the treatment is topical, this comparison is made in the tissue or cells of the individual or patient that have not yet been treated with the composition disclosed herein.

[0202]

[0189] The compositions disclosed herein can be administered to a subject in need thereof, or to cells, tissues or organs, or to the patient, for at least 1 day, 1 week, 1 month, 6 months, 1 year, or for a longer period.

[0203]

[0190] Accordingly, there is provided a composition disclosed herein for use by a subject in need thereof. In some embodiments, this composition is used as a medicament in the prevention, delay or treatment of a condition when the condition of the subject is associated with Cutibacterium acnes.

[0204]

[0191] Further provided is a composition disclosed herein for systemic or topical administration to a subject.

[0205]

[0192] In some embodiments, topical administration is used locally at the site of a condition or infection, or is used locally in relation to surgery, or is used locally at the site of transplantation. The medical uses disclosed herein can be formulated as products disclosed herein for use as medicaments for treating the stated conditions, but similarly, the products disclosed herein, the products disclosed herein used in the preparation of medicaments for treating the stated conditions, and the use of the products disclosed herein for treating the stated conditions can be formulated as methods of treating the stated conditions using the use of the products disclosed herein. All such medical uses are contemplated by the present disclosure. A subject in need of treatment, delay, and / or prevention of the listed conditions can be any animal subject, preferably a mammal, more preferably a domestic animal such as a cow, dog or cat, or a human subject.

[0206]

[0193] Further provided is the in vitro use of a composition disclosed herein, or a nucleic acid construct disclosed herein, or a host cell disclosed herein, as an antibacterial or disinfectant.

[0207]

[0194] Also provided is the use of the compositions or nucleic acid constructs disclosed herein, or expression constructs disclosed herein, or host cells disclosed herein, or compositions disclosed herein, for binding to and / or detecting Cutibacterium acnes in ex vivo diagnostic applications. [Examples]

[0208] Examples

[0209] [Table 40]

[0210] [Table 41]

[0211] [Table 42]

[0212] I. Examples 1-4: Novel Endorphins of the genus Cutibacterium Example 1: Identification of novel CLC1 family endolysins, including amidasemain, in species of the genus Cutibacterium.

[0195] Previous attempts to identify endolysins that target C. acnes have focused on exploring the genomes of C. acnes strains, including C. acnes phages or prophages. However, this approach has only identified a single, well-conserved endolysin, as exemplified by protein sequence NCBI accession ID: YP_006907103.1 (referred to as CaLys1 in this application) (SEQ ID NO: 72).

[0213]

[0196] To identify novel endolysins targeting C. acnes, bioinformatics analyses were performed on a broader range of Cutibacterium species. While we do not wish to be bound by any particular theory, it is hypothesized that putative endolysins and cell wall hydrolases were found within the genomes of a broader range of Cutibacterium sp. species, and that phages infecting a broader range of Cutibacterium sp. species may also possess activity against C. acnes.

[0214]

[0197] These genomes were bioinformatics-analyzed for proteins containing the N-acetylmuramoyl-L-alanine amidase domain (PFAM:amidase_2), which is commonly found in endolysin across a wide range of bacteria, including the CaLys1 family. This search identified a novel family of amidases found in many Cutibacterium sp. species, such as C. avidum, C. granulosum, and C. porci, including 19 proteins which the inventors have named CLC1 to CLC19 (all referred to in this application as the "CLC1 family" amidases). The full-length amino acid sequences of CLC1 to CLC19 are included in SEQ ID NOs: 1 to 19, respectively. These amidases show similarities to the CaLys1 endolysin family (approximately 70% amino acid identity), but they form a distinct phylogenetic group from the CaLys1 family found in C. acnes phage / prophage (Figure 1A). Table 9 lists the GenBank ID numbers of the non-CLC1 family branches of the phylogenetic tree shown in Figure 1A. Phylogenetic comparison of only the amidase EADs (SEQ ID NOs. 20-36) of these proteins also confirmed that the amidase EADs of the CLC1 family are different from those of the CaLys1 family (Figure 1B). Table 10 lists the GenBank ID numbers and amino acid regions of the EADs of the non-CLC family branches of the phylogenetic tree shown in Figure 1B. Using multiple sequence alignment tools (Clustal Omega and EMBOSS Cons, see Madeira et al., Nucleic Acids Research 2024 Jul; 52(W1): W521-W525), we also determined that the EADs of the CLC1 family share a significant core of conserved residues (SEQ ID NO: 2939). Figure 27 shows the results of further motif analysis identifying conserved motifs in the EADs of the CLC1 family.

[0215] [Table 43]

[0216] [Table 44]

[0217] [Table 45]

[0218] [Table 46]

[0219] [Table 47]

[0220] Example 2: The CLC1 protein exhibits high solubility and significant anti-C. acnes lytic activity.

[0198] To test whether the putative endolysin CLC1 family possesses lytic activity against C. acnes, CLC1 was cloned into an inducible expression plasmid, and this protein was expressed in BL21 E. coli cells. A 6×His tag was added to this protein during these cloning steps. The cell lysates were then tested for activity in a clearing assay. The activity was tested using cell lysates. Briefly, C. acnes cells were embedded in soft agar to create an opaque plate. Candidate enzymes (either purified or derived from E. coli lysates) could be spotted onto this plate, and if the enzyme possessed anti-C. acnes activity, clearing / halo would occur. In this halo assay, clearing / halo was observed when lysates derived from BL21 E. coli cells expressing CLC1 were spotted, indicating anti-C. acnes activity (Figure 2).

[0221]

[0199] Importantly, the observed anti-C. acnes activity by CLC1 did not require any additional steps to resolubilize the protein. In contrast, lysates derived from BL21 cells expressing CaLys1 showed no clearing in this halo assay, which is consistent with previous reports that the CaLys1 protein is primarily insoluble (and therefore inactive) when expressed in Escherichia coli without further manipulation.

[0222]

[0200] To further clarify the activity characteristics of CLC1, CLC1 was purified from cell lysates using a 6×His tag and then tested in a turbidity reduction assay. In this assay, C. acnes cells were subjected to an OD of approximately 1.0. 600 The solution was resuspended and then tested with the purified enzyme. If this enzyme has C. acnes soluble activity, then OD 600The activity level decreased over time. This result confirmed that CLC1 possesses anti-C. acnes activity (Figure 3). In particular, CLC1 exhibited activity in a lower concentration range (6 μg / mL to 24 μg / mL) compared to that reported for CaLys1 (100 μg / mL). Furthermore, the CLC1 protein remained soluble after purification using a 6xHis tag. Therefore, CLC1 represents a novel endolysin that is solublely expressed in Escherichia coli (E. coli) and possesses lytic activity against C. acnes.

[0223] Example 3: CLC1 truncation exhibits improved solubility and solubility activity compared to the full-length protein.

[0201] Using SMART and CD-Search, we identified the bioinformatically predicted protein domains in the CLC1 family enzymes. Using these tools, we found that most members of the CLC1 family have an N-terminal amidase domain (EAD) followed by a C-terminal region of approximately 100 amino acids that does not contain the predicted protein domain. The exception is CLC15, which is predicted to have a longer repeating domain of unknown function.

[0224]

[0202] Despite the absence of all predicted protein domains, this C-terminal region is conserved among members of the CLC1 family and in CaLys1, suggesting that this C-terminal region may have several biological functions. To evaluate the effect of this C-terminal domain on CLC1 activity, we generated a truncated form of CLC1 lacking 85 amino acids at the C-terminus ("CLC1-truncation"). This protein was expressed in BL21 Escherichia coli (E. coli), purified, and tested using a turbidity reduction assay. Surprisingly, we found that CLC1-truncation increased solubility compared to full-length CLC1, as evidenced by the significantly higher yield of soluble protein after expression in E. coli. In addition, CLC1-truncation was found to be significantly more active than full-length CLC1 in a turbidity reduction assay measuring lytic activity against C. acnes (Figure 4). These data indicate that removing the C-terminal portion of the CLC1 protein has a positive effect on both solubility and activity, resulting in a more stable protein with much stronger lytic activity against C. acnes.

[0225] Example 4: Solubility and activity can also be improved by removing other CLC1 family proteins and the C-terminal region of CaLys1.

[0203] Full-length CLC2 and CLC3, when purified, exhibited relatively weak C. acnes lytic activity and lower soluble protein yields compared to other CLC proteins. The inventors removed approximately 85 amino acids from the C-terminus of both proteins to generate CLC2-truncations and CLC3-truncations, respectively. Full-length CLC2, full-length CLC3, CLC2-truncations, and CLC3-truncations were purified and tested for C. acnes lytic activity in a turbidity reduction assay. The truncated proteins showed both higher soluble protein yields and significantly higher C. acnes lytic activity in the turbidity reduction assay (Figures 5A-5B). C-terminal truncations were also generated for CLC16 and CaLys1 and tested in a turbidity reduction assay against their full-length counterparts. In all cases, the truncations showed improved solubility and lytic activity compared to their full-length counterparts (Figures 5C-5D). These data demonstrate that removal of the C-terminal region of CLC1 family proteins and CaLys1 results in a positive effect on solubility and activity in turbidity reduction assays. The improved solubility and activity of CaLys1-truncations are particularly surprising considering the known problems with the solubility and activity of full-length CaLys1. Table 5 provides the sequences of truncations of the CLC1 family and CaLys1 truncations, all of which are expected to show improved solubility and lytic activity compared to their full-length counterparts, as observed with CLC1, CLC2, CLC3, CLC16, and CaLys1.

[0226] II. Examples 5-7: Novel C. acnes cell wall binding domains Example 5: Identification of CLB1-CLB4 as putative cell wall hydrolases derived from the genera Cutibacterium sp. and Propionimicrobium sp., containing the CW_7 cell wall binding domain.

[0227]

[0204] Many endolysins (particularly those targeting Gram-positive bacteria) contain both an enzyme-active domain (EAD) and a cell wall-binding domain (CBD). This cell wall-binding domain is involved in the recognition and binding of epitopes in the peptidoglycan of target bacteria, thereby transporting the EAD to its target substrate. In this way, the CBD can increase specificity and / or activity. No known CBDs have been shown to recognize and bind to C. acnes. To find CBDs that can recognize C. acnes, the genomes of Cutibacterium sp. species and a broader range of bacteria from the Propionibacteriaceae family were subjected to bioinformatics analysis for putative cell wall hydrolases containing potential cell wall-binding domains. Four putative proteins (CLB1-CLB4) with unknown characteristics were identified from C. avidum (CLB1), C. acnes (CLB2), and Propionimicrobium lymphophilum (CLB3 and CLB4). All four proteins contain an N-terminal amidase domain. CLB1, CLB3, and CLB4 have a putative C-terminal cell wall-binding domain containing two CW_7 domain repeats, while CLB2 has a putative C-terminal cell wall-binding domain containing a single CW_7 repeat (Figure 6).

[0228] Example 6: Identification of a large family of CBD with broad taxonomic diversity, including the CW_7 repeat.

[0205] CLB1-4 CBDs contain one or two CW_7 repeats. The CW_7 repeats are relatively small (37-40aa) and are thought to bind to the peptidoglycan chain. See Bustamante et al., Sci Rep 2017 Nov 28; 7(1):16494. To characterize the taxonomic diversity of the CW_7 repeat, the Uniprot database was searched for proteins containing the CW_7 domain using the Interpro domain entry IPR013168. UniProt contains approximately 1,333 proteins annotated with one or more CW_7 repeats found in a wide range of bacterial species, mainly bacteria of the phyla Actinobacteria and Firmicutes, as well as in related bacteriophage viruses, where they are expressed more small in the phylum Bacteriodota (Figure 7A-7B). The BLASTP search was also performed in a non-redundant protein database using the CLB2-CBD CW_7 repeat sequence as the query. Using this search, 2656 protein sequences were identified with an E value < 0.05. The CW_7 repeat sequences in these proteins ranged from 43.59% to 100% amino acid identity with respect to the CLB2-CBD CW_7 query, and again, these originated from a diverse range of bacterial species and associated viruses. For each protein, the GenBank accession number, sequence number, and the identity percentage of the top CW_7 matches are listed in Table 7. The sequences of CW_7 CBD containing the proteins identified in this search are provided under sequence numbers 282–2938.

[0229] Example 7: CLB1-CBD and CLB2-CBD can target chimeric cell wall hydrolases in C. acnes.

[0206] To demonstrate that newly identified CBD binds to C. acnes, the inventors investigated the possibility of using CLB1 and CLB2 CBD to create chimeric proteins that have activity against C. acnes. To test this idea, the inventors selected the following two endolysins that in nature target other genera of bacteria and do not target C. acnes: CD27L (typically targeting Clostridium difficle) (Mayer et al., J Bacteriol. 2008 Oct; 190(20):6734-40); and PlyGVE2 (derived from a thermophilic phage known to infect Geobacillus sp.) (Jin et al., Microbiology (Reading). 2013 Aug; 159(Pt 8):1597-1605). These two endolysin-derived EADs were fused to either CLB1-CBD or CLB2-CBD. The resulting chimeric proteins were expressed, purified, and tested for their activity against C. acnes in a turbidity reduction assay. Natural CD27L and PlyGVE2 proteins were also expressed, purified, and tested. While natural CD27L and PlyGVE2 showed no lytic activity against C. acnes, the four chimeric CWHs were able to lyse C. acnes, a bacterium well outside the natural target genera of the two originating endolysins. It has been demonstrated that by fusing EADs to CLB1-CBD and CLB2-CBD, chimeric cell wall hydrolases targeting C. acnes can be created (Figures 8A-8B).

[0230] III. Examples 8-19: Novel CLC1 family EAD and CW_7 CBD chimeras Example 8: Chimeras containing CLC1-3 EAD combined with CLB1-4 CBD exhibit anti-C. acnes activity.

[0207] The CLC1 family of amidases consists of an N-terminal amidase with a conserved sequence at its C-terminus. However, although conserved, there is no predictive protein domain encoded by this sequence, and therefore it is not a clear CBD (Figure 4). Therefore, although we do not wish to be bound by any particular theory, we hypothesized that the activity of the CLC1 family amidases could be modified by fusing the CLC1 family's EAD to a CBD that can recognize and bind to C. acnes.

[0231]

[0208] A set of eight chimeric proteins was created, consisting of combinations of EAD derived from CLC1, CLC2, and CLC3 and CBD derived from CLB1, CLB2, CLB3, and CLB4, to test whether CBD from CLB1-CLB4 could target C. acnes. The designs of these proteins are shown in Table 11 below.

[0232] [Table 48]

[0233]

[0209] These eight chimeric proteins were expressed, purified, and tested using a turbidity reduction assay. As shown in Figures 9A and 9B, the natural CLC1 protein was compared with chimeric proteins in which the CLC1 EAD was linked to various candidate CBDs (CLC1-EAD+CLB1-CBD, CLC1-EAD+CLB2-CBD, CLC1-EAD+CLB3-CBD, and CLC1-EAD+CLB4-CBD). The results showed that all four chimeric proteins exhibited higher C. acnes lytic activity in the turbidity reduction assay compared to the natural full-length CLC1 protein. These data suggest that chimeric cell wall hydrolases containing the CLC1 EAD in combination with CBDs from CLB1 to CBL4 can recognize and bind to C. acnes, and can exhibit higher activity against C. acnes compared to natural CLC1. Further turbidity reduction assays demonstrated that the CLC1-EAD+CLB2-CBD chimera exhibited superior anti-C. acnes lytic activity compared to both full-length CLC1 and more active CLC1-truncations (Figure 9C).

[0234]

[0210] Figures 10A and 11A show the results of lysis assays comparing native CLC2 and CLC3 proteins with CLC2-EAD chimeras (CLC2-EAD+CLB1-CBD and CLC2-EAD+CLB2-CBD) and CLC3-EAD chimeras (CLC3-EAD+CLB1-CBD and CLC3-EAD+CLB2-CBD). Both full-length CLC2 and CLC3 exhibited relatively low C. acnes lysis activity. Linking either CLB1 CBD or CLB2 CBD to CLC2 or CLC3 EAD dramatically increased lysis activity compared to native CLC2 or CLC3, similar to the results observed for CLC1. Further turbidity reduction assays demonstrated that the CLC2-EAD+CLB2-CBD chimera performed better than both full-length CLC2 and more active CLC2-truncations (Figure 10B). Similarly, the CLC3-EAD+CLB2-CBD chimera showed improved lysis activity compared to both full-length CLC3 and more active CLC3-truncations (Figure 11B).

[0235] Example 9: Chimeric proteins containing EAD and CW_7 CBD from the CLC1 family exhibit solubility over a wide range of pH values.

[0211] The enzymatic characteristics of two types of CLC1 EAD chimeric proteins (CLC1-EAD+CLB1-CBD and CLC1-EAD+CLB2-CBD) and two types of CLC3 EAD chimeric proteins (CLC3-EAD+CLB1-CBD and CLC3-EAD+CLB2-CBD) were further clarified at various pH levels.

[0236]

[0212] Enzyme activity may be affected by pH. For commercial applications, it is important that the enzyme functions at the pH relevant to that application. For example, in the case of topical skin application, the normal pH of healthy skin is slightly acidic (approximately pH 5-6). In addition, many topical skin products are formulated to be slightly acidic. Therefore, enzymes used in these types of applications should ideally function at pH 5-6. However, it has been reported that well-preserved C. acnes endolysin CaLys1 exhibits maximum activity at pH 7 (see International Publication No. 2021 / 175606). The following four chimeras of this application were tested by turbidity reduction assay in the pH range of pH 4.2 to pH 8.0: CLC1-EAD+CLB1-CBD, CLC1-EAD+CLB2-CBD, CLC2-EAD+CLB1-CBD, and CLC2-EAD+CLB2-CBD. Surprisingly, all four enzymes exhibited maximum solubility at approximately pH 6.0 (Figure 12). Activity increased approximately threefold at pH 6 compared to approximately pH 7.3. Furthermore, these enzymes remained active over a very wide pH range, including the highest (8.0) and lowest (4.2) pH levels tested in this study.

[0237] Example 10: Chimeric proteins containing EAD and CW_7 CBD from the CLC1 family exhibit strong selectivity for C. acnes.

[0213] The following four chimeric enzymes were tested for genus selectivity: CLC1-EAD+CLB1-CBD, CLC1-EAD+CLB2-CBD, CLC3-EAD+CLB1-CBD, and CLC3-EAD+CLB2-CBD. Specifically, these four chimeras were tested for activity against symbiotic skin bacteria of other genera, such as Corynebacterium xerosis, Corynebacterium striatum, and Staphylococcus epidermidis, using a turbidity reduction assay. In all four cases, potent lytic activity against C. acnes was observed, but not against the other symbiotic skin bacteria tested. This demonstrates that these chimeric proteins have the ability to lyse target bacteria associated with acne vulgaris without damaging important symbiotic skin bacteria (Figures 13A-13D).

[0238] Example 11: Chimeric proteins containing EAD and CW_7 CBD from the CLC1 family exhibit remarkable thermal stability.

[0214] Thermal stability is an important factor in commercial applications. The thermal stability of these chimeric enzymes was tested by incubating aliquots of the protein at a range of temperatures for 30 minutes, and then measuring the residual lysis activity in these aliquots in a turbidity reduction assay at room temperature (Figures 14A-14D). The results showed that the CLC1-EAD+CLB1-CBD chimera retained activity up to incubation at 50°C. The CLC1-EAD+CLB2-CBD chimera showed higher thermal stability, retaining some activity up to incubation at 54°C. The two CLC3 EAD chimeric enzymes exhibited even higher thermal stability; the CLC3-EAD+CLB1-CBD chimera retained activity up to incubation at 58°C, and the CLC3-EAD+CLB2-CBD chimera retained some activity even at the highest temperature tested (58°C).

[0239] Example 12: A quantitative elimination assay demonstrated remarkable C. acnes solubility activity by the CLC1-EAD+CLB1-CBD chimera.

[0215] The solubility of C. acnes is measured by a turbidity reduction assay through the observed reduction in optical density. A more direct method for measuring antimicrobial activity is to use a quantitative cell death assay. This assay measures antimicrobial activity by reducing the number of viable cells after incubation with an enzyme. Specifically, approximately 1 × 10⁻⁶ 6 C. acnes cells were incubated with or without the enzyme for 2–6 hours, at which point serial dilutions of the mixture were seeded onto BHI agar plates and the CFU was quantified. This assay tested the CLC1-EAD+CLB1-CBD chimera. The results showed a three-order-of-magnitude decrease in the number of viable C. acnes cells after 6 hours of incubation (Figure 15).

[0240] Example 13: A chimeric CWH containing EAD from the broader CLC1 family in combination with CLB2 CBD exhibits solubility against C. acnes.

[0216] To test the activity levels of a broader CLC1 family of amidase proteins and their ability to function as components of chimeric cell wall hydrolases, chimeric enzymes were constructed by ligating CLC4-CLC17 EADs with CLB2 CBDs. CLC18 EADs and CLC19 EADs have the same amino acid sequence as CLC13 EADs and were therefore not included independently.

[0241]

[0217] Expression vectors containing the cloned CLC1 family's EAD+CLB2 CBD chimeric CWH were used to transform BL21 Escherichia coli (E. coli) cells. Protein expression was induced, and cell lysates were isolated for each chimeric enzyme. These cell lysates were tested for C. acnes lytic activity using a clearing / halo assay. In this assay, six of the tested lysates showed significant clearing, indicating C. acnes lytic activity. These lysates contained chimeric CWHs containing CLB2 CBD combined with EAD derived from CLC4, CLC5, CLC8, CLC10, CLC14, or CLC16. To confirm these results, these chimeric proteins were purified and tested using a turbidity reduction assay (Figures 16A-16F). Proteins were tested at 12 μg / mL. All six chimeric proteins exhibited potent lytic activity against C. acnes, supporting the results of the clearing assay. Further turbidity reduction assays demonstrated that the CLC16-EAD+CLB2-CBD chimera was superior to both full-length CLC16 and the more active CLC16-truncation in terms of anti-C. acnes lytic activity (Figure 16G).

[0242]

[0218] These active CLC1 family EADs exhibit a range of 77% to 96% amino acid identity with respect to one another (Figure 17), demonstrating the broader utility of the CLC1 family proteins as a source of more highly active EADs that can target C. acnes.

[0243] Example 14: Chimeric CWH containing EAD and CLB2 CBD from the CLC1 family exhibits remarkable thermal stability.

[0219] The thermal stability characteristics of chimeric proteins containing EAD from the CLC1 family were elucidated. The thermal stability of these proteins was measured by incubating aliquots of purified chimeric proteins at a specified temperature for 30 minutes. Subsequently, the lysis activity of the incubated enzymes was measured using a turbidity reduction assay against C. acnes and calculated by comparing it with the activity after incubation at room temperature.

[0244]

[0220] The results are shown in Figures 18A-18F. All six proteins retained significant activity after exposure to 45°C. Chimeric CWHs containing EAD derived from CLC4, CLC5, CLC8, CLC10, and CLC14 showed loss of activity after 30 minutes of incubation at 50-55°C, similar to the CLC1-EAD, CLC2-EAD, and CLC3-EAD chimeric proteins. Surprisingly, the CLC16-EAD+CLB2-CBD chimeric protein showed exceptional thermal stability, retaining over 80% of its activity after 30 minutes of incubation at 75°C and over 40% of its activity even after 30 minutes of incubation at 95°C (Figure 18F).

[0245] Example 15: The CLC16 EAD+CLB2 CBD chimera shows stronger selectivity against C. acnes than against symbiotic skin bacteria.

[0221] The CLC16-EAD+CLB2-CBD chimeric CWH was tested for its lytic activity against C. acnes and three symbiotic skin bacteria (C. xerosis, C. striatum, and S. epidermidis) in a turbidity reduction assay. The CLC16-EAD+CLB2-CBD chimeric CWH showed potent selective activity against C. acnes compared to the other three bacteria (Figure 19).

[0246] Example 16: The CLC1-EAD+CLB2-CBD and CLC16-EAD+CLB2-CBD chimeric enzymes are active against a broad panel of Cutibacterium acnes strains and lineages.

[0222] Based on phylogenetic analysis, including the use of polylocus sequencing (MLST) schemes, C. acnes strains can be classified into five major lineages (IA1, IA2, IB, II, and III). See, for example, McDowell et al., Plos One 2012; 7(7): e41480. Lineages IA1 and IA2 are most often associated with acne lesions.

[0247]

[0223] To evaluate the activity of CLC1-EAD+CLB2-CBD and CLC16-EAD+CLB2-CBD chimeric enzymes against various strains of C. acnes, these enzymes were tested in a turbidity reduction assay against a panel of C. acnes strains from various lineages (Table 12).

[0248] [Table 49]

[0249]

[0224] The results are shown in Figures 20A-20D. Both the CLC1-EAD+CLB2-CBD and CLC16-EAD+CLB2-CBD chimeric enzymes showed potent lytic activity against all C. acnes strains tested, indicating that these chimeric enzymes are active against a broad set of C. acnes strains and lineages.

[0250] Example 17: CBD derived from CLB2 increases the enzymatic activity of CaLys1 EAD.

[0225] The well-conserved CaLys1 endolysin commonly found in the C. acnes phage genome is characterized by weak lytic activity, often requiring concentrations above 100 mg / mL to confirm its activity. This activity has also been observed to be largely nonspecific; for example, its activity against S. aureus has been shown to be at a similar level to its activity against C. acnes. See Varotsou C et al., Int J Mol Sci 2023 May 10; 24(10):8523. To test whether CLB2-CBD can improve the activity and specificity of CaLys1-EAD, a CaLys1-EAD+CLB2-CBD chimeric CWH was generated. The CaLys1-EAD+CLB2-CBD chimeric CWH and the native CaLys1 protein were expressed and purified. The lytic activity of this chimera was compared to that of native proteins in turbidity reduction assays against C. acnes, C. striatum, and S. aureus. These assays utilized a concentration of 12 μg / mL for each protein.

[0251]

[0226] The results are shown in Figures 21A-21C. At these low concentrations, CaLys1 did not exhibit detectable lytic activity against any of the three bacteria. In contrast, the CaLys1-EAD+CLB2-CBD chimeric CWH showed potent lytic activity against C. acnes, but no detectable lytic activity against C. striatum and S. aureus. These data demonstrate that CBD derived from CLB1-CLB4 proteins can increase the C. acnes-specific lytic activity of standard CaLys1 EAD in the chimeric CWH. While CaLys1 has been reported to have equivalent lytic activity against C. acnes and other bacteria (e.g., S. aureus), a chimeric CWH containing CLB2-CBD in combination with CaLys1-EAD showed high C. acnes-specific lytic activity without increasing C. striatum or S. aureus lytic activity. Further turbidity reduction assays also demonstrated that the CaLys1-EAD+CLB2-CBD chimera exhibited higher anti-C. acnes-lytic activity compared to both full-length CaLys1 and more active CaLys1-truncations (Figure 22).

[0252] Example 18: Diverse CW_7 repeats can promote the binding and lysis of C. acnes in chimeric CWH during whole cell lysate screening.

[0227] To test the ability of diverse CW_7-containing CBDs to target C. acnes in chimeric CWHs, 67 CBDs were selected from a wide range of bacterial species and viruses identified in Example 6. These CBDs contained 1 to 4 CW_7 repeats with amino acid identity ranging from 58.5% to 89% relative to the CLB2-CBD CW_7 sequence. The EADs associated with these CBDs contained both amidase and glycohydrolase. Chimeric proteins containing CLC2-EAD were generated by combining each of these CW_7-containing CBDs. These proteins were expressed in BL21 Escherichia coli (E. coli) cells. The whole cell culture lysates were tested for C. acnes-lytic activity in a turbidity reduction assay. The activity was measured in comparison to the activity of the CLC2-EAD+CLB2-CBD chimeric CWH. The results are shown in Figures 23A-23B. Of the 67 CBDs tested, 9 were classified as not having detectable activity in this assay (relative activity < 0.4), likely due to solubility or assay format; 9 had "detectable activity" (relative activity between 0.4 and 0.6); and 49 had "potent activity" (relative activity > 0.6).

[0253]

[0228] CBD found to have "potent" or "detectable" activity consisted of CW_7 repeats ranging from 58.5% to 89% AA ID to CLB2-CBD CW_7, and originated from a wide range of bacterial species (Table 13). These diverse CBD-containing chimeras showed similar activity to that observed in the more highly active CLC2-EAD+CLB2-CBD chimeras. Surprisingly, these data suggest that the C. acnes-specific solubility of EAD can be improved by combining EAD with CBD containing a diverse range of CW_7 repeats, regardless of taxonomic origin or amino acid identity to CLB2-CBD.

[0254]

[0229] CBD-derived CW_7 repeat sequences with potent or detectable activity were edited and bioinformatics analyzed using MEME (see Bailey et al., “The MEME Suite,” Nucleic Acids Research, 2015; 43(W1): W39-W49), a bioinformatics tool for discovering novel gapless sequence motifs in nucleotide and protein sequences. MEME identified two similar amino acid motifs characteristic of the analyzed CBDS. The 21 amino acid motifs contained 12 highly conserved residues, while the 19 amino acid motifs contained 10 highly conserved residues, illustrating the CW_7 repeats found in CBD that can be utilized by chimeric cell wall hydrolases to increase activity against C. acnes. Figure 23C visually represents the relative frequencies of amino acids at each position in this conserved stretch of the CW_7 repeat sequence characterized in this embodiment. The identified motif sequences are as follows:

[0255]

[0230] CW_7-21 motif: LAXXVI / LXGXXGN / SGXXRK / RXXLG (Sequence ID 2940); and

[0256]

[0231] CW_7-19 motif: AXXVI / LXGXXGN / SGXXRK / RXXL (Sequence ID 2941).

[0257] [Table 50]

[0258] [Table 51]

[0259] [Table 52]

[0260] [Table 53]

[0261] [Table 54]

[0262] [Table 55]

[0263] [Table 56]

[0264] Example 19: CW_7 CBD from Actinobacteria, viruses, and Firmicute promotes the binding and lysis of C. acnes in chimeric CWH in a turbidity reduction assay.

[0232] From the diverse CW_7-containing CBDs identified in Example 6 and investigated in Example 18, six CBDs were selected for protein purification and activity testing, with emphasis on selection from diverse classifications and similarity ranges to the CLB2-CBD CW_7 sequence. Thus, two candidates were selected from each of the Actinobacteria diversity, viral diversity, and Firmicute (also known as "bacillota") diversity. These six CBDs contained 2 to 4 CW_7 repeats, and the top CW_7 sequence matching ranged from 63% to 89% amino acid identity to the CLB2-CBD CW_7 sequence, as listed in Table 14. Chimeric proteins containing CLC2-EAD were generated by combining each of these CW_7-containing CBDs. These proteins were expressed in BL21 Escherichia coli (E. coli) cells, purified using a C-terminal 6xHis tag, and assayed for C. acnes lytic activity in a turbidity reduction assay. For control, the results were compared to full-length CLC2 protein, CLC2-truncation, and CLC2-EAD+CLB2-CBD chimeric protein. As shown in Figures 24A-24B, all six diverse CW_7 chimeric proteins exhibited potent C. acnes lytic activity compared to full-length CLC2 and the more active CLC2-truncation, indicating that these CW_7 repeat-containing CBDs enhance the C. acnes lytic activity of CLC1 family EADs. These data suggest that CW_7 repeat-containing CBDs from diverse biological sources can promote C. acnes binding and lysis.

[0265] [Table 57]

[0266] Example 20: A single CW_7 repeat sufficient to increase anti-C. acnes activity in a chimeric combination of the CLC1 family with EAD

[0233] CLB1-CBD contains two CW_7 repeats, CLB1-CW7-1 (SEQ ID NO: 45) and CLB1-CW7-2 (SEQ ID NO: 46), which are 82% identical at the amino acid level. To test whether either single CW_7 repeat is sufficient to improve the activity of the native CLC1 protein, two chimeric proteins were constructed by linking CLC1-EAD to individual CW_7 repeats of CLB1: CLC1-EAD+CLB1-CW7-1-CBD (SEQ ID NO: 2942) and CLC1-EAD+CLB1-CW7-2-CBD (SEQ ID NO: 2943). The sequences of these chimeric proteins are shown in Table 15.

[0267] [Table 58]

[0268]

[0234] These chimeric proteins were expressed and purified, and then tested in a turbidity reduction assay against C. acnes. The results are shown in Figure 25, along with the activity of the natural full-length CLC1 enzyme, which is shown for comparison. In both cases, the chimeric proteins containing a single CW_7 repeat from CLB1-CBD showed significantly improved activity compared to the natural CLC1 protein, demonstrating that a single CW_7 repeat from CBD containing multiple CW_7 repeats is sufficient to improve the activity of the CLC1 family of enzymes.

[0269] Example 21: The topical hydrogel formulation of the chimeric protein retained potent solubility against C. acnes.

[0235] CLC1-EAD+CLB2-CBD chimeric enzyme (SEQ ID NO: 53) and CLC16-EAD+CLB2-CBD chimeric enzyme (SEQ ID NO: 106) were formulated at a concentration of 12 μg / mL in a hydroxypropyl methylcellulose (HPMC)-based hydrogel. HPMC is a commonly used component in skincare formulations. These formulations were incubated at room temperature for 7 days, and then aliquots were used in a quantitative killing assay for C. acnes. In short, approximately 1 × 10⁶ C. acnes were killed. 6 Cells were added to either a hydrogel control (without enzyme) or a hydrogel containing the enzyme. After 6 hours of incubation, the amount of surviving C. acnes cells was measured by serial dilution on a BHI plate incubated at 37°C under anaerobic conditions for 4-5 days. The results are shown in Figure 26. The preparations containing either enzyme showed a reduction of 3-4 orders of magnitude in the size of surviving C. acnes cells compared to the preparation control, demonstrating that these chimeric enzymes were stable and retained their enzymatic activity in the topical preparations.

[0270] Materials and methods

[0236] The main materials and methods used in the examples are described below.

[0271] Phylogenetic analysis of CLC1 family proteins and their amidase domains

[0237] Multiple sequence alignments of CLC1-CLC19 compared to well-conserved members of the CaLys1 endolysin family found in C. acnes and C. acnes phage were generated using MUSCLE (world wide web at ebi.ac.uk / Tools / msa / muscle / ) and visualized as a rootless tree using standard parameters with the Interactive Tree of Life online tool (world wide web at academic.oup.com / nar / article / 49 / W1 / W293 / 6246398). Similarly, to specifically analyze the amidase domain, the amino acid sequences encoding the amidase domain in these 100 proteins were plotted using SMART or CDD, and multiple sequence alignments and phylogenetic trees were generated as described above.

[0272] Bacterial strains and culture conditions

[0238] In this study, Cutibacterium acnes (ATCC11827) was grown at 37°C with shaking in Brain Heart Infusion (BHI) Broth containing Oxyrase® for broth to remove dissolved oxygen, or on a BHI plate containing 2% (w / v) agar placed in a GasPak EZ pouch (Becton Dickinson) at 37°C to create an anaerobic environment. Corynebacterium xerosis (ATCC 373) and Corynebacterium striatum (ATCC BAA-1293) were grown in BHI Broth at 37°C. Staphylococcus epidermidis (NRLL B4268) was grown in trypsin soy broth (TSB; Difco, Frankling Lakes, NJ) at 37°C with shaking. Escherichia coli DH10B (Invitrogen, Carlsbad, CA) was used for cloning and preservation. Escherichia coli BL21 (DE3) (EMD Biosciences, San Diego, CA) was used for protein expression. All Escherichia coli (E. coli) strains were grown at 37°C with shaking in 2×YT medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl) or on plates containing LB (10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract) supplemented with 2% (w / v) agar. 50 μg / ml kanamycin was used for appropriate selection of E. coli clones.

[0273] Construction of full-length cell wall hydrolase, truncated cell wall hydrolase, and chimeric cell wall hydrolase.

[0239] Full-length cell wall hydrolase was synthesized at the NdeI / HindIII site and liated to the NdeI / HindIII site of pET24a(+). The truncated form of cell wall hydrolase was amplified from a full-length wild-type gene containing the NdeI / HindIII site and liated to the NdeI / HindIII site of pET24a(+). In the case of chimeric enzymes, individual enzyme domains containing up to 50 amino acids upstream and downstream were synthesized at the NdeI / SpeI site. Individual cell wall binding domains containing up to 25 amino acids upstream and downstream were synthesized at the SpeI / HindIII site. Chimeric lysin was constructed by liating both the enzyme domain and the cell wall binding domain to the NdeI / HindIII site of pET24a(+).

[0274]

[0240] An expression vector containing a chimeric cell wall hydrolase was chemically transformed into BL21 Escherichia coli (E. coli) for downstream protein expression and purification.

[0275] Protein production and purification

[0241] BL21 cells containing the appropriate expression plasmid were grown in 2×YT medium at 37°C overnight with shaking. The following morning, the cells were returned to a flask containing 50 mL of ZYM-5052 autoinducible medium (Fisher Scientific catalog number NC1093977), diluted to 1:1000, and incubated at 37°C for 2-3 hours with shaking. The flask was then transferred to 22°C and incubated overnight with shaking. The culture was centrifuged to precipitate, the supernatant was drained, and the pellet was stored at -80°C for at least 30 minutes. Each frozen pellet was resuspended in 5 mL of NPI-10 (100 μM Tris pH 8, 300 mM NaCl, 10 mM imidazole) containing 5 mg of lysozyme and 100 units of DNAseI, and incubated at 30°C for 30 minutes with gentle shaking. The cells were then centrifuged until a clear lysate was obtained and a solid pellet was formed. The clear lysate was transferred to a column containing Nickel-NTA Agarose Resin (Gold Bio) suspended in NPI-10. The column was inverted several times to completely resuspend the resin, and incubated at 4°C for at least 1 hour to enable protein binding. Once the resin had completely settled, the lysate was drained from the column, and the column was washed with two column volumes of NPI-20 (100 μM Tris pH 8, 300 mM NaCl, 20 mM imidazole). Then, NPI-250 (100 μM Tris) was added. The protein was eluted by adding 3 mL of pH 8, 300 mM NaCl, and 250 mM imidazole, and the fraction was collected. The protein was quantified, and its purity was confirmed by the Bradford assay, as well as by SDS-PAGE gel and Coomassi staining. The protein was then concentrated and the buffer was changed to protein preservation buffer (50 mM Tris pH 6.8, 300 mM NaCl) using an Amicon Ultra-15 Filter Unit. For long-term storage, the protein was stored at -80°C with 30% glycerol.

[0276] Clearing assay

[0242] Candidate cell wall hydrolases were tested for their lytic activity against C. acnes by a clearing assay. C. acnes was grown on BHI agar plates at 37°C under anaerobic conditions for 4 days. The cells were then harvested from the plates and washed twice with 25 mM HEPES buffer pH 7.3. The washed cells were resuspended in 25 mM HEPES buffer pH 7.3 at 10 OD / mL. Next, 1 mL of cells was mixed with 20 mL of 0.5% agar and poured into a Petri dish. After setting the agar, 15 μL of purified protein from the candidate cell wall hydrolase was spotted onto the agar. The plates were incubated at room temperature, and clearing around the spots was confirmed after 24 hours.

[0277] Turbidity reduction assay

[0243] Purified candidate cell wall hydrolases were tested for their lytic activity against C. acnes by a turbidity reduction assay. C. acnes was grown on BHI agar plates at 37°C under anaerobic conditions for 4 days. The cells were then harvested from the plates and washed twice with 25 mM HEPES pH 7.3. The OD of the cells was measured. 600 The pH was adjusted to approximately 1.0, and in a flat-bottom microtiter plate, the purified protein was mixed with a 2-fold dilution (e.g., 12 μg / mL to 0.75 μg / mL) and 200 μl of 25 mM HEPES pH 7.3 up to a final volume. Then, the OD of each well was measured. 600The specific activity was measured every 2 minutes for 1 hour using a microplate reader, and then every 5 minutes. The specific activity was calculated as previously described (Briers et al., J Biochem Biophys Methods. 2007 Apr 10; 70(3):531-3). To test the thermal stability of the protein, aliquots of the protein were incubated at a temperature in the range of 37°C to 58°C for 30 minutes. The protein was then immediately tested with the turbidity reduction assay described above. To test the pH range of the protein, turbidity reduction assays were performed using 25 mM Tris HCl pH 8.0, 25 mM HEPES pH 7.3, 25 mM citrate buffer pH 6.5, 25 mM citrate buffer pH 6.2, 25 mM citrate buffer pH 5.7, 25 mM citrate buffer pH 5.3, 25 mM citrate buffer pH 4.9, 25 mM citrate buffer pH 4.5, and 25 mM citrate buffer pH 4.2.

[0278] Quantitative death experiment

[0244] The antimicrobial activity of candidate cell wall hydrolases was measured by a quantitative cell death assay. C. acnes was grown on BHI agar plates at 37°C under anaerobic conditions for 4 days. The cells were then harvested from the plates and washed twice with 25 mM HEPES pH 7.3. Approximately 1 × 10 cells per reaction were then measured. 6 The cells were mixed with the desired amount of protein in a final volume of 200 μL of 25 mM HEPES pH 7.3 and incubated at room temperature. At appropriate time points (e.g., 0 and 6 hours), 20 μL of the reaction mixture was taken out, and serial dilutions were plated onto BHI agar plates and grown under anaerobic conditions at 37°C for 4–5 days. The number of viable cells was then calculated by counting the CFU.

[0279] References The following references are incorporated herein by reference in their entirety and used for any purpose. 1. Bickers, D. R. et al. The burden of skin diseases: 2004. Journal of the American Academy of Dermatology 55, 490-500 (2006). 2. Gallitano, S. M. & Berson, D. S. How Acne Bumps Cause the Blues: The Influence of Acne Vulgaris on Self-Esteem. Int J Womens Dermatol 4, 12-17 (2018). 3. Mias, C., Mengeaud, V., Bessou‐Touya, S. & Duplan, H. Recent advances in understanding inflammatory acne: Deciphering the relationship between Cutibacterium acnes and Th17 inflammatory pathway. Acad Dermatol Venereol 37, 3-11 (2023). 4. Fitz-Gibbon, S. et al. Propionibacterium acnes strain populations in the human skin microbiome associated with acne. J Invest Dermatol 133, 2152-2160 (2013). 5. Lomholt, H. B., Scholz, C. F. P., Brueggemann, H., Tettelin, H. & Kilian, M. A comparative study of Cutibacterium (Propionibacterium) acnes clones from acne patients and healthy controls. Anaerobe 47, 57-63 (2017). 6. Zhang, N., Yuan, R., Xin, K. Z., Lu, Z. & Ma, Y. Antimicrobial Susceptibility, Biotypes and Phylotypes of Clinical Cutibacterium (Formerly Propionibacterium) acnes Strains Isolated from Acne Patients: An Observational Study. Dermatol Ther (Heidelb) 9, 735-746 (2019). 7. Brueggemann, H., Salar-Vidal, L., Gollnick, H. P. M. & Lood, R. A Janus-Faced Bacterium: Host-Beneficial and -Detrimental Roles of Cutibacterium acnes. Front Microbiol 12, 673845 (2021). 8. Zaenglein, A. L. et al. Guidelines of care for the management of acne vulgaris. Journal of the American Academy of Dermatology 74, 945-973.e33 (2016). 9. Chien, A. L. et al. Association of Systemic Antibiotic Treatment of Acne With Skin Microbiota Characteristics. JAMA Dermatol 155, 425-434 (2019). 10. Patangia, D. V., Anthony Ryan, C., Dempsey, E., Paul Ross, R. & Stanton, C. Impact of antibiotics on the human microbiome and consequences for host health. Microbiologyopen 11, e1260 (2022). 11. Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 399, 629-655 (2022). 12. Dams, D. & Briers, Y. Enzybiotics: Enzyme-Based Antibacterials as Therapeutics. Adv Exp Med Biol 1148, 233-253 (2019). 13. Gerstmans, H., Criel, B. & Briers, Y. Synthetic biology of modular endolysins. Biotechnology Advances 36, 624-640 (2018). 14. Natarelli, N., Gahoonia, N. & Sivamani, R. K. Bacteriophages and the Microbiome in Dermatology: The Role of the Phageome and a Potential Therapeutic Strategy. Int J Mol Sci 24, 2695 (2023).

[0280] Incorporation by reference

[0246] All references, articles, publications, patents, patent gazettes, and patent applications cited herein are incorporated by reference in whole for all purposes. However, no reference to any reference, article, publication, patent, patent gazette, or patent application cited herein shall be construed, and shall not be construed, as an acknowledgment or in any form of suggestion that they constitute prior art in effect in any country of the world or form part of common general knowledge.

[0281] Embodiments of the present invention, numbered

[0247] Notwithstanding the attached claims, the present disclosure illustrates embodiments numbered as follows:

[0282] I. Novel truncated proteins 1. Enzymatically active recombinant CLC1 family proteins with a truncated C-terminus.

[0283] 2. The recombinant protein according to Embodiment 1, wherein the recombinant protein is truncated compared to the corresponding full-length natural CLC1 family protein sequence.

[0284] 3. The recombinant protein according to Embodiment 1 or 2, wherein the recombinant protein exhibits higher solubility and / or solubility compared to the corresponding full-length natural CLC1 family protein sequence.

[0285] 4. The truncation is a truncation of the conserved C-terminal region of a full-length native CLC1 family protein sequence, a recombinant protein in any one of Embodiments 1 to 3.

[0286] 5. The recombinant protein according to any one of Embodiments 1 to 4, wherein the truncation is a truncation of the entire C-terminal region of a CLC1 family protein following the enzymatically active domain of a full-length native CLC1 family protein sequence.

[0287] 6. The recombinant protein according to any one of Embodiments 1 to 5, wherein the truncation is a truncation of residue numbers 195-205 and all subsequent residues derived from the full-length natural CLC1 family protein amino acid sequence.

[0288] 7. The recombinant protein according to any one of Embodiments 1 to 6, wherein the truncation is a truncation of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids derived from the C-terminus of a full-length native CLC1 family protein.

[0289] 8. The CLC1 family protein is CLC1 (SEQ ID NO: 1), a recombinant protein according to any one of Embodiments 1 to 7.

[0290] 9. The CLC1 family protein is CLC2 (SEQ ID NO: 2), a recombinant protein according to any one of Embodiments 1 to 7.

[0291] 10. The CLC1 family protein is CLC3 (SEQ ID NO: 3), a recombinant protein according to any one of Embodiments 1 to 7.

[0292] 11. The CLC1 family protein is CLC4 (SEQ ID NO: 4), a recombinant protein according to any one of Embodiments 1 to 7.

[0293] 12. The CLC1 family protein is CLC5 (SEQ ID NO: 5), a recombinant protein according to any one of Embodiments 1 to 7.

[0294] 13. The CLC1 family protein is CLC6 (SEQ ID NO: 6), a recombinant protein according to any one of Embodiments 1 to 7.

[0295] 14. The CLC1 family protein is CLC7 (SEQ ID NO: 7), a recombinant protein according to any one of Embodiments 1 to 7.

[0296] 15. The CLC1 family protein is CLC8 (SEQ ID NO: 8), a recombinant protein according to any one of Embodiments 1 to 7.

[0297] 16. The CLC1 family protein is CLC9 (SEQ ID NO: 9), a recombinant protein according to any one of Embodiments 1 to 7.

[0298] 17. The CLC1 family protein is CLC10 (SEQ ID NO: 10), a recombinant protein according to any one of Embodiments 1 to 7.

[0299] 18. The CLC1 family protein is CLC11 (SEQ ID NO: 11), a recombinant protein according to any one of Embodiments 1 to 7.

[0300] 19. The CLC1 family protein is CLC12 (SEQ ID NO: 12), a recombinant protein according to any one of Embodiments 1 to 7.

[0301] 20. The CLC1 family protein is CLC13 (SEQ ID NO: 13), a recombinant protein according to any one of Embodiments 1 to 7.

[0302] 21. The CLC1 family protein is CLC14 (SEQ ID NO: 14), a recombinant protein according to any one of Embodiments 1 to 7.

[0303] 22. The CLC1 family protein is CLC15 (SEQ ID NO: 15), a recombinant protein according to any one of Embodiments 1 to 7.

[0304] 23. The CLC1 family protein is CLC16 (SEQ ID NO: 16), a recombinant protein according to any one of Embodiments 1 to 7.

[0305] 24. The CLC1 family protein is CLC17 (SEQ ID NO: 17), a recombinant protein according to any one of Embodiments 1 to 7.

[0306] 25. The CLC1 family protein is CLC18 (SEQ ID NO: 18), a recombinant protein according to any one of Embodiments 1 to 7.

[0307] 26. The CLC1 family protein is CLC19 (SEQ ID NO: 19), a recombinant protein according to any one of Embodiments 1 to 7.

[0308] 27. The CLC1 family protein is CLC1, CLC2, CLC3, CLC4, CLC5, CLC6, CLC7, CLC8, CLC9, CLC10, CLC11, CLC12, CLC13, CLC14, CLC16, CLC18, or CLC19, and the C-terminal truncation is a truncation of approximately 70 to 90 amino acids compared to the full-length native CLC1 family protein sequence, as described in any one of Embodiments 1 to 7.

[0309] 28. The recombinant protein according to any one of Embodiments 1 to 7, wherein the CLC1 family protein is CLC1, CLC2, CLC3, CLC4, CLC5, CLC6, CLC7, CLC8, CLC9, CLC10, CLC11, CLC12, CLC13, CLC14, CLC16, CLC18, or CLC19, and the C-terminal truncation is a truncation of about 80, 81, or 82 amino acids compared to the full-length native CLC1 family protein sequence.

[0310] 29. The recombinant protein according to any one of Embodiments 1 to 7, wherein the CLC1 family protein is CLC15, and the C-terminal truncation is a truncation of approximately 115 to 135 amino acids compared to the full-length native CLC1 family protein sequence.

[0311] 30. The recombinant protein according to any one of Embodiments 1 to 7, wherein the CLC1 family protein is CLC15, and the C-terminal truncation is a truncation of approximately 123 amino acids compared to the full-length native CLC1 family protein sequence.

[0312] 31. The recombinant protein according to any one of Embodiments 1 to 7, wherein the CLC1 family protein is CLC17, and the C-terminal truncation is a truncation of approximately 55 to 75 amino acids compared to the full-length native CLC1 family protein sequence.

[0313] 32. The recombinant protein according to any one of Embodiments 1 to 7, wherein the CLC1 family protein is CLC17, and the C-terminal truncation is a truncation of approximately 66 amino acids compared to the full-length native CLC1 family protein sequence.

[0314] 33. The recombinant protein according to any one of Embodiments 1 to 32, wherein the recombinant protein comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to an amino acid sequence selected from the group consisting of SEQ ID NOs. 75 to 93.

[0315] 34. The recombinant protein according to any one of Embodiments 1 to 33, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 75 to 93.

[0316] 35. The recombinant protein according to any one of Embodiments 1 to 34, wherein the recombinant protein comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to an amino acid sequence selected from the group consisting of SEQ ID NOs. 20 to 36.

[0317] 36. The recombinant protein according to any one of Embodiments 1 to 35, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 20 to 36.

[0318] 37. Enzymatically active recombinant CLC16 protein with a truncated C-terminus.

[0319] 38. The recombinant protein according to Embodiment 37, wherein the C-terminal truncation is a truncation of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 amino acids compared to the full-length native CLC16 protein sequence (SEQ ID NO: 16).

[0320] 39. The recombinant protein according to Embodiment 37 or 38, wherein the C-terminal truncation is a truncation of approximately 81 amino acids compared to the full-length native CLC16 protein sequence (SEQ ID NO: 16).

[0321] 40. The recombinant protein according to any one of embodiments 37 to 39, wherein the truncation is a truncation of residues 195-210 to 282 of the full-length natural CLC16 protein amino acid sequence (SEQ ID NO: 16).

[0322] 41. The recombinant protein is the recombinant protein according to any one of Embodiments 37 to 40, comprising the amino acid sequence of SEQ ID NO: 90.

[0323] 42. The recombinant protein is the recombinant protein according to any one of embodiments 37 to 41, comprising the amino acid sequence of SEQ ID NO: 35.

[0324] 43. The recombinant protein according to any one of embodiments 37 to 42, wherein the recombinant protein exhibits higher solubility and / or solubility compared to full-length natural CLC16.

[0325] 44. Enzymatically active recombinant CLC2 protein with a truncated C-terminus.

[0326] 45. The recombinant protein according to Embodiment 44, wherein the C-terminal truncation is a truncation of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 amino acids compared to the full-length native CLC2 protein sequence (SEQ ID NO: 2).

[0327] 46. ​​The recombinant protein according to Embodiment 44 or 45, wherein the C-terminal truncation is a truncation of approximately 82 amino acids compared to the full-length native CLC2 protein sequence (SEQ ID NO: 2).

[0328] 47. The recombinant protein according to any one of embodiments 44 to 46, wherein the truncation is a truncation of residues 195-210 to 282 of the full-length natural CLC2 protein amino acid sequence (SEQ ID NO: 2).

[0329] 48. The recombinant protein is the recombinant protein according to any one of embodiments 44 to 47, comprising the amino acid sequence of SEQ ID NO: 76.

[0330] 49. The recombinant protein is the recombinant protein according to any one of embodiments 44 to 48, comprising the amino acid sequence of SEQ ID NO: 21.

[0331] 50. The recombinant protein according to any one of Embodiments 44 to 49, wherein the recombinant protein exhibits higher solubility and / or solubility compared to full-length natural CLC2.

[0332] 51. Recombinant proteins containing a sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 75-93.

[0333] 52. Recombinant proteins containing a sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 20-36.

[0334] 53. The recombinant protein according to any one of Embodiments 1 to 52, wherein the recombinant protein exhibits higher anti-Cutibacterium acnes lytic activity compared to the corresponding full-length natural CLC1 family protein.

[0335] 54. The recombinant protein according to any one of Embodiments 1 to 53, wherein the recombinant protein has improved solubility compared to the corresponding full-length natural CLC1 family protein.

[0336] 55. Enzymatically active recombinant CaLys1 family proteins with a truncated C-terminus.

[0337] 56. The recombinant protein according to Embodiment 55, wherein the recombinant protein is truncated compared to the corresponding full-length natural CaLys1 family protein sequence.

[0338] 57. The recombinant protein according to Embodiment 55 or 56, wherein the truncation is a truncation of a conserved C-terminal region of a full-length native CaLys1 family protein sequence.

[0339] 58. The recombinant protein according to any one of embodiments 55 to 57, wherein the truncation is a truncation of the entire C-terminal region of a CaLys1 family protein following the enzymatically active domain of the full-length native CaLys1 family protein acid sequence.

[0340] 59. The recombinant protein according to any one of embodiments 55 to 58, wherein the truncation is a truncation of residue numbers 195-205 and all subsequent residues derived from the full-length natural CaLys1 family protein amino acid sequence.

[0341] 60. The recombinant protein according to any one of embodiments 55 to 59, wherein the truncation is a truncation of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids derived from the C-terminus of a full-length native CaLys1 family protein.

[0342] 61. The CaLys1 family protein is CaLys1 (SEQ ID NO: 72), a recombinant protein according to any one of embodiments 55 to 60.

[0343] 62. The CaLys1 family protein is CaLys1, and the C-terminal truncation is a truncation of approximately 70 to 90 amino acids compared to the full-length native CaLys1 sequence, as described in any one of Embodiments 55 to 61.

[0344] 63. The CaLys1 family protein is CaLys1, and the C-terminal truncation is a truncation of approximately 83 amino acids compared to the full-length native CaLys1 sequence, as described in any one of Embodiments 55 to 62.

[0345] 64. The recombinant protein according to any one of Embodiments 55 to 63, wherein the recombinant protein contains an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 74.

[0346] 65. The recombinant protein is the recombinant protein according to any one of embodiments 55 to 64, comprising the amino acid sequence of SEQ ID NO: 74.

[0347] 66. The recombinant protein according to any one of Embodiments 55 to 65, wherein the recombinant protein comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 73.

[0348] 67. The recombinant protein is the recombinant protein according to any one of embodiments 55 to 66, comprising the amino acid sequence of SEQ ID NO: 73.

[0349] 68. Recombinant proteins containing an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 74.

[0350] 69. Recombinant protein containing the amino acid sequence of SEQ ID NO: 74.

[0351] 70. Recombinant proteins containing an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 73.

[0352] 71. Recombinant protein containing the amino acid sequence of SEQ ID NO: 73.

[0353] 72. The recombinant protein according to any one of Embodiments 1 to 71, wherein the recombinant protein is enzymatically active against Cutibacterium acnes.

[0354] 73. The recombinant protein according to any one of embodiments 55 to 72, wherein the recombinant protein has higher anti-Cutibacterium acnes lytic activity compared to the corresponding native full-length CaLys1 family protein sequence.

[0355] 74. The recombinant protein according to any one of Embodiments 1 to 73, wherein the recombinant protein has higher anti-Cutibacterium acnes lytic activity compared to full-length natural CaLys1.

[0356] 75. The recombinant protein according to any one of embodiments 55 to 74, wherein the recombinant protein exhibits improved solubility compared to the corresponding native full-length CaLys1 family protein sequence.

[0357] 76. The recombinant protein according to any one of Embodiments 1 to 75, wherein the recombinant protein has improved solubility compared to the natural full-length CaLys1.

[0358] II. Novel EAD Embodiments 1. Recombinant proteins containing the enzyme-active domain (EAD) of the CLC1 family.

[0359] 2. EAD is a recombinant protein according to Embodiment 1, derived from CLC1, CLC2, CLC3, CLC4, CLC5, CLC6, CLC7, CLC8, CLC9, CLC10, CLC11, CLC12, CLC13, CLC14, CLC15, CLC16, CLC17, CLC18, or CLC19.

[0360] 3. EAD is a recombinant protein of Embodiment 1 or 2, derived from Sequence ID No. 1 to 19.

[0361] 4. The recombinant protein according to any one of Embodiments 1 to 3, wherein EAD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 20 to 36.

[0362] 5. Recombinant proteins containing an enzyme activity domain (EAD) having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 20-36.

[0363] 6.EAD is a recombinant protein according to any one of Embodiments 1 to 5, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 20 to 36.

[0364] 7. The recombinant protein according to any one of Embodiments 1 to 6, wherein EAD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 35.

[0365] 8. EAD is a recombinant protein according to any one of Embodiments 1 to 6, comprising the amino acid sequence of SEQ ID NO: 35.

[0366] 9. The recombinant protein according to any one of Embodiments 1 to 6, wherein EAD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 21.

[0367] 10.EAD is a recombinant protein according to any one of Embodiments 1 to 6, comprising the amino acid sequence of SEQ ID NO: 21.

[0368] 11. The recombinant protein is the recombinant protein according to any one of Embodiments 1 to 10, which exhibits lytic activity against Cutibacterium acnes.

[0369] 12. The recombinant protein according to any one of Embodiments 1 to 11, which exhibits lytic activity against a strain of Cutibacterium acnes selected from the list consisting of IA1, IA2, IB, II, and III.

[0370] 13. The recombinant protein according to any one of Embodiments 1 to 12, which exhibits lytic activity against the IA1, IA2, IB, and II strains of Cutibacterium acnes.

[0371] 14. The recombinant protein according to any one of Embodiments 1 to 13, which exhibits improved solubility and / or anti-Cutibacterium acnes activity compared to natural CaLys1 (SEQ ID NO: 72).

[0372] 14.1. The recombinant protein is the recombinant protein according to any one of Embodiments 1 to 14, comprising the CLC1 family EAD motif (SEQ ID NO: 2939).

[0373] 15. The recombinant protein is the recombinant protein according to any one of Embodiments 1 to 14.1, comprising a heterogeneous cell wall binding domain (CBD).

[0374] 16. The recombinant protein according to Embodiment 15, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 47.

[0375] 17. The recombinant protein according to Embodiment 15 or 16, wherein CBD comprises a CW_7 repeat having the amino acid sequence of SEQ ID NO: 47.

[0376] 18. The recombinant protein according to any one of Embodiments 15 to 17, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to an amino acid sequence selected from the group consisting of SEQ ID NOs. 45 to 51.

[0377] 19. The recombinant protein according to any one of Embodiments 15 to 18, wherein CBD comprises a CW_7 repeat having an amino acid sequence selected from the group consisting of SEQ ID NOs. 45 to 51.

[0378] 20. The recombinant protein according to any one of Embodiments 15 to 19, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CW_7 repeat composed of an amino acid sequence selected from the group consisting of SEQ ID NOs. 166 to 223.

[0379] 21. The recombinant protein according to any one of Embodiments 15 to 20, wherein CBD comprises a CW_7 repeat consisting of an amino acid sequence selected from the group comprising SEQ ID NOs. 166 to 223.

[0380] 22. The recombinant protein according to any one of Embodiments 15 to 21, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CW_7 repeat composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 188, 198, 205, 211, and 221.

[0381] 23. The recombinant protein according to any one of Embodiments 15 to 22, wherein CBD comprises a CW_7 repeat consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 188, 198, 205, 211, and 221.

[0382] 24. The recombinant protein according to any one of Embodiments 15 to 23, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CW_7 repeat composed of an amino acid sequence selected from the group consisting of SEQ ID NOs. 24. The recombinant protein according to any one of Embodiments 15 to 23, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CW_7 repeat composed of an amino acid sequence selected from the group consisting of S

[0383] 25. The recombinant protein according to any one of Embodiments 15 to 24, wherein CBD comprises a CW_7 repeat consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938.

[0384] 26. The recombinant protein according to any one of Embodiments 15 to 25, wherein CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to CW_7, which is composed of the proteins listed in Table 7.

[0385] 27. CBD is a recombinant protein according to any one of Embodiments 15 to 26, comprising a CW_7 repeat composed of the proteins listed in Table 7.

[0386] 28. The recombinant protein according to any one of Embodiments 15 to 27, wherein CBD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 41 to 44.

[0387] 29. CBD is a recombinant protein according to any one of Embodiments 15 to 27, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 44.

[0388] 30. The recombinant protein according to any one of Embodiments 15 to 27, wherein the CBD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 42.

[0389] 31. CBD is a recombinant protein according to any one of Embodiments 15 to 27, comprising the amino acid sequence of SEQ ID NO: 42.

[0390] 32. The recombinant protein according to any one of Embodiments 15 to 27, wherein CBD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 166 to 223.

[0391] 33. CBD is a recombinant protein according to any one of embodiments 15 to 27, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 166 to 223.

[0392] 34. The recombinant protein according to any one of Embodiments 15 to 27, wherein the CBD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CW_7 repeat composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 188, 198, 205, 211, and 221.

[0393] 35. The recombinant protein according to any one of Embodiments 15 to 27, wherein CBD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 188, 198, 205, 211, and 221.

[0394] 36. The recombinant protein according to any one of Embodiments 15 to 27, comprising CBD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to CBD composed of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938.

[0395] 37. The recombinant protein according to any one of embodiments 15 to 27, comprising a CBD consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938.

[0396] 38. The recombinant protein according to any one of Embodiments 15 to 27, comprising CBD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to CBD composed of the proteins listed in Table 7.

[0397] 39. The recombinant protein is a recombinant protein according to any one of Embodiments 15 to 27, comprising CBD composed of the proteins listed in Table 7.

[0398] 40. CBD is a recombinant protein according to any one of Embodiments 15 to 39, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CW_7 repeats.

[0399] 41. The recombinant protein is a chimeric cell wall hydrolase (CWH), as described in any one of Embodiments 1 to 40.

[0400] 42. The recombinant protein is a recombinant protein according to any one of Embodiments 1 to 41, which binds to Cutibacterium acnes.

[0401] 43. The recombinant protein is the recombinant protein according to any one of Embodiments 1 to 42, having lytic activity against Cutibacterium acnes.

[0402] 44. The recombinant protein according to any one of Embodiments 1 to 43, wherein the recombinant protein includes a cell wall-binding domain (CBD), the CBD being located at the C-terminus of the EAD.

[0403] 45. The recombinant protein according to any one of Embodiments 1 to 44, which exhibits higher solubility against C. acnes compared to natural CaLys1 (SEQ ID NO: 72).

[0404] 46. ​​The recombinant protein according to any one of Embodiments 1 to 45, which exhibits higher solubility against C. acnes compared to the natural protein derived from EAD.

[0405] 47. The recombinant protein according to any one of Embodiments 1 to 46, wherein the recombinant protein exhibits minimal or no lytic activity toward Corynebacterium xerosis, Corynebacterium striatum, and / or Staphylococcus epidermidis.

[0406] 48. The recombinant protein according to any one of Embodiments 1 to 47, wherein the recombinant protein has lytic activity against Cutibacterium acnes, and the lytic activity is measured using a turbidity reduction assay.

[0407] 49. The recombinant protein according to any one of Embodiments 1 to 48, wherein the recombinant protein has lytic activity against Cutibacterium acnes over a pH range of 4.2 to 8.0.

[0408] 50. The recombinant protein according to any one of Embodiments 1 to 49, wherein the recombinant protein exhibits maximum C. acnes solubility at a pH of less than 7.

[0409] 51. The recombinant protein according to any one of Embodiments 1 to 50, wherein the recombinant protein exhibits maximum C. acnes solubility at a pH of 5.5–6.5, 5.8–6.2, 5.9–6.1, or approximately 6.0.

[0410] 52. The recombinant protein according to any one of Embodiments 1 to 51, wherein the recombinant protein retains at least 50% of the activity of the recombinant protein at 25°C after exposure to a temperature of up to 45°C, 50°C, 55°C, or 58°C for 30 minutes.

[0411] 53. The recombinant protein according to any one of Embodiments 1 to 52, wherein the recombinant protein comprises CLC16 EAD (SEQ ID NO: 35) and retains at least 50% of the recombinant protein activity at 25°C after exposure to a temperature of up to 75°C for 30 minutes.

[0412] 54. The recombinant protein according to any one of Embodiments 1 to 53, wherein the recombinant protein comprises CLC16 EAD (SEQ ID NO: 35) and retains at least 40% of the recombinant protein activity at 25°C after exposure to a temperature of up to 90°C for 30 minutes.

[0413] 55. The recombinant protein according to any one of Embodiments 1 to 54, wherein the solubility of the recombinant protein is at least twice, at least five times, at least ten times, or at least 100 times higher than the solubility of CaLys1 (SEQ ID NO: 72).

[0414] III. Novel Embodiments of CBD 1. Recombinant protein containing the CW_7 cell wall-binding domain (CBD).

[0415] 2. The recombinant protein according to Embodiment 1, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 47.

[0416] 3. The recombinant protein according to Embodiment 1 or 2, wherein CBD comprises a CW_7 repeat having the amino acid sequence of SEQ ID NO: 47.

[0417] 4. The recombinant protein according to any one of Embodiments 1 to 3, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 45 to 51.

[0418] 5. The recombinant protein according to any one of Embodiments 1 to 4, wherein CBD comprises a CW_7 repeat having an amino acid sequence selected from the group consisting of SEQ ID NOs. 45 to 51.

[0419] 6. The recombinant protein according to any one of Embodiments 1 to 5, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CW_7 repeat composed of an amino acid sequence selected from the group consisting of SEQ ID NOs. 166 to 223.

[0420] 7. The recombinant protein according to any one of Embodiments 1 to 6, wherein CBD contains a CW_7 repeat consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 166 to 223.

[0421] 8. The recombinant protein according to any one of Embodiments 1 to 7, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CW_7 repeat composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 188, 198, 205, 211, and 221.

[0422] 9. The recombinant protein according to any one of Embodiments 1 to 8, wherein the CBD comprises a CW_7 repeat consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 188, 198, 205, 211, and 221.

[0423] 10. The recombinant protein according to any one of Embodiments 1 to 9, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CW_7 repeat composed of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938.

[0424] 11. The recombinant protein according to any one of Embodiments 1 to 10, wherein CBD contains a CW_7 repeat consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938.

[0425] 12. The recombinant protein according to any one of Embodiments 1 to 11, wherein CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to CW_7 composed of the proteins listed in Table 7.

[0426] 13. CBD is a recombinant protein according to any one of Embodiments 1 to 12, comprising a CW_7 repeat composed of the proteins listed in Table 7.

[0427] 14. The recombinant protein according to any one of Embodiments 1 to 13, wherein CBD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to an amino acid sequence selected from the group consisting of SEQ ID NOs. 41 to 44.

[0428] 15. CBD is a recombinant protein according to any one of Embodiments 1 to 13, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 44.

[0429] 16. The recombinant protein according to any one of Embodiments 1 to 13, wherein the CBD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 42.

[0430] 17. CBD is a recombinant protein according to any one of Embodiments 1 to 13, comprising the amino acid sequence of SEQ ID NO: 42.

[0431] 18. The recombinant protein according to any one of Embodiments 1 to 13, wherein CBD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 166 to 223.

[0432] 19. CBD is a recombinant protein according to any one of Embodiments 1 to 13, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 166 to 223.

[0433] 20. A recombinant protein according to any one of Embodiments 1 to 13, wherein CBD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CW_7 repeat composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 188, 198, 205, 211, and 221.

[0434] 21. CBD is a recombinant protein according to any one of Embodiments 1 to 13, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 188, 198, 205, 211, and 221.

[0435] 22. The recombinant protein according to any one of Embodiments 1 to 13, wherein the recombinant protein contains CBD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to CBD composed of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938.

[0436] 23. The recombinant protein according to any one of Embodiments 1 to 13, comprising a CBD consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938.

[0437] 24. The recombinant protein according to any one of Embodiments 1 to 13, comprising CBD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to CBD composed of the proteins listed in Table 7.

[0438] 25. The recombinant protein is a recombinant protein according to any one of Embodiments 1 to 13, comprising CBD composed of the proteins listed in Table 7.

[0439] 26. CBD is a recombinant protein according to any one of Embodiments 1 to 25, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CW_7 repeats.

[0440] 27. The recombinant protein is an enzyme, as described in any one of Embodiments 1 to 26.

[0441] 28. The recombinant protein is a chimeric protein, as described in any one of Embodiments 1 to 27.

[0442] 29. The recombinant protein is a chimeric cell wall hydrolase (CWH), as described in any one of Embodiments 1 to 28.

[0443] 29.1. The recombinant protein is the recombinant protein according to any one of Embodiments 1 to 29, comprising the CW_7-21 motif (SEQ ID NO: 2940).

[0444] 29.2. The recombinant protein is the recombinant protein according to any one of Embodiments 1 to 29.1, comprising the CW_7-19 motif (SEQ ID NO: 2941).

[0445] 30. The recombinant protein is the recombinant protein according to any one of Embodiments 1 to 29.2, comprising an enzyme-active domain (EAD).

[0446] 31. EAD is a recombinant protein according to Embodiment 30, which is heterologous to CBD.

[0447] 32. The recombinant protein according to Embodiment 30 or 31, wherein EAD has at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with EAD of the CaLys1 family.

[0448] 33. EAD is a recombinant protein according to any one of embodiments 30 to 32, comprising EAD of the CaLys1 family.

[0449] 34. The recombinant protein according to any one of Embodiments 30 to 33, wherein EAD has at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 73.

[0450] 35.EAD is a recombinant protein according to any one of embodiments 30 to 34, comprising the amino acid sequence of SEQ ID NO: 73.

[0451] 36. The recombinant protein according to any one of Embodiments 30 to 31, wherein EAD has at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with EAD of the CLC1 family.

[0452] 37. EAD is a recombinant protein according to any one of embodiments 30-31 and 36, comprising EAD of the CLC1 family.

[0453] 38.EAD is a recombinant protein according to any one of embodiments 30-31 and 36-37, having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to an amino acid sequence selected from the list consisting of SEQ ID NOs.

[0454] 39.EAD is a recombinant protein according to any one of embodiments 30-31 and 36-38, comprising an amino acid sequence selected from the list consisting of SEQ ID NOs. 20-36.

[0455] 40.EAD is a recombinant protein according to any one of embodiments 30-31 and 36-39, having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 21.

[0456] 41.EAD is a recombinant protein according to any one of embodiments 30-31 and 36-40, comprising the amino acid sequence of SEQ ID NO: 21.

[0457] 42.EAD is a recombinant protein according to any one of embodiments 30-31 and 36-39, having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 35.

[0458] 43.EAD is a recombinant protein according to any one of embodiments 30-31 and 36-39, comprising the amino acid sequence of SEQ ID NO: 35.

[0459] 44. The recombinant protein is a recombinant protein according to any one of Embodiments 1 to 43, which binds to Cutibacterium acnes.

[0460] 45. The recombinant protein is the recombinant protein according to any one of Embodiments 1 to 44, having lytic activity against Cutibacterium acnes.

[0461] 46. ​​The recombinant protein according to any one of Embodiments 1 to 45, wherein the recombinant protein comprises an enzyme-active domain (EAD), and the CBD is located at the C-terminus of the EAD.

[0462] 47. The recombinant protein according to any one of Embodiments 1 to 46, which exhibits higher solubility against C. acnes compared to the natural protein derived from CW_7 and / or CBD.

[0463] 48. The recombinant protein according to any one of Embodiments 1 to 47, wherein the recombinant protein exhibits minimal or no lytic activity toward Corynebacterium xerosis, Corynebacterium striatum, and / or Staphylococcus epidermidis.

[0464] 49. The recombinant protein according to any one of Embodiments 1 to 48, wherein the recombinant protein has lytic activity against Cutibacterium acnes, and the lytic activity is measured using a turbidity reduction assay.

[0465] 50. The recombinant protein according to any one of Embodiments 1 to 49, wherein the recombinant protein has lytic activity against Cutibacterium acnes over a pH range of 4.2 to 8.0.

[0466] 51. The recombinant protein according to any one of Embodiments 1 to 50, wherein the recombinant protein exhibits maximum C. acnes solubility at a pH of less than 7.

[0467] 52. The recombinant protein according to any one of Embodiments 1 to 51, wherein the recombinant protein exhibits maximum C. acnes solubility at a pH of 5.5–6.5, 5.8–6.2, 5.9–6.1, or approximately 6.0.

[0468] 53. The recombinant protein according to any one of Embodiments 1 to 52, wherein the recombinant protein retains at least 50% of the activity of the recombinant protein at 25°C after exposure to a temperature of up to 45°C, 50°C, 55°C, or 58°C for 30 minutes.

[0469] 54. The recombinant protein according to any one of Embodiments 1 to 53, wherein the solubility of the recombinant protein is at least twice, at least five times, at least ten times, or at least 100 times higher than that of natural CaLys1 (SEQ ID NO: 72).

[0470] IV. Embodiments of Novel Chimeric Cell Wall Hydrolases 1. Chimeric cell wall hydrolase (CWH), a) CW_7 cell wall binding domain (CBD), b) The enzyme-active domain (EAD) of the CLC1 family and A chimeric CWH containing

[0471] 2. The chimera CWH according to Embodiment 1, wherein EAD is derived from CLC1, CLC2, CLC3, CLC4, CLC5, CLC6, CLC7, CLC8, CLC9, CLC10, CLC11, CLC12, CLC13, CLC14, CLC15, CLC16, CLC17, CLC18, or CLC19.

[0472] 3.EAD is a chimera CWH according to Embodiment 1 or 2, derived from Sequence IDs 1 to 19.

[0473] 4. The chimeric CWH according to any one of Embodiments 1 to 3, wherein EAD includes an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs.

[0474] 5. EAD is a chimeric CWH according to any one of Embodiments 1 to 4, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 20 to 36.

[0475] 6. A chimeric CWH according to any one of Embodiments 1 to 5, wherein EAD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 35.

[0476] 7.EAD is a chimeric CWH according to any one of Embodiments 1 to 5, comprising the amino acid sequence of SEQ ID NO: 35.

[0477] 8. A chimeric CWH according to any one of Embodiments 1 to 5, wherein EAD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 21.

[0478] 9.EAD is a chimeric CWH according to any one of Embodiments 1 to 5, comprising the amino acid sequence of SEQ ID NO: 21.

[0479] 10. Chimeric cell wall hydrolase (CWH), a) CW_7 cell wall binding domain (CBD), b) Enzyme-active domain (EAD) derived from CLC16 (SEQ ID NO: 35) A chimeric CWH containing

[0480] 11. Chimeric cell wall hydrolase (CWH), a) CW_7 cell wall binding domain (CBD), b) Enzyme-active domain (EAD) derived from CLC2 (SEQ ID NO: 21) A chimeric CWH containing

[0481] 12. Chimeric cell wall hydrolase (CWH), a) CW_7 cell wall binding domain (CBD), b) The enzyme-active domain (EAD) of the CaLys1 family and A chimeric CWH containing

[0482] 13. The chimeric CWH according to Embodiment 12, wherein EAD has at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to sequence number 73.

[0483] 14.EAD is a chimeric CWH according to Embodiment 12 or 13, comprising the amino acid sequence of SEQ ID NO: 73.

[0484] 14.1. Chimeric cell wall hydrolase (CWH), a) CW_7 cell wall binding domain (CBD), b) PlyGVE2 enzyme active domain (EAD) (SEQ ID NO: 64) or CD27L EAD (SEQ ID NO: 63) A chimeric CWH containing

[0485] 15. Chimeric cell wall hydrolase (CWH), a) CW_7 cell wall binding domain (CBD), b) Enzyme-active domain (EAD) derived from CaLys1 (SEQ ID NO: 73) A chimeric CWH containing

[0486] 16. A chimeric CWH according to any one of Embodiments 1 to 15, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 47.

[0487] 17. CBD is a chimeric CWH according to any one of Embodiments 1 to 16, comprising a CW_7 repeat having the amino acid sequence of SEQ ID NO: 47.

[0488] 18. A chimeric CWH according to any one of Embodiments 1 to 17, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 45 to 51.

[0489] 19. CBD is a chimeric CWH according to any one of Embodiments 1 to 18, comprising a CW_7 repeat having an amino acid sequence selected from the group consisting of SEQ ID NOs. 45 to 51.

[0490] 20. A chimeric CWH according to any one of Embodiments 1 to 19, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CW_7 repeat composed of an amino acid sequence selected from the group consisting of SEQ ID NOs. 166 to 223.

[0491] 21. A chimeric CWH according to any one of Embodiments 1 to 20, wherein the CBD comprises a CW_7 repeat consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 166 to 223.

[0492] 22. A chimeric CWH according to any one of Embodiments 1 to 21, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CW_7 repeat composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 188, 198, 205, 211, and 221.

[0493] 23. A chimeric CWH according to any one of Embodiments 1 to 22, wherein the CBD comprises a CW_7 repeat consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 188, 198, 205, 211, and 221.

[0494] 24. A chimeric CWH according to any one of Embodiments 1 to 23, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CW_7 repeat composed of an amino acid sequence selected from the group consisting of SEQ ID NOs. 24. A chimeric CWH according to any one of Embodiments 1 to 23, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CW_7 repeat composed of an amino acid sequence selected from the group consisting

[0495] 25. A chimeric CWH according to any one of Embodiments 1 to 24, wherein the CBD comprises a CW_7 repeat consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938.

[0496] 26. A chimeric CWH according to any one of Embodiments 1 to 25, wherein the CBD comprises a CW_7 repeat having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to CW_7 composed of the proteins listed in Table 7.

[0497] 27. CBD is a chimeric CWH according to any one of Embodiments 1 to 26, comprising a CW_7 repeat composed of the proteins listed in Table 7.

[0498] 28. A chimeric CWH according to any one of Embodiments 1 to 27, wherein the CBD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 41 to 44.

[0499] 29. CBD is a chimeric CWH according to any one of Embodiments 1 to 27, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 44.

[0500] 30. A chimeric CWH according to any one of Embodiments 1 to 27, wherein the CBD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 42.

[0501] 31. CBD is a chimeric CWH according to any one of Embodiments 1 to 27, comprising the amino acid sequence of SEQ ID NO: 42.

[0502] 32. A chimeric CWH according to any one of Embodiments 1 to 27, wherein the CBD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 166 to 223.

[0503] 33. CBD is a chimeric CWH according to any one of Embodiments 1 to 27, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 166 to 223.

[0504] 34. A chimeric CWH according to any one of Embodiments 1 to 27, wherein the CBD comprises an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CW_7 repeat composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 188, 198, 205, 211, and 221.

[0505] 35. A chimeric CWH according to any one of Embodiments 1 to 27, wherein the CBD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 188, 198, 205, 211, and 221.

[0506] 36. A chimeric CWH according to any one of Embodiments 1 to 27, wherein the recombinant protein comprises a CBD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CBD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938.

[0507] 37. A chimeric CWH according to any one of Embodiments 1 to 27, comprising a CBD whose recombinant protein consists of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938.

[0508] 38. A chimeric CWH according to any one of Embodiments 1 to 27, wherein the recombinant protein comprises a CBD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to a CBD composed of the proteins listed in Table 7.

[0509] 39. A chimeric CWH according to any one of Embodiments 1 to 27, comprising a CBD composed of the proteins listed in Table 7.

[0510] 40. CBD is a chimera CWH according to any one of Embodiments 1 to 39, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CW_7 repeats.

[0511] 41. Chimeric cell wall hydrolase (CWH), a) CLB2-derived cell wall-binding domain (CBD) (SEQ ID NO: 42), b) Enzyme-active domain (EAD) derived from CLC16 (SEQ ID NO: 35) A chimeric CWH containing

[0512] 42. A chimeric cell wall hydrolase (CWH) comprising an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 106.

[0513] 43. Chimeric cell wall hydrolase (CWH) containing the amino acid sequence of SEQ ID NO: 106.

[0514] 44. Chimeric cell wall hydrolase (CWH), a) CLB2-derived cell wall-binding domain (CBD) (SEQ ID NO: 42), b) Enzyme-active domain (EAD) derived from CLC2 (SEQ ID NO: 21) A chimeric CWH containing

[0515] 45. A chimeric cell wall hydrolase (CWH) comprising an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 57.

[0516] 46. ​​Chimeric cell wall hydrolase (CWH) containing the amino acid sequence of SEQ ID NO: 57.

[0517] 47. A chimeric cell wall hydrolase (CWH) comprising an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the list consisting of SEQ ID NOs. 52-59.

[0518] 48. A chimeric cell wall hydrolase (CWH) containing an amino acid sequence selected from the list consisting of SEQ ID NOs. 52-59.

[0519] 49. A chimeric cell wall hydrolase (CWH) comprising an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the list consisting of SEQ ID NOs. 53, 57, 59, and 94-107.

[0520] 50. A chimeric cell wall hydrolase (CWH) containing an amino acid sequence selected from the list consisting of SEQ ID NOs. 52-59.

[0521] 51. A chimeric cell wall hydrolase (CWH) comprising an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the list consisting of SEQ ID NOs. 224-281.

[0522] 52. A chimeric cell wall hydrolase (CWH) containing an amino acid sequence selected from the list consisting of SEQ ID NOs. 224-281.

[0523] 53. A chimeric cell wall hydrolase (CWH) comprising an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the list consisting of SEQ ID NOs. 66-69.

[0524] 54. A chimeric cell wall hydrolase (CWH) containing an amino acid sequence selected from the list consisting of SEQ ID NOs. 66-69.

[0525] 55. A chimeric CWH according to any one of embodiments 1 to 54, wherein the chimeric CWH is bound to Cutibacterium acnes.

[0526] 56. A chimeric CWH according to any one of Embodiments 1 to 55, which exhibits solubility activity against Cutibacterium acnes.

[0527] 57. A chimeric CWH according to any one of Embodiments 1 to 56, which exhibits lytic activity against a lineage of Cutibacterium acnes selected from the list consisting of IA1, IA2, IB, II, and III.

[0528] 58. A chimeric CWH according to any one of Embodiments 1 to 57, which exhibits solubility activity against the IA1 and IA2 lineages of Cutibacterium acnes.

[0529] 59. A chimeric CWH according to any one of Embodiments 1 to 58, which exhibits solubility activity against the IA1, IA2, IB, and II lineages of Cutibacterium acnes.

[0530] 60. A chimeric CWH according to any one of embodiments 1 to 59, exhibiting improved anti-Cutibacterium acnes activity compared to natural CaLys1 (SEQ ID NO: 72).

[0531] 61. A chimeric CWH according to any one of embodiments 1 to 60, wherein CBD is located at the C-terminus of EAD.

[0532] 62. The chimeric CWH according to any one of Embodiments 1 to 61, which exhibits higher solubility against C. acnes compared to the natural protein derived from CBD.

[0533] 63. The chimeric CWH according to any one of Embodiments 1 to 62, which exhibits higher solubility against C. acnes compared to the natural protein derived from EAD.

[0534] 64. A chimeric CWH according to any one of Embodiments 1 to 63, which exhibits minimal or no soluble activity toward Corynebacterium xerosis, Corynebacterium striatum, and / or Staphylococcus epidermidis.

[0535] 65. The chimeric CWH according to any one of Embodiments 1 to 64, wherein the chimeric CWH has solubility activity against Cutibacterium acnes, and the solubility activity is measured using a turbidity reduction assay.

[0536] 66. A chimeric CWH according to any one of Embodiments 1 to 65, having solubility activity against Cutibacterium acnes over a pH range of 4.2 to 8.0.

[0537] 67. A chimeric CWH according to any one of embodiments 1 to 66, wherein the chimeric CWH exhibits maximum C. acnes solubility at a pH of less than 7.

[0538] 68. The chimeric CWH according to any one of Embodiments 1 to 67, wherein the C. acnes solubility is maximized at a pH of 5.5 to 6.5, 5.8 to 6.2, 5.9 to 6.1, or approximately 6.0.

[0539] 69. The chimeric CWH according to any one of Embodiments 1 to 68, wherein the chimeric CWH retains at least 50% of the activity of the chimeric CWH at 25°C after exposure to a maximum temperature of 45°C, 50°C, 55°C, or 58°C for 30 minutes.

[0540] 70. The chimeric CWH according to any one of Embodiments 1 to 69, wherein the chimeric CWH comprises CLC16 EAD (SEQ ID NO: 35) or an EAD having an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to CLC16 EAD (SEQ ID NO: 35), and the chimeric CWH retains at least 50% of the activity of the chimeric CWH at 25°C after exposure to a temperature of up to 75°C for 30 minutes.

[0541] 71. The chimeric CWH according to any one of Embodiments 1 to 70, wherein the chimeric CWH comprises CLC16 EAD (SEQ ID NO: 35) or an EAD having an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to CLC16 EAD (SEQ ID NO: 35), and the chimeric CWH retains at least 40% of the activity of the chimeric CWH at 25°C after exposure to a temperature of up to 90°C for 30 minutes.

[0542] 72. Chimeric CWH as described in any one of Embodiments 1 to 71, exhibiting improved solubility compared to natural CaLys1 (SEQ ID NO: 72).

[0543] 73. The chimeric CWH according to any one of Embodiments 1 to 72, wherein the solubility of the chimeric CWH is at least twice, at least five times, at least ten times, or at least 100 times higher than the solubility of natural CaLys1 (SEQ ID NO: 72).

[0544] V. Embodiments of Novel Formulations and Methods 1. A formulation comprising a recombinant protein or chimeric CWH of any of the embodiments described above.

[0545] 2. A preparation containing chimeric cell wall hydrolase (CWH), wherein CWH is a) A cell wall-binding domain (CBD) having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with CBD derived from CLB2 (SEQ ID NO: 42), b) An enzyme activity domain (EAD) having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with the EAD derived from CLC16 (SEQ ID NO: 35) A formulation containing the above.

[0546] 3. A preparation containing chimeric cell wall hydrolase (CWH), wherein CWH is a) CLB2-derived cell wall-binding domain (CBD) (SEQ ID NO: 42), b) Enzyme-active domain (EAD) derived from CLC16 (SEQ ID NO: 35) A formulation containing the above.

[0547] 4. A formulation containing a chimeric CWH having an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 106.

[0548] 5. A formulation containing a chimeric CWH that includes the amino acid sequence of SEQ ID NO: 106.

[0549] 6. The formulation is a topical formulation, as described in any one of Embodiments 1 to 5.

[0550] 7. The formulation according to any one of Embodiments 1 to 6, wherein the formulation is a hydrogel, lotion, cream, gel-cream, colloidal patch, or microneedle patch.

[0551] 8. The formulation is a hydrogel, as described in any one of Embodiments 1 to 7.

[0552] 9. The formulation is the formulation according to any one of Embodiments 1 to 8, comprising a wetting agent.

[0553] 10. The formulation according to any one of Embodiments 1 to 9, comprising a humectant, wherein the humectant is selected from the list consisting of aloe vera, betaine, butylene glycol, caprylyl glycol, dimethicone, fructose, glucomannan, glucose, glycerin, glyceryl glucoside, honey, hyaluronic acid, lactic acid, panthenol, polyethylene glycol, propylene glycol, propanediol, sodium hyaluronate, sodium lactate, sodium pyrrolidone carboxylate, sorbitol, and urea.

[0554] 11. The formulation is the formulation according to any one of Embodiments 1 to 10, comprising 0.1 to 50 w / v% of a wetting agent.

[0555] 12. The formulation is the formulation according to any one of Embodiments 1 to 11, comprising 0.5 to 10 w / v% of a wetting agent.

[0556] 13. The formulation is the formulation according to any one of Embodiments 1 to 12, comprising a cellulose polymer.

[0557] 14. The formulation according to any one of Embodiments 1 to 13, wherein the formulation comprises a cellulose polymer, the cellulose polymer being selected from the list consisting of hydroxyethylcellulose, methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, microcrystalline cellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, and cellulose acetate.

[0558] 15. The formulation is the formulation according to any one of Embodiments 1 to 14, comprising 0.5 to 10 w / v% of a cellulose polymer.

[0559] 16. The formulation is the formulation according to any one of Embodiments 1 to 15, comprising 1 to 5 w / v% of a cellulose polymer.

[0560] 17. The formulation is the formulation according to any one of Embodiments 1 to 16, comprising a salt.

[0561] 18. The formulation according to any one of Embodiments 1 to 17, wherein the formulation comprises a salt, the salt being selected from the list consisting of calcium chloride, Dead Sea salt, Epsom salt, Himalayan pink salt, magnesium chloride, sea salt, and sodium chloride.

[0562] 19. The formulation is the formulation according to any one of Embodiments 1 to 18, comprising 10 to 500 mM of salt.

[0563] 20. The formulation is the formulation according to any one of Embodiments 1 to 19, comprising a salt of 50 to 250 mM.

[0564] 21. The formulation is the formulation according to any one of Embodiments 1 to 20, comprising a buffer solution.

[0565] 22. The formulation according to any one of Embodiments 1 to 21, comprising a buffer, the buffer being selected from the list consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, acetic acid, ammonium acetate, boric acid, citric acid, glycine, phosphoric acid, potassium hydroxide, potassium phosphate, sodium acetate, sodium bicarbonate, sodium borate, sodium carbonate, sodium citrate, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium hydroxide, sodium phosphate, sodium tetraborate, tris(hydroxymethyl)aminomethane, and trisodium phosphate.

[0566] 23. The formulation is the formulation according to any one of Embodiments 1 to 22, comprising a buffer solution of 5 to 50 mM.

[0567] 24. The formulation is the formulation according to any one of Embodiments 1 to 23, comprising a surfactant.

[0568] 25. The formulation comprises a surfactant, the formulation according to any one of Embodiments 1 to 24, wherein the surfactant is selected from the list consisting of ceteareth-20, cocamidopropyl betaine, cocoglucoside, decyl glucoside, decyl polyglucose, disodium laureth sulfosuccinate, glycereth-26, lauryl glucoside, lauryl polyglucose, sodium cocoyl glutamate, sodium cocoyl isethionate, sodium laureth sulfate, and sodium lauryl sulfate.

[0569] 26. The formulation is the formulation according to any one of Embodiments 1 to 25, comprising 0.1 to 20 w / v% of a surfactant.

[0570] 27. The formulation is the formulation according to any one of Embodiments 1 to 26, comprising 1 to 10 w / v% of a surfactant.

[0571] 28. The formulation is the formulation according to any one of Embodiments 1 to 27, comprising a free amino acid.

[0572] 29. The preparation according to any one of Embodiments 1 to 28, wherein the preparation comprises a free amino acid, the free amino acid being selected from the list consisting of alanine, arginine, cysteine, glutamine, glycine, histidine, lysine, methionine, proline, serine, and threonine.

[0573] 30. The formulation is the formulation according to any one of Embodiments 1 to 29, comprising 10 to 250 mM of free amino acids.

[0574] 31. The formulation is the formulation according to any one of Embodiments 1 to 30, comprising oil.

[0575] 32. The formulation according to any one of Embodiments 1 to 31, wherein the formulation comprises an oil, the oil being selected from the list consisting of argan oil, avocado oil, baobab oil, camellia oil, carrot seed oil, coconut oil, evening primrose oil, grape seed oil, hemp seed oil, jojoba oil, macadamia nut oil, marula oil, mineral oil, olive oil, pomegranate seed oil, raspberry seed oil, rosehip seed oil, squalane oil, sunflower seed oil, sweet almond oil, and tamanu oil.

[0576] 33. The formulation is the formulation according to any one of Embodiments 1 to 32, comprising 0.1 to 20 w / v% oil.

[0577] 34. The formulation is the formulation according to any one of Embodiments 1 to 33, comprising alcohol.

[0578] 35. The formulation according to any one of Embodiments 1 to 34, wherein the formulation comprises an alcohol, the alcohol being selected from the list consisting of cetyl alcohol, ethyl alcohol, isopropyl alcohol, and stearyl alcohol.

[0579] 36. The formulation is the formulation according to any one of Embodiments 1 to 35, comprising 0.1 to 20 w / v% alcohol.

[0580] 37. The formulation is the formulation according to any one of Embodiments 1 to 36, comprising 1 to 10 w / v% alcohol.

[0581] 38. The formulation is the formulation according to any one of Embodiments 1 to 37, comprising glycerol.

[0582] 39. The formulation is one of the formulations according to any one of Embodiments 1 to 38, comprising 0.5 to 50 w / v% glycerol, 1 to 30 w / v% glycerol, or 1 to 5 w / v% glycerol.

[0583] 40. The formulation is the formulation according to any one of Embodiments 1 to 39, comprising petrolatum.

[0584] 41. The formulation is the formulation according to any one of Embodiments 1 to 40, comprising 0.1 to 20 w / v% petrolatum.

[0585] 42. The formulation according to any one of Embodiments 1 to 41, wherein the formulation is thermally stable at 45°C or 50°C for at least 4 weeks or at least 2 months.

[0586] 43. The formulation according to any one of Embodiments 1 to 42, wherein the formulation is active in a pH range of 6 to 8 and optionally active in a pH range of 5 to 8.

[0587] 44. A method for treating a condition associated with Cutibacterium acnes (C. acnes), comprising administering a composition comprising the recombinant protein or chimeric CWH described in any one of the above embodiments.

[0588] 45. A method for treating a condition associated with Cutibacterium acnes (C. acnes), comprising administering a formulation according to any one of Embodiments 1 to 43.

[0589] 46. ​​The method according to Embodiment 44 or 45, wherein the condition is acne vulgaris.

[0590] 47. The method according to any one of embodiments 44 to 46, wherein the condition is a C. acnes infection.

[0591] 48. A method for restoring phylogenetic diversity of Cutibacterium acnes (C. acnes), comprising administering a composition comprising a recombinant protein or chimeric CWH as described in any one of the embodiments described above.

[0592] 49. A method for restoring the phylogenetic diversity of Cutibacterium acnes (C. acnes), comprising administering a formulation according to any one of Embodiments 1 to 43.

[0593] 50. The method according to any one of embodiments 44 to 49, wherein the condition is related to an excess abundance of C. acnes lineage IA1 and / or IA2.

[0594] 51. The method according to any one of embodiments 44 to 50, wherein the relative abundance of C. acnes lineage IA1 and / or IA2 is reduced by the method.

[0595] 52. The method according to any one of Embodiments 44 to 51, wherein the composition or preparation is administered topically, enterally, or parenterally.

[0596] 53. The method according to any one of Embodiments 44 to 52, wherein the composition or formulation is administered topically.

[0597] 54. The method according to any one of embodiments 44 to 53, further comprising administering an antibiotic.

[0598] 55. The method according to any one of Embodiments 44 to 54, wherein the number and / or size of acne lesions is reduced by the method.

[0599] 56. The method according to any one of Embodiments 44 to 55, wherein skin redness and / or pain is reduced by the method.

[0600] 57. The method according to any one of embodiments 44 to 56, wherein the amount of C. acnes present is reduced by the method.

[0601] 58. A method for identifying a novel CW_7 cell wall binding domain used for binding, targeting, and / or lysis of Cutibacterium acnes, a) A step of searching a gene database using a known CW_7 query sequence; b) A step of identifying sequences that exceed the amino acid sequence identity cutoff for the query sequence; c) A step of cloning a CW_7 sequence or a CW_7-containing CBD sequence into a chimeric cell wall hydrolase in combination with an enzyme activity domain (EAD) to form a CW_7 chimera; and d) Assaying the CW_7 chimera for its binding activity, targeting activity, and / or lysis activity against Cutibacterium acnes. A method that includes this.

[0602] 59. The CW_7 query array is, a) CW_7 sequences disclosed herein; b) CW_7 sequences disclosed in Table 7; or c) CW_7 array derived from CLB1~4 The method according to embodiment 58.

[0603] 60. The method of Embodiment 58, wherein the CW_7 query sequence is a CW_7 sequence derived from CLB2.

[0604] 61.EAD is, a) EADs disclosed herein; b) EAD in Table 2; or c) EAD in Table 4 The method according to any one of embodiments 58 to 60.

[0605] 62. The method according to any one of embodiments 58 to 61, wherein the cutoff is any number selected from the range of 30 to 99%.

[0606] 63. The method according to any one of embodiments 58 to 62, wherein the cutoff is 40%.

Claims

1. Chimeric cell wall hydrolase (CWH), a) CW_7 cell wall binding domain (CBD), b) The enzyme-active domain (EAD) of the CLC1 family and Chimera CWH, including

2. The aforementioned CBD is, a) Sequence ID 47; b) Amino acid sequences selected from the group consisting of SEQ ID NOs: 45, 46, 48, 49, 50, and 51; c) A CW_7 sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 166 to 223; d) A CW_7 sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938; or e) CW_7 sequence composed of amino acid sequences selected from Table 7 The chimeric CWH according to claim 1, having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to the sequence, or comprising a CW_7 amino acid sequence having the sequence.

3. The aforementioned EAD is, a) Sequence ID 35; b) Sequence ID 21; c) An amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 36; d) EAD derived from Sequence IDs 1-19; or e) EAD derived from CLC16, CLC2, CLC1, CLC3, CLC4, CLC5, CLC6, CLC7, CLC8, CLC9, CLC10, CLC11, CLC12, CLC13, CLC14, CLC15, CLC17, CLC18, or CLC19 The chimeric CWH according to claim 1, having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to the sequence, or comprising an amino acid sequence having said sequence.

4. Chimeric cell wall hydrolase (CWH), a) It is a cell wall binding domain (CBD), i) Sequence ID 47; ii) Amino acid sequences selected from the group consisting of Sequence IDs 45, 46, 48, 49, 50, and 51; iii) CW_7 sequences consisting of amino acid sequences selected from the group consisting of SEQ ID NOs: 166-223; iv) A CW_7 sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938; or v) CW_7 sequence composed of amino acid sequences selected from Table 7 A CBD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to the sequence, or a CBD containing a CW_7 amino acid sequence having said sequence; b) Enzyme-active domain (EAD), i) Sequence ID 35; ii) Sequence ID 21; iii) Sequence ID 73; iv) An amino acid sequence selected from the group consisting of SEQ ID NOs: 20-36; v) EADs derived from Sequence IDs 1-19; vi) CLC16, CLC2, CLC1, CLC3, CLC4, CLC5, CLC6, CLC7, CLC8, CLC9, CLC10, CLC11, CLC12, CLC13, CLC14, CLC15, CLC17, CLC18, or CLC19; vii) Sequence ID 64; or viiii) Sequence ID 63 An EAD having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to the sequence, or an EAD containing an amino acid sequence having the aforementioned sequence. Chimeric CWH including

5. The chimeric CWH according to any one of claims 1 to 4, wherein the EAD contains an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 35 or 21, or the EAD contains the amino acid sequence of SEQ ID NO: 35 or 21.

6. The aforementioned chimeric CWH is a) An amino acid sequence selected from the list consisting of SEQ ID NOs. 52 to 59; b) An amino acid sequence selected from the list consisting of SEQ ID NOs: 66-69; c) An amino acid sequence selected from the list consisting of SEQ ID NOs: 94-107; d) an amino acid sequence selected from the list consisting of SEQ ID NOs: 224-281; ​​or e) Amino acid sequence selected from the list consisting of SEQ ID NOs: 2942-2943 A chimeric CWH according to any one of claims 1 to 4, comprising an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to, or comprising the aforementioned amino acid sequence.

7. The aforementioned CBD is, a) Sequence ID 42; b) An amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 43, and 44; c) An amino acid sequence selected from the group consisting of SEQ ID NOs: 166 to 223; d) CBD consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938; or e) CBD composed of amino acid sequences selected from Table 7 A chimeric CWH according to any one of claims 1 to 4, having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to the sequence, or comprising an amino acid sequence having the sequence.

8. The chimeric CWH according to any one of claims 1 to 4, wherein the CBD has at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to SEQ ID NO: 42, or the CBD contains the amino acid sequence of SEQ ID NO:

42.

9. Recombinant proteins containing the enzyme-active domain (EAD) of the CLC1 family.

10. The aforementioned EAD is, a) Whether it originates from CLC1, CLC2, CLC3, CLC4, CLC5, CLC6, CLC7, CLC8, CLC9, CLC10, CLC11, CLC12, CLC13, CLC14, CLC15, CLC16, CLC17, CLC18, or CLC19; b) Is it derived from sequence numbers 1-19? c) an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 20 to 36; or d) The recombinant protein according to claim 9, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 36.

11. The recombinant protein according to claim 9, wherein the EAD contains an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO: 35 or SEQ ID NO: 21, or the EAD contains the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO:

21.

12. The recombinant protein according to any one of claims 9 to 11, wherein the recombinant protein comprises a heterogeneous cell wall binding domain (CBD).

13. The CBD includes a CW_7 repeat, and the CW_7 repeat is a) Sequence ID 47; b) Amino acid sequences selected from the group consisting of SEQ ID NOs: 45, 46, 48, 49, 50, and 51; c) CW_7 repeats consisting of amino acid sequences selected from the group consisting of SEQ ID NOs: 166-223; d) A CW_7 repeat consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938; or e) CW_7 composed of the proteins listed in Table 7 The recombinant protein according to claim 12, having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to the sequence, or comprising an amino acid sequence having said sequence.

14. The aforementioned CBD is, a) Sequence ID 42; b) An amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 43, and 44; c) An amino acid sequence selected from the group consisting of SEQ ID NOs: 166 to 223; d) CBD consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938; or e) CBD composed of amino acid sequences selected from Table 7 The recombinant protein according to claim 12, having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to the sequence, or comprising an amino acid sequence having said sequence.

15. A recombinant protein containing the CW_7 cell wall-binding domain (CBD).

16. The CBD includes a CW_7 sequence, and the CW_7 sequence is a) Sequence ID 47; b) Amino acid sequences selected from the group consisting of SEQ ID NOs: 45, 46, 48, 49, 50, and 51; c) CW_7 repeats consisting of amino acid sequences selected from the group consisting of SEQ ID NOs: 166-223; d) A CW_7 repeat consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938; or e) CW_7 composed of the proteins listed in Table 7 The recombinant protein according to claim 15, having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to the sequence, or comprising an amino acid sequence having the sequence.

17. The aforementioned CBD is a) Sequence ID 42; b) An amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 43, and 44; c) An amino acid sequence selected from the group consisting of SEQ ID NOs: 166 to 223; d) CBD consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 282 to 2938; or e) CBD composed of amino acid sequences selected from Table 7 The recombinant protein according to claim 15, having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to the sequence, or comprising an amino acid sequence having the sequence.

18. The recombinant protein according to any one of claims 15 to 17, wherein the recombinant protein comprises an enzyme-active domain (EAD).

19. The aforementioned EAD is, a) Sequence ID 35; b) Sequence ID 21; c) Sequence ID 73; d) An amino acid sequence selected from the group consisting of SEQ ID NOs: 20-36; e) EADs derived from Sequence IDs 1-19; f) EAD derived from CLC16, CLC2, CLC1, CLC3, CLC4, CLC5, CLC6, CLC7, CLC8, CLC9, CLC10, CLC11, CLC12, CLC13, CLC14, CLC15, CLC17, CLC18, or CLC19; g) Sequence ID 64; or h) Sequence ID 63 The recombinant protein according to claim 18, having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with respect to the sequence, or comprising an amino acid sequence having said sequence.

20. The EAD comprises the EAD motif of the CLC1 family (SEQ ID NO: 2939), as described in any one of claims 1 to 14 and 18 to 19, for a chimeric CWH or recombinant protein.

21. The CBD comprises the CW_7-21 motif (SEQ ID NO: 2940), as described in any one of claims 1 to 8 and 12 to 20, a chimeric CWH or recombinant protein.

22. The CBD comprises the CW_7-19 motif (SEQ ID NO: 2941), as described in any one of claims 1 to 8 and 12 to 20, a chimeric CWH or recombinant protein.

23. The recombinant protein according to any one of claims 9 to 20, wherein the recombinant protein is a chimeric cell wall hydrolase (CWH).

24. The chimeric CWH or recombinant protein according to any one of claims 1 to 8, 12 to 14, and 18 to 23, wherein the CBD is located at the C-terminus of the EAD.

25. The chimeric CWH or recombinant protein according to any one of claims 1 to 24, wherein the chimeric CWH or recombinant protein binds to Cutibacterium acne and / or has lytic activity against Cutibacterium acnes.

26. The chimeric CWH or recombinant protein according to any one of claims 1 to 25, wherein the chimeric CWH or recombinant protein exhibits lytic activity against Cutibacterium acnes, and optionally exhibits lytic activity against strains selected from the list consisting of IA1, IA2, IB, II, and III.

27. The chimeric CWH or recombinant protein according to any one of claims 1 to 26, exhibiting improved solubility and / or anti-Cutibacterium acnes activity compared to natural CaLys1 (SEQ ID NO: 72).

28. The chimeric CWH or recombinant protein according to any one of claims 1 to 27 exhibits higher lytic activity against Cutibacterium acnes compared to the natural protein from which each domain originates.

29. The chimeric CWH or recombinant protein according to any one of claims 1 to 28, wherein the chimeric CWH or recombinant protein exhibits minimal lytic activity toward Corynebacterium xerosis, Corynebacterium striatum, and / or Staphylococcus epidermidis, or does not exhibit such lytic activity.

30. The chimeric CWH or recombinant protein according to any one of claims 1 to 28, wherein the chimeric CWH or recombinant protein has solubility against Cutibacterium acnes over a pH range of 4.2 to 8.0, and optionally exhibits maximum C. acnes solubility at a pH of less than 7.

31. The chimeric CWH or recombinant protein according to any one of claims 1 to 28, wherein the chimeric CWH or recombinant protein exhibits maximum C. acnes solubility at a pH of 5.5–6.5, 5.8–6.2, 5.9–6.1, or about 6.

0.

32. The chimeric CWH or recombinant protein according to any one of claims 1 to 28, wherein the chimeric CWH or recombinant protein retains at least 50% of the activity of the chimeric CWH or recombinant protein at 25°C after exposure to a maximum temperature of 45°C, 50°C, 55°C, or 58°C for 30 minutes.

33. The chimeric CWH or recombinant protein according to any one of claims 1 to 28, comprising an EAD having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to CLC16 EAD (SEQ ID NO: 35), or comprising CLC16 EAD (SEQ ID NO: 35), and retaining at least 50% of its anti-Cutibacterium acnes activity at 25°C after exposure to a maximum temperature of 75°C for 30 minutes, and optionally retaining at least 40% of its anti-Cutibacterium acnes activity at 25°C after exposure to a maximum temperature of 90°C for 30 minutes.

34. The solubility of the chimeric CWH or recombinant protein is at least twice, at least five times, at least ten times, or at least 100 times higher than that of CaLys1 (SEQ ID NO: 72), according to any one of claims 1 to 28.

35. Enzymatically active recombinant CLC1 family protein or CaLys1 protein with a truncated C-terminus.

36. The aforementioned truncation is a) The conserved C-terminal region of the full-length native CLC1 family protein sequence; b) The entire C-terminal region of the CLC1 family protein following the enzyme-active domain of the full-length native CLC1 family protein sequence; c) Residue numbers 195-205 and all subsequent residues derived from the amino acid sequence of the full-length native CLC1 family protein; or d) Approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acids at the C-terminus of the full-length native CLC1 family protein. The truncated protein according to claim 35, which is a truncation of the protein.

37. The truncated protein according to claim 35 or 36, wherein the CLC1 family protein is selected from CLC1 to CLC19 (SEQ ID NOs: 1 to 19).

38. The CLC1 family protein is CLC1, CLC2, CLC3, CLC4, CLC5, CLC6, CLC7, CLC8, CLC9, CLC10, CLC11, CLC12, CLC13, CLC14, CLC16, CLC18, or CLC19, or the protein is CaLys1, and the C-terminal truncation is a) A truncation of approximately 70 to 90 amino acids compared to the full-length natural protein sequence; or b) A truncation of approximately 80, 81, or 82 amino acids compared to the full-length natural protein sequence, The truncated protein according to any one of claims 35 to 37.

39. The CLC1 family protein is CLC15, and the C-terminal truncation is a) A truncation of approximately 115 to 135 amino acids compared to the full-length natural CLC1 family protein sequence; or b) A truncation of approximately 123 amino acids compared to the full-length natural CLC1 family protein sequence, The truncated protein according to any one of claims 35 to 37.

40. The CLC1 family protein is CLC17, and the C-terminal truncation is a) A truncation of approximately 55 to 75 amino acids compared to the full-length natural CLC1 family protein sequence; or b) A truncation of approximately 66 amino acids compared to the full-length natural CLC1 family protein sequence, The truncated protein according to any one of claims 35 to 37.

41. The truncated protein according to any one of claims 35 to 40, wherein the truncated protein exhibits higher solubility and / or solubility compared to the corresponding full-length native protein sequence.

42. The truncated protein is a) Amino acid sequences having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to an amino acid sequence selected from the group consisting of Sequence IDs 74 to 93, b) An amino acid sequence selected from the group consisting of SEQ ID NOs: 74-93; c) an amino acid sequence having at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-36 and 73; or d) Amino acid sequence selected from the group consisting of SEQ ID NOs: 20-36 and 73 A truncated protein according to any one of claims 35 to 41, comprising:

43. A formulation comprising a chimeric CWH, recombinant protein, or truncated protein as described in any one of the embodiments described above, wherein the formulation is optionally a topical formulation.

44. The formulation according to claim 43, wherein the formulation is a hydrogel, lotion, cream, gel-cream, colloidal patch, or microneedle patch, and optionally the formulation is a hydrogel.

45. The aforementioned formulation is a) Wetters optionally selected from the list consisting of aloe vera, betaine, butylene glycol, caprylyl glycol, dimethicone, fructose, glucomannan, glucose, glycerin, glyceryl glucoside, honey, hyaluronic acid, lactic acid, panthenol, polyethylene glycol, propylene glycol, propanediol, sodium hyaluronate, sodium lactate, sodium pyrrolidone carboxylate, sorbitol, and urea; b) Cellulose polymers, optionally selected from the list consisting of hydroxyethylcellulose, methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, microcrystalline cellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, and cellulose acetate; c) Salts, optionally selected from the list consisting of calcium chloride, Dead Sea salt, Epsom salt, Himalayan pink salt, magnesium chloride, sea salt, and sodium chloride; d) A buffer solution optionally selected from the list consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, acetic acid, ammonium acetate, boric acid, citric acid, glycine, phosphoric acid, potassium hydroxide, potassium phosphate, sodium acetate, sodium bicarbonate, sodium borate, sodium carbonate, sodium citrate, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium hydroxide, sodium phosphate, sodium tetraborate, tris(hydroxymethyl)aminomethane, and trisodium phosphate; e) A surfactant optionally selected from the list consisting of ceteareth-20, cocamidopropyl betaine, cocoglucoside, decyl glucoside, decyl polyglucose, disodium laureth sulfosuccinate, glycereth-26, lauryl glucoside, lauryl polyglucose, sodium cocoyl glutamate, sodium cocoyl isethionate, sodium laureth sulfate, and sodium lauryl sulfate; f) Free amino acids, optionally selected from the list consisting of alanine, arginine, cysteine, glutamine, glycine, histidine, lysine, methionine, proline, serine, and threonine; g) Oils, optionally selected from the list consisting of argan oil, avocado oil, baobab oil, camellia oil, carrot seed oil, coconut oil, evening primrose oil, grape seed oil, hemp seed oil, jojoba oil, macadamia nut oil, marula oil, mineral oil, olive oil, pomegranate seed oil, raspberry seed oil, rosehip seed oil, squalane oil, sunflower seed oil, sweet almond oil, and tamanu oil; h) an alcohol, optionally selected from the list consisting of cetyl alcohol, ethyl alcohol, isopropyl alcohol, and stearyl alcohol; i) Glycerol; and / or j) Vaseline The formulation according to claim 43 or 44, including the formulation described in claim 43 or 44.

46. The aforementioned formulation is a) A humectant in a concentration of 0.1 to 50 w / v%, with an optional concentration of 0.5 to 10 w / v; b) A cellulose polymer in an amount of 0.5 to 10 w / v%, optionally comprising 1 to 5 w / v% of a cellulose polymer; c) A salt with a concentration of 10 to 500 mM, optionally a salt with a concentration of 50 to 250 mM; d) 5-50 mM buffer solution; e) A surfactant in an amount of 0.1 to 20 w / v%, wherein optionally a surfactant in an amount of 1 to 10 w / v; f) Free amino acids in concentrations of 10–250 mM; g) 0.1 to 20 w / v% oil; h) an alcohol in a concentration of 0.1 to 20 w / v%, optionally including an alcohol in a concentration of 1 to 10 w / v%; i) 0.5 to 50 w / v% glycerol, 1 to 30 w / v% glycerol, or 1 to 5 w / v% glycerol; and / or j) 0.1-20 w / v% petrolatum A formulation according to any one of claims 43 to 45, including the formulation described in any one of claims 43 to 45.

47. The formulation according to any one of claims 43 to 46, wherein the formulation is thermally stable at 45°C or 50°C for at least four weeks or at least two months, and / or the formulation is active in a pH range of 6 to 8, and optionally active in a pH range of 5 to 8.

48. A method for treating a condition associated with Cutibacterium acnes (C. acnes), comprising administering a composition comprising a recombinant protein, chimeric CWH, or truncated protein as described in any one of claims 1 to 42, or a formulation as described in any one of claims 43 to 47.

49. The method according to claim 48, wherein the condition is associated with acne vulgaris, C. acnes infection, or an excess of C. acnes.

50. A method for restoring phylogenetic diversity of Cutibacterium acnes, comprising administering a composition comprising a recombinant protein, chimeric CWH, or truncated protein according to any one of claims 1 to 42, or a formulation according to any one of claims 43 to 47.

51. The method according to any one of claims 48 to 50, wherein the aforementioned state is related to an excess abundance of C. acnes lineage IA1 and / or IA2, and optionally, the relative abundance of C. acnes lineage IA1 and / or IA2 is reduced by the method.

52. The method according to any one of claims 48 to 51, wherein the composition or formulation is administered topically, enterally, or parenterally, and optionally, the composition or formulation is administered topically.

53. The method according to any one of claims 44 to 49, wherein the number, severity, and / or size of acne lesions are reduced, and / or skin redness and / or pain are reduced.

54. A method for identifying a novel CW_7 cell wall binding domain for use in binding, targeting, and / or lysis of Cutibacterium acnes, a) A step of searching a gene database using a known CW_7 query sequence; b) A step of identifying sequences that exceed the amino acid sequence identity cutoff for the query sequence; c) A step of cloning a CW_7 sequence or a CW_7-containing CBD sequence into a chimeric cell wall hydrolase in combination with an enzyme activity domain (EAD) to form a CW_7 chimera; and d) The step of assaying the CW_7 chimera for its binding activity, targeting activity, and / or lysis activity against Cutibacterium acnes. Methods that include...

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