Peptidoglycan hydrolases having multiple enzymatically active domains

Abstract

The present invention is generally in the fields of pharmaceuticals, in particular antibacterials, and protein engineering. In particular, the present invention relates to peptidoglycan hydrolases such as endolysins and nucleic acids, e.g., RNAs, encoding the peptidoglycan hydrolases of the invention, as well as medical uses thereof, for example, for treating diseases caused by and / or associated with a Staphylococcus (e.g., S. aureus) infection.

Description

[0001] Peptidoglycan hydrolases having multiple enzymatically active domains

[0002] Field of the invention

[0003] The present invention is generally in the fields of pharmaceuticals, in particular antibacterials, and protein engineering. In particular, the present invention relates to peptidoglycan hydrolases such as endolysins and nucleic acids, e.g., RNAs, encoding the peptidoglycan hydrolases of the invention, as well as medical uses thereof, for example, for treating diseases caused by and / or associated with a Staphylococcus e.g., S. aureus) infection.

[0004] Background

[0005] Staphylococci, in particular Staphylococcus aureus strains, are major human pathogens responsible for a vast array of pathologies, both acute and chronic, varying from mild to life threatening, including skin and soft tissue infections, bone-related infections, pneumonia, and sepsis. For example, S. aureus is a leading cause of mortality among antibiotic resistant bacterial pathogens, with ~ 700.000 deaths per year due to antibiotic resistant S. aureus globally; Antimicrobial Resistance Collaborators (2022), The Lancet, 399. The pathogenesis of S. aureus infection involves several critical steps: invasion of host tissues, evasion of the immune system, adhesion to surfaces, and biofilm formation. For example, by persisting in biofilm, bacteria evade neutrophil killing and display decreased susceptibility to antibiotics. Despite decades of research and promising preclinical data, there is no available vaccine against S. aureus.

[0006] Peptidoglycan hydrolases (belonging to the class of "enzybiotics") such as bacteriophage-encoded endolysins, are a promising alternative 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, in particular endolysins, towards the end of the lytic cycle. The enzymes cleave peptidoglycans (PG) in the bacterial cell wall, thus lysing the cells and releasing the progeny phages. Peptidoglycan hydrolases, in particular endolysins (also abbreviated as "lysins"), have several advantages over antibiotics; especially, their narrow host specificity, which is often limited to a single genus or even a single species (Fischetti (2010), International Journal of Medical Microbiology, 300(6)), and their rather low propensity for generating resistance in their hosts (Schuch (2014), The Journal of Infectious Diseases 209(9)). Bacteriophages that invade Gram-positive bacteria encode a variety of highly diverse endolysins. Typically, endolysins have a modular structure consisting of one or more enzymatically active domains (EADs) connected by a flexible interdomain linker to at least one cell wall-binding domain (CBD). Both domains may contribute to the specificity for a given genus or species of bacteria; Oliveira (2013), J. Virol. 87. However, bacteriophage derived endolysins containing solely an enzymatically active domain, e.g., a cysteine, histidine-dependent amidohydrolase / peptidase (CHAP) domain (but no separate CBD), also exist. Such lysins break down peptidoglycan, in particular, from outside the bacterial cell.

[0007] A native (i.e. natural) phage lysin targeting S. aureus, CF-301 (ContraFect, also known as exebacase), delivered intravenously, showed therapeutic benefit in methicillin-resistant Staphylococcus aureus (MRSA) blood stream infections in a phase II clinical study (Fowler (2020), J. Clin. Invest., 130(7)), but the results could not be recapitulated in a phase III clinical study. Another native endolysin, SAL200, administrated intravenously to patients with persistent S. aureus bacteremia in a Phase II clinical study, resulted in serious adverse effects including pneumonia and respiratory failure (NCT03089697; Danis-Wlodarczyk (2021), Antibiotics, 10(12)); moreover, a very short half-life and an immune response against the enzyme was believed 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).

[0008] On the other hand, certain peptidoglycan hydrolases delivered locally (intranasally and / or topically) have shown some success in S. aureus decolonization, e.g. ectolysin P128 and endolysin SA.100 (Danis-Wlodarczyk (2021), Antibiotics, 10(12)) as well as lysostaphin (Jayakumar (2020), J. Appl. Microbiol.)). Exposure of S. aureus to lysostaphin, a glycylglycine endopeptidase, was however shown to generate rapid resistance both in vitro and in vivo, due to loss of function mutations in femA, which is required for the incorporation of the second and third glycine in the cross-bridges (Climo (2001), Antimicrobial Agents and Chemotherapy, 45 (5).

[0009] In view of the caveats of native phage endolysins, it has been tried to improve drug-like properties of endolysins by protein engineering; De Maesschalck V (2020) Crit Rev Microbiol, 46(5). The modular architecture of endolysins has served as a basis for lysin engineering, via domain shuffling, truncation, as well as random and / or site-directed mutagenesis; Gerstmans (2020), Sci Adv. 6(23). So far, lysin optimization focused on optimizing either functional activity (Gerstmans (2020), Sci Adv. 6(23)) or stability (Ritter (2019), Appl Environ Microbiol., 85(10)). Moreover, for the latter, only marginal improvements have been achieved, i.e., 4°C increase in melting temperatures for the best engineered variants. Furthermore, chimeric endolysins comprising two enzymatically active domains (EADs; catalytic domains), in particular a CHAP domain and a PepM23 domain (e.g., from lysostaphin) have been described in WO 2021 / 213898. However, the bactericidal activity of these chimeric endolysins is not optimal. This might be due to steric problems in effectively accessing and hydrolyzing peptidoglycan in bacterial cell walls as result of two relatively large catalytic domains being fused to each other.

[0010] It has been reported in WO 2023 / 083916 that endolysins of various architectures can be expressed from a nucleic acid such as an mRNA in human cells and be secreted from these cells. It has been also demonstrated in WO 2023 / 083916 that at least some of the tested endolysins are secreted in an active from human cells to kill target bacteria. When a nucleic acid construct encoding a peptidoglycan hydrolase (e.g., an endolysin) is introduced into cells in a patient, said cells can continuously produce and secrete the peptidoglycan hydrolase protein. In particular, administration of an endolysin in form of an mRNA ("ribolysin") may provide a safe and more efficient treatment of the bacterial infection as compared to direct protein administration and, for example, provide a higher efficacy in treating difficult to treat bacterial infections such as bacterial biofilms. However, the endolysins employed in WO 2023 / 083916, in particular those of the LYSM-CHAP architecture, do not have optimal pharmaceutical properties such as an optimized killing activity of target bacteria, an optimized ability of being secreted from human cells and / or an optimized stability.

[0011] Hence, there is still a need for improved antibacterials, in particular peptidoglycan hydrolases with improved pharmaceutical properties.

[0012] Summary of the invention

[0013] The invention relates to the embodiments as characterized in the claims and described herein below.

[0014] Accordingly, the present invention provides peptidoglycan hydrolases comprising a cysteine, histidine-dependent amidohydrolase / peptidase (CHAP) domain having a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1. Specifically, the present invention relates to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises

[0015] (i) a CHAP domain that has a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; and

[0016] (ii) a PepM23 domain that has a sequence identity of at least 60% to the sequence from position 1 to position 143 in SEQ ID NO: 407.

[0017] Preferably, the peptidoglycan hydrolase of the present invention has a killing activity against a Staphylococcus species or strain, preferably Staphylococcus aureus. Furthermore, the peptidoglycan hydrolase of the invention has preferably the ability of being secreted from a eukaryotic cell, more preferably a human cell. Furthermore, the peptidoglycan hydrolase of the invention is, preferably, stable up to a temperature of at least about 40°C, more preferably of at least about 50°C. Furthermore, the peptidoglycan hydrolase of the present invention is, preferably, an endolysin.

[0018] In context of the present invention, the peptidoglycan hydrolase of the invention is, preferably, contained in a pharmaceutical composition and / or, preferably, used for the treatment of a disease. Herein and in context of the present invention, the disease is, in particular, a bacterial disease, preferably, a disease caused by and / or associated with a Staphylococcus infection, e.g., an S. aureus infection.

[0019] Combination of two enzymatically active domains (EADs / catalytic domains)

[0020] The invention is, inter alia, based on the surprising finding, as illustrated in the appended non-limiting Examples, that fusing the catalytic domain, in particular the CHAP domain, from a LYSM-CHAP (i.e. L0482) variant to lysostaphin enhances the S. aureus peptidoglycan hydrolase activity in comparison to lysostaphin; see, e.g., Example 12 and Figure 14. It was not expected that the addition of a large second catalytic domain derived from a barely established endolysin family (i.e., a CHAP domain of about 144 amino acids in length derived from L0482) to lysostaphin would retain the peptidoglycan hydrolase activity of the endolysin, let alone enhance it.

[0021] In particular, the inventors found that fusing a GH15 CHAP domain as described in WO 2021 / 213898 to lysostaphin strongly reduced the killing activity against S. aureus, as compared to the parental lysostaphin. In contrast, fusion of a CHAP domain according to the present invention to lysostaphin, surprisingly, rather enhanced the killing activity against S. aureus, as compared to the corresponding parental lysostaphin; see, e.g., Example 12 and Table 8.

[0022] Hence, it has been surprisingly found in context of the present invention that the chimeric endolysin comprising a CHAP domain from a L0482 variant in addition to lysostaphin (L1079; SEQ ID NO: 409) according to the present invention had a higher killing activity against S. aureus in comparison to a chimeric CHAP-lysostaphin endolysin (L1055; SEQ ID NO: 410) described in the prior art, i.e., WO 2021 / 213898, having a GH15 CHAP domain. In addition, such an enhanced killing activity has been also observed vis a vis further CHAP-PepM3-SH3 chimera described in WO 2021 / 213898; see, e.g., Example 12 and Table 8.

[0023] Since replacement of the CHAP domain vis a vis a comparable CHAP-lysostaphin construct (L1055) described in WO 2021 / 213898 enhanced the killing activity against S. aureus, it is plausible that such an effect is mainly due to the CHAP-PepM23 fusion and may be independent from the cell wall binding domain(s). Furthermore, it is evident that both, the CHAP domain and the PepM23 domain, were functional and positively contributed to the peptidoglycan hydrolase activity and / or bactericidal activity of the Gl-14 CHAP-lysostaphin fusion (L1079) because (i) the peptidoglycan hydrolase activity was enhanced and the MIC slightly reduced as compared to the parental lysostaphin alone (cf. Figure 14B) and (ii) the MIC was considerably reduced as compared to Gl-14 alone (cf. Tables 4 and 8).

[0024] Thus, the inventors surprisingly found a chimeric peptidoglycan hydrolase which is thought to synergistically reduce the propensity of generating resistance in a therapeutic setting by combining two catalytic domains (i.e., CHAP and PepM23) having different peptidoglycan hydrolase activities / mechanisms and different substrate specificities, while retaining an excellent enzymatic and bactericidal activity that is overall about as good as in lysostaphin (and even slightly enhanced as compared to the corresponding parental lysostaphin variant employed). In addition, it was surprisingly found that the CHAP-lysostaphin fusion (L1079; SEQ ID NO: 409) showed an excellent secretion from human cells which was strongly enhanced as compared to WT L0482 (SEQ ID NO: 1) and the aglycosylated variant thereof (SEQ ID NO: 2), as illustrated in the appended non-limiting Examples.

[0025] Selection of the LYSM-CHAP domain architecture as starting point for further protein engineering and directed evolution

[0026] As illustrated in the appended Examples, it has been surprisingly found in context of the present invention that lysins with a LYSM-CHAP domain architecture such as L0482 (SEQ ID NO: 1) reliably have a good killing activity against Staphylococci, in particular Staphylococcus aureus (S. aureus) including methicillin-resistant Staphylococcus aureus (MRSA) strains such as ATCC43300. Therefore, lysins with a LYSM-CHAP domain architecture such as L0482 provide a particularly good starting point for protein engineering and directed evolution approaches. In particular, it has been surprisingly found in context of the present invention that 12 out of 13 lysins with a LYSM-CHAP domain architecture (i.e., >92%) effectively killed S. aureus cells, whereas lysins with other domain architectures did often not show considerable killing activity against S. 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 a good killing activity against many different S. aureus strains and also against other Staphylococcus species such as S. warned and S. capitis and coagulate-negative Staphylococci 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 clinically most developed lysins, i.e., exebacase (also known as CF-301, ContraFect or described as "L0466" herein) which has a CHAP-SH3 domain architecture; see Figure 2. Of note, the CHAP domain of LYSM-CHAP lysins such as L0482 is very different to the CHAP domain of exebacase. In particular, the sequence identity of the CHAP domain of exebacase (i.e. the sequence from position 19 to position 164 in SEQ ID NO: 304) to the CHAP domain of L0482 (i.e. the sequence from position 72 to position 215 in SEQ ID NO: 1) is merely about 20%.

[0027] While it is known from WO 2023 / 083916 that LysM-CHAP lysins such as L0482 (SEQ ID NO: 1) can be secreted in active form by eukaryotic cells in order to kill S. aureus; see, it has been observed by the present inventors that the stability and solubility of the wild-type (WT) L0482 (SEQ ID NO: 1), as disclosed in WO 2023 / 083916, and its secretion from eukaryotic cells as well as its killing activity upon secretion from eukaryotic cells is still not optimal; see, e.g., Example 3 and Figure 4, Example 4 and Figure 5, and Example 11 and Figure 9. Yet, a high killing efficiency against target bacteria (e.g., S. aureus), a good solubility in aqueous solutions, a high stability (including a sufficient thermostability and a low tendency for aggregation) are important properties of anti-bacterial compounds, in particular, enzybiotics 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, administration of a peptidoglycan hydrolase in form of a nucleic acid (e.g., an mRNA) encoding the peptidoglycan hydrolase to cells in a subject (e.g., a human) has, inter alia, the advantage of continuous production, i.e., secretion, of the peptidoglycan hydrolase from the cells of the subject, for example in the liver or at the site of a bacterial infection. Hence, the ability of being efficiently secreted from eukaryotic cells, e.g., human cells, is another desired property of peptidoglycan hydrolases, in particular, when they are to be administered in form of a nucleic acid such as an mRNA ("ribolysin").

[0028] L0482 variants with improved pharmaceutical properties

[0029] By protein engineering and directed evolution, as illustrated, e.g, in Examples 3 to 8, the inventors surprisingly found L0482 variants which had several improved pharmaceutical properties at the same time. In particular, the inventors found L0482 variants (e.g. H5) which had an enhanced killing activity against S. aureus, an enhanced protein stability and an enhanced ability of being secreted from human cells, as illustrated e.g., in Examples 7 and 8 and Figures 12 and 13. Almost all of the beneficial mutations contained in various L0482 variants showing such improved pharmaceutical properties were contained in the CHAP domain. Thus, CHAP domains having one or more amino acid substitutions as compared to the amino acid sequence from position 72 to position 215 in SEQ ID NO: 1, as described herein, are preferably used in context of the present invention, e.g., in the chimeric CHAP-PepM23 proteins.

[0030] Enhanced killii

[0031] As described in more detail herein below and as also illustrated in the appended Examples, an enhanced killing activity against a target bacterium such as S. aureus corresponds to a lower minimal inhibitory concentration (MIC). In particular, herein and in context of the present invention, the MIC is defined as the minimal concentration at which a bacterial (e.g. S. aureus) liquid culture comprising 5xl05cfu / ml of bacterial (e.g. S. aureus) cells stays translucent (i.e., OD620 < 0.1) for at least 18 h, preferably at least 24h, at 37°C, as judged by the naked eye, or, preferably, by measuring the optimal density at 620 nm (OD620). Thus, the inventive peptidoglycan hydrolases provided herein may be used at a lower concentration for treating a bacterial disease than comparable peptidoglycan hydrolases, which may increase the efficiency and safety of the treatment.

[0032] For example, introduction of aglycosylation mutations into the WT L0482 (SEQ ID NO: 1) resulting in L0482ag (SEQ ID NO: 2), strongly increased the bactericidal activity of the endolysin against target cells (e.g., S. aureus) upon production in eukaryotic cells (e.g., human cells), as described herein.

[0033] Furthermore, the hit variant of L0482ag named H5 (SEQ ID NO: 11) found in context of the present invention upon directed evolution has an at least about 2-8-fold lower MIC than WT L0482 (SEQ ID NO: 1) when produced in E coli; of note, when being secreted from human cells the difference is even much higher because the hit variants contain the same aglycosylation mutations as L0482ag. Furthermore, L0482 variants with engineered active sites based on G1 or H5 found in context of the present invention such as Gl-14 (SEQ ID NO: 386), H5-14 (SEQ ID NO: 387), L2 (SEQ ID NO: 388), L14 (SEQ ID NO: 389), L2DR (SEQ ID NO: 390) and L14DR (SEQ ID NO: 391) surprisingly also show effective killing of target bacteria (e.g. S. aureus), e.g. when secreted from human cells ; see, e.g., Table 4. In addition, fusing the CHAP domain of Gl-14 (SEQ ID NO: 394) to an aglycosylated lysostaphin (SEQ ID NO: 444) also provided an excellent killing activity against S. aureus,- see, e.g., Example 12 and Table 8. Notably, the effective MICs have been observed with the methicillin-resistant Staphylococcus aureus (MRSA) strain "ATCC43300" further highlighting that the peptidoglycan hydrolases of the invention may be particularly suitable as antibacterials, in particular, for medical uses.

[0034] The enhancement of the killing activity of peptidoglycan hydrolases according to invention was further confirmed with an OD reduction assay showing that, for example, H5 (SEQ ID NO: 11) killed S. aureus cells with faster kinetics than wild-type L0482 (SEQ ID NO: 1); see, e.g., Figure 11.

[0035] Enhanced protein stability

[0036] An enhanced protein stability, e.g., an enhanced thermostability and / or a reduced propensity for aggregation, further improves the pharmaceutical properties of a peptidoglycan hydrolase. For example, a sufficient thermostability, in particular, the ability of being stable of up to a temperature of at least about 40°C, e.g., at least 37°C, is critical for the use as a pharmaceutical in many mammals including humans (in view of the body temperatures). Furthermore, an enhanced thermostability facilitates storage and distribution of a pharmaceutical. As illustrated in the non-limiting appended Examples, e.g., in Table 4, L0482 variants (with improved CHAP domains) have been generated in context of the present invention which are stable of up to a temperature of at least about 50°C, e.g., of about 51°C - 54°C. Thus, the inventive peptidoglycan hydrolases provided herein may be particularly suitable as pharmaceuticals. Furthermore, a decreased propensity for aggregation enhances the manufacturability of a peptidoglycan hydrolase. Thus, the inventive peptidoglycan hydrolases provided herein may be more efficiently manufactured than comparable peptidoglycan hydrolases. Moreover, a decreased propensity for aggregation further reduces the immunogenicity of a peptidoglycan hydrolase in a mammalian subject, e.g., a human, which, in turn, may further increase the efficiency and / or safety of a treatment.

[0037] Enhanced ability of being secreted from human cells

[0038] An enhanced ability of being secreted from human cells, is particularly important for the expression of a peptidoglycan hydrolase from a nucleic acid, e.g., a RNA. As described herein, when a suitable nucleic acid construct encoding a peptidoglycan hydrolase of the invention is introduced into cells in a subject (e.g. a human patient), said cells can continuously produce and secrete the peptidoglycan hydrolase protein. This may provide a more efficient treatment of the bacterial infection and, for example, provide a higher efficacy in treating difficult to treat bacterial infections such as bacterial biofilms. As illustrated in the non-limiting appended Examples, L0482 variants found in context of the present invention upon directed evolution such as H5 show an enhanced ability of being secreted from human cells as compared to WT L0482 (SEQ ID NO: 1) or its acglycosylated variant L0482ag (SEQ ID NO: 2); see, e.g., Examples 4-6 and 12 and Figures 8, 10 and 6. Unexpectedly, engineering of the active site of H5 (or another variant obtained by directed evolution, i.e. Gl), led to L0482 variants (having mutations within the active site, e.g., Y94F, Y117D, G118Y, and / or W189E), such as Gl-14, H5-14, L2, L14, L2DR and L14DR, which show a further drastically enhanced ability of being secreted from human cells; see, e.g., Table 4. Strikingly, the secretion from HEK cells reached about 10 - 87 pg / ml. Further surprisingly, a fusion protein comprising the CHAP domain from Gl-14 in addition to an aglycosylated lysostaphin, i.e., L1079 (SEQ ID NO: 409), also showed an excellent secretion from HEK cells of about 63 pg / ml. Therefore, the peptidoglycan hydrolases of the invention may be particularly suitable for delivery as a nucleic acid, e.g., as RNA. Moreover, the present invention provides more efficient means, e.g., nucleic acids encoding a peptidoglycan hydrolase of the invention, for treating bacterial infections. An enhanced ability of being secreted from a human cell means, in particular, that the peptidoglycan hydrolase shows an enhanced secretion from a human cell when being expressed in said cell. Enhanced solubility

[0039] An enhanced stability (e.g. a reduced propensity for aggregation) and an enhanced ability of being secreted from human cells may be associated with each other as well as with an enhanced solubility in an aqueous solution such as PBS. Indeed, it has been observed in context of the present invention that many of the generated L0482 variants which were found to have an enhanced stability and an enhanced ability of being secreted from human cells also showed an enhanced solubility in PBS. Thus, the inventive peptidoglycan hydrolases provided herein may have a better solubility in aqueous solutions than comparable peptidoglycan hydrolases. This is highly beneficial, in particular, for the manufacturability and useability of the peptidoglycan hydrolases. Moreover, an enhanced solubility in aqueous solutions may increase the efficiency and / or safety of a peptidoglycan hydrolases in the treatment of a disease.

[0040] Reduced propensity of generating resistance

[0041] A CHAP domain (contained in L0482 and L0482 variants as described herein) may have a dual enzymatic activity, i.e., an amidase activity and a peptidase activity; Frankel (2012), J Biol Chem. 23;287(13). Both, the amidase activity and the peptidase activity, may contribute to the hydrolysis / cleavage of peptidoglycan in the cell wall of bacteria, as described herein. As illustrated in the appended Examples, L0482 variants, e.g., H5, L2, L2DR, L14 and L14DR have been found which have an increased peptidase activity as compared to the WT L0482 lysin; see, e.g., Figure 14. Moreover, L0482 variants with no or low peptidase activity such as Gl-14 and H5-14 still showed an effective killing of S. aureus; see, e.g., Figure 13 and Table 4. This suggests that L0482 variants show at least an amidase activity, and at least some of the L0482 variants found in context of the present invention show a strong peptidase activity.

[0042] Combination of a CHAP domain according to the invention with a PepM23 domain from lysostaphin results in a peptidoglycan hydrolase having two or three different mechanisms / activities for hydrolyzing peptidoglycan, i.e., a D-alanyl-glycyl peptidase activity and / or an N-acetylmuramyl L-Ala amidase activity provided by the CHAP domain and a glycyl-glycyl peptidase activity provided by the PepM23 domain. Thus, CHAP-PepM23 fusions according to the invention may have an even further reduced propensity for generating resistance as compared to an endolysin having either a CHAP domain or a PepM23 domain.

[0043] Therefore, a peptidoglycan hydrolase of the present invention comprising a CHAP domain of the invention, and preferably an additional PepM23 domain, may have a reduced propensity of generating resistance in target bacteria, e.g., S. aureus, compared to other peptidoglycan hydrolases such as lysostaphin, in particular in a therapeutic setting. Thus, the peptidoglycan hydrolases of the invention may be particularly effective for treating bacterial infections for this additional reason.

[0044] Active site mutations

[0045] As described in the appended non-limiting illustrative Examples, the inventors, unexpectedly, found an L0482ag (SEQ ID NO: 2)-derived variant having (as the only mutations vis a vis L0482ag) five mutations in the active catalytic site (i.e., Y94F, Y117D, G118Y, N186G and W189E; SEQ ID NO: 414) which was secreted from eukaryotic cells and showed peptidoglycan hydrolase activity; see, e.g., Example 7 and Figure 12A. Surprisingly, only one of these five mutations (i.e. N186G) was contained among the L0482 hits variants (i.e. among G1-G4, H1-H10 and 11-130) obtained by an earlier directed evolution screening, e.g. H5 (SEQ ID NO: 11); see Examples 4-6. The five mutations Y94F, Y117D, G118Y, N186G and W189E are also referred to herein as "active site mutations". The mutation "N186G" can be considered in context of the present invention not only as "active site mutation", but also as a "G1 / H5 mutation", "H5 mutation" or "H5-specific mutation", as described herein. In certain contexts and embodiments, e.g. relating to H5 or derivatives thereof, the active site mutations can also refer to the four mutations not already contained in H5, i.e., Y94F, Y117D, G118Y, and W189E.

[0046] The inventors then introduced the active site mutations into two hit variants obtained by the directed evolution (further having two aglycosylation mutations as described herein), i.e., G1 (SEQ ID NO: 3) and H5 (SEQ ID NO: 11), resulting in Gl-14 (SEQ ID NO: 386) and H5-14 (SEQ ID NO: 387), respectively.

[0047] Surprisingly, the active site mutations in the CHAP domain of G1 or H5 strongly enhanced the secretion of the endolysins from human cells while providing the endolysins with a high thermostability and effective killing activity against target bacteria including biofilms; see, e.g., Example 7, Table 4 and Figure 12B.

[0048] The inventors further found that each of the following active site mutations, i.e., Y117D, G118Y, W189E, had individually a strong contribution to the drastically enhanced ability of the endolysins of being secreted from human cells, while the combination of Y117D and G118Y showed a particularly pronounced effect; see, e.g., Example 7 and Table 3. In addition, the active site mutation Y94F also had some positive impact of secretability, in particular in context of Gl-14, albeit to a lower extent; see Table 3.

[0049] As further described below, a CHAP domain having one or more amino acid substitutions and / or deletions as compared to the amino acid sequence from position 72 to position 215 in SEQ ID NO: 1 is considered herein and in context of the present invention as a variant of the CHAP domain of L0482, i.e., a variant of the sequence from position 72 to position 215 in SEQ ID NO: 1.

[0050] Preferably, the CHAP domain according to the invention has a sequence identity of at least 60%, preferably at least 70%, to the sequence from position 72 to position 215 in SEQ ID NO: 1 and one or more amino acid substitutions at positions 94, 117, 118, 186 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions. Preferably, the amino acid residue at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and / or the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0051] Furthermore, the present invention relates to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a CHAP domain that has a sequence identity of at least 60%, preferably at least 70%, to the sequence from position 72 to position 215 in SEQ ID NO: 1 and one or more amino acid substitutions at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions. Preferably, the amino acid residue at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0052] In preferred embodiments, the CHAP domain of the invention has one, two or three amino acid substitutions at positions 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0053] Surprisingly, the inventors further found that certain active site mutations, WT amino acid residues and / or combinations thereof in the CHAP domain conferred peptidase activity to L0482 variants. For example, the backmutation D117Y strongly enhanced peptidase activity in combination with four active site mutations (Y94F, G118Y, N186G and W189E); see, e.g., Example 8, Figure 13C, D and Tables 3 and 4. Moreover, the active site mutation Y117D in combination with the WT residue G118, the active site mutation G118Y in combination with the WT residue Y117, the two active site mutations Y117G and G118Y in combination, as well as the two active site mutations Y94F and W189E in combination with the two WT residues Y117 and G118, also provided peptidase activity.

[0054] Hence, when the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid in a CHAP domain of the invention, the glycine at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is, in some embodiments, preferably, not deleted or substituted with another amino acid residue.

[0055] When the glycine at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine in a CHAP domain of the invention, the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is, in some embodiments, preferably, not deleted or substituted with another amino acid residue.

[0056] When the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine in a CHAP domain of the invention, the glycine at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is, in some embodiments, preferably, substituted with tyrosine. Similarly, when the glycine at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine in a CHAP domain of the invention, the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is, in some embodiments, preferably, substituted with glycine.

[0057] When the tyrosine at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine and the tryptophan at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid in a CHAP domain of the invention, the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position and the glycine at position 118 in SEQ ID NO: 1 or at a position corresponding to this position are, in some embodiments, preferably, not deleted or substituted with other amino acid residues.

[0058] Preferably, a CHAP domain of the invention having at least one active site mutation as described herein further has an aglycosylation mutation at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein, and / or at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, H5 / G1 mutation(s) at positions 82, 85, 86, 130, 136, 155, 169, 173, 175, 185, 186 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein. As explained further in the following, said aglycosylation mutation and said H5 / G1 mutations also improve the pharmaceutical properties of L0482 variants.

[0059] The CHAP domain according to the invention may be combined with a PepM23 domain that has a sequence identity of at least 60% to the sequence from position 1 to position 143 in SEQ ID NO: 407, and, preferably, at least one cell wall binding domain such as an SH3 domain and / or a LYSM domain, in particular, in one peptidoglycan hydrolase (e.g. endolysin) protein, as described herein.

[0060] Aglycosylation mutations enhance bactericidal activity upon expression in eukaryotic cells

[0061] As shown in Example 3 and Figure 4A, it has been found in context of the present invention that WT L0482 (SEQ ID NO: 1) has a good killing activity against S. aureus, i.e., a minimum inhibitory concentration (MIC) of 4 pg / ml, when produced in Escherichia coli (E. coli) but only a moderate killing activity (i.e. a MIC of 352 pg / ml when secreted from Pichia pastoris (P. pastoris), i.e., yeast.

[0062] The inventors reasoned that N-and O-glycosylation of WT L0482 during its maturation in the secretory pathway in eukaryotic cells might have negatively affected its bactericidal activity. In particular, the inventors speculated that transgenes such as lysins may be glycosylated at sites essential for folding and activity, which could potentially lead to the secretion of a less active protein (while secretory proteins are normally evolutionary adapted to these modifications).

[0063] It has been found that L0482 (SEQ ID NO: 1) contains two motifs of N-glycosylation at residues N68 (which is in the linker sequence) and N73 (which is in the CHAP domain). Therefore, the inventors removed the two N- glycosylation motifs in L0482 in a degenerate codon screen by employing the YODA method as described in Example 3 and Figure 4B.

[0064] The 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). It was confirmed that all three L0482 mutants (i.e., having the N68K / N73G, N68K / N73Y and N68A / N73H substitutions, respectively) were aglycosylated; see, e.g., Example 3 and Figure 4C. In addition, the further mutation pairs N68N73 to ML, RE, KL and AA were also found in active L0482 mutants by YODA. Moreover, it has been surprisingly found by the inventors that the aglycosylated N68K / N73G (i.e., KG) mutant, also called L0482ag herein, had a killing activity against S. aureus which was almost as good as for the WT L0482 upon production in E coir, see, e.g., Example 3 and Figure 4C.

[0065] Accordingly, the present invention further relates to aglycosylated variants of L0482 (SEQ ID NO: 1) as illustrated by but not limited to the N68K / N73G (i.e., L0482ag; SEQ ID NO: 2) mutant described herein. Herein and in context of the invention, e.g., in the context of a peptidoglycan hydrolase that has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, an aglycosylation mutation refers to an amino acid substitution or deletion (preferably to an amino acid substitution) at position 68 or 73 in SEQ ID NO: 1 or at any position corresponding to these positions. In context of the CHAP domain of the invention, an aglycosylation mutation refers to an amino acid substitution or deletion (preferably to an amino acid substitution) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position.

[0066] In particular, an amino acid substitution at position 68 in SEQ ID NO: 1 or at a position corresponding to this position means that the asparagine ("N") at position 68 in SEQ ID NO: 1 is substituted with another amino acid, as described herein.

[0067] Following the same logic, an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position means that the asparagine ("N") at position 73 in SEQ ID NO: 1 or the asparagine corresponding to position 73 in SEQ ID NO: 1 is substituted with another amino acid, as described herein.

[0068] The following amino acid substitutions are considered herein and in context of the present invention as particularly effective aglycosylation mutations or aglycosylation amino acid substitutions: N68K, N68A, N68M, N68R, N73G, N73Y, N73H, N73L, N73E, and N73A in SEQ ID NO: 1 or at positions corresponding to these positions. Since the CHAP domain is of most relevance herein and in context of the present invention, an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, e.g. N73G (as contained in L0482ag; SEQ ID NO: 2), is of particular relevance herein and in context of the present invention. Preferably, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than phenylalanine or lysine such as glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, preferably glycine.

[0069] L0482 variants having at least one aglycosylation mutation (e.g., an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position), as described herein and as illustrated by L0482ag (SEQ ID NO: 2), may be a particularly good starting point for further protein engineering and / or directed evolution, as described herein. As illustrated in the appended Examples, and as further described herein, additional beneficial amino acid substitutions (e.g. G1 and / or H5 mutations) and / or active site mutations may be introduced into L0482 variants having at least one aglycosylation mutation, in particular in the CHAP domain thereof, to further enhance the pharmaceutical properties of the L0482 variants (e.g. the stability solubility and ability of being secreted from a human cell but also the killing activity against a target bacterium, e.g. S. Aureus), as described herein.

[0070] Thus, an aglycosylation mutation (preferably an amino acid substitution) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position (more preferably N73G), is, preferably, combined with at least one of the amino acid substitutions described herein in context of the L0482ag variants obtained upon directed evolution, in particular with at least one G1 / H5 mutation and / or at least one active site mutation, as described herein. In particular, an aglycosylation mutation (preferably an amino acid substitution) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is preferably combined with at least one H5 / G1 amino acid substitution, i.e. at least one of the amino acid substitutions selected from the group consisting of T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, K173N, M175Q, N185Y, N186G and A192Q in reference to SEQ ID NO: 1, and / or at least one active site mutation, i.e., at least one of the amino acid substitutions selected from the group consisting of Y94F, Y117D, G118Y, W189E in reference to SEQ ID NO: 1.

[0071] Beneficial mutations contained in hits variants obtained by directed evolution

[0072] Consensus mutations / the variant H3

[0073] By directed evolution, the inventors unexpectedly found a L0482 variant, i.e., H3 (SEQ ID NO: 9), which (i) had all of the beneficial properties assayed (i.e. an enhanced killing activity against S. aureus, an enhanced stability and an enhanced ability of being secreted from human cells) compared to the parental L0482 variant L0482ag (SEQ ID NO: 2), and which (ii) contained exclusively amino acid substitutions that were deemed to be beneficial as they were consistently observed (in slightly different combinations) among the best hits obtained by the three rounds of directed evolution, i.e., G1-G4, H1-H10 and 11-130; see, e.g., Examples 5 and 6 and Figures 7 and 8.

[0074] H3 (SEQ ID NO: 9) has been obtained by removing two glycosylation sites in L0482 (SEQ ID NO: 1) to yield the aglycosylated variant L0482ag (SEQ ID NO: 2) (see Example 3), followed by two rounds of directed evolution with L0482ag as a starting point; see Example 4. H3 (SEQ ID NO: 9) contains in addition to the two aglycosylation mutations of L0482ag, i.e. N68K, N73G, exclusively the following mutations, i.e., amino acid substitutions: T82S, N85G, R86K, S130N, H136K, D169N, N185Y and N186G, in reference to the wild-type L0482 sequence SEQ ID NO: 1.

[0075] Furthermore, it has been found that several of the hit variants identified upon directed evolution of L0482ag (SEQ ID NO: 2) contained the amino acid substitution H136R instead of H136K. Hence, H136R is considered herein and in context of the present invention as a very good alternative to H136K.

[0076] Thus, H3 (SEQ ID NO: 9) reflects the consensus sequence for improved L0482 variants obtained upon directed evolution, and the corresponding amino acid substitutions, i.e. T82S, N85G, R86K, S130N, H136K / R, D169N, N185Y and N186G, in reference to the wild-type L0482 sequence SEQ ID NO: 1, are considered herein as consensus mutations, in particular, consensus amino acid substitutions.

[0077] The variant H5

[0078] Furthermore, it has been surprisingly found in context of the present invention that the L0482 variant H5 (SEQ ID NO: 11), obtained in the same way as H3, had an even more enhanced stability, killing activity against S. aureus, and ability of being secreted from human cells compared to H3 (SEQ ID NO: 9); see, e.g., Example 5. 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 found in context of the present invention, e.g., F155Y, can improve several pharmaceutical properties of L0482, as described herein, at the same time.

[0079] H5 further showed enhanced killing kinetics against S. aureus in an OD reduction assay than WT L0482 (SEQ ID NO: 1); see Example 5 and Figure 10. Hence, the amino acid substitutions contained in H5 (in addition to the two aglycosylation mutations N68K and N73G), i.e., the so-called "H5 mutations", namely T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y and N186G (in reference to SEQ ID NO: 1) are beneficial amino acid substitutions for enhancing the killing activity against a target bacterium, e.g., S. aureus, and / or other pharmaceutical properties (e.g., the stability, solubility and secretion from human cells) of L0482 derived peptidoglycan hydrolases (L0482 variants).

[0080] Moreover, it needs to be emphasized that it has been surprisingly found in context of the present invention that all of the H5 mutations occurred in the CHAP domain of L0482, i.e., positions 72 to 215 in SEQ ID NO: 1. It is therefore considered herein and in context of the present invention that the CHAP domain is the most important domain of L0482 derived peptidoglycan hydrolases. Again, the CHAP domain according to the invention may be combined with a PepM23 domain that has a sequence identity of at least 60% to the sequence from position 1 to position 143 in SEQ ID NO: 407, and, preferably, at least one cell wall binding domain such as an SH3 domain and / or a LYSM domain, in particular, in one peptidoglycan hydrolase (e.g. endolysin) protein, as described herein.

[0081] Since H5 was the most promising candidate obtained by the directed evolution, the active site engineering, described herein, was performed also with H5, resulting in H5-14 and derivatives thereof.

[0082] The variant G1

[0083] G1 (SEQ ID NO: 3) is a hit variant obtained in the first round of the directed evolution, which showed an increased thermostability and an enhanced secretion from eukaryotic cells as compared to the starting for the directed evolution (L0482ag); see Figure 7. Furthermore, G1 had an effective killing activity against S. aureus.

[0084] G1 (SEQ ID NO: 3) contains the following amino acid substitutions in the CHAP domain as compared to L0482ag (SEQ ID NO: 2): T82S, N85G, R86K, D169N, K173N, M175Q and A192Q, which are called the "Gl mutations" herein.

[0085] Since G1 was another candidate obtained by the directed evolution, the active site engineering, described herein, was performed also with Gl, resulting in Gl-14 and derivatives thereof.

[0086] G1 / H5 mutations

[0087] The two L0482 variants employed for active site engineering, i.e., Gl and H5, have four mutations in common with reference to the WT L0482 (SEQ ID NO: 1), namely T82S, N85G, R86K and D169N. These four mutations common between Gl and H5 are also called the "stem mutations" herein.

[0088] The mutations contained in Gl and / or H5 vis a vis the L0482ag (SEQ ID NO: 2), namely T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, K173N, M175Q, N185Y, N186G and A192Q, are also called "G1 / H5 mutations" or"H5 / Gl mutations" herein.

[0089] Mutations only contained in Gl but not in H5, namely K173N, M175Q and A192Q, are also called "Gl-specific mutations herein".

[0090] Mutations only contained H5 but not in Gl, namely S130N, H136K, F155Y, N185Y and N186G, are also called "H5- specific mutations herein". The most recurrent amino acid substitution: R.86K

[0091] It has been further surprisingly found in context of the present invention that the amino acid substitution R86K was contained in all 44 identified hit variants, i.e., G1-G4, H1-H10 and 11-130, and in 96.08% of all L0482 variants that were secreted from eukaryotic cells and that were determined to have a killing activity against S. aureus, as described herein and as illustrated in the appended Examples; see, e.g., Figure 8.

[0092] Therefore, R86K in SEQ ID NO: 1 is, herein and in context of the present invention, a particularly preferred amino acid substitution among the G1 / H5 mutations. Accordingly, in preferred embodiments, the CHAP domain of the present invention has an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with lysine.

[0093] Recurrent pairs of amino acid substitutions

[0094] It has been further surprisingly found in 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 11-130; see, e.g., Figure 8. These mutation pairs are specifically: (i) T82S and N85G, (ii) S130N and H136K / R and (iii) N185Y and N186G. Each of said 44 hit variants contained at least one of said mutation pairs. Of note, it is beneficial but not necessary that these amino acid substitutions, i.e., T82S and N85G, S130N and H136K / R, and N185Y and N186G, occur in pairs since among the 252 active L0482 variants secreted from yeast cells found in context of the present invention, variants having only one of these paired mutations (but not the other) were found as well.

[0095] Therefore, a CHAP domain of the invention having an amino acid substitution at position 82 in SEQ ID NO: 1 or at a position corresponding to this position as described herein, has preferably, in addition, an amino acid substitution at position 85 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein. Similarly, a CHAP domain of the invention having an amino acid substitution at position 85 in SEQ ID NO: 1 or at a position corresponding to this position as described herein, has preferably, in addition, an amino acid substitution at position 82 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

[0096] Furthermore, a CHAP domain of the invention having an amino acid substitution at position 130 in SEQ ID NO: 1 or at a position corresponding to this position as described herein, has preferably, in addition, an amino acid substitution at position 136 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein. Similarly, a CHAP domain of the invention having an amino acid substitution at position 136 of SEQ ID NO: 1 or at a position corresponding to this position as described herein, has preferably, in addition, an amino acid substitution at position 130 of SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

[0097] Furthermore, a CHAP domain of the invention having an amino acid substitution at position 185 in SEQ ID NO: 1 or at a position corresponding to this position as described herein, has preferably, in addition, an amino acid substitution at position 186 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein. Similarly, a CHAP domain of the invention having an amino acid substitution at position 186 of SEQ ID NO: 1 or at a position corresponding to this position as described herein, has preferably, in addition, an amino acid substitution at position 185 of SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

[0098] Consensus mutation units

[0099] As already mentioned herein above, the hit variant H3 (SEQ ID NO: 9) reflects the consensus sequence of the hit variants obtained by the directed evolution. H3 contains exclusively, the following amino acid substitutions, also referred to herein as "consensus mutations": T82S, N85G, R86K, S130N, H136K / R (esp. H136K), D169N, N185Y and N186G in reference to SEQ ID NO: 1.

[0100] Furthermore, the inventors found five consensus mutation units based on the 44 L0482ag hit variants and the consensus sequence reflected by H3 (SEQ ID NO: 9) found in context of the present invention. These consensus mutation units consist of 1 or 2 amino acid substitutions, i.e.: (i) R86K (which is particularly preferred), (ii) T82S and N85G, (iii) S130N and H136K / R (preferably H136K), (iv) D169N, and (v) N185Y and N186G.

[0101] Accordingly, in preferred embodiments, the CHAP domain of the present invention has at least one amino acid substitution or substitution pair (i.e. at least one consensus mutation unit) selected from the group consisting of the following (i) to (v):

[0102] (i) an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with lysine;

[0103] (ii) an amino acid substitution at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, and the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine;

[0104] (iii) an amino acid substitution at positions 130 and 136 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, and the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine;

[0105] (iv) an amino acid substitution at position 169 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with asparagine; and

[0106] (v) an amino acid substitution at positions 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0107] Preferably, said CHAP domain has at least two, preferably at least three, more preferably at least four, most preferably all of said consensus mutation units.

[0108] Preferably, said CHAP domain further has at least one active site mutation as described herein, in particular, Y94F, Y117D, G118Y, and / or W189E, more preferably Y117D, G118Y, and / or W189E, in reference to SEQ ID NO: 1.

[0109] Preferably, said CHAP domain has further an aglycosylation mutation as described herein, preferably an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein, more preferably the substitution N73G.

[0110] In addition, said CHAP domain has preferably the amino acid substitution F155Y.

[0111] A single amino acid substitution improving several pharmaceutical properties at once: F155Y

[0112] As further indicated herein above, the best performing L0482 variant found obtained by the directed evolution (prior to active site engineering), i.e. H5 (SEQ ID NO: 11), surprisingly contained exclusively one additional amino acid substitution vis a vis H3 (SEQ ID NO: 9), i.e., F155Y. As further described herein, said amino acid substitution improved several pharmaceutical properties compared to H3 (SEQ ID NO: 9) at the same time, i.e. the killing activity against S. aureus, the protein stability and the ability of being secreted from human cells; see, e.g., Figure 7.

[0113] Furthermore, it was surprisingly found in context of the present invention that the amino acid substitution F155Y enhanced the peptidase activity when introduced into a Gl-based L0482 variant, i.e., L2 (SEQ ID NO: 388), resulting in L2DR (SEQ ID NO: 390); see e.g., Example 8 and Figure 13.

[0114] Therefore, F155Y in reference to SEQ ID NO: 1 is considered herein and in context of the present invention as a further particularly preferred amino acid substitution. Accordingly, in preferred embodiments, the CHAP domain of the present invention has an amino acid substitution at position 155 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with tyrosine.

[0115] Preferably, said CHAP domain further has at least one consensus mutation, as 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, as 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; at least one active site mutation, as described herein (i.e., Y94F, Y117D, G118Y and / or W189E); and / or an aglycosylation substitution as described herein, i.e., an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein, preferably N73G.

[0116] In some embodiments, the CHAP domain of the present invention has a sequence identity of at least 93% (in particular, at least 93.2%), at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the sequence from position 72 to position 215 in SEQ ID NO: 11 (H5). Preferably, said CHAP domain has a) (i) at least one of the most beneficial amino acid substitutions and / or at least one consensus mutation unit, as described herein and (ii) at least one aglycosylation mutation, preferably an amino acid substitution at position 73, as described herein, and / or b) at least two of the most beneficial amino acid substitutions, e.g., at least one amino acid substitution pair, as described herein. Furthermore, in context of these embodiments or in similar embodiments, the CHAP domain of the present invention has, preferably, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 amino acid substitutions in the sequence from position 72 to position 215 in SEQ ID NO: 11 (H5) or in the sequence from a position corresponding to position 72 in SEQ ID NO: 11 to a position corresponding to position 215 in SEQ ID NO: 11.

[0117] In some embodiments, the peptidoglycan hydrolase of the present invention has a sequence identity of at least 95% (in particular at least 95.0%), at least 96%, at least 97%, at least 98% or at least 99% to the sequence of SEQ ID NO: 11 (H5). Furthermore, in context of these embodiments or in similar embodiments, the peptidoglycan hydrolase of the present 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, or at most 1 amino acid substitutions in SEQ ID NO: 11. Preferably, said peptidoglycan hydrolase comprises a CHAP domain according to the present invention.

[0118] Single H5 / G1 mutations enhancing peptidase activity

[0119] As already mentioned above, the H5-specific mutation F155Y enhanced the peptidase activity when introduced into a Gl-based L0482 variant, i.e., L2 (SEQ ID NO: 388), resulting in L2DR (SEQ ID NO: 390); see e.g., Example 8 and Figure 13. Introducing a further H5-specific mutation, i.e., N185Y, into the Gl-based L0482 variant L2DR, resulting in L2TR (having a CHAP domain with the sequence shown in SEQ ID NO: 401), surprisingly, further enhanced the peptidase activity. Specifically, the peptidase activity of L2TR was as high as the H5-based L0482 variant L14DR (SEQ ID NO: 391) having the same active site mutations as L2DR, see, e.g., Figure 13E. Without being bound by theory, the full peptidase activity (relative to L14DR) in L2TR might have been established by perfecting the H3 / H5 mutation pair N185Y and N186G found in the consensus sequence (and H5) also in the G1 background.

[0120] Therefore, N185Y in reference to SEQ ID NO: 1 is considered herein and in context of the present invention as a further preferred amino acid substitution, especially in combination with N186G.

[0121] In addition, the Gl-specific mutation M175Q, surprisingly, enhanced the peptidase activity when introduced into a H5-based L0482 variant, i.e., L14 (SEQ ID NO: 389), resulting in L14DR (SEQ ID NO: 391); see e.g., Example 8 and Figure 13.

[0122] Hence, M175Q in reference to SEQ ID NO: 1 is also considered herein and in context of the present invention as a further preferred amino acid substitution.

[0123] Furthermore, the peptidoglycan hydrolase of the present invention is, in particular, a single polypeptide, i.e., a single amino acid chain.

[0124] The surprising finding of L0482 variants (e.g., H3, H5, Gl, Gl-14, H5-15, and derivatives thereof), wherein several pharmaceutical properties were improved at the same time, is partly based on the improved means and methods for protein engineering and directed evolution developed in context of the present invention and described herein and illustrated in the appended non-limiting Examples.

[0125] Means and methods for screening peptidoglycan hydrolases for bactericidal activity

[0126] As illustrated in the appended Examples, the inventors surprisingly found that solidified yeast culture media such as agar plates containing autoclaved (i.e. dead) target bacteria, e.g. dead S. aureus cells, are particularly suitable for screening yeast cells for the secretion of an active peptidoglycan hydrolase, i.e., for identifying peptidoglycan hydrolases with a good killing activity against target bacteria, e.g., S. aureus. The corresponding screening method called "Yeast on dead aureus" (YODA) is based on the inventive concept that only an active (but not an inactive) peptidoglycan hydrolase secreted from a yeast colony cultured on the solidified yeast culture medium of the invention is able to break down the peptidoglycan of the dead bacterial cells in vicinity of the colony. Hence, only a secreted and active peptidoglycan hydrolase renders the previously turbid culture medium locally translucent and thereby generates a halo around the colony secreting said peptidoglycan hydrolase; see Example 2 and Figure 3. YODA-derived methods are extremely simple and efficient methods which allows to easily distinguish yeast cells / colonies expressing peptidoglycan hydrolases with a good killing activity against a target bacterium from yeast cells / colonies expressing inactive peptidoglycan hydrolases. Furthermore, YODA-derived methods are very sensitive since the lysins are constantly secreted from the cells. Thus, small secretion rates may be sufficient to see a halo (when the peptidoglycan hydrolase is active). Moreover, as described herein, e.g., in Example 2, the YODA method or derivatives thereof as described herein (e.g., YODB) have additional advantages over prior art screening methods, e.g., the double agar layer (DAL) assay described in Zhao (2014). Appl Environ Microbiol 80(9). Methods for screening peptidoglycan hydrolases having several improved pharmaceutical properties / directed evolution

[0127] As already indicated herein above, the inventors evaluated in context of the present invention whether it would be possible to not only improve the bactericidal activity of lysins but also further pharmaceutical properties of the lysins at the same time, (e.g., the solubility, secretion from eukaryotic cells and / or stability). As already indicated above and as further described in the following, the inventors surprisingly found that several pharmaceutical properties (including the bactericidal activity) of L0482 variants, in particular L0482ag, could be further improved.

[0128] To this end, the inventors developed a combinatorial screening method based on the inventive YODA-derived method which combines eukaryotic cell display, e.g., yeast display, with YODA; see, e.g., Example 4. As illustrated in the appended Examples, it has been surprisingly found that this inventive combinatorial screening method, allows 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 derivatives thereof such as L0482ag (SEQ ID NO: 2). The inventors reasoned that the display (e.g. yeast display) step allows, in particular, to screen for peptidoglycan hydrolase variants which are adapted to the eukaryotic secretory pathway, and, hence, have an improved expression and secretion profile in eukaryotic cells. Moreover, the ability of being efficiently secreted is often associated with a good stability and good solubility of the protein, as described herein. Hence these biophysical and pharmaceutically relevant properties can be also improved by the inventive method. The YODA step, in particular, allows to screen for secreted peptidoglycan hydrolase variants which have good bactericidal activity, in particular against the desired target bacterium, as described herein. Hence, the combinatorial screening method developed in context of the invention allows to screen for improved peptidoglycan hydrolase variants which are adapted to the eukaryotic secretory pathway and effectively kill a target bacterium, e.g. S. aureus, and which may have further beneficial pharmaceutical properties such as an enhanced stability and an enhanced solubility as described herein and as illustrated in the appended Examples.

[0129] Administration in form of a nucleic acid

[0130] As already indicated above, administration of a peptidoglycan hydrolase of the invention in form of a nucleic acid (e.g., an mRNA) encoding said peptidoglycan hydrolase to a subject (e.g., a human), in particular introducing the nucleic acid into cells in a subject, has certain advantages. For example, when a nucleic acid (e.g., an mRNA) encoding a peptidoglycan hydrolase of the invention is introduced and expressed in cells in a subject, e.g., in a patient that has a bacterial, e.g. Staphylococcus, infection, the cells can continuously produce and secrete the peptidoglycan hydrolase protein. This may provide a more efficient treatment of the bacterial infection and, for example, provide a higher efficacy in treating difficult to treat bacterial infections such as bacterial biofilms, e.g., Staphylococcus biofilms. Furthermore, the nucleic acid may be introduced into cells at a particular location, e.g. in the liver or at the site of a bacterial infection, and / or into specific cell types which may further improve the efficiency and / or safety of the treatment. Furthermore, nucleic acids, in particular RNAs, have further certain practical advantages over proteins with respect to their manufacturing, safety profile and / or adaptability.

[0131] It has been further found by the inventors that all hit variants found in context of the invention and further tested, i.e., G1 to G4 and Hl to H6, were efficiently secreted from human cells when expressed from an RNA in these cells. In particular, it has been found that the secretion from human cells was highly enhanced compared to a control RNA expressing WT L0482 (SEQ ID NO: 1); see Examples 10 and 11 and Figure 9. It is plausible that L0482 variants having at least one active site mutation, as described herein, may show an even further enhanced secretion from human cells when expressed from an RNA in these cells, as such an effect was already observed in context of the present invention when L0482 variants with active site mutations were expressed from a DNA in human cells; see, e.g., Table 4.

[0132] Therefore, the present invention further relates to a nucleic acid (e.g. DNA or RNA) encoding the peptidoglycan hydrolase of the invention, preferably comprising a CHAP domain that has at least one of the active site mutations, i.e. Y94F, Y117D, G118Y, N186G, W189E, preferably Y117D, G118Y, and / or W189E, in reference to SEQ ID NO: 1), as described herein. Preferably said CHAP further comprises an aglycoslyation mutation and / or at least one G1 / H5 mutation, as described herein. In some embodiments, the nucleic acid of the invention encodes a peptidoglycan hydrolase according to the invention comprising a CHAP domain according to the invention and a PepM23 domain that has a sequence identity of at least 60% to the sequence from position 1 to position 143 in SEQ ID NO: 407, as described herein.

[0133] Preferably, the nucleic acid of the invention is a RNA, preferably a mRNA. Preferably, the RNA, e.g., the mRNA, of the present invention comprises at least one modified nucleoside such as pseudouridine (i ), Nl-methyl- pseudouridine (mli ) or 5-methyl-uridine (m5U), preferably Nl-methyl-pseudouridine (mli ), in place of at least one uridine, preferably in place of multiple, more preferably all uridines.

[0134] Detailed description of the invention

[0135] Peptidoglycan hydrolases

[0136] The term "peptidoglycan hydrolase", as used herein and in context of the present invention, refers to a polypeptide (i.e., a single amino acid chain) which is 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. A peptidoglycan hydrolase of the invention may comprise naturally occurring amino acids and / or non- naturally occurring amino acids as well as modifications such as, but not limited to, glycosylation (in particular 0- glycosylation and / or N-glycosylation), acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, or a protective group. Preferably, the peptidoglycan hydrolase of the invention is comprised 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 comprise modifications or not), as described herein. The term "peptidoglycan hydrolase" encompasses, for example, endolysins, lysozymes, tail-spike depolymerases, Virion- associated peptidoglycan hydrolases (VAPGH), bacteriocins and autolysins. In preferred embodiments, the term "peptidoglycan hydrolase" refers to an endolysin. The terms "endolysin" and "lysin" are used interchangeably herein and in context of the present invention.

[0137] Isolated peptidoglycan hydrolases

[0138] The peptidoglycan hydrolase of the present invention may be an isolated peptidoglycan hydrolase, e.g., an isolated endolysin. In particular, herein, "isolated” means removed (e.g., purified) from the natural state. For example, a nucleic acid, peptide or polypeptide naturally present 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 substantially purified form, or can exist in a non-native environment such as, for example, a host cell. Preferably, an isolated peptidoglycan hydrolase, as used herein, refers to a peptidoglycan hydrolase which is isolated (e.g. purified) from its natural context, e.g., from a bacterial cell where it occurs in nature.

[0139] Engineered and natural peptidoglycan hydrolases

[0140] In context of the present invention, a "peptidoglycan hydrolase" or an "endolysin", is, preferably, a modified, i.e., an engineered, "peptidoglycan hydrolase" or "endolysin", respectively. In particular, a "modified" (i.e., an "engineered", synthetic", "recombinant", or "artificial") peptidoglycan hydrolase or endolysin does not occur in nature and thus may be also considered herein and in context of the present invention as a "non-natural" (i.e., "non-native") peptidoglycan hydrolase or endolysin, respectively.

[0141] Thus, in certain embodiments, the peptidoglycan hydrolase of the invention is an engineered peptidoglycan hydrolase.

[0142] Furthermore, in certain embodiments, the peptidoglycan hydrolase of the invention is a non-natural peptidoglycan hydrolase.

[0143] However, in certain embodiments of the invention, the peptidoglycan hydrolases may also include natural (i.e. native) peptidoglycan hydrolases, in particular, natural endolysins, for example, in context of pharmaceutical compositions, medical uses, fusion proteins of a peptidoglycan hydrolase and another (poly)peptide (e.g. a PK tag), RNA constructs, and / or modified nucleic acids, e.g. RNAs containing a modified nucleoside such as Nl-methyl- pseudouridine (mli ) in place of at least one uridine, as described herein.

[0144] Endolysins

[0145] Herein, and in context of the present invention, the term "endolysin" refers, in particular, to natural peptidoglycan hydrolases encoded by bacteriophages (or bacterial viruses), or engineered peptidoglycan hydrolases, in particular modified endolysins, derived from such natural peptidoglycan hydrolases (e.g., from L0482; SEQ ID NO: 1). Natural endolysins act, in particular, by hydrolyzing the host cell wall and subsequently allow the release of bacteriophage progenies. The peptidoglycan hydrolases, e.g., the endolysins, of the present invention are also capable of hydrolyzing peptidoglycan in the cell wall of a bacterial species or strain (e.g., S. aureus) and, thus, have a killing activity against said bacterial species or strain (e.g., S. aureus), as described herein.

[0146] The term "cell wall" as used herein refers to all components that form the outer cell enclosure of bacteria (containing, in particular, peptidoglycan), as commonly understood in the art.

[0147] Natural endolysins usually have a molecular weight ranging from 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 invention may also have a different, e.g., higher, molecular weight, for example when they further comprise additional domains or tags such as a PK tag, as described herein. Moreover, natural endolysins typically have a modular configuration. Herein and in context of the present invention, an endolysin refers, in particular, to a modular endolysin, i.e., a polypeptide (i.e., a single amino acid chain) comprising one or more enzymatically active domains (EADs) and, preferably, additionally one or more cell wall-binding domains (CBDs). Moreover, it is possible, also in context of a modular endolysin, that the endolysin only comprises one module, namely an EAD, e.g. a CHAP domain of the invention, but not a separate cell wall-binding domain. Nevertheless, it is preferred that the peptidoglycan hydrolase of the invention comprises at least one EAD, in particular the CHAP domain of the invention, and additionally at least one CBD. Furthermore, the various domains in a modular endolysin, e.g., an EAD and a CBD, may be separated by linker regions, in particular, by short and flexible linkers, as described herein. An EAD may be N-terminally or C-terminally of a CBD. In L0482 (SEQ ID NO: 1), a single cell wall-binding domain (CBD), i.e. the LYSM domain, is N-terminally of a single enzymatically active domain (EAD), i.e. the CHAP domain. Furthermore, in L0482, the LYSM and CHAP domains are separated by a linker region.

[0148] L0482

[0149] Herein, and in context of the present invention, L0482 (i.e., wild-type L0482; also called "lytN") has the following sequence:

[0150] REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTN NIAN PN LIFIGQKLKVPMTPLVEPKPKTVSSN N KSNSNSSTLNYLKTLEN R GWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNSEAKIYHNTPTFKAEPGDLWFSGRFGGGYGHTAIVLNG DYDGKLMKFQSLDQNWNNGGWRKAEVAHKWHNYENDMIFIRPFKKA (SEQ ID NO: 1)

[0151] In the following version of SEQ ID NO: 1, the LYSM domain (positions 1 to 51) is underlined, the linker (positions 52 to 71) is in italics, and the CHAP domain (positions 72 to 215) is in bold:

[0152] REAPKTOIYTVKKGDTLSAIALKYKTTVSNIONTNNIANPNLIFIGOKLKVPMTPLl / EP / <P / <7VSS / WW<S / \ / SNSSTLNYLKnLE71 EG WDFDGS G WQCFDLVNVYWN H LYGHG LKG||iiAKDIPYAN N FN0EAKrY[I]NTPTFKAEPGDLWFSGR[3GG GYGHTAIVLNGfTYDGHLnnKFQSLDQNWTniGGtlRK^EVAHKWHNYENDMIFIRPFKKA (SEQ ID NO: 1)

[0153] Furthermore, in the above sequence, the two glycosylation positions in SEQ ID NO: 1, i.e., positions 68 and 73, which may be deleted or, preferably, substituted with another amino acid (e.g., lysine and glycine, respectively) in order to obtain "aglycosylated" L0482 variants (e.g., L0482ag; SEQ ID NO: 2), as described herein, are further highlighted in black on grey background. Furthermore, the positions which may be substituted with amino acid residues contained in H5 (SEQ ID NO: 11) and / or G1 (SEQ ID NO: 3), as described herein, i.e., positions 82, 85, 86, 130, 136, 155, 169, 173, 175, 185, 186 and 192 in SEQ ID NO: 1, are shown in white on black background. In addition, the four positions which may be substituted with the active site mutations found in context of the invention (e.g., in H5-14; SEQ ID NO: 387) not already contained in H5, i.e. positions 94, 117, 118 and 189, are shown in white on grey background.

[0154] The CHAP domain of L0482 (SEQ ID NO: 1) is further shown in SEQ ID NO: 301; the LYSM domain of of L0482 (SEQ ID NO: 1) is further shown in SEQ ID NO: 302; and the linker region of L0482 (SEQ ID NO: 1) is further shown in SEQ ID NO: 303.

[0155] In context of the present invention, a natural peptidoglycan hydrolase, in particular the endolysin L0482 (SEQ ID NO: 1), is usually 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. A modified L0482 protein is also considered herein as an L0482 variant. Furthermore, the peptidoglycan hydrolase according to the present invention, in particular a modified L0482 variant, may lack the LYSM domain and / or the linker region, as described herein. Alternatively, the LYSM domain and / or the linker region, may be replaced by another cell-wall binding domain and / or or linker region, respectively, as described herein. In context of the present invention, lysostaphin (also called "LSN") has the following sequence:

[0156] AATHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKIVEAGWSNYGGGNQIGLIEN

[0157] DGVHRQWYMHLSKYNVKVGDYVKAGQIIGWSGSTGYSTAPHLHFQRMVNSFSNSTAQDPMPFLKSAGYGKA

[0158] GG7V P7~P / V7GWKTNKYGTLYKSESASFTPNTDirrRTTGPFRSMPOSGVLKAGOT[HYDEVMKODGHVWVGYTGNSGORIYL

[0159] PVRTWN KSTNTLGVLWGTIK ID NO: 407)

[0160] In the above lysostaphin sequence, the PepM23 domain (positions 1 to 143) is in bold, the SH3 domain is underlined and the linker (positions 144 to 153) is in italics. The glycosylation positions which are, preferably, at least partially aglycosylated in context of the present invention are highlighted in grey. In particular, the serine at position 126 and the asparagine at position 232 highlighted in grey are substituted by proline and glutamine, respectively, in the aglycosylated lysostaphin variant (S126P, N232Q; SEQ ID NO: 444), and the asparagines at positions 125 and 232 highlighted in grey are substituted by glutamines in another aglycosylated lysostaphin variant (N125Q, N232Q; SEQ ID NO: 408). The aglycosylated PepM23 domain contained in SEQ ID NO: 444 has the sequence shown in SEQ ID NO: 441, the PepM23-SH3 linker has the sequence shown in SEQ ID NO: 442, and the aglycosylated SH3 domain contained in SEQ ID NO: 444 has the sequence shown in SEQ ID NO: 443.

[0161] Domains of in particular, of modular

[0162] As already indicated above, the enzymatically active domains (EAD) of modular endolysins function, in particular, to cleave certain peptidoglycan bonds in the murein (i.e. peptidoglycan) layer of a host bacterium. Cell wall-binding domains (CBD) are typically enzymatically inactive, and, in particular, recognize and bind to certain epitopes in the cell wall of the host bacterium for proper fixation of the catalytic effect of the EAD. Preferably, herein and in context of the present invention, the cell wall binding domain is a peptidoglycan binding domain which binds, in particular, to the peptidoglycan structure of a target bacterium. The different domains of an endolysin can be connected by a peptide linker, also called "domain linker". Moreover, as described herein, an EAD (e.g. a CHAP domain or a PepM23 domain) may hydrolyse the peptidoglycan of a target bacterium e.g., the host bacterium, by itself, i.e., without the need for interacting with a separate CBD. For example, an EAD such as a CHAP domain may have (in addition to the peptidoglycan hydrolase activity) some intrinsic cell wall-binding activity and, therefore, have bactericidal activity by itself. This has been already demonstrated, e.g., for some peptidoglycan hydrolases such as lysozyme. In particular, binding of a substrate in the cell wall of a target bacterium to or close to the catalytic pocket of an EAD may be sufficient to initiate docking and subsequent lysis of the bacterium. Therefore, a CHAP domain of the invention may be also considered herein and in context of the present invention as a peptidoglycan hydrolase which may have a bactericidal activity by itself.

[0163] Accordingly, a peptidoglycan hydrolase according to the present invention comprises at least one enzymatically active domain (EAD - also called "catalytic domain" herein), in particular, at least a CHAP domain of the invention, as described herein, i.e., a CHAP domain that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1. In some embodiments said peptidoglycan hydrolase further has a PepM23 domain that has a sequence identity of at least 60% to the sequence from position 1 to position 143 in SEQ ID NO: 407, as described herein. Enzymatic peptidoglycan cleavage mechanisms

[0164] As used herein, the term "peptidoglycan hydrolase" is, in general, not restricted to a specific enzymatic cleavage mechanism. In particular, an enzymatically active domain of a peptidoglycan hydrolase may function as a glycosidase, as an amidase (i.e. an amidohydrolase) and / or as a peptidase. Hence, a peptidoglycan hydrolase comprising one or more EADs as described herein and in context of the present invention, e.g., an endolysin, may function as a glycosidase, an amidase and / or a peptidase, preferably as an amidase and / or peptidase. As used herein, glycosidases such as acetylmuramidases, lytic transglycosylases or glucosaminidases generally cleave the backbone of glycan. In particular glycosidases may cleave the p-1,4 glycosidic bonds linking alternating polymeric structures of N-acetylmuramic acids (MurNAc) and N-acetylglucosamines (GIcNAc) in a peptidoglycan layer. Amidases (i.e. amidohydrolases) generally cleave the side-chain peptide, in particular they may catalyze the cleavage of amide bonds between the MurNAc and the first amino acid in the peptide stem moiety, i.e., L-alanine. Peptidases (in particular, endopeptidases and carboxypeptidases) generally cleave within the peptide side-chain, in particular they may cleave bonds between two amino acids of the stem peptide of peptidoglycan, whereby bond cleavage can either occur within interpeptide bridge or stem peptide-interpeptide bridge.

[0165] The PepM23 domain of lysostaphin (SEQ ID NO: 407) is a peptidase of family M23 having a peptidase activity, specifically a glycyl-glycyl peptidase activity, as is well known in the art.

[0166] Herein and in context of the present invention, the term "CHAP domain" refers to a cysteine, histidine-dependent amidohydrolase / peptidase domain which is an enzymatically active domain (EAD) of a peptidoglycan hydrolase (in particular of a L0482 variant), as described herein. A CHAP domain may be further considered herein and in context of the present invention as a peptidoglycan hydrolase.

[0167] Moreover, the CHAP domain of L0482 (SEQ ID NO: 1) may function as an amidase and as a peptidase in order to hydrolyse peptidoglycan. In particular, it has been reported that the CHAP domain has an N-acetylmuramyl L-Ala amidase activity and a D-Ala-Gly endopeptidase activity; Frankel (2012), J Biol Chem. 23;287(13). Hence, the CHAP domain of the invention may also function, in particular, as an amidase and / or as a peptidase, preferably as an amidase and as a peptidase. Accordingly, the peptidoglycan hydrolase of the present invention functions, preferably, as an amidase (i.e. it has, preferably, an amidase activity) and / or as a peptidase (i.e. it has, preferably, a peptidase activity). In particular, the peptidoglycan hydrolase of the present invention may have an N- acetylmuramyl L-Ala amidase activity and / or a D-Ala-Gly endopeptidase activity. More preferably the peptidoglycan hydrolase of the present invention functions as an amidase and as a peptidase, in particular, wherein it may have an N-acetylmuramyl L-Ala amidase activity and a D-Ala-Gly endopeptidase activity.

[0168] Moreover, a CHAP domain which functions as an amidase and / or as a peptidase in order to hydrolyse peptidoglycan, may be also considered herein and in context of the present invention as a peptidoglycan hydrolase with amidase and / or peptidase activity, respectively. Thus, the CHAP domain of the invention may be a peptidoglycan hydrolase with amidase (e.g. N-acetylmuramyl L-Ala amidase activity) and / or peptidase activity (e.g. D-Ala-Gly endopeptidase activity). As described herein and as illustrated in the non-limited appended Examples, the inventors have identified L0482 variants and specific amino acids and amino acid combinations (including G1 / H5 mutations, WT amino acids and active site mutations) which confer peptidase activity, preferably in addition to amidase activity. Accordingly, a peptidoglycan hydrolase of the present invention comprising the CHAP domain of the invention has, preferably, an amidase and / or peptidase activity, as described herein. Peptidase activity can be measured, e.g., by tracking the cleavage of a fluorometric substrate analogue of the S.aureus stem peptide (sequence: L-Ala - yD-Glu - L-Lys - D-Ala - Gly - Gly - Gly - Gly - Gly; SEQ ID NO: 416), containing an N-terminal fluorophore (such as Glu(EDANS)) and a C-terminal quencher (such as Lys(DABCYL)) (Example 8 and Figure 13A); Frankel (2011); J Biol Chem 286(37)). The close proximity between the fluorophore and quencher in the peptide results in quenching of EDANS fluorescence. Peptidase cleavage of the peptide results in the release of the quencher from the fluorophore and thus in an increase in fluorescence. Rates of peptide hydrolysis can thus be determined from the of rate of fluorescence increase. The peptide can be synthesized using solid phase peptide chemistry and purified to homogeneity by high performance liquid chromatography.

[0169] For the detection of amidase activity, peptidoglycan fragments treated with amidase can be analyzed by high performance liquid chromatography followed by mass spectrometry; Frankel (2011); J Biol Chem 286(37).

[0170] While the catalytic activity of an enzymatically active domain, such as amidase or peptidase activity of a CHAP domain, is essential for bacterial killing, this killing activity may be also affected by the rest of the lysin, such as the cell wall binding domain and the linker between enzymatically active domain and cell wall binding domain.

[0171] Herein, and in context of the present invention, hydrolyzing peptidoglycan in the cell wall of a bacterium may refer also to breaking down and / or cleaving said peptidoglycan.

[0172] Bactericidal activity

[0173] Hydrolyzing, in particular breaking down and / or cleaving, peptidoglycan in the cell wall of a bacterium, usually kills the bacterium. Therefore, the peptidoglycan hydrolase of the present invention has, in particular, a bactericidal activity. Herein, and in 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, in particular at least one target bacterium (i.e., a bacterial species or strain to be killed). Preferably, a peptidoglycan hydrolase of the invention has the ability to kill at least one gram-positive bacterium, more preferably a Staphylococcus species or strain, most preferably at least Staphylococcus aureus, preferably, including methicillin-resistant Staphylococcus aureus (MRSA) strains. A peptidoglycan hydrolase which has the ability to kill a certain bacterium, e.g., S. aureus, is also referred to herein as a peptidoglycan hydrolase which has a "killing activity" against said bacterium, e.g., S. aureus.

[0174] Herein and in context of the present invention, the killing activity of a peptidoglycan hydrolase against a certain bacterium, e.g., S. aureus, is preferably measured by determining the minimum concentration at which the peptidoglycan hydrolase growth-inhibits a liquid culture of said bacterium, e.g., S. aureus. Said minimum concentration is also referred to herein as "minimal inhibitory concentration" (MIC). Herein and in context of the present invention, the "minimal inhibitory concentration" (MIC) is, in particular, the minimal concentration at which a bacterial (e.g. S. aureus) liquid culture comprising 5xl05cfu / ml of bacterial (e.g. S. aureus) cells stays translucent (i.e., OD620 < 0.1) for at least 18 h, preferably at least 24h, at 37°C, as judged by the naked eye, or, preferably, by measuring the optimal density at 620 nm (OD620). Preferably, the culture medium of said liquid culture is cation adjusted Muller-Hinton broth (caMHB) medium supplemented with 20-25% heat inactivated horse serum, in particular, when the target bacterium is a Staphylococcus species or strain such as S. aureus. Optionally, the peptidoglycan hydrolase is diluted in a buffer containing 20-25% inactivated horse serum (e.g., with the culture medium) before combining it with the bacterial (e.g. S. aureus) culture. When the killing activity against S. aureus is measured, the S. aureus cells in the liquid culture correspond, preferably, to 5xl05cfu / ml of ATCC43300 which is a methicillin-resistant Staphylococcus aureus (MRSA) strain. The term"cfu" is the abbreviation of "colony forming units", and refers to the estimated number of viable bacterial cells as commonly understood in the art.

[0175] A detailed assay for measuring the killing activity against a target bacterium, e.g., a Staphylococcus species or strain such as S. aureus, is provided in Example 5: First, a peptidoglycan hydrolase (e.g., a L0482 variant of the present invention) is produced in E coll, as described in Example 5 under the heading "Production of L0482 variants in E coif. Then, the peptidoglycan hydrolase (e.g., the L0482 variant of the present invention) is purified from E coll, as described under the heading "Purification of L0482 variants from E coif in Example 5. Alternatively, the peptidoglycan hydrolyase is produced in a human cell line, preferably the human embryonic kidney (HEK) cell line "Expi293" (ThermoFisher), as described in Example 7. The peptidoglycan hydrolase (e.g., the L0482 variant of the present invention) is then purified from supernatants of HEK cells, as described in Example 7. Production in a human cell line is preferably employed when the endolysin is very effectively secreted from human cells (e.g., for L0482 variants such as Gl-14, H5-14, L2, L14, L2DR, L14DR or L2TR).

[0176] Finally, the bactericidal activity of the peptidoglycan hydrolase (e.g., the L0482 variant or CHAP-lysostaphin fusion protein of the present invention) against a target bacterium (e.g. S. aureus) is determined as described in Example 5 under the heading "Determination of bactericidal activity of L0482 variants", optionally modified by diluting the peptidoglycan hydrolase in a buffer containing 20-25% inactivated horse serum before combining it with the bacterial (e.g. S. aureus) culture, as described in Example 7.

[0177] Detailed assays for measuring the killing activity of a peptidoglycan hydrolase against a biofilm or free-floating aggregate of a target bacterium, e.g., a biofilm or free-floating aggregate of Staphylococcus species or strain such as S. aureus, are provided in Example 5 under the heading "Determination of anti-biofilm activity of L0482 variants" which may be preferably employed. One of these assays makes use of a peg biofilm in plasma (PBA) and is particularly well suitable for determining the killing activity of a peptidoglycan hydrolase against a classical biofilm that is attached to a surface. The other assay makes use of a free-floating aggregate in synovial fluid (FBA) and is particularly well suitable for determining the killing activity of a peptidoglycan hydrolase against a free-floating (biofilm-like) aggregate.

[0178] As used herein and in context of the present invention, the term "activity" of a peptidoglycan hydrolase refers, in particular, to the bactericidal activity of the peptidoglycan hydrolase, as described herein. Furthermore, because the bactericidal activity of a peptidoglycan hydrolase against a certain bacterial species or strain, e.g. S. aureus as described herein, is tightly correlated to its capability of hydrolyzing (and breaking down and / or cleaving) 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 according to the present invention to hydrolyze, breakdown and / or cleave peptidoglycan by an enzymatic assay; it is sufficient to determine the bactericidal activity of the peptidoglycan hydrolase, i.e., its killing activity against the bacterial species or strain, e.g. S. aureus, as described herein. In other words, a peptidoglycan hydrolase (e.g. an endolysin) according to the present invention is considered to implicitly have the ability to hydrolase peptidoglycan in a target bacterium when it is able to kill said target bacterium, as described herein. However, to further corroborate that a peptidoglycan hydrolase functions indeed as a "peptidoglycan hydrolase", it is also possible to perform additional assays. For example, the reduction of purified peptidoglycan at OD620 nm in the present of a peptidoglycan hydrolase of the invention may be easily measured. Furthermore, purified peptidoglycan may be incubated with a peptidoglycan hydrolase of the invention followed by analysis and identification of cleavage products by mass spectrometry. Furthermore, the YODA-derived method of screening yeast cells for the secretion of an active peptidoglycan hydrolase according to the invention, as described herein may be employed. As described herein, in said screening method, the "activity" of a peptidoglycan hydrolase, i.e., its killing activity against a certain bacterium, is determined by measuring its ability to break down peptidoglycan contained in dead bacterial cells of said bacterium and / or fragments thereof or corresponding peptidoglycan particles (in particular via hydrolysis of the peptidoglycan).

[0179] Gram-positive and gram-negative bacteria

[0180] In gram-positive bacteria, the cytoplasmic membrane is surrounded by a peptidoglycan layer. A main purpose of the cell wall of Gram-positive bacteria is to maintain the shape of the bacteria and counteract the pressure inside the bacterial cells. Peptidoglycan or murein is a polymer composed of sugar and amino acid. The sugar component is composed of N-acetylglucosamine residues and a N-acetylmuramic acid residues that are p-( 1,4) linked. A peptide chain consisting of 3 to 5 amino acids is bound to N-acetylmuramic acid. Peptide chains can be cross-linked to peptide chains of other chains to form a 3D mesh-like layer. The peptide chain can contain D- and L-amino acid residues, and its composition can vary depending on the type of bacteria.

[0181] In contrast to gram-positive bacteria, gram-negative bacteria have an outer membrane with a characteristic asymmetric bilayer. The outer membrane bilayer consists of an inner monolayer containing phospholipids (primarily phosphatidylethanolamine) and an outer monolayer composed primarily of lipopolysaccharide (LPS). This outer membrane overlays a peptidoglycan layer which is normally much thinner than in gram-positive bacteria.

[0182] As described herein, the peptidoglycan hydrolase of the invention has, in particular, a killing activity against at least one gram-positive bacterium, preferably a Staphylococcus species or strain, more preferably at least Staphylococcus aureus, as described herein.

[0183] Variants and sequence identity to a reference sequence

[0184] Herein and in context of the present invention, a polypeptide (e.g., a peptidoglycan hydrolase of the invention) or a part thereof (e.g., a CHAP domain of the invention) which is derived from a certain polypeptide (e.g. L0482) or a part thereof (e.g. the CHAP domain of L0482) is considered herein and in context of the present invention as a "variant" of said polypeptide or part thereof, respectively, (e.g. a L0482 variant or CHAP domain variant, respectively). Furthermore, a peptidoglycan hydrolase of the invention comprising a CHAP domain of the invention, i.e., a CHAP domain derived from the CHAP domain of L0482, is also considered herein and in context of the present invention as an L0482 variant.

[0185] Herein, and in context of the present invention, the terms "variant" and "mutant" may be used interchangeably.

[0186] A variant (e.g. the CHAP domain of the invention) has, in particular, a sequence identity of at least n % (i.e. at least 60%) to a corresponding reference sequence (e.g. the sequence from position 72 to position 215 in SEQ ID NO: 1). Herein, a reference sequence, usually, refers to the sequence of the (poly)peptide or part thereof from which the variant is derived (e.g. the CHAP domain of L0482). Moreover, it is possible that a variant itself is employed as a reference sequence (e.g. H5; SEQ ID NO: 11) for describing another variant.

[0187] Furthermore, a variant, i.e. a polypeptide (e.g., a peptidoglycan hydrolase of the invention) or a part thereof (e.g., a CHAP domain of the invention) which is derived from a certain polypeptide (e.g. L0482) or a part thereof (e.g. the CHAP domain of L0482) that is used as a reference sequence, has at least one mutation, i.e., at least one amino J acid substitution, deletion, insertion and / or addition, preferably at least one amino acid substitution, relative to the reference sequence (e.g. at least one mutation in SEQ ID NO: 1, or in the sequence from position 72 to position 215 in SEQ ID NO: 1, respectively).

[0188] As commonly understood in the art and as also used herein and in context of the present invention, the term "sequence identity", refers to the extent to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment. Typically, the "sequence identity" is expressed as a percentage. Moreover, one of the two sequences may be considered as a reference sequence to which the other sequence has a sequence identity of at least n %.

[0189] Thus, herein and in context of the present invention, a certain (poly)peptide (e.g., a peptidoglycan hydrolase of the invention) or a part thereof (e.g., a CHAP domain of the invention) can be structurally defined by having a sequence identity of at least n % to a corresponding reference sequence, with n being 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 just mentioned above, said reference sequence refers to a (poly)peptide (e.g. SEQ ID NO: 1) or a part thereof (e.g. the sequence from position 72 to position 215 in SEQ ID NO: 1) from which said (poly)peptide or part thereof is derived, respectively.

[0190] Furthermore, it may be specifically indicated herein and in context of the present invention that a certain polypeptide or part thereof has a higher minimal sequence identity to a corresponding reference sequence than "at least 60%", i.e., a sequence identity of at least n %, with n being an integer between 61 and 99. For example, when the sequence identity is at least 80%, n is an integer between 80 and 99.

[0191] Furthermore, when it is indicated herein that a certain polypeptide or part thereof has a minimal sequence identity of at least 90% to a corresponding reference sequence, i.e., a sequence identity of at least n %, with n being an integer between 90 and 99, n may also refer to a decimal number with one decimal place 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 the minimal sequence identity is indicated by an integer, i.e., at least n % with n being an integer (e.g. at least 95%), n may also refer to a decimal number with one decimal place that can be rounded by conventional rounding rules to said integer. For example, a sequence identity of at least 95% may also refer to a sequence identity of 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%.

[0192] Generally, herein and in context of the present invention, the higher the sequence identity to a reference sequence, the more preferred it is. Furthermore, certain minimal sequence identities (other than "at least 60% ") are indicated herein in specific contexts and thus are considered as preferred minimal sequence identities in these contexts. However, other minimal sequence identities to a reference sequence, in particular, a sequence identity of at least n %, with n being an integer between 60 and 99, or n % with n being a decimal number with one decimal place between 89.5 and 99.9, are considered herein as well in context of any (poly)peptide or part thereof described herein. Of note, the term "(poly)peptide", as used herein, can refer to a polypeptide or a peptide, as commonly understood in the art.

[0193] The degree of sequence identity can be determined according to methods well known in the art using, preferably, suitable computer algorithms such as BLAST and / or CLUSTAL Omega. In particular, BLAST may be used in combination with CLUSTAL Omega. Furthermore, such computer algorithms such as BLAST allow to identify and compare peptidoglycan hydrolase variants having at certain sequence coverage (e.g. at least 80%) and a sequence identity (e.g. at least 60%) to a reference sequence. The coverage is a filter that selects sequences with the same architecture as the reference sequence, e.g., LYSM-CHAP endolysins.

[0194] For example, in context of the present invention and as illustrated in the appended Examples, endolysin sequences were isolated from the NCBI nucleotide database using BLAST, wherein the sequences were first sorted with a cutoff of 80% sequence coverage and then with a cutoff of 60% identity to L0482 (SEQ ID NO: 1) or a certain variant thereof. CLUSTAL Omega was then employed to align the sequences.

[0195] When using the Clustal Omega analysis method (e.g. in combination with BLAST) to determine whether a particular sequence is, for instance, at least 60% identical to a reference sequence default settings may be used.

[0196] 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(Wl):W636-W641. DOI: 10.1093 / nar / gkz268. PMID: 30976793; PMCID: PMC6602479) is used for the comparison of amino acid sequences in context of the present invention. In the case of pairwise comparisons / alignments, the following default settings are preferably chosen: 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 iteration: true; Number of iterations: 0; Maximum guide tree iterations: -1; Maximum HMM iterations: -1; Output alignment format: clustal_num; Output order: aligned; Sequence Type: protein. Preferably, the degree of sequence identity is calculated over the complete length of the reference sequence.

[0197] Amino acid residues located at a position corresponding to a position in a reference sequence, e.g., the amino acid sequence shown in SEQ ID NO:1, can be easily identified by the skilled person by methods known in the art. For example, such amino acid residues can be identified by aligning the sequence in question with the reference sequence, e.g., the sequence shown in SEQ ID NO: 1, and by identifying the positions which correspond to the indicated positions in the reference sequence, e.g. in SEQ ID NO: 1. The alignment can be done with means and methods known to the skilled person, e.g. by using a known computer algorithm such as the Lipman-Pearson method (Science 227 (1985), 1435) or the CLUSTAL algorithm. It is preferred that in such an alignment maximum homology is assigned to conserved amino acid residues present in the amino acid sequences.

[0198] Preferably, Clustal Omega is used for the comparison of amino acid sequences in context of the present invention, and hence for determining positions corresponding to positions in a reference sequence. In the case of pairwise comparisons / alignments, the following default settings are preferably chosen: 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 iteration: true; Number of iterations: 0; Maximum guide tree iterations: -1; Maximum HMM iterations: -1; Output alignment format: clustal_num; Output order: aligned; Sequence Type: protein. When the amino acid sequences (e.g. of L0482 variants) are aligned by means of such a method, regardless of insertions, deletions or additions that occur in the amino acid sequences, the positions of the corresponding amino acid residues can be determined (e.g., in each of the L0482 variants).

[0199] For example, SEQ ID NO: 1 (WT L0482) has a length of 215 amino acids and an arginine ("R") at position 86. The L0482 hit variant H3 (SEQ ID NO: 9), for example, does not show any deletions, insertions or additions compared to the sequence of SEQ ID NO: 1 but has a lysine f'K") at position 86. Thus, H3 (SEQ ID NO: 9) has an amino acid substitution at position 86 in reference to SEQ ID NO: 1, wherein the residue at said position (i.e., the arginine) is substituted with lysine. In short, H3 has the R86K mutation in reference to SEQ ID NO: 1, as described herein. In the L0482 hit variant "Hl" (SEQ ID NO: 7), however, five amino acid residues are deleted at the N-terminus. Thus, Hl only has a length of 210 amino acid residues. However, by aligning SEQ ID NO: 1 and SEQ ID NO: 7 by standard means as described herein and as illustrated in the appended Examples, it is immediately evident that Hl (SEQ ID NO: 7) has a lysine f'K") at a position corresponding to position 86 in SEQ ID NO: 1, similarly as H3; see, e.g., Figure 8A. Thus, Hl (SEQ ID NO: 7) is considered herein and in context to the present invention to have an amino acid substitution at a position corresponding to position 86 in SEQ ID NO: 1, wherein the residue at said position (i.e., the arginine) is substituted with lysine. In short, Hl has the same R86K mutation in reference to SEQ ID NO: 1 as other hit variants such as H3.

[0200] A similar logic also applies to any other mutations, e.g., amino acid substitutions, described herein and in context of the present invention.

[0201] Herein and in the context of the present invention, an "amino acid substitution" or short "substitution" at a certain position in a reference amino acid sequence or at a position corresponding to a certain position in a reference sequence (e.g. in SEQ ID NO: 1) means that the amino acid residue at said position is substituted with another amino acid residue, as described herein. In particular, the terms "amino acid substitution" or "substituted with another amino acid residue" mean that the respective amino acid residue at the indicated position can be substituted with any other possible amino acid residue, e.g. a naturally occurring amino acid or a non-naturally occurring amino acid (Brustad and Arnold, Curr. Opin. Chem. Biol. 15 (2011), 201-210), preferably with 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.

[0202] Herein and in the context of the present invention, in particular with respect to mutations within amino acid sequences, "deleted" or "deletion" means that the amino acid at the indicated position is deleted.

[0203] Herein and in the context of the present invention, in particular with respect to mutations within amino acid sequences, "inserted" or "insertion" means that at the respective position at least one amino acid residue, e.g., one or two, preferably one residue, is inserted after the indicated position.

[0204] Herein and in the context of the present invention, in particular with respect to mutations within amino acid sequences, "added" or "addition" means that at least one amino acid residue is added at the N-terminus and / or the C-terminus of the reference sequence.

[0205] Furthermore, a standard tool, preferably Clustal Omega, is used for determining the sequence identity of a nucleic acid sequence to a corresponding reference nucleic acid sequence in context of the present invention, preferably by using default settings.

[0206] CHAP domain of the invention and peptidoglycan hydrolases comprising a CHAP domain of the invention

[0207] As described herein above, the present invention relates, in particular, to a peptidoglycan hydrolase comprising a CHAP domain according to the present invention, and, optionally, comprising additionally a PepM23 domain that has a sequence identity of at least 60% to the sequence from position 1 to position 143 in SEQ ID NO: 407. In particular, said peptidoglycan hydrolase has bactericidal activity, preferably a killing activity against a gram-positive bacterium, more preferably against Staphylococcus species or strain, most preferably against Staphylococcus aureus, preferably including methicillin-resistant Staphylococcus aureus (MRSA) strains, as described herein. In some embodiments, e.g., when no PepM23 domain is fused to a CHAP domain, the peptidoglycan hydrolase of the invention may have a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, as described herein.

[0208] In particular, herein and in context of the present invention, the CHAP domain of the invention has a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1 (WT L0482), to the sequence from position 72 to position 215 in SEQ ID NO: 2 (L0482ag), to the sequence from position 72 to position 215 in SEQ ID NO: 3 (Gl) and / or to the sequence from position 72 to position 215 in SEQ ID NO: 11 (H5), preferably, to the sequence from position 72 to position 215 in SEQ ID NO: 1. Preferably, the CHAP domain of the invention further has one or more amino acid substitutions as compared to the sequence from position 72 to position 215 in SEQ ID NO: 1, in particular, at least one active site mutation, at least one H5 / G1 mutation and / or at least one acglycosylation mutation, as described herein.

[0209] Thus, the CHAP domain according to the present invention has, in particular, (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1, and preferably the following (i), (ii) and / or (iii):

[0210] (i) one or more active site mutations, i.e., one or more amino acid substitutions at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid;

[0211] (ii) one or more H5 / G1 mutations, i.e., one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 173, 175, 185, 186 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and / or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine;

[0212] (iii) an aglycosylation mutation, i.e., an amino acid substitution or a deletion at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, preferably wherein the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid, preferably with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, more preferably with glycine.

[0213] Furthermore, the CHAP domain of the invention may have one or more amino acid deletions, insertions or additions as compared to the sequence from position 72 to position 215 in SEQ ID NO: 1. However, the extent of the amino acid substitutions, deletions, insertions and / or additions may not be such that the sequence identity of the CHAP domain of the invention to the sequence from position 72 to position 215 in SEQ ID NO: 1 is below 60%.

[0214] In particular, the CHAP domain according to the invention functions as a peptidoglycan hydrolase and, preferably, has bactericidal activity, as described herein.

[0215] As already mentioned above, the sequence from position 72 to position 215 in SEQ ID NO: 1 is also shown in SEQ ID NO: 301.

[0216] Thus, the CHAP domain according to the invention has, in other words, a sequence identity of at least 60% to the sequence of SEQ ID NO: 301 and, preferably, one or more amino acid substitutions as compared to the sequence of SEQ ID NO: 301.

[0217] As described herein, the comparison to a reference sequence comprises, in particular, performing a sequence alignment. Thus, regardless of the occurrence of any amino acid deletions, insertions or additions, positions in the CHAP domain of the invention corresponding to positions in SEQ ID NO: 1 can be readily identified. In particular, in the absence of any deletions, insertions or additions, the CHAP domain of the invention has one or more amino acid substitutions in the sequence from position 72 to position 215 in SEQ ID NO: 1. Otherwise, the CHAP domain of the invention may have one or more amino acid substitutions in the sequence from a position corresponding to position 72 in SEQ ID NO: 1 to a position corresponding to position 215 in SEQ ID NO: 1, i.e., in the sequence which corresponds to the sequence from position 72 to position 215 in SEQ ID NO: 1 in a sequence alignment (and which may have deletions, insertions and / or additions relative to the sequence from position 72 to position 215 in SEQ ID NO: 1). In this context, it is not necessary that positions corresponding to positions 72 and 215 are present in the CHAP domain of the invention because other positions between these positions are sufficient to perform a sequence alignment.

[0218] In certain embodiments, the peptidoglycan hydrolase of the invention consists of the CHAP domain of the invention, in particular, a sequence that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; and that has (ii) one or more amino acid substitutions as compared to the sequence from position 72 to position 215 in SEQ ID NO: 1. In particular, said CHAP domain (and hence said peptidoglycan hydrolase) has bactericidal activity, preferably a killing activity against a Staphylococcus species or strain, more preferably against Staphylococcus aureus, as described herein.

[0219] Thus, in some aspects, the invention relates to the CHAP domain of the invention, i.e. a CHAP domain which has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1, and which has (ii) one or more amino acid substitutions as compared to the sequence from position 72 to position 215 in SEQ ID NO: 1, as described herein. In particular, the CHAP domain of the invention may be advantageously employed in a peptidoglycan hydrolase of the invention, e.g., in combination with a cell wall binding domain such as an LYSM or SH3 domain, and / or in combination with an additional enzymatically active domain, preferably a PepM23 domain which, preferably, has a sequence identity of at least 60% to the sequence from position 1 to position 143 in SEQ ID NO: 407, as described herein. . Alternatively, the CHAP domain of the invention may be considered itself as a peptidoglycan hydrolase of the invention.

[0220] In certain embodiments, the peptidoglycan hydrolase of the invention (i.e., a peptidoglycan hydrolase comprising or consisting of the CHAP domain of the invention), has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1. This sequence identity is, in particular, calculated over the full length of the sequence of SEQ ID NO: 1 (and not over the full length of the sequence of the peptidoglycan hydrolase of the invention). Thus, any additional domains, peptides or tags that may be comprised in (or fused to) the peptidoglycan hydrolase of the invention, e.g., a signal peptide or a PK tag, should not be considered when determining the sequence identity of the peptidoglycan hydrolase of the invention to the sequence of SEQ ID NO: 1. Hence, the peptidoglycan hydrolase of the invention may comprise (I) an amino acid sequence having a sequence identity of at least 60% to the sequence of SEQ ID NO: 1 (comprising the CHAP domain of the invention, and optionally the LYSM domain and / or peptide linker described herein), and, optionally, (II) one or more further domains, peptides or tags, e.g. a signal peptide, a PK tag, a further peptide linker etc., as described herein. In particular, said CHAP domain has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1, and, preferably, has (ii) one or more amino acid substitutions as compared to the sequence from position 72 to position 215 in SEQ ID NO: 1, as described herein.

[0221] Preferably, the peptidoglycan hydrolase of the invention, preferably, comprises a CHAP domain according to the present invention, i.e., a CHAP domain that has a sequence identity of at least 60% to the amino acid sequence from position 72 to position 215 in SEQ ID NO: 1, and, preferably, one or more amino acid substitutions at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, and, preferably, wherein the amino acid residue at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0222] Aalvcosylation mutations

[0223] As described herein and as illustrated in the appended Examples, the removal of glycosylation sites in L0482 (i.e. at positions 68 and 73 in SEQ ID NO: 1) resulting in so-called "aglycosylated" L0482 variants strongly enhanced the bactericidal activity upon expression in eukaryotic cells; see, e.g., Example 3.

[0224] Position 68 in SEQ ID NO: 1 is in the linker sequence of L0482, whereas position 73 in SEQ ID NO: 1 is in the CHAP domain of L0482. As already mentioned above, the CHAP domain is considered herein and in context of the invention as particularly important for the function of the peptidoglycan hydrolases of the invention, especially more important than the linker. Thus, an aglycosylation mutation at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is more preferred in context of the present invention than an aglycosylation mutation at position 68 in SEQ ID NO: 1 or at a position corresponding to this position.

[0225] Thus, in some embodiments, e.g., in context of aglycosylation mutations and aglycosylated L0482 variants, the inventive CHAP domain of the invention has a mutation, in particular, an amino acid substitution or a deletion, at position 73 in SEQ ID NO: 1 or at a position corresponding to this position as described herein, e.g., as specified in the subsequent embodiments. In other words, in the CHAP domain of the invention, the arginine f'N") at position 73 in SEQ ID NO: 1 or the arginine at a position corresponding to position 73 in SEQ ID NO: 1 may be deleted or, preferably, substituted with another amino acid residue, as described herein. As further described herein, said position 73 may be considered herein and in context of the present invention as a glycosylation position which is, preferably, aglycosylated. Therefore, the CHAP domain of the invention has preferably an aglycosylation mutation, preferably an amino acid substitution, at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

[0226] Thus, in some preferred embodiments, the CHAP domain of the invention has an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, in particular, an aglycosylation substitution as described herein.

[0227] In certain embodiments, e.g., in context of a mutation or substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, the peptidoglycan hydrolase of the invention does not have a sequence as shown in any one of SEQ ID NO: 276 to 278. Herein and in context of the present invention, in particular in context of the inventive CHAP domain, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position may be, for example, substituted with another amino acid residue than methionine, phenylalanine or lysine.

[0228] In certain embodiments, e.g., with respect to peptidoglycan hydrolases consisting of the inventive CHAP domain and / or lacking a cell wall binding domain as described herein, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than methionine.

[0229] In further embodiments of the inventive peptidoglycan hydrolase or CHAP domain, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than phenylalanine or lysine.

[0230] Preferably herein and in context of the present invention, in particular in context of the inventive CHAP domain and / or aglycosylation substitutions, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, for example, glycine, tyrosine, leucine, glutamic acid, alanine, or histidine.

[0231] In some embodiments, in particular in context of the inventive peptidoglycan hydrolase and / or the inventive CHAP domain, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than phenylalanine, lysine or serine.

[0232] Preferably herein and in context of the present invention, in particular in context of the inventive CHAP domain and / or aglycosylation substitutions, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine. For example, the CHAP domain of the invention may have a sequence as shown in positions 72 to 215 in SEQ ID NO: 2.

[0233] In certain embodiments, in particular in context of a peptidoglycan hydrolase that has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, the peptidoglycan hydrolase of the invention further comprises a deletion or an amino acid substitution (preferably an amino acid substitution) at position 68 in SEQ ID NO: 1 or at a position corresponding to this position. In other words, the arginine at position 68 in SEQ ID NO: 1 or at 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 in SEQ ID NO: 1 or at a position corresponding to this position may be considered herein and in context of the present invention as an aglycosylation mutation, in particular in context of a peptidoglycan hydrolase having a sequence identity of at least 60% to the sequence of SEQ ID NO: 1.

[0234] In certain embodiments, in particular in context of the inventive peptidoglycan hydrolase that has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted, for example, with another amino acid residue than threonine, serine or lysine, e.g., another amino acid residue than threonine or serine.

[0235] In certain embodiments, in particular in context of the inventive peptidoglycan hydrolase that has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than threonine, serine or lysine, e.g. another amino acid residue than threonine or serine, and / or the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid residue than phenylalanine, lysine or serine, e.g., another amino acid residue than phenylalanine or lysine.

[0236] In certain preferred embodiments, in particular in context of the inventive peptidoglycan hydrolase that has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, methionine, arginine or alanine, preferably lysine. In addition, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position may be substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, preferably glycine.

[0237] In certain preferred embodiments, in particular in context of the inventive peptidoglycan hydrolase that has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, the peptidoglycan hydrolase has a pair of amino acid substitutions at positions 68 and 73 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein a) the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, serine, tyrosine or leucine, b) the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with methionine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with leucine, c) the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with arginine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, or d) the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with alanine or histidine.

[0238] In certain particularly preferred embodiments, in particular in context of the inventive peptidoglycan hydrolase that has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, the residue at position 68 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, and the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0239] As already mentioned above, a peptidoglycan hydrolase of the invention having at least one aglycosylation mutation, in particular in the CHAP domain, (e.g. an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position such as K73G, for example as show in SEQ ID NO: 2) is, inter alia, a particularly good starting point for further protein engineering and / or directed evolution.

[0240] Thus, the peptidoglycan hydrolase of the invention may have instead or in addition (preferably in addition) to the mutations at positions 68 and / or 73 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein, for example in the embodiments just described above, at least one active site mutation and / or at least one G1 / H5 mutation, as described herein. H5 / G1 mutations

[0241] H5 mutations

[0242] As described herein and as illustrated in the appended Examples, the inventors found by directed evolution a particular set of beneficial mutations, i.e., T82S, N85G, R86K, S130N, H136K / R (in particular H136K), F155Y, D169N, N185Y and N186G in reference to SEQ ID NO: 1. As described herein, the variant H5 (SEQ ID NO: 11) which has been found to have overall the best pharmaceutical properties among the L0482 variants obtained by two rounds of directed evolution, contains exclusively these 9 beneficial amino acid substitutions; see, e.g., Figures 7 and 8. In particular, H5 (SEQ ID NO: 11) has an enhanced ability of being secreted from human cells, an enhanced killing activity against S. aureus, and an improved (thermo)stability as compared to the parental L0482ag (SEQ ID NO: 2) and also as compared to WT L0482 (SEQ ID NO: 1); see, e.g., Examples 3-5 and 11 and Figures 7, 9 and 5. In particular, H5 (SEQ ID NO: 11) showed an at least about 2-8-fold lower MIC than WT L0482 (SEQ ID NO: 1). Moreover, H5 (SEQ ID NO: 11) showed the strongest killing activity against S. aureus (together with H7; SEQ ID NO: 13), the best stability (in particular thermostability), and a good secretion from human cells among the hit variant obtained by the directed evolution; see Figure 7.

[0243] H5 (SEQ ID NO: 11) further showed enhanced (i.e. faster) killing kinetics against S. aureus in an OD reduction assay than WT L0482 (SEQ ID NO: 1); see Example 5 and Figure 10. In addition, H5 (SEQ ID NO: 11) effectively killed S. aureus biofilms; see Example 5 and Figure 11. This is in line with the particularly low MIC observed for H5.

[0244] Thus, the "H5 mutations", i.e., T82S, N85G, R86K, S130N, H136K / R (in particular H136K), F155Y, D169N, N185Y and N186G in reference to SEQ ID NO: 1, are beneficial for enhancing the desired pharmaceutical properties of peptidoglycan hydrolases, in particular, the bactericidal activity (e.g., against S. aureus including S. aureus biofilms), the stability and / or the ability of being secreted from eukaryotic cells, e.g. human cells.

[0245] As already mentioned above, it has been further found in context of the present invention that the L0482 variant H3 (SEQ ID NO: 9) obtained by the directed evolution contained 8 out of the 9 most beneficial amino acid substitutions and exclusively these 8 mutations. Since the 8 "H3" amino acid substitutions occurred recurrently in slightly different combinations among the 44 hit variants obtained by three rounds of directed evolution, they are considered herein and in context of the present invention as the consensus mutations. Furthermore, H3 reflects the consensus sequence. In particular, the 8 consensus mutations refer to T82S, N85G, R86K, S130N, H136K / R, D169N, N185Y and N186G, in reference to the sequence of SEQ ID NO: 1.

[0246] Furthermore, as described herein and as illustrated in the appended Examples, H3 (SEQ ID NO: 9) has improved pharmaceutical properties, e.g., an improved ability of being secreted from eukaryotic cells compared to WT L0482 and L0482ag as well as a good killing activity against S. aureus; see, e.g., Figures 7 and 9. This further demonstrates the advantageous effects of the consensus mutations with respect to the pharmaceutical properties of L0482 variants, as described herein.

[0247] Thus, in some preferred embodiments, e.g., in context of the consensus mutations, the CHAP domain of the present invention has one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions. Preferably, in these embodiments, the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0248] In other words, the CHAP domain of the invention has, in some preferred embodiments, 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 in SEQ ID NO: 1.

[0249] Preferably, said CHAP domain (i.e. a CHAP domain comprising at least one of the consensus mutations described herein) further comprises (i) an aglycosylation mutation at position 73 or at a position corresponding to this position, as described herein, for example, wherein the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, preferably glycine or serine, more preferably glycine; (ii) an amino acid substitution at position 155 or at a position corresponding to this position, wherein the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine; and / or (iii) one or more amino acid substitutions at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0250] As also already mentioned above, it has been surprisingly found in context of the invention that the L0482 variant H5 (SEQ ID NO: 11) which had an enhanced killing activity against S. aureus, an enhanced stability (e.g thermostability / melting temperature) and an enhanced ability of being secreted from human cells (Figure 7) as compared to H3, differed from H3 by only one amino acid, i.e., it had, in addition to the consensus mutations, the amino acid substitution F155Y in reference to SEQ ID NO: 1 (Figure 8). Furthermore, it has been found that the amino acid substitution H136R occurred in many of the hit variants (i.e.

[0251] G1-G4, H1-H10 and II to 130) instead of H136K and thus is a very good alternative to H136K.

[0252] In particular, the L0482 variant H3 has the following sequence, wherein the LYSM domain foositions 1 to 51) is underlined, the linker (positions 52 to 71) is in italics, and the CHAP domain (positions 72 to 215) is in bold:

[0253] REAPKTOIYTVKKGDTLSAIALKYKTTVSNIONTNNIANPNLIFIGOKLKVPMTPZ.l / E'PX'PX'TVSS / Vm / VSGSSTLNYLKSLE^ EGWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNHEAKIYENTPTFKAEPGDLWFSGRFGG GYGHTAIVLNGIUYDGKLMKFQSLDQNWEEGGWRKAEVAHKWHNYENDMIFIRPFKKA (SEQ ID NO: 9).

[0254] Furthermore, the L0482 variant H5 has the following sequence, wherein the LYSM domain f positions 1 to 51) is underlined, the linker (positions 52 to 71) is in italics, and the CHAP domain (positions 72 to 215) is in bold:

[0255] REAPKTOIYmKGDTLSAIALKYKTTVSNIONTNNIANPNLIFIGOKLKVPMTPLl / EP / fP / fmS / Vm / VSGSSTLNYLKSLES EGWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFN[I]EAKIYENTPTFKAEPGDLWFSGRnGG

[0256] GYGHTAIVLNGEIYDGKLMKFQSLDQNWEEGGWRKAEVAHKWHNYENDMIFIRPFKKA (SEQ ID NO: 11).

[0257] 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, wherein aglycosylation amino acid residues have been introduced in H3 and H5 (i.e. lysine at position 68 and glycine at position 73) are highlighted in grey.

[0258] The amino acid residues in SEQ ID NO: 9 shown in white on black background refer to the consensus mutations, and the amino acid residues in SEQ ID NO: 11 shown in white on black background refer to the H5 mutations, as described herein and in context of the present invention. Of note, H3 (SEQ ID NO: 9) contains all the H5 mutations except F155Y. Also see Figure 8A for the alignments.

[0259] Notably, all H5 mutations, i.e. T82S, N85G, R86K, S130N, H136K / R (preferably H136K), F155Y, D169N, N185Y and N186G in reference to SEQ ID NO: 1, occurred within the CHAP domain of L0482, i.e. within the sequence from position 72 to position 215 in SEQ ID NO: 1. Herein and in context of the present invention any of the H5 mutations or any combination thereof is, preferably, combined with an aglycosylation mutation at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein, e.g., with the amino acid substitution N73G in SEQ ID NO: 1, and / or one or more amino acid substitutions at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0260] In certain embodiments, e.g. in context of a peptidoglycan hydrolase which has a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, any of the H5 mutations or any combination thereof is further combined with at least one aglycosylation mutation at positions 68 and 73 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein, e.g., with the amino acid substitutions N68K and / or N73G in SEQ ID NO: 1.

[0261] It is by no means necessary that the peptidoglycan hydrolase or CHAP domain of the invention contains all of the 9 H5 mutations described herein at once. For example, it has been found that the L0482 variant Hl (SEQ ID NO: 7) did not show any mutations at positions corresponding to positions 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 and, nevertheless, had enhanced pharmaceutical properties compared to the parental L0482ag lysin (SEQ ID NO: 2), in particular, an enhanced killing activity against S. aureus, an enhanced stability (e.g. thermostability / melting temperature), and an enhanced ability of being secreted from human cells. As a further example, it has been found that the L0482 variant H9 (SEQ ID NO: 15) did not show any mutations at positions corresponding to positions 82, 85, 155, 185 and 185 in SEQ ID NO: 1, and also showed enhanced pharmaceutical properties compared to the parental L0482ag lysin (SEQ ID NO: 2); see, e.g., Figures 7 and 8. In fact, the amino acid substitution R86K is the only one among the H5 mutations which Hl (SEQ ID NO: 7) and H9 (SEQ ID NO: 15) have in common. This clearly demonstrates that the H5 mutations found in context of the present invention may be employed in various combinations and reliably enhance the pharmaceutical properties of L0482-derived peptidoglycan hydrolases, as described herein. However, this finding does, in no way, contradict the notion that the group of H5 mutations described herein has been purposefully and carefully selected by the present inventors in order to improve the pharmaceutical properties of L0482-derived peptidoglycan hydrolases. For example, when the additional amino acid substitutions N185Y and N186G are introduced in H9 (SEQ ID NO: 15) resulting, e.g., in 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 pharmaceutical properties assayed can be further improved; see Figure 7 and 8. Furthermore, as already mentioned above, the single amino acid substitution F155Y (occurring, e.g., in H5) improved all pharmaceutical properties compared to H3, further demonstrating that individual H5 mutations can already have highly beneficial effects. In fact, the L0482 variant H5 (SEQ ID NO: 11) showed the best pharmaceutical properties among all hit variants obtained by the directed evolution and analyzed, as described herein.

[0262] Thus, in some preferred embodiments, the CHAP domain of the present invention has one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions. Preferably, in these embodiments, the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0263] In other words, the CHAP domain of the invention has, in some preferred embodiments, at least one 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.

[0264] Preferably, said CHAP domain (i.e. a CHAP domain comprising at least one H5 mutation) further comprises an aglycosylation mutation at position 73 or at a position corresponding to this position, as described herein, for example, wherein the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, preferably glycine or serine, more preferably glycine.

[0265] G1 mutations

[0266] G1 (SEQ ID NO: 3) is a hit variant obtained in the first round of the directed evolution, which showed an increased thermostability, an enhanced secretion from eukaryotic cells as compared to the starting for the directed evolution (L0482ag); see Figure 7. Furthermore, G1 is effective in killing target bacteria such as S. aureus.

[0267] G1 (SEQ ID NO: 3) contains the following amino acid substitutions in the CHAP domain as compared to L0482ag (SEQ ID NO: 2): T82S, N85G, R86K, D169N, K173N, M175Q and A192Q.

[0268] Thus, in some embodiments, the CHAP domain of the invention has one or more amino acid substitutions at positions 82, 85, 86, 169, 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position (i.e. threonine) is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position (i.e. asparagine) is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position (i.e. arginine) is substituted with lysine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position (i.e. aspartic acid) is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position (i.e. lysin) is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position (i.e. methionine) is substituted with glutamine, and / or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position (i.e. alanine) is substituted with glutamine.

[0269] Furthermore, the CHAP domain may of the invention have a sequence identity of at least 95% to the sequence from position 72 to position 215 in SEQ ID NO: 3. In some embodiments, the CHAP domain of the invention has

[0270] (a) an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine;

[0271] (b) an amino acid substitution pair at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, and the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine; and / or

[0272] (c) an amino acid substitution at position 169 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine.

[0273] Furthermore, said CHAP domain may comprise one or more amino acid substitutions at positions 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, and / or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

[0274] Preferably, the CHAP domain further has an aglycosylation mutation at position 73 or at a position corresponding to this position, as described herein, preferably wherein the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, more preferably with glycine, and / or one or more amino acid substitutions at positions 94, 117, 118, 186 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and / or the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0275] In some embodiments of the present invention, the peptidoglycan hydrolase comprises a CHAP domain that has (i) a sequence identity of at least 80% to the sequence from position 72 to position 215 in SEQ ID NO: 1; (ii) an amino acid substitution or a deletion at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and (iii) one or more amino acid substitutions at positions 82, 85, 86, 169, 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, and / or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine; with the proviso that said CHAP domain does not have a sequence as set forth in SEQ ID NO: 381, 382, 383, 384 or 385.

[0276] In some embodiments of the present invention, the peptidoglycan hydrolase comprises a CHAP domain that has (i) a sequence identity of at least 91% to the sequence from position 72 to position 215 in SEQ ID NO: 1; (ii) an amino acid substitution or a deletion at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and (iii) one or more amino acid substitutions at positions 82, 85, 86, 169, 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, and / or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine; with the proviso that said CHAP domain does not have a sequence as set forth in SEQ ID NO: 381 or 382. In some further embodiments of the invention, the peptidoglycan hydrolase comprises a CHAP domain that has (i) a sequence identity of at least 80% to the sequence from position 72 to position 215 in SEQ ID NO: 1; (ii) an amino acid substitution or a deletion at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and (iii) at least two amino acid substitutions at positions 82, 85, 86, 169, 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, and / or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

[0277] In some embodiments of the invention, the peptidoglycan hydrolase comprises a CHAP domain that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; (ii) an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine; and (iii) one or more amino acid substitutions at positions 82, 85, 86, 169, 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, and / or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

[0278] Combination of H5 and Gl mutations

[0279] As illustrated in the appended non-limiting Examples, introducing H5-specific mutations (not contained in Gl) into lysins derived from Gl, e.g., L2 (SEQ ID NO: 388), leads to effective endolysins which may even have improved pharmaceutical properties, e.g., an enhanced peptidase activity. Similarly, introducing Gl-specific mutations (not contained in H5) into lysins derived from H5, e.g., L14 (SEQ ID NO: 389), also leads to effective endolysins which may even have improved pharmaceutical properties, e.g., an enhanced peptidase activity; see, e.g., Example 8 and Figure 13.

[0280] Hence, H5-specific mutations and Gl-specific mutations can be combined in the same CHAP domain and thus the same peptidoglycan hydrolase.

[0281] Therefore, the CHAP domain of the invention has, preferably, one or more (preferably a plurality, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 173, 175, 185, 186 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and / or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

[0282] In some preferred embodiments, the CHAP domain of the invention comprises at least one of the stem mutations which are common to G1 and H5, i.e., T82S, N85G, R86K, and / or D169N.

[0283] Accordingly, in some preferred embodiments, the CHAP domain of the invention has one or more amino acid substitutions at positions 82, 85, 86 and 169 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, and / or the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine.

[0284] In some embodiments, the CHAP domain (optionally having at least one stem mutation), has further at least one Gl-specific mutations, i.e., one or more amino acid substitutions at positions 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, and / or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

[0285] In some embodiments, the CHAP domain (optionally having at least one stem mutation), has further at least one H5-specific mutation, i.e., one or more amino acid substitutions at positions 130, 136, 155, 185 and 186, wherein the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0286] Amino acid substitution pairs and consensus mutation units

[0287] As further described herein above, the inventors further surprisingly found that certain amino acid substitutions, in particular some of the consensus mutations (i.e. T82S, N85G, R86K, S130N, H136K / R (esp. H136K), D169N, N185Y and N186G in reference to SEQ ID NO: 1) occurred very often as substitution pairs in the active and secreted L0482 variants obtained by the directed evolution, in particular in the hits variants, i.e., G1 to G4, Hl to H10 and II to 130. Thus, the consensus mutations may be further grouped as consensus mutation units herein and in context of the present invention. As mentioned above, these consensus mutation units consist of 1 or 2 amino acid substitutions and refer to: (i) R86K (which is particularly preferred), (ii) T82S and N85G, (iii) S130N and H136K / R (preferably H136K), (iv) D169N, and (v) N185Y and N186G.

[0288] Thus, in some preferred embodiments, the CHAP domain of the invention has at least one pair of amino acid substitutions a) at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, b) at positions 130 and 136 in SEQ ID NO: 1 or at positions corresponding to these positions, and / or c) at positions 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions.

[0289] In context of these embodiments, in a), preferably, the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, and the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine; in b), preferably, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, and the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine; and / or in c), preferably, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0290] In other words, the CHAP domain of the invention has, in some preferred embodiments, 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.

[0291] Furthermore, said CHAP domain (i.e. a CHAP domain having at least one substitution pair) further has, preferably, an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

[0292] Furthermore, said CHAP domain (i.e. a CHAP domain having at least one substitution pair, and preferably an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position) further has, preferably, an amino acid substitution at position 155 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

[0293] Furthermore, said CHAP domain, has, preferably, an aglycosylation mutation at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein.

[0294] Furthermore, said CHAP domain, has, preferably, at least one active site mutation, in particular, one or more amino acid substitutions at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0295] In some further preferred embodiments, the CHAP domain of the present invention has at least one consensus mutation unit, i.e., at least one amino acid substitution or substitution pair selected from the group consisting of the following (i) to (v):

[0296] (i) an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at said position is substituted with lysine (i.e. R86K);

[0297] (ii) an amino acid substitution at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine (i.e. T82S), and the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine (i.e. N85G);

[0298] (iii) an amino acid substitution at positions 130 and 136 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine (i.e. S130N), and the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine (i.e. H136K / R), preferably lysine (H136K);

[0299] (iv) an amino acid substitution at position 169 (i.e. the aspartic acid) in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with asparagine (i.e. D169N); and

[0300] (v) an amino acid substitution at positions 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine (i.e. N185Y), and the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine (i.e. N186G). Preferably, in context of these embodiments, the CHAP domain of the invention has at least 2, preferably at least 3, more preferably at least 4 of the amino acid substitutions or substitution pairs (i) to (v) (i.e. consensus mutation units), as just described herein above.

[0301] In some further preferred embodiments, the CHAP domain of the invention has

[0302] (i) an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at said position is substituted with lysine; and has at least one, preferably at least two, more preferably at least three of the following amino acid substitutions or substitution pairs (ii) to (v) (i.e. consensus mutation units):

[0303] (ii) an amino acid substitution at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, and the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine;

[0304] (iii) an amino acid substitution at positions 130 and 136 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, and the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine;

[0305] (iv) an amino acid substitution at position 169 (i.e. the aspartic acid) in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with asparagine; and / or

[0306] (v) an amino acid substitution at positions 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0307] Generally, herein and in context of 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, in 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 position 155 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein. Preferably, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine (i.e. F155Y), as described herein.

[0308] Furthermore, generally herein and in context of the present invention, a consensus mutation or consensus mutation unit or any combination thereof (which may be further combined with F155Y as just described) is, preferably, combined with at least one aglycosylation mutation (preferably at least one aglycosylation substitution) as described herein, preferably an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position. Preferably, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, more preferably, with glycine, as described herein.

[0309] Furthermore, generally herein and in context of the present invention, a consensus mutation or consensus mutation unit or any combination thereof (which may be further combined with F155Y and / or an aglycoslylation mutation as just described) is, preferably, combined with at least one active site mutation, in particular, Y94F, Y117D, G118Y, and / or W189E in reference to SEQ ID NO: 1.

[0310] What has been just described herein above in context of an amino acid substitution at position 155 in SEQ ID NO: 1 or at a position corresponding to this position, an aglycosylation mutation, and an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is, in particular, also the case for the following embodiments:

[0311] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitution R86K, as described herein.

[0312] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S and N85G, as described herein.

[0313] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, S130N and H136K / R, as described herein.

[0314] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K and D169N, as described herein.

[0315] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K and N185Y and N186, as described herein.

[0316] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, S130N and H136K / R, as described herein.

[0317] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, and D169N, as described herein.

[0318] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, and N185Y and N186G, as described herein.

[0319] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, D169N, N185Y and N186G, as described herein.

[0320] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, S130N, H136K / R, N185Y and N186G, as described herein.

[0321] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, S130N, H136K / R, and D169N, as described herein. In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, N185Y and N186G, as described herein.

[0322] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, S130N, H136K / R and D169N, as described herein.

[0323] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, S130N, H136K / R, N185Y and N186, as described herein.

[0324] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, S130N, H136K / R, D169N, N185Y and N186G, as described herein.

[0325] In some preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, D169N, N185Y and N186G, as described herein.

[0326] In some of particularly preferred embodiments, the CHAP domain of the invention has the amino acid substitutions R86K, T82S, N85G, S130N, H136K / R, D169N, N185Y and N186G, as described herein.

[0327] In some embodiments, the CHAP domain of the invention has the amino acid substitutions T82S and N85G, as described herein.

[0328] In some embodiments, the CHAP domain of the invention has the amino acid substitutions T82S, N85G, S130N and H136K / R, as described herein.

[0329] In some embodiments, the CHAP domain of the invention has the amino acid substitutions T82S, N85G and D169N, as described herein.

[0330] In some embodiments, the CHAP domain of the invention has the amino acid substitutions T82S, N85G, N185Y and N186G, as described herein.

[0331] In some embodiments, the CHAP domain of the invention has the amino acid substitutions T82S, N85G, S130N, H136K / R and D169N, as described herein.

[0332] In some embodiments, the CHAP domain of the invention has the amino acid substitutions T82S, N85G, S130N, H136K / R, N185Y and N186G, as described herein.

[0333] In some embodiments, the CHAP domain of the invention has the amino acid substitutions T82S, N85G, S130N, H136K / R, D169N, N185Y and N186G, as described herein.

[0334] In some embodiments, the CHAP domain of the invention has the amino acid substitutions S130N and H136K / R, as described herein.

[0335] In some embodiments, the CHAP domain of the invention has the amino acid substitutions S130N, H136K / R and D169N, as described herein.

[0336] In some embodiments, the CHAP domain of the invention has the amino acid substitutions S130N, H136K / R, N185Y and N186G, as described herein.

[0337] In some embodiments, the CHAP domain of the invention has the amino acid substitutions S130N, H136K / R, D169N, N185Y and N186G, as described herein. In some embodiments, the CHAP domain of the invention has the amino acid substitution D169N, as described herein.

[0338] In some embodiments, the CHAP domain of the invention has the amino acid substitutions D169N, N185Y and N186G as described herein.

[0339] In some embodiments, the CHAP domain of the invention has the amino acid substitutions N185Y and N186G, as described herein.

[0340] Furthermore, herein and context of the present invention, and hence, also in the above embodiments, the substitution H136K is preferred over the substitution H136R.

[0341] In certain embodiments, the CHAP domain of the present invention has a sequence identity of at least 94% (in particular, at least 93.9%), at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the sequence from position 72 to position 215 in SEQ ID NO: 9 (H3). Preferably, said CHAP domain has a) (i) at least one of the consensus mutations or consensus mutation units described herein and (ii) at least one aglycosylation mutation, preferably an amino acid substitution at position 73 as described herein, and / or b) at least two of the consensus mutations, e.g., at least one amino acid substitution pair, as described herein. Furthermore, in context of these embodiments or in similar embodiments, the CHAP domain of the present invention has, preferably, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 amino acid substitutions in the sequence from position 72 to position 215 in SEQ ID NO: 9 (H3) or in the sequence from a position corresponding to position 72 in SEQ ID NO: 9 to a position corresponding to position 215 in SEQ ID NO: 9. Furthermore, said CHAP domain has preferably the amino acid substitution F155Y.

[0342] In certain embodiments, the peptidoglycan hydrolase of the present invention has a sequence identity of at least 95% (in particular at least 95.4%), at least 96%, at least 97%, at least 98% or at least 99% to the sequence of SEQ ID NO: 9 (H3). Furthermore, in context of these embodiments or in similar embodiments, the peptidoglycan hydrolase of the present invention has, preferably, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 amino acid substitutions in SEQ ID NO: 9. Preferably, said peptidoglycan hydrolase comprises a CHAP domain according to the present invention.

[0343] Particularly important individual amino acid substitutions: R86K and F155Y

[0344] As already mentioned above and as illustrated in the appended Examples, all hit variants, i.e. G1-G4, H1-H10 and 11-130, had the amino acid substitution R86K in reference to SEQ ID NO: 1. Thus, this amino acid substitution is considered herein and in context of the present invention as a particularly important substitution among the H5 / G1 mutations, as described herein.

[0345] Thus, in some of the most preferred embodiments, e.g., in context of H5 / G1 mutations, the CHAP domain of the present invention has an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position. For example, in context of these embodiments, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position may be substituted with another amino acid residue than serine. Preferably, in context of these embodiments, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or methionine. More preferably, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine. Furthermore, said CHAP domain, has, preferably, an aglycosylation mutation at position 73 in SEQ ID NO 1 or at a position corresponding to this position, as described herein. Furthermore, said CHAP domain has, preferably, an active site mutation, in particular, one or more amino acid substitutions at positions 94, 117, 118, 186 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and / or the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0346] As also already mentioned above, the amino acid substitution F155Y further improved several pharmaceutical properties (e.g. the killing activity against S. aureus, the stability and the ability of being secreted from human cells) in H5 (SEQ ID NO: 11) compared to H3 (SEQ ID NO: 9) containing only the consensus mutations. Furthermore, the amino acid substitution F155Y improved the peptidase activity, e.g., in L2DR (SEQ ID NO: 390) as compared to L2 (SEQ ID NO: 388). The mutation F155Y also further enhanced the activity against biofilms (esp. of S. aureus) when introduced into L2.

[0347] Therefore, in some of the most preferred embodiments, the CHAP domain of the invention has an amino acid substitution at position 155 in SEQ ID NO: 1 or at a position corresponding to this position. Preferably, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

[0348] Further important individual amino acid substitutions: N185Y, N186G and M175Q

[0349] As described above, and as illustrated in the appended non-limiting Examples, the amino acid substitution N186G in reference to SEQ ID NO: 1 is contained in the L0482 variant H5 which was the best performing tested variant obtained by the directed evolution. In addition, N186G was independently found in an active site mutation screen. Thus, N186G is considered as a further important amino acid substitution in the CHAP domain of the invention.

[0350] Therefore, in some preferred embodiments, the CHAP domain of the invention has an amino acid substitution at position 186 in SEQ ID NO: 1 or at a position corresponding to this position. Preferably, the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0351] Furthermore, it has been surprisingly found that introducing the amino acid substitution N185Y into a Gl-based variant, i.e., L2DR (SEQ ID NO: 390) further enhanced the peptidase activity.

[0352] Therefore, in some preferred embodiments, the CHAP domain of the invention has an amino acid substitution at position 185 in SEQ ID NO: 1 or at a position corresponding to this position. Preferably, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

[0353] Of note, L2DR further contained the amino acid substitution N186G (due to the active site engineering), and N185Y and N186G are one of the consensus mutation units found by the directed evolution. It is thus plausible that N185Y and N186G have an additive or synergistic effect, e.g., synergistically enhance peptidase activity and / or bactericidal activity.

[0354] Therefore, in some preferred embodiments, the CHAP domain of the invention has an amino acid substitution at position 185 in SEQ ID NO: 1 or at a position corresponding to this position and an amino acid substitution at position 186 in SEQ ID NO: 1 or at a position corresponding to this position. Preferably, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0355] In addition, it has been surprisingly found that introducing the amino acid substitution M175Q into a H5-based variant, i.e., L14 (SEQ ID NO: 389) also enhanced the peptidase activity.

[0356] Therefore, in some preferred embodiments, the CHAP domain of the invention has an amino acid substitution at position 175 in SEQ ID NO: 1 or at a position corresponding to this position. Preferably, the amino acid residue (i.e. the methionine) at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

[0357] Generally, herein and in context of the present invention, the CHAP domain, has, preferably, an aglycosylation mutation at position 73 in SEQ ID NO 1 or at a position corresponding to this position, as described herein. Furthermore, generally herein and in context of the present invention, the CHAP domain, has, preferably, an active site mutation, in particular one or more amino acid substitutions at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid. This is also true, inter alia, for all the following embodiments:

[0358] Further embodiments

[0359] In some embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 91% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 86 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; and has (iii) one or more amino acid substitutions at positions 82, 85, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0360] In some embodiments, the peptidoglycan hydrolase of the invention has (i) a sequence identity of at least 84% to the sequence of SEQ ID NO: 1; has (ii) an amino acid substitution at position 86 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; and has (iii) one or more amino acid substitutions at positions 82, 85, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0361] In some embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 78% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 86 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; and has (iii) at least two amino acid substitutions at positions 82, 85, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0362] In some embodiments, the peptidoglycan hydrolase of the invention has (i) a sequence identity of at least 73% to the sequence in SEQ ID NO: 1; has (ii) an amino acid substitution at position 86 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; and has (iii) at least two amino acid substitutions at positions 82, 85, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0363] In some embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 78% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 86 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; and has (iii) one or more amino acid substitutions at positions 82, 85, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0364] In some embodiments, the peptidoglycan hydrolase of the invention has (i) a sequence identity of at least 73% to the sequence in SEQ ID NO: 1; has (ii) an amino acid substitution at position 86 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; and has (iii) one or more amino acid substitutions at positions 82, 85, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0365] In some embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 86 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; and has (iii) at least six amino acid substitutions at positions 82, 85, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0366] In some embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 86 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; has (iii) an amino acid substitution at position 82 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine; and has (iv) an amino acid substitution at position 85 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine. In other words, said CHAP domain has the R86K substitution and a substitution pair consisting of T82S and N85G in reference to SEQ ID NO: 1.

[0367] In some embodiments, the peptidoglycan hydrolase of the invention has (i) a sequence identity of at least 60% to the sequence of SEQ ID NO: 1; has (ii) an amino acid substitution at position 86 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine; has (iii) an amino acid substitution at position 82 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine; and has (iv) an amino acid substitution at position 85 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine. In other words, said peptidoglycan hydrolase has the R86K substitution and the substitution pair consisting of T82S and N85G in reference to SEQ ID NO: 1.

[0368] In some embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 83% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution or a deletion (preferably, an amino acid substitution) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and has (iii) one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0369] In some embodiments, the peptidoglycan hydrolase of the invention has (i) a sequence identity of at least 73% to the sequence of SEQ ID NO: 1; has (ii) an amino acid substitution or a deletion (preferably, an amino acid substitution) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and has (iii) one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0370] In some embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; and (ii) an amino acid substitution at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue (i.e. the asparagine) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine. Preferably, said CHAP domain further has (iii) one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0371] In some embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution or a deletion (preferably, an amino acid substitution) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and has (iii) an amino acid substitution at position 82 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine.

[0372] In some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution or a deletion (preferably, an amino acid substitution) at position 73 in SEQ ID NO: 1 or at a position corresponding to this position; and has (iii) an amino acid substitution at position 130 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine.

[0373] In some particular embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 73 or a deletion (preferably, an amino acid substitution) in SEQ ID NO: 1 or at a position corresponding to this position; and has (iii) an amino acid substitution at position 185 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

[0374] In some embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 130 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine; and has (iii) one or more amino acid substitutions at positions 82, 85, 86, 136, 155, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0375] In some embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 78% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 155 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine; and has (iii) at least two amino acid substitutions at positions 82, 85, 86, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0376] In some embodiments, the peptidoglycan hydrolase of the invention has (i) a sequence identity of at least 73% to the sequence of SEQ ID NO: 1; has (ii) an amino acid substitution at position 155 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine; and has (iii) at least two amino acid substitutions at positions 82, 85, 86, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0377] In some embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 78% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 155 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine; and has (iii) one or more amino acid substitutions at positions 82, 85, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0378] In some embodiments, the peptidoglycan hydrolase of the invention has (i) a sequence identity of at least 73% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 155 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine; and has (iii) one or more amino acid substitutions at positions 82, 85, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0379] In some embodiments, the CHAP domain of the invention has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; has (ii) an amino acid substitution at position 155 in SEQ ID NO: 1, or at a position corresponding to this position, wherein the amino acid residue (i.e. the phenylalanine) at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine; and has (iii) at least six amino acid substitutions at positions 82, 85, 86, 130, 136, 169, 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. the threonine) at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue (i.e. the asparagine) at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue (i.e. the arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue (i.e. the serine) at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the histidine) at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, preferably lysine, the amino acid residue (i.e. the aspartic acid) at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue (i.e. the asparagine) at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. the asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0380] In some embodiments, e.g., in context of L0482 variants obtained by the directed evolution, permissive or beneficial mutations, particularly beneficial mutations, most beneficial mutations, consensus mutations or consensus mutation units, the peptidoglycan hydrolase of the invention does not have a sequence as shown in any one of SEQ ID NO: 279 to 293.

[0381] In some embodiments, the CHAP domain of the present invention has a sequence identity of at least 90%, preferably at least 95%, more preferably at least 97% or at least 98%, e.g. at least 97.3%, to the CHAP domain of a hit variant as described herein, i.e., to the CHAP domain of any one of G1 to G4, Hl to H10 and II to 130 (SEQ ID NO: 3 to 46, respectively), as described herein. In particular, the CHAP domain of a hit variant (e.g. Hl) refers to the sequence from a position in the sequence of said hit variant (e.g. SEQ ID NO: 7) which corresponds to position 72 in SEQ ID NO: 1 to a position in the sequence of said hit variant (e.g. SEQ ID NO: 7) which corresponds to position 215 in SEQ ID NO: 1. In particular, said CHAP domain has at least one of the amino acid substitutions selected from the group consisting of: T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1, i.e. with respect to the sequence from position 72 to position 215 in SEQ ID NO: 1 as a reference sequence, as described herein.

[0382] In some further embodiments, the peptidoglycan hydrolase of the present invention has a sequence identity of at least 90%, preferably at least 95%, more preferably at least 97%, e.g. at least 96.8%, to a hit variant as described herein, i.e., to any one of SEQ ID NO: 3 to 46. In particular, said peptidoglycan hydrolase has at least one of the amino acid substitutions selected from the group consisting of: T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1, i.e. with respect to SEQ ID NO: 1 as a reference sequence, as described herein.

[0383] In some preferred embodiments, the CHAP domain of the invention has a sequence identity of at least 93.2% or at least 94% to the sequence from position 72 to position 215 in SEQ ID NO: 11. In particular, said CHAP domain has at least one of the amino acid substitutions selected from the group consisting of: T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1, i.e. with respect to the sequence from position 72 to position 215 in SEQ ID NO: 1 as a reference sequence, as described herein.

[0384] In some preferred embodiments, the peptidoglycan hydrolase of the invention has a sequence identity of at least 95.0% or at least 96% to the sequence of SEQ ID NO: 11. In particular, said peptidoglycan hydrolase has at least one of the amino acid substitutions selected from the group consisting of: T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1, i.e. with respect to SEQ ID NO: 1 as a reference sequence, as described herein. In some preferred embodiments, the CHAP domain of the invention has a sequence identity of at least 93.9% or at least 94% to the sequence from position 72 to position 215 in SEQ ID NO: 9. In particular, said CHAP domain has at least one of the amino acid substitutions selected from the group consisting of: T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1, i.e. with respect to the sequence from position 72 to position 215 in SEQ ID NO: 1 as a reference sequence, as described herein.

[0385] In some preferred embodiments, the peptidoglycan hydrolase of the invention has a sequence identity of at least 95.4% or at least 96% to the sequence of SEQ ID NO: 9. In particular, said peptidoglycan hydrolase has at least one of the amino acid substitutions selected from the group consisting of: T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, N185Y and N186G in SEQ ID NO: 1, i.e. with respect to SEQ ID NO: 1 as a reference sequence, as described herein.

[0386] Active site mutations

[0387] As described above and in the non-limiting illustrative Examples, the inventors further, unexpectedly, found five mutations in the active site of the CHAP domain of L0482 variants which strongly enhanced the secretion of the endolysins (i.e. L0482 variants) from human cells while providing the endolysins with a high thermostability and effective killing activity against target bacteria including biofilms; see, e.g., Example 7, Table 4 and Figure 12B.

[0388] The active site of the CHAP domain of L0482 or variants thereof, as used herein and in context of the present invention, refers to the active site pocket / region including and surrounding the catalytic cysteine of the CHAP domain of L0482 (position 98 in SEQ ID NO: 1). In particular, the active site is located at positions 94, 97, 99, 117, 118, 119, 156, 159, 160, 182, 183, 184, 186, and 189 of SEQ ID NO: 1, or at positions corresponding to these positions, and is thus formed by the amino acid residues at these positions.

[0389] The active site mutations according to the present invention are: Y94F, Y117D / G (preferably Y117D), G118Y, N186G and W189E.

[0390] Therefore, in some embodiments, the CHAP domain of the present invention has one or more (i.e at least one) amino acid substitutions at positions 94, 117, 118, 186 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably, wherein the amino acid residue (i.e. tyrosine) at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue (i.e. tyrosine) at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue (i.e. glycine) at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and / or the amino acid residue (i.e. tryptophan) at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid. Thus, a peptidoglycan hydrolase comprising an inventive CHAP domain that has one or more active site mutations, i.e., amino acid substitutions at positions 94, 117, 118, 186 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein, may have an enhanced ability of being secreted by a human cell and / or an enhanced stability (e.g. thermostability), preferably at least an enhanced ability of being secreted by a human cell, in comparison to the peptidoglycan hydrolase of SEQ ID NO: 1.

[0391] Furthermore, a peptidoglycan hydrolase comprising an inventive CHAP domain that has one or more active site mutations, i.e., amino acid substitutions at positions 94, 117, 118, 186 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein, may be stable up to a temperature of about 40°C, preferably of about 50°C. In other words, said peptidoglycan hydrolase may have a thermostability of about 40°C, preferably of about 50°C.

[0392] As mentioned above, the active site mutation N186G is also contained in some hit variants obtained by directed evolution, e.g., in H5 (SEQ ID NO: 11) but not in G1 (SEQ ID NO: 3), and thus is also considered herein and in context of the present invention as a G1 / H5 mutation (or H5 / G1 mutation), a H5 mutation or a H5-specific mutation.

[0393] Thus, in further embodiments, e.g., in context of G1 / H5 mutations or H5 mutations, the CHAP domain of the invention has one or more amino acid substitutions at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 94 (i.e. tyrosine) in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue (i.e. tyrosine) at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue (i.e. glycine) at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. tryptophan) at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0394] The inventors further found that each of the following active site mutations, i.e., Y117D, G118Y, W189E, had individually a strong contribution to the drastically enhanced ability of the endolysins of being secreted from human cells, while the combination of Y117D and G118Y showed a particularly pronounced effect; see, e.g., Example 7 and Table 3.

[0395] Therefore, in some preferred embodiments, the CHAP domain of the invention has one or more amino acid substitutions at positions 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. tyrosine) at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue (i.e. glycine) at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. tryptophan) at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0396] In some embodiments, the CHAP domain of the invention has one or two amino acid substitutions at positions 117 and 118 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. tyrosine) at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably glycine, and / or the amino acid residue (i.e. glycine) at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

[0397] Modulation of peptidase activity

[0398] While the active site mutations according to the invention strongly enhance the ability of L0482 variants of being secreted from human, the inventors surprisingly found that certain active site mutations or combinations thereof with each other or WT residues reduce the peptidase activity of the endolysin. A reduction or loss of peptidase activity is not critical because L0482 variants showing such a reduction or loss still are highly effective in killing target bacteria, e.g., S. aureus, as illustrated in the appended non-limiting Examples, most likely due to their amidase activity. Nevertheless, it is preferred that the peptidoglycan hydrolase (e.g. endolysin) also has a peptidase activity, in particular, in addition to amidase activity. Such a double peptidase / amidase activity might further reduce the risk of resistance formation in target bacteria, e.g., S. aureus, as described herein.

[0399] Specifically, the inventors found that the introduction of the active site mutations Y94F, Y117D G118Y and W189E into H5 (SEQ ID NO: 11), resulting in H5-14 (SEQ ID NO: 387), led to a reduction of peptidase activity (and a slightly reduced but still good killing activity against S. aureus. Surprisingly, the backmutation D117Y in H5-14, resulting in L14 (SEQ ID NO: 389) strongly enhanced peptidase activity; see, e.g., Examples 7 and 8, Table 4 and Figure 13.

[0400] Therefore, in some preferred embodiments, the CHAP domain has one or more amino acid substitutions at positions 94, 118, 186 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably, wherein the amino acid residue (i.e. tyrosine) at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue (i.e. glycine) at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and / or the amino acid residue (i.e. tryptophan) at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0401] Preferably, the amino acid residue (i.e. tyrosine) at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is not deleted or substituted with another amino acid in these embodiments. In other words, the CHAP domain according to these embodiments, preferably, has a tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position. In further preferred embodiments, e.g., in context of G1 / H5 mutations or H5 mutations, the CHAP domain of the invention has one or more amino acid substitutions at positions 94, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 94 (i.e. tyrosine) in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue (i.e. glycine) at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. tryptophan) at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0402] Preferably, the amino acid residue (i.e. tyrosine) at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is not deleted or substituted with another amino acid in these embodiments. In other words, the CHAP domain according to these embodiments, preferably, has a tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position.

[0403] In some embodiments, the CHAP domain of the invention has one or two amino acid substitutions at positions 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue (i.e. glycine) at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue (i.e. tryptophan) at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0404] Furthermore, the inventors found that, the active site mutation Y117D in combination with the WT residue G118, the active site mutation G118Y in combination with the WT residue Y117, the two active site mutations Y117G and G118Y in combination, as well as the two active site mutations Y94F and W189E in combination with the two WT residues Y117 and G118, also provided peptidase activity; see e.g., Examples 7 and 8 and Table 3.

[0405] Thus, in some embodiments, the CHAP domain has an amino acid substitution at position 117 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, and, preferably, wherein the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is a glycine. In other words, the glycine at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is, preferably, not deleted or substituted with another amino acid in these embodiments. Furthermore, the CHAP domain according to these embodiments, preferably, has one, two or three amino acid substitutions at positions 94, 186 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein.

[0406] In some embodiments, the CHAP domain has an amino acid substitution at position 118 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and, preferably, wherein the amino acid residue at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is a tyrosine. In other words, the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is, preferably, not deleted or substituted with another amino acid in these embodiments. Furthermore, the CHAP domain according to these embodiments, preferably, has one, two or three amino acid substitutions at positions 94, 186 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein.

[0407] In some embodiments, the CHAP domain has two amino acid substitutions at positions 117 and 118 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine. Furthermore, the CHAP domain according to these embodiments, preferably, has one, two or three amino acid substitutions at positions 94, 186 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, as described herein.

[0408] In some embodiments, the CHAP domain has two amino acid substitutions at positions 94 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, and the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid; and, preferably, wherein the amino acid residue at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is a tyrosine, and the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is a glycine. In other words, the tyrosine at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is, preferably, not deleted or substituted with another amino acid and the glycine at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is, preferably, not deleted or substituted with another amino acid in these embodiments. Furthermore, the CHAP domain according to these embodiments, preferably, has an amino acid substitution at position 186 in SEQ ID NO: 1 or at a position corresponding to this positions, as described herein.

[0409] Furthermore, said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions) has, in particular, an amidase activity and / or a peptidase activity, preferably at least an amidase activity, more preferably an amidase activity and a peptidase activity. Preferably, the amidase activity comprises peptidoglycan cleavage between N-acetylmuramyl and L-Alanine (N-acetylmuramyl L-Ala amidase activity), and / or the peptidase activity comprises peptidoglycan cleavage between D-alanine and glycine (D-Ala-Gly endopeptidase activity).

[0410] Moreover, the CHAP domain of the invention (e.g., said CHAP domain having at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions) may have an enhanced peptidoglycan hydrolase activity as compared to the CHAP domain consisting of the sequence from position 72 to position 215 in SEQ ID NO: 1. Preferably said peptidoglycan hydrolase activity is a hydrolase activity against peptidoglycan of Staphylococcus aureus. In particular, said CHAP domain may have an enhanced peptidase activity as compared to the CHAP domain consisting of the sequence from position 72 to position 215 in SEQ ID NO: 1. Preferably, said enhanced peptidase activity comprises an enhanced D-Ala-Gly endopeptidase activity as compared to the CHAP domain consisting of the sequence from position 72 to position 215 in SEQ ID NO: 1. Preferably, said peptidase activity may be measured by the cleavage of a fluorigenic substrate analogue of the S.aureus stem peptide, as described herein.

[0411] Thus, any one of (i) mutation F155Y, (ii) mutation M175Q, (iii) WT residue Y117, (iv) the combination of mutation Y117D with WT residue G118, (v) the combination of mutation G118Y with WT residue Y117, (vi) the combination of mutations Y117G and G118Y, and (vii) the combination of mutations Y94F and W189E with WT residues Y117 and G118, in reference to SEQ ID NO: 1, conferred peptidase activity to L0482 variants. Thus, a CHAP domain of the invention having at least one of said mutations or WT residues or said combinations thereof, may have a peptidase activity and, preferably, an additional amidase activity. Preferably, a CHAP domain of the invention having at least one of said mutations or WT residues or said combinations thereof, may have an enhanced peptidoglycan hydrolase activity, in particular an enhanced peptidase activity (preferably an enhanced D-Ala-Gly endopeptidase activity) as compared to the CHAP domain consisting of the sequence from position 72 to position 215 in SEQ ID NO: 1.

[0412] Combinations of active site mutations with H5 / G1 mutations and / or an aglycosylation mutation

[0413] The invention further relates to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a CHAP domain that has a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1 and one or more (i.e at least one) amino acid substitutions at positions 94, 117, 118, 186 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably, wherein the amino acid residue (i.e. tyrosine) at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue (i.e. tyrosine) at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, the amino acid residue (i.e. glycine) at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue (i.e. asparagine) at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and / or the amino acid residue (i.e. tryptophan) at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid.

[0414] Furthermore, said peptidoglycan hydrolase may additionally comprise a PepM23 domain that has a sequence identity of at least 60% to the sequence from position 1 to position 143 in SEQ ID NO: 407, as described herein.

[0415] Furthermore, the present invention relates to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a CHAP domain that has a sequence identity of at least 60%, preferably at least 70%, to the sequence from position 72 to position 215 in SEQ ID NO: 1 and one or more amino acid substitutions at positions 189, 94, 117 and 118 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably, wherein the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid. the amino acid residue at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, and / or the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

[0416] Furthermore, said peptidoglycan hydrolase may additionally comprise a PepM23 domain that has a sequence identity of at least 60% to the sequence from position 1 to position 143 in SEQ ID NO: 407, as described herein.

[0417] In some embodiments, the CHAP domain of the invention, e.g., said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions), does not have the sequence shown in SEQ ID NO: 417.

[0418] Preferably, said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions) has additionally an aglycosylation mutation, as described herein. Said aglycosylation mutation is particularly beneficial for an effective killing activity of the peptidoglycan hydrolase against target bacteria (e.g. S. aureus) upon secretion of the peptidoglycan hydrolase from eukaryotic cells, preferably mammalian cells, more preferably human cells, as described herein.

[0419] Therefore, said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions) has, preferably, additionally an amino acid substitution or a deletion at position 73 in SEQ ID NO: 1 or at a position corresponding to this position. Preferably, the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with another amino acid, preferably glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, more preferably with glycine.

[0420] A peptidoglycan hydrolase comprising an inventive CHAP domain with an aglycosylation mutation (i.e. a substitution or a deletion at position 73 in SEQ ID NO: 1 or at a position corresponding to this position), as described herein, may have an enhanced killing activity against Staphylococcus aureus in comparison to the peptidoglycan hydrolase of SEQ ID NO: 1, in particular upon secretion from a eukaryotic cell, preferably a mammalian cell, more preferably a human cell.

[0421] Preferably, said CHAP domain (having at least one active site mutation and, optionally an aglycoslyation mutation) has additionally at least one H5 / G1 mutation, as described herein. In particular, said H5 / G1 mutation(s) is / are beneficial for further improving the pharmaceutical properties of the peptidoglycan hydrolase, e.g., the killing activity against target bacteria (e.g. S. aureus), the stability (e.g. thermostability) and / or the ability of being secreted from eukaryotic cells, preferably mammalian cells, more preferably human cells, as described herein.

[0422] Therefore, said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, and, optionally, an a aglycosylation mutation at position 73 in SEQ ID NO: 1 or at a position corresponding to this position) has, preferably, additionally one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 173, 175, 185, 186 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and / or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

[0423] A peptidoglycan hydrolase comprising an inventive CHAP domain with at least one H5 / G1 mutation (i.e. one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 173, 175, 185, 186 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions), as described herein, may have in comparison to the peptidoglycan hydrolase of SEQ ID NO: 1 at least one of the following:

[0424] (i) an enhanced killing activity against Staphylococcus aureus,

[0425] (ii) an enhanced thermostability, and / or

[0426] (iii) an enhanced ability of being secreted by a human cell. Furthermore, it may be preferred that the H5 mutation N186G is combined with at least one of the other four active site mutations found in context of the present invention. Again, it also preferred that any active site mutation(s) and / or any H5 / G1 mutation(s) are combined with an aglycosylation position at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, as described. This is also the case for the following embodiments.

[0427] Thus, in some embodiments, said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions) has, preferably, additionally an amino acid substitution at position 186 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0428] In some embodiments, said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions) has additionally at least one of the stem mutations (i.e. mutations that are common to G1 and H5). Therefore, in some embodiments, said CHAP domain has additionally one or more amino acid substitutions at positions 82, 85, 86 and 169 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and / or the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine.

[0429] As mentioned above and as illustrated in the appended Examples, it has been further surprisingly found in context of the present invention that the amino acid substitution R86K was contained in all 44 identified hit variants, i.e., G1-G4, H1-H10 and 11-130, and in 96.08% of all L0482 variants that were secreted from eukaryotic cells and that were determined to have a killing activity against S. aureus, as described herein and as illustrated in the appended Examples; see, e.g., Figure 8. Thus, R86K is considered herein and in context of the present invention as a preferred stem mutation and as a particularly preferred mutation in the CHAP domain of L0482 variants in general.

[0430] Thus, in preferred embodiments, said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1) further has an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein. Preferably, the amino acid (i.e. arginine) at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine.

[0431] In some embodiments, said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions and, optionally, at least one of the stem mutations) has further at least one Gl-specific mutation, i.e., one or more amino acid substitutions at positions 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, and / or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

[0432] In some embodiments, said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions and, optionally, at least one of the stem mutations) has further at least one H5-specific mutation, i.e., one or more amino acid substitutions at positions 130, 136, 155, 185 and 186, wherein the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0433] In some embodiments, said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions) further has at least one, preferably at least two or more, of the consensus mutation units, as described herein, in particular the following consensus mutation units I) to V):

[0434] I) an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine;

[0435] II) an amino acid substitution pair at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, and the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine;

[0436] III) an amino acid substitution pair at positions 130 and 136 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, and the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine;

[0437] IV) an amino acid substitution at position 169 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine; and / or V) an amino acid substitution pair at positions 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

[0438] In some embodiments, said CHAP domain has at least one, preferably at least two or all of said consensus mutation units I), II) and IV), i.e., comprising the stem mutations.

[0439] As described herein and as illustrated in the appended Examples, the H5-specific mutation F155Y enhanced the pharmaceutical properties, including the killing activity against S. aureus, the ability of being secreted from human cells, the thermostability and / or the peptidase activity, of L0482 variants in various contexts (e.g., H5 (SEQ ID NO: 11) vs. H3 (SEQ ID NO: 9), and L2DR (SEQ ID NO: 390) vs. L2 (SEQ ID NO: 388)). The mutation F155Y also further enhanced the activity against biofilms (esp. of S. aureus) when introduced into L2.

[0440] Thus, in preferred embodiments, said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, and optionally at least one stem mutation) further has an amino acid substitution at position 155 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

[0441] A peptidoglycan hydrolase comprising an inventive CHAP domain with an F155Y mutation in reference to SEQ ID NO: 1, as described herein, may have in comparison to the peptidoglycan hydrolase of SEQ ID NO: 1 at least one of the following:

[0442] (i) an enhanced killing activity against Staphylococcus aureus (e.g., against S. aureus biofilms),

[0443] (ii) an enhanced thermostability,

[0444] (iii) an enhanced ability of being secreted by a human cell, and / or

[0445] (iv) an enhanced peptidase activity.

[0446] Furthermore, as described herein as illustrated in the appended non-limiting Examples, it has been surprisingly found that the H5-specific mutation N185Y further enhances peptidase activity, e.g., in an endolysin of the Gl- lineage, i.e., L2DR.

[0447] Thus, in some embodiments, said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, and optionally at least one stem mutation and / or at least one Gl-specific mutation) further has an amino acid substitution at position 185 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine. Since N185Y and N186G usually occurred together in the hits variants upon directed evolution (and form a consensus mutation unit), and L2DR already contained the N186G due to active site engineering, said CHAP domain, preferably, further has an amino acid substitution at position 186 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine. Additionally, as also described herein as illustrated in the appended non-limiting Examples, it has been surprisingly found that the Gl-specific mutation M175Q further enhances peptidase activity, e.g., in an endolysin of the H5- lineage, i.e., L14.

[0448] Thus, in some embodiments, said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, and optionally at least one stem mutation and / or at least one H5-specific mutation) further has an amino acid substitution at position 175 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

[0449] A peptidoglycan hydrolase comprising an inventive CHAP domain with an F155Y mutation, an N185Y mutation and / or an M175Q mutation in reference to SEQ ID NO: 1, as described herein, may have an enhanced peptidase activity in comparison to the peptidoglycan hydrolase of SEQ ID NO: 1.

[0450] Furthermore, said CHAP domain (having, e.g., at least one active site mutation at positions 94, 117, 118 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions) may have a sequence identity of at least 95% to the sequence from position 72 to position 215 in SEQ ID NO: 3, and / or a sequence identity of at least 94% to the sequence from position 72 to position 215 in SEQ ID NO: 11.

[0451] Specific CHAP domains having at least one active site mutation and peptidoglycan hydrolases comprising such a CHAP domain

[0452] Gl-14

[0453] In some particular embodiments, the CHAP domain of the invention has, in reference to SEQ ID NO: 1, (i) the aglycosylation mutation N73G, (ii) one, two, three or four stem mutation(s) selected from the group consisting of T82S, N85G, R86K and D169N, (iii) one, two or three Gl-specific mutation(s) selected from the group consisting of K173N, M175Q, A192Q, and / or (iv) one, two, three, four or five active site mutations selected from the group consisting of Y94F, Y117D, G118Y, N186G and W189E. Preferably, said CHAP domain has all said mutations, i.e., N73G, T82S, N85G, R86K, D169N, K173N, M175Q, A192Q, Y94F, Y117D, G118Y, N186G and W189E. Furthermore, the CHAP domain may have a sequence identity of at least 97%, at least 98% or at least 99% to the sequence from position 72 to position 215 in SEQ ID NO: 386. More preferably, said CHAP domain has the sequence shown in SEQ ID NO: 394 (i.e., the sequence from position 72 to position 215 in SEQ ID NO: 386). The CHAP domain according to these embodiments may have an enhanced ability of being secreted from a human cell in comparison to the CHAP domain defined by the sequence from position 72 to position 215 in SEQ ID NO: 1. A peptidoglycan hydrolase of the invention having a CHAP domain according to these embodiments, may further have at least one cell wall binding domain (preferably a LYSM domain), and optionally a peptide linker (preferably a LYSM-CHAP linker), as described herein. Furthermore, said peptidoglycan hydrolase may have a sequence identity of at least 60% to the sequence of SEQ ID NO: 1. The peptidoglycan hydrolase according to these embodiments may have in comparison to the peptidoglycan hydrolase of SEQ ID NO: 1 (i) an enhanced killing activity against Staphylococcus aureus (e.g. against a biofilm of S. aureus), (ii) an enhanced thermostability, and / or (iii) an enhanced ability of being secreted by a human cell. In some embodiments, the peptidoglycan hydrolase comprises an amino acid sequence that has a sequence identity of at least 98% or at least 99% to the sequence of SEQ ID NO: 386. In some specific embodiments, the peptidoglycan hydrolase comprises the sequence shown in SEQ ID NO: 386.

[0454] Gl-14 (SEQ ID NO: 386) has the following amino acid sequence: REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKSLEGKG

[0455] WDFDGSFGWQCFDLVNVYWNHLYGHGLKGDYAKDIPYANNFNSEAKIYHNTPTFKAEPGDLWFSGRFGGGYGHTAIVLNGN

[0456] YDGNLQKFQSLDQNWNGGGERKQEVAHKWHNYENDMIFIRPFKKA

[0457] H5-14

[0458] In some particular embodiments, the CHAP domain of the invention has, in reference to SEQ ID NO: 1, (i) the aglycosylation mutation N73G, (ii) one, two, three or four stem mutation(s) selected from the group consisting of T82S, N85G, R86K and D169N, (iii) one, two, three, four or five H5-specific mutation(s) selected from the group consisting of S130N, H136K, F155Y, N185Y and N186G, and / or (iv) one, two, three or four active site mutations selected from the group consisting of Y94F, Y117D, G118Y and W189E. Preferably, said CHAP domain has all said mutations, i.e., N73G, T82S, N85G, R86K, D169N, S130N, H136K, F155Y, N185Y, N186G, Y94F, Y117D, G118Y and W189E. Furthermore, the CHAP domain may have a sequence identity of at least 98% or at least 99% to the sequence from position 72 to position 215 in SEQ ID NO: 387. More preferably, said CHAP domain has the sequence shown in SEQ ID NO: 396 (i.e., the sequence from position 72 to position 215 in SEQ ID NO: 387). The CHAP domain according to these embodiments may have an enhanced ability of being secreted from a human cell in comparison to the CHAP domain defined by the sequence from position 72 to position 215 in SEQ ID NO: 1. A peptidoglycan hydrolase of the invention having a CHAP domain according to these embodiments, may further have at least one cell wall binding domain (preferably a LYSM domain), and optionally a peptide linker (preferably a LYSM-CHAP linker), as described herein. Furthermore, said peptidoglycan hydrolase may have a sequence identity of at least 60% to the sequence of SEQ ID NO: 1. The peptidoglycan hydrolase according to these embodiments may have in comparison to the peptidoglycan hydrolase of SEQ ID NO: 1 (i) an enhanced killing activity against Staphylococcus aureus (e.g. against a biofilm of S. aureus), (ii) an enhanced thermostability, and / or (iii) an enhanced ability of being secreted by a human cell. In some embodiments, the peptidoglycan hydrolase comprises an amino acid sequence that has a sequence identity of at least 99% to the sequence of SEQ ID NO: 387. In some specific embodiments, the peptidoglycan hydrolase comprises the sequence shown in SEQ ID NO: 387.

[0459] H5-14 (SEQ ID NO: 387) has the following amino acid sequence:

[0460] REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKSLEGKG WDFDGSFGWQCFDLVNVYWNHLYGHGLKGDYAKDIPYANNFNNEAKIYKNTPTFKAEPGDLWFSGRYGGGYGHTAIVLNGN YDGKLMKFQSLDQNWYGGGERKAEVAHKWHNYENDMIFIRPFKKA

[0461] L2

[0462] In some particular embodiments, the CHAP domain of the invention has, in reference to SEQ ID NO: 1, (i) the aglycosylation mutation N73G, (ii) one, two, three or four stem mutation(s) selected from the group consisting of T82S, N85G, R86K and D169N, (iii) one, two or three Gl-specific mutation(s) selected from the group consisting of K173N, M175Q, A192Q, and / or (iv) one, two, three or four active site mutations selected from the group consisting of Y94F, G118Y, N186G and W189E. Preferably, said CHAP domain has all said mutations, i.e., N73G, T82S, N85G, R86K, D169N, K173N, M175Q, A192Q, Y94F, G118Y, N186G and W189E. Preferably, said CHAP domain has the WT residue Y117. Furthermore, the CHAP domain may have a sequence identity of at least 98% or at least 99% to the sequence from position 72 to position 215 in SEQ ID NO: 388. More preferably, said CHAP domain has the sequence shown in SEQ ID NO: 397 (i.e., the sequence from position 72 to position 215 in SEQ ID NO: 388). The CHAP domain according to these embodiments may have an enhanced ability of being secreted from a human cell and / or an enhanced peptidase activity in comparison to the CHAP domain defined by the sequence from position 72 to position 215 in SEQ ID NO: 1. A peptidoglycan hydrolase of the invention having a CHAP domain according to these embodiments, may further have at least one cell wall binding domain (preferably a LYSM domain), and optionally a peptide linker (preferably a LYSM-CHAP linker), as described herein. Furthermore, said peptidoglycan hydrolase may have a sequence identity of at least 60% to the sequence of SEQ ID NO: 1. The peptidoglycan hydrolase according to these embodiments may have in comparison to the peptidoglycan hydrolase of SEQ ID NO: 1 (i) an enhanced killing activity against Staphylococcus aureus (e.g. against a biofilm of S. aureus), (ii) an enhanced thermostability, (iii) an enhanced ability of being secreted by a human cell, and / or (iv) an enhanced peptidase activity. In some embodiments, the peptidoglycan hydrolase comprises an amino acid sequence that has a sequence identity of at least 99% to the sequence of SEQ ID NO: 388. In some specific embodiments, the peptidoglycan hydrolase comprises the sequence shown in SEQ ID NO: 388.

[0463] L2 (SEQ ID NO: 388) has the following amino acid sequence:

[0464] REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKSLEGKG WDFDGSFGWQCFDLVNVYWNHLYGHGLKGYYAKDIPYANNFNSEAKIYHNTPTFKAEPGDLWFSGRFGGGYGHTAIVLNGN YDGNLQKFQSLDQNWNGGGERKQEVAHKWHNYENDMIFIRPFKKA

[0465] L14

[0466] In some particular embodiments, the CHAP domain of the invention has, in reference to SEQ ID NO: 1, (i) the aglycosylation mutation N73G, (ii) one, two, three or four stem mutation(s) selected from the group consisting of T82S, N85G, R86K and D169N, (iii) one, two, three, four or five H5-specific mutation(s) selected from the group consisting of S130N, H136K, F155Y, N185Y and N186G, and / or (iv) one, two or three active site mutations selected from the group consisting of Y94F, G118Y and W189E. Preferably, said CHAP domain has all said mutations, i.e., N73G, T82S, N85G, R86K, D169N, S130N, H136K, F155Y, N185Y, N186G, Y94F, G118Y and W189E. Preferably, said CHAP domain has the WT residue Y117. Furthermore, the CHAP domain may have a sequence identity of at least 98% or at least 99% to the sequence from position 72 to position 215 in SEQ ID NO: 389. More preferably, said CHAP domain has the sequence shown in SEQ ID NO: 398 (i.e., the sequence from position 72 to position 215 in SEQ ID NO: 389). The CHAP domain according to these embodiments may have an enhanced ability of being secreted from a human cell and / or an enhanced peptidase activity in comparison to the CHAP domain defined by the sequence from position 72 to position 215 in SEQ ID NO: 1. A peptidoglycan hydrolase of the invention having a CHAP domain according to these embodiments, may further have at least one cell wall binding domain (preferably a LYSM domain), and optionally a peptide linker (preferably a LYSM-CHAP linker), as described herein. Furthermore, said peptidoglycan hydrolase may have a sequence identity of at least 60% to the sequence of SEQ ID NO: 1. The peptidoglycan hydrolase according to these embodiments may have in comparison to the peptidoglycan hydrolase of SEQ ID NO: 1 (i) an enhanced killing activity against Staphylococcus aureus (e.g. against a biofilm of S. aureus), (ii) an enhanced thermostability, and / or (iii) an enhanced ability of being secreted by a human cell, and / or (iv) an enhanced peptidase activity. In some embodiments, the peptidoglycan hydrolase comprises an amino acid sequence that has a sequence identity of at least 99% to the sequence of SEQ ID NO: 389. In some specific embodiments, the peptidoglycan hydrolase comprises the sequence shown in SEQ ID NO: 389. L14 (SEQ ID NO: 389) has the following amino acid sequence:

[0467] REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKSLEGKG WDFDGSFGWQCFDLVNVYWNHLYGHGLKGYYAKDIPYANNFNNEAKIYKNTPTFKAEPGDLWFSGRYGGGYGHTAIVLNGN YDGKLMKFQSLDQNWYGGGERKAEVAHKWHNYENDMIFIRPFKKA

[0468] L2DR

[0469] In some of the most preferred embodiments, the CHAP domain of the invention has, in reference to SEQ ID NO: 1, (i) the aglycosylation mutation N73G, (ii) one, two, three or four stem mutation(s) selected from the group consisting of T82S, N85G, R86K and D169N, (iii) one, two or three Gl-specific mutation(s) selected from the group consisting of K173N, M175Q, A192Q, (iv) the H5-specific mutation F155Y, and / or (v) one, two, three or four active site mutations selected from the group consisting of Y94F, G118Y, N186G and W189E. Furthermore, said CHAP domain may have the additional H5-specific mutation N185Y. Preferably, said CHAP domain has all said mutations, i.e., N73G, T82S, N85G, R86K, D169N, K173N, M175Q, A192Q, F155Y, Y94F, G118Y, N186G and W189E, and optionally further N185Y. Preferably, said CHAP domain has the WT residue Y117. Furthermore, the CHAP domain may have a sequence identity of at least 97%, at least 98% or at least 99% to the sequence from position 72 to position 215 in SEQ ID NO: 390. More preferably, said CHAP domain has the sequence shown in SEQ ID NO: 399 (i.e., the sequence from position 72 to position 215 in SEQ ID NO: 390). The CHAP domain according to these embodiments may have an enhanced ability of being secreted from a human cell and / or an enhanced peptidase activity in comparison to the CHAP domain defined by the sequence from position 72 to position 215 in SEQ ID NO: 1. A peptidoglycan hydrolase of the invention having a CHAP domain according to these embodiments, may further have at least one cell wall binding domain (preferably a LYSM domain), and optionally a peptide linker (preferably a LYSM-CHAP linker), as described herein. Furthermore, said peptidoglycan hydrolase may have a sequence identity of at least 60% to the sequence of SEQ ID NO: 1. The peptidoglycan hydrolase according to these embodiments may have in comparison to the peptidoglycan hydrolase of SEQ ID NO: 1 (i) an enhanced killing activity against Staphylococcus aureus (e.g. against a biofilm of S. aureus), (ii) an enhanced thermostability, (iii) an enhanced ability of being secreted by a human cell, and / or (iv) an enhanced peptidase activity. In some embodiments, the peptidoglycan hydrolase comprises an amino acid sequence that has a sequence identity of at least 98% or at least 99% to the sequence of SEQ ID NO: 390. In some specific embodiments, the peptidoglycan hydrolase comprises the sequence shown in SEQ ID NO: 390.

[0470] L2DR (SEQ ID NO: 390) has the following amino acid sequence:

[0471] REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKSLEGKG WDFDGSFGWQCFDLVNVYWNHLYGHGLKGYYAKDIPYANNFNSEAKIYHNTPTFKAEPGDLWFSGRYGGGYGHTAIVLNGN YDGNLQKFQSLDQNWNGGGERKQEVAHKWHNYENDMIFIRPFKKA

[0472] L14DR (SEO ID NO: 391)

[0473] In some further of the most preferred embodiments, the CHAP domain of the invention has, in reference to SEQ ID NO: 1, (i) the aglycosylation mutation N73G, (ii) one, two, three or four stem mutation(s) selected from the group consisting of T82S, N85G, R86K and D169N, (iii) one, two, three, four or five H5-specific mutation(s) selected from the group consisting of S130N, H136K, F155Y, N185Y and N186G, (iv) the Gl-specific mutation M175Q, and / or (v) one, two or three active site mutations selected from the group consisting of Y94F, G118Y and W189E. Preferably, said CHAP domain has all said mutations, i.e., N73G, T82S, N85G, R86K, D169N, S130N, H136K, F155Y, N185Y, N186G, M175Q, Y94F, G118Y and W189E. Preferably, said CHAP domain has the WT residue Y117. Furthermore, the CHAP domain may have a sequence identity of at least 98% or at least 99% to the sequence from position 72 to position 215 in SEQ ID NO: 391. More preferably, said CHAP domain has the sequence shown in SEQ ID NO: 400 (i.e., the sequence from position 72 to position 215 in SEQ ID NO: 391). The CHAP domain according to these embodiments may have an enhanced ability of being secreted from a human cell and / or an enhanced peptidase activity in comparison to the CHAP domain defined by the sequence from position 72 to position 215 in SEQ ID NO: 1. A peptidoglycan hydrolase of the invention having a CHAP domain according to these embodiments, may further have at least one cell wall binding domain (preferably a LYSM domain), and optionally a peptide linker (preferably a LYSM-CHAP linker), as described herein. Furthermore, said peptidoglycan hydrolase may have a sequence identity of at least 60% to the sequence of SEQ ID NO: 1. The peptidoglycan hydrolase according to these embodiments may have in comparison to the peptidoglycan hydrolase of SEQ ID NO: 1 (i) an enhanced killing activity against Staphylococcus aureus (e.g. against a biofilm of S. aureus), (ii) an enhanced thermostability, and / or (iii) an enhanced ability of being secreted by a human cell, and / or (iv) an enhanced peptidase activity. In some embodiments, the peptidoglycan hydrolase comprises an amino acid sequence that has a sequence identity of at least 99% to the sequence of SEQ ID NO: 391. In some specific embodiments, the peptidoglycan hydrolase comprises the sequence shown in SEQ ID NO: 391.

[0474] L14DR (SEQ ID NO: 391) has the following amino acid sequence:

[0475] REAPKTQIYTVKKGDTLSAIALKYKTTVSNIQNTNNIANPNLIFIGQKLKVPMTPLVEPKPKTVSSNKKSNSGSSTLNYLKSLEGKG WDFDGSFGWQCFDLVNVYWNHLYGHGLKGYYAKDIPYANNFNNEAKIYKNTPTFKAEPGDLWFSGRYGGGYGHTAIVLNGN YDGKLQKFQSLDQNWYGGGERKAEVAHKWHNYENDMIFIRPFKKA

[0476] L2TR

[0477] In some particular embodiments, the CHAP domain of the invention has, in reference to SEQ ID NO: 1, (i) the aglycosylation mutation N73G, (ii) one, two, three or four stem mutation(s) selected from the group consisting of T82S, N85G, R86K and D169N, (iii) one, two or three Gl-specific mutation(s) selected from the group consisting of K173N, M175Q, A192Q, (iv) one or two H5-specific mutations selected from the group consisting of F155Y and N185Y, and / or (v) one, two, three or four active site mutations selected from the group consisting of Y94F, G118Y, N186G and W189E. Preferably, said CHAP domain has all said mutations, i.e., N73G, T82S, N85G, R86K, D169N, K173N, M175Q, A192Q, F155Y, N185Y, Y94F, G118Y, N186G and W189E. Preferably, said CHAP domain has the WT residue Y117. Furthermore, the CHAP domain may have a sequence identity of at least 96%, at least 97%, at least 98% or at least 99% to the sequence from position 72 to position 215 in SEQ ID NO: 401. More preferably, said CHAP domain has the sequence shown in SEQ ID NO: 401. The CHAP domain according to these embodiments may have an enhanced ability of being secreted from a human cell and / or an enhanced peptidase activity in comparison to the CHAP domain defined by the sequence from position 72 to position 215 in SEQ ID NO: 1. A peptidoglycan hydrolase of the invention having a CHAP domain according to these embodiments, may further have at least one cell wall binding domain (preferably a LYSM domain), and optionally a peptide linker (preferably a LYSM-CHAP linker), as described herein. Furthermore, said peptidoglycan hydrolase may have a sequence identity of at least 60% to the sequence of SEQ ID NO: 1. The peptidoglycan hydrolase according to these embodiments may have in comparison to the peptidoglycan hydrolase of SEQ ID NO: 1 (i) an enhanced killing activity against Staphylococcus aureus (e.g. against a biofilm of S. aureus), (ii) an enhanced thermostability, (iii) an enhanced ability of being secreted by a human cell, and / or (iv) an enhanced peptidase activity.

[0478] The CHAP domain of L2TR (SEQ ID NO: 401) has the following sequence:

[0479] SGSSTLNYLKSLEGKGWDFDGSFGWQCFDLVNVYWNHLYGHGLKGYYAKDIPYANNFNSEAKIYHNTPTFKAEPGDLWFSGR YGGGYGHTAIVLNGNYDGNLQKFQSLDQNWYGGGERKQEVAHKWHNYENDMIFIRPFKKA

[0480] Furthermore, it has been surprisingly found that Gl-14 has an enhanced peptidoglycan hydrolase activity, in particular an enhanced peptidoglycan hydrolase kinetics, as compared to lysostaphin, e.g., the peptidoglycan hydrolase of SEQ ID NO: 408; see, Figure 14B.

[0481] Thus, the peptidoglycan hydrolase of the invention (having an inventive CHAP domain), e.g., according to the above embodiments relating to Gl-14, H5-14, L2, L14, L2DR, L14DR or L2TR, in particular to Gl-14 or derivatives thereof, may have in comparison to the peptidoglycan hydrolase of SEQ ID NO: 407,408 or 444 (preferably SEQ ID NO: 408), an enhanced peptidoglycan hydrolase activity, in particular enhanced peptidoglycan hydrolase kinetics.

[0482] Conserved positions and conserved segment

[0483] In some embodiments, a segment of the inventive CHAP domain has a sequency identity of at least 80%, preferably at least 90%, to the sequence from position 87 to position 128 in SEQ ID NO: 1. In particular, said segment is contained in the inventive CHAP domain at positions corresponding to positions 87 to 128 in SEQ ID NO: 1, i.e., it is a corresponding segment. As shown, e.g., in Example 6, said segment may be considered herein and in context of the present invention as a particularly conserved CHAP segment.

[0484] Furthermore, in some of these embodiments or in other embodiments, the CHAP domain of the invention has at most six, five, four, three or two, more preferably at most one, 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 at positions corresponding to these positions. As shown, e.g., in Example 6, said positions may be considered herein and in context of the present invention as conserved positions within the CHAP domain.

[0485] Furthermore, in context of the present invention, it should be avoided to delete the catalytic cysteine at position 98 in SEQ ID NO: 1 or at a position corresponding to this position or to substitute said cysteine with another amino acid residue.

[0486] It is thus preferred herein and in context of the present invention that the CHAP domain of the invention has a cysteine at position 98 in SEQ ID NO: 1 or at a position corresponding to this position.

[0487] In context of the present invention, the CHAP domain of the invention has, preferably, one or more amino acid substitutions at other positions than at the conserved positions described herein. Preferably said CHAP domain has an aglycosylation mutation, at least one H5 / G1 mutation and / or at least one active site mutation, as described herein.

[0488] Moreover, a conserved CHAP segment has in context of the present invention, preferably, less mutations than other segments of the CHAP domain of the invention. Thus, a corresponding segment of the CHAP domain of the invention has, preferably, a higher sequency identity (e.g. about 90%) to the sequence from position 87 to position 128 in SEQ ID NO: 1, as compared to the sequence identity of the CHAP domain of the invention to the sequence from position 72 to position 215 in SEQ ID NO: 1 (which may be in this example, e.g., about 60% to 80%).

[0489] Hence, the present invention further relates to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises a cysteine, histidine-dependent amidohydrolase / peptidase (CHAP) domain that has (i) a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; and that has (ii) one or more amino acid substitutions as compared to the sequence from position 72 to position 215 in SEQ ID NO: 1, and wherein

[0490] (a) a corresponding segment of said CHAP domain has a sequency identity of at least 80%, preferably at least 90%, to the sequence from position 87 to position 128 in SEQ ID NO: 1, and / or

[0491] (b) said CHAP domain has at most six, five, four, three or two, preferably at most one, 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 at positions corresponding to these positions.

[0492] Preferably, said CHAP domain comprises (i) at least one aglycosylation mutation at position 73 in SEQ ID NO: 1 or at a position corresponding to this position as described herein, preferably the substitution N73G, (ii) at least one amino acid substitution described herein in context of the L0482ag variants obtained upon directed evolution, preferably at least one G1 / H5 mutation, as described herein, i.e., at least amino acid substitution selected from the group consisting of T82S, N85G, R86K, S130N, H136K / R, F155Y, D169N, K173N, M175Q, N185Y, N186G and A192Q in reference to SEQ ID NO: 1, and / or (iii) at least one active site mutation, in particular, at least one amino acid substitution selected from the group consisting of Y94F, Y117D, G118Y, and W189E.

[0493] Additional cell wall bindina domain(s)

[0494] Preferably, herein and in context of the present invention, the peptidoglycan hydrolase of the invention comprises, in addition to the CHAP domain of the invention, and the optional PepM23 domain described herein in context of the invention, at least one cell wall binding domain.

[0495] As mentioned above, endolysins often have at least one cell wall-binding domain (CBD) which recognize and bind to certain epitopes in the cell wall of the host bacterium for proper fixation of the catalytic effect of an enzymatically active domain (EAD). Normally, CBDs are enzymatically inactive by themselves. Thus, a domain which has catalytic activity and is able to recognize and bind to certain epitopes in the cell wall of a bacterium is rather considered herein as a an enzymatically active domain (EAD). Preferably, herein and in context of the present invention, the cell wall binding domain is a peptidoglycan binding domain which binds, in particular, to the peptidoglycan structure of a target bacterium, preferably S. aureus.

[0496] Suitable cell wall binding domains to be used in context of the present invention, include, inter alia: a LYSM domain, a SH3 domain and a choline binding domain.

[0497] Preferably, in context of the present invention, the at least one cell wall binding domain comprises a LYSM domain and / or a SH3 domain. In some preferred embodiments, the peptidoglycan hydrolase comprises a LYSM domain, as described herein. In further embodiments, e.g., in context of CHAP-PepM23 fusions, the peptidoglycan hydrolase comprises an SH3 domain, more preferably an SH3 domain from lysostaphin, as described herein. Preferably, herein and in context of the present invention, the cell wall binding domain is derived from an endolysin, in particular a cell wall binding domain thereof. Preferably, said endolysin has a killing activity against a Staphylococcus species or strain, more preferably against Staphylococcus aureus.

[0498] In context of the present invention, the cell wall binding domain is, preferably, derived from an endolysin comprising a LYSM domain or a SH3 domain.

[0499] In some preferred embodiments, e.g., in context of peptidoglycan hydrolases having no PepM23 domain, the cell wall binding domain is derived from an endolysin comprising a LYSM domain. Preferably, said LYSM domain is derived from an endolysin comprising a LYSM domain and a CHAP domain, wherein the LYSM domain is, preferably, N-terminally of the CHAP domain. In other words, the LYSM domain of the invention is, in some preferred embodiments, derived from an endolysin having a LYSM-CHAP architecture, as described herein, e.g. L0482 (SEQ ID NO: 1) or L0499 (SEQ ID NO: 47).

[0500] Hence, in some preferred embodiments, the peptidoglycan of the invention comprises, in addition, to the CHAP domain of the invention, a LYSM domain, as described herein.

[0501] In some embodiments, the cell wall binding domain, e.g. the LYSM domain, is N-terminally of the CHAP domain.

[0502] Preferably herein, the LYSM domain according to the invention is defined by the sequence from position 1 to position 51 in SEQ ID NO: 1 or it has a sequence identity of at least 60% to the sequence from position 1 to position 51 in SEQ ID NO: 1. In other words, the LYSM domain according to the invention is, preferably, defined by the sequence of SEQ ID NO: 302 or has a sequence identity of at least 60% to the sequence of SEQ ID NO: 302.

[0503] In some embodiments, the LYSM domain is defined by the sequence from position 1 to position 47 in SEQ ID NO: 47 or it has a sequence identity of at least 60% to the sequence from position 1 to position 47 in SEQ ID NO: 47.

[0504] In some embodiments, the SH3 domain is defined by the sequence of SEQ ID NO: 376 or has a sequence identity of at least 60% to the sequence of SEQ ID NO: 376.

[0505] In some embodiments, the SH3 domain is defined by the sequence of SEQ ID NO: 377 or has a sequence identity of at least 60% to the sequence of SEQ ID NO: 377.

[0506] In some embodiments, e.g., in context of CHAP-PepM23 fusions, the cell wall binding domain is derived from an endolysin comprising an SH3 domain. Preferably, said SH3 domain is derived from an endolysin comprising a PepM23 domain and an SH3 domain, wherein the SH3 domain is, preferably, C-terminally of the PepM23 domain. In other words, the SH3 domain of the invention is, in some preferred embodiments, derived from an endolysin having a PepM23-SH3 architecture, as described herein, preferably from lysostaphin (SEQ ID NO: 407) or a variant thereof (e.g., SEQ ID NO: 444), as described herein.

[0507] In some embodiments, e.g., in context of CHAP-PepM23 fusions, the SH3 domain has a sequence identity of at least 60% to the sequence from position 154 to position 246 in SEQ ID NO: 407.

[0508] Furthermore, the cell wall binding domain of the invention, e.g. the LYSM domain or SH3 domain according to the invention, has, preferably, the ability to bind to the cell wall of a Staphylococcus species or strain, more preferably to Staphylococcus aureus, as described herein. Additional linker

[0509] Preferably, herein and in context of the present invention, in particular in context of peptidoglycan hydrolase comprising a CHAP domain and a CBD, the peptidoglycan hydrolase comprises a peptide linker between the CHAP domain and the cell wall binding domain.

[0510] The peptide linker is, generally, not limited to any specific linkers and any linkers used in the art for connecting different domains or parts of proteins such as fusion proteins may be used herein and in context of the present invention. However, preferred peptide linkers are described herein in specific contexts.

[0511] As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence which connects two or more domains (e.g., the CHAP domain or the invention and a cell wall binding domain, and / or the CHAP of the invention and an extended pharmacokinetic (PK) peptide) in a linear amino acid sequence of a polypeptide chain. In context of a CBD-CHAP linker, e.g., LYSM-CHAP linker, linker is, preferably, a flexible linker. Exemplary linkers include glycine-serine-polypeptide linkers, glycine-proline-polypeptide linkers, and proline-alanine polypeptide linkers. An exemplary linker with a furin cleavage site (which may be also used in context of the present invention) is shown in SEQ ID NO: 370.

[0512] In some embodiments, the linker is a glycine-serine linker, i.e., a peptide that predominantly, essentially or exclusively consists of glycine and serine residues. Herein and in context of the present invention, a glycine-serine linker may comprise, for example, one or multiple copies (e.g. 2 to 5 copies) of the sequence shown in SEQ ID NO:

[0513] 297 (i.e. GGGGS). Preferably, said copies are directly adjacent to each other, for example, as shown in SEQ ID NO:

[0514] 298 or 299 (i.e. GGGGSGGGGS (GS2: 2x GGGGS), or GGGGSGGGGSGGGGSGGGGS (GS4: 4x GGGGS), respectively. Moreover, GS3 (3x GGGGS) or G5 (5x GGGGS) may be equally used. Further suitable glycine-serine linkers are shown in SEQ ID NO: 363 to 369. Another suitable glycine-serine linker has the sequence "GGS".

[0515] In certain embodiments, the peptide linker is derived from an endolysin, in particular, a linker sequence thereof. Preferably, said endolysin has a killing activity against a Staphylococcus species or strain, preferably Staphylococcus aureus.

[0516] In some preferred embodiments, e.g., in context of a LYSM-CHAP linker, the peptide linker is a L0482-derived linker, i.e. a linker which has a sequence identity of at least 60% to the sequence from position 52 to position 71 in SEQ ID NO: 1.

[0517] Preferably, said peptide linker, in particular said linker which has a sequence identity of at least 60% to the sequence from position 52 to position 71 in SEQ ID NO: 1, has an aglycosylation mutation, e.g., a deletion (preferably and amino acid substitution) at position 68 in SEQ ID NO: 1 or at a position corresponding to this position, as described herein. Preferably, the L0482-derived peptide linker has an amino acid substitution at position 68 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid residue at said position is substituted with lysine, methionine, arginine or alanine, preferably lysine.

[0518] Furthermore, a peptidoglycan hydrolase comprising a CHAP domain, and optionally a LYSM domain and / or an L0482-derived peptide linker, as described herein, has, in some embodiments, a sequence identity of at least 60% to the sequence of SEQ ID NO: 1, as described herein. In some embodiments, e.g., in context of lysostaphin or CHAP-PepM23 fusions, the PepM23-SH3 linker has a sequence identity of at least 60% to the sequence from position 144 to position 153 in SEQ ID NO: 407.

[0519] CHAP-PepM23 fusions

[0520] As mentioned above and as illustrated in the appended non-limiting Examples, the inventors surprisingly found a peptidoglycan hydrolase / endolysin having two enzymatically active domains, i.e., a CHAP domain from an L0482 variant (i.e., Gl-14) and a PepM23 domain from lysostaphin, which showed an enhanced peptidoglycan hydrolase activity and an enhanced killing activity against S. aureus in comparison to the parental lysostaphin; see, e.g., Example 12 and Figure 14. Furthermore, said chimeric endolysin / CHAP-lysostaphin fusion protein (L1079, SEQ ID NO: 409) showed further an enhanced killing activity against S. aureus in comparison to Gl-14, or in comparison to CHAP-PepM23-SH3 fusion proteins (e.g., CHAP-lysostaphin fusions) described in the prior art, i.e., WO 2021 / 213898.

[0521] Therefore, the CHAP domain of the invention may be combined with / fused to / connected with a PepM23 domain that, preferably, has a sequence identity of at least 60% to the sequence from position 1 to position 143 in SEQ ID NO: 407.

[0522] Accordingly, the present invention also relates to a peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises

[0523] (i) a CHAP domain that has a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; and

[0524] (ii) a PepM23 domain that has a sequence identity of at least 60% to the sequence from position 1 to position 143 in SEQ ID NO: 407.

[0525] Preferably, said peptidoglycan hydrolase further comprises at least one cell wall binding domain, as described herein. Preferably, said cell wall binding domain(s) is / are derived from an endolysin. More preferably said cell wall binding domain is a LYSM domain and / or an SH3 domain, most preferably an SH3 domain, in particular, an SH3 domain that has a sequence identity of at least 60% to the sequence from position 154 to position 246 in SEQ ID NO: 407.

[0526] A peptidoglycan hydrolase comprising a CHAP domain of the invention and a PepM23 domain from lysostaphin or a lysostaphin variant as described herein, is also referred to herein as "CHAP-PepM23 fusion" or "CHAP-PepM23 chimera".

[0527] In some embodiments, the peptidoglycan hydrolase comprises a LYSM domain that has a sequence identity of at least 60% to the sequence from position 1 to position 51 in SEQ ID NO: 1, as described herein.

[0528] Preferably herein, also in context of CHAP-PepM23 fusions, the cell wall binding domain(s) (e.g. said SH3 domain and / or said LYSM domain), bind(s) to at least one peptidoglycan structure of a Staphylococcus species or strain, preferably of Staphylococcus aureus.

[0529] In some preferred embodiments, said peptidoglycan hydrolase (comprising a CHAP domain and a PepM23 domain), further comprises an SH3 domain that has a sequence identity of at least 60% to the sequence from position 154 to position 246 in SEQ ID NO: 407 located C-terminally of the PepM23 domain. Preferably, in these embodiments, the CHAP domain is located C-terminally of the SH3-domain, N-terminally of the PepM23 domain, or between the PepM23 domain and the SH3 domain. More preferably the CHAP domain is located N-terminally of the PepM23 domain in these embodiments.

[0530] Thus, in some preferred embodiments, in particular in context of CHAP-PepM23 fusions comprising an SH3 domain, the domains are oriented as follows (from N- to C-terminus): PepM23-SH3-CHAP; CHAP-PepM23-SH3; or PepM23- CHAP-SH3. Preferably, in these embodiments, the domains are oriented as follows (from N- to C-terminus): CHAP- PepM23-SH3.

[0531] Furthermore, the peptidoglycan hydrolase may comprise a peptide linker connecting the CHAP domain and the PepM23 domain. Preferably, said peptide linker consists of one to four amino acids. More preferably, said peptide linker connecting the CHAP domain and the PepM23 domain consists of two glycines. Such a "GG" linker provided particularly good results in terms of peptidoglycan hydrolase activity and / or killing of target bacteria (e.g. S. aureus) in context of the CHAP-PepM23 fusions. Preferably, the CHAP domain is located N-terminally of the PepM23 domain.

[0532] Thus, in some particularly preferred embodiments, the peptidoglycan hydrolase comprises a CHAP domain according to the invention N-terminally of the PepM23 domain described herein, wherein the CHAP domain and PepM23 domain are connected via a peptide linker consisting of two glycines. Furthermore, said peptidoglycan hydrolase may comprise a cell wall binding domain as described herein, preferably an SH3 domain that has a sequence identity of at l...

Claims

1. Claims1. A peptidoglycan hydrolase having bactericidal activity, wherein the peptidoglycan hydrolase comprises(i) a CHAP domain that has a sequence identity of at least 60% to the sequence from position 72 to position 215 in SEQ ID NO: 1; and(ii) a PepM23 domain that has a sequence identity of at least 60% to the sequence from position 1 to position 143 in SEQ ID NO: 407.

2. The peptidoglycan hydrolase of claim 1 which comprises 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, most preferably an SH3 domain.

3. The peptidoglycan hydrolase of claim 1 or 2 which comprises an SH3 domain that has a sequence identity of at least 60% to the sequence from position 154 to position 246 in SEQ ID NO: 407.

4. The peptidoglycan hydrolase of any one of claims 1 to 3 which comprises a LYSM domain that has a sequence identity of at least 60% to the sequence from position 1 to position 51 in SEQ ID NO: 1.

5. The peptidoglycan hydrolase of any one of claims 2 to 4, wherein the cell wall binding domain(s), preferably said SH3 domain and / or said LYSM domain, bind(s) to at least one peptidoglycan structure of a Staphylococcus species or strain, preferably Staphylococcus aureus.

6. The peptidoglycan hydrolase of any one of claims 1 to 5, which comprises an SH3 domain that has a sequence identity of at least 60% to the sequence from position 154 to position 246 in SEQ ID NO: 407 located C-terminally of the PepM23 domain.

7. The peptidoglycan hydrolase of claim 6, wherein the CHAP domain is located C-terminally of the SH3- domain, N-terminally of the PepM23 domain, or between the PepM23 domain and the SH3 domain, preferably N-terminally of the PepM23 domain.

8. The peptidoglycan hydrolase of any one of claims 1 to 7, wherein the peptidoglycan hydrolase comprises a peptide linker connecting the CHAP domain and the PepM23 domain, wherein said peptide linker consists of one to four amino acids, preferably of two glycines; and, preferably, wherein the CHAP domain is located N-terminally of the PepM23 domain.

9. The peptidoglycan hydrolase of any one of claims 1 to 8, wherein the PepM23 domain has one or more amino acid substitutions at positions 125, 126, and 127, and 232 in SEQ ID NO: 407 or at positions corresponding to these positions, wherein the amino acid residue at position 125 in SEQ ID NO: 407 or at a position corresponding to this position is substituted with glutamine, the amino acid residue at position 126 in SEQ ID NO: 407 or at a position corresponding to this position is substituted with proline, and / orthe amino acid residue at position 127 in SEQ ID NO: 407 or at a position corresponding to this position is substituted with alanine.

10. The peptidoglycan hydrolase of any one of claims 1 to 9, wherein the PepM23 domain has an amino acid substitution at position 126 in SEQ ID NO: 407 or at a position corresponding to this position, wherein the amino acid residue at position 126 in SEQ ID NO: 407 or at a position corresponding to this position is substituted with proline.

11. The peptidoglycan hydrolase of any one of claims 3 to 10, wherein the SH3 domain has an amino acid substitution at position 232 in SEQ ID NO: 407 or at a position corresponding to this position, wherein the amino acid residue at position 232 in SEQ ID NO: 407 or at a position corresponding to this position is substituted with glutamine.

12. The peptidoglycan hydrolase of any one of claims 1 to 11, which has an enhanced peptidoglycan hydrolase activity, in particular an enhanced peptidoglycan hydrolase kinetics, as compared to the peptidoglycan hydrolase of SEQ ID NO: 407.

13. The peptidoglycan hydrolase of anyone of claims 1 to 12, wherein the CHAP domain has one or more amino acid substitutions at positions 189, 94, 117 and 118 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably wherein the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid, the amino acid residue at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, the amino acid residue at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid or glycine, preferably aspartic acid, and / or the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

14. The peptidoglycan hydrolase of any one of claims 1 to 13, wherein the CHAP domain has an amino acid substitution or a deletion at position 73 in SEQ ID NO: 1 or at a position corresponding to this position, preferably wherein the residue at position 73 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, tyrosine, leucine, glutamic acid, alanine, histidine or serine, more preferably with glycine.

15. The peptidoglycan hydrolase of any one of claims 1 to 14, wherein the CHAP domain has an amino acid substitution at position 117 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with aspartic acid, and, preferably, wherein the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is a glycine.

16. The peptidoglycan hydrolase of any one of claims 1 to 14, wherein the CHAP domain has an amino acid substitution at position 118 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and, preferably, wherein the amino acid residue at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is a tyrosine.

17. The peptidoglycan hydrolase of any one of claims 1 to 16, wherein the CHAP domain has two amino acid substitutions at positions 117 and 118 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

18. The peptidoglycan hydrolase of any one of claims 1 to 17, wherein the CHAP domain has two amino acid substitutions at positions 94 and 189 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 94 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with phenylalanine, and the amino acid residue at position 189 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamic acid; and, preferably, wherein the amino acid residue at position 117 in SEQ ID NO: 1 or at a position corresponding to this position is a tyrosine, and the amino acid residue at position 118 in SEQ ID NO: 1 or at a position corresponding to this position is a glycine.

19. The peptidoglycan hydrolase of any one of claims 1 to 18, wherein the CHAP domain has one or more amino acid substitutions at positions 82, 85, 86, 130, 136, 155, 169, 173, 175, 185, 186 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, preferably wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine,the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and / or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

20. The peptidoglycan hydrolase of any one of claims 1 to 19, wherein the CHAP domain has an amino acid substitution at position 186 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

21. The peptidoglycan hydrolase of any one of claims 1 to 20, wherein the CHAP domain has one or more amino acid substitutions at positions 82, 85, 86 and 169 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine, the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine, and / or the amino acid residue at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine.

22. The peptidoglycan hydrolase of any one of claims 1 to 21, wherein the CHAP domain has one or more amino acid substitutions at positions 173, 175 and 192 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 173 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine, and / or the amino acid residue at position 192 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

23. The peptidoglycan hydrolase of any one of claims 1 to 22, wherein the CHAP domain has one or more amino acid substitutions at positions 130, 136, 155, 185 and 186, whereinthe amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine, the amino acid residue at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and / or the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

24. The peptidoglycan hydrolase of any one of claims 1 to 23, wherein said CHAP domain has at least one, preferably at least two or more, of the following consensus mutation units I) to V):I) an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine;II) an amino acid substitution pair at positions 82 and 85 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 82 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with serine, and the amino acid residue at position 85 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine;III) an amino acid substitution pair at positions 130 and 136 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 130 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine, and the amino acid residue at position 136 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine or arginine;IV) an amino acid substitution at position 169 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 169 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with asparagine; and / orV) an amino acid substitution pair at positions 185 and 186 in SEQ ID NO: 1 or at positions corresponding to these positions, wherein the amino acid residue at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine, and the amino acid residue at position 186 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glycine.

25. The peptidoglycan hydrolase of claim 24, wherein said CHAP domain has at least one, preferably at least two or all of said consensus mutation units I), II) and IV).

26. The peptidoglycan hydrolase of any one of claims 1 to 25, wherein the CHAP domain has an amino acid substitution at position 155 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 155 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

27. The peptidoglycan hydrolase of any one of claims 1 to 26, wherein the CHAP domain has an amino acid substitution at position 185 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 185 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with tyrosine.

28. The peptidoglycan hydrolase of any one of claims 1 to J, wherein the CHAP domain has an amino acid substitution at position 175 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 175 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with glutamine.

29. The peptidoglycan hydrolase of any one of claims 1 to 28, wherein said CHAP domain has an amino acid substitution at position 86 in SEQ ID NO: 1 or at a position corresponding to this position, wherein the amino acid at position 86 in SEQ ID NO: 1 or at a position corresponding to this position is substituted with lysine.

30. The peptidoglycan hydrolase of any one of claims 1 to 29, wherein said CHAP domain has a sequence identity of at least 95% to the sequence from position 72 to position 215 in SEQ ID NO: 3.

31. The peptidoglycan hydrolase of any one of claims 1 to 30, wherein said CHAP domain has a sequence identity of at least 94% to the sequence from position 72 to position 215 in SEQ ID NO: 11.

32. The peptidoglycan hydrolase of any one of claims 1 to 31, wherein said CHAP domain has an amidase activity and / or a peptidase activity, preferably at least an amidase activity, more preferably an amidase activity and a peptidase activity.

33. The peptidoglycan hydrolase of claim 32, wherein the amidase activity comprises peptidoglycan cleavage between N-acetylmuramyl and L-Alanine (N-acetylmuramyl L-Ala amidase activity), and / or the peptidase activity comprises peptidoglycan cleavage between D-alanine and glycine (D-Ala-Gly endopeptidase activity).

34. The peptidoglycan hydrolase of any one of claims 1 to 33, wherein said CHAP domain has an enhanced peptidoglycan hydrolase activity as compared to the CHAP domain consisting of the sequence from position 72 to position 215 in SEQ ID NO: 1, preferably wherein said peptidoglycan is from Staphylococcus aureus.

35. The peptidoglycan hydrolase of any one of claims 1 to 34, wherein said CHAP domain has an enhanced peptidase activity as compared to the CHAP domain consisting of the sequence from position 72 to position 215 in SEQ ID NO: 1, preferably wherein said CHAP domain has at positions 94, 117, 118 and / or 189 in SEQ ID NO: 1 or at positions corresponding to these positions the amino acids as defined in any one of claims 15 to 18; and, preferably wherein the peptidoglycan hydrolase activity is measured by the cleavage of a fluorigenic substrate analogue of the S.aureus stem peptide.

36. The peptidoglycan hydrolase of claim 35, wherein said enhanced peptidase activity comprises an enhanced D-Ala-Gly endopeptidase activity as compared to the CHAP domain consisting of the sequence from position 72 to position 215 in SEQ ID NO: 1.

37. The peptidoglycan hydrolase of any one of claims 1 to 36, wherein said PepM23 domain has a peptidase activity, preferably a glycyl-glycyl peptidase activity.

38. The peptidoglycan hydrolase of any one of claims 1 to 37, which has (i) a glycyl-glycyl peptidase activity and additionally (ii) a D-alanyl-glycyl peptidase activity and / or an N-acetylmuramyl L-Ala amidase activity.

39. The peptidoglycan hydrolase of any one of claims 1 to 38 which has a killing activity against at least one Staphylococcus species or strain, preferably against Staphylococcus aureus, more preferably against a Staphylococcus aureus strain that is resistant to at least one antibiotic such as methicillin.

40. The peptidoglycan hydrolase of claim 39, wherein said Staphylococcus species or strain, preferably said Staphylococcus aureus, is present in form of a biofilm or is suspected of forming a biofilm.

41. The peptidoglycan hydrolase of any one of claims 1 to 40 which is stable up to a temperature of about 40°C, preferably of about 50°C.

42. The peptidoglycan hydrolase of any one of claims 1 to 41 which has the ability of being secreted from a human cell.

43. The peptidoglycan hydrolase of any one of claims 1 to 42, which shows in comparison to the peptidoglycan hydrolase of SEQ ID NO: 1 at least one of the following:(i) an enhanced killing activity against Staphylococcus aureus,(ii) an enhanced thermostability, and / or(iii) an enhanced ability of being secreted by a human cell.

44. The peptidoglycan hydrolase of any one of claims 1 to 43, which has an enhanced killing activity against Staphylococcus aureus and / or Staphylococcus epidermidis as compared to at least one peptidoglycan hydrolase consisting of a sequence selected from the group consisting of: SEQ ID NO: 410, 411, 412 and 413; and which has, preferably, an enhanced killing activity against Staphylococcus aureus as compared to each peptidoglycan hydrolase consisting of a sequence selected from the group consisting of: SEQ ID NO: 410, 411, 412 and 413.

45. The peptidoglycan hydrolase of any one of claims 1 to 44 further comprising a human FC domain.

46. The peptidoglycan hydrolase of any one of claims 1 to 45 further comprising a signal peptide, preferably at the N-terminus.

47. A nucleic acid encoding the peptidoglycan hydrolase of any one of claims 1 to 46.

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

49. The nucleic acid of claim 47 or 48 which is a DNA or an RNA, preferably an RNA.

50. The nucleic acid of any one of claims 47 to 49 which is an RNA construct comprising in 5' to 3' order:(i) a 5' UTR that comprises or consists of a modified human alpha-globin 5'-UTR;(ii) a sequence encoding a peptidoglycan hydrolase of any one of claims 1 to 46;(iii) a 3' UTR that comprises or consists of a first sequence from the amino terminal enhancer of split(AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA; and(iv) a poly-A sequence; and, preferably, wherein said RNA construct further comprises(v) a 5' cap and / or(vi) a modified nucleoside selected from pseudouridine (i ), Nl-methyl-pseudouridine (mli ), and 5- methyl-uridine (m5U), preferably Nl-methyl-pseudouridine (mli ), in place of uridine, preferably in place of each uridine.

51. A pharmaceutical composition comprising the peptidoglycan hydrolase of any one of claims 1 to 46 and / or the nucleic acid of any one of claims 47 to 50; and, preferably, a pharmaceutically acceptable excipient.

52. The peptidoglycan hydrolase of any one of claims 1 to 46, the nucleic acid of any one of claims 47 to 50 or the pharmaceutical composition of claim 51 for use in treating a disease caused by and / or associated with a Staphylococcus infection and / or a subject that has or is suspected of having a Staphylococcus infection.

53. The peptidoglycan hydrolase, nucleic acid or pharmaceutical composition for use according to claim 52, wherein said infection is a Staphylococcus aureus infection.

54. The peptidoglycan hydrolase, nucleic acid or pharmaceutical composition for use according to claim 52 or 53, wherein the Staphylococcus is present in form of a biofilm and / or a free-floating aggregate, or is suspected of forming a biofilm and / or a free-floating aggregate.

55. The peptidoglycan hydrolase, nucleic acid or pharmaceutical composition for use according to any one of claims 52 to 54, wherein said Staphylococcus infection is a Staphylococcus aureus infection of a skin, soft tissue, bone, lung, sinus and / or urinary tract.

56. The peptidoglycan hydrolase, nucleic acid or pharmaceutical composition for use according to any one of claims 52 to 55, wherein said disease is selected from the group consisting of: pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia, sepsis, a respiratory infection such as sinusitis, pimples, impetigo, boils, cellulitis, folliculitis, carbuncles, scalded skin syndrome, abscesses, foodpoisoning, necrotizing fasciitis, pyomyositis, mediastinitis, infected dermatitis, wound infection, diabetic foot ulcer, septic arthritis, osteoarticular infections, prosthetic infection such as infection of a prosthetic joint or a cardiac device, and urinary tract infections.

57. The peptidoglycan hydrolase, nucleic acid or pharmaceutical composition for use according to any one of claims 52 to 56, wherein said disease is pneumonia, bacteremia, endocarditis, or a prosthetic infection.

58. The peptidoglycan hydrolase, nucleic acid or pharmaceutical composition for use according to any one of claims 52 to 57, which is to be administered systemically, preferably intravenously.

59. A method of sterilizing a device in vitro or in a subject, comprising a step of contacting the device with a peptidoglycan hydrolase of any one of claims 1 to 46.

60. The method of claim 59, wherein said device is a catheter, a pacemaker or a prosthetic joint.

61. The method of claim 59 or 60, wherein said device is contaminated by a Staphylococcus species or strain, preferably Staphylococcus aureus,- and, preferably, wherein said Staphylococcus species or strain is present in form of a biofilm or suspected of forming a biofilm.

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