An antibacterial enzyme effective against staphylococcus aureus for use in treating se-producing staphylococcus aureus in subjects suffering from atopic dermatitis

Abstract

The invention relates to the field of medicine, more specifically to treatment of a condition associated with CD4+ T cell-mediated overactivation of the immune system.

Description

[0001] Treatment of a condition associated with CD4+T cell-mediated overactivation of the immune system

[0002] Field of the invention

[0003] The invention relates to the field of medicine, more specifically to treatment of a condition associated with CD4+T cell-mediated overactivation of the immune system.

[0004] Background of the invention

[0005] Atopic Dermatitis (AD) or eczema is a chronic inflammatory skin disorder that is associated with severe itching and skin damage with development of often severe skin lesions and symptoms which compromise the quality of life (1 ,2). There is no cure for eczema, but there are many treatments, ranging from special diets to emollients and immunosuppressive ointments like e.g. corticosteroid ointment. While corticosteroids, such as hydrocortisone or clobetasol propionate (topical, oral or intradermal administration) usually bring about improvements, they also may have side effects. Prolonged use of topical corticosteroids is thought to increase the risk of side effects, the most common of which is the skin becoming thin and fragile (atrophy). Because of this, if used on the face or other delicate skin, a low-strength steroid should be used or applied less frequently. Additionally, high-strength steroids used over large areas, or under occlusion, may be absorbed into the body, causing hypothalamic-pituitary-adrenal axis suppression (HPA axis suppression). Due to the impaired skin barrier in AD an increase in skin colonization with and infections with bacteria such as Staphylococcus aureus might be the result. For more severe cases, dermatologists may also prescribe either topical or oral conventional antibiotics such as penicillin, streptomycin and chloramphenicol. The antibiotics help prevent colonization and infection that can result from impaired skin barrier such as cracked skin. S. aureus colonization or infection is the most common cause of increased eczema severity. The effectiveness of antibiotic treatments varies from person to person. The well-known disadvantages of conventional antibiotics are a-specificity, i.e. also non-pathogenic and / or beneficial bacteria are killed, and the risk of developing resistance, not only by the target bacterial cells but possibly also by other pathogenic bacteria. In addition, antibiotic treatment comes with the risk of gastro-intestinal side effects and dysbiosis as well as the risk of sensibilization and potentially dangerous allergic side-effects. Furthermore, conventional, systemic antibiotic treatment can interact with other drugs, including contraceptive pills. Certain antibiotics cannot be combined with the use of alcohol.

[0006] In AD, CD4+T cells are believed to play a key role in the pathogenesis by producing a series of cytokines that triggers inflammation, itching, and damage to the skin barrier which in turn leads to skin colonization and infection with bacteria such as Staphylococcus aureus (2,3). Staphylococcus aureus colonization and infection have been linked to disease flare because increased amounts of these bacteria are detected during flare-ups (4). Although the mechanisms by which Staphylococcus aureus aggravates the pathogenesis and fuels disease flares are not known in detail, multiple staphylococcal toxins and factors have been implicated. For instance, the staphylococcal alpha-toxin seems to elicit damage to keratinocytes and therefore hypothesized tocompromise the structure of the epidermis while a staphylococcal V8 protease stimulate itching and scratching leading to subsequent skin damage (5). A large fraction of AD patients harbours Staphylococcus aureus that produce Staphylococcus aureus enterotoxins (SE) (4-6), which are also known as superantigens because they are potent activators of CD4+T cells (7). Several studies on peripheral blood T cells from AD patients indicate that SE induces increased production of T helper cell (TH) type 2 (TH2) cytokines such as IL-4 and IL-13, which have been implicated in the pathogenesis in AD (8). Yet, it remains unknown how SE-producing Staphylococcus aureus may impact AD. The inventors have addressed this question in primary lesional CD4+T cells directly expanded from freshly biopsied lesional AD skin.

[0007] As will be clear from the above, there is a continuing need for improved treatment of eczema.

[0008] Description of the invention

[0009] The inventors have established that Staphylococcus aureus (S. aureus) and its toxins (staphylococcal enterotoxins, herein interchangeably used with the term S. aureus superantigens (SE)) induce activation and survival of CD4+T cells from inflamed skin of a subject suffering from Atopic Dermatitis (AD).

[0010] Accordingly, in a first aspect, the invention provides for an antibacterial enzyme effective against Staphylococcus aureus for use in the treatment of a subject suffering from Atopic Dermatitis (AD), wherein the subject’s AD lesions are colonized or infected with Staphylococcus aureus producing Staphylococcus aureus superantigen (SE).

[0011] In the embodiments herein, the subject may have an overactivation of the immune system characterized by CD4+T cells that are refractive to an agent that inhibits IL-4 and / or IL-13 signalling. In the embodiments herein, the term “CD4+T cells that are refractive to an agent that inhibits IL-4 and / or IL-13 signalling” means that proliferation of the CD4+T cells in the presence of S. aureus superantigen (SE), IL-4 and / or IL-13 and further in the presence of an agent that inhibits IL-4 and / or IL-13 signalling, respectively, is higher compared to said CD4+T cells in the presence of IL-4 and / or IL-13 and further in the presence of an agent that inhibits IL-4 and / or IL-13 signalling, respectively. This is clearly demonstrated in the examples herein, where SE-producing Staphylococcal aureus enhances survival of pathogenetic IL-4 / IL-13-responsive CD4+T cells. A preferred assay to determine whether CD4+T cells are refractive to an agent that inhibits IL-4 and / or IL-13 signalling is the assay set forth in the examples herein. The person skilled in the art will comprehend that IL-4 and / or IL-13 signalling can be inhibited in various ways. IL-4 and / or IL-13 may e.g. be blocked themselves by an IL-4 and / or IL-13 antagonist or -neutralizing agent that binds to IL-4 and / or IL-13 and blocks or hinders binding of IL-4 and / or IL-13 to the IL-4 and / or IL-13 receptor. Such IL-4 and / or IL-13 antagonist or neutralizing agent may be - but is not limited to - an antibody capable of binding to IL-4 and / or IL-13. Another way of inhibiting IL-4 and / or IL-13 signalling is e.g. by an IL-4 and / or IL-13 receptor antagonist. Such receptor antagonist is an agent (e.g. a ligand or drug) that binds to an IL-4 and / or IL-13 receptor and by its binding, blocks or inhibits IL-4 and / or IL-13 mediated responses. Such the IL-4 and / or IL-13 receptor antagonist does not activate the IL-4 and / or IL-13 receptor.In the embodiments herein, the SE may be any staphylococcal enterotoxin. In the embodiments herein, the SE may be a staphylococcal enterotoxin produced by Staphylococcus aureus, such as SE type A, SE type B, SE type C1 , SE type C2, SE type C3 SE type D, SE type E, SE type F, SE type G, SE type H, and / or SE type I. In the embodiments herein, the SE may preferably be staphylococcal enterotoxin (SE) type A.

[0012] In the embodiments herein, the antibacterial enzyme effective against Staphylococcus aureus for use is referred to as the antibacterial enzyme effective against Staphylococcus aureus for use according to the invention, or plainly the antibacterial enzyme.

[0013] In the embodiments herein, the subject suffering from Atopic Dermatitis (AD), may be any subject suffering from any form or grade of AD known to the person skilled in the art, as long as the subject’s AD lesions are colonized or infected with Staphylococcus aureus producing Staphylococcus aureus superantigen (SE). The person skilled in the art knows Staphylococcus aureus and knows Staphylococcus aureus producing Staphylococcus aureus superantigen (SE). Such Staphylococcus aureus producing Staphylococcus aureus superantigen (SE), may be any Staphylococcus aureus producing Staphylococcus aureus superantigen (SE) known to the person skilled in the art.

[0014] In the embodiments herein, the treatment of the subject suffering from Atopic Dermatitis (AD), wherein the subject’s AD lesions are colonized or infected with Staphylococcus aureus producing Staphylococcus aureus superantigen (SE), comprises administration of the antibacterial enzyme to the subject. In the embodiments herein, said treatment preferably comprises administration of an effective amount of the antibacterial enzyme to the subject.

[0015] In the embodiments herein, an effective amount of the antibacterial enzyme may be construed as a therapeutically effective amount, meaning an amount of the antibacterial enzyme that is sufficient, when administered to the subject, to treat, delay or improve symptoms of a condition, preferably AD, in the subject. Preferably, the effective amount abrogates the refractiveness of the CD4+T cells that are refractive to an agent that inhibits IL-4 and / or IL-13 signalling, such that the amount of refractive CD4+T cells in the subject’s AD lesions is decreased with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, at least 1 log, 2 log, 3 log, 4 log, 5 log, 6 log, 7 log, 8 log, 9 log, or at least 10 log increased compared to when the antibacterial enzyme is not administered to the subject.

[0016] In the embodiment’s herein, further to the antibacterial enzyme, an agent that inhibits IL-4 and / or IL-13 signalling, may be administered to the subject.

[0017] In the embodiment herein, the agent that inhibits IL-4 and / or IL-13 signalling in CD4+T cells, may be any such agent known to the person skilled in the art. In the embodiments herein, the agent that inhibits IL-4 and / or IL-13 signalling may be an IL-4 receptor antagonist or an IL-4 neutralizing agent and / or an IL-13 receptor antagonist or an IL-4 neutralizing agent. The agent may also be an agent that interferes upstream of downstream in the IL-4 or IL-13 signalling pathway, such as a Signal Transducer and activator of Transcription-6 (STAT-6). A STAT-6 agent may be, but is not limited to, KP-273 or KT-621.In the embodiments herein, the agent may be an antibody specific for IL-4 and / or IL-13, such as the monoclonal antibody Dupilumab. A preferred agent that inhibits IL-4 and / or IL-13 signalling is Dupilumab. Dupilumab is a monoclonal antibody targeting the IL-14Ra receptor, thereby inhibiting both the IL-4 and IL-13 pathways. Another agent may be Pascolizumab, which is a humanized monoclonal antibody targeting the IL-4 cytokine. Binding to the cytokine inhibits receptor binding, thus preventing the downstream effects of the IL-4 pathway. Another agent may be Pitrakinra, which is a synthetic protein targeting the IL-4Ra receptor. Like Dupilumab, Pitrakinra inhibits both the IL-4 and IL-13 pathways. Another agent may be Lebrikizumab, which is a monoclonal antibody that targets IL-13 cytokines, thereby blocking the downstream pathway. Another agent is Anrukisumab, which is a monoclonal antibody that also targets the IL-13 cytokine, like Lebrikizumab. Another agent may be Tralokinumab, which targets IL-13. The person skilled in the art will comprehend that biosimilar agents to the ones listed herein may be used.

[0018] Further agents that inhibit IL-4 and / or IL-13 signalling are Nemolizumab, Abrocitinib, Upadacitinib, Crisaborole, Roflumilast, Fezakinumab, Rocatinlimab, Amlitelimab, Tezepelumab, Brepocitinib, ZPL-389, and Brutininib.

[0019] In the embodiments herein, the antibacterial enzyme is preferably selected from the group consisting of: a bacteriocin or a functional part thereof, a bacterial lysin or autolysin or a functional part thereof, a bacteriophage lysin or a functional part thereof, and a chimeric lysin or a functional part thereof. In the embodiments herein, the antibacterial enzyme has peptidoglycan hydrolase activity; hydrolysis of the peptidoglycan results in lysis of the bacterium. In the embodiments herein, the antibacterial enzyme may have more than one peptidoglycan hydrolase activity.

[0020] In the embodiments herein, the antibacterial enzyme may be a chimeric lysin, such as a recombinant chimeric endolysin comprising one or more heterologous domains.

[0021] In the embodiments herein, a functional part of an antibacterial enzyme means that the part of the functional antibacterial enzyme still is able to lyse its native target bacterium by hydrolysis of the peptidoglycan. Preferably, the activity of a functional part is at least 20%, 30%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the complete antibacterial enzyme.

[0022] In the embodiments herein, the bacteriocin may be any bacteriocin with peptidoglycan hydrolase activity, such as Class Illa bacteriocins. Preferred bacteriocins herein are Lysostaphin, Mutanolysin and Enterolysin.

[0023] In the embodiments herein, the bacterial lysin or autolysin may be any bacterial lysin or autolysin with peptidoglycan hydrolase activity, such as AltA from Enterococcus faecalis (Mesnage et al., 2008) or Acm A, Acm B, Acm C, and Acm D from Lactococcus lactis (Steen et al, 2005).

[0024] In the embodiments herein, the bacteriophage lysin is also referred to as endolysin and may be any bacteriophage lysin with peptidoglycan hydrolase activity. Bacteriophage lysins are known to the person skilled in the art, see e.g. Bacteriophage endolysins as novel antimicrobials. Schmelcher M, Donovan DM, Loessner MJ. Future Microbiol. 2012 Oct;7(10): I 147-7.

[0025] In the embodiments herein, the endolysin may be, but is not limited to: PhiV10p30 of phage OV10 (Sequence analysis of Escherichia coli 0157:H7 bacteriophage PhiVIO and identification of aphage-encoded immunity protein that modifies the 0157 antigen. Perry LL, SanMiguel P, Minocha U, Terekhov Al, Shroyer ML, Farris LA, Bright N, Reuhs BL, Applegate BM. FEMS Microbiol Lett.

[0026] 2009 Mar;292(2):182- 6); STM0907.FelsO of phage FELS-1 (Nature. 2001 Oct 25;413(6858):852-6. Complete genome sequence of Salmonella enterica serovarTyphimurium LT2. McClelland Ml, Sanderson KE, Spieth J, Clifton SW, Latreille P, Courtney L, Porwollik S, Ali J, Dante M, Du F, Hou S, Layman D, Leonard S, Nguyen C, Scott K, Holmes A, Grewal N, Mulvaney E, Ryan E, Sun H, Florea L, Miller W, Stoneking T, Nhan M, Waterston R, Wilson RK); epsilonl5p25 of phage s15 (Virology. 2007 Dec 20;369(2):234-44. Epub 2007 Sep 7. The genome of epsilonl5, a serotypeconverting, Group El Salmonella enterica-specific bacteriophage. Kropinski AMI, Kovalyova IV, Billington SJ, Patrick AN, Butts BD, Guichard JA, Pitcher TJ, Guthrie CC, Sydlaske AD, Barnhill LM, Havens KA, Day KR, Falk DR, McConnell MR); YuA20 of phage YUA (NCBI Reference Sequence: YP 001595885.1 and J Bacteriol. 2008 Feb;190(4): 1429-35. The genome and structural proteome of YuA, a new Pseudomonas aeruginosa phage resembling M6. Ceyssens PJ1 , Mesyanzhinov V, Sykilinda N, Briers Y, Roucourt B, Lavigne R, Robben J, Domashin A, Miroshnikov K, Volckaert G, Hertveldt K); ORF23 of phage B3 (Complete genomic sequence of bacteriophage B3, a Mu-like phage of Pseudomonas aeruginosa. Braid MD, Silhavy JL, Kitts CL, Cano RJ, Howe MM. J Bacteriol. 2004 Oct;186(19):6560-74); BcepMu22 of phage BcepMu (J Mol Biol. 2004 Jun 25;340(l):49-65. Burkholderia cenocepacia phage BcepMu and a family of Mu-like phages encoding potential pathogenesis factors. Summer EJ1 , Gonzalez CF, Carlisle T, Mebane LM, Cass AM, Sawa CG, LiPuma J, Young R); F116p62 of phage Fl 16 (Gene. 2005 Feb 14;346: 187-94. The genome of the Pseudomonas aeruginosa generalized transducing bacteriophage Fl 16. Byrne Ml, Kropinski AM.); STM2715.S.Fels2 of phage Fels2 (Nature. 2001 Oct 25;413(6858):852-6. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. McClelland Ml, Sanderson KE, Spieth J, Clifton SW, Latreille P, Courtney L, Porwollik S, Ali J, Dante M, Du F, Hou S, Layman D, Leonard S, Nguyen C, Scott K, Holmes A, Grewal N, Mulvaney E, Ryan E, Sun H, Florea L, Miller W, Stoneking T, Nhan M, Waterston R, Wilson RK); gp76 of phage ESI 8 (Casjens.S.R., Gilcrease.E.B., Winn-Stapley,D.A., Schicklmaier.P., Schmieger.H., Pedulla.M.L., Ford.M.E., HoutzJ.M., Hatfull, G.F. and Hendrix, R.W. The generalized transducing Salmonella bacteriophage ESI 8: complete genome sequence and DNA packaging strategy J. Bacteriol. 187 (3), 1091-1104 (2005)); SPSV3_gp23 of phage SETP3 (J Med Microbiol. 2009 Jan;58(Pt l):86-93. Characterization of bacteriophages used in the Salmonella enterica serovar Enteritidis phage-typing scheme. De Lappe Nl, Doran G, O'Connor J, O'Hare C, Cormican M); phi32_17 of phage OEC032 (Genomic and proteomic analysis of phiEco32, a novel Escherichia coll bacteriophage. Savalia D, Westblade LF, Goel M, Florens L, Kemp P, Akulenko N, Pavlova O, Padovan JC, Chait BT, Washburn MP, Ackermann HW, Mushegian A, Gabisonia T, Molineux I, Severinov K. J Mol Biol. 2008 Mar28;377(3):774-89); HK022p54 of phage HK022 (J Mol Biol. 2000 May 26;299(1):27-51. Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoid bacteriophages. Juhala RJ1 , Ford ME, Duda RL, Youlton A, Hatfull GF, Hendrix RW); HK97p58 of phage HK97 (J Mol Biol. 2000 May 26;299(1):27-51. Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoidbacteriophages. Juhala RJI, Ford ME, Duda RL, Youlton A, Hatfull GF, Hendrix RW); HK620p36 of phage HK620 (Nucleotide sequence of coliphage HK620 and the evolution of lambdoid phages. Clark AJ, Inwood W, Cloutier T, Dhillon TS. J Mol Biol. 2001 Aug 24;311(4):657-79); VIP0007 of phage El (Molecular characterization of the Salmonella enterica serovar Typhi Vi-typing bacteriophage El . Pickard D, Thomson NR, Baker S, Wain J, Pardo M, Goulding D, Hamlin N, Choudhary J, Threfall J, Dougan G. J Bacteriol. 2008 Apr; 190(7) :2580-7); Sf6p62 of phage SF6 (J Mol Biol. 2004 May 28;339(2):379-94. The chromosome of Shigella flexneri bacteriophage Sf6: complete nucleotide sequence, genetic mosaicism, and DNA packaging. Casjens SI, Winn-Stapley DA, Gilcrease EB, Morona R, Kuhlewein C, Chua JE, Manning PA, Inwood W, Clark AJ); R (SfVp40) of phage SFV (J Bacteriol. 2002 Apr; 184(7): 1974-87. Complete genomic sequence of SfV, a serotype- converting temperate bacteriophage of Shigella flexneri. Allison GE1 , Angeles D, Tran-Dinh N, Verma NK); gp22 of phage BCEPC6B (Summer, E.J., Christian, B.N., Collins, J., Morrison, W., Patel, P., Wells, W., Mebane, L., Gonzalez, C.F. and Young, R.F. GenBank: AAT38381.1); Nazgul38 of phage BCEPNAZGUL (Summer, E.J., Peek.M.L., HaliburtonJ.R., Hall.E., Heusinkveld.K., SimserJ., No,E.G., Gonzalez, C.F. and Young, R.F. NCBI Reference Sequence: NP 918971.2); K (P2p09) of phage P2 (Christie, G.E., Haggard- Ljungquist.E. and Calendar, R. NCBI Reference Sequence: NP 046765.1); K (Wphi09) of phage WO (Esposito, D., Schmidt, B. J., Bloom, F.R. and Christie, G.E. GenBank: AAN28227.1); rv5_gp085 of phage RV5 (Virol J. 2013 Mar 6;10:76. The host-range, genomics and proteomics of Escherichia coll 0157:H7 bacteriophage rV5. Kropinski AMI, Waddell T, Meng J, Franklin K, Ackermann HW, Ahmed R, Mazzocco A, Yates J 3rd, Lingohr EJ, Johnson RP); EpJS98_gpl 16 of phage JS98 (Zuber, S., Ngom-Bru,C, Barretto.C, Bruttin.A., Brussow, H. and Denou.E. Genome analysis of phage JS98 defines a fourth major subgroup of T4-like phages in Escherichia coll J. Bacteriol. 189 (22), 8206-8214 (2007)); gp3.5 of phage 13A (Savalia.D., Severinov.K. and Molineux,!. NCBI Reference Sequence: YP 002003950.1); gp3.5 of phage BAM (Savalia.D., Severinov.K. and Molineux, I. GenBank: ACF15743.1); gp3.5 of phage ECODS1 (Savalia.D., Severinov.K. and Molineux, I. GenBank: ACF15800.1); CKVIF_gpl6 of phage K1F (Scholl, D. and Merril.C. The Genome of Bacteriophage K1 F, a T7-Like Phage That Has Acquired the Ability To Replicate on KI Strains of Escherichia coli J. Bacterid. 187 (24), 8499-8503 (2005)); T3pl8 of phage T3 (Pajunen.M.L, Elizondo, M.R., Skurnik.M., KieleczawaJ. and Molineux, I. J. Complete nucleotide sequence and likely recombinatorial origin of bacteriophage T3 J. Mol. Biol. 319 (5), 1115-1132); gh-lpl2 of phage GH-1 (Kovalyovai.V. and Kropinski, A. M. The complete genomic sequence of lytic bacteriophage gh-1 infecting Pseudomonas putida — evidence for close relationship to the T7 group Virology 311 (2), 305-315 (2003)); gp3.5 of phage KI I (Savalia.D., Severinov.K. and Molineux, I. NCBI Reference Sequence: YP 002003804.1); ORF12 of phage OCTX (Nakayama, K., Kanaya.S., Ohnishi.M., Terawaki.Y. and Hayashi, T. The complete nucleotide sequence of phi CTX, a cytotoxin-converting phage of Pseudomonas aeruginosa-, implications for phage evolution and horizontal gene transfer via bacteriophages Mol. Microbiol. 31 (2), 399-419 (1999)); Bcep43-27 of phage BCEP43 (Summer, E. J., Gonzalez.C.F., Borner, M., Carlile, T., Embry, A., Kucherka.A.M., Lee, J., Mebane, L., Morrison, W.C., Mark.L., King.M.D., LiPumaJ.J., Vidaver, A. K. and Young, R. Divergence andmosaicism among virulent soil phages of the Burkholderia cepacia complex J. Bacteriol. 188 (1), 255-268 (2006)); Bcep781-27 of phage BCEP781 (Summer, E. J., Gonzalez, C.F., Borner, M., Carlile, T., Embry, A., Kucherka.A.M., Lee, J., Mebane, L., Morrison, W.C., Mark,L., King.M.D., LiPumaJ.J., Vidaver, A. K. and Young, R. Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex J. Bacteriol. 188 (1), 255- 268 (2006)); Bcepl-28 of phage BCEP1 (Summer, E. J., Gonzalez.C.F., Borner, M., Carlile, T., Embry, A., Kucherka.A.M., Lee, J., Mebane, L., Morrison, W.C, Mark.L., King.M.D., LiPumaJ.J., Vidaver, A. K. and Young, R. Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex J. Bacteriol. 188 (1), 255-268 (2006)); BcepNY3gene26 of phage BCEPNY3 (GenBank: ABR10561.1 Summer, E.J., Orchard, R.C, Attenhofer.K., Coffey, A., GillJ.J., Gonzalez.C.F. and Young, R.); gp45 of phage <t>E12-2 (NCBI Reference Sequence: YP 001111195.1 DeShazer.D., Ronning.C.M., Brinkac.L.M. and Merman, W.C.); gp28 of phage <t>52237 (DeShazer, D and Merman, W.C. NCBI Reference Sequence: YP 293741.1 DeShazer.D., Ronning.C.M., Brinkac.L.M. and Merman, W.C); P27p30 of phage <t>P27 (RecktenwaldJ. and Schmidt, H. The nucleotide sequence of Shiga toxin (Stx) 2e-encoding phage phiP27 is not related to other Stx phage genomes, but the modular genetic structure is conserved Infect. Immun. 70 (4), 1896-1908 (2002)); RB49plO2 of phage RB49 (Monod.C, Repoila.F., Kutateladze, M., Tetart.F. and Krisch.H.M. The genome of the pseudo T-even bacteriophages, a diverse group that resembles T4 J. Mol. Biol. 267 (2), 237-249 (1997)); phil-pl02 of phage <t>1 (Arbiol.C, Comeau, A. M., Kutateladze, M., Adamia.R. and Krisch.H.M. Mobile regulatory cassettes mediate modular shuffling in t4-type phage genomes Genome Biol Evol 2010, 140-152 (2010)); lys (T5.040) of phage T5 (NCBI Reference Sequence: YP 006868.1 Ksenzenko.V.N., Kaliman, A.V., Krutilina.A.I. and Shlyapnikov.M.G.); YP 001956952.1 of phage 201 phi2-l (ThomasJ.A., Rolando, M.R., Carroll, C.A., Shen.P.S., Belnap.D.M., Weintraub, S.T., Serwer.P. and Hardies, S.C. 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Briers Yl, Volckaert G, Cornelissen A, Lagaert S, Michiels CW, Hertveldt K, Lavigne R), of the phage LUZ24 (NCBI Reference Sequence: YP 001671940.1) as well as of the E. coli phage N4gp61 (J Mol Biol. 2007 Feb 16;366(2):406-19. Coliphage N4 N-acetylmuramidase defines a new family of murein hydrolases. Stojkovic EA1 , Rothman- Denes LB), STM0016 endolysin (NCBI Reference Sequence: NP 459021.1), PSP3 endolysin (NP 958065.1) and endolysin of Salmonella enteritis phage PVPSE1 (PVP-SEIgpl46 (YP 004893953.1)); Listeria phage endolysins PlyA118 (NCBI Reference Sequence: YP 008666952.1), PlyA500 (NCBI Reference Sequence: YP 001468411.1), PlyPSA (GenBank: CAC85577.1 and J Mol Biol. 2006Dec 8;364(4):678-89. The crystal structure of the bacteriophage PSA endolysin reveals a unique fold responsible for specific recognition of Listeria cell walls. 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Inducible Clostridium perfringens bacteriophages OS9 and OS63: Different genome structures and a fully functional sigK intervening element. Kim KP1 , Born Y, Lurz R, Eichenseher F, Zimmer M, Loessner MJ, Klumpp J.), Ply3626 (Zimmer, M., Scherer, S. and Loessner, M.J. Genomic analysis of Clostridium perfringens bacteriophage phi3626, which integrates into guaA and possibly affects sporulation J. Bacteriol. 184 (16), 4359-4368 (2002)), Clostridium difficile-. CD27L endolysin (J Bacteriol. 2008 0ct;190(20):6734-40. Molecular characterization of a Clostridium difficile bacteriophage and its cloned biologically active endolysin. Mayer MJ1 , Narbad A, Gasson MJ), Streptococcus: B30 endolysin (The bifunctional peptidoglycan lysin of Streptococcus agalactiae bacteriophage B30. Pritchard DG, Dong S, Baker JR, Engler JA. Microbiology. 2004 Jul;150(Pt 7):2079-87), phage Dp-1 encoded Pal amidase (J Biol Chem. 2004 Oct 15;279(42):43697-707. Structural and thermodynamic characterization of Pal, a phage natural chimeric lysin active against pneumococci. Varea JI, Monterroso B, Saiz JL, Lopez-Zumel C, Garcia JL, Laynez J, Garcia P, Menendez M.), Cl endolysin PlyC (PlyC: a multimeric bacteriophage lysin. Nelson D, Schuch R, Chahales P, Zhu S, Fischetti VA. Proc Natl Acad Sci U S A. 2006 Jul 11 ;103(28): 10765-70), Cpl-1 endolysin (Gene. 1990 Jan 31 ;86(1):81-8. Modular organization of the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. Garcia PI, Garcia JL, Garcia E, Sanchez-Puelles JM, Lopez R.), PlyGBS (Antimicrob Agents Chemother. 2005 Jan;49(l): 1 11-7. Removal of group B streptococci colonizing the vagina and oropharynx of mice with a bacteriophage lytic enzyme. Cheng QI, Nelson D, Zhu S, Fischetti VA.), Enterococccus: PlyV12 (J Bacteriol. 2004 Jul; 186(14):4808- 12. Identification of a broadly active phage lytic enzyme with lethal activity against antibiotic-resistant Enterococcus faecalis and Enterococcus faecium. Yoong PI, Schuch R, Nelson D, Fischetti VA.).

[0027] Many antibacterial enzymes are comprised of different domains, such as a cell wall-binding domain and one or more lytic domains exhibiting peptidoglycan hydrolase activity, such as an amidase domain, a peptidase_M23 domain, and a CHAP (cysteine, histidine-dependent amidohydrolases / peptidases) domain. Such domains can be used for the design of chimeric endolysins that do not occur in nature. Such endolysins all are explicitly within the scope of the antibacterial enzymes of the embodiments herein.In the embodiments herein, the chimeric endolysin may be a chimeric endolysin comprising or consisting of a polypeptide having at least about 70% sequence identity or at least 70% sequence identity with an endolysin as set forward in WO2012 / 150858, W02013 / 169104, WO2016 / 142445, WO2017 / 046021 , WO2015155244, WO2015005787, WO2011 / 023702, WO2012 / 146738, W02003 / 082184, WO2010 / 011960, WO2010 / 149795, WO2010 / 149792, WO2012 / 094004, WO2011 / 023702, WO2011 / 065854, WO2011 / 076432, WO2011 / 134998, WO2012 / 059545, WO2012 / 085259, WO2012146738, WO2018 / 091707. Further preferred endolysins are endolysins comprising or consisting of a polypeptide having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by one of the following sequences: SEQ ID NO:s 1 to 81 in Table 1.

[0028] The person skilled in the art will comprehend that individual domains of endolysins disclosed and / or referenced herein may be combined with other domains to result in a chimeric endolysin. Such chimeric endolysins are explicitly within the scope of the antibacterial enzymes of the embodiments herein, such as but not limited to the domains as represented by one of the following sequences: SEQ ID NO:s 82 to 95, or a functional part thereof.

[0029] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 1.

[0030] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 2.

[0031] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 3.

[0032] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 4.

[0033] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 5.

[0034] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 6.In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 7.

[0035] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 8.

[0036] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 9.

[0037] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 10.

[0038] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 11.

[0039] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 12.

[0040] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 13.

[0041] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 14.

[0042] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 15.

[0043] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 16.In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 17.

[0044] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 18.

[0045] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 19.

[0046] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO:20.

[0047] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 21.

[0048] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 22.

[0049] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 23.

[0050] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 24.

[0051] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 25.

[0052] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 26.In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 27.

[0053] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 28.

[0054] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 29.

[0055] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 30.

[0056] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 31.

[0057] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 32.

[0058] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 33.

[0059] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 34.

[0060] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 35.

[0061] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 36.In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 37.

[0062] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 38.

[0063] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 39.

[0064] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 40.

[0065] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 41.

[0066] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 42.

[0067] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 43.

[0068] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 44.

[0069] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 45.

[0070] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 46.In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 47.

[0071] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 48.

[0072] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 49.

[0073] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 50.

[0074] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 51.

[0075] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 52.

[0076] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 53.

[0077] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 54.

[0078] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 55.

[0079] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 56.In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 57.

[0080] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 58.

[0081] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 59.

[0082] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 60.

[0083] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 61.

[0084] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 62.

[0085] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 63.

[0086] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 64.

[0087] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 65.

[0088] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 66.In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 67.

[0089] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 68.

[0090] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 69.

[0091] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 70.

[0092] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 71.

[0093] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 72.

[0094] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 73.

[0095] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 74.

[0096] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 75.

[0097] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 76.In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 77.

[0098] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 78.

[0099] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 79.

[0100] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 80.

[0101] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide, or a functional part thereof, having at least about 70% sequence identity or at least 70% sequence identity with an endolysin, or a functional part thereof, as represented by SEQ ID NO: 81.

[0102] In the embodiments herein, the antibacterial enzyme may or may not have a tag attached to it, such as a peptide tag useful to facilitate expression and / or affinity purification of the enzyme, to immobilize the enzyme to a surface or to serve as a marker or a label moiety for detection of the enzyme, e.g., by antibody binding in different ELISA assay formats. Such tag may be a His-tag, such as a C-terminal 6x His-Tag. The person skilled in the art knows how to incorporate a His-tag into an mRNA encoding an antibacterial enzyme.

[0103] In the embodiments herein, the antibacterial enzyme may or may not have an antibacterial peptide attached to it, such as a cationic or polycationic peptide, an amphipathic peptide, a sushi peptide, a defensin and a hydrophobic peptide. Examples of these antibacterial peptides are the ones set forward in WQ2015 / 155244 and WQ2016 / 142445.

[0104] In the embodiments herein, the domains of an antibacterial enzyme, may or may not be separated by a linker. Such linker may be a linker native to the domain or may be a linker foreign to the domain. The person skilled in the art will comprehend that polypeptides may lack one or more encoded amino acids, especially at the C-terminus or N-terminus of the polypeptide. Accordingly, in the embodiments herein, the antibacterial enzyme may lack one or more amino acids at the C-terminus or N-terminus, such as the N-terminal methionine.

[0105] In the embodiments herein, the Staphylococcus aureus may be an antibiotic-resistant Staphylococcus aureus, such as a methicillin resistant Staphylococcus aureus (MRSA).

[0106] In the embodiments herein, the antibacterial enzyme may be present as such or may be present as a source of the antibacterial enzyme, such as a polynucleotide encoding for the antibacterialenzyme. Such polynucleotide encoding for the antibacterial enzyme may conveniently be an mRNA. Said mRNA may be present in a delivery vehicle.

[0107] In the embodiments herein, the antibacterial enzyme may be present in a composition, such as a pharmaceutical composition, wherein the composition further comprises a pharmaceutically acceptable excipient or carrier. Such a pharmaceutically acceptable carrier or herein interchangeably depicted as pharmaceutically acceptable excipient, may be any such carrier known to the person skilled in the art. Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, colouring agents, coating agents, sweetening, flavouring, and / or performing agents can be present in the composition, according to the judgment of the formulator,

[0108] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulphate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and / or combinations thereof.

[0109] Exemplar / granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose, methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminium silicate, sodium lauryl sulphate, quaternary ammonium compounds, etc., and / or combinations thereof,

[0110] Exemplary surface active agents and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatine, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays {e.g. bentonite [aluminium silicate] and magnesium aluminium silicate]), long chain amino acid derivatives, high molecular weight alcohols {e.g. stearyl alcohol, cetyl alcohol, oieyl alcohol, triacetin monostearate, ethylene glycol di-stearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), ear homers {e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan, polyoxyethylene sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, glyceryl monooleate, sorbitan monooleate, polyoxyethylene esters (e.g. polyoxyethylene monostearate, polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, sucrose fatty acid esters, polyethylene glycol fatty acid esters, polyoxyethylene ethers, (e.g.polyoxyethylene lauryl ether), polyfvinyl-pyrrolidone), diethylene glycol monolaurate, triethanolaniine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulphate, Poloxamer*188, cetrimonium bromide, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof.

[0111] Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatine; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, micro crystalline cellulose, cellulose acetate, poly(vmyl-pyrrolidone), magnesium aluminium silicate, and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts: silicic acid; poiyniethaerylates; waxes; water; alcohol; etc.; and combinations thereof.

[0112] Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxy toluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulphite, sodium metabisulphite, and / or sodium sulphite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, henzethonium chloride, benzyl alcohol bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl, alcohol, glycerine, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenyimercuric nitrate, propylene glycol, and / or thimerosal.

[0113] Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta- carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulphate (SLS), sodium lauryl ether sulphate (SLES), sodium bisulphite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, methyl paraben.

[0114] Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentadienoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminium hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and / or combinations thereof.

[0115] Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulphate, sodium lauryl sulphate, etc., and combinations thereof.

[0116] Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macadamia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughie, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, Shaquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, Tsubaki, vetiver, walnut, and wheat germ oils. Exemplar oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof. In the embodiments herein the composition may be a solid composition, such as a dry powder composition, or a liquid composition, such as an aqueous composition which may be a solution and / or a suspension. Liquid dosage forms for e.g. oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetra hydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof, Besides inert, diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavouring, and / or perfuming agents. In the embodiments herein for parenteral administration, compositions may be mixed with solubilizing agents such as Cremophor\ alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0117] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions,and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[0118] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0119] Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatine capsules using such excipients as lactose or milk sugar as well, as high molecular weight polyethylene glycols and the like.

[0120] Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard- filled gelatine capsules using such excipients as lactose or milk sugar as weli as high molecular weight polyethylene glycols and the like.

[0121] Dosage forms for topical, and / or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and / or patches. Generally, an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and / or any needed preservatives and / or buffers as may be required. Additionally, within the scope of the invention is the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms may be prepared, for example, b dissolving and / or dispensing the compound in the proper medium. Alternatively, or additionally, rate may be controlled by either providing a rate controlling membrane and / or by dispersing the compound in a polymer matrix and / or gel.

[0122] In the embodiments herein, the subject may be any animal subject, preferably a mammal, livestock animal such as cattle, a domestic animal like a dog or a cat, or a human subject. Most preferably, the subject is a human.

[0123] The medical use disclosed in the embodiments herein may be formulated as a product for use as a medicament for treatment of the stated conditions but can equally be formulated as a method of treatment of the stated conditions using the product, a product for use in the preparation of a medicament to treat the stated conditions and use of the product for the treatment of the stated conditions. Such medical uses are all envisaged by the present invention.

[0124] Accordingly, herein, there is provided for the use of an antibacterial enzyme effective against Staphylococcus aureus in the treatment of a subject suffering from a condition associated withoveractivation of the immune system by CD4+T cells that are refractive to an IL-4 receptor antagonist and / or an IL-13 receptor antagonist. All features are as described herein above.

[0125] In addition, herein, there is provided fora method of treatment of a subject suffering from a condition associated with overactivation of the immune system by CD4+T cells that are refractive to an IL-4 receptor antagonist and / or an IL-13 receptor antagonist, said treatment comprising administration of an effective amount of the antibacterial enzyme effective against Staphylococcus aureus to the subject. All features are as described herein above.

[0126] In addition, herein, there is provided for the use of an antibacterial enzyme effective against Staphylococcus aureus for the preparation of a medicament for the treatment of a subject suffering from a condition associated with overactivation of the immune system by CD4+T cells that are refractive to an IL-4 receptor antagonist and / or an IL-13 receptor antagonist. All features are as described herein above.

[0127] In a second aspect, there is provided for a method of blocking CD4+T cell refractiveness to an IL-4 receptor antagonist and / or an IL-13 receptor antagonist, comprising contacting the CD4+T cell with an antibacterial enzyme effective against Staphylococcus aureus. All features are as described herein above in the first aspect.

[0128] Table 1 : Overview of sequences

[0129]

[0130] Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898 Chimeric endolysin in WO 2021 / 213898

[0131]

[0132]

[0133]

[0134] Figure legends

[0135] Figure 1.

[0136] Figure 1 depicts the workflow of the examples herein. Firstly (1), Staphylococcus aureus (S. aureus) was isolated from patient skin and validated for SE expression in our previous study (9) (upper section of the figure). Secondly (2), punch biopsies were collected from the lesional skin of AD patients and cultured ex-vivo in 24-well plates in RPMI 1640 media supplemented with 10% pooled human serum and 1% antibiotics (penicillin and streptomycin) along with IL-2 and IL-4 (10 ng / mL). Cells were either cryopreserved or used subsequently for experiments, (middle section of the figure). Thirdly (3), cells were washed and cultured in RPMI 1640 media with and without IL-4 and IL-13 and with and without a pool of SE (SEA, SEB, SEC, SED and SEI) followed by treatment with or without Dupilumab. Cells were cultured for 3 and 6 days prior to flow cytometric analysis.

[0137] Figure 2.

[0138] Figure 2 depicts the flow cytometry results showing flow cytometric contour plots and quantifications of percentage of viable CD4+ T cells expanded from lesional AD skin. (A) Cells were, from left to right: not treated (control), treated with supernatant from SE-producing S. aureus, treated with supernatant from endolysin (XZ.700)-treated SE-producing S. aureus, and, treated with IL-4 and IL-13. (B) Cells were treated with and without SE-pool, and with and without IL-4 and IL-13 (upper part “control”) and with and without Dupilumab and with and without SE-pool and IL-4 and IL-13 (lower part “Dupilumab”)

[0139] Figure 3.

[0140] Figure 3 depicts the flow cytometry results showing flow cytometric contour plots and quantifications of percentage of viable CD4+ T cells expanded from lesional AD skin from four patients. (A) and (B) Cells from two additional AD patients were treated with and without SE-pool, and with and without IL-4 and IL-13 (upper part “control”) and with and without Dupilumab and with and without SE-pool and IL-4 and IL-13 (lower part “Dupilumab”). (C) Cells (from the patient used in Figure 2) were treated with and without IL-2, and with and without IL-4 and IL-13 and with and without Dupilumab. (D) Cells from a fourth AD patient were treated with (from left to right): vehicle (control), supernatant from SE-producing S. aureus, and supernatant from endolysin (MEndoB)-treated SE-producing S. aureus and without (upper panel) and with Dupilumab (lower panel).

[0141] Figure 4.

[0142] Figure 4 depicts cytokine production by CD4+ T cells expanded from lesional AD skin and cultured for three days with vehicle (PBS)(grey bars), supernatant from SE-producing S. aureus (red bars), and supernatant from endolysin (MEndoB) treated SE-producing S. aureus (green bars). Cytokine release was measured using MSD human cytokine analysis kits and measures are expressed as pg / ml in culture supernatants.Figure 5.

[0143] Figure 5 depicts the flow cytometry results showing flow cytometric gating-strategy, contour plots, and quantifications of percentage of viable CD4+ T cells isolated from blood from two healthy donors and treated with vehicle (control), supernatant from SE-producing S. aureus, supernatant from endolysin (MEndoB)-treated SE-producing S. aureus, and supernatant from SE-producing S. aureus treated with an inactive (mutated MEndoB) endolysin. (A and B) Cells from donor 1 (A) and donor 2 (B) treated with and without supernatant from a standard culture of patients derived S. aureus and with and without MEndoB and mutated MEndoB. (C and D) Cells from donor 1 (C) and donor 2 (D) treated with and without supernatant from an additional AD patient derived S. aureus culture treated with and without MEndoB and mutated MEndoB. (E and F) Cells from donor 1 (E) and donor 2 (F) treated with and without supernatant from S. aureus from a new independent cohort of AD patients treated with and without MEndoB and mutated MEndoB.

[0144] Definitions

[0145] "Sequence identity" is herein defined as a relationship between two or more amino acid (peptide, polypeptide, or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide or polypeptide to the sequence of a second peptide or polypeptide. In a preferred embodiment, identity or similarity is calculated over the whole SEQ ID NO as identified herein. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

[0146] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S„ et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S„ et al., J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, Wl. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps). Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons. Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalaninetyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu ortyr; Serto thr; Thrto ser; Trp to tyr; Tyrto trp or phe; and, Val to ile or leu.

[0147] A “nucleic acid molecule” or “polynucleotide” (the terms are used interchangeably herein) is represented by a nucleotide sequence. A “polypeptide” is represented by an amino acid sequence. A “nucleic acid construct” is defined as a nucleic acid molecule which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acids which are combined or juxtaposed in a manner which would not otherwise exist in nature. A nucleic acid molecule is represented by a nucleotide sequence. Optionally, a nucleotide sequence present in a nucleic acid construct is operably linked to one or more control sequences, which direct the production or expression of said peptide or polypeptide in a cell or in a subject.

[0148] “Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the nucleotide sequence coding for the polypeptide of the invention such that the control sequence directs the production / expression of the peptide or polypeptide of the invention in a cell and / or in a subject. “Operably linked” may also be used for defining aconfiguration in which a sequence is appropriately placed at a position relative to another sequence coding for a functional domain such that a chimeric polypeptide is encoded in a cell and / or in a subject.

[0149] “Expression” is construed as to include any step involved in the production of the peptide or polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion.

[0150] A “control sequence” is defined herein to include all components which are necessary or advantageous for the expression of a polypeptide. At a minimum, the control sequences include a promoter and transcriptional and translational stop signals. Optionally, a promoter represented by a nucleotide sequence present in a nucleic acid construct is operably linked to another nucleotide sequence encoding a peptide or polypeptide as identified herein.

[0151] The term "transformation" refers to a permanent or transient genetic change induced in a cell following the incorporation of new DNA (i.e. DNA exogenous to the cell). When the cell is a bacterial cell, as is intended in the present invention, the term usually refers to an extrachromosomal, selfreplicating vector which harbors a selectable antibiotic resistance.

[0152] An “expression vector” may be any vector which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of a nucleotide sequence encoding a polypeptide of the invention in a cell and / or in a subject. As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes or nucleic acids, located upstream with respect to the direction of transcription of the transcription initiation site of the gene. It is related to the binding site identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites, and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. Within the context of the invention, a promoter preferably ends at nucleotide -1 of the transcription start site (TSS).

[0153] A “polypeptide” as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules.

[0154] Sequence identity herein of a polynucleotide, polynucleotide construct or of a polypeptide is preferably at least 70%. Preferably at least 70% is defined as preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, more preferably at least 98%, more preferably at least 99%, or most preferably 100% sequence identity. In case of 100%sequence identity, the polynucleotide or polypeptide has exactly the sequence of the depicted SEQ ID NO:. Sequence identity is preferably determined over the entire length of the subject sequence. The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.

[0155] In this document and in its claims, the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of’ meaning that a product or a composition or a nucleic acid molecule or a peptide or polypeptide of a nucleic acid construct or vector or cell as defined herein may comprise additional component(s) than the ones specifically identified; said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 10% of the value.

[0156] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0157] Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.

[0158] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

[0159] Further embodiments

[0160] Further embodiments of the invention are listed here below.

[0161] 1. An antibacterial enzyme effective against Staphylococcus aureus for use in the treatment of a subject suffering from Atopic Dermatitis (AD), wherein the subject’s AD lesions are colonized with or infected with Staphylococcus aureus producing Staphylococcus aureus superantigen (SE).

[0162] 2. An antibacterial enzyme effective against Staphylococcus aureus for use according to embodiment 1 , wherein the subject has an overactivation of the immune system characterized by CD4+T cells that are refractive to an agent that inhibits IL-4 and / or IL-13 signalling.

[0163] 3. An antibacterial enzyme effective against Staphylococcus aureus for use according to embodiment 1 of 2, wherein said treatment comprises administration of an effective amount of the antibacterial enzyme to the subject.4. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of embodiments 1 to 3, wherein, further to the antibacterial enzyme, an agent that inhibits IL-4 and / or IL-13 signalling, is administered.

[0164] 5. An antibacterial enzyme effective against Staphylococcus aureus for use according to embodiment 4, wherein the agent that inhibits IL-4 and / or IL-13 signalling is an IL-4 receptor antagonist or an IL-4 neutralizing agent and / or an IL-13 receptor antagonist or an IL-4 neutralizing agent.

[0165] 6. An antibacterial enzyme effective against Staphylococcus aureus for use according to embodiment 5, wherein the further administered agent is an antibody specific for IL-4 and / or IL-13, such as the monoclonal antibody Dupilumab.

[0166] 7. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of the embodiments 1 to 6, wherein the antibacterial enzyme is selected from the group consisting of: a bacteriocin or a functional part thereof, a bacterial lysin or autolysin or a functional part thereof, a bacteriophage lysin or a functional part thereof, and a chimeric lysin or a functional part thereof.

[0167] 8. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of embodiments 1 to 7, wherein the antibacterial enzyme is a chimeric lysin, preferably a recombinant chimeric endolysin comprising one or more heterologous domains

[0168] 9. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of embodiments 1 to 8, wherein the Staphylococcus aureus is an antibiotic-resistant Staphylococcus aureus, such as a methicillin resistant Staphylococcus aureus (MRSA).

[0169] 10. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of embodiments 1 to 9, wherein the antibacterial enzyme is present in a composition, such as a pharmaceutical composition, and wherein the composition further comprises a pharmaceutically acceptable excipient.

[0170] 11. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of embodiments 1 to 10, wherein the subject is a mammal, such as a livestock animal, a domestic animal or a human.

[0171] 12. Use of an antibacterial enzyme effective against Staphylococcus aureus in the treatment of a subject suffering from a condition associated with overactivation of the immune system by CD4+T cells that are refractive to an IL-4 receptor antagonist and / or an IL-13 receptor antagonist.13. A method of treatment of a subject suffering from a condition associated with overactivation of the immune system by CD4+T cells that are refractive to an IL-4 receptor antagonist and / or an IL-13 receptor antagonist, said treatment comprising administration of an effective amount of the antibacterial enzyme to the subject.

[0172] 14. A method of blocking CD4+T cell refractiveness to an IL-4 receptor antagonist and / or an IL-13 receptor antagonist, comprising contacting the CD4+T cell with an antibacterial enzyme effective against Staphylococcus aureus.

[0173] Example 1 : SE-producing Staphylococcal aureus enhances survival of pathogenetic IL-4 / IL-13-responsive CD4+T cells

[0174] Abstract

[0175] Cytokine-producing CD4+ T helper (TH) cells play a key role in the pathogenesis of Atopic Dermatitis (AD) driving inflammation, skin barrier impairment, and itching through the release of cytokines such as IL-4 and IL-13 (TH2-type cytokines), IL-22, and other factors. Staphylococcus aureus colonization is common in AD skin and is hypothesized to be associated with flares and disease progression. Yet, no studies so far have demonstrated that Staphylococcus aureus and staphylococcal enterotoxins may affect survival and death of cytokine (IL-4 and IL-13) responsive CD4+T cell isolated from AD skin lesions.

[0176] Here, we used IL-2 and IL-4 to expand primary CD4+T cells from lesional skin from AD and demonstrated for the first time that most of the expanded CD4+ T cells die (1) following treatment with an antibody (Dupilumab) that blocks IL-4 / IL-13 receptor-binding and (2) following IL-4 / IL-13 depravation. Surprisingly, treatment with supernatant from enterotoxin-producing Staphylococcus and purified Staphylococcus aureus enterotoxins (SE) rescued a large fraction of primary CD4+T cells from cell death following IL-4 / IL-13 depravation. In addition, purified SE induced survival in a large fraction of lesional CD4+T cells treated with (1) dupilumab and (2) following IL-4 and IL-13 depravation.

[0177] Importantly, the effect of SE-producing Staphylococcus aureus could be inhibited by an endolysin effective against the Staphylococcus.

[0178] In conclusion, our findings show for the first time that SE can induce survival of pathogenetic IL-4 / IL-13 responsive CD4+T cells isolated from AD skin and thereby circumvent the effect of Dupilumab and removal of IL-4 and IL-13. Accordingly, our findings provide first evidence that SE-producing Staphylococcal aureus can enhance survival of pathogenetic IL-4 / IL-13 responsive CD4+T cells despite treatments that inhibit the IL-4 / IL-13 environment. As a remedy for AD patients that are refractive to an agent that inhibits IL-4 and / or IL-13 signalling, these patients can be treated with an antibacterial enzyme, such as an endolysin, effective against Staphylococcal aureus.

[0179] IntroductionAD is a relatively common chronic inflammatory skin disorder that is associated with severe itching and skin damage with development of often severe skin lesions and symptoms which compromise the quality of life (1 ,2). CD4+T cells are believed to play a key role in the pathogenesis by producing a series of cytokines that triggers inflammation, itching, and damage to the skin barrier which in turn leads to skin colonization and infection with bacteria such as Staphylococcus aureus (2,3). Staphylococcus aureus colonization or infection has been linked to disease flare because increased amounts of these bacteria are detected during flare-ups (4). Although the mechanisms by which Staphylococcus aureus aggravates the pathogenesis and fuels disease flares are not known in detail, multiple staphylococcal toxins and factors have been implicated. For instance, the staphylococcal alpha-toxin seems to elicit damage to keratinocytes and therefore hypothesized to compromise the structure of the epidermis while a staphylococcal V8 protease stimulate itching and scratching leading to subsequent skin damage (5). A large fraction of AD patients harbours Staphylococcus aureus that produce SE (4-6), which are also known as superantigens because they are potent activators of CD4+T cells (7). Several studies on peripheral blood T cells from AD patients indicate that SE induces increased production of T helper cell (TH) type 2 (TH2) cytokines such as IL-4 and IL-13, which have been implicated in the pathogenesis in AD (8). Yet, it remains unknown how SE-producing Staphylococcus aureus impact AD. Accordingly, we herein examined this question in primary lesional CD4+T cells directly expanded from freshly biopsied lesional AD skin.

[0180] Materials and Methods

[0181] Biopsy and cell culture

[0182] Punch biopsies were collected from the lesional skin of AD patients and shipped in RPMI 1640 media containing IL-2 (2 x 103U / mL). Biopsy from each patient were cultured ex-vivo in 24-well plates in RPMI 1640 media supplemented with 10% pooled human serum and 1% antibiotics (penicillin and streptomycin) along with IL-2 and IL-4 (10 ng / mL). Cells were either cryopreserved or used subsequently for experiments. Cells were cryopreserved in vials with a preservative media (RPMI 1640 media with 30% human serum and 20% dimethyl sulfoxide) and stored in either liquid nitrogen tanks or -150° C freezer. For experiments, cryopreserved vials were taken out of the freezer, quickly thawed in a 37° C water bath, and washed twice in RPMI media by centrifugation and then resuspended in culture media.

[0183] For experiments, cells were washed and cultured in RPMI 1640 media with and without IL-4 and IL-13 (10 ng / mL each). For Staphylococcal enterotoxins (SE) experiments, cells were treated in the presence or absence of 10 to 50 ng / mL of a pool of SE (SEA, SEB, SEC, SED and SEI) followed by treatment with or without Dupilumab. Cells were cultured for 3 and 6 days and flow cytometry was performed.

[0184] For supernatant experiments, cells were treated as above with and without supernatant from patient-derived S. aureus treated with and without endolysin (XZ.700 (Figure 2A) or MEndoB (Figures 3D, 4, 5) and an enzymatically inactive (mutated) form of endolysin (mutated MEndoB) (Figure 5) prior to flow cytometric analysis as above. In control experiments, peripheral bloodmononuclear cells (PBMC) were isolated from healthy volunteers and cultured as described herein above.

[0185] Bacterial isolation and culture

[0186] Staphylococcus aureus (S. aureus) from skin was isolated and validated for SE expression in our previous study (9). S aureus was prepared from an overnight culture diluted to an optical density 600 nm of 0.01 in tryptic soy broth and regrown for 4 hours with or without endolysin. For XZ.700 experiments, 1 pg / mL XZ.700 endolysin (an engineered S aureus-specific endolysin known in the art as extensively described in e.g. WO2017 / 046021 and Eichenseher et al, 2022) was added. For MEndoB experiments, 1 pg / mL MEndoB endolysin (known in the art and extensively described in e.g. WO2024 / 133850 and Roehrig et al, 2024) or 1 pg / mL of an inactive form of MEndoB (mutated MendoB (inactive)), wherein active site residues were knocked out in the CHAP domain and in the central M23-peptidase domain. All bacterial supernatants were then centrifuged (10.000g for 10 minutes) and filter sterilized (0.22 pm filter). For cell culture experiments, cells were cultured in the presence or absence of the bacterial supernatants treated with and without XZ.700, MEndoB, and mutated MEndoB.

[0187] Flow cytometry

[0188] At the end of cell culture, flow cytometry was performed to assess cell death / viability. Cultured cells were pelleted by centrifugation, washed using FACS-PBS buffer (phosphate-buffered saline [PBS] + 1% fetal bovine serum + 0.02% NaNs). Cell surface staining (30 minutes on ice) was performed using Brilliant Stain buffer. After cell surface staining, cells were stained with propidium Iodide and annexin V suspended in annexin V binding buffer to study cell viability / death and apoptosis. Flow cytometric analysis was conducted using a 5-laser BD LSR-Fortessa. Data was analyzed using FlowJo (TreeStar).

[0189] Results

[0190] Figure 1 illustrates the experimental workflow: Staphylococcus aureus was isolated from skin swabs and cultured with or without endolysin prior to harvest of the supernatant (Figure 1 , upper first row). In parallel, skin biopsies were taken from AD patient skin lesions and cultured ex vivo with medium containing cytokines (Figure 1 , second middle row). When expanded sufficiently, cells were treated with or without cytokines, Staphylococcus aureus supernatant, and Dupilumab (which inhibits IL-4 and IL-13 binding to the shared IL-4 receptor) for 3 and 6 days prior to flow cytometric analysis of CD4+T cells (Figure 1 , third row). As shown in Figure 2A, the majority of CD4+T cells died in cultures without cytokines (IL-4 and IL-13). Thus, only 16% CD4+T cells were alive following cytokine depravation as judged from annexin V and propidium iodide staining of CD4+T cells (Figure 2A, left). In contrast, approximately 50% survived in IL-4 and IL-13 treated cultures (Figure 2A, right). Of note, supernatant from SE-producing Staphylococcus aureus induced survival in 35% of CD4+T cells deprived of IL-4 and IL-13 (Figure 2A, second plot) - an effect which was inhibited by pretreatment of the bacteria with endolysin (XZ.700) (Figure 2A, third plot). To address whether theeffect could be ascribed to SE, the cells were treated with or without SE, cytokines, and dupilumab prior to analysis for survival of CD4+T cells. As shown in Figure 2B (upper row), SE, IL-4 and IL-13, and the combination of both significantly increased the survival of CD4+T cells from 19 % in the untreated cultures to approximately 50% in cytokine treated cultures and >60% in SE treated cultures. As expected, Dupilumab completely blocked the effect of IL-4 and IL-13 on CD4+T cells survival (Figure 2B, upper versus lower 3rdplots). Importantly, SE treatment rescued more than 50% of the Dupilumab treated CD4+T cells from cell death irrespectively of the presence or absence of IL-4 and IL-13 (Figure 2B, lower row).

[0191] Essentially similar results were obtained in independent experiments using CD4+ T cells derived from skin lesions from other AD patients (Figure 3). Thus, in lesional CD4+ T cells from an additional patient, treatment with SE, IL-4 and IL-13, (and the combination) significantly increased the survival of CD4+T cells (up to 7-fold) compared with untreated cultures (Figure 3A, upper row). Importantly, Dupilumab completely blocked the effect of IL-4 and IL-13 on CD4+T cells survival (Figure 3A, upper versus lower 3rdplots) whereas SE treatment rescued approximately 50% of the Dupilumab treated CD4+T cells from cell death irrespectively of the presence or absence of IL-4 and IL-13 (Figure 3A, lower row). A similar response pattern was observed in a second additional patient shown in Figure 3B, where the effect of Dupilumab on cell death was bypassed by SE irrespectively of the presence or absence of IL-4 and IL-13 (Figure 3B). As SE is known to induce secretion of various IL-2 family cytokines including IL-2 (a classical growth and survival factor in T cells), we tested survival of lesional CD4+ T cell in cultures (from the patient used in the experiments in Figure 2) treated with and without IL-2 and with and without IL-4 / IL-13 and with and without Dupilumab. As expected, Dupilumab had no effect on cellular survival induced by IL-2 (Figure 3C). Importantly, however, exogenous IL-2 bypassed Dupilumab-induced cell death in T cell cultures treated with IL-4 and IL-13 (Figure 3C). Figure 3D shows S. aureus supernatant induced survival in lesional CD4+ T cells from a third additional patient. Thus, S. aureus supernatant increased cellular survival from 46% to 62% in IL-4 and IL-13 cultures (Figure 3D, upper row). This effect was absent in T cell cultures treated with supernatant from endolysin (MEndoB) treated S. aureus (Figure 3D, upper right panel). As shown in the lower panel of Figure 3D, Dupilumab inhibited survival from 46 to 32 % (Figure 3D upper left versus lower left). However, the effect of Dupilumab was blocked in cultures treated with supernatant from patient-derived S. aureus (Figure 3A). In line with the data above, supernatant from endolysin (MEndoB) treated S. aureus did not rescue from Dupilumab-induced cell death (Figure 3A, lower panel). As IL-2 can bypass the Dupilumab effect we used a multiplexcytokine detection assay (V-PLEX Human Biomarker 39-Plex Kit, MesoScale Discovery) to examine whether supernatant from enterotoxin-producing S. aureus induced secretion of IL-2 and other potential survival factors in lesional CD4+ T cells. Indeed, as shown in Figure 4, lesional CD4+ T cells produced multiple cytokines including IL-2, IL-4, and IL-13 when exposed to supernatant from SE-producing S. aureus (Figure 4). Importantly, induction of IL-2 and other cytokines was almost completely blocked using MEndoB-treated S. aureus supernatant (Figure 4). Control experiments with healthy donor PBMC stimulated with supernatant from S. aureus derived from skin lesions from different patient cohorts confirmed the potential of patient-derived enterotoxinproducing S. aureus to activate CD4+ T cells and showed that MEndoB efficiently block the S. aureus effect (Figure 5A-F)) whereas mutated MEndoB did not (Figure 5 A-F, lower lanes) indicating that the effect of endolysin was critically dependent on the enzymatic activity (Figure 5).

[0192] Discussion

[0193] The present data show for the first time that supernatant from SE-producing Staphylococcus aureus and purified SE can increase survival of primary CD4+T cells derived from AD skin lesions and can rescue AD derived CD4+T cells from cell death following IL-4 and IL-13 depravation. Importantly, the rescue effect by the bacteria was inhibited by pre-treatment of the bacteria with a Staphylococcus aureus specific endolysin (XZ.700 and MEndoB). Thus, these findings provide evidence that SE- producing Staphylococcus aureus can induce prolonged survival of pathogenetic CD4+T cells in patients - even when treated with modalities that target (i) IL-4 and IL-13 and / or, (ii) IL-4 / IL-13 receptors, and / or (iii) signal transduction downstream of IL-4 / IL-13 receptors. In support, SE induced an enhanced survival in lesional CD4+T cells in the presence of Dupilumab in concentrations that blocked IL-4 and IL-13 induced survival. In support, SE-producing Staphylococcus aureus induced secretion by lesional CD4+ T cells of a series of cytokines in addition to induction of the disease associated cytokines (IL-4, IL-13, IL- 5) in CD4+ T cells from lesional AD. Of note, SE-producing Staphylococcus aureus induced IL-2 secretion in lesional AD CD4+ T cells, an effect blocked when S. aureus was treated with an endolysin. As IL-2 is a potent survival and proliferation factor that is not inhibited by dupilumab, and IL-2 rescued lesional CD4+ T cells from Dupilumab-induced cell death in cultures with IL-4 and IL-13, the data also provide evidence for a molecular mechanism whereby SE-producing Staphylococcus aureus induce resistance to Dupilumab. They also provide an explanation for the mechanism whereby treatment with endolysin inhibits S. aureus mediated resistance and reestablishes the sensitivity to dupilumab-induced cell death. Taken together, these findings indicate that SE circumvents the blocking effect of Dupilumab and dupilumab-induced cell-death in pathogenetic CD4+T cells. Thus, our findings provide novel insight into the mechanism by which SE-producing Staphylococcus aureus may both fuel the pathogenesis, increase flare-ups, and inhibit the efficacy of IL-4 / IL-13 targeting treatments like dupilumab. Moreover, they indicate that SE-producing Staphylococcus aureus can induce dupilumab resistance in AD patients.

[0194] Indirect evidence for an involvement of SE in the generation of TH2 responses in AD patients came from analysis of TCRVp repertoires in peripheral blood of AD patients. Thus, higher frequences of T cells expressing SE-responsive TCRVp families were detected in peripheral blood from AD patients compared with healthy controls (10,11). A series of functional studies have also reported on cytokine expression in CD4+T cells isolated from peripheral blood from AD patients - thus, it was concluded that peripheral CD4+T cells from AD patients have a TH2 bias with higher expression of IL-4 and IL-13 and a TH22 bias with cells producing IL-22 than their healthy counterpart (8). Histological and transcriptomics studies have confirmed a TH2 and TH22 bias in situ in AD lesions (8,12,13) supporting the notion that TH2 CD4+T cells play a key role in thepathogenesis of AD. Importantly, some animal studies have indicated that SE promote an atopylike phenotype and dermatitis in mice and studies on human peripheral T cells show enhanced cytokine expression in peripheral T cells upon SE stimulation, while others suggest that staphylococcal toxins and factors drive TH2 responses via indirect mechanisms involving keratinocytes and dendritic cells. In a recent report, Orfali et al showed that SE trigger anergy in peripheral AD CD4+T cells and downregulated active effector T cell responses (14). While much is known about the pathological role of TH2 and TH22 cytokines in relation to skin damage and pruritus, little is known about the role of these cytokines as survival factors to CD4+T cells in situ in AD lesions. Likewise, until the present investigation, it was unknown that SE can modulate survival of lesional CD4+T cells and impact the effect of Dupilumab on survival of IL-4 and / or IL-13 responsive CD4+T cells derived from AD skin lesions. Because our findings contrast with the findings of SE induced anergy in peripheral blood CD4+ T cells from AD patients (14), it seems that crucial differences exist between lesional CD4+T cells and peripheral blood CD4+T cells. Importantly, our findings demonstrate that SE-producing Staphylococcal aureus can sustain survival of pathogenetic lesional CD4+T cells and can induce resistance to IL-4 / IL-13 blockage.

[0195] In conclusion, our findings show for the first time that SE can induce survival of pathogenetic IL-4 / IL-13 responsive CD4+T cells isolated from AD skin and thereby circumvent the effect of Dupilumab and of removal of IL-4 and IL-13. Accordingly, our findings provide first evidence that SE-producing Staphylococcal aureus enhances survival of pathogenetic IL-4 / IL-13-responsive CD4+T cells despite treatments that inhibit IL-4 / IL-13, IL-4 / IL-13 receptors, and downstreamsignalling pathways. As a remedy for AD patients that are refractive to an agent that inhibits IL-4 and / or IL-13 signalling, these patients can be treated with an antibacterial enzyme, such as an endolysin, effective against Staphylococcal aureus, as demonstrated in the non-limiting examples herein using chimeric endolysins XZ.700 and MEndoB. The person skilled in the art will understand that these results can be extrapolated to any antibacterial enzyme, such as an endolysin, effective against Staphylococcal aureus.References

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Claims

39Claims1. An antibacterial enzyme effective against Staphylococcus aureus for use in the treatment of a subject suffering from Atopic Dermatitis (AD), wherein the subject’s AD lesions are colonized with or infected with Staphylococcus aureus producing Staphylococcus aureus superantigen (SE).

2. An antibacterial enzyme effective against Staphylococcus aureus for use according to claim 1 , wherein the subject has an overactivation of the immune system characterized by CD4+T cells that are refractive to an agent that inhibits IL-4 and / or IL-13 signalling.

3. An antibacterial enzyme effective against Staphylococcus aureus for use according to claim 1 of 2, wherein said treatment comprises administration of an effective amount of the antibacterial enzyme to the subject.

4. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of claims 1 to 3, wherein, further to the antibacterial enzyme, an agent that inhibits IL-4 and / or IL-13 signalling, is administered.

5. An antibacterial enzyme effective against Staphylococcus aureus for use according to claim 4, wherein the agent that inhibits IL-4 and / or IL-13 signalling is an IL-4 receptor antagonist or an IL-4 neutralizing agent and / or an IL-13 receptor antagonist or an IL-4 neutralizing agent.

6. An antibacterial enzyme effective against Staphylococcus aureus for use according to claim 5, wherein the further administered agent is an antibody specific for IL-4 and / or IL-13, such as the monoclonal antibody Dupilumab.

7. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of the claims 1 to 6, wherein the antibacterial enzyme is selected from the group consisting of: a bacteriocin or a functional part thereof, a bacterial lysin or autolysin or a functional part thereof, a bacteriophage lysin or a functional part thereof, and a chimeric lysin or a functional part thereof.

8. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of claims 1 to 7, wherein the antibacterial enzyme is a chimeric lysin, preferably a recombinant chimeric endolysin comprising one or more heterologous domains9. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of claims 1 to 8, wherein the Staphylococcus aureus is an antibiotic-resistant Staphylococcus aureus, such as a methicillin resistant Staphylococcus aureus (MRSA).4010. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of claims 1 to 9, wherein the antibacterial enzyme is present in a composition, such as a pharmaceutical composition, and wherein the composition further comprises a pharmaceutically acceptable excipient.

11. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of claims 1 to 10, wherein the subject is a mammal, such as a livestock animal, a domestic animal or a human.

12. Use of an antibacterial enzyme effective against Staphylococcus aureus in the treatment of a subject suffering from a condition associated with overactivation of the immune system by CD4+T cells that are refractive to an IL-4 receptor antagonist and / or an IL-13 receptor antagonist.

13. A method of treatment of a subject suffering from a condition associated with overactivation of the immune system by CD4+T cells that are refractive to an IL-4 receptor antagonist and / or an IL-13 receptor antagonist, said treatment comprising administration of an effective amount of the antibacterial enzyme as defined in any one of claims 1 to 13 to the subject.

14. A method of blocking CD4+T cell refractiveness to an IL-4 receptor antagonist and / or an IL-13 receptor antagonist, comprising contacting the CD4+T cell with an antibacterial enzyme effective against Staphylococcus aureus.

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