Peptidoglycan hydrolases with bactericidal activity

Abstract

The present invention is generally in the field of pharmacology, particularly antibacterial agents and protein engineering. In particular, the present invention relates to peptidoglycan hydrolases, such as endolysins, and nucleic acids, e.g., RNA, encoding the peptidoglycan hydrolases of the present invention, and their medical uses, for example, to treat diseases caused by and / or associated with Staphylococcus (e.g., S. aureus) infections. Furthermore, the present invention relates to solidified yeast culture media and corresponding screening methods for screening yeast cells for the secretion of peptidoglycan hydrolases with bactericidal activity.

Description

[Technical Field]

[0001] The present invention is generally in the field of pharmacology, particularly antibacterial agents and protein engineering. In particular, the present invention relates to peptidoglycan hydrolases, such as endolysins, and nucleic acids, e.g., RNA, encoding the peptidoglycan hydrolases of the present invention, and their medical uses, for example, to treat diseases caused by and / or associated with Staphylococcus (e.g., S. aureus) infections. Furthermore, the present invention relates to solidified yeast culture media and corresponding screening methods for screening yeast cells for the secretion of peptidoglycan hydrolases with bactericidal activity. [Background technology]

[0002] Staphylococci, particularly strains of Staphylococcus aureus, are major human pathogens responsible for a wide range of acute and chronic conditions, ranging from mild to life-threatening, including skin and soft tissue infections, bone-related infections, pneumonia, and sepsis. For example, S. aureus is the leading cause of death among antibiotic-resistant bacterial pathogens, with approximately 700,000 deaths worldwide each year due to antibiotic-resistant S. aureus (Antimicrobial Resistance Collaborators (2022) The Lancet, 399). The pathogenesis of S. aureus infection involves several key steps: invasion of host tissues, evasion of the immune system, adhesion to surfaces, and biofilm formation. For example, by persisting in biofilms, bacteria evade killing by neutrophils and exhibit reduced susceptibility to antibiotics. Despite decades of research and promising preclinical data, no vaccine against S. aureus is available.

[0003] Bacteriophage-encoded peptidoglycan hydrolases, such as endolysins (belonging to the class of "enzybiotics"), are promising alternatives to antibiotics; Fischetti (2010), International Journal of Medical Microbiology, 300(6); Schmelcher (2012), Future Microbiology, 7; Hojckova (2013), BMC Microbiol. 13. Bacteriophages produce these enzymes, especially endolysins, toward the end of their lytic cycle. The enzymes cleave the peptidoglycan (PG) of the bacterial cell wall, thus lysing the cell and releasing progeny phages. Peptidoglycan hydrolases, especially endolysins (also abbreviated as "lysins"), have several advantages over other antibiotics; in particular, their narrow host specificity, often restricted to a single genus or even a single species (Fischetti (2010), International Journal of Medical Microbiology, 300(6)), and their rather low tendency to generate resistance in their hosts (Schuch (2014), The Journal of Infectious Diseases, 209(9)). Bacteriophages that invade Gram-positive bacteria encode a very wide variety of endolysins. Typically, endolysins have a modular structure consisting of one or more enzymatically active domains (EADs) connected to at least one cell wall-binding domain (CBD) by a flexible interdomain linker. Both domains may contribute to the specificity for a given genus or species of bacteria; Oliveira (2013), J. Virol. 87. However, bacteriophage-derived endolysins (without a separate CBD) also exist that contain only enzymatically active domains, such as the cysteine, histidine-dependent amidohydrolase / peptidase (CHAP) domain. Such lysins specifically disrupt peptidoglycan from the exterior of bacterial cells.

[0004] Intravenous delivery of CF-301 (ContraFect, also known as exebacase), a natural (i.e., naturally occurring) phage lysin that targets Staphylococcus aureus, demonstrated therapeutic benefit in methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections in a phase II clinical trial (Fowler (2020), J. Clin. Invest., 130(7)), but the results could not be replicated in a phase III clinical trial. Another natural endolysin, SAL200, when administered intravenously to patients with persistent S. aureus bacteremia in a phase II clinical trial, resulted in severe adverse effects, including pneumonia and respiratory failure (NCT03089697; Danis-Wlodarczyk (2021), Antibiotics, 10(12)); moreover, the very short bactericidal half-life and immune response to the enzyme were thought to limit its usefulness (WHO technical document, January 15, 2022: 2019 antibacterial agents in clinical development: an analysis of the antibacterial clinical development pipeline, https: / / www.who.int / publications / i / item / 9789240000193).

[0005] On the other hand, local delivery (e.g., intranasally and / or to topical surfaces) of certain peptidoglycan hydrolases, such as ectolysin P128 and endolysin SA.100 (Danis-Wlodarczyk (2021), Antibiotics, 10(12)) and lysostaphin (Jayakumar (2020), J. Appl. Microbiol.), has shown some success in eradicating S. aureus. However, exposure of S. aureus to the glycylglycine endopeptidase lysostaphin has been shown to rapidly generate resistance both in vitro and in vivo due to loss-of-function mutations in femA, which are required for the incorporation of the second and third glycines into the cross-bridge (Climo (2001), Antimicrobial Agents and Chemotherapy, 45(5)).

[0006] Given the caveats regarding natural phage endolysins, attempts have been made to improve their drug-like properties through protein engineering; De Maesschalck V (2020) Crit Rev Microbiol, 46(5). The modular architecture of endolysins has served as the basis for engineering lysins through domain shuffling, truncation, and random and / or site-directed mutagenesis; Gerstmans (2020), Sci Adv. 6(23). To date, lysin optimization has focused on optimizing either functional activity (Gerstmans (2020), Sci Adv. 6(23)) or stability (Ritter (2019), Appl Environ Microbiol., 85(10)). Furthermore, only modest improvements have been achieved in terms of stability, i.e., the melting temperature of the best engineered variants increased by 4°C. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2007 / 036366

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[0008] [Non-licensed document 1] Antimicrobial Resistance Collaborators (2022), The Lancet, 399 [Non-licensed document 2] Fischetti (2010), International Journal of Medical Microbiology, 300(6) [Non-licensed document 3] Schmelcher (2012), Future Microbiology, 7

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[0009] Therefore, there is a need for improved antibacterial agents, in particular peptidoglycan hydrolases, with improved pharmaceutical properties, as well as improved means and methods for producing them. [Means for solving the problem]

[0010] Thus, the present invention relates to a peptidoglycan hydrolase having bactericidal activity, the peptidoglycan hydrolase comprising a cysteine, histidine-dependent amidohydrolase / peptidase (CHAP) domain that (i) has at least 60% sequence identity with the amino acid sequence of positions 72 to 215 of SEQ ID NO:1; and (ii) has one or more amino acid substitutions compared to the amino acid sequence of positions 72 to 215 of SEQ ID NO:1.

[0011] As described in detail below, the CHAP domain is considered herein, and in the context of the present invention, to be a variant of the CHAP domain of L0482, ie, a variant of the sequence from positions 72 to 215 of SEQ ID NO:1.

[0012] Preferably, the peptidoglycan hydrolase of the present invention has killing activity against Staphylococcus species or strains, preferably Staphylococcus aureus. Furthermore, the peptidoglycan hydrolase of the present invention is preferably capable of being secreted from eukaryotic cells. Furthermore, the peptidoglycan hydrolase of the present invention is preferably stable up to a temperature of at least about 40°C, for example, at least 37°C. Furthermore, the peptidoglycan hydrolase of the present invention is preferably an endolysin.

[0013] In the context of the present invention, the peptidoglycan hydrolase of the present invention is preferably contained in a pharmaceutical composition and / or is preferably used for the treatment of a disease, wherein the disease is particularly a bacterial disease, preferably a disease caused by and / or associated with a staphylococcal infection, such as a Staphylococcus aureus infection.

[0014] Selection of LYSM-CHAP domain constructs as starting points for further protein engineering and directed evolution The present invention is based, in part, on the surprising finding that lysins having a LYSM-CHAP domain structure, such as L0482 (SEQ ID NO: 1), reliably possess good killing activity against Staphylococcus, particularly Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA) strains such as ATCC 43300, as illustrated in the accompanying Examples. Lysins having a LYSM-CHAP domain structure, such as L0482, therefore provide particularly good starting points for protein engineering and directed evolution approaches. In particular, in the context of the present invention, it has been surprisingly found that 12 of 13 lysins (i.e., >92%) having a LYSM-CHAP domain structure effectively killed Staphylococcus aureus cells, while lysins having other domain structures often did not exhibit appreciable killing activity against Staphylococcus aureus; see, e.g., Example 1 and Figure 1. Moreover, it has been found that a representative of these LYSM-CHAP lysins, i.e., L0482 (SEQ ID NO: 1), had good killing activity against many different Staphylococcus aureus strains, as well as against other Staphylococcus species such as S. warneri and S. capitis, and coagulase-negative Staphylococcus species such as S. epidermidis; see, e.g., Example 1 and Figure 2. Surprisingly, the killing activity against the tested Staphylococcus species and strains was even increased compared to one of the most clinically developed lysins, i.e., exevacase (CF-301, also known as ContraFect, or referred to herein as "L0466"), which has a CHAP-SH3 domain structure; see Figure 2. Notably, the CHAP domain of LYSM-CHAP lysins such as L0482 is significantly different from the CHAP domain of exevacase. In particular, the sequence identity of the CHAP domain of execase (ie, the sequence from positions 19 to 164 of SEQ ID NO: 304) to the CHAP domain of L0482 (ie, the sequence from positions 72 to 215 of SEQ ID NO: 1) is only about 20%.

[0015] Furthermore, in the context of the present invention, it has been found that LysM-CHAP lysins, such as L0482 (SEQ ID NO: 1), can be secreted in an active form by eukaryotic cells to kill Staphylococcus aureus; see, e.g., Example 2 and FIG. 3. Nevertheless, the present inventors have also observed that the stability and solubility of wild-type (WT) L0482 (SEQ ID NO: 1), as well as its secretion from and killing activity upon secretion from eukaryotic cells, are still suboptimal; see, e.g., Example 4 and FIG. 5, Example 5 and FIG. 6, and Example 12 and FIG. 10. However, high killing efficiency against target bacteria (e.g., Staphylococcus aureus), good solubility in aqueous solution, and high stability (including sufficient thermostability and low aggregation tendency) are important properties of antibacterial compounds, particularly antibacterial enzymes such as peptidoglycan hydrolases. These pharmaceutical properties are particularly important for the treatment of bacterial infections in mammals, e.g., humans, as described herein. Furthermore, administering peptidoglycan hydrolase to cells of a subject (e.g., human) in the form of a nucleic acid (e.g., mRNA) encoding the peptidoglycan hydrolase has the advantage of continuous production, i.e., secretion, of the peptidoglycan hydrolase from the subject's cells, particularly at the site of bacterial infection. Thus, the ability to be efficiently secreted from eukaryotic cells, e.g., human cells, is another beneficial property of peptidoglycan hydrolases.

[0016] Briefly, the inventors have surprisingly found that L0482 (SEQ ID NO: 1) is an optimal starting point for further protein engineering and directed evolution.

[0017] Thus, the present invention further relates to a peptidoglycan hydrolase having bactericidal activity, which has at least 60% sequence identity with the sequence of SEQ ID NO: 1 and which comprises one or more amino acid substitutions compared to the sequence of SEQ ID NO: 1. This sequence identity is calculated specifically over the entire length of the sequence of SEQ ID NO: 1 (not over the entire length of the sequence of the peptidoglycan hydrolase of the present invention). Thus, any additional domains, peptides, or tags, such as a signal peptide or PK tag, that may be included in (or fused to) the peptidoglycan hydrolase of the present invention should not be taken into account when determining the sequence identity of the peptidoglycan hydrolase of the present invention to the sequence of SEQ ID NO: 1. Thus, the peptidoglycan hydrolase of the present invention may comprise an amino acid sequence that has at least 60% sequence identity with the sequence of SEQ ID NO: 1, and optionally one or more additional domains, peptides, or tags described herein, such as a signal peptide, PK tag, additional peptide linker, etc.

[0018] Preferably, the peptidoglycan hydrolase of the present invention comprises a CHAP domain according to the present invention, i.e., a CHAP domain that (i) has at least 60% sequence identity with the amino acid sequence of positions 72 to 215 of SEQ ID NO: 1; and (ii) has one or more amino acid substitutions compared to the amino acid sequence of positions 72 to 215 of SEQ ID NO: 1.

[0019] L0482 variants with improved pharmaceutical properties (brief overview) Through protein engineering as exemplified in Example 4 and subsequent directed evolution as exemplified in Example 5, the present inventors have surprisingly discovered L0482 variants with simultaneously improved pharmaceutical properties. In particular, the present inventors have discovered L0482 variants with enhanced killing activity against Staphylococcus aureus, enhanced protein stability, and enhanced secretion ability from human cells, as exemplified, for example, in Example 6 and Figure 8.

[0020] Enhanced killing activity As described in more detail herein below and also illustrated in the accompanying examples, enhanced killing activity against target bacteria, e.g., Staphylococcus aureus, corresponds to a lower minimum inhibitory concentration (MIC). In particular, herein and in the context of the present invention, an MIC is defined as a concentration of 5×10 5 A peptidoglycan hydrolase activity is defined as the minimum concentration that maintains the optical density at 620 nm (OD620) of a bacterial (e.g., Staphylococcus aureus) liquid culture containing cfu / ml of bacterial (e.g., Staphylococcus aureus) cells below 0.1 for at least 24 hours at 37°C. Thus, the peptidoglycan hydrolases of the present invention provided herein can be used at lower concentrations than comparable peptidoglycan hydrolases for the treatment of bacterial diseases, which may increase the efficacy and safety of the treatment. For example, the hit variants G1 (SEQ ID NO: 3) and H5 (SEQ ID NO: 11) found in the context of the present invention have MICs that are 4-fold and 8-fold lower than WT L0482 (SEQ ID NO: 1), respectively, i.e., the MIC for G1 is 1 μg / ml and the MIC for H5 is 0.5 μg / ml, compared to the MIC of 4 μg / ml for WT L0482; see, e.g., Figure 8. In particular, lower MICs were observed for the methicillin-resistant Staphylococcus aureus (MRSA) strain "ATCC43300," further highlighting that the peptidoglycan hydrolases of the present invention may be particularly suitable as antibacterial agents, particularly for medical use.

[0021] The enhanced killing activity of peptidoglycan hydrolases according to the present invention was further confirmed in OD reduction assays showing, for example, that H5 (SEQ ID NO: 11) killed Staphylococcus aureus cells with faster kinetics than wild-type L0482 (SEQ ID NO: 1); see, e.g., Figure 11.

[0022] Enhanced protein stability Enhanced protein stability, e.g., enhanced thermostability and / or reduced aggregation tendency, further improves the pharmaceutical properties of peptidoglycan hydrolases. For example, sufficient thermostability, particularly the ability to be stable up to temperatures of at least about 40°C, e.g., at least 37°C, is crucial for use as a pharmaceutical in many mammals, including humans (considering body temperature). Furthermore, enhanced thermostability facilitates storage and distribution of the pharmaceutical. Thus, the peptidoglycan hydrolases of the present invention provided herein may be particularly suitable as pharmaceuticals. Furthermore, a reduced aggregation tendency enhances the manufacturability of the peptidoglycan hydrolase. Thus, the peptidoglycan hydrolases of the present invention provided herein may be produced more efficiently than comparable peptidoglycan hydrolases. Moreover, a reduced aggregation tendency further reduces the immunogenicity of the peptidoglycan hydrolase in mammalian subjects, e.g., humans, which in turn may further increase the efficacy and / or safety of treatment.

[0023] Enhancement of secretion capacity from human cells Enhanced secretion capacity from human cells is particularly important for expression of peptidoglycan hydrolases from nucleic acids, e.g., RNA. As described herein, when a suitable nucleic acid construct encoding the peptidoglycan hydrolase of the present invention is introduced into cells of a subject (e.g., a human patient), the cells can continuously produce and secrete the peptidoglycan hydrolase protein. This provides a more efficient treatment of bacterial infections and provides greater efficacy in treating hard-to-treat bacterial infections, such as bacterial biofilms. Thus, the peptidoglycan hydrolases of the present invention may be particularly suitable for delivery as nucleic acids, e.g., RNA. Furthermore, the present invention provides more efficient means for treating bacterial infections, such as nucleic acids encoding the peptidoglycan hydrolases of the present invention.

[0024] Enhanced solubility Enhanced solubility (e.g., reduced aggregation tendency) and enhanced secretion ability from human cells may be related to each other and to enhanced solubility in aqueous solutions such as PBS. Indeed, in the context of the present invention, it has been observed that many of the generated L0482 variants found to have enhanced stability and enhanced secretion ability from human cells also exhibited enhanced solubility in PBS. Thus, the peptidoglycan hydrolases of the present invention provided herein may have better solubility in aqueous solutions than comparable peptidoglycan hydrolases. This is particularly beneficial for the manufacturability and usability of peptidoglycan hydrolases. Furthermore, enhanced solubility in aqueous solutions may increase the efficiency and / or safety of peptidoglycan hydrolases in treating diseases.

[0025] Reduced tendency to develop resistance The CHAP domain (contained in L0482 and L0482 variants described herein) may have dual enzymatic activity, i.e., amidase activity and peptidase activity; Frankel (2012), J Biol Chem. 23;287(13). Both amidase activity and peptidase activity may contribute to the hydrolysis / cleavage of peptidoglycan in bacterial cell walls, as described herein. Thus, peptidoglycan hydrolases of the present invention containing the CHAP domain of the present invention may have a reduced tendency to develop resistance in target bacteria, such as Staphylococcus aureus, compared to other peptidoglycan hydrolases, such as lysostaphin. Thus, the peptidoglycan hydrolases of the present invention may be particularly effective for treating bacterial infections for this additional reason.

[0026] Consensus mutation / variant H3 The inventors unexpectedly found an L0482 variant, namely H3 (SEQ ID NO: 9), that (i) possesses all of the beneficial properties assayed (i.e., enhanced killing activity against Staphylococcus aureus, enhanced stability, and enhanced secretion from mammalian cells) compared to the parent L0482 variant L0482ag (SEQ ID NO: 2), and (ii) contains only the amino acid substitutions, namely G1-G4, H1-H10, and I1-I30, that appear to be the most beneficial substitutions because they were consistently observed (in slightly different combinations) among the best hits obtained through three rounds of directed evolution; see, e.g., Examples 6 and 7 and Figures 8 and 9.

[0027] H3 (SEQ ID NO: 9) was derived by removing two glycosylation sites in L0482 (SEQ ID NO: 1) to generate the deglycosylated variant L0482ag (SEQ ID NO: 2) (see Example 4), followed by two rounds of directed evolution starting from L0482ag; see Example 5. H3 (SEQ ID NO: 9) contains the two deglycosylation mutations of L0482ag, i.e., N68K and N73G, as well as only the following mutations, i.e., amino acid substitutions, with reference to the wild-type L0482 sequence of SEQ ID NO: 1: T82S, N85G, R86K, S130N, H136K, D169N, N185Y, and N186G.

[0028] Furthermore, it was found that some of the hit variants identified during directed evolution of L0482ag (SEQ ID NO: 2) contained the amino acid substitution H136R rather than H136K, which is therefore considered herein and in the context of the present invention to be a very good alternative to H136K.

[0029] Thus, H3 (SEQ ID NO: 9) reflects a consensus sequence for particularly improved L0482 variants, and with reference to the wild-type L0482 sequence of SEQ ID NO: 1, the corresponding amino acid substitutions, i.e., T82S, N85G, R86K, S130N, H136K / R, D169N, N185Y, and N186G, are considered herein to be consensus mutations, particularly consensus amino acid substitutions.

[0030] Most beneficial amino acid substitutions / variants H5 Furthermore, it has surprisingly been found in the context of the present invention that the L0482 variant H5 (SEQ ID NO: 11), obtained by the same method as H3, had even more enhanced stability, killing activity against Staphylococcus aureus, and secretion ability from human cells compared to H3 (SEQ ID NO: 9); see, e.g., Example 6. Surprisingly, the only difference between H5 (SEQ ID NO: 11) and H3 (SEQ ID NO: 9) was an additional amino acid substitution in H5, namely F155Y. This further demonstrates that a single amino acid substitution, e.g., F155Y, found in the context of the present invention can simultaneously improve several pharmaceutical properties of L0482 as described herein.

[0031] Therefore, the amino acid substitutions T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y, and N186G in SEQ ID NO: 1 are considered herein, and in the context of the present invention, to be the most beneficial amino acid substitutions for enhancing the killing activity of L0482-derived peptidoglycan hydrolase (L0482 variant) against target bacteria, e.g., Staphylococcus aureus, and / or other pharmaceutical properties (e.g., stability, solubility, and secretion from human cells).

[0032] Moreover, it should be emphasized that, surprisingly, in the context of the present invention, all of the most beneficial amino acid substitutions were found to have occurred in the CHAP domain of L0482, i.e., positions 72 to 215 of SEQ ID NO: 1. Therefore, in the present specification and in the context of the present invention, the CHAP domain is considered to be the most important domain of the L0482-derived peptidoglycan hydrolase.

[0033] Therefore, the present invention further provides a peptidoglycan hydrolase, particularly having bactericidal activity, having (i) at least 60% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:1, and (ii) one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185, and 186 of SEQ ID NO:1, or positions corresponding to these positions, the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; the amino acid residue at position 130 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding thereto is substituted with lysine or arginine, preferably lysine; the amino acid residue at position 155 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 is substituted with tyrosine, and / or the amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; The present invention relates to a peptidoglycan hydrolase comprising a CHAP domain having one or more amino acid substitutions.

[0034] Preferably, the CHAP domain further comprises a deglycosylation mutation as described herein, i.e., an amino acid substitution or deletion, preferably an amino acid substitution, at or corresponding to position 73 of SEQ ID NO:1, wherein the residue at or corresponding to position 73 of SEQ ID NO:1 is preferably substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, preferably glycine.

[0035] The surprising discovery of L0482 variants (e.g., H3 and H5) with simultaneously improved pharmaceutical properties is based, in part, on improved protein engineering and directed evolution tools and methods developed in the context of the present invention and described herein.

[0036] Means and methods for screening peptidoglycan hydrolases for bactericidal activity As illustrated in the accompanying examples, the present inventors have surprisingly found that solidified yeast culture media, such as agar plates containing autoclaved (i.e., dead) target bacteria, e.g., dead Staphylococcus aureus cells, are particularly suitable for screening yeast cells secreting active peptidoglycan hydrolases, i.e., for identifying peptidoglycan hydrolases with good killing activity against target bacteria, e.g., Staphylococcus aureus. A corresponding screening method, termed "Yeast on dead aureus" (YODA), is based on the inventive concept that only active (but not inactive) peptidoglycan hydrolases secreted from yeast colonies cultured on the inventive solidified yeast culture media can destroy peptidoglycan in dead bacterial cells near the colony. Therefore, only secreted active peptidoglycan hydrolases locally clear the turbid culture medium, thereby generating a halo around the colonies secreting the peptidoglycan hydrolases; see Example 3 and Figure 4. The YODA-derived method is a very simple and efficient method that allows yeast cells / colonies expressing peptidoglycan hydrolases with good killing activity against target bacteria to be easily distinguished from yeast cells / colonies expressing inactive peptidoglycan hydrolases. Furthermore, the YODA-derived method is highly sensitive because lysin is continuously secreted from the cells. Thus, a low secretion rate may be sufficient to see a halo (if the peptidoglycan hydrolase is active). Furthermore, as described herein, for example, in Example 3, the YODA method described herein or its derivatives (e.g., YODB) have additional advantages over prior art screening methods, such as the double agar layer (DAL) assay described in Zhao (2014). Appl Environ Microbiol 80(9).

[0037] Therefore, the present invention further relates to a solidified yeast culture medium comprising substrate particles mixed with the solidified yeast culture medium, wherein said substrate particles comprise killed bacterial cells and / or fragments thereof, and / or peptidoglycan particles.

[0038] The present invention further provides a method for screening yeast cells for secretion of an active peptidoglycan hydrolase (i.e., a peptidoglycan hydrolase having bactericidal activity against a target bacterium, e.g., Staphylococcus aureus), comprising: a) providing a solidified yeast culture medium comprising substrate particles mixed with the solidified yeast culture medium, wherein the substrate particles comprise killed bacterial cells and / or fragments thereof, and / or peptidoglycan particles; b) culturing yeast cells expressing peptidoglycan hydrolase on the surface of said solidified medium until at least one yeast colony is detectable, in particular the yeast cells being capable of secreting peptidoglycan hydrolase; c) assessing whether a halo is visible around the yeast colony, in particular a halo corresponding to a localized decrease in absorbance of the solidified medium around the colony, for example in a radius of about 0.1 to 1 cm from the colony, in particular when compared to an area of ​​the solidified medium that does not contain a yeast colony; d) determining that the yeast colony secretes active peptidoglycan hydrolase if a halo is visible around the colony, or determining that the yeast colony does not secrete active peptidoglycan hydrolase if no halo is visible around the colony. The present invention relates to a method comprising:

[0039] As illustrated in the accompanying Examples, the YODA-derived screening methods of the invention provided herein have proven highly useful for identifying improved peptidoglycan hydrolases having bactericidal activity against, for example, Staphylococcus aureus; see, e.g., Examples 4-6.

[0040] Deglycosylation mutations enhance bactericidal activity when expressed in eukaryotic cells As shown in Example 4 and Figure 5A, in the context of the present invention, WT L0482 (SEQ ID NO: 1) has been found to have good killing activity against Staphylococcus aureus when produced in Escherichia coli (E. coli), i.e., a minimum inhibitory concentration (MIC) of 4 μg / ml, but only moderate killing activity (i.e., an MIC of 352 μg / ml) when secreted from the yeast Pichia pastoris (P. pastoris).

[0041] We reasoned that N- and O-glycosylation of WT L0482 during its maturation in the secretory pathway in eukaryotic cells negatively affected its bactericidal activity. In particular, we speculated that a transgene such as lysin could be glycosylated at sites essential for folding and activity, potentially resulting in the secretion of a less active protein (although secreted proteins are usually evolutionarily adapted to these modifications).

[0042] We found that L0482 (SEQ ID NO: 1) contains two N-glycosylation motifs at residues N68 (which reside in the linker sequence) and N73 (which reside in the CHAP domain). Therefore, we removed the two N-glycosylation motifs in L0482 in a degenerate codon screen by using the YODA method, as described in Example 4 and Figure 5B.

[0043] The present inventors surprisingly found that the most frequent amino acid substitution pairs contained in active L0482 mutants were N68K / N73G (i.e., N68N73 to KG), N68K / N73Y (i.e., N68N73 to KY), and N68A / N73H (i.e., N68N73 to AH). All three L0482 mutants (i.e., with N68K / N73G, N68K / N73Y, and / or N68A / N73H substitutions, respectively) were confirmed to be deglycosylated; see, e.g., Example 4 and Figure 5C. In addition, further mutation pairs from N68N73 to ML, RE, KL, and AA were also found in L0482 mutants active by YODA.

[0044] Moreover, it has surprisingly been found by the inventors that the deglycosylated N68K / N73G (i.e., KG) mutant, also referred to herein as L0482ag, had killing activity against Staphylococcus aureus nearly as good as WT L0482 when produced in E. coli; see, e.g., Example 4 and Figure 5C.

[0045] Thus, the present invention further relates to deglycosylated variants of L0482 (SEQ ID NO: 1), exemplified by, but not limited to, the N68K / N73G (i.e., L0482ag; SEQ ID NO: 2) mutant described herein. As used herein and in the context of the present invention, for example in the context of a peptidoglycan hydrolase having at least 60% sequence identity to the sequence of SEQ ID NO: 1, a deglycosylated mutation refers to an amino acid substitution or deletion (preferably, an amino acid substitution) at position 68 or 73 of SEQ ID NO: 1 or any position corresponding to these positions. In the context of the CHAP domain of the present invention, a deglycosylated mutation refers to an amino acid substitution or deletion (preferably, an amino acid substitution) at position 73 of SEQ ID NO: 1 or a position corresponding to this position.

[0046] In particular, an amino acid substitution at position 68 of SEQ ID NO:1 or a position corresponding to this position means that the asparagine ("N") at position 68 of SEQ ID NO:1 has been replaced with another amino acid described herein.

[0047] Following the same logic, an amino acid substitution at position 73 of SEQ ID NO:1 or a position corresponding to this position means that the asparagine ("N") at position 73 of SEQ ID NO:1 or the asparagine corresponding to position 73 of SEQ ID NO:1 has been substituted with another amino acid described herein.

[0048] The following amino acid substitutions are considered to be particularly effective deglycosylating mutations or deglycosylating amino acid substitutions herein and in the context of the present invention: N68K, N68A, N68M, N68R, N73G, N73Y, N73H, N73L, N73E, and N73A at SEQ ID NO: 1 or a position corresponding to these positions. The CHAP domain is most suitable herein and in the context of the present invention, and an amino acid substitution at position 73 of SEQ ID NO: 1 or a position corresponding to this position, for example, N73G (L0482ag; contained in SEQ ID NO: 2), is particularly suitable herein and in the context of the present invention.

[0049] L0482 variants having at least one deglycosylation mutation (e.g., an amino acid substitution at position 73 of SEQ ID NO: 1 or a position corresponding to this position) described herein and exemplified by L0482ag (SEQ ID NO: 2) may be particularly good starting points for further protein engineering and / or directed evolution as described herein. As illustrated in the accompanying Examples and described in detail herein, additional permissible beneficial, particularly beneficial, or most beneficial amino acid substitutions can be introduced into L0482 variants having at least one deglycosylation mutation, particularly in their CHAP domain, to further enhance the pharmaceutical properties of the L0482 variants described herein (e.g., stability, solubility, and ability to be secreted from human cells, as well as killing activity against target bacteria, e.g., Staphylococcus aureus).

[0050] Thus, a deglycosylation mutation (preferably an amino acid substitution) at position 73 of SEQ ID NO: 1 or a position corresponding to this position (more preferably N73G) is preferably combined with at least one of the amino acid substitutions described herein, in particular at least one tolerable or beneficial amino acid substitution, preferably at least one particularly beneficial amino acid substitution, more preferably at least one of the most beneficial amino acid substitutions detailed herein, i.e. more preferably at least one amino acid substitution selected from the group consisting of, with reference to SEQ ID NO: 1: T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y, and N186G, in the context of the L0482 variant obtained during directed evolution.

[0051] Therefore, the present invention further relates to a peptidoglycan hydrolase, particularly having bactericidal activity, comprising a CHAP domain that (i) has at least 60% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO: 1, and (ii) has an amino acid substitution at or corresponding to position 73 of SEQ ID NO: 1. Preferably, the residue at or corresponding to position 73 of SEQ ID NO: 1 is substituted with another amino acid residue other than phenylalanine or lysine, such as glycine, tyrosine, leucine, glutamic acid, alanine, histidine, or serine, preferably glycine.

[0052] Additionally, the present invention relates to a peptidoglycan hydrolase having bactericidal activity, which (i) has at least 60% sequence identity with the sequence of SEQ ID NO: 1, and (ii) has at least one amino acid substitution at positions 68 and 73 of SEQ ID NO: 1 or a corresponding position, preferably at least at position 73 of SEQ ID NO: 1 or a corresponding position. Preferably, the residue at position 68 (i.e., "N") of SEQ ID NO: 1 or a corresponding position is substituted with another amino acid residue other than threonine or serine, such as lysine, methionine, arginine, or alanine, preferably lysine. Furthermore, the residue at position 73 (i.e., "N") of SEQ ID NO: 1 or a corresponding position is preferably substituted with another amino acid residue other than phenylalanine or lysine, such as glycine, tyrosine, leucine, glutamic acid, alanine, histidine, or serine, preferably glycine. Preferably, the peptidoglycan hydrolase comprises a CHAP domain according to the present invention.

[0053] A method / directed evolution for screening peptidoglycan hydrolases with improved pharmaceutical properties As already indicated herein above, the inventors evaluated in the context of the present invention whether it was possible not only to improve the bactericidal activity of lysin, but also to simultaneously improve further pharmaceutical properties of lysin (e.g., solubility, secretion from eukaryotic cells, and / or stability). As already indicated above and as will be described in more detail below, the inventors surprisingly found that some pharmaceutical properties of L0482 variants, in particular L0482ag, including bactericidal activity, can be further improved.

[0054] To this end, the present inventors have developed a combinatorial screening method based on the YODA-derived method of the present invention, which combines eukaryotic cell display, e.g., yeast display, with YODA; see, e.g., Example 5. As illustrated in the accompanying examples, it has surprisingly been found that the combinatorial screening method of the present invention makes it possible to simultaneously improve, inter alia, the solubility, bactericidal activity, and eukaryotic secretion of peptidoglycan hydrolases, e.g., endolysins such as L0482 (SEQ ID NO: 1) or their derivatives such as L0482ag (SEQ ID NO: 2). The present inventors reasoned that the display (e.g., yeast display) step is particularly compatible with the eukaryotic secretory pathway and therefore makes it possible to screen for peptidoglycan hydrolase variants with improved expression and secretion profiles in eukaryotic cells. Moreover, efficient secretion ability is often associated with good protein stability and good solubility, as described herein. Therefore, these biophysical and pharmaceutically relevant properties can also be improved by the methods of the present invention. The YODA step makes it possible to screen for secreted peptidoglycan hydrolase variants that have good bactericidal activity, particularly against desired target bacteria as described herein. Thus, the combinatorial screening method of the present invention makes it possible to screen for improved peptidoglycan hydrolase variants that are compatible with the eukaryotic secretory pathway, effectively kill target bacteria, such as Staphylococcus aureus, and may have additional beneficial pharmaceutical properties, such as enhanced stability and solubility, as described herein and illustrated in the accompanying examples.

[0055] As already mentioned above, the YODA-based method of the present invention is a method for screening yeast cells for secretion of active peptidoglycan hydrolase, comprising the steps of: a) providing a solidified yeast culture medium comprising substrate particles mixed with the solidified yeast culture medium, wherein the substrate particles comprise killed bacterial cells and / or fragments thereof, and / or peptidoglycan particles; b) culturing peptidoglycan hydrolase-expressing yeast cells on the surface of the solidified medium until at least one yeast colony is detectable; c) assessing whether a halo is visible around the yeast colonies; d) determining that the yeast colony secretes active peptidoglycan hydrolase if a halo is visible around the colony, or determining that the yeast colony does not secrete active peptidoglycan hydrolase if no halo is visible around the colony. This refers to a method including:

[0056] Herein and in the context of the present invention, said YODA-based method can be combined with eukaryotic cell display methods, in particular said method of screening yeast cells for secretion of active peptidoglycan hydrolase can be implemented in step III) of the method according to the present invention, in particular as described below, for identifying active peptidoglycan hydrolase variants optimized for secretion by eukaryotic cells.

[0057] Therefore, the present invention further provides a method for identifying an active peptidoglycan hydrolase variant optimized for secretion by a eukaryotic cell (i.e., a peptidoglycan hydrolase variant having bactericidal activity against a target bacterium, such as Staphylococcus aureus), comprising: I) preparing a library of eukaryotic cells, preferably yeast cells, expressing peptidoglycan hydrolase variants on the cell surface; II) selecting eukaryotic cells, e.g., yeast cells, based on high levels of peptidoglycan hydrolase on their cell surface relative to other cells in the library; e.g., selecting 10%, 5%, 1%, or 0.5% of the cells in the library that have the highest peptidoglycan levels on their cell surface; III) performing the method of the present invention for screening yeast cells for secretion of active peptidoglycan hydrolase (i.e., the YODA-derived method) provided herein to culture the yeast cells capable of secreting the peptidoglycan hydrolase variant expressed in the eukaryotic cells selected in step II) in step b) of the method for screening yeast cells for secretion of active peptidoglycan hydrolase; and IV) determining that the yeast colonies determined to secrete an active peptidoglycan hydrolase in step d) of the method of screening yeast cells for secretion of an active peptidoglycan hydrolase produce an active peptidoglycan hydrolase variant that is optimized for secretion by a eukaryotic cell. The present invention relates to a method comprising:

[0058] Moreover, said method, i.e. the combinatorial screening method of the present invention, can be used for the directed evolution of peptidoglycan hydrolases, e.g. endolysins, such as L0482 (SEQ ID NO: 1) or derivatives thereof, e.g. L0482ag (SEQ ID NO: 2), as illustrated in the accompanying examples.

[0059] Thus, in certain embodiments, at least two rounds of steps I) to IV) of the method of the invention for identifying active peptidoglycan hydrolase variants optimized for secretion by eukaryotic cells are carried out, and in step I) of each subsequent round, a further library of eukaryotic cells is prepared, the cells in the library expressing a different set of peptidoglycan variants compared to the library used in the preceding round.

[0060] Summary of beneficial and tolerated amino acid substitutions found by deglycosylation screening or directed evolution As already indicated above, and as described in detail herein, and as illustrated in the accompanying Examples, the screening method of the present invention provided herein contributed to the discovery of the peptidoglycan hydrolases of the present invention, in particular L0482 variants, which surprisingly simultaneously have improved pharmaceutical properties. In particular, 252 L0482 variants, in particular deglycosylated L0482 variants derived from L0482ag (SEQ ID NO: 2), have been generated in three rounds of directed evolution; see, for example, Examples 5 to 7. Importantly, all of these L0482 variants are capable of secretion by eukaryotic cells, in particular yeast cells, and appear to have killing activity against Staphylococcus aureus, as determined by the YODA method described in Example 3. Based on this large and highly informative dataset, the inventors were able to not only discover the most beneficial mutations described herein, but also many additional beneficial or at least tolerable amino acid substitutions at many positions in SEQ ID NO: 1 (or SEQ ID NO: 2) contained in the active, secreted L0482ag variants; see, e.g., Example 7 and Table 3. It is reliably determined that essentially all of the amino acid substitutions found in the active, secreted L0482ag variants are beneficial or at least tolerable with respect to the desired pharmaceutical properties of the L0482ag variants. In particular, it is reliably determined that essentially all or at least the majority of these amino acid substitutions, alone or in combination, confer upon LYSM-CHAP lysin, and in particular the L0482ag variant, sufficient killing activity against Staphylococcus species or strains, in particular Staphylococcus aureus, sufficient secretion ability from eukaryotic cells, sufficient stability, and / or sufficient solubility.

[0061] As illustrated in the accompanying Examples, e.g., Examples 4 to 7, amino acid substitutions in the CHAP domain of L0482 that may confer, enhance, or at least maintain (i) killing activity against Staphylococcus species or strains, particularly Staphylococcus aureus, and (ii) the ability to be secreted from eukaryotic cells, and preferably (iii) sufficient solubility and / or stability, include, inter alia, amino acid substitutions 72 to 76, 78, 81, 82, 85, 86, 93, 96, 104, 107, 108, 110, 112, 114, 116, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 1 They have been identified to occur at positions 8, 111, 113, 115, 117, 121, 124, 125, 129, 130, 133-136, 138, 140-142, 144, 145, 148, 149, 152, 153, 155, 157, 159, 169, 173, 175-178, 185, 186, 190-194, 197, 198, 199, 201, 203, 204, 207, and 212-215, or positions corresponding to these positions.

[0062] Accordingly, the present invention provides a peptidoglycan hydrolase having bactericidal activity, which (i) has at least 60% sequence identity with the sequence of positions 72 to 215 of SEQ ID NO: 1, and (ii) has at least one of positions 72 to 76, 78, 81, 82, 85, 86, 93, 96, 104, 107, 108, 111, 113, 115, 117, 121, 124, 125, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 185, 186, 93, 96, 104, 107, 108, 111, 113, 115, 117, 121, 124, 125, 129, 130, 178, 179 , 133-136, 138, 140-142, 144, 145, 148, 149, 152, 153, 155, 157, 159, 169, 173, 175-178, 185, 186, 190-194, 197, 198, 199, 201, 203, 204, 207, and 212-215, or one or more amino acid substitutions at positions corresponding to these positions; the amino acid residue at position 72 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine; the amino acid residue at position 73 of SEQ ID NO: 1 or a position corresponding thereto is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine, or serine, preferably glycine or serine, more preferably glycine; the amino acid residue at position 74 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine or proline; the amino acid residue at position 75 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine or proline; the amino acid residue at position 76 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 78 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine or tyrosine; the amino acid residue at position 81 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid, arginine, or serine; the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding thereto is substituted with lysine or methionine, preferably lysine; the amino acid residue at position 93 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or leucine, the amino acid residue at position 96 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 104 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 107 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine or tyrosine; the amino acid residue at position 108 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 111 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 113 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 115 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 117 of SEQ ID NO: 1 or a position corresponding to this position is substituted with histidine; the amino acid residue at position 121 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine; the amino acid residue at position 124 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine or histidine, the amino acid residue at position 125 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine; the amino acid residue at position 129 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine or serine; the amino acid residue at position 130 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine, isoleucine, or asparagine, preferably asparagine; the amino acid residue at position 133 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine or threonine; the amino acid residue at position 134 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine or valine; the amino acid residue at position 135 of SEQ ID NO: 1 or a position corresponding thereto is substituted with cysteine ​​or histidine; the amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding thereto is substituted with lysine or arginine, preferably lysine; the amino acid residue at position 138 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 140 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or aspartic acid; the amino acid residue at position 141 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine; the amino acid residue at position 142 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid or arginine; the amino acid residue at position 144 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid; the amino acid residue at position 145 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or leucine; the amino acid residue at position 148 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine or valine; the amino acid residue at position 149 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or threonine; the amino acid residue at position 152 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 153 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine or serine; the amino acid residue at position 155 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 157 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid; the amino acid residue at position 159 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine; the amino acid residue at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 of SEQ ID NO: 1 or a position corresponding to this position is substituted with histidine, asparagine, or arginine; the amino acid residue at position 175 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, glutamine, or threonine; the amino acid residue at position 176 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid or arginine; the amino acid residue at position 177 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine; the amino acid residue at position 178 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine or arginine; the amino acid residue at position 185 of SEQ ID NO: 1 or a position corresponding thereto is substituted with phenylalanine, histidine, serine, or tyrosine, preferably tyrosine; the amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 190 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine; the amino acid residue at position 191 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid or arginine; the amino acid residue at position 192 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine or threonine; the amino acid residue at position 193 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid; the amino acid residue at position 194 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, the amino acid residue at position 197 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 198 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 199 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, the amino acid residue at position 201 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid or serine; the amino acid residue at position 203 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid or valine; the amino acid residue at position 204 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, serine, or tyrosine; the amino acid residue at position 207 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine or valine; the amino acid residue at position 212 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 213 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine or threonine; the amino acid residue at position 214 of SEQ ID NO: 1 or a position corresponding thereto is substituted with glutamine or threonine; and / or The amino acid residue at position 215 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine, valine, or serine. The present invention relates to a peptidoglycan hydrolase comprising a CHAP domain having one or more amino acid substitutions.

[0063] Furthermore, the CHAP domain may have a deglycosylation substitution, i.e., an amino acid substitution, at position 73 of SEQ ID NO: 1 as described herein or a position corresponding to this position, and in addition, may have a deglycosylation substitution, i.e., an amino acid substitution, at other positions as described herein above, i.e., 72, 74-76, 78, 81, 82, 85, 86, 93, 96, 104, 107, 108, 111, 113, 115, 117, 121, 124, 125, 129, 130, 133-136, 138, 140-142, 144, 145, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 2 It is preferred that the polypeptide has at least one amino acid substitution at positions 152, 153, 155, 157, 159, 169, 173, 175-178, 185, 186, 190-194, 197, 198, 199, 201, 203, 204, 207, and 212-215, or positions corresponding to these positions, and preferably at least one amino acid substitution at positions 82, 85, 86, 130, 136, 155, 169, 185, and 186 of SEQ ID NO: 1, or positions corresponding to these positions.

[0064] Accordingly, the present invention further provides a peptidoglycan hydrolase having bactericidal activity, the peptidoglycan hydrolase comprising: (i) at least 60% sequence identity with the sequence of positions 72 to 215 of SEQ ID NO:1; and (ii) a peptidoglycan hydrolase selected from the group consisting of positions 72, 74 to 76, 78, 81, 82, 85, 86, 93, 96, 104, 107, 108, 111, 113, 115, 117, 121, 124, 125, 129, 130, 133 to 136, 140 to 142, 144, 145, 148, 149, 152, 153, 155, 157, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 The present invention relates to a peptidoglycan hydrolase comprising a cysteine, histidine-dependent amidohydrolase / peptidase (CHAP) domain having one or more amino acid substitutions at positions 169, 173, 175-178, 185, 186, 190-192, 194, 197, 198, 199, 201, 203, 204, 207, and 212-215, or positions corresponding to these positions, preferably at positions 82, 85, 86, 130, 136, 155, 169, 185, and 186, or positions corresponding to these positions, of SEQ ID NO: 1. Preferably, the CHAP domain further comprises a deglycosylation mutation, i.e., an amino acid substitution or deletion, preferably a substitution, at position 73 of SEQ ID NO: 1 as described herein, or a position corresponding to this position.

[0065] Particularly beneficial amino acid substitutions / selected hit variants As already shown herein above, the present inventors found several hit variants of L0482ag (SEQ ID NO: 2) in each round of directed evolution, namely, G1 to G4 (SEQ ID NOs: 3 to 6) in round 1, H1 to H10 (SEQ ID NOs: 7 to 16) in round 2, and I1 to I30 (SEQ ID NOs: 17 to 46) in round 3. The various amino acid substitutions contained in these hit variants (in addition to the two deglycosylation substitutions N68K and N73G) are considered to be particularly beneficial mutations, i.e., amino acid substitutions, in the context of this specification and the present invention; see, for example, Examples 5 to 7.

[0066] As illustrated in the accompanying examples, particularly beneficial amino acid substitutions in the CHAP domain of L0482 that may confer, enhance, or at least maintain (i) killing activity against Staphylococcus species or strains, particularly Staphylococcus aureus, and (ii) the ability to be secreted from eukaryotic cells, and (iii) preferably sufficient solubility and stability, have been identified as occurring at positions 73, 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169, 173, 175, 178, 185, 186, 191-194, 198, 204, 212 and 215 of SEQ ID NO: 1, or positions corresponding to these positions, as described herein.

[0067] Accordingly, the present invention further provides a peptidoglycan hydrolase having bactericidal activity, the peptidoglycan hydrolase having (i) at least 60% sequence identity with the sequence of positions 72 to 215 of SEQ ID NO:1, and (ii) one or more amino acid substitutions at positions 73, 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169, 173, 175, 178, 185, 186, 191, 192, 194, 198, 204, 212, and 215 of SEQ ID NO:1, or positions corresponding to these positions; the amino acid residue at position 73 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine or serine, preferably glycine; the amino acid residue at position 75 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine; the amino acid residue at position 78 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 81 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid; the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; the amino acid residue at position 104 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 107 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 115 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 124 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine; the amino acid residue at position 125 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine; the amino acid residue at position 130 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 133 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 135 of SEQ ID NO: 1 or a position corresponding to this position is substituted with histidine, the amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding thereto is substituted with lysine or arginine, preferably lysine; the amino acid residue at position 140 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, the amino acid residue at position 141 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine; the amino acid residue at position 155 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine; the amino acid residue at position 178 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine; the amino acid residue at position 185 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 191 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 192 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine; the amino acid residue at position 194 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, the amino acid residue at position 198 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 204 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 212 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; and / or the amino acid residue at position 215 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; The present invention relates to a peptidoglycan hydrolase comprising a CHAP domain having one or more amino acid substitutions.

[0068] Furthermore, the CHAP domain preferably comprises a deglycosylation mutation, preferably an amino acid substitution at or corresponding to position 73 of SEQ ID NO:1 as described herein, more preferably N73G, and in addition at least one particularly beneficial amino acid substitution at any of the other positions described herein above, i.e., positions 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169, 173, 175, 178, 185, 186, 191, 192, 194, 198, 204, 212 and 215 of SEQ ID NO:1, or positions corresponding to these positions, preferably positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 of SEQ ID NO:1, or positions corresponding to these positions.

[0069] Accordingly, the present invention further provides a peptidoglycan hydrolase having bactericidal activity, the peptidoglycan hydrolase having (i) at least 60% sequence identity with the sequence of positions 72 to 215 of SEQ ID NO:2, and (ii) any of positions 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169, 173, 175, 178, 179, 180, 181, 182, 185, 186, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169, 173, 175, 178 ... The present invention relates to a peptidoglycan hydrolase comprising a CHAP domain having one or more amino acid substitutions at positions 85, 186, 191, 192, 194, 198, 204, 212 and 215, or positions corresponding to these positions, preferably one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 of SEQ ID NO: 1 described herein, or positions corresponding to these positions.

[0070] Most beneficial amino acid substitutions As already indicated above, based on the characteristics of the hit variants of L0482 found in the context of the present invention (see, e.g., Example 6 and Figure 8) and the comparative analysis of the sequences of these hits (see, e.g., Example 7), the inventors were able to find the most beneficial amino acid substitutions in SEQ ID NO: 1, namely T82S, N85G, R86K, S130N, H136K / R (preferably H136K), F155Y, D169N, N185Y and N186G.

[0071] As mentioned above, the most beneficial amino acid substitutions refer only to the amino acid substitutions contained in the hit variant H5 (SEQ ID NO: 11) relative to the parent L0482ag variant (SEQ ID NO: 2); see, for example, Figure 9A. H5 is considered herein to be the variant with the best pharmaceutical properties obtained by two rounds of directed evolution. As mentioned above, the most beneficial amino acid substitutions in H5 strongly enhanced killing activity against S. aureus, including S. aureus biofilms, protein stability, and secretion ability from human cells; see, for example, Figures 8, 9, 11, and 12.

[0072] Thus, as already mentioned above, the present invention further provides a peptidoglycan hydrolase, in particular having bactericidal activity, which has (i) at least 60% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO: 1, and (ii) one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 of SEQ ID NO: 1 or at positions corresponding to these positions, the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; the amino acid residue at position 130 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding thereto is substituted with lysine or arginine, preferably lysine; the amino acid residue at position 155 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; The amino acid residue at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine. the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 is substituted with tyrosine, and / or the amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; The present invention relates to a peptidoglycan hydrolase comprising a CHAP domain having one or more amino acid substitutions.

[0073] Preferably, the CHAP domain has a plurality of said most beneficial amino acid substitutions (e.g., 2, 3, 4, 5, 6, 7, 8, or 9), preferably at least 6 or 7, more preferably at least 8, and most preferably all of said most beneficial amino acid substitutions. Furthermore, the CHAP domain preferably further has a deglycosylation mutation, preferably an amino acid substitution, more preferably N73G, at position 73 of SEQ ID NO: 1 as described herein or a position corresponding to this mutation.

[0074] The most frequent substitution of the most beneficial amino acid substitution: R86K Furthermore, it has surprisingly been found in the context of the present invention that the amino acid substitution R86K is contained in all 44 identified hit variants, i.e., G1-G4, H1-H10, and I1-I30, and is contained in 96.08% of all L0482 variants secreted from eukaryotic cells, and has been determined to have killing activity against Staphylococcus aureus, as described herein and illustrated in the accompanying examples; see, e.g., Example 7 and Figure 9.

[0075] Thus, R86K in SEQ ID NO: 1 is a particularly preferred amino acid substitution herein, and in the context of the present invention, among the most beneficial amino acid substitutions identified. Thus, in a particularly preferred embodiment, the CHAP domain of the present invention has an amino acid substitution at position 86 of SEQ ID NO: 1 or a position corresponding to this position, in which the amino acid residue at said position is replaced with lysine.

[0076] Frequent pairs of amino acid substitutions in the most beneficial amino acid substitutions Furthermore, it was surprisingly found in the context of the present invention that certain amino acid substitutions occurred together in pairs in the identified hit variants, i.e., G1-G4, H1-H10, and I1-I30; see, e.g., Example 7 and FIG. 9. These mutation pairs are specifically (i) T82S and N85G, (ii) S130N and H136K / R, and (iii) N185Y and N186G. Each of the 44 hit variants contained at least one of the mutation pairs. Notably, these amino acid substitutions, i.e., T82S and N85G, S130N and H136K / R, and N185Y and N186G, advantageously but not necessarily occur in pairs, since variants carrying only one of these paired mutations (but not the other) were also found among the 252 active L0482 variants secreted from yeast cells found in the context of the present invention.

[0077] Thus, a CHAP domain of the invention having an amino acid substitution at or corresponding to position 82 of SEQ ID NO: 1 described herein preferably also has an amino acid substitution at or corresponding to position 85 of SEQ ID NO: 1 described herein. Similarly, a CHAP domain of the invention having an amino acid substitution at or corresponding to position 85 of SEQ ID NO: 1 described herein preferably also has an amino acid substitution at or corresponding to position 82 of SEQ ID NO: 1 described herein.

[0078] Furthermore, a CHAP domain of the present invention having an amino acid substitution at or corresponding to position 130 of SEQ ID NO: 1 described herein preferably also has an amino acid substitution at or corresponding to position 136 of SEQ ID NO: 1 described herein. Similarly, a CHAP domain of the present invention having an amino acid substitution at or corresponding to position 136 of SEQ ID NO: 1 described herein preferably also has an amino acid substitution at or corresponding to position 130 of SEQ ID NO: 1 described herein.

[0079] Furthermore, a CHAP domain of the present invention having an amino acid substitution at or corresponding to position 185 of SEQ ID NO: 1 described herein preferably also has an amino acid substitution at or corresponding to position 186 of SEQ ID NO: 1 described herein. Similarly, a CHAP domain of the present invention having an amino acid substitution at or corresponding to position 186 of SEQ ID NO: 1 described herein preferably also has an amino acid substitution at or corresponding to position 185 of SEQ ID NO: 1 described herein.

[0080] Consensus mutation unit As already mentioned herein above, the hit variant H3 (SEQ ID NO: 9) reflects the consensus sequence of particularly improved L0482 variants. H3 contains only the following amino acid substitutions, also referred to herein as "consensus mutations", with reference to SEQ ID NO: 1: T82S, N85G, R86K, S130N, H136K / R (particularly H136K), D169N, N185Y and N186G.

[0081] Furthermore, based on the consensus sequence reflected by the 44 L0482ag hit variants and H3 (SEQ ID NO: 9) found in the context of the present invention, the inventors have found five consensus mutation units of particularly improved L0482 variants, which consist of one or two amino acid substitutions: (i) R86K (which is particularly preferred), (ii) T82S and N85G, (iii) S130N and H136K / R (preferably H136K), (iv) D169N, and (v) N185Y and N186G.

[0082] Thus, in a preferred embodiment, the CHAP domain of the present invention has at least one amino acid substitution or pair of substitutions (i.e., at least one consensus mutation unit) selected from the group consisting of: (i) an amino acid substitution at position 86 of SEQ ID NO: 1 or a position corresponding to this position, wherein the amino acid residue at said position is replaced with a lysine; (ii) amino acid substitutions at positions 82 and 85 of SEQ ID NO: 1 or a position corresponding thereto, wherein the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding thereto is substituted with serine and the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding thereto is substituted with glycine; (iii) amino acid substitutions at positions 130 and 136 of SEQ ID NO: 1 or a position corresponding thereto, wherein the amino acid residue at position 130 of SEQ ID NO: 1 or a position corresponding thereto is substituted with asparagine, and the amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding thereto is substituted with lysine or arginine, preferably lysine; (iv) an amino acid substitution at position 169 of SEQ ID NO: 1 or a position corresponding to this position, wherein the amino acid residue at said position is replaced with asparagine; and (v) Amino acid substitutions at positions 185 and 186 of SEQ ID NO: 1 or a position corresponding thereto, in which the amino acid residue at position 185 of SEQ ID NO: 1 or a position corresponding thereto is substituted with tyrosine and the amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto is substituted with glycine.

[0083] Preferably, said CHAP domain has at least two, preferably at least three, more preferably at least four, and most preferably all of said consensus mutation units.

[0084] Preferably, the CHAP domain further comprises a deglycosylation mutation as described herein, preferably an amino acid substitution at or corresponding to position 73 of SEQ ID NO:1 as described herein, more preferably the substitution N73G.

[0085] Furthermore, the CHAP domain preferably has the amino acid substitution F155Y.

[0086] In certain preferred embodiments, a CHAP domain of the invention has at least 94% (particularly at least 93.9%), at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence from positions 72 to 215 of SEQ ID NO: 9 (H3). Preferably, the CHAP domain has a) (i) at least one consensus mutation or consensus mutation unit described herein, and (ii) at least one deglycosylation mutation, preferably an amino acid substitution, at position 73 described herein, and / or b) at least two of the consensus mutations described herein, e.g., at least one amino acid substitution pair. Furthermore, in the context of these preferred embodiments or similar preferred embodiments, the CHAP domain of the present invention has at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, at most 1, or no amino acid substitutions in the sequence from positions 72 to 215 of SEQ ID NO: 9 (H3), or in the sequence from the position corresponding to position 72 of SEQ ID NO: 9 to the position corresponding to position 215 of SEQ ID NO: 9.

[0087] Furthermore, the CHAP domain preferably has the amino acid substitution F155Y.

[0088] In certain preferred embodiments, the peptidoglycan hydrolase of the invention has at least 95% (particularly at least 95.4%), at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of SEQ ID NO:9 (H3). Furthermore, in the context of these preferred embodiments or similar preferred embodiments, the peptidoglycan hydrolase of the invention has at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, at most 1, or no amino acid substitutions in SEQ ID NO:9.

[0089] Preferably, said peptidoglycan hydrolase comprises a CHAP domain according to the present invention.

[0090] A single amino acid substitution that improves several pharmaceutical properties at once: F155Y As detailed herein above, the best-performing L0482 variant found and characterized in the context of the present invention, namely H5 (SEQ ID NO: 11), surprisingly contained only one additional amino acid substitution relative to H3 (SEQ ID NO: 9), namely F155Y. As detailed herein, said amino acid substitution simultaneously improved several pharmaceutical properties compared to H3 (SEQ ID NO: 9), namely killing activity against Staphylococcus aureus, protein stability, and ability to be secreted from human cells; see, e.g., Figure 8.

[0091] Therefore, F155Y of SEQ ID NO: 1 is considered herein, and in the context of the present invention, to be a further particularly preferred amino acid substitution among the most beneficial amino acid substitutions identified. Thus, in a particularly preferred embodiment, the CHAP domain of the present invention has an amino acid substitution at or corresponding to position 155 of SEQ ID NO: 1, in which the amino acid residue at said position is replaced with tyrosine.

[0092] Preferably, the CHAP domain further comprises at least one consensus mutation described herein (i.e., T82S, N85G, R86K, S130N, H136K / R (preferably H136K), D169N, N185Y and / or N186G), or more preferably at least one consensus mutation unit described herein, i.e., (i) R86K (particularly preferred), (ii) T82S and N85G, (iii) S130N and H136K / R (preferably H136K), (iv) D169N, and / or (v) N185Y and N186G; and / or a deglycosylation substitution described herein, i.e., an amino acid substitution, preferably N73G, at position 73 of SEQ ID NO: 1 described herein or a position corresponding to this position.

[0093] In a further preferred embodiment, the CHAP domain of the invention has at least 93% (particularly at least 93.2%), at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence from positions 72 to 215 (H5) of SEQ ID NO: 11. Preferably, the CHAP domain has a) (i) at least one of the most beneficial amino acid substitutions and / or at least one consensus mutation unit described herein, and (ii) at least one deglycosylation mutation, preferably an amino acid substitution, at position 73 described herein, and / or b) at least two of the most beneficial amino acid substitutions described herein, e.g., at least one amino acid substitution pair. Furthermore, in the context of these preferred embodiments, or in similar preferred embodiments, the CHAP domain of the present invention has at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, at most 1, or no amino acid substitutions in the sequence from positions 72 to 215 of SEQ ID NO: 11 (H5) or in the sequence from the position corresponding to position 72 of SEQ ID NO: 11 to the position corresponding to position 215 of SEQ ID NO: 11.

[0094] In certain preferred embodiments, the peptidoglycan hydrolase of the invention has at least 95% (particularly at least 95.0%), at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of SEQ ID NO: 11 (H5). Furthermore, in the context of these preferred embodiments or similar preferred embodiments, the peptidoglycan hydrolase of the invention has at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, at most 1, or no amino acid substitutions in SEQ ID NO: 11.

[0095] Preferably, said peptidoglycan hydrolase comprises a CHAP domain according to the present invention.

[0096] Furthermore, the peptidoglycan hydrolases of the present invention are particularly single polypeptides, ie, single amino acid chains.

[0097] Administration in the form of nucleic acids As already indicated above, administering the peptidoglycan hydrolase of the present invention to a subject (e.g., a human) in the form of a nucleic acid (e.g., mRNA) encoding the peptidoglycan hydrolase, particularly by introducing the nucleic acid into the subject's cells, has certain advantages. For example, when a nucleic acid (e.g., mRNA) encoding the peptidoglycan hydrolase of the present invention is introduced into and expressed in the cells of a subject, e.g., a patient with a bacterial, e.g., staphylococcal, infection, the cells can continuously produce and secrete the peptidoglycan hydrolase protein. This can provide a more efficient treatment of bacterial infections, for example, providing greater efficacy in treating hard-to-treat bacterial infections such as bacterial biofilms, e.g., staphylococcal biofilms. Furthermore, the nucleic acid may be introduced into a specific location, e.g., cells at the site of a bacterial infection, and / or into a specific cell type, which may further improve the efficacy and / or safety of the treatment. Furthermore, nucleic acids, particularly RNA, have certain practical advantages over other proteins in terms of their manufacturing, safety profile, and / or suitability.

[0098] It has further been found by the present inventors that all hit variants discovered and further tested in the context of the present invention, i.e., G1-G4 and H1-H6, were efficiently secreted from human cells when expressed from RNA in these cells. In particular, it has been found that secretion from human cells was greatly enhanced compared to a control RNA expressing WT L0482 (SEQ ID NO: 1); see Examples 11 and 12 and Figure 10.

[0099] Thus, the present invention further relates to a nucleic acid encoding the peptidoglycan hydrolase of the present invention, preferably comprising a CHAP domain having at least one of the most beneficial mutations described herein (i.e., T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y, and N186G in SEQ ID NO: 1) and / or at least one consensus mutation unit. Preferably, the nucleic acid of the present invention is RNA, preferably mRNA. Preferably, the RNA of the present invention, e.g., mRNA, comprises at least one modified nucleoside, such as pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U), preferably N1-methyl-pseudouridine (m1ψ), in place of at least one uridine, preferably multiple uridines, more preferably all uridines. DETAILED DESCRIPTION OF THE INVENTION

[0100] peptidoglycan hydrolase The term "peptidoglycan hydrolase," as used herein and in the context of the present invention, refers to a polypeptide (i.e., a single amino acid chain) capable of hydrolyzing peptidoglycan (also called "murein") of at least one bacterial species or strain, preferably at least one Staphylococcus species or strain, more preferably Staphylococcus aureus. Peptidoglycan hydrolases of the present invention may contain naturally occurring and / or non-naturally occurring amino acids and modifications, such as, but not limited to, glycosylation (particularly O-glycosylation and / or N-glycosylation), acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, or protecting groups. Preferably, peptidoglycan hydrolases of the present invention are composed of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% naturally occurring amino acids (which may or may not contain modifications) as described herein. The term "peptidoglycan hydrolase" encompasses, for example, endolysins, lysozymes, tail-spike depolymerases, virion-associated peptidoglycan hydrolases (VAPGH), bacteriocins, and autolysins. In a preferred embodiment, the term "peptidoglycan hydrolase" refers to endolysins. The terms "endolysin" and "lysin" are used interchangeably herein and in the context of the present invention.

[0101] Isolated peptidoglycan hydrolases The peptidoglycan hydrolase of the present invention may be an isolated peptidoglycan hydrolase, such as an isolated endolysin. In particular, as used herein, "isolated" means removed from its natural state (e.g., purified). For example, a nucleic acid, peptide, or polypeptide naturally occurring in a living animal is not "isolated," but the same nucleic acid, peptide, or polypeptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid, peptide, or polypeptide can exist in a substantially purified form or can exist in a non-natural environment, such as a host cell. Preferably, as used herein, an isolated peptidoglycan hydrolase refers to a peptidoglycan hydrolase that has been isolated (e.g., purified) from its natural context, for example, from bacterial cells in which it naturally occurs.

[0102] Engineered and natural peptidoglycan hydrolases In the context of the present invention, a "peptidoglycan hydrolase" or "endolysin" is preferably a modified, i.e., engineered, "peptidoglycan hydrolase" or "endolysin," respectively. In particular, a "modified" (i.e., "engineered," "synthetic," "recombinant," or "artificial") peptidoglycan hydrolase or endolysin does not occur in nature and thus may be considered herein, and in the context of the present invention, to be a "non-natural" (i.e., "non-natural") peptidoglycan hydrolase or endolysin, respectively.

[0103] Thus, in certain embodiments, the peptidoglycan hydrolases of the invention are engineered peptidoglycan hydrolases.

[0104] Furthermore, in certain embodiments, the peptidoglycan hydrolases of the invention are non-naturally occurring peptidoglycan hydrolases.

[0105] However, in certain embodiments of the invention, peptidoglycan hydrolase may also include naturally occurring (i.e., natural) peptidoglycan hydrolases, particularly, for example, in the context of pharmaceutical compositions, medical uses, naturally occurring endolysins, fusion proteins of peptidoglycan hydrolase with another (poly)peptide (e.g., PK tag), RNA constructs, and / or modified nucleic acids, for example, RNAs containing modified nucleosides such as N1-methyl-pseudouridine (m1ψ) in place of at least one uridine as described herein.

[0106] Endolysin As used herein and in the context of the present invention, the term "endolysin" refers to a naturally occurring peptidoglycan hydrolase, particularly one encoded by a bacteriophage (or bacterial virus), or an engineered peptidoglycan hydrolase, particularly a modified endolysin, derived from such a naturally occurring peptidoglycan hydrolase (e.g., derived from L0482; SEQ ID NO: 1). Natural endolysins act, in particular, by hydrolyzing the host cell wall, which then allows the release of bacteriophage progeny. The peptidoglycan hydrolases, e.g., endolysins, of the present invention are also capable of hydrolyzing peptidoglycan in the cell wall of bacterial species or strains (e.g., Staphylococcus aureus) and thus have killing activity against said bacterial species or strains (e.g., Staphylococcus aureus) described herein.

[0107] The term "cell wall," as used herein, as commonly understood in the art, refers to all components that form the outer cell enclosure of bacteria (especially containing peptidoglycan).

[0108] Naturally occurring endolysins typically have molecular weights in the range of about 15 to about 60 kDa, which is also a preferred range for the peptidoglycan hydrolases (e.g., modified endolysins) of the present invention. However, the peptidoglycan hydrolases of the present invention may also have different, e.g., higher, molecular weights, for example, if they further comprise an additional domain or tag, such as a PK tag, as described herein. Moreover, natural endolysins typically have a modular organization. Herein and in the context of the present invention, endolysin refers specifically to modular endolysins, i.e., polypeptides (i.e., single amino acid chains) comprising one or more enzymatically active domains (EADs) and, preferably, further comprising one or more cell wall-binding domains (CBDs). Furthermore, in the context of similarly modular endolysins, it is possible for an endolysin to comprise only one module, i.e., an EAD, e.g., a CHAP domain of the present invention, but not a separate cell wall-binding domain. Nevertheless, it is preferred that the peptidoglycan hydrolases of the present invention comprise at least one EAD, particularly a CHAP domain of the present invention, and further comprising at least one CBD. Furthermore, various domains in a modular endolysin, such as the EAD and CBD, may be separated by a linker region as described herein, particularly a short flexible linker. The EAD may be N-terminal or C-terminal to the CBD. In L0482 (SEQ ID NO: 1), the single cell wall binding domain (CBD), i.e., the LYSM domain, is N-terminal to the single enzymatically active domain (EAD), i.e., the CHAP domain. Furthermore, in L0482, the LYSM and CHAP domains are separated by a linker region.

[0109] L0482 As used herein, and in the context of the present invention, L0482 (i.e., wild-type L0482; also referred to as "lytN") has the following sequence, where the LYSM domain (positions 1-51) is underlined, the linker (positions 52-71) is italicized, and the CHAP domain (positions 72-215) is bolded: [ka]

[0110] In the above sequence, two glycosylation positions in SEQ ID NO: 1, namely positions 68 and 73, which may be deleted or preferably substituted with another amino acid (e.g., lysine and glycine, respectively) to obtain the "deglycosylated" L0482 variant described herein (e.g., L0482ag; SEQ ID NO: 2), are further highlighted in grey. Furthermore, positions 82, 85, 86, 130, 136, 155, 169, 185, and 186 of SEQ ID NO: 1, which may be substituted with the most beneficial amino acid residues contained in the best-performing characterized variant described herein, i.e., H5 (SEQ ID NO: 11), are shown in white on a black background.

[0111] The CHAP domain of L0482 (SEQ ID NO: 1) is further set forth in SEQ ID NO: 301; the LYSM domain of L0482 (SEQ ID NO: 1) is further set forth in SEQ ID NO: 302; and the linker region of L0482 (SEQ ID NO: 1) is further set forth in SEQ ID NO: 303.

[0112] In the context of the present invention, naturally occurring peptidoglycan hydrolases, particularly endolysin L0482 (SEQ ID NO: 1), are typically modified by at least one amino acid substitution as described herein. Furthermore, one or more deletions, insertions, and / or additions of amino acid residues may also occur. Modified L0482 proteins are also considered to be L0482 variants herein. Furthermore, peptidoglycan hydrolases according to the present invention, particularly modified L0482 variants, may lack the LYSM domain or CHAP domain of L0482 and the linker region described herein. Alternatively, the LYSM domain or CHAP domain and the linker region may be replaced with another cell wall-binding domain or enzymatically active domain, or linker region, respectively, as described herein.

[0113] Thus, the peptidoglycan hydrolase according to the present invention comprises: (I) a CHAP domain (i) having at least 60% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO: 1; and (ii) having one or more amino acid substitutions compared to the sequence from positions 72 to 215 of SEQ ID NO: 1 described herein; and / or (II) a LYSM domain (i) having at least 60% sequence identity with the sequence from positions 1 to 51 of SEQ ID NO: 1, and (ii) having one or more amino acid substitutions compared to the sequence from positions 1 to 51 of SEQ ID NO: 1 described herein.

[0114] Peptidoglycan hydrolases, especially modular endolysin domains As already indicated above, the enzymatically active domain (EAD) of a modular endolysin functions to specifically cleave certain peptidoglycan bonds in the murein (i.e., peptidoglycan) layer of a host bacterium. The cell wall-binding domain (CBD) is typically enzymatically inactive and specifically recognizes and binds to certain epitopes in the cell wall of a host bacterium, in order to properly anchor the catalytic effect of the EAD. Preferably, herein and in the context of the present invention, the cell wall-binding domain is a peptidoglycan-binding domain that specifically binds to the peptidoglycan structure of a target bacterium. Different domains of an endolysin can be connected by a peptide linker, also referred to as a "domain linker." Moreover, as described herein, an EAD (e.g., a CHAP domain) can hydrolyze the peptidoglycan of a target bacterium, e.g., a host bacterium, alone, i.e., without the need to interact with a separate CBD. For example, an EAD, such as a CHAP domain, may have some intrinsic cell wall-binding activity (in addition to peptidoglycan hydrolase activity) and thus may have bactericidal activity alone. This has already been demonstrated for some peptidoglycan hydrolases, such as, for example, lysozyme. Binding of a substrate, particularly a substrate in the cell wall of a target bacterium, or near the catalytic pocket of the EAD, may be sufficient to initiate docking and subsequent lysis of the bacterium. Thus, the CHAP domain of the invention may also be considered herein and in the context of the present invention to be a peptidoglycan hydrolase that may have bactericidal activity alone.

[0115] Thus, the peptidoglycan hydrolase according to the present invention comprises at least one enzymatically active domain (EAD), also referred to herein as a "catalytic domain." Preferably, the at least one enzymatically active domain comprises at least a CHAP domain of the present invention described herein, i.e., a CHAP domain (i) having at least 60% sequence identity with the sequence of positions 72 to 215 of SEQ ID NO: 1; and (ii) having one or more amino acid substitutions compared to the sequence of positions 72 to 215 of SEQ ID NO: 1.

[0116] Enzymatic peptidoglycan cleavage mechanism As used herein, the term "peptidoglycan hydrolase" is generally not limited to a specific enzymatic cleavage mechanism. In particular, the enzymatically active domain of a peptidoglycan hydrolase can function as a glycosidase, an amidase (i.e., an amidohydrolase), and / or a peptidase. Thus, a peptidoglycan hydrolase containing one or more EADs, such as an endolysin, as described herein and in the context of the present invention, can function as a glycosidase, an amidase, and / or a peptidase. As used herein, glycosidases, such as acetylmuramidase, lytic transglycosylase, or glucosaminidase, generally cleave the backbone of a glycan. In particular, glycosidases can cleave the β-1,4 glycosidic bond that connects the alternating polymeric structure of N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in the peptidoglycan layer. Amidases (i.e., amidohydrolases) generally cleave peptide side chains, and in particular, they can catalyze the cleavage of the amide bond between MurNAc and the first amino acid in the peptide stem, i.e., L-alanine. Peptidases (especially endopeptidases and carboxypeptidases) generally cleave within peptide side chains, and in particular, they cleave the bond between two amino acids in the stem peptide of peptidoglycan, whereby bond cleavage can occur either within the interpeptide bridge or the stem peptide-interpeptide bridge.

[0117] As used herein and in the context of the present invention, the term "CHAP domain" refers to the cysteine, histidine-dependent amidohydrolase / peptidase domain that is the enzymatically active domain (EAD) of the peptidoglycan hydrolases described herein (particularly the L0482 variant). The CHAP domain may also be considered, as used herein and in the context of the present invention, to be a peptidoglycan hydrolase.

[0118] Furthermore, the CHAP domain of L0482 (SEQ ID NO: 1) can function as an amidase and a peptidase to hydrolyze peptidoglycan. In particular, the CHAP domain has been reported to have N-acetylmuramyl L-Ala amidase activity and D-Ala-Gly endopeptidase activity; Frankel (2012), J Biol Chem. 23;287(13). Therefore, the CHAP domain of the present invention can also function, in particular, as an amidase and / or a peptidase, preferably as an amidase and a peptidase. Thus, the peptidoglycan hydrolase of the present invention preferably functions as an amidase (i.e., it preferably has amidase activity) and / or as a peptidase (i.e., it preferably has peptidase activity). In particular, the peptidoglycan hydrolase of the present invention may have N-acetylmuramyl L-Ala amidase activity and / or D-Ala-Gly endopeptidase activity. More preferably, the peptidoglycan hydrolase of the present invention functions as both an amidase and a peptidase, and in particular, it may have N-acetylmuramyl L-Ala amidase activity and D-Ala-Gly endopeptidase activity.

[0119] Furthermore, a CHAP domain that functions as an amidase and / or a peptidase to hydrolyze peptidoglycan may also be considered herein, and in the context of the present invention, to be a peptidoglycan hydrolase having amidase and / or peptidase activity, respectively. Thus, a CHAP domain of the present invention may be a peptidoglycan hydrolase having amidase (e.g., N-acetylmuramyl L-Ala amidase activity) and / or peptidase activity (e.g., D-Ala-Gly endopeptidase activity). Thus, a peptidoglycan hydrolase of the present invention comprising a CHAP domain of the present invention preferably has the amidase and / or peptidase activity described herein.

[0120] Herein, and in the context of the present invention, hydrolyzing peptidoglycan in bacterial cell walls may also refer to breaking and / or cleaving said peptidoglycan.

[0121] Bactericidal activity Hydrolysis, particularly destruction and / or cleavage of peptidoglycan in bacterial cell walls, typically kills bacteria. Therefore, the peptidoglycan hydrolases of the present invention have particularly bactericidal activity. As used herein and in the context of the present invention, the term "bactericidal activity" refers to the ability of a peptidoglycan hydrolase to kill at least one bacterium, i.e., at least one bacterial species or strain, particularly at least one target bacterium (i.e., the bacterial species or strain to be killed). Preferably, the peptidoglycan hydrolase of the present invention has the ability to kill at least one Gram-positive bacterium, more preferably a Staphylococcus species or strain, and most preferably at least Staphylococcus aureus, preferably including methicillin-resistant Staphylococcus aureus (MRSA) strains. A peptidoglycan hydrolase having the ability to kill a specific bacterium, e.g., Staphylococcus aureus, is also referred to herein as a peptidoglycan hydrolase having "killing activity" against said bacterium, e.g., Staphylococcus aureus.

[0122] Herein, and in the context of the present invention, the killing activity of a peptidoglycan hydrolase against a certain bacterium, e.g., Staphylococcus aureus, is preferably measured by determining the minimum concentration at which the peptidoglycan hydrolase inhibits the growth of a liquid culture of said bacterium, e.g., Staphylococcus aureus. Said minimum concentration is also referred to herein as "minimum inhibitory concentration" (MIC). Herein, and in the context of the present invention, "minimum inhibitory concentration" (MIC) refers in particular to a concentration of 5 x 10 5The concentration of staphylococcus aureus is defined as the minimum concentration that maintains an optical density at 620 nm (OD620) of less than 0.1 for at least 24 hours of incubation at 37°C for a liquid culture containing cfu / ml of target bacteria (e.g., Staphylococcus aureus). Preferably, the culture medium for said liquid culture is cation-adjusted Mueller-Hinton broth (caMHB) medium supplemented with 25% horse serum, especially when the target bacteria is a staphylococcus species or strain, e.g., Staphylococcus aureus. When measuring killing activity against Staphylococcus aureus, the Staphylococcus aureus cells in the liquid culture are preferably at least 5 x 10 5 cfu / ml corresponds to methicillin-resistant Staphylococcus aureus (MRSA) strain ATCC 43300. The term "cfu" is an abbreviation for "colony forming units" and refers to an estimated number of viable bacterial cells, as commonly understood in the art.

[0123] A detailed assay for measuring the killing activity against a target bacterium, such as a Staphylococcus species or strain, e.g., Staphylococcus aureus, is provided in Example 6: First, a peptidoglycan hydrolase (e.g., an L0482 variant of the present invention) is produced in E. coli as described in Example 6 under the heading "Production of an L0482 variant in E. coli." Next, the peptidoglycan hydrolase (e.g., an L0482 variant of the present invention) is purified from E. coli as described in Example 6 under the heading "Purification of an L0482 variant from E. coli." Finally, the killing activity of the peptidoglycan hydrolase (e.g., an L0482 variant of the present invention) against a target bacterium (e.g., Staphylococcus aureus) is determined as described in Example 6 under the heading "Determination of the killing activity of an L0482 variant."

[0124] Detailed assays for measuring the killing activity of peptidoglycan hydrolases against biofilms or planktonic aggregates of target bacteria, e.g., biofilms or planktonic aggregates of a Staphylococcus species or strain, e.g., Staphylococcus aureus, are provided in Example 6 under the heading "Determination of Antibiofilm Activity of L0482 Variants," which may be preferably used. One of these assays utilizes PEGylated biofilms (PBAs) in plasma and is particularly well suited for determining the killing activity of peptidoglycan hydrolases against surface-attached classical biofilms. Another assay utilizes planktonic aggregates (FBAs) in synovial fluid and is particularly well suited for determining the killing activity of peptidoglycan hydrolases against planktonic (biofilm-like) aggregates.

[0125] As used herein and in the context of the present invention, the term "activity" of a peptidoglycan hydrolase particularly refers to the bactericidal activity of the peptidoglycan hydrolase described herein. Furthermore, since the bactericidal activity of a peptidoglycan hydrolase against a particular bacterial species or strain, such as Staphylococcus aureus described herein, is strictly correlated with its ability to hydrolyze (and destroy and / or cleave) peptidoglycan in the cell wall of said bacterial species or strain, it is not necessary to further measure or determine the ability of the peptidoglycan hydrolase of the present invention to hydrolyze, destroy, and / or cleave peptidoglycan by enzyme assay; it is sufficient to determine the bactericidal activity of the peptidoglycan hydrolase, i.e., its killing activity against the bacterial species or strain described herein, such as Staphylococcus aureus. In other words, if a peptidoglycan hydrolase (e.g., endolysin) according to the present invention can kill the target bacteria described herein, it is considered to implicitly have the ability to hydrolyze peptidoglycan in the target bacteria. However, additional assays can be performed to further confirm that the peptidoglycan hydrolase actually functions as a "peptidoglycan hydrolase." For example, the reduction in OD620nm of purified peptidoglycan in the presence of the peptidoglycan hydrolase of the present invention can be easily measured. Furthermore, purified peptidoglycan can be incubated with the peptidoglycan hydrolase of the present invention, followed by analysis and identification of cleavage products by mass spectrometry. Furthermore, the YODA-derived method described herein for screening yeast cells for secretion of active peptidoglycan hydrolases according to the present invention can be used. As described herein, in the screening method, the "activity" of a peptidoglycan hydrolase, i.e., its killing activity against a particular bacterium, is determined by measuring its ability to destroy peptidoglycan contained in killed bacterial cells and / or fragments thereof or corresponding peptidoglycan particles (particularly via peptidoglycan hydrolysis).

[0126] Gram-positive and Gram-negative bacteria In Gram-positive bacteria, the cell membrane is surrounded by a layer of peptidoglycan. The main purpose of the cell wall of Gram-positive bacteria is to maintain the shape of the bacterium and resist pressure within the bacterial cell. Peptidoglycan, or murein, is a polymer composed of sugars and amino acids. The sugar components are composed of N-acetylglucosamine and N-acetylmuramic acid residues linked in a β-(1,4) fashion. Peptide chains consisting of 3-5 amino acids are attached to the N-acetylmuramic acid. Peptide chains can be crosslinked to other peptide chains to form 3D mesh-like layers. Peptide chains can contain both D- and L-amino acid residues, and their composition can vary depending on the type of bacteria.

[0127] In contrast to Gram-positive bacteria, Gram-negative bacteria have a characteristic asymmetric outer membrane bilayer consisting of an inner monolayer containing phospholipids (mainly phosphatidylethanolamine) and an outer monolayer composed primarily of lipopolysaccharide (LPS). This outer membrane is usually overlain by a peptidoglycan layer that is much thinner than that of Gram-positive bacteria.

[0128] As described herein, the peptidoglycan hydrolases of the invention have, in particular, killing activity against at least one Gram-positive bacterium, preferably a Staphylococcus species or strain, more preferably at least Staphylococcus aureus, as described herein.

[0129] Sequence identity to variant and reference sequence As used herein and in the context of the present invention, a polypeptide (e.g., a peptidoglycan hydrolase of the present invention) or a portion thereof (e.g., a CHAP domain of the present invention) derived from a particular polypeptide (e.g., L0482) or a portion thereof (e.g., a CHAP domain of L0482) is considered to be a "variant" of the polypeptide (e.g., a L0482 variant or a CHAP domain variant, respectively) or a portion thereof, as used herein and in the context of the present invention. Furthermore, a peptidoglycan hydrolase of the present invention comprising a CHAP domain derived from the CHAP domain of L0482 is also considered to be a L0482 variant as used herein and in the context of the present invention.

[0130] Herein, and in the context of the present invention, the terms "variant" and "mutant" may be used interchangeably.

[0131] A variant (e.g., a CHAP domain of the present invention) in particular has at least n% (i.e., at least 60%) sequence identity with a corresponding reference sequence (e.g., the sequence from positions 72 to 215 of SEQ ID NO: 1). As used herein, a reference sequence generally refers to the sequence of a (poly)peptide or a portion thereof from which a variant is derived (e.g., the CHAP domain of L0482). Moreover, a variant itself can be used as a reference sequence (e.g., H5; SEQ ID NO: 11) to describe other variants.

[0132] Furthermore, variants derived from a particular polypeptide (e.g., L0482) or a portion thereof (e.g., the CHAP domain of L0482) used as a reference sequence, i.e., a polypeptide (e.g., a peptidoglycan hydrolase of the present invention) or a portion thereof (e.g., a CHAP domain of the present invention), have at least one mutation, i.e., at least one amino acid substitution, deletion, insertion, and / or addition, preferably at least one amino acid substitution, compared to the reference sequence (e.g., at least one mutation in SEQ ID NO: 1 or in the sequence from positions 72 to 215 of SEQ ID NO: 1, respectively).

[0133] As generally understood in the art, and as used herein, and in the context of the present invention, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment. Typically, "sequence identity" is expressed as a percentage. Moreover, one of the two sequences can be considered a reference sequence to which the other sequence has at least n% sequence identity.

[0134] Thus, herein, and in the context of the present invention, a particular (poly)peptide (e.g., a peptidoglycan hydrolase of the invention) or portion thereof (e.g., a CHAP domain of the invention) can be structurally defined by having at least n% sequence identity to a corresponding reference sequence, where n is an integer between 60 and 99, in particular 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 or 99. As mentioned immediately above, the reference sequence refers to a (poly)peptide (e.g., SEQ ID NO: 1) or a portion thereof (e.g., the sequence from positions 72 to 215 of SEQ ID NO: 1) from which the (poly)peptide or portion thereof, respectively, is derived.

[0135] Furthermore, herein, and in the context of the present invention, a particular polypeptide or portion thereof may be specifically designated as having a minimum sequence identity of more than "at least 60%" to a corresponding reference sequence, i.e., at least n% sequence identity, where n is an integer between 61 and 99. For example, if the sequence identity is at least 80%, then n is an integer between 80 and 99.

[0136] Furthermore, when it is stated herein that a particular polypeptide or portion thereof has a minimum sequence identity of at least 90% with a corresponding reference sequence, i.e., at least n% sequence identity, where n is an integer between 90 and 99, n may refer to a tenth of a decimal point between 89.5 and 99.9, in particular 89.5, 89.6, 89.7, 89.9, 90.0, 90.1, 90.2, etc., or 99.9. In particular, when a minimum sequence identity is stated by an integer, i.e., at least n% where n is an integer (e.g., at least 95%), n may also refer to a tenth of a decimal point that can be rounded to said integer by conventional rounding rules. For example, at least 95% sequence identity can also refer to at least 94.5%, at least 94.6%, at least 94.7%, at least 94.8%, at least 94.9%, at least 95.0%, at least 95.1%, at least 95.2%, at least 95.3%, or at least 95.4% sequence identity.

[0137] Generally, herein, and in the context of the present invention, higher sequence identity to a reference sequence is preferred. Furthermore, certain minimum sequence identities (other than "at least 60%) are indicated in certain contexts herein and are thus considered preferred minimum sequence identities in those contexts. However, other minimum sequence identities to a reference sequence are also considered herein in the context of any (poly)peptide or portion thereof described herein, particularly at least n% sequence identity, where n is an integer between 60 and 99, or n% sequence identity, where n is a decimal point between 89.5 and 99.9. Notably, the term "(poly)peptide," as used herein, may refer to a polypeptide or peptide as generally understood in the art.

[0138] The degree of sequence identity can be determined according to methods well known in the art, preferably using a suitable computer algorithm such as BLAST and / or CLUSTAL Omega. In particular, BLAST may be used in combination with CLUSTAL Omega.

[0139] Furthermore, computer algorithms such as BLAST allow for the identification and comparison of peptidoglycan hydrolase variants with a certain sequence coverage (e.g., at least 80%) and sequence identity (e.g., at least 60%) to a reference sequence. Coverage is a filter that selects sequences with the same structure as a reference sequence, e.g., LYSM-CHAP endolysin.

[0140] For example, in the context of the present invention, and as illustrated in the accompanying examples, endolysin sequences were isolated from the NCBI nucleotide database using BLAST, and sequences were first filtered with a cutoff of 80% sequence coverage to L0482 (SEQ ID NO: 1) or certain variants thereof, followed by a cutoff of 60% identity, and then aligned using CLUSTAL Omega.

[0141] When using the Clustal Omega analysis method (eg, in combination with BLAST) to determine whether a particular sequence is, for example, at least 60% identical to a reference sequence, default settings may be used.

[0142] Preferably, Clustal Omega (Madeira F, Park YM, Lee J et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Research. 2019 Jul;47(W1):W636-W641. DOI: 10.1093 / nar / gkz268. PMID: 30976793; PMCID: PMC6602479) is used for comparing amino acid sequences in the context of the present invention. For paired comparison / alignment, the following default settings are preferably selected: Program: clustalo; Version: 1.2.4; Input parameters: Output guide tree: True; Output distance matrix: False; Dealign input sequences: False; mBed-like clustering guide tree: True; mBed-like clustering iterations: True; Number of iterations: 0; Max guide tree iterations: -1; Max HMM iterations: -1; Output alignment format: clustal_num; Output order: Aligned order; Sequence type: Protein. Preferably, the degree of sequence identity is calculated over the entire length of the reference sequence.

[0143] Amino acid residues located at positions corresponding to positions in a reference sequence, for example, the amino acid sequence shown in SEQ ID NO: 1, can be easily identified by those skilled in the art using methods known in the art. For example, such amino acid residues can be identified by aligning the sequence with a reference sequence, for example, the sequence shown in SEQ ID NO: 1, and identifying positions corresponding to the indicated positions in the reference sequence, for example, SEQ ID NO: 1. Alignment can be performed using means and methods known to those skilled in the art, for example, the Lipman-Pearson method (Science 227 (1985), 1435) or known computer algorithms such as the CLUSTAL algorithm. In such alignments, it is preferable to assign maximum homology to conserved amino acid residues present in the amino acid sequences.

[0144] Preferably, in the context of the present invention, Clustal Omega is used for comparing amino acid sequences and therefore for determining positions corresponding to positions in a reference sequence. For pairwise comparison / alignment, the following default settings are preferably selected: Program: clustalo; Version: 1.2.4; Input parameters: Output guide tree: True; Output distance matrix: False; Dealign input sequence: False; mBed-like clustering guide tree: True; mBed-like clustering iterations: True; Number of iterations: 0; Maximum guide tree iterations: -1; Maximum HMM iterations: -1; Output alignment format: clustal_num; Output order: Aligned order; Sequence type: Protein. When amino acid sequences (e.g., of L0482 variants) are aligned by such a method, the positions of corresponding amino acid residues can be determined (e.g., in each of the L0482 variants) regardless of insertions, deletions, or additions that occur in the amino acid sequences.

[0145] For example, SEQ ID NO: 1 (WT L0482) has a length of 215 amino acids and has an arginine ("R") at position 86. For example, L0482 hit variant H3 (SEQ ID NO: 9) does not exhibit any deletions, insertions, or additions compared to the sequence of SEQ ID NO: 1 and has a lysine ("K") at position 86. Thus, H3 (SEQ ID NO: 9) has an amino acid substitution at position 86 with reference to SEQ ID NO: 1, where the residue at said position (i.e., arginine) has been replaced with a lysine. In short, H3 has an R86K mutation with reference to SEQ ID NO: 1 described herein. In L0482 hit variant "H1" (SEQ ID NO: 7), however, five amino acid residues have been deleted from the N-terminus. Thus, only H1 has a length of 210 amino acid residues. However, by aligning SEQ ID NO:1 and SEQ ID NO:7 by standard means described herein and illustrated in the accompanying Examples, it is immediately apparent that H1 (SEQ ID NO:7), like H3, has a lysine ("K") at the position corresponding to position 86 of SEQ ID NO:1; see, e.g., Figure 9A. Thus, H1 (SEQ ID NO:7) is considered herein, and in the context of the present invention, to have an amino acid substitution at the position corresponding to position 86 of SEQ ID NO:1, where the residue at said position (i.e., arginine) has been replaced with a lysine. In short, H1 has the same R86K mutation as other hit variants, such as H3, with reference to SEQ ID NO:1.

[0146] Similar logic also applies to any other mutations, such as amino acid substitutions, described herein and in the context of the present invention.

[0147] In the context of the present specification and the present invention, an "amino acid substitution" or, simply, "substitution" at a specific position in a reference amino acid sequence or at a position corresponding to a specific position in a reference sequence (e.g., SEQ ID NO: 1) means that the amino acid residue at that position is replaced with another amino acid residue as described herein. In particular, the term "amino acid substitution" or "substituted with another amino acid residue" means that the respective amino acid residue at the indicated position can be replaced with any other possible amino acid residue, such as a naturally occurring amino acid or a non-naturally occurring amino acid (Brustad and Arnold, Curr. Opin. Chem. Biol. 15 (2011), pp. 201-210), preferably a naturally occurring amino acid, i.e., an amino acid residue selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0148] As used herein and in the context of the present invention, particularly with respect to mutations in amino acid sequences, "deleted" or "deletion" means that an amino acid is missing at the indicated position.

[0149] As used herein, and in the context of the present invention, particularly with respect to mutations within an amino acid sequence, "inserted" or "insertion" means that at each position at least one amino acid residue, e.g., one or two, preferably one residue, has been inserted after the indicated position.

[0150] As used herein, and in the context of the present invention, particularly with respect to variations in amino acid sequences, "added" or "addition" means that at least one amino acid residue is added to the N-terminus and / or C-terminus of the reference sequence.

[0151] Furthermore, standard tools, preferably Clustal Omega, are used to determine the sequence identity of a nucleic acid sequence to a corresponding reference nucleic acid sequence in the context of the present invention, preferably using default settings.

[0152] CHAP domain of the present invention and peptidoglycan hydrolase comprising the CHAP domain of the present invention As described hereinabove, the present invention relates in some aspects to a peptidoglycan hydrolase comprising a CHAP domain according to the present invention. In particular, the peptidoglycan hydrolase has bactericidal activity, preferably against Gram-positive bacteria, more preferably against Staphylococcus species or strains, most preferably against Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA) strains, preferably as described herein. The peptidoglycan hydrolase of the present invention may have at least 60% sequence identity with the sequence of SEQ ID NO: 1 as described herein.

[0153] In particular, in the context of this specification and the present invention, a CHAP domain of the present invention (i) has at least 60% sequence identity with the sequence of positions 72 to 215 of SEQ ID NO: 1, and (ii) has one or more amino acid substitutions compared to the sequence of positions 72 to 215 of SEQ ID NO: 1 described herein. Furthermore, a CHAP domain of the present invention may have one or more amino acid deletions, insertions, or additions compared to the sequence of positions 72 to 215 of SEQ ID NO: 1. However, the degree of amino acid substitution, deletion, insertion, and / or addition is not such that the sequence identity of the CHAP domain of the present invention to the sequence of positions 72 to 215 of SEQ ID NO: 1 is less than 60%.

[0154] In particular, the CHAP domains of the present invention function as peptidoglycan hydrolases and preferably have bactericidal activity as described herein.

[0155] As already mentioned above, the sequence from positions 72 to 215 of SEQ ID NO: 1 is also shown in SEQ ID NO: 301.

[0156] Thus, a CHAP domain according to the invention, in other words, has at least 60% sequence identity with the sequence of SEQ ID NO:301 and has one or more amino acid substitutions compared to the sequence of SEQ ID NO:301.

[0157] As described herein, comparison with a reference sequence particularly involves performing a sequence alignment. In this way, positions in a CHAP domain of the present invention corresponding to positions in SEQ ID NO: 1 can be easily identified, regardless of the presence of any amino acid deletions, insertions, or additions. In particular, in the absence of any deletions, insertions, or additions, a CHAP domain of the present invention has one or more amino acid substitutions in the sequence from positions 72 to 215 of SEQ ID NO: 1. Otherwise, a CHAP domain of the present invention may have one or more amino acid substitutions in the sequence from position 72 to position 215 of SEQ ID NO: 1 in a sequence alignment (and may have deletions, insertions, and / or additions compared to positions 72 to 215 of SEQ ID NO: 1). In this context, these positions do not necessarily need to be present in a CHAP domain of the present invention, since other positions between positions 72 and 215 are sufficient for performing a sequence alignment.

[0158] In certain embodiments, the peptidoglycan hydrolase of the invention consists of a CHAP domain of the invention, in particular a sequence that (i) has at least 60% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO: 1; and (ii) has one or more amino acid substitutions compared to the sequence from positions 72 to 215 of SEQ ID NO: 1. In particular, the CHAP domain (and therefore the peptidoglycan hydrolase) has bactericidal activity, preferably against a Staphylococcus species or strain described herein, more preferably Staphylococcus aureus.

[0159] Thus, in some aspects, the present invention relates to CHAP domains of the invention, i.e., CHAP domains that (i) have at least 60% sequence identity with the sequence of positions 72 to 215 of SEQ ID NO: 1, and (ii) have one or more amino acid substitutions as described herein compared to the sequence of positions 72 to 215 of SEQ ID NO: 1. In particular, the CHAP domains of the invention may be advantageously used in peptidoglycan hydrolases of the invention (e.g., in combination with a cell wall-binding domain), or are themselves considered to be peptidoglycan hydrolases of the invention.

[0160] In certain embodiments, the peptidoglycan hydrolase of the invention (i.e., the peptidoglycan hydrolase comprising or consisting of the CHAP domain of the invention) has at least 60% sequence identity with the sequence of SEQ ID NO: 1. This sequence identity is specifically calculated over the entire length of the sequence of SEQ ID NO: 1 (not over the entire length of the sequence of the peptidoglycan hydrolase of the invention). Thus, any additional domains, peptides, or tags, such as a signal peptide or PK tag, that may be included in (or fused to) the peptidoglycan hydrolase of the invention should not be taken into account when determining the sequence identity of the peptidoglycan hydrolase of the invention to the sequence of SEQ ID NO: 1. Thus, the peptidoglycan hydrolase of the invention may comprise (I) an amino acid sequence having at least 60% sequence identity with the sequence of SEQ ID NO: 1 (including the CHAP domain of the invention, and optionally the LYSM domain and / or the peptide linker of the invention), and optionally (II) one or more additional domains, peptides, or tags, such as a signal peptide, a PK tag, an additional peptide linker, etc., as described herein. In particular, the CHAP domain (i) has at least 60% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO: 1, and (ii) has one or more amino acid substitutions compared to the sequence from positions 72 to 215 of SEQ ID NO: 1 described herein.

[0161] Deglycosylation mutations As described herein and illustrated in the accompanying Examples, removal of the glycosylation sites in L0482 (i.e., positions 68 and 73 of SEQ ID NO: 1), resulting in a so-called "deglycosylated" L0482 variant, strongly enhanced bactericidal activity when expressed in eukaryotic cells; see, e.g., Example 4.

[0162] Position 68 of SEQ ID NO: 1 is in the linker sequence of L0482, whereas position 73 of SEQ ID NO: 1 is in the CHAP domain of L0482. As already mentioned above, the CHAP domain is considered herein to be particularly important, and in particular more important than the linker, for the function of the peptidoglycan hydrolase of the invention, and in the context of the present invention. Thus, a deglycosylation mutation at or corresponding to position 73 of SEQ ID NO: 1 is preferred, in the context of the present invention, to a deglycosylation mutation at or corresponding to position 68 of SEQ ID NO: 1.

[0163] Thus, in some embodiments, for example, in the context of deglycosylation mutations and deglycosylated L0482 variants, the CHAP domain of the invention has a mutation, in particular an amino acid substitution or deletion, at position 73 of SEQ ID NO:1 or a position corresponding to this position, as described herein, for example, as specified in subsequent embodiments. In other words, in the CHAP domain of the invention, the arginine ("N") at position 73 of SEQ ID NO:1 or the arginine at a position corresponding to position 73 of SEQ ID NO:1 may be deleted as described herein, or preferably substituted with another amino acid residue. As described in detail herein, said position 73 may be considered herein, and in the context of the present invention, to be a glycosylation position that is preferably deglycosylated. Therefore, the CHAP domain of the invention preferably has a deglycosylation mutation, preferably an amino acid substitution, at position 73 of SEQ ID NO:1 or a position corresponding to this position, as described herein.

[0164] Thus, in some preferred embodiments, the CHAP domains of the invention have an amino acid substitution at position 73 of SEQ ID NO:1 or a position corresponding to this position, particularly a deglycosylation substitution as described herein.

[0165] In certain embodiments, for example, in the context of a mutation or substitution at position 73 of SEQ ID NO: 1 or a position corresponding to this position, the peptidoglycan hydrolase of the invention does not have a sequence set forth in any one of SEQ ID NOs: 276-278.

[0166] Herein, and in the context of the present invention, particularly in the context of the CHAP domain of the present invention, the residue at or corresponding to position 73 of SEQ ID NO: 1 may be substituted with another amino acid residue other than, for example, methionine, phenylalanine or lysine.

[0167] In certain embodiments, for example, with respect to peptidoglycan hydrolases consisting of a CHAP domain of the invention and / or lacking a cell wall binding domain as described herein, the residue at or corresponding to position 73 of SEQ ID NO:1 is substituted with another amino acid residue other than methionine.

[0168] In a further embodiment of the peptidoglycan hydrolase or CHAP domain of the invention, the residue at or corresponding to position 73 of SEQ ID NO:1 is substituted with another amino acid residue other than phenylalanine or lysine.

[0169] Preferably, herein, and in the context of the present invention, particularly in the context of the CHAP domain and / or deglycosylation substitutions of the present invention, the residue at or corresponding to position 73 of SEQ ID NO: 1 is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine, or serine, e.g., glycine, tyrosine, leucine, glutamic acid, alanine, or histidine.

[0170] In some embodiments, particularly in the context of the peptidoglycan hydrolases of the invention and / or the CHAP domains of the invention, the residue at or corresponding to position 73 of SEQ ID NO: 1 is substituted with another amino acid residue other than phenylalanine, lysine, or serine.

[0171] Preferably, herein, and in the context of the present invention, particularly in the context of the CHAP domain and / or deglycosylation substitutions of the present invention, the residue at or corresponding to position 73 of SEQ ID NO: 1 is substituted with glycine. For example, a CHAP domain of the present invention may have the sequence shown at positions 72 to 215 of SEQ ID NO: 2.

[0172] In certain embodiments, particularly in the context of peptidoglycan hydrolases having at least 60% sequence identity with the sequence of SEQ ID NO: 1, the peptidoglycan hydrolases of the invention further comprise a deletion or amino acid substitution (preferably an amino acid substitution) at position 68 of SEQ ID NO: 1 or a position corresponding to this position. In other words, the arginine at position 68 of SEQ ID NO: 1 or a position corresponding to this position may be deleted or, preferably, substituted with another amino acid residue as described herein. As already indicated above, a mutation at position 68 of SEQ ID NO: 1 or a position corresponding to this position may be considered a deglycosylating mutation herein and in the context of the present invention, particularly in the context of peptidoglycan hydrolases having at least 60% sequence identity with the sequence of SEQ ID NO: 1.

[0173] In certain embodiments, particularly in the context of peptidoglycan hydrolases of the invention having at least 60% sequence identity to the sequence of SEQ ID NO: 1, the residue at or corresponding to position 68 of SEQ ID NO: 1 is substituted with another amino acid residue other than, for example, threonine, serine, or lysine, e.g., another amino acid residue other than threonine or serine.

[0174] In certain embodiments, particularly in the context of peptidoglycan hydrolases of the invention having at least 60% sequence identity with the sequence of SEQ ID NO: 1, the residue at or corresponding to position 68 of SEQ ID NO: 1 is substituted with another amino acid residue other than threonine, serine, or lysine, for example, another amino acid residue other than threonine or serine, and / or the residue at or corresponding to position 73 of SEQ ID NO: 1 is substituted with another amino acid residue other than phenylalanine, lysine, or serine, for example, another amino acid residue other than phenylalanine or lysine.

[0175] In certain preferred embodiments, particularly in the context of peptidoglycan hydrolases of the invention having at least 60% sequence identity with SEQ ID NO: 1, the residue at or corresponding to position 68 of SEQ ID NO: 1 is substituted with lysine, methionine, arginine, or alanine, preferably lysine. Additionally, the residue at or corresponding to position 73 of SEQ ID NO: 1 may be substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine, or serine, preferably glycine.

[0176] In certain preferred embodiments, particularly in the context of peptidoglycan hydrolases of the invention having at least 60% sequence identity to the sequence of SEQ ID NO: 1, the peptidoglycan hydrolase has a pair of amino acid substitutions at positions 68 and 73 of SEQ ID NO: 1 or positions corresponding to these positions, a) the residue at or corresponding to position 68 of SEQ ID NO: 1 is substituted with lysine, and the residue at or corresponding to position 73 of SEQ ID NO: 1 is substituted with glycine, serine, tyrosine, or leucine; b) the residue at or corresponding to position 68 of SEQ ID NO: 1 is substituted with methionine, and the residue at or corresponding to position 73 of SEQ ID NO: 1 is substituted with leucine; c) the residue at or corresponding to position 68 of SEQ ID NO: 1 is substituted with arginine, and the residue at or corresponding to position 73 of SEQ ID NO: 1 is substituted with glutamic acid; or d) The residue at or corresponding to position 68 of SEQ ID NO: 1 is substituted with alanine, and the residue at or corresponding to position 73 of SEQ ID NO: 1 is substituted with alanine or histidine.

[0177] In certain preferred embodiments, particularly in the context of peptidoglycan hydrolases of the invention having at least 60% sequence identity to the sequence of SEQ ID NO: 1, the residue at or corresponding to position 68 of SEQ ID NO: 1 is substituted with lysine, and the residue at or corresponding to position 73 of SEQ ID NO: 1 is substituted with glycine.

[0178] As already mentioned above, the peptidoglycan hydrolases of the invention, especially those having at least one deglycosylating mutation in the CHAP domain (e.g., an amino acid substitution at position 73 of SEQ ID NO: 1 or a position corresponding to this position, e.g., K73G as shown in SEQ ID NO: 2), are particularly good starting points for further protein engineering and / or directed evolution.

[0179] Thus, the peptidoglycan hydrolases of the invention may have at least one amino acid substitution described herein in the context of beneficial / tolerated, particularly beneficial, or most beneficial amino acid substitutions found in L0482 variants obtained by directed evolution (see, e.g., Examples 5-7) or found in the context of deimmunization analyses or experiments (see, e.g., Example 10), instead of or in addition to (preferably in addition to) mutations at positions 68 and / or 73 of SEQ ID NO: 1 or positions corresponding to these positions, as described herein, e.g., in the immediately preceding embodiment. Beneficial / tolerated, particularly beneficial, most beneficial amino acid or deimmunization amino acid substitutions are described herein, e.g., below.

[0180] Beneficial and tolerated amino acid substitutions found in deglycosylation screens, directed evolution, or in silico deimmunization screens As described herein and illustrated in the accompanying Examples, the inventors have found 252 L0482 variants that are active and secreted from eukaryotic cells. These variants contained various amino acid substitutions that are believed herein to be beneficial or at least tolerable with respect to the pharmaceutical properties of the peptidoglycan hydrolases of the invention; see, e.g., Example 7 and Table 3. In particular, the amino acid substitutions may be beneficial (or at least tolerable) with respect to the bactericidal activity (e.g., against Staphylococcus aureus) and secretion ability from eukaryotic cells (e.g., human cells) of the L0482 variants described herein.

[0181] Furthermore, the deimmunizing amino acid substitutions identified in silico may be considered beneficial with respect to the safety and / or efficacy of the L0482 variant in the context of medical use, as well as at least tolerable with respect to other pharmaceutical properties such as bactericidal activity; see, e.g., Example 10 and Table 5.

[0182] Furthermore, the inventors have discovered an amino acid substitution at position 73 of SEQ ID NO: 1, a deglycosylation substitution in the CHAP domain of L0482, that enhances bactericidal activity when expressed in eukaryotic cells; see, e.g., Example 4.

[0183] The term "at least tolerable" means that the introduction of the corresponding amino acid substitution does not abolish the functionality of the L0482 variant as a drug. In other words, a tolerable amino acid substitution, as used herein, does not deteriorate the pharmaceutical properties of the L0482 variant to the extent that it cannot be used as a drug. As a minimum requirement for being useful as a drug, an L0482 variant must have the bactericidal activity described herein. Furthermore, an L0482 variant ideally should have sufficient stability when used as a drug, as described herein. When an L0482 variant is provided in the form of a nucleic acid (e.g., RNA) for medical use, the L0482 variant ideally should also be capable of being secreted from mammalian cells. However, a tolerable amino acid substitution alone does not necessarily render an L0482 variant suitable for pharmaceutical use or improve the pharmaceutical properties of the L0482 variant. Thus, a tolerable amino acid substitution may rather be used in combination with at least one particularly beneficial amino acid substitution, or preferably in combination with at least one of the most beneficial amino acid substitutions described herein.

[0184] A "beneficial" amino acid substitution, as used herein, may improve or contribute to the improvement of at least one pharmaceutical property of the L0482 variant, such as bactericidal activity, secretion ability from mammalian cells, stability, and / or reduced immunogenicity. Beneficial amino acid substitutions may be used alone, but they are preferably used in combination with at least one particularly beneficial amino acid substitution, or more preferably, in combination with at least one of the most beneficial amino acid substitutions described herein.

[0185] Thus, in some embodiments, particularly in the context of tolerant / beneficial and / or deimmunizing mutations and active, secreted L0482 variants obtained by directed evolution, the CHAP domains of the invention comprise one or more amino acid substitutions at positions 72-83, 85, 86, 93, 96, 99, 102-104, 107, 108, 111, 113-115, 117, 121-125, 129-131, 133-145, 148, 149, 152, 153, 155, 157, 159, 166, 169, 170, 173-178, 185, 186, 190-194, 196-199, 201, 203-207, and 212-215 of SEQ ID NO: 1, or positions corresponding to these positions. Preferably, in these embodiments, the amino acid residue at or corresponding to position 72 of SEQ ID NO: 1 is substituted with phenylalanine, aspartic acid, histidine, or threonine; the amino acid residue at position 73 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine, serine, aspartic acid, or threonine; the amino acid residue at position 74 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine or proline; the amino acid residue at position 75 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine, proline, aspartic acid, or glutamine; the amino acid residue at position 76 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine, aspartic acid, or asparagine; the amino acid residue at position 77 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, aspartic acid, glycine, histidine, lysine, asparagine, glutamine, serine, or threonine; the amino acid residue at position 78 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine or tyrosine; the amino acid residue at position 79 of SEQ ID NO: 1 or a position corresponding to this position is substituted with histidine or tryptophan; the amino acid residue at position 80 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, aspartic acid, methionine, or asparagine; the amino acid residue at position 81 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid, arginine, serine, alanine, or glycine; the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine or glycine; the amino acid residue at position 83 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine; the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine, methionine, or tyrosine; the amino acid residue at position 93 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or leucine; the amino acid residue at position 96 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine; the amino acid residue at position 99 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine, methionine, glutamine, threonine, or valine; the amino acid residue at position 102 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, glycine, serine, or threonine; the amino acid residue at position 103 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or glycine; the amino acid residue at position 104 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine, aspartic acid, methionine, glutamine, or tyrosine; the amino acid residue at position 107 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine, tyrosine, or alanine; the amino acid residue at position 108 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine, glycine, lysine, or tyrosine; the amino acid residue at position 111 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 113 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 114 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine or threonine; the amino acid residue at position 115 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine, aspartic acid, glycine, or serine; the amino acid residue at position 117 of SEQ ID NO: 1 or a position corresponding thereto is substituted with histidine, aspartic acid, or asparagine; the amino acid residue at position 121 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine; the amino acid residue at position 122 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine; the amino acid residue at position 123 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, serine, threonine, or tryptophan; the amino acid residue at position 124 of SEQ ID NO: 1 or a position corresponding thereto is substituted with phenylalanine, histidine, glutamine, or threonine; the amino acid residue at position 125 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine; the amino acid residue at position 129 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine or serine; the amino acid residue at position 130 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine, isoleucine, asparagine, or tyrosine; the amino acid residue at position 131 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, glutamine, or tyrosine; the amino acid residue at position 133 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine, threonine, or glutamine; the amino acid residue at position 134 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine, valine, lysine, or glutamine; the amino acid residue at position 135 of SEQ ID NO: 1 or a position corresponding to this position is substituted with cysteine, histidine, or serine; the amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine, arginine, glutamic acid, or threonine; the amino acid residue at position 137 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 138 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine; the amino acid residue at position 139 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, aspartic acid, glutamine, or serine; the amino acid residue at position 140 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or aspartic acid; the amino acid residue at position 141 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine, aspartic acid, histidine, or threonine; the amino acid residue at position 142 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid or arginine; the amino acid residue at position 143 of SEQ ID NO: 1 or a position corresponding to this position is substituted with proline; the amino acid residue at position 144 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid or glutamine; the amino acid residue at position 145 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or leucine; the amino acid residue at position 148 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine or valine; the amino acid residue at position 149 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or threonine; the amino acid residue at position 152 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine; the amino acid residue at position 153 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine or serine; the amino acid residue at position 155 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 157 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid; the amino acid residue at position 159 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine; the amino acid residue at position 166 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, glutamic acid, or threonine; the amino acid residue at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine or aspartic acid; the amino acid residue at position 170 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, histidine, or serine; the amino acid residue at position 173 of SEQ ID NO: 1 or a position corresponding to this position is substituted with histidine, asparagine, or arginine; the amino acid residue at position 174 of SEQ ID NO: 1 or a position corresponding thereto is substituted with alanine, histidine, lysine, arginine, serine, or threonine; the amino acid residue at position 175 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, glutamine, or threonine; the amino acid residue at position 176 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid, arginine, alanine, glutamine, serine, or threonine; the amino acid residue at position 177 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine; the amino acid residue at position 178 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine, arginine, serine, or threonine; the amino acid residue at position 185 of SEQ ID NO: 1 or a position corresponding thereto is substituted with phenylalanine, histidine, serine, tyrosine, aspartic acid, or asparagine; the amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 190 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine, alanine, cysteine, aspartic acid, histidine, asparagine, serine, or threonine; the amino acid residue at position 191 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid, arginine, aspartic acid, or asparagine; the amino acid residue at position 192 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine, threonine, glutamic acid, glycine, or lysine; the amino acid residue at position 193 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid; the amino acid residue at position 194 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, glycine, proline, or threonine; the amino acid residue at position 196 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine or threonine; the amino acid residue at position 197 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine or proline; the amino acid residue at position 198 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine; the amino acid residue at position 199 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, asparagine, serine, or threonine; the amino acid residue at position 201 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, serine, or lysine; the amino acid residue at position 203 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid or valine; the amino acid residue at position 204 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, serine, or tyrosine; the amino acid residue at position 205 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine or valine; the amino acid residue at position 206 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine; the amino acid residue at position 207 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine, valine, histidine, or tryptophan; the amino acid residue at position 212 of SEQ ID NO: 1 or a position corresponding thereto is substituted with tyrosine, aspartic acid, glutamic acid, histidine, lysine, or asparagine; the amino acid residue at position 213 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine, tyrosine, or threonine; the amino acid residue at position 214 of SEQ ID NO: 1 or a position corresponding thereto is substituted with glutamine or threonine; and / or The amino acid residue at position 215 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine, valine, or serine.

[0186] The amino acid substitutions listed above correspond to the amino acid residues not enclosed in parentheses, i.e., not marked with "()," shown in the "Beneficial / Tolerant Residues" column of Table 3 in Example 7, and those shown in the "Deimmunization" column of Table 5 in Example 10. In particular, the positions (abbreviated as "pos") in Tables 3 and 5 correspond to positions in SEQ ID NO: 1. Thus, an amino acid residue listed at a particular position in these tables means that the amino acid at that position or at a position corresponding to that position in SEQ ID NO: 1 is substituted with the amino acid residue shown at that position in the "Beneficial / Tolerant Residues" column of Table 3 and / or the "Deimmunization" column of Table 5. As an illustrative example in this context, "ER(H)" in the "Beneficial / Tolerated Residues" column of Table 3 (Table 5) means that the amino acid residue at position 176 of SEQ ID NO: 1 or the position corresponding to position 176 of SEQ ID NO: 1 (i.e., lysine; "K") is substituted with glutamic acid ("E") or arginine ("R") in the L0482ag variant, which was found to be secreted from yeast cells and active by YODA, as described herein and illustrated in the accompanying Examples. Thus, said mutation, i.e., amino acid substitution, is considered herein and in the context of the present invention to be "beneficial" or at least "tolerated" with respect to the desired pharmaceutical properties of peptidoglycan hydrolase, in particular sufficient killing activity (e.g., against Staphylococcus aureus), sufficient stability, and sufficient ability to be secreted from eukaryotic cells. Additionally, amino acid residues indicated in parentheses in the "Beneficial / Acceptable Residues" column of Table 3 (e.g., histidine "(H)" at position 176 in the illustrative example above) may also be at least acceptable with respect to the pharmaceutical properties.

[0187] Table 3 (Table 5) shows positions in SEQ ID NO: 1 where beneficial or tolerant amino acid substitutions have been found. These are positions where the "Beneficial / Tolerant Residue" column in Table 3 (Table 5) shows at least one amino acid residue, regardless of whether the residue is shown without parentheses or within parentheses. These positions are also referred to herein as "tolerant positions." Furthermore, positions 72-215 in Table 3 (Table 5), where the "Beneficial / Tolerant Residue" column shows at least one amino acid residue, regardless of whether the residue is shown without parentheses or within parentheses, are considered to be "tolerant positions" in the CHAP domain herein. In the context of this specification and the present invention, an amino acid residue in SEQ ID NO: 1 corresponding to a beneficial or tolerant position may be substituted with an amino acid residue shown in the "Beneficial / Tolerant Residue" column in Table 3 (Table 5) for that position, i.e., a residue shown without parentheses or within parentheses in that column of Table 3, preferably a residue shown without parentheses in that column of Table 3.

[0188] In some embodiments, particularly in the context of permissive / beneficial mutations and Table 3, the CHAP domain of the invention comprises at least one amino acid substitution at a permissive position in the CHAP domain or at a position corresponding to said permissive position in the CHAP domain. Preferably, in these embodiments, at least one amino acid residue at said permissive position in the CHAP domain or at a position corresponding to said permissive position in the CHAP domain is substituted with the amino acid residue shown for said position in the "Beneficial / Permissive Residue" column of Table 3, more preferably with the residue shown without brackets in said column of Table 3.

[0189] In some embodiments, particularly in the context of peptidoglycan hydrolases having at least 60% sequence identity with the sequence of SEQ ID NO: 1, tolerated / beneficial mutations, and Table 3, the peptidoglycan hydrolases of the invention have at least one amino acid substitution at a permissive position or a position corresponding to said permissive position. Preferably, in these embodiments, at least one amino acid residue at said permissive position or a position corresponding to said permissive position is substituted with the amino acid residue shown in the "Beneficial / Tolerated Residue" column of Table 3 for said position, more preferably with the residue shown without parentheses in said column of Table 3.

[0190] Thus, in certain embodiments, particularly in the context of permissive / beneficial mutations as well as active, secreted L0482 variants obtained by directed evolution, the CHAP domain of the present invention may comprise any of the sequences 72-76, 78, 81, 82, 85, 86, 93, 96, 104, 107, 108, 111, 113, 115, 117, 121, 124, 125, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 2 and one or more amino acid substitutions at positions 30, 133-136, 138, 140-142, 144, 145, 148, 149, 152, 153, 155, 157, 159, 169, 173, 175-178, 185, 186, 190-194, 197, 198, 199, 201, 203, 204, 207, and 212-215, or positions corresponding to these positions. Preferably, in these embodiments, the amino acid residue at position 72 of SEQ ID NO: 1, or a position corresponding to this position, is substituted with phenylalanine, the amino acid residue at position 73 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine or serine; the amino acid residue at position 74 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine or proline; the amino acid residue at position 75 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine or proline; the amino acid residue at position 76 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 78 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine or tyrosine; the amino acid residue at position 81 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid, arginine, or serine; the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine or methionine; the amino acid residue at position 93 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or leucine; the amino acid residue at position 96 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine; the amino acid residue at position 104 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine; the amino acid residue at position 107 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine or tyrosine; the amino acid residue at position 108 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 111 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 113 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 115 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 117 of SEQ ID NO: 1 or a position corresponding to this position is substituted with histidine; the amino acid residue at position 121 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine; the amino acid residue at position 124 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine or histidine; the amino acid residue at position 125 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine; the amino acid residue at position 129 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine or serine; the amino acid residue at position 130 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine, isoleucine, or asparagine; the amino acid residue at position 133 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine or threonine; the amino acid residue at position 134 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine or valine; the amino acid residue at position 135 of SEQ ID NO: 1 or a position corresponding to this position is substituted with cysteine ​​or histidine; the amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine or arginine; the amino acid residue at position 138 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine; the amino acid residue at position 140 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or aspartic acid; the amino acid residue at position 141 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine; the amino acid residue at position 142 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid or arginine; the amino acid residue at position 144 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid; the amino acid residue at position 145 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or leucine; the amino acid residue at position 148 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine or valine; the amino acid residue at position 149 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or threonine; the amino acid residue at position 152 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine; the amino acid residue at position 153 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine or serine; the amino acid residue at position 155 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 157 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid; the amino acid residue at position 159 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine; the amino acid residue at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine; the amino acid residue at position 173 of SEQ ID NO: 1 or a position corresponding to this position is substituted with histidine, asparagine, or arginine; the amino acid residue at position 175 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, glutamine, or threonine; the amino acid residue at position 176 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid or arginine; the amino acid residue at position 177 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine; the amino acid residue at position 178 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine or arginine; the amino acid residue at position 185 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine, histidine, serine, or tyrosine; the amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 190 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine; the amino acid residue at position 191 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid or arginine; the amino acid residue at position 192 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine or threonine; the amino acid residue at position 193 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid; the amino acid residue at position 194 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine; the amino acid residue at position 197 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 198 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine; the amino acid residue at position 199 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine; the amino acid residue at position 201 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid or serine; the amino acid residue at position 203 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid or valine; the amino acid residue at position 204 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, serine, or tyrosine; the amino acid residue at position 207 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine or valine; the amino acid residue at position 212 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 213 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine or threonine; the amino acid residue at position 214 of SEQ ID NO: 1 or a position corresponding thereto is substituted with glutamine or threonine; and / or The amino acid residue at position 215 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine, valine, or serine.

[0191] The amino acid substitutions described immediately herein refer to all substitutions found in sequenced L0482 variants secreted from yeast cells and determined to be active by the YODA method described herein and illustrated in the accompanying Examples; see, e.g., Example 7 and Table 3, particularly the "beneficial / tolerated residues" shown without parentheses in Table 3.

[0192] Particularly beneficial amino acid substitutions Furthermore, the present inventors have determined hit variants among all of these L0482 variants that are particularly well suited for production in eukaryotic cells and that may have particularly beneficial pharmaceutical properties as described herein; see, for example, Examples 5 to 7, Table 4, and Figures 8 and 9. As described herein, the amino acid substitutions found in these hit variants, i.e., G1 to G4, H1 to H10, and I1 to I30, are considered herein, and in the context of the present invention, to be "particularly beneficial mutations" or "particularly beneficial amino acid substitutions" that may be particularly beneficial for maintaining or enhancing desirable pharmaceutical properties of peptidoglycan hydrolase, in particular bactericidal activity (e.g., against Staphylococcus aureus), stability, and / or secretion ability from eukaryotic cells, e.g., human cells.

[0193] Herein, and in the context of the present invention, particularly beneficial amino acid substitutions are preferred over amino acid substitutions described herein as "beneficial" or "tolerated."

[0194] Thus, in some preferred embodiments, the CHAP domains of the invention have one or more amino acid substitutions at positions 73, 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169, 173, 175, 178, 185, 186, 191, 192, 194, 198, 204, 212 and 215 of SEQ ID NO: 1, or positions corresponding to these positions. Preferably, in these embodiments, the amino acid residue at position 73 of SEQ ID NO: 1, or a position corresponding to this position, is substituted with glycine or serine, the amino acid residue at position 75 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine; the amino acid residue at position 78 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 81 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid; the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; the amino acid residue at position 104 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine; the amino acid residue at position 107 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 115 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 124 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine; the amino acid residue at position 125 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine; the amino acid residue at position 130 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine; the amino acid residue at position 133 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 135 of SEQ ID NO: 1 or a position corresponding to this position is substituted with histidine; the amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine or arginine; the amino acid residue at position 140 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine; the amino acid residue at position 141 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine; the amino acid residue at position 155 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine; the amino acid residue at position 173 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine; the amino acid residue at position 175 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine; the amino acid residue at position 178 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine; the amino acid residue at position 185 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 191 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 192 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine; the amino acid residue at position 194 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine; the amino acid residue at position 198 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine; the amino acid residue at position 204 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 212 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; and / or The amino acid residue at position 215 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine.

[0195] In a further preferred embodiment, the CHAP domain of the invention comprises a deglycosylation mutation at or corresponding to position 73 as described herein, and further comprises one or more amino acid substitutions at or corresponding to positions 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169, 173, 175, 178, 185, 186, 191-194, 198, 204, 212 and 215 of SEQ ID NO: 1 as described herein, e.g., immediately above.

[0196] G1 as an exemplary first-round hit variant containing particularly beneficial amino acid substitutions G1 (SEQ ID NO: 3) was a hit variant obtained in the first round of directed evolution, which showed increased thermostability, enhanced bactericidal activity, and enhanced secretion from eukaryotic cells compared to the starting point of directed evolution (L0482ag); see Figure 8.

[0197] G1 (SEQ ID NO: 3) contains the following amino acid substitutions in the CHAP domain compared to L0482ag (SEQ ID NO: 2): T82S, N85G, R86K, D169N, K173N, M175Q and A192Q.

[0198] Thus, in some embodiments, a CHAP domain of the invention comprises one or more amino acid substitutions at positions 82, 85, 86, 169, 173, 175 and 192 of SEQ ID NO:1, or positions corresponding to these positions, the amino acid residue at or corresponding to position 82 of SEQ ID NO: 1 (i.e., threonine) is substituted with serine; the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) is substituted with lysine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 173 of SEQ ID NO: 1 (i.e., lysine) is substituted with asparagine; the amino acid residue at or corresponding to position 175 of SEQ ID NO: 1 (i.e., methionine) is substituted with glutamine; and / or The amino acid residue at position 192 of SEQ ID NO: 1 or a position corresponding to this position (i.e., alanine) is substituted with glutamine. It has one or more amino acid substitutions.

[0199] Furthermore, the CHAP domain may have at least 95% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:3.

[0200] In some embodiments, the CHAP domain is: (a) an amino acid substitution at or corresponding to position 86 of SEQ ID NO: 1, wherein the amino acid at or corresponding to position 86 of SEQ ID NO: 1 is substituted with lysine; (b) an amino acid substitution pair at positions 82 and 85 of SEQ ID NO: 1 or a corresponding position, wherein the amino acid residue at position 82 of SEQ ID NO: 1 or a corresponding position is substituted with serine and the amino acid residue at position 85 of SEQ ID NO: 1 or a corresponding position is substituted with glycine; and / or (c) an amino acid substitution at or corresponding to position 169 of SEQ ID NO: 1, wherein the amino acid at or corresponding to position 169 of SEQ ID NO: 1 is substituted with asparagine; It has amino acid substitutions.

[0201] Additionally, the CHAP domain may comprise one or more amino acid substitutions at positions 173, 175, and 192 of SEQ ID NO: 1, or positions corresponding to these positions, the amino acid residue at position 173 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at or corresponding to position 175 of SEQ ID NO: 1 is substituted with glutamine; and / or the amino acid residue at position 192 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine; It may contain one or more amino acid substitutions.

[0202] Preferably, the CHAP domain further comprises a deglycosylation mutation at or corresponding to position 73 as described herein, preferably in which the residue at or corresponding to position 73 of SEQ ID NO:1 is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, more preferably glycine.

[0203] In some embodiments of the invention, the peptidoglycan hydrolase has (i) at least 80% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution or deletion at position 73 of SEQ ID NO:1 or a position corresponding to this position; and (iii) one or more amino acid substitutions at positions 82, 85, 86, 169, 173, 175, and 192 of SEQ ID NO:1 or a position corresponding to these positions, the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; the amino acid residue at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at or corresponding to position 175 of SEQ ID NO: 1 is substituted with glutamine; and / or The amino acid residue at position 192 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine. a CHAP domain with one or more amino acid substitutions; However, the CHAP domain does not have the sequence set forth in SEQ ID NO: 381, 382, ​​383, 384 or 385.

[0204] In some embodiments of the invention, the peptidoglycan hydrolase has (i) at least 91% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution or deletion at position 73 of SEQ ID NO:1, or a position corresponding to this position; and (iii) one or more amino acid substitutions at positions 82, 85, 86, 169, 173, 175, and 192 of SEQ ID NO:1, or a position corresponding to these positions, the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; the amino acid residue at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at or corresponding to position 175 of SEQ ID NO: 1 is substituted with glutamine; and / or The amino acid residue at position 192 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine. a CHAP domain with one or more amino acid substitutions; However, the CHAP domain does not have the sequence set forth in SEQ ID NO: 381 or 382.

[0205] In some further embodiments of the invention, the peptidoglycan hydrolase has (i) at least 80% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution or deletion at position 73 of SEQ ID NO:1, or a position corresponding to this position; and (iii) at least two amino acid substitutions at positions 82, 85, 86, 169, 173, 175, and 192 of SEQ ID NO:1, or positions corresponding to these positions, the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; the amino acid residue at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at or corresponding to position 175 of SEQ ID NO: 1 is substituted with glutamine; and / or the amino acid residue at position 192 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine; It contains a CHAP domain with amino acid substitutions.

[0206] In some embodiments of the invention, the peptidoglycan hydrolase has (i) at least 60% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution at or at a position corresponding to position 73 of SEQ ID NO:1, wherein the amino acid residue at or at a position corresponding to position 73 of SEQ ID NO:1 is replaced with glycine; and (iii) one or more amino acid substitutions at or at positions corresponding to positions 82, 85, 86, 169, 173, 175, and 192 of SEQ ID NO:1, the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; the amino acid residue at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at or corresponding to position 175 of SEQ ID NO: 1 is substituted with glutamine; and / or the amino acid residue at position 192 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine; It comprises a CHAP domain with one or more amino acid substitutions.

[0207] Most beneficial amino acid substitutions As described herein and illustrated in the accompanying Examples, the inventors have identified a set of most beneficial mutations, in particular a set of nine most beneficial amino acid substitutions, namely T82S, N85G, R86K, S130N, H136K / R (preferably H136K), F155Y, D169N, N185Y, and N186G, with reference to SEQ ID NO: 1. As described herein, variant H5 (SEQ ID NO: 11), which has been found to have the best overall pharmaceutical properties among the L0482 variants obtained by two rounds of directed evolution, contains only these nine most beneficial amino acid substitutions; see, for example, Figures 8 and 9. In particular, H5 (SEQ ID NO: 11) exhibits strongly enhanced secretion from human cells, strongly enhanced killing activity against S. aureus, and improved (thermo)stability compared to the parent L0482ag (SEQ ID NO: 2), as well as compared to WT L0482 (SEQ ID NO: 1); see, e.g., Examples 4-6 and 12 and Figures 8, 10, and 6. Notably, H5 (SEQ ID NO: 11) exhibited an 8-fold lower MIC than WT L0482 (SEQ ID NO: 1), i.e., WT L0482 had an MIC of 4 μg / ml, while H5 had an MIC of 0.5 μg / ml. Furthermore, H5 (SEQ ID NO: 11) exhibited the strongest killing activity against S. aureus (together with H7; SEQ ID NO: 13), the best stability (especially a melting temperature of 47°C), and good secretion from human cells; see Figure 8. H5 also exhibited enhanced secretion from human cells, enhanced killing activity against Staphylococcus aureus, and improved (thermo)stability compared to G1 (SEQ ID NO: 3), an improved L0482 variant obtained by the first round of directed evolution; see Figure 8. Notably, G1 had an MIC of 1 μg / ml, while H5 had an MIC of 0.5 μg / ml.

[0208] H5 (SEQ ID NO: 11) also exhibited enhanced (i.e., more rapid) kill kinetics against S. aureus than WT L0482 (SEQ ID NO: 1) in an OD reduction assay; see Example 6 and Figure 11. In addition, H5 (SEQ ID NO: 11) effectively killed S. aureus biofilms and exhibited even greater anti-biofilm activity than G1 (SEQ ID NO: 3); see Example 6 and Figure 12. This is consistent with the particularly low MIC observed for H5, which was lower than the MIC for G1.

[0209] Thus, the most beneficial amino acid substitutions (i.e., the "H5" mutations) are particularly beneficial for enhancing the desired pharmaceutical properties of the peptidoglycan hydrolase, particularly bactericidal activity (e.g., against Staphylococcus aureus, including S. aureus biofilms), stability, and / or secretion ability from eukaryotic cells, e.g., human cells.

[0210] In the context of the present invention, the most beneficial amino acid substitutions are preferred over amino acid substitutions described as "particularly beneficial", and even more preferred than amino acid substitutions described as "beneficial" or "tolerated".

[0211] As already mentioned above, in the context of the present invention, it was further found that the L0482 variant H3 (SEQ ID NO: 9) obtained by directed evolution contained eight of the nine most beneficial amino acid substitutions and only contained these eight mutations. Since the eight "H3" amino acid substitutions occurred repeatedly in slightly different combinations among the 44 hit variants obtained by three rounds of directed evolution, they are considered to be consensus mutations in the present specification and in the context of the present invention. Furthermore, H3 reflects a consensus sequence. In particular, the eight consensus mutations refer to T82S, N85G, R86K, S130N, H136K / R, D169N, N185Y, and N186G with reference to the sequence of SEQ ID NO: 1.

[0212] Furthermore, as described herein and illustrated in the accompanying Examples, H3 (SEQ ID NO: 9) has improved pharmaceutical properties compared to WT L0482 and L0482ag, such as improved secretion ability from eukaryotic cells and better killing activity against Staphylococcus aureus; see, e.g., Figures 8 and 10. This further demonstrates the beneficial effect of the consensus mutations on the pharmaceutical properties of the L0482 variants described herein.

[0213] Thus, in some particularly preferred embodiments, for example in the context of consensus mutations, the CHAP domains of the invention have one or more amino acid substitutions at or corresponding to positions 82, 85, 86, 130, 136, 169, 185, and 186 of SEQ ID NO: 1. Preferably, in these embodiments, the amino acid residue at or corresponding to position 82 of SEQ ID NO: 1 (i.e., threonine) is substituted with serine, the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) is substituted with lysine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with a tyrosine; and / or The amino acid residue at or corresponding to position 186 of SEQ ID NO: 1 (ie, asparagine) is substituted with glycine.

[0214] In other words, in some particularly preferred embodiments, the CHAP domain of the present invention has at least one consensus mutation, i.e., at least one amino acid substitution selected from the group consisting of T82S, N85G, R86K, S130N, H136K / R, D169N, N185Y and N186G of SEQ ID NO:1.

[0215] Preferably, the CHAP domain (i.e., a CHAP domain comprising at least one of the consensus mutations described herein) further comprises: (i) a deglycosylation mutation at or corresponding to position 73 described herein, e.g., a deglycosylation mutation in which the residue at or corresponding to position 73 of SEQ ID NO:1 has been substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine, or serine, preferably glycine or serine, more preferably glycine; and / or (ii) an amino acid substitution at or corresponding to position 155, in which the amino acid residue at or corresponding to position 155 of SEQ ID NO:1 (i.e., phenylalanine) has been substituted with tyrosine.

[0216] Also as already mentioned above, it has surprisingly been found in the context of the present invention that the L0482 variant H5 (SEQ ID NO: 11), which has enhanced killing activity against Staphylococcus aureus, enhanced stability (e.g. thermostability / melting temperature) and enhanced secretion ability from human cells compared to H3 (Figure 8), differs from H3 by only one amino acid, namely it has, in addition to the consensus mutation, the amino acid substitution F155Y with reference to SEQ ID NO: 1 (Figure 9).

[0217] Furthermore, the amino acid substitution H136R was found to occur instead of H136K in many of the hit variants (i.e., G1-G4, H1-H10, and I1-I30) and is thus a very good alternative to H136K.

[0218] In particular, L0482 variant H3 has the following sequence, where the LYSM domain (positions 1-51) is underlined, the linker (positions 52-71) is italicized, and the CHAP domain (positions 72-215) is bolded: [ka]

[0219] Furthermore, L0482 variant H5 has the following sequence, where the LYSM domain (positions 1-51) is underlined, the linker (positions 52-71) is italicized, and the CHAP domain (positions 72-215) is bolded: [ka]

[0220] In the above H3 and H5 sequences (i.e., SEQ ID NO: 9 and SEQ ID NO: 11, respectively), the two glycosylation positions, i.e., positions 68 and 73, where deglycosylated amino acid residues have been introduced into H3 and H5 (i.e., lysine at position 68 and glycine at position 73), are highlighted in gray.

[0221] The amino acid residues in SEQ ID NO:9 shown in white on a black background refer to consensus mutations, and the amino acid residues in SEQ ID NO:11 shown in white on a black background refer to the most beneficial amino acid substitutions described herein and in the context of the present invention. Notably, H3 (SEQ ID NO:9) contains all of the most beneficial amino acid substitutions except for F155Y. See also Figure 9A for alignment.

[0222] Notably, the most beneficial amino acid substitutions with reference to SEQ ID NO: 1, namely T82S, N85G, R86K, S130N, H136K / R (preferably H136K), F155Y, D169N, N185Y and N186G, all occurred within the CHAP domain of L0482, i.e. within the sequence from position 72 to 215 of SEQ ID NO: 1. Herein, and in the context of the present invention, any of the most beneficial mutations or any combination thereof is preferably combined with a deglycosylation mutation at position 73 of SEQ ID NO: 1 or a position corresponding to this position as described herein, such as the amino acid substitution N73G of SEQ ID NO: 1. In certain embodiments, for example, in the context of a peptidoglycan hydrolase having at least 60% sequence identity with the sequence of SEQ ID NO:1, any of these most beneficial mutations or any combination thereof is further combined with at least one deglycosylation mutation at positions 68 and 73 of SEQ ID NO:1 or positions corresponding to these positions as described herein, e.g., the amino acid substitutions N68K and / or N73G of SEQ ID NO:1.

[0223] However, a peptidoglycan hydrolase or CHAP domain of the present invention need not necessarily contain all nine of the most beneficial mutations described herein at once. For example, L0482 variant H1 (SEQ ID NO: 7) did not exhibit any mutations at positions corresponding to 130, 136, 155, 169, 185, and 186 of SEQ ID NO: 1, yet was found to have enhanced pharmaceutical properties compared to the parent L0482ag lysin (SEQ ID NO: 2), particularly enhanced killing activity against Staphylococcus aureus, enhanced stability (e.g., thermostability / melting temperature), and enhanced secretion ability from human cells. As a further example, L0482 variant H9 (SEQ ID NO: 15) did not exhibit any mutations at positions corresponding to 82, 85, 155, 185, and 185 of SEQ ID NO: 1, yet was also found to exhibit enhanced pharmaceutical properties compared to the parent L0482ag lysin (SEQ ID NO: 2); see, e.g., Figures 8 and 9. Indeed, the amino acid substitution R86K is the only most beneficial substitution shared by H1 (SEQ ID NO: 7) and H9 (SEQ ID NO: 15). This clearly demonstrates that the most beneficial amino acid substitutions found in the context of the present invention can be used in various combinations and can reliably enhance the pharmaceutical properties of the L0482-derived peptidoglycan hydrolases described herein. However, this finding in no way contradicts the idea that the group of most beneficial amino acid substitutions described herein was intentionally and carefully selected by the inventors to improve the pharmaceutical properties of the L0482-derived peptidoglycan hydrolases. For example, when additional amino acid substitutions N185Y and N186G are introduced into H9 (SEQ ID NO: 15) to yield, for example, G4 (SEQ ID NO: 6), H2 (SEQ ID NO: 8), H8 (SEQ ID NO: 14), or H10 (SEQ ID NO: 16), some or all of the assayed pharmaceutical properties can be further improved; see Figures 8 and 9. Furthermore, as already mentioned above, the single amino acid substitution F155Y (for example occurring in H5) improves all pharmaceutical properties compared to H3, further demonstrating that the most beneficial individual amino acid substitutions can already have a very beneficial effect.Indeed, L0482 variant H5 (SEQ ID NO: 11), which contains all of the most beneficial mutations described herein and only these mutations, exhibited the best pharmacological properties of all hit variants analyzed as described herein.

[0224] Thus, in some particularly preferred embodiments, e.g., in the context of the most beneficial amino acid substitutions, the CHAP domains of the invention have one or more amino acid substitutions at or corresponding to positions 82, 85, 86, 130, 136, 155, 169, 185, and 186 of SEQ ID NO: 1. Preferably, in these embodiments, the amino acid residue at or corresponding to position 82 of SEQ ID NO: 1 (i.e., threonine) is substituted with serine; the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) is substituted with lysine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 155 of SEQ ID NO: 1 (i.e., phenylalanine) is substituted with tyrosine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with a tyrosine; and / or The amino acid residue at or corresponding to position 186 of SEQ ID NO: 1 (ie, asparagine) is substituted with glycine.

[0225] In other words, in some particularly preferred embodiments, the CHAP domain of the present invention has at least one amino acid substitution selected from the group consisting of T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y and N186G of SEQ ID NO:1.

[0226] Preferably, the CHAP domain (i.e., a CHAP domain comprising at least one of the most beneficial amino acid substitutions described herein) further comprises a deglycosylation mutation at or corresponding to position 73 described herein, e.g., a deglycosylation mutation in which the residue at or corresponding to position 73 of SEQ ID NO: 1 is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine, or serine, preferably glycine or serine, more preferably glycine.

[0227] Amino acid substitution pairs and consensus mutation units As described in detail herein above, the inventors have further surprisingly found that certain amino acid substitutions, in particular some of the consensus mutations (i.e., T82S, N85G, R86K, S130N, H136K / R (particularly H136K), D169N, N185Y and N186G with reference to SEQ ID NO: 1), occurred very frequently as substitution pairs in the active secreted L0482 variants obtained by directed evolution, in particular the hit variants, i.e., G1 to G4, H1 to H10 and I1 to I30. Thus, the consensus mutations (which are particularly preferred amino acid substitutions in the context of the present invention) may be further classified as consensus mutation units herein and in the context of the present invention. As mentioned above, these consensus mutation units consist of one or two amino acid substitutions and refer to (i) R86K (particularly preferred), (ii) T82S and N85G, (iii) S130N and H136K / R (preferably H136K), (iv) D169N, and (v) N185Y and N186G.

[0228] Thus, in some preferred embodiments, for example, in the context of the most beneficial amino acid substitutions and / or consensus mutation units, the CHAP domain of the invention has at least one pair of amino acid substitutions at a) positions 82 and 85 of SEQ ID NO:1 or corresponding to these positions, b) positions 130 and 136 of SEQ ID NO:1 or corresponding to these positions, and / or c) positions 185 and 186 of SEQ ID NO:1 or corresponding to these positions.

[0229] In the context of these embodiments, in a), preferably the amino acid residue at or at a position corresponding to 82 of SEQ ID NO: 1 is substituted with serine, and the amino acid residue at or at a position corresponding to 85 of SEQ ID NO: 1 is substituted with glycine; In b), preferably, the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 is substituted with asparagine, and the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 is substituted with lysine or arginine, preferably lysine; and / or In c), preferably, the amino acid residue at position 185 of SEQ ID NO: 1 or a position corresponding thereto is substituted with tyrosine, and the amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto is substituted with glycine.

[0230] In other words, in some preferred embodiments, the CHAP domain of the present invention has at least one substitution pair selected from the group consisting of: a) T82S and N85G, b) S130N and H136K / R (preferably H136K), and c) N185Y and N186G.

[0231] Furthermore, the CHAP domain (i.e., a CHAP domain having at least one substitution pair) preferably further comprises an amino acid substitution at position 86 of SEQ ID NO: 1 or a position corresponding to this position, as described herein.

[0232] Furthermore, the CHAP domain (i.e., a CHAP domain having at least one substitution pair, preferably having an amino acid substitution at or corresponding to position 86 of SEQ ID NO:1) further preferably has an amino acid substitution at or corresponding to position 155 of SEQ ID NO:1, as described herein.

[0233] Additionally, the CHAP domain preferably comprises a deglycosylation mutation at position 73 of SEQ ID NO: 1 or a position corresponding to this position, as described herein.

[0234] In some further preferred embodiments, the CHAP domain of the present invention comprises at least one consensus mutation unit, i.e., one of the following (i) to (v): (i) an amino acid substitution at position 86 of SEQ ID NO: 1 or a position corresponding to this position, in which the amino acid residue at said position (i.e., arginine) is replaced with lysine (i.e., R86K); (ii) amino acid substitutions at or corresponding to positions 82 and 85 of SEQ ID NO:1, in which the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) is substituted with serine (i.e., T82S), and the amino acid residue at or corresponding to position 85 of SEQ ID NO:1 (i.e., asparagine) is substituted with glycine (i.e., N85G); (iii) amino acid substitutions at positions 130 and 136 of SEQ ID NO: 1 or corresponding thereto, in which the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine (i.e., S130N), and the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine (i.e., H136K / R), preferably lysine (H136K); (iv) an amino acid substitution at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid), where the amino acid residue at said position is replaced with asparagine (i.e., D169N); and (v) Amino acid substitutions at positions 185 and 186 of SEQ ID NO: 1 or corresponding positions, in which the amino acid residue at position 185 of SEQ ID NO: 1 or corresponding position (i.e., asparagine) is replaced with tyrosine (i.e., N185Y), and the amino acid residue at position 186 of SEQ ID NO: 1 or corresponding position (i.e., asparagine) is replaced with glycine (i.e., N186G). and having at least one amino acid substitution or pair of substitutions selected from the group consisting of:

[0235] Preferably, in the context of these embodiments, the CHAP domain of the invention has at least two, preferably at least three, and more preferably at least four of the amino acid substitutions or substitution pairs (i) to (v) (i.e., consensus mutation units) as described immediately above in the present invention.

[0236] In some particularly preferred embodiments, the CHAP domain of the invention comprises: (i) an amino acid substitution at position 86 of SEQ ID NO: 1 or a position corresponding to this position, in which the amino acid residue at said position (i.e., arginine) is replaced with lysine; and the following amino acid substitutions or substitution pairs (ii) to (v) (i.e., consensus mutation units): (ii) amino acid substitutions at or corresponding to positions 82 and 85 of SEQ ID NO:1, wherein the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine, and the amino acid residue at or corresponding to position 85 of SEQ ID NO:1 (i.e., asparagine) is replaced with glycine; (iii) amino acid substitutions at or corresponding to positions 130 and 136 of SEQ ID NO:1, wherein the amino acid residue at or corresponding to position 130 of SEQ ID NO:1 (i.e., serine) is replaced with asparagine, and the amino acid residue at or corresponding to position 136 of SEQ ID NO:1 (i.e., histidine) is replaced with lysine or arginine, preferably lysine; (iv) an amino acid substitution at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid), where the amino acid residue at said position is replaced with asparagine; and / or (v) Amino acid substitutions at positions 185 and 186 of SEQ ID NO: 1 or corresponding positions, in which the amino acid residue at position 185 of SEQ ID NO: 1 or corresponding positions (i.e., asparagine) is replaced with tyrosine, and the amino acid residue at position 186 of SEQ ID NO: 1 or corresponding positions (i.e., asparagine) is replaced with glycine. It has at least one, preferably at least two, and more preferably at least three of the above.

[0237] Generally, in the context of the present specification and the present invention, a consensus mutation or any combination thereof (i.e., at least one substitution selected from the group consisting of T82S, N85G, R86K, S130N, H136K / R, D169N, N185Y and N186G of SEQ ID NO: 1) or a consensus mutation unit or any combination thereof (i.e., at least one consensus mutation unit selected from the group consisting of (i) R86K, (ii) T82S and N85G, (iii) S130N and H136K / R, (iv) D169N, (v) N185Y and N186G) is preferably combined with an amino acid substitution at or corresponding to position 155 of SEQ ID NO: 1, as described herein. Preferably, the amino acid residue at or corresponding to position 155 of SEQ ID NO: 1 is substituted with tyrosine (i.e., F155Y) as described herein.

[0238] Furthermore, generally, in the context of this specification and the present invention, the consensus mutation or consensus mutation unit or any combination thereof (which may further be combined with F155Y as just described herein) is preferably combined with at least one deglycosylation mutation (preferably at least one deglycosylation substitution) as described herein, preferably an amino acid substitution at or corresponding to position 73 of SEQ ID NO: 1. Preferably, the residue at or corresponding to position 73 of SEQ ID NO: 1 is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine as described herein, more preferably glycine.

[0239] What has just been described above in the context of the amino acid substitution at or corresponding to position 155 of SEQ ID NO: 1, the deglycosylation mutation at or corresponding to position 73 of SEQ ID NO: 1, and the amino acid substitution also applies specifically to the following embodiments.

[0240] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitution R86K described herein.

[0241] In some preferred embodiments, the CHAP domains of the invention have the amino acid substitutions R86K, T82S, and N85G described herein.

[0242] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, S130N and H136K / R described herein.

[0243] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K and D169N described herein.

[0244] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K and N185Y and N186 described herein.

[0245] In some preferred embodiments, the CHAP domains of the invention have the amino acid substitutions R86K, T82S, N85G, S130N and H136K / R described herein.

[0246] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, and D169N described herein.

[0247] In some preferred embodiments, the CHAP domains of the invention have the amino acid substitutions R86K, T82S, N85G, and N185Y and N186G described herein.

[0248] In some preferred embodiments, the CHAP domains of the invention have the amino acid substitutions R86K, D169N, N185Y and N186G described herein.

[0249] In some preferred embodiments, the CHAP domains of the invention have the amino acid substitutions R86K, S130N, H136K / R, N185Y and N186G described herein.

[0250] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, S130N, H136K / R, and D169N described herein.

[0251] In some preferred embodiments, the CHAP domains of the invention have the amino acid substitutions R86K, T82S, N85G, N185Y and N186G described herein.

[0252] In some particularly preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, S130N, H136K / R and D169N described herein.

[0253] In some particularly preferred embodiments, the CHAP domains of the invention have the amino acid substitutions R86K, T82S, N85G, S130N, H136K / R, N185Y and N186 as described herein.

[0254] In some particularly preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, S130N, H136K / R, D169N, N185Y and N186G described herein.

[0255] In some particularly preferred embodiments, the CHAP domains of the invention have the amino acid substitutions R86K, T82S, N85G, D169N, N185Y and N186G described herein.

[0256] In some of the most preferred embodiments, the CHAP domains of the invention have the amino acid substitutions R86K, T82S, N85G, S130N, H136K / R, D169N, N185Y and N186G described herein.

[0257] In some embodiments, a CHAP domain of the invention has the amino acid substitutions T82S and N85G described herein.

[0258] In some embodiments, a CHAP domain of the invention has the amino acid substitutions T82S, N85G, S130N, and H136K / R described herein.

[0259] In some embodiments, a CHAP domain of the invention has the amino acid substitutions T82S, N85G, and D169N described herein.

[0260] In some embodiments, a CHAP domain of the invention has the amino acid substitutions T82S, N85G, N185Y, and N186G described herein.

[0261] In some embodiments, a CHAP domain of the invention has the amino acid substitutions T82S, N85G, S130N, H136K / R and D169N described herein.

[0262] In some embodiments, a CHAP domain of the invention has the amino acid substitutions T82S, N85G, S130N, H136K / R, N185Y, and N186G described herein.

[0263] In some embodiments, a CHAP domain of the invention has the amino acid substitutions T82S, N85G, S130N, H136K / R, D169N, N185Y, and N186G described herein.

[0264] In some embodiments, a CHAP domain of the invention has the amino acid substitutions S130N and H136K / R described herein.

[0265] In some embodiments, a CHAP domain of the invention has the amino acid substitutions S130N, H136K / R and D169N described herein.

[0266] In some embodiments, a CHAP domain of the invention has the amino acid substitutions S130N, H136K / R, N185Y, and N186G described herein.

[0267] In some embodiments, a CHAP domain of the invention has the amino acid substitutions S130N, H136K / R, D169N, N185Y, and N186G described herein.

[0268] In some embodiments, a CHAP domain of the invention has the amino acid substitution D169N described herein.

[0269] In some embodiments, a CHAP domain of the invention has the amino acid substitutions D169N, N185Y, and N186G described herein.

[0270] In some embodiments, a CHAP domain of the invention has the amino acid substitutions N185Y and N186G described herein.

[0271] Furthermore, herein and in the context of the present invention, and therefore also in the above embodiment, the substitution H136K is preferred over the substitution H136R.

[0272] Particularly important individual amino acid substitutions: R86K and F155Y As already mentioned above and illustrated in the appended examples, all hit variants, i.e. G1 to G4, H1 to H10 and I1 to I30, have the amino acid substitution R86K with reference to SEQ ID NO: 1. As such, this amino acid substitution is considered herein and in the context of the present invention to be a particularly important substitution among the most beneficial amino acid substitutions described herein.

[0273] Thus, in some of the most preferred embodiments, for example, in the context of the most beneficial amino acid substitutions, the CHAP domain of the invention has an amino acid substitution at or corresponding to position 86 of SEQ ID NO: 1. For example, in the context of these embodiments, the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) may be substituted with another amino acid residue other than serine. Preferably, in the context of these embodiments, the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) is substituted with lysine or methionine. More preferably, the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) is substituted with lysine.

[0274] Additionally, the CHAP domain preferably comprises a deglycosylation mutation at position 73 of SEQ ID NO: 1 or a position corresponding to this position, as described herein.

[0275] Also as already mentioned above, the amino acid substitution F155Y further improved several pharmaceutical properties (e.g., killing activity against Staphylococcus aureus, stability and secretion ability from human cells) in H5 (SEQ ID NO: 11) compared to H3 (SEQ ID NO: 9), which contains only the consensus mutation.

[0276] Thus, in some preferred embodiments, the CHAP domain of the invention has an amino acid substitution at or corresponding to position 155 of SEQ ID NO: 1. Preferably, the amino acid residue at or corresponding to position 155 of SEQ ID NO: 1 (i.e., phenylalanine) is substituted with tyrosine.

[0277] Generally, herein and in the context of the present invention, the CHAP domain preferably has a deglycosylation mutation at or corresponding to position 73 of SEQ ID NO: 1 as described herein. This also applies, inter alia, to all of the following embodiments.

[0278] Further embodiments In certain embodiments, the CHAP domain of the present invention has (i) at least 91% sequence identity with the sequence of positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution at or corresponding to position 86 of SEQ ID NO:1, wherein the amino acid residue at or corresponding to position 86 of SEQ ID NO:1 (i.e., arginine) is replaced with lysine; and (iii) one or more amino acid substitutions at or corresponding to positions 82, 85, 130, 136, 155, 169, 185, and 186 of SEQ ID NO:1, wherein the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine, the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 155 of SEQ ID NO: 1 (i.e., phenylalanine) is substituted with tyrosine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has one or more amino acid substitutions.

[0279] In certain embodiments, the peptidoglycan hydrolase of the invention has (i) at least 84% sequence identity with the sequence of SEQ ID NO:1; (ii) an amino acid substitution at or corresponding to position 86 of SEQ ID NO:1, wherein the amino acid residue at or corresponding to position 86 of SEQ ID NO:1 (i.e., arginine) is replaced with lysine; and (iii) one or more amino acid substitutions at or corresponding to positions 82, 85, 130, 136, 155, 169, 185, and 186 of SEQ ID NO:1, wherein the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine. the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 155 of SEQ ID NO: 1 (i.e., phenylalanine) is substituted with tyrosine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has one or more amino acid substitutions.

[0280] In certain embodiments, the CHAP domain of the present invention has (i) at least 78% sequence identity with the sequence of positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution at or corresponding to position 86 of SEQ ID NO:1, where the amino acid residue at or corresponding to position 86 of SEQ ID NO:1 (i.e., arginine) is replaced with lysine; and (iii) at least two amino acid substitutions at or corresponding to positions 82, 85, 130, 136, 155, 169, 185, and 186 of SEQ ID NO:1, where the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine, the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 155 of SEQ ID NO: 1 (i.e., phenylalanine) is substituted with tyrosine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has at least two amino acid substitutions.

[0281] In certain embodiments, the peptidoglycan hydrolase of the invention has (i) at least 73% sequence identity with the sequence of SEQ ID NO:1; (ii) an amino acid substitution at or corresponding to position 86 of SEQ ID NO:1, where the amino acid residue at or corresponding to position 86 of SEQ ID NO:1 (i.e., arginine) is replaced with lysine; and (iii) at least two amino acid substitutions at or corresponding to positions 82, 85, 130, 136, 155, 169, 185, and 186 of SEQ ID NO:1, where the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine, the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 155 of SEQ ID NO: 1 (i.e., phenylalanine) is substituted with tyrosine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has at least two amino acid substitutions.

[0282] In certain embodiments, the CHAP domain of the present invention has (i) at least 78% sequence identity with the sequence of positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution at or corresponding to position 86 of SEQ ID NO:1, wherein the amino acid residue at or corresponding to position 86 of SEQ ID NO:1 (i.e., arginine) is replaced with lysine; and (iii) one or more amino acid substitutions at or corresponding to positions 82, 85, 130, 136, 169, 185, and 186 of SEQ ID NO:1, wherein the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine, the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has one or more amino acid substitutions.

[0283] In certain embodiments, the peptidoglycan hydrolase of the invention has (i) at least 73% sequence identity with the sequence of SEQ ID NO:1; (ii) an amino acid substitution at or corresponding to position 86 of SEQ ID NO:1, wherein the amino acid residue at or corresponding to position 86 of SEQ ID NO:1 (i.e., arginine) is replaced with lysine; and (iii) one or more amino acid substitutions at or corresponding to positions 82, 85, 130, 136, 169, 185, and 186 of SEQ ID NO:1, wherein the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine. the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; The amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding thereto (i.e., histidine) is substituted with lysine or arginine, preferably lysine. the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has one or more amino acid substitutions.

[0284] In certain embodiments, the CHAP domain of the present invention has (i) at least 60% sequence identity with the sequence of positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution at or corresponding to position 86 of SEQ ID NO:1, where the amino acid residue at or corresponding to position 86 of SEQ ID NO:1 (i.e., arginine) is replaced with lysine; and (iii) at least six amino acid substitutions at or corresponding to positions 82, 85, 130, 136, 155, 169, 185, and 186 of SEQ ID NO:1, where the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine, the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 155 of SEQ ID NO: 1 (i.e., phenylalanine) is substituted with tyrosine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has at least six amino acid residues.

[0285] In certain embodiments, the CHAP domain of the present invention has (i) at least 60% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO: 1; (ii) an amino acid substitution at or corresponding to position 86 of SEQ ID NO: 1, in which the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) is replaced with lysine; (iii) an amino acid substitution at or corresponding to position 82 of SEQ ID NO: 1, in which the amino acid residue at or corresponding to position 82 of SEQ ID NO: 1 (i.e., threonine) is replaced with serine; and (iv) an amino acid substitution at or corresponding to position 85 of SEQ ID NO: 1, in which the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is replaced with glycine. In other words, the CHAP domain has an R86K substitution and a substitution pair consisting of T82S and N85G with reference to SEQ ID NO: 1.

[0286] In certain embodiments, the peptidoglycan hydrolase of the present invention (i) has at least 60% sequence identity with the sequence of SEQ ID NO: 1; (ii) has an amino acid substitution at or corresponding to position 86 of SEQ ID NO: 1, where the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) is replaced with lysine; (iii) has an amino acid substitution at or corresponding to position 82 of SEQ ID NO: 1, where the amino acid residue at or corresponding to position 82 of SEQ ID NO: 1 (i.e., threonine) is replaced with serine; and (iv) has an amino acid substitution at or corresponding to position 85 of SEQ ID NO: 1, where the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is replaced with glycine. In other words, the peptidoglycan hydrolase has an R86K substitution and a substitution pair consisting of T82S and N85G with reference to SEQ ID NO: 1.

[0287] In certain embodiments, the CHAP domain of the present invention has (i) at least 83% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution or deletion (preferably an amino acid substitution) at position 73 of SEQ ID NO:1 or a position corresponding thereto; and (iii) one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185, and 186 of SEQ ID NO:1 or a position corresponding thereto, wherein the amino acid residue at position 82 of SEQ ID NO:1 or a position corresponding thereto (i.e., threonine) is replaced with serine, the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) is substituted with lysine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; The amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding thereto (i.e., histidine) is substituted with lysine or arginine, preferably lysine. the amino acid residue at or corresponding to position 155 of SEQ ID NO: 1 (i.e., phenylalanine) is substituted with tyrosine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has one or more amino acid substitutions.

[0288] In certain embodiments, the peptidoglycan hydrolase of the invention has (i) at least 73% sequence identity with the sequence of SEQ ID NO:1; (ii) an amino acid substitution or deletion (preferably an amino acid substitution) at or corresponding to position 73 of SEQ ID NO:1; and (iii) one or more amino acid substitutions at or corresponding to positions 82, 85, 86, 130, 136, 155, 169, 185, and 186 of SEQ ID NO:1, wherein the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine, the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) is substituted with lysine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 155 of SEQ ID NO: 1 (i.e., phenylalanine) is substituted with tyrosine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has one or more amino acid substitutions.

[0289] In certain embodiments, the CHAP domain of the present invention (i) has at least 60% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO: 1; and (ii) an amino acid substitution at or corresponding to position 73 of SEQ ID NO: 1, in which the amino acid residue at or corresponding to position 73 of SEQ ID NO: 1 (i.e., asparagine) is replaced with glycine. Preferably, the CHAP domain further comprises (iii) one or more amino acid substitutions at or corresponding to positions 82, 85, 86, 130, 136, 155, 169, 185, and 186 of SEQ ID NO: 1, in which the amino acid residue at or corresponding to position 82 of SEQ ID NO: 1 (i.e., threonine) is replaced with serine. the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) is substituted with lysine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 155 of SEQ ID NO: 1 (i.e., phenylalanine) is substituted with tyrosine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has one or more amino acid substitutions.

[0290] In certain embodiments, the CHAP domain of the present invention (i) has at least 60% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:1; (ii) has an amino acid substitution or deletion (preferably an amino acid substitution) at or corresponding to position 73 of SEQ ID NO:1; and (iii) has an amino acid substitution at or corresponding to position 82 of SEQ ID NO:1, in which the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) has been replaced with serine.

[0291] In certain embodiments, the CHAP domain of the present invention (i) has at least 60% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:1; (ii) has an amino acid substitution or deletion (preferably an amino acid substitution) at or corresponding to position 73 of SEQ ID NO:1; and (iii) has an amino acid substitution at or corresponding to position 130 of SEQ ID NO:1, in which the amino acid residue at or corresponding to position 130 of SEQ ID NO:1 (i.e., serine) has been replaced with asparagine.

[0292] In certain embodiments, the CHAP domain of the present invention (i) has at least 60% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:1; (ii) has an amino acid substitution or deletion (preferably an amino acid substitution) at or corresponding to position 73 of SEQ ID NO:1; and (iii) has an amino acid substitution at or corresponding to position 185 of SEQ ID NO:1, in which the amino acid residue at or corresponding to position 185 of SEQ ID NO:1 (i.e., asparagine) has been replaced with tyrosine.

[0293] In certain embodiments, the CHAP domain of the present invention has (i) at least 60% sequence identity with the sequence of positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution at or corresponding to position 130 of SEQ ID NO:1, where the amino acid residue at or corresponding to position 130 of SEQ ID NO:1 (i.e., serine) is replaced with asparagine; and (iii) one or more amino acid substitutions at or corresponding to positions 82, 85, 86, 136, 155, 169, 185, and 186 of SEQ ID NO:1, where the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine, the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) is substituted with lysine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 155 of SEQ ID NO: 1 (i.e., phenylalanine) is substituted with tyrosine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has one or more amino acid substitutions.

[0294] In certain embodiments, the CHAP domain of the present invention has (i) at least 78% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution at or corresponding to position 155 of SEQ ID NO:1, in which the amino acid residue at or corresponding to position 155 of SEQ ID NO:1 (i.e., phenylalanine) is replaced with tyrosine; and (iii) at least two amino acid substitutions at or corresponding to positions 82, 85, 86, 130, 136, 169, 185, and 186 of SEQ ID NO:1, in which the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine, the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) is substituted with lysine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has at least two amino acid substitutions.

[0295] In certain embodiments, the peptidoglycan hydrolase of the invention has (i) at least 73% sequence identity with the sequence of SEQ ID NO:1; (ii) an amino acid substitution at or corresponding to position 155 of SEQ ID NO:1, where the amino acid residue at or corresponding to position 155 of SEQ ID NO:1 (i.e., phenylalanine) is replaced with tyrosine; and (iii) at least two amino acid substitutions at or corresponding to positions 82, 85, 86, 130, 136, 169, 185, and 186 of SEQ ID NO:1, where the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine, the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) is substituted with lysine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding to this position (i.e., asparagine) is substituted with glycine. It has at least two amino acid substitutions.

[0296] In some embodiments, the CHAP domain of the present invention has (i) at least 78% sequence identity with the sequence of positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution at, or a position corresponding to, position 155 of SEQ ID NO:1, where the amino acid residue at, or a position corresponding to, position 155 of SEQ ID NO:1 (i.e., phenylalanine) is replaced with tyrosine; and (iii) one or more amino acid substitutions at, or positions corresponding to, positions 82, 85, 130, 136, 169, 185, and 186 of SEQ ID NO:1, where the amino acid residue at, or a position corresponding to, position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine, the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has one or more amino acid substitutions.

[0297] In some embodiments, the peptidoglycan hydrolase of the invention has (i) at least 73% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution at, or a position corresponding to, position 155 of SEQ ID NO:1, where the amino acid residue at, or a position corresponding to, position 155 of SEQ ID NO:1 (i.e., phenylalanine) is replaced with tyrosine; and (iii) one or more amino acid substitutions at, or positions corresponding to, positions 82, 85, 130, 136, 169, 185, and 186 of SEQ ID NO:1, where the amino acid residue at, or a position corresponding to, position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine. the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has one or more amino acid substitutions.

[0298] In certain embodiments, the CHAP domain of the present invention has (i) at least 60% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution at or corresponding to position 155 of SEQ ID NO:1, in which the amino acid residue at or corresponding to position 155 of SEQ ID NO:1 (i.e., phenylalanine) is replaced with tyrosine; and (iii) at least six amino acid substitutions at or corresponding to positions 82, 85, 86, 130, 136, 169, 185, and 186 of SEQ ID NO:1, in which the amino acid residue at or corresponding to position 82 of SEQ ID NO:1 (i.e., threonine) is replaced with serine, the amino acid residue at or corresponding to position 85 of SEQ ID NO: 1 (i.e., asparagine) is substituted with glycine; the amino acid residue at or corresponding to position 86 of SEQ ID NO: 1 (i.e., arginine) is substituted with lysine; the amino acid residue at or corresponding to position 130 of SEQ ID NO: 1 (i.e., serine) is substituted with asparagine; the amino acid residue at or corresponding to position 136 of SEQ ID NO: 1 (i.e., histidine) is substituted with lysine or arginine, preferably lysine; the amino acid residue at or corresponding to position 169 of SEQ ID NO: 1 (i.e., aspartic acid) is substituted with asparagine; the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 (i.e., asparagine) is substituted with tyrosine; and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding thereto (i.e., asparagine) is substituted with glycine. It has at least six amino acid substitutions.

[0299] In some embodiments, for example, in the context of a L0482 variant obtained by directed evolution, a tolerated or beneficial mutation, a particularly beneficial mutation, a most beneficial mutation, a consensus mutation, or a consensus mutation unit, a peptidoglycan hydrolase of the invention does not have a sequence set forth in any one of SEQ ID NOs: 279-293.

[0300] In some embodiments, the CHAP domain of the present invention has at least 90%, preferably at least 95%, more preferably at least 97% or at least 98%, e.g., at least 97.3%, sequence identity to the CHAP domain of a hit variant described herein, i.e., the CHAP domain of any one of G1 to G4, H1 to H10, and I1 to I30 (SEQ ID NOs: 3 to 46, respectively) described herein. In particular, the CHAP domain of a hit variant (e.g., H1) refers to the sequence from the position in the sequence of the hit variant (e.g., SEQ ID NO: 7) corresponding to position 72 of SEQ ID NO: 1 to the position in the sequence of the hit variant (e.g., SEQ ID NO: 7) corresponding to position 215 of SEQ ID NO: 1. In particular, the CHAP domain has at least one amino acid substitution selected from the group consisting of T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y, and N186G with respect to SEQ ID NO: 1, i.e., the sequence from positions 72 to 215 of SEQ ID NO: 1 as the reference sequence described herein.

[0301] In some further embodiments, the peptidoglycan hydrolase of the present invention has at least 90%, preferably at least 95%, more preferably at least 97%, e.g., at least 96.8%, sequence identity to any one of the hit variants described herein, i.e., SEQ ID NOs: 3 to 46. In particular, the peptidoglycan hydrolase has at least one amino acid substitution in SEQ ID NO: 1, i.e., with respect to SEQ ID NO: 1 as the reference sequence described herein, selected from the group consisting of T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y, and N186G.

[0302] In some preferred embodiments, the CHAP domain of the present invention has at least 93.2% or at least 94% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO: 11. In particular, the CHAP domain has at least one amino acid substitution selected from the group consisting of T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y, and N186G relative to the sequence from positions 72 to 215 of SEQ ID NO: 1, i.e., as the reference sequence described herein.

[0303] In some preferred embodiments, the peptidoglycan hydrolase of the present invention has at least 95.0% or at least 96% sequence identity to the sequence of SEQ ID NO: 11. In particular, the peptidoglycan hydrolase has at least one amino acid substitution in SEQ ID NO: 1 selected from the group consisting of T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y, and N186G with respect to SEQ ID NO: 1 as the reference sequence described herein.

[0304] In some preferred embodiments, the CHAP domain of the present invention has at least 93.9% or at least 94% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO: 9. In particular, the CHAP domain has at least one amino acid substitution selected from the group consisting of T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y, and N186G relative to the sequence from positions 72 to 215 of SEQ ID NO: 1, i.e., as the reference sequence described herein.

[0305] In some preferred embodiments, the peptidoglycan hydrolase of the present invention has at least 95.4% or at least 96% sequence identity to the sequence of SEQ ID NO: 9. In particular, the peptidoglycan hydrolase has at least one amino acid substitution in SEQ ID NO: 1 selected from the group consisting of T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y, and N186G, with respect to SEQ ID NO: 1 as the reference sequence described herein.

[0306] Saved Positions and Saved Segments In some embodiments, a segment of a CHAP domain of the invention has at least 80%, preferably at least 90%, sequence identity with the sequence from positions 87 to 128 of SEQ ID NO: 1. In particular, the segment is contained in a CHAP domain of the invention at a position corresponding to positions 87 to 128 of SEQ ID NO: 1, i.e., it is a corresponding segment. For example, as shown in Example 7, the segment may be considered herein and in the context of the present invention to be a particularly conserved CHAP segment.

[0307] Furthermore, in some or other of these embodiments, the CHAP domain of the invention has at most 6, 5, 4, 3 or 2, more preferably at most 1, or most preferably no amino acid substitutions or deletions at positions 80, 87, 88, 98, 99, 103, 106, 110, 114, 122, 126, 128, 137, 182, 202, and 208 of SEQ ID NO: 1 or positions corresponding to these positions. For example, as shown in Example 7, such positions may be considered to be conserved positions within a CHAP domain herein and in the context of the present invention.

[0308] In the context of the present invention, the CHAP domain of the present invention preferably has one or more amino acid substitutions at positions other than the conserved positions described herein. Nevertheless, the CHAP domain may also contain one or more mutations, e.g., amino acid substitutions, at conserved positions. For example, the in-silico deimmunization screen illustrated in Example 10 revealed possible amino acid substitutions at many different positions, including "conserved positions." Notably, this deimmunization screen is designed to maintain advantageous pharmaceutical properties, particularly bactericidal activity, stability, and secretion ability from eukaryotic cells, upon deimmunization. Thus, mutations such as the deimmunization mutations found in the context of the present invention may also be introduced at positions considered "conserved" herein.

[0309] Moreover, the conserved CHAP segments, in the context of the present invention, preferably have fewer mutations than other segments of the CHAP domain of the present invention. Thus, the corresponding segments of the CHAP domain of the present invention preferably have a higher sequence identity (e.g., about 90%) to the sequence from positions 87 to 128 of SEQ ID NO: 1 compared to the sequence identity of the CHAP domain of the present invention to the sequence from positions 72 to 215 of SEQ ID NO: 1 (which in this example may be, for example, about 60% to 80%).

[0310] Therefore, the present invention further provides a peptidoglycan hydrolase having bactericidal activity, the peptidoglycan hydrolase having (i) at least 60% sequence identity with the sequence of positions 72 to 215 of SEQ ID NO: 1; and (ii) one or more amino acid substitutions compared to the sequence of positions 72 to 215 of SEQ ID NO: 1, and (a) the corresponding segment of the CHAP domain has at least 80%, preferably at least 90%, sequence identity with the sequence from positions 87 to 128 of SEQ ID NO: 1; and / or (b) a peptidoglycan hydrolase comprising a cysteine, histidine-dependent amidohydrolase / peptidase (CHAP) domain, wherein the CHAP domain has at most 6, 5, 4, 3, or 2, preferably at most 1, amino acid substitutions or deletions, or more preferably no amino acid substitutions or deletions, at positions 80, 87, 88, 98, 99, 103, 106, 110, 114, 122, 126, 128, 137, 182, 202, and 208 of SEQ ID NO: 1 or positions corresponding to these positions.

[0311] Preferably, said CHAP domain comprises at least one deglycosylation mutation at position 73 of SEQ ID NO: 1 as described herein or a position corresponding to this position, preferably the substitution N73G as described herein in the context of the L0482ag variant obtained during directed evolution, and / or at least one amino acid substitution, preferably at least one of the most beneficial mutations as described herein, i.e. at least an amino acid substitution selected from the group consisting of T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1.

[0312] Additional cell wall-binding domains Preferably, herein and in the context of the present invention, the peptidoglycan hydrolase of the present invention comprises at least one cell wall binding domain in addition to the CHAP domain of the present invention.

[0313] As mentioned above, endolysins often have at least one cell wall binding domain (CBD) that recognizes and binds to a specific epitope on the cell wall of a host bacterium to properly fix the catalytic effect of the enzymatically active domain (EAD). Usually, the CBD alone is enzymatically inactive. Thus, a domain that has catalytic activity and can recognize and bind to a specific epitope on the cell wall of a bacterium is considered herein to be the enzymatically active domain (EAD). Preferably, in this specification and in the context of the present invention, the cell wall binding domain is a peptidoglycan binding domain that specifically binds to the peptidoglycan structure of the target bacterium.

[0314] Suitable cell wall binding domains for use in the context of the present invention include, inter alia, LYSM domains, SH3 domains and choline binding domains.

[0315] Preferably, in the context of the present invention, the at least one cell wall binding domain comprises a LYSM domain and / or an SH3 domain as described herein, more preferably a LYSM domain.

[0316] Preferably, herein and in the context of the present invention, the cell wall binding domain is derived from an endolysin, in particular the cell wall binding domain thereof. Preferably, said endolysin has killing activity against Staphylococcus species or strains, more preferably Staphylococcus aureus.

[0317] In the context of the present invention, the cell wall-binding domain is preferably derived from an endolysin comprising a LYSM domain or an SH3 domain, more preferably an endolysin comprising a LYSM domain. Preferably, the LYSM domain is derived from an endolysin comprising a LYSM domain and a CHAP domain, and the LYSM domain is preferably N-terminal to the CHAP domain. In other words, the LYSM domain of the present invention is preferably derived from an endolysin having the LYSM-CHAP structure described herein, for example, L0482 (SEQ ID NO: 1) or L0499 (SEQ ID NO: 47).

[0318] Therefore, in a preferred embodiment, the peptidoglycan of the invention comprises, in addition to the CHAP domain of the invention, a LYSM domain as described herein.

[0319] Preferably, in the context of the present invention, the cell wall binding domain, eg the LYSM domain, is N-terminal to the CHAP domain.

[0320] Preferably, herein, a LYSM domain according to the invention is defined by, or has at least 60% sequence identity with, the sequence of positions 1 to 51 of SEQ ID NO: 1. In other words, a LYSM domain according to the invention is preferably defined by, or has at least 60% sequence identity with, the sequence of SEQ ID NO: 302.

[0321] In some embodiments, the LYSM domain is defined by the sequence from positions 1 to 47 of SEQ ID NO:47, or has at least 60% sequence identity with the sequence from positions X to Y of SEQ ID NO:47.

[0322] In some embodiments, the SH3 domain is defined by or has at least 60% sequence identity with the sequence of SEQ ID NO:376.

[0323] In some embodiments, the SH3 domain is defined by or has at least 60% sequence identity with the sequence of SEQ ID NO:377.

[0324] Furthermore, a cell wall binding domain of the invention, such as a LYSM domain of the invention, preferably has the ability to bind to the cell wall of a Staphylococcus species or strain described herein, more preferably Staphylococcus aureus.

[0325] Beneficial and tolerated amino acid substitutions in the LYSM domain discovered by directed evolution or in silico deimmunization screening As described herein and illustrated in the accompanying Examples, the inventors have discovered a variety of amino acid substitutions contained in the LYSM domain of L0482ag variants obtained by directed evolution; see, for example, the "Beneficial / Tolerated Residues" column in Table 3. Notably, all L0482ag variants containing these amino acid substitutions were determined to be well secreted by eukaryotic cells and active by the YODA method. Furthermore, as also described herein and illustrated in the accompanying Examples, the inventors have discovered deimmunizing substitutions in the LYSM domain of L0482 variants that may reduce the immunogenicity of the LYSM domain of the invention and peptidoglycan hydrolases containing the LYSM domain of the invention; see, for example, the "Deimmunizing" column in Table 5.

[0326] Thus, the LYSM domain of the present invention (preferably having at least 60% sequence identity to positions 1 to 51 of SEQ ID NO: 1) comprises one or more amino acid substitutions at positions 1 to 6, 8, 10 to 13, 16 to 20, 22 to 30, 32 to 45 and 47 to 51 of SEQ ID NO: 1, or positions corresponding to these positions, and preferably the amino acid residue at position 1 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tryptophan, glutamine, or asparagine; the amino acid residue at position 2 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 3 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 4 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 5 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 6 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine or proline, the amino acid residue at position 8 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 10 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine or alanine, the amino acid residue at position 11 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or isoleucine, the amino acid residue at position 12 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, glycine, glutamine, or serine; the amino acid residue at position 13 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid, aspartic acid, asparagine, proline, or arginine; the amino acid residue at position 16 of SEQ ID NO: 1 or a position corresponding to this position is substituted with methionine, the amino acid residue at position 17 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, isoleucine, methionine, or phenylalanine; the amino acid residue at position 18 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine or proline; the amino acid residue at position 19 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, histidine, lysine, asparagine, glutamine, serine, or valine; the amino acid residue at position 20 of SEQ ID NO: 1 or a position corresponding to this position is substituted with methionine or valine; the amino acid residue at position 22 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, aspartic acid, glycine, histidine, lysine, glutamine, arginine, or tryptophan; the amino acid residue at position 23 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine, asparagine, glutamine, or tryptophan; the amino acid residue at position 24 of SEQ ID NO: 1 or a position corresponding to this position is substituted with cysteine, histidine, threonine, or asparagine; the amino acid residue at position 25 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid, aspartic acid, glycine, histidine, glutamine, serine, or arginine; the amino acid residue at position 26 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, the amino acid residue at position 27 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, the amino acid residue at position 28 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, glutamic acid, glutamine, serine, threonine, or alanine; the amino acid residue at position 29 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid; the amino acid residue at position 30 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, alanine, glutamic acid, glutamine, or serine; the amino acid residue at position 32 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, lysine, methionine, or arginine; the amino acid residue at position 33 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, alanine, glutamic acid, glycine, lysine, glutamine, or serine; the amino acid residue at position 34 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, tryptophan, or isoleucine; The amino acid residue at position 36 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, glycine, glutamine, isoleucine, or serine, the amino acid residue at position 37 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine, aspartic acid, lysine, methionine, asparagine, glutamine, or valine; the amino acid residue at position 38 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine, serine, or glycine; the amino acid residue at position 39 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine, alanine, aspartic acid, glycine, or isoleucine; the amino acid residue at position 40 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine, aspartic acid, glycine, asparagine, serine, leucine, or glutamine; the amino acid residue at position 41 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine, aspartic acid, or threonine; the amino acid residue at position 42 of SEQ ID NO: 1 or a position corresponding thereto is substituted with aspartic acid, histidine, lysine, methionine, asparagine, glutamine, or serine; the amino acid residue at position 43 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine, valine, or threonine; the amino acid residue at position 44 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, glutamic acid, histidine, serine, tryptophan, or leucine; the amino acid residue at position 45 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, aspartic acid, glutamic acid, lysine, proline, glutamine, or threonine; the amino acid residue at position 47 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine; the amino acid residue at position 48 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, glutamic acid, asparagine, glutamine, isoleucine, or arginine; the amino acid residue at position 49 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine, methionine, or asparagine; the amino acid residue at or corresponding to position 50 of SEQ ID NO: 1 is substituted with aspartic acid, histidine, asparagine, threonine or isoleucine; and / or The amino acid residue at position 51 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine. It may have one or more amino acid substitutions.

[0327] The above amino acid substitutions, when described in the context of the beneficial or tolerable amino acid substitutions described herein, e.g., in the CHAP domain, may be considered beneficial, or at least tolerable, with respect to the desired pharmaceutical properties of the peptidoglycan hydrolases of the invention.

[0328] Furthermore, the LYSM domain of the present invention (preferably having at least 60% sequence identity to positions 1 to 51 of SEQ ID NO: 1) may comprise one or more amino acid substitutions at positions 1, 8, 10, 12, 13, 17, 19, 22 to 25, 28 to 30, 32 to 45, and 48 to 51 of SEQ ID NO: 1, or positions corresponding to these positions, preferably the amino acid residue at position 1 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tryptophan or glutamine, the amino acid residue at position 8 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 10 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine; the amino acid residue at position 12 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, glycine, glutamine, or serine; the amino acid residue at position 13 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid, aspartic acid, asparagine, or proline; the amino acid residue at position 17 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, isoleucine, or methionine; the amino acid residue at position 19 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, histidine, lysine, asparagine, or glutamine; the amino acid residue at position 22 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, aspartic acid, glycine, histidine, lysine, glutamine, arginine, or tryptophan; the amino acid residue at position 23 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine, asparagine, glutamine, or tryptophan; the amino acid residue at position 24 of SEQ ID NO: 1 or a position corresponding to this position is substituted with cysteine, histidine, or threonine; the amino acid residue at position 25 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid, aspartic acid, glycine, histidine, glutamine, or serine; the amino acid residue at position 28 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, glutamic acid, glutamine, serine, or threonine; the amino acid residue at position 29 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid; the amino acid residue at position 30 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, alanine, glutamic acid, or glutamine; the amino acid residue at position 32 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, lysine, or methionine; the amino acid residue at position 33 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, alanine, glutamic acid, glycine, lysine, or glutamine; the amino acid residue at position 34 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or tryptophan; the amino acid residue at position 36 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, glycine, or glutamine; the amino acid residue at position 37 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine, aspartic acid, lysine, methionine, asparagine, or glutamine; the amino acid residue at position 38 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine or serine, the amino acid residue at position 39 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine, alanine, aspartic acid, or glycine; the amino acid residue at position 40 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine, aspartic acid, glycine, asparagine, or serine; the amino acid residue at position 41 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine or aspartic acid; the amino acid residue at position 42 of SEQ ID NO: 1 or a position corresponding thereto is substituted with aspartic acid, histidine, lysine, methionine, asparagine, glutamine, or serine; the amino acid residue at position 43 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine or valine; the amino acid residue at position 44 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, glutamic acid, histidine, serine, or tryptophan; the amino acid residue at position 45 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, aspartic acid, glutamic acid, lysine, proline, glutamine, or threonine; the amino acid residue at position 48 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, glutamic acid, asparagine, or glutamine; the amino acid residue at position 49 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine, methionine, or asparagine; The amino acid residue at or corresponding to position 50 of SEQ ID NO: 1 is substituted with aspartic acid, histidine, asparagine, or threonine; and / or The amino acid residue at position 51 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine. It may have one or more amino acid substitutions.

[0329] The above amino acid substitutions may be considered beneficial, or at least tolerable, with respect to the desired pharmaceutical properties of the peptidoglycan hydrolases of the present invention, as described herein.

[0330] As already mentioned above, e.g., in the context of the CHAP domain of the present invention, Table 3 (Table 5) shows positions in SEQ ID NO: 1 where beneficial or tolerant amino acid substitutions have been found. These are positions where the "Beneficial / Tolerant Residues" column of Table 3 (Table 5) shows at least one amino acid residue, regardless of whether that residue is shown without or within parentheses. These positions are also referred to herein as "tolerant positions." Furthermore, positions 1 to 51 in Table 3 (Table 5), where the "Beneficial / Tolerant Residues" column of Table 3 shows at least one amino acid residue, regardless of whether that residue is shown without or within parentheses, are considered to be "tolerant positions" in the LYSM domains herein.

[0331] As already mentioned above, in the present specification and in the context of the present invention, amino acid residues of SEQ ID NO: 1 corresponding to advantageous or tolerant positions may be substituted with the amino acid residues shown in the column "Advantageous / Tolerant Residues" in Table 3 for said positions, i.e. with the residues shown in said column of Table 3 without brackets or within brackets, preferably with the residues shown in said column of Table 3 without brackets.

[0332] Thus, in some embodiments, particularly in the context of tolerant / beneficial mutations and Table 3, the LYSM domain has at least one amino acid substitution at a permissive position in the LYSM domain or at the corresponding permissive position in the LYSM domain. Preferably, in these embodiments, at least one amino acid residue at the permissive position in the LYSM domain or at a position corresponding to the permissive position in the LYSM domain is substituted with the amino acid residue shown in the "Beneficial / Tolerated Residue" column of Table 3 for that position, more preferably with the residue shown without parentheses in that column of Table 3.

[0333] Particularly beneficial amino acid substitutions in the LYSM domain Furthermore, as described herein, the inventors have found hit variants containing certain amino acid substitutions in the LYSM domain, namely, G1-G4, H1-H10, and I1-I30; see, e.g., Table 4. These amino acid substitutions are thus considered, in the context of the present invention, to be particularly beneficial mutations, i.e., amino acid substitutions, in the LYSM domain. As described herein, for example, in the context of the CHAP domain of the present invention, a "particularly beneficial amino acid substitution" may be particularly beneficial for maintaining or enhancing the desired pharmaceutical properties of the peptidoglycan hydrolase, in particular, bactericidal activity (e.g., against Staphylococcus aureus), stability, and / or secretion ability from eukaryotic cells, e.g., human cells. This also applies to particularly beneficial amino acid substitutions in the LYSM domain.

[0334] Therefore, the LYSM domain of the present invention (preferably having at least 60% sequence identity to positions 1 to 51 of SEQ ID NO: 1) preferably comprises one or more amino acid substitutions at positions 1, 8, 10, 13, 23 to 25, 30, 33, 37, and 39 to 41 of SEQ ID NO: 1 or positions corresponding to these positions, preferably the amino acid residue at position 1 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tryptophan; the amino acid residue at position 8 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 10 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine; the amino acid residue at position 13 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid; the amino acid residue at position 23 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 24 of SEQ ID NO: 1 or a position corresponding to this position is substituted with cysteine, the amino acid residue at position 25 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid; the amino acid residue at position 30 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid; the amino acid residue at position 33 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid; the amino acid residue at position 37 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine, the amino acid residue at position 39 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 40 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine, and / or The amino acid residue at position 41 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine. It has one or more amino acid substitutions.

[0335] As mentioned above, the cell wall binding domain of the invention, such as the LYSM domain of the invention, preferably has the ability to bind to the cell wall of a Staphylococcus species or strain, more preferably Staphylococcus aureus.

[0336] Furthermore, the peptidoglycan hydrolases comprising a CHAP domain and / or a LYSM domain described herein preferably have at least 60% sequence identity with the sequence of SEQ ID NO: 1, as described herein.

[0337] Additional Linkers Preferably, herein, and in the context of the present invention, particularly in the context of peptidoglycan hydrolases comprising a CHAP domain and a CBD, the peptidoglycan hydrolase comprises a peptide linker between the CHAP domain and the cell wall binding domain.

[0338] The peptide linker is not limited to any particular linker, and any linker used in the art to connect different domains or portions of proteins, such as fusion proteins, may be used herein and in the context of the present invention.

[0339] As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence that connects two or more domains (e.g., a CHAP domain and a cell wall-binding domain of the invention, and / or a CHAP and a pharmacokinetic-extending (PK) peptide of the invention) in the linear amino acid sequence of a polypeptide chain. Preferably, the linker is a flexible linker. Exemplary linkers include a glycine-serine-polypeptide linker, a glycine-proline-polypeptide linker, and a proline-alanine polypeptide linker. An exemplary linker having a furin cleavage site (which may also be used in the context of the present invention) is set forth in SEQ ID NO: 370.

[0340] In some embodiments, the linker is a glycine-serine linker, i.e., a peptide consisting predominantly, essentially, or exclusively of glycine and serine residues. Herein, and in the context of the present invention, a glycine-serine linker may comprise one or more copies (e.g., 2-5 copies) of the sequence set forth in SEQ ID NO: 297 (i.e., GGGGS). Preferably, the copies are immediately adjacent to each other, as set forth in SEQ ID NO: 298 or 299 (i.e., GGGGSGGGGS (GS2: 2xGGGGS) or GGGGSGGGGSGGGGSGGGGS (GS4: 4xGGGGS), respectively). Furthermore, GS3 (3xGGGGS) or G5 (5xGGGGS) can equally be used. Further suitable glycine-serine linkers are set forth in SEQ ID NOs: 363-369. Another suitable glycine-serine linker has the sequence "GGS."

[0341] In certain embodiments, the peptide linker is derived from an endolysin, in particular its linker sequence. Preferably, said endolysin has killing activity against Staphylococcus species or strains, preferably Staphylococcus aureus.

[0342] In some preferred embodiments, the peptide linker is a L0482-derived linker, ie, a linker having at least 60% sequence identity with the sequence from positions 52 to 71 of SEQ ID NO:1.

[0343] Beneficial and tolerated amino acid substitutions in the L0482 linker found by deglycosylation screening, directed evolution, or in silico deimmunization screening As described herein and illustrated in the accompanying Examples, the inventors have found a variety of amino acid substitutions contained in the linker sequences of L0482 variants obtained by directed evolution; see, for example, the "Beneficial / Tolerated Residues" column in Table 3. Notably, all L0482 variants containing these amino acid substitutions were successfully secreted from eukaryotic cells and were determined to be active by the YODA method. Furthermore, as also described herein and illustrated in the accompanying Examples, the inventors have found deimmunizing substitutions in the linker sequences of L0482 variants that can reduce the immunogenicity of linker sequences and peptidoglycan hydrolases containing such linkers; see, for example, the "Deimmunizing" column in Table 5.

[0344] Furthermore, the inventors have discovered an amino acid substitution at position 68 of SEQ ID NO: 1, i.e., a deglycosylation substitution in the linker of L0482, that enhances bactericidal activity when expressed in eukaryotic cells as described herein; see, e.g., Example 4.

[0345] Thus, the L0482-derived linker (which may have at least 60% sequence identity with the sequence of positions 52 to 71 of SEQ ID NO: 1) may contain one or more amino acid substitutions at positions 52 to 56, 58, 60 to 66, and 68 to 71 of SEQ ID NO: 1 or positions corresponding to these positions, preferably the amino acid residue at position 52 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 53 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine, alanine, glycine, lysine, glutamine, or serine; The amino acid residue at position 54 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, glutamic acid, histidine, asparagine, alanine, or isoleucine; the amino acid residue at position 55 of SEQ ID NO: 1 or a position corresponding to this position is substituted with histidine, aspartic acid, or serine; the amino acid residue at position 56 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine or isoleucine, the amino acid residue at position 58 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; the amino acid residue at position 60 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid or isoleucine, the amino acid residue at position 61 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine or glutamine, the amino acid residue at position 62 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid or asparagine, the amino acid residue at position 63 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine, alanine, or methionine; the amino acid residue at position 64 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine or leucine, the amino acid residue at position 65 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine, aspartic acid, asparagine, or arginine; the amino acid residue at position 66 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 68 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine, methionine, arginine, alanine, or serine; the amino acid residue at position 69 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine, histidine, asparagine, or glutamine; the amino acid residue at position 70 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; and / or The amino acid residue at position 71 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine or aspartic acid. It may have one or more amino acid substitutions.

[0346] The above amino acid substitutions may be considered beneficial or at least tolerable with respect to the desired pharmaceutical properties of the peptidoglycan hydrolases of the invention, as described herein, e.g., in the context of beneficial or tolerable amino acid substitutions in the CHAP domain.

[0347] Furthermore, the peptide linker (which may have at least 60% sequence identity with the sequence of positions 52 to 71 of SEQ ID NO: 1) may comprise one or more amino acid substitutions at positions 52 to 56, 58, 63, 65, 68, 69 and 71 of SEQ ID NO: 1 or positions corresponding to these positions, preferably the amino acid residue at position 52 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 53 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine, alanine, glycine, lysine, glutamine, or serine; the amino acid residue at position 54 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid, glutamic acid, histidine, or asparagine; the amino acid residue at position 55 of SEQ ID NO: 1 or a position corresponding to this position is substituted with histidine or aspartic acid; the amino acid residue at position 56 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 58 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; the amino acid residue at position 63 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine; the amino acid residue at position 65 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine or aspartic acid; the amino acid residue at position 68 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine, methionine, arginine, alanine, or serine; The amino acid residue at or corresponding to position 69 of SEQ ID NO: 1 is substituted with glycine, histidine, asparagine, or glutamine; and / or The amino acid residue at position 71 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine. It may have one or more amino acid substitutions.

[0348] The above amino acid substitutions may be considered beneficial, or at least tolerable, with respect to the desired pharmaceutical properties of the peptidoglycan hydrolases of the invention described herein.

[0349] As already mentioned above, for example, in the context of the CHAP domain of the present invention, Table 3 (Table 5) shows positions in SEQ ID NO: 1 where beneficial or tolerant amino acid substitutions have been found. These are positions where the "Beneficial / Tolerant Residues" column in Table 3 (Table 5) shows at least one amino acid residue, whether that residue is shown without or within parentheses. These positions are also referred to herein as "tolerant positions." Furthermore, positions 52-71 in Table 3 (Table 5), where the "Beneficial / Tolerant Residues" column shows at least one amino acid residue, whether that residue is shown without or within parentheses, are considered herein to be "tolerant positions" in the linker of L0482.

[0350] Herein, and in the context of the present invention, an amino acid residue in SEQ ID NO: 1 corresponding to a beneficial or tolerant position may be substituted with an amino acid residue shown in the "Beneficial / Tolerant Residue" column of Table 3 for said position, i.e., a residue shown in said column of Table 3 without brackets or within brackets, preferably a residue shown in said column of Table 3 without brackets.

[0351] In some embodiments, particularly in the context of tolerant / beneficial mutations and Table 3, the L0482-derived peptide linker has at least one amino acid substitution at a permissive position in the linker region of L0482 or at a position corresponding to said permissive position in the linker region of L0482. Preferably, in these embodiments, at least one amino acid residue at said permissive position in the linker region of L0482 or at a position corresponding to said permissive position in the linker region of L0482 is substituted with the amino acid residue shown in the "Beneficial / Tolerated Residue" column of Table 3 for said position, more preferably with the residue shown without parentheses in said column of Table 3.

[0352] Particularly beneficial amino acid substitutions in the L0482 linker Furthermore, as described herein, the inventors have found hit variants, namely, G1-G4, H1-H10, and I1-I30, containing certain amino acid substitutions in the linker region of the L0482 variant; see, e.g., Table 4. These amino acid substitutions are thus considered, in the context of the present invention, to be particularly beneficial mutations, i.e., amino acid substitutions, in the L0482-derived peptide linker. As used herein, for example, in the context of the CHAP domain of the present invention, a "particularly beneficial amino acid substitution" may be particularly beneficial for maintaining or enhancing the desired pharmaceutical properties of the peptidoglycan hydrolase, in particular, bactericidal activity (e.g., against Staphylococcus aureus), stability, and / or secretion ability from eukaryotic cells, e.g., human cells. This also applies to particularly beneficial amino acid substitutions in the linker of L0482.

[0353] Thus, in some preferred embodiments, particularly in the context of L0482-derived peptide linkers, the peptide linker has at least 60% sequence identity with the sequence of positions 52-71 of SEQ ID NO:1, and one or more amino acid substitutions at or corresponding to positions 53, 55, 56, 58, 63, 65 and 68 of SEQ ID NO:1, preferably the amino acid residue at position 53 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine; the amino acid residue at position 55 of SEQ ID NO: 1 or a position corresponding to this position is substituted with histidine, the amino acid residue at position 56 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 58 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; the amino acid residue at position 63 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine; the amino acid residue at or corresponding to position 65 of SEQ ID NO: 1 is substituted with glycine; and / or The amino acid residue at position 68 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine. It has one or more amino acid substitutions.

[0354] Preferably, the peptide linker comprises a deglycosylation mutation, e.g., a deletion (and preferably an amino acid substitution), at or corresponding to position 68 of SEQ ID NO: 1, as described herein. Preferably, the L0482-derived peptide linker comprises an amino acid substitution at or corresponding to position 68 of SEQ ID NO: 1, where the amino acid residue at said position is substituted with lysine, methionine, arginine, or alanine, preferably lysine. Furthermore, the peptide linker (in addition to the deglycosylation mutation) preferably comprises at least one amino acid substitution at or corresponding to positions 53, 55, 56, 58, 63, and 65 of SEQ ID NO: 1, as described herein, e.g., as described immediately above.

[0355] Furthermore, the peptidoglycan hydrolases described herein comprising a CHAP domain, a LYSM domain and / or an L0482-derived peptide linker preferably have at least 60% sequence identity with the sequence of SEQ ID NO: 1, as described herein.

[0356] LYSM domains of the present invention and peptidoglycan hydrolases comprising the LYSM domains of the present invention As described herein and illustrated in the accompanying Examples, the inventors have found particularly beneficial amino acid substitutions in the LYSM domain of L0482.

[0357] Thus, in some aspects, the present invention provides a polypeptide comprising: (i) at least 60% sequence identity with the sequence of positions 1 to 51 of SEQ ID NO:1; and (ii) one or more amino acid substitutions at positions 1, 8, 10, 13, 23-25, 30, 33, 37, and 39-41 of SEQ ID NO:1, or positions corresponding to these positions, the amino acid residue at position 1 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tryptophan; the amino acid residue at position 8 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 10 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine; the amino acid residue at position 13 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid; the amino acid residue at position 23 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 24 of SEQ ID NO: 1 or a position corresponding to this position is substituted with cysteine, the amino acid residue at position 25 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid; the amino acid residue at position 30 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid; the amino acid residue at position 33 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid; the amino acid residue at position 37 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine, the amino acid residue at position 39 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 40 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine, and / or The amino acid residue at position 41 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine. The present invention relates to a LYSM domain having one or more amino acid substitutions.

[0358] In particular, said LYSM domain has the ability to bind to the cell wall of a Staphylococcus species or strain, more preferably Staphylococcus aureus, as described herein.

[0359] Moreover, the LYSM domain is particularly advantageous for use in peptidoglycan hydrolases with bactericidal activity as described herein and in the context of the present invention.

[0360] Thus, the present invention further provides a peptidoglycan hydrolase having bactericidal activity, e.g., in the context of a LYSM domain of the present invention, comprising: (a) an enzymatically active domain; and (b) (i) at least 60% sequence identity with the sequence of positions 1 to 51 of SEQ ID NO:1; and (ii) one or more amino acid substitutions at positions 1, 8, 10, 13, 23-25, 30, 33, 37, and 39-41 of SEQ ID NO:1, or positions corresponding to these positions, the amino acid residue at position 1 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tryptophan; the amino acid residue at position 8 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 10 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine; the amino acid residue at position 13 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid; the amino acid residue at p...

Claims

1. 1. A peptidoglycan hydrolase having bactericidal activity, comprising: (i) at least 83% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:1; (ii) an amino acid substitution or deletion at position 73 of SEQ ID NO:1 or a position corresponding thereto; and (iii) one or more amino acid substitutions at positions 86, 82, 85, 130, 136, 155, 169, 185, and 186 of SEQ ID NO:1 or a position corresponding thereto; the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 130 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine or arginine; the amino acid residue at position 155 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 is substituted with tyrosine, and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine. One or more amino acid substitutions A peptidoglycan hydrolase containing a cysteine, histidine-dependent amidohydrolase / peptidase (CHAP) domain.

2. 2. The peptidoglycan hydrolase of claim 1, having at least 60% sequence identity with the sequence of SEQ ID NO:

1.

3. 3. The peptidoglycan hydrolase of claim 2, further comprising an amino acid substitution or deletion at position 68 of SEQ ID NO: 1 or a position corresponding to this position, preferably in which the amino acid residue at position 68 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine, methionine, arginine, or alanine, more preferably lysine.

4. 4. The peptidoglycan hydrolase according to claim 1, wherein the residue at position 73 of SEQ ID NO: 1 or a position corresponding thereto is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine, or serine, preferably glycine.

5. The CHAP domain comprises the following consensus mutation units I) to V): I) an amino acid substitution at position 86 of SEQ ID NO: 1 or a position corresponding to this position, in which the amino acid at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; II) an amino acid substitution pair at positions 82 and 85 of SEQ ID NO: 1 or a position corresponding to these positions, in which the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine, and the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; III) an amino acid substitution pair at positions 130 and 136 of SEQ ID NO: 1 or a position corresponding to these positions, in which the amino acid residue at position 130 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, and the amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine or arginine; IV) an amino acid substitution at or corresponding to position 169 of SEQ ID NO: 1, in which the amino acid at or corresponding to position 169 of SEQ ID NO: 1 is replaced with asparagine; and / or V) An amino acid substitution pair at positions 185 and 186 of SEQ ID NO: 1 or a position corresponding to these positions, in which the amino acid residue at position 185 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine, and the amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine. The peptidoglycan hydrolase according to claim 1 , having at least one, preferably at least two or more of the following:

6. 6. The peptidoglycan hydrolase of claim 5, wherein the CHAP domain further comprises an amino acid substitution at position 155 of SEQ ID NO: 1 or a position corresponding thereto, in which phenylalanine at position 155 of SEQ ID NO: 1 or a position corresponding thereto is replaced with tyrosine.

7. 7. The peptidoglycan hydrolase according to claim 1, wherein the CHAP domain has an amino acid substitution at position 86 of SEQ ID NO: 1 or a position corresponding to this position, in which the amino acid at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine.

8. The peptidoglycan hydrolase according to any one of claims 1 to 7, wherein the CHAP domain has at least 94% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:

11.

9. the CHAP domain: (a) an amino acid substitution at position 86 of SEQ ID NO: 1 or a position corresponding to this position, in which the amino acid at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; (b) at least one, preferably two, amino acid substitutions at positions 82 and 85 of SEQ ID NO: 1 or a corresponding position, wherein the amino acid residue at position 82 of SEQ ID NO: 1 or a corresponding position is substituted with serine, and the amino acid residue at position 85 of SEQ ID NO: 1 or a corresponding position is substituted with glycine; and / or (c) an amino acid substitution at position 169 of SEQ ID NO: 1 or a position corresponding to this position, in which the amino acid at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine; The peptidoglycan hydrolase according to any one of claims 1 to 7, having the formula:

10. the CHAP domain further comprises one or more amino acid substitutions at positions 173, 175, and 192 of SEQ ID NO: 1, or positions corresponding to these positions, the amino acid residue at position 173 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at or corresponding to position 175 of SEQ ID NO: 1 is substituted with glutamine, and / or The amino acid residue at position 192 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine.

10. The peptidoglycan hydrolase of claim 9, comprising one or more amino acid substitutions.

11. 11. The peptidoglycan hydrolase according to claim 1, further comprising at least one cell wall binding domain, preferably at least one cell wall binding domain derived from an endolysin, more preferably a LYSM domain and / or an SH3 domain.

12. The peptidoglycan hydrolase according to any one of claims 1 to 11, comprising a LYSM domain having at least 60% sequence identity with the sequence from positions 1 to 51 of SEQ ID NO:

1.

13. the LYSM domain comprises one or more amino acid substitutions at positions 1, 8, 10, 13, 23-25, 30, 33, 37, and 39-41 of SEQ ID NO: 1, or positions corresponding to these positions; the amino acid residue at position 1 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tryptophan; the amino acid residue at position 8 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 10 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine; the amino acid residue at position 13 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid; the amino acid residue at position 23 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 24 of SEQ ID NO: 1 or a position corresponding to this position is substituted with cysteine, the amino acid residue at position 25 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid; the amino acid residue at position 30 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid; the amino acid residue at position 33 of SEQ ID NO: 1 or a position corresponding to this position is substituted with aspartic acid; the amino acid residue at position 37 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine, the amino acid residue at position 39 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 40 of SEQ ID NO: 1 or a position corresponding to this position is substituted with threonine, and / or The amino acid residue at position 41 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine.

13. The peptidoglycan hydrolase of claim 12, having one or more amino acid substitutions.

14. 14. The peptidoglycan hydrolase of any one of claims 1 to 13, having killing activity against at least one Staphylococcus species or strain, preferably Staphylococcus aureus, more preferably a Staphylococcus aureus strain that is resistant to at least one antibiotic, such as methicillin.

15. 15. The peptidoglycan hydrolase of claim 14, wherein the Staphylococcus species or strain, preferably the Staphylococcus aureus, is present in the form of a biofilm or is suspected of forming a biofilm.

16. 16. The peptidoglycan hydrolase of claim 1, which is stable up to a temperature of about 40°C.

17. 17. The peptidoglycan hydrolase of claim 1, which has enhanced killing activity against Staphylococcus aureus, enhanced secretion ability by human cells, and / or enhanced thermostability compared to the peptidoglycan hydrolase of SEQ ID NO:

1.

18. 18. The peptidoglycan hydrolase of claim 1, further comprising a pharmacokinetic-extending (PK) peptide, such as a human FC domain, a C-terminal peptide of human chorionic gonadotropin, or human lysozyme.

19. 19. The peptidoglycan hydrolase of claim 1, further comprising a signal peptide, preferably at the N-terminus.

20. 20. A nucleic acid encoding the peptidoglycan hydrolase of any one of claims 1 to 19.

21. 21. The nucleic acid of claim 20, wherein the peptidoglycan hydrolase comprises a signal peptide, preferably at the N-terminus.

22. 22. The nucleic acid of claim 20 or 21, which is RNA.

23. In order from 5' to 3', (i) a 5'UTR comprising or consisting of a modified human alpha-globin 5'-UTR; (ii) a sequence encoding a peptidoglycan hydrolase according to any one of claims 1 to 19; (iii) a 3'UTR comprising or consisting of a first sequence from an amino terminal enhancer of split (AES) messenger RNA and a second sequence from a mitochondrial-encoded 12S ribosomal RNA; and (iv) Poly(A) sequence an RNA construct comprising: Preferably, the RNA construct comprises: (v) a 5' cap, and / or (vi) in place of uridine, preferably in place of each uridine, a modified nucleoside selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U), preferably N1-methyl-pseudouridine (m1ψ); 23. The nucleic acid of any one of claims 20 to 22, further comprising:

24. A pharmaceutical composition comprising a peptidoglycan hydrolase according to any one of claims 1 to 19 and / or a nucleic acid according to any one of claims 20 to 23; and preferably a pharmaceutically acceptable excipient.

25. A peptidoglycan hydrolase described in any one of claims 1 to 19, a nucleic acid described in any one of claims 20 to 23, or a pharmaceutical composition described in claim 24, for use in treating a disease caused by and / or associated with a staphylococcal infection and / or a subject having or suspected of having a staphylococcal infection.

26. 26. The peptidoglycan hydrolase, nucleic acid, or pharmaceutical composition for use according to claim 25, wherein the infection is a Staphylococcus aureus infection.

27. A pharmaceutical composition comprising a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase (i) has at least 60% sequence identity with the amino acid sequence of positions 72 to 215 of SEQ ID NO: 1; and (ii) comprises a cysteine, histidine-dependent amidohydrolase / peptidase (CHAP) domain having one or more amino acid substitutions compared to the amino acid sequence of positions 72 to 215 of SEQ ID NO: 1, and preferably further comprises a pharmaceutically acceptable excipient.

28. 28. The pharmaceutical composition of claim 27, wherein the CHAP domain has an amino acid substitution or deletion at or corresponding to position 73 of SEQ ID NO: 1, preferably wherein the residue at or corresponding to position 73 of SEQ ID NO: 1 is replaced with glycine, tyrosine, leucine, glutamic acid, alanine, histidine, or serine, more preferably glycine.

29. 29. The pharmaceutical composition of claim 27 or 28, wherein the peptidoglycan hydrolase has at least 60% sequence identity with the sequence of SEQ ID NO:

1.

30. 30. The pharmaceutical composition of claim 29, wherein the peptidoglycan hydrolase further comprises an amino acid substitution or deletion at position 68 of SEQ ID NO: 1 or a position corresponding to this position, preferably wherein the amino acid residue at position 68 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine, methionine, arginine, or alanine, more preferably lysine.

31. the CHAP domain comprises one or more amino acid substitutions at positions 73, 75, 78, 81, 82, 85, 86, 104, 107, 115, 124, 125, 130, 133, 135, 136, 140, 141, 155, 169, 173, 175, 178, 185, 186, 191, 192, 194, 198, 204, 212 and 215 of SEQ ID NO: 1, or positions corresponding to these positions; the amino acid residue at position 73 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine or serine, the amino acid residue at position 75 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine; the amino acid residue at position 78 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 81 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamic acid; the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; the amino acid residue at position 104 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 107 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 115 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 124 of SEQ ID NO: 1 or a position corresponding to this position is substituted with phenylalanine; the amino acid residue at position 125 of SEQ ID NO: 1 or a position corresponding to this position is substituted with valine; the amino acid residue at position 130 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 133 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 135 of SEQ ID NO: 1 or a position corresponding to this position is substituted with histidine, the amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine or arginine; the amino acid residue at position 140 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, the amino acid residue at position 141 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine; the amino acid residue at position 155 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine; the amino acid residue at position 178 of SEQ ID NO: 1 or a position corresponding to this position is substituted with leucine; the amino acid residue at position 185 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 191 of SEQ ID NO: 1 or a position corresponding to this position is substituted with arginine; the amino acid residue at position 192 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glutamine; the amino acid residue at position 194 of SEQ ID NO: 1 or a position corresponding to this position is substituted with alanine, the amino acid residue at position 198 of SEQ ID NO: 1 or a position corresponding to this position is substituted with isoleucine, the amino acid residue at position 204 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 212 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; and / or The amino acid residue at position 215 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine.

31. The pharmaceutical composition of any one of claims 27 to 30, having one or more amino acid substitutions.

32. the CHAP domain comprises one or more amino acid substitutions at positions 86, 82, 85, 130, 136, 155, 169, 185 and 186 of SEQ ID NO: 1, or positions corresponding to these positions; the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding to this position is substituted with serine; the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine; the amino acid residue at position 130 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding thereto is substituted with lysine or arginine, preferably lysine; the amino acid residue at position 155 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine; the amino acid residue at position 169 of SEQ ID NO: 1 or a position corresponding to this position is substituted with asparagine, the amino acid residue at or corresponding to position 185 of SEQ ID NO: 1 is substituted with tyrosine, and / or The amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine.

32. The pharmaceutical composition of any one of claims 27 to 31, having one or more amino acid substitutions.

33. 33. The pharmaceutical composition of any one of claims 27 to 32, wherein the CHAP domain has an amino acid substitution at position 86 of SEQ ID NO: 1 or a position corresponding to this position, preferably wherein the amino acid residue at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine.

34. The CHAP domain comprises the following consensus mutation units I) to V): I) an amino acid substitution at position 86 of SEQ ID NO: 1 or a position corresponding to this position, in which the amino acid at position 86 of SEQ ID NO: 1 or a position corresponding to this position is substituted with lysine; II) an amino acid substitution pair at positions 82 and 85 of SEQ ID NO: 1 or a position corresponding thereto, in which the amino acid residue at position 82 of SEQ ID NO: 1 or a position corresponding thereto is substituted with serine, and the amino acid residue at position 85 of SEQ ID NO: 1 or a position corresponding thereto is substituted with glycine; III) an amino acid substitution pair at positions 130 and 136 of SEQ ID NO: 1 or a position corresponding thereto, in which the amino acid residue at position 130 of SEQ ID NO: 1 or a position corresponding thereto is substituted with asparagine, and the amino acid residue at position 136 of SEQ ID NO: 1 or a position corresponding thereto is substituted with lysine or arginine; IV) an amino acid substitution at or corresponding to position 169 of SEQ ID NO: 1, in which the amino acid at or corresponding to position 169 of SEQ ID NO: 1 is replaced with asparagine; and / or V) An amino acid substitution pair at positions 185 and 186 of SEQ ID NO: 1 or a position corresponding to these positions, in which the amino acid residue at position 185 of SEQ ID NO: 1 or a position corresponding to this position is substituted with tyrosine, and the amino acid residue at position 186 of SEQ ID NO: 1 or a position corresponding to this position is substituted with glycine.

34. The pharmaceutical composition according to any one of claims 27 to 33, comprising at least one, preferably at least two or more of:

35. 35. The pharmaceutical composition of any one of claims 27 to 34, wherein the CHAP domain has an amino acid substitution at position 155 of SEQ ID NO: 1 or a position corresponding to this position, preferably wherein the amino acid residue at position 155 of SEQ ID NO: 1 or a position corresponding to this position is replaced with tyrosine.

36. (i) the corresponding segment of the CHAP domain has at least 80%, preferably at least 90%, sequence identity with the sequence from positions 87 to 128 of SEQ ID NO: 1; and / or (ii) the CHAP domain has at most 6, 5, 4, 3, or 2, preferably at most 1, amino acid substitutions or deletions, and more preferably no amino acid substitutions or deletions, at positions 80, 87, 88, 98, 99, 103, 106, 110, 114, 122, 126, 128, 137, 182, 202, and 208 of SEQ ID NO: 1 or positions corresponding to these positions; 36. A pharmaceutical composition according to any one of claims 27 to 35.

37. 37. The pharmaceutical composition of any one of claims 27 to 36, wherein the peptidoglycan hydrolase further comprises at least one cell wall binding domain as defined in claim 11, preferably a LYSM domain as defined in claim 12 or 13.

38. 38. The pharmaceutical composition of any one of claims 27 to 37, wherein the peptidoglycan hydrolase has killing activity against at least one Staphylococcus species or strain, preferably Staphylococcus aureus, more preferably a Staphylococcus aureus strain that is resistant to at least one antibiotic, such as methicillin.

39. 39. The pharmaceutical composition of claim 38, wherein said Staphylococcus species or strain, preferably said Staphylococcus aureus, is present in the form of a biofilm or is suspected of forming a biofilm.

40. 40. The pharmaceutical composition of any one of claims 27 to 39, wherein the peptidoglycan hydrolase is stable up to a temperature of about 40°C.

41. 41. The pharmaceutical composition of any one of claims 27 to 40, wherein the peptidoglycan hydrolase has enhanced killing activity against Staphylococcus aureus, enhanced ability to be secreted by human cells, and / or enhanced thermostability compared to the peptidoglycan hydrolase of SEQ ID NO:

1.

42. 42. The pharmaceutical composition of any one of claims 27 to 41, wherein the CHAP domain has at least 80% sequence identity with the amino acid sequence of positions 72 to 215 of SEQ ID NO:

1.

43. 43. The pharmaceutical composition of any one of claims 27 to 42, wherein the CHAP domain has at least 90% sequence identity with the amino acid sequence of positions 72 to 215 of SEQ ID NO:

1.

44. 44. The pharmaceutical composition of any one of claims 27 to 43, wherein the CHAP domain has at least 94% sequence identity with the sequence from positions 72 to 215 of SEQ ID NO:

11.

45. 45. The pharmaceutical composition of any one of claims 27 to 44, wherein the peptidoglycan hydrolase further comprises a pharmacokinetic-prolonging (PK) peptide, such as a human FC domain, a C-terminal peptide of human chorionic gonadotropin, or human lysozyme.

46. 46. ​​The pharmaceutical composition of any one of claims 27 to 45, wherein the peptidoglycan hydrolase further comprises a signal peptide, preferably at the N-terminus.

47. 47. A pharmaceutical composition comprising a nucleic acid encoding a peptidoglycan hydrolase as defined in any one of claims 27 to 46.

48. 48. The pharmaceutical composition of claim 47, wherein the peptidoglycan hydrolase comprises a signal peptide, preferably at the N-terminus.

49. 49. The pharmaceutical composition of claim 47 or 48, wherein the nucleic acid is RNA.

50. The nucleic acid is, in 5' to 3' order: (i) a 5'UTR comprising or consisting of a modified human alpha-globin 5'-UTR; (ii) a sequence encoding a peptidoglycan hydrolase as defined in any one of claims 25 to 45; (iii) a 3'UTR comprising or consisting of a first sequence from an amino terminal enhancer of split (AES) messenger RNA and a second sequence from a mitochondrial-encoded 12S ribosomal RNA; and (iv) Poly(A) sequence an RNA construct comprising: Preferably, the RNA construct further comprises: (v) a 5' cap and / or (vi) in place of uridine, preferably in place of each uridine, a modified nucleoside selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U), preferably N1-methyl-pseudouridine (m1ψ); 50. The pharmaceutical composition of any one of claims 47 to 49, comprising:

51. 51. A pharmaceutical composition according to any one of claims 27 to 50 for use in treating a disease caused by and / or associated with a staphylococcal infection and / or a subject having or suspected of having a staphylococcal infection.

52. 52. The pharmaceutical composition for use according to claim 51, wherein the infection is a Staphylococcus aureus infection.

53. A peptidoglycan hydrolase, nucleic acid, or pharmaceutical composition for use as described in claim 25 or 26, or a pharmaceutical composition for use as described in claim 51 or 52, wherein the staphylococcus is present in the form of a biofilm and / or floating aggregates or is suspected of forming a biofilm and / or floating aggregates.

54. A peptidoglycan hydrolase, nucleic acid, or pharmaceutical composition for use according to any one of claims 25, 26, and 53, or a pharmaceutical composition for use according to any one of claims 51 to 53, wherein the staphylococcal infection is a Staphylococcus aureus infection of the skin, soft tissue, bone, lung, sinuses, and / or urinary tract.

55. 55. The peptidoglycan hydrolase, nucleic acid, or pharmaceutical composition for use according to any one of claims 25, 26, 53, and 54, or the pharmaceutical composition for use according to any one of claims 51 to 54, wherein the disease is selected from the group consisting of respiratory infections such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia, sepsis, sinusitis, acne, impetigo, boils, cellulitis, folliculitis, carbuncles, scalded skin syndrome, abscesses, food poisoning, necrotizing fasciitis, pyomyositis, mediastinitis, infectious dermatitis, wound infections, diabetic foot ulcers, septic arthritis, osteoarticular infections, periprosthetic infections such as infections of artificial joints or cardiac devices, and urinary tract infections.

56. 56. The peptidoglycan hydrolase, nucleic acid, or pharmaceutical composition for use according to claim 55, wherein the disease is pneumonia, bacteremia, endocarditis, or periprosthetic infection.

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