Treatment of lymphoma

Abstract

The invention relates to the field of medicine, more specifically to treatment of lymphoma.

Description

[0001] Treatment of lymphoma

[0002] Field of the invention

[0003] The invention relates to the field of medicine, more specifically to treatment of lymphoma.

[0004] Background of the invention

[0005] Sezary syndrome (SS) is a malignant disorder of T cells that belongs to the spectrum of Cutaneous T cell lymphoma (CTCL), a non-Hodgkin lymphoma. SS is a leukemic variant of cutaneous T cell lymphoma (1-4). SS patients are prone to infection with Staphylococcus aureus (S. aureus) and have a poor prognosis due to treatment-resistance.

[0006] There is thus a need to improve treatment and outcome of treatment of Sezary syndrome.

[0007] Description of the invention

[0008] The inventors have established that S. aureus and its toxins (staphylococcal enterotoxins (SE)) induce drug resistance in malignant T cells, specifically resistance against histone deacetylase (HDAC) inhibitors. These findings provide one of the missing links between S. aureus and cancer and grant an explanation how skin colonization by SE-producing bacteria can fuel disease activity in CTCL.

[0009] Accordingly, in a first aspect, there is provided for an antibacterial enzyme effective against Staphylococcus aureus for use in the treatment of a subject suffering from cancer refractive to a chemotherapeutic drug, preferably a histone deacetylase (HDAC) inhibitor, said treatment comprising administration of an effective amount of the antibacterial enzyme to the subject.

[0010] In the embodiments herein, the antibacterial enzyme effective against Staphylococcus aureus for use is referred to as “an” or “the antibacterial enzyme effective against Staphylococcus aureus for use according to the invention”, “an” or “the antibacterial enzyme as disclosed herein” or plainly “an” or “the antibacterial enzyme”.

[0011] 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 SS, in the subject. Preferably, the effective amount abrogates the resistance of the cancer cells to the chemotherapeutic drug, preferably a histone deacetylase (HDAC) inhibitor, such that the effectiveness of the chemotherapeutic drug is 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.

[0012] In the embodiments herein, the term “cancer refractive to a chemotherapeutic drug” means that the cancer cells are less sensitive to the chemotherapeutic drug, preferably a histone deacetylase (HDAC) inhibitor, due to interference of the SE, see Figure 7 for hypothesized mechanisms. Less sensitive herein means that the cancer cells are 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 less sensitive to the chemotherapeutic drug, preferably a histone deacetylase (HDAC) inhibitor, compared to when no SE is present.

[0013] In the embodiments herein, administration of the antibacterial enzyme or composition comprising the antibacterial enzyme, to the subject, may be performed in any way known to the person skilled in the art, such as systemic, local, intravenous, topical, oral, via a dressing, such as a wound dressing, via a bandage, rectal, vaginal, intramuscular, trans-arterial, intraperitoneal, intranasal, subcutaneous, endoscopic, transdermal, mucosal and intrathecal administration.

[0014] In the embodiment herein, the chemotherapeutic drug where the cancer is refractive to, may be an anthracycline such as Doxorubin, a topoisomerase II inhibitor such as Etoposide, and / or preferably a histone deacetylase (HDAC) inhibitor, such as Vorinostat, Romidepsin, Resminostat, Panobinostat, and Belinostat. In an embodiment, the histone deacetylase (HDAC) inhibitor is Vorinostat, In an embodiment, the histone deacetylase (HDAC) inhibitor is Romidepsin. In an embodiment, the histone deacetylase (HDAC) inhibitor is Resminostat. In an embodiment, the histone deacetylase (HDAC) inhibitor is Panobinostat. In an embodiment, the histone deacetylase (HDAC) inhibitor is Belinostat.

[0015] In the embodiments herein, the treatment comprising administration of an effective amount of the antibacterial enzyme to the subject may comprise that further to the antibacterial enzyme, a chemotherapeutic drug is administered to the subject.

[0016] In the embodiments herein, the cancer may be any type of cancer that is refractive to a chemotherapeutic drug, preferably a histone deacetylase (HDAC) inhibitor, such as, but not limited to, lymphoma, such as cutaneous T cell lymphoma (CTCL), such as the leukemic variant Sezary syndrome (SS), which are extensively described elsewhere herein. Accordingly, in the embodiments herein, the cancerthat is refractive to a chemotherapeutic drug, preferably a histone deacetylase (HDAC) inhibitor, may be the leukemic variant Sezary syndrome of CTCL.

[0017] In the embodiments herein, the chemotherapeutic drug further administered may be any chemotherapeutic drug where a cancer may be refractive to. Accordingly, in the embodiments herein, the chemotherapeutic drug may be an anthracycline such as Doxorubin, a topoisomerase II inhibitor such as Etoposide, and / or a histone deacetylase (HDAC) inhibitor such as Vorinostat, Romidepsin, Resminostat, Panobinostat, and Belinostat. In the embodiments herein, the further administered chemotherapeutic drug preferably is a histone deacetylase (HDAC) inhibitor and may be Vorinostat. In the embodiments herein, the further administered chemotherapeutic drug may be Romidepsin. In the embodiments herein, the further administered chemotherapeutic drug may be Resminostat. In the embodiments herein, the further administered chemotherapeutic drug may be Panobinostat. In the embodiments herein, the further administered chemotherapeutic drug may be Belinostat.

[0018] In the embodiments herein, the cancer may be refractive to a chemotherapeutic drug, preferably a histone deacetylase (HDAC) inhibitor, due to a staphylococcal enterotoxin (SE), such as a SE produced by Staphylococcus aureus. Hypothesized mechanisms of how a staphylococcal enterotoxin (SE), such as a SE produced by Staphylococcus aureus is involved in refractiveness of the cancer to a chemotherapeutic drug, preferably a histone deacetylase (HDAC) inhibitor, is extensively described in the examples herein. Herein, the SE may be any staphylococcal enterotoxin. Herein, the SE may be a staphylococcal enterotoxin produced by Staphylococcus aureus, such as SE type A, SE type B, SE type C2, SE type D and / or SE type I. Herein , the SE may be staphylococcal enterotoxin (SE) type A.

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

[0020] 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.

[0021] 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.

[0022] 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).

[0023] 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.

[0024] 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 a phage-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. 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 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); 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); F1 16p62 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 coli 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 Mar 28;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 lambdoid bacteriophages. 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 coli 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 coli 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 K1 F (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), 1 115-1132); 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 and mosaicism 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. Characterization of Pseudomonas chlororaphis myo virus 201varphi2- I via genomic sequencing, mass spectrometry, and electron microscopy Virology 376 (2), 330-338 (2008)); Aehlp339 of phage Aehl (NCBI Reference Sequence: NP 944217.1 Petrov, V., NolanJ., Bertrand, C, Letarov.A.V., Krisch.H.M. and KaramJ.D); YYZgp45 of phage YYZ-2008 (GenBank: ACI32381.1 Zhang, Y., Laing, C.R., Kropinski.A. and Gannon, V.J.P.); the endolysin of the Pseudomonas aeruginosa phages <t>KZ, g I44 (J Biol Chem. 2008 Mar 14;283(11):7242-50. Structure of the bacteriophage phi KZ lytic transglycosylase gpl44. Fokine A, Miroshnikov KA, Shneider MM, Mesyanzhinov VV, Rossmann MG.), and EL, El. 1 88 (Mol Microbiol. 2007 Sep;65(5 ): 1334-44. Mura lytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages phiKZ and EL. 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. 2006 Dec 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. Korndorfer IP1 , Danzer J, Schmelcher M, Zimmer M, Skerra A, Loessner MJ), PlyA511 (NCBI Reference Sequence: YP 001468459.1), PlyP35 (GenBank: AAY53213.1), PlyP40 (NCBI Reference Sequence: YP 002261442.1), Staphylococcal phage Phi 11 endolysin (Lytic activity of recombinant bacteriophage phi 11 and phi 12 endolysins on whole cells and bio films of Staphylococcus aureus. Sass P, Bierbaum G. Appl Environ Microbiol. 2007 Jan;73(l):347-52), Phi MRU endolysin (NCBI Reference Sequence: YP 001604156.1), LysK (The recombinant phage lysin LysK has a broad spectrum of lytic activity against clinically relevant staphylococci, including methicillin-resistant Staphylococcus aureus. O'Flaherty S, Coffey A, Meaney W, Fitzgerald GF, Ross RP.J Bacteriol. 2005 Oct;187(20):7161-4), Clostridium perfringens PlyS9 (WO2010003943 (Al; Bacteriophage. 2012 Apr l;2(2):89-97. 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.).

[0025] 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.

[0026] 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, WO201 1 / 023702, WO2012 / 146738, WG2003 / 082184, WO2010 / 011960, WO2010 / 149795, WO2010 / 149792, WO2012 / 094004, WO201 1 / 023702, WO201 1 / 065854, WO201 1 / 076432, WO2011 / 134998, WO2012 / 059545,

[0027] 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.

[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: 7.

[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: 8.

[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: 9. 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.

[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: 17.

[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: 18.

[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: 19. 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.

[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: 27.

[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: 28.

[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: 29. 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.

[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: 37.

[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: 38.

[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: 39. 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.

[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: 47.

[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: 48.

[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: 49. 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.

[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: 57.

[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: 58.

[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: 59. 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.

[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: 67.

[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: 68.

[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: 69. 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.

[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: 77.

[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: 78.

[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: 79. 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 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 WO2015 / 155244 and WO2016 / 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 M-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 antibacterial enzyme. Such polynucleotide encoding for the antibacterial enzyme may conveniently be an mRNA. Said mRNA may be present in a delivery vehicle. Such mRNA constructs and delivery vehicles comprising such mRNA encoding the antibacterial enzyme are extensively described in EP 23190916.9, which is herein incorporated by reference. Use of the constructs described in EP 23190916.9 is explicitly included in the embodiments herein.

[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. In the embodiments herein, the antibacterial enzyme may be present in 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, 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. 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,

[0108] 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.

[0109] Exemplar / 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.

[0110] 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.

[0111] 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.

[0112] 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. 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.

[0113] 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.

[0114] 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.

[0115] 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. 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.

[0116] Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well, as high molecular weight polyethylene glycols and the like.

[0117] 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 ingredients) 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] Accordingly, in the embodiments herein, there is provided for a method of treatment of a subject suffering from a cancer refractive to a chemotherapeutic drug, preferably a histone deacetylase (HDAC) inhibitor, preferably a histone deacetylase (HDAC) inhibitor as described herein above, said treatment comprising administration of an effective amount of an antibacterial enzyme effective against Staphylococcus aureus to the subject.

[0122] Accordingly, in the embodiments herein, there is provided for the use of an antibacterial enzyme effective against Staphylococcus aureus for the manufacture of a medicament for the treatment of a cancer refractive to a chemotherapeutic drug, preferably a histone deacetylase (HDAC) inhibitor, preferably a histone deacetylase (HDAC) inhibitor as described herein above, said treatment comprising administration of an effective amount of an antibacterial enzyme effective against Staphylococcus aureus to the subject.

[0123] In a second aspect, there is provided for a method for blocking drug-resistance in primary malignant T-cells comprising contacting the malignant T-cells with an antibacterial enzyme effective against Staphylococcus aureus. The features of this aspect are preferably the features of the embodiments of the first aspect. The malignant T-cells may be from any subject suffering from a cancer that is refractive to a chemotherapeutic drug. The chemotherapeutic drug where the cancer is refractive to, may be an anthracycline such as Doxorubin, a topoisomerase II inhibitor such as Etoposide, and / or preferably a histone deacetylase (HDAC) inhibitor, such as Vorinostat, Romidepsin, Resminostat, Panobinostat, and Belinostat. In an embodiment, the histone deacetylase (HDAC) inhibitor is Vorinostat, In an embodiment, the histone deacetylase (HDAC) inhibitor is Romidepsin. In an embodiment, the histone deacetylase (HDAC) inhibitor is Resminostat. In an embodiment, the histone deacetylase (HDAC) inhibitor is Panobinostat. In an embodiment, the histone deacetylase (HDAC) inhibitor is Belinostat. Preferably, the malignant T-cells are malignant T-cells from SS patients.

[0124] Table 1 : Overview of sequences Figure legends

[0125] Figure 1. SE rescues malignant cells from Romidepsin induced apoptosis while endolysin abrogates this recue.

[0126] A. Flow cytometric plots depicting apoptotic populations of gated malignant cells from SS patient PBMC following 72hr treatment with Romidepsin and supernatant of S. aureus culture (alone or treated with endolysin) isolated from a different CTCL patient.

[0127] B. Western blot depicting PARP expression following 24 and 48 hr treatment of SS patient PBMC with either SE and / or Romidepsin. GAPDH is used as a loading control.

[0128] C. Plots depicting apoptotic populations of malignant cells gated from PBMC from SS patients (n=5) following 72hr and 144hr stimulation without SE (PBS) and with SE and treated with different concentrations of Romidepsin. A two-way ANOVA followed by Sidak's multiple comparisons test was performed. **P <0.01 , ***P<0.0005.

[0129] D. Plot depicting that SE rescues malignant cells from Romidepsin induced apoptosis treated at high concentration of Romidepsin (as mentioned in Romidepsin Pulse experiments in materials and methods).

[0130] E. Representative flow cytometric plots from SS patient PBMC showing apoptotic populations of gated malignant cells following 72hr treatment with either SEA or mutant SEA (SEAF47A / D227A) and treated with Romidepsin.

[0131] F. Bar plot representation of Figure 1 E. depicting that SEA but not the mutant SEA rescues malignant cells from Romidepsin induced apoptosis in SS patients (n=5).

[0132] G. Representative flow cytometric plots from SS patient PBMC showing apoptotic populations of gated malignant cells following 72hr treatment with either SEA or SEA and anti-SEA antibody and treated with Romidepsin.

[0133] H. Bar plot representation of Figure 1 G. depicting that SEA rescues malignant cells but that SEA blocking antibody inhibits rescue of malignant from Romidepsin induced apoptosis in SS patients (n=4).

[0134] For F. and H., RM one-way ANOVA followed by Tukey’s multiple comparison test was performed. *P<0.05.

[0135] Figure 2. SE largely overrides the effect of Romidepsin in malignant T cells.

[0136] A. Representative flow cytometric plots on gated malignant cells from a SS patient PBMC showing SE induced cell survival following 2 nM Romidepsin treatment for 72 hr.

[0137] B. Bar plot representation of Figure 2A. depicting that SE induces significant survival of malignant cells following Romidepsin treatment in SS patients. (n=11). Red dot shows the SS patient PBMC which did not respond to Romidepsin treatment. For statistical analysis, ordinary one-way ANOVA followed by Tukey’s multiple comparison test was performed. **P<0.01 , ***P<0.0005, ****P <0.0001.

[0138] C. - H. Single-cell cellular indexing of transcriptomes and epitopes sequencing (CITE-seq) analysis of SS PBMCs treated with romidepsin in the presence or absence of SE. Figure 3. SE rescues malignant cells from cell death induced by different drugs used in CTCL treatment.

[0139] Representative flow cytometric plots on gated malignant cells from a SS patient PBMC and bar plots depicting live malignant cell populations in more SS patients’ PBMC with and without SE stimulation following treatment with:

[0140] A. - B. Vorinostat (n=5),

[0141] C. - D. Resminostat (n=3),

[0142] E. - F. Doxorubicin (n=4) (Mitotracker is used instead of PI, otherwise the gating strategy for flow cytometry is similar to other drug treatments),

[0143] G. - H. Etoposide (n=5)

[0144] I. - J. Bortezomib (n=4).

[0145] For statistical analyses, ordinary one-way ANOVA followed by Dunnett's multiple comparisons test were performed. *P<0.05, **P<0.005, ***P<0.0005.

[0146] Figure 4. SE mediated rescue from Romidepsin can be blocked by targeting LCK and PKC kinases involved in TCR signaling.

[0147] Representative flow cytometric plots on gated malignant cells from a SS patient PBMC and bar plots depicting live malignant cell populations in more SS patients’ PBMC with and without SE stimulation and Romidepsin following treatment with:

[0148] A. - B. A-419259 (Src inhibitor) (n=8),

[0149] C. - D. Dasatinib (n=6),

[0150] E. - F. Sotrastaurin (PKC inhibitor) (n=5).

[0151] For statistical analyses, RM one-way ANOVA followed by Tukey’s multiple comparison test was performed. *P<0.05, **P<0.01 , **P<0.0005, ****P <0.0001.

[0152] Figure 5. IL-2 family cytokines rescue malignant cells from Romidepsin mediated cell death.

[0153] A. Representative flow cytometric plots on gated malignant cells from a SS patient PBMC depicting that treatment with cytokines (IL2, IL4, IL7, IL15) induces malignant cell survival following Romidepsin treatment for 72 hr.

[0154] B. Bar plot representation of Figure 5A. depicting the percent live malignant cell population following different treatments where cytokines treatment induced significant cell survival following Romidepsin treatment (n=7). For statistical analysis, ordinary one-way ANOVA followed by Tukey’s multiple comparison test was performed. ****P <0.0001 .

[0155] C. Representative flow cytometric plots post 72 hr study from a SS patient treated with Romidepsin depicting that SRC inhibitor (A-419259) and JAK inhibitor (Tofacitinib) only blocks SE stimulated or cytokine treated malignant cells respectively. Control shows untreated live cell population post 72hr. E. Representative flow cytometric plots from a JAK dependent SS patient depicting that Tofacitinib could inhibit SE induced malignant cell survival following Romidepsin treatment. D. and F. Bar plots depicting the percent malignant cell survival following SE stimulation for 72 hr and treatment with Romidepsin alone or with Tofacitinib. (Figure 5 D.: JAK dependent = SS Patients where tofacitinib + Romidepsin blocked SE mediated survival (n=2) and Figure 5 F.: JAK independent = SS patients where Tofacitinib couldn’t abrogate SE mediated survival following Romidepsin treatment (n=3)).

[0156] Figure 6. IL-2 family cytokines rescue malignant cells from Romidepsin mediated cell death.

[0157] A. Representative flow cytometric plots depicting the percentages of malignant cell populations pre- and post- sorting of SS patient PBMC.

[0158] B. Representative flow cytometric plots depicting malignant cell survival following 72 hr treatment on sorted malignant cells stimulated SE and treated with Romidpesin and JAK inhibitor (Tofacitinib) couldn’t block SE mediated survival (JAK independent).

[0159] C. Representative flow cytometric plots depicting that JAK inhibitor (Tofacitinib) blocked SE mediated survival of sorted malignant cells co-treated with Romidepsin (JAK dependent).

[0160] Figure 7. SE rescues Malignant T cells via direct and indirect TCR activation

[0161] Illustration of the putative mechanisms by which Staphylococcus aureus and it’s SE can induce drug resistance in malignant T cells - a direct TCR-driven, cytokine-independent pathway (Figure 7, upper part) and an indirect, cytokine-driven pathway which may involve bystander CD4 T cells (Figure 7, lower part). SRC inhibitor blocks SE mediated TCR dependent survival (both direct and indirect) whereas Tofacitinib only blocks cytokine dependent survival mediated via indirect activation.

[0162] Figure 8. SE rescues malignant cells from Romidepsin induced apoptosis while endolysin abrogates this recue.

[0163] A. Representative flow cytometric plots from a SS patient’s PBMC depicting that varying concentrations of Romidepsin induce apoptosis in malignant but not in nonmalignant T cells post 72 hr.

[0164] B. Representative flow cytometric plots depicting the apoptotic populations of gated malignant cells from an additional SS patient’s PBMC following 72hr treatment with Romidepsin and supernatant of S. aureus culture (alone or treated with endolysin) isolated from a different CTCL patient.

[0165] C. Representative flow cytometric plots depicting the apoptotic populations of gated malignant cells from an additional SS patient PBMC following 72hr treatment with Romidepsin and supernatant of two different cultures of S. aureus strains which produce (S. aureus sup (SEA)) or didn’t produce SEA (S. aureus sup (no SEA)).

[0166] D - E. Representative flow cytometric plots depicting apoptotic populations of gated malignant cells from two SS patients’ PBMC (left & right) following Romidepsin treatment for 24, 48 and 72 hr.

[0167] Definitions "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).

[0168] 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.

[0169] 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).

[0170] 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 or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

[0171] 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.

[0172] “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 a configuration 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.

[0173] “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.

[0174] 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.

[0175] 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. 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).

[0176] 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.

[0177] 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.

[0178] 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.

[0179] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0180] Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.

[0181] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

[0182] Further embodiments

[0183] Further embodiments of the invention are listed here below.

[0184] 1 . An antibacterial enzyme effective against Staphylococcus aureus for use in the treatment of a subject suffering from cancer refractive to a chemotherapeutic drug, said treatment comprising administration of an effective amount of the antibacterial enzyme to the subject.

[0185] 2. An antibacterial enzyme effective against Staphylococcus aureus for use according to embodiment 1 , wherein the chemotherapeutic drug where the cancer is refractive to, is an anthracycline such as Doxorubin, a topoisomerase II inhibitor such as Etoposide, and / or a histone deacetylase (HDAC) inhibitor,

[0186] 3. An antibacterial enzyme effective against Staphylococcus aureus for use according to embodiment 1 or 2 wherein the histone deacetylase (HDAC) inhibitor is Vorinostat, Romidepsin Resminostat, Panobinostat, or Belinostat.

[0187] 4. An antibacterial enzyme effective against Staphylococcus aureus for use according to anyone of embodiments 1 to 3, wherein, further to the antibacterial enzyme, a chemotherapeutic drug is administered to the subject.

[0188] 5. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of embodiments 1 to 4, wherein the cancer is lymphoma, such as cutaneous T cell lymphoma (CTCL), such as the leukemic variant Sezary syndrome (SS).

[0189] 6. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of any one of embodiments 4 and 5, wherein the further administered chemotherapeutic drug is an anthracycline such as Doxorubin, a topoisomerase II inhibitor such as Etoposide, and / or a histone deacetylase (HDAC) inhibitor such as Vorinostat, Romidepsin, Resminostat, Panobinostat, and Belinostat. 7. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of the preceding embodiments, wherein the cancer is refractive due to a staphylococcal enterotoxin (SE), such as a SE produced by Staphylococcus aureus.

[0190] 8. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of the preceding embodiments, 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.

[0191] 9. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of the preceding embodiments, wherein the antibacterial enzyme is a chimeric lysin, preferably a recombinant chimeric endolysin comprising one or more heterologous domains

[0192] 10. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of the preceding embodiments, wherein the Staphylococcus aureus is an antibiotic-resistant Staphylococcus aureus, such as a methicillin resistant Staphylococcus aureus (MRSA).

[0193] 11 . An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of the preceding embodiments, wherein the antibacterial enzyme is present in a composition, such as a pharmaceutical composition, and wherein the composition further comprises a pharmaceutically acceptable excipient.

[0194] 12. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of the preceding embodiments, wherein the subject is a mammal, such as a live stock animal, a domestic animal or a human.

[0195] 13. A method of treatment of a subject suffering from a cancer refractive to a chemotherapeutic drug, said treatment comprising administration of an effective amount of an antibacterial enzyme effective against Staphylococcus aureus to the subject.

[0196] 14. A method of treatment according to embodiment 13, wherein the chemotherapeutic drug where the cancer is refractive to, is an anthracycline such as Doxorubin, a topoisomerase II inhibitor such as Etoposide, and / or a histone deacetylase (HDAC) inhibitor.

[0197] 15. A method of treatment according to embodiment 14, wherein the histone deacetylase (HDAC) inhibitor is Vorinostat, Romidepsin Resminostat, Panobinostat, or Belinostat.

[0198] 16. Use of an antibacterial enzyme effective against Staphylococcus aureus for the manufacture of a medicament for the treatment of a cancer refractive to a chemotherapeutic drug, said treatment comprising administration of an effective amount of an antibacterial enzyme effective against Staphylococcus aureus to the subject.

[0199] 17. Use according to embodiment 16, wherein the chemotherapeutic drug where the cancer is refractive to, is an anthracycline such as Doxorubin, a topoisomerase II inhibitor such as Etoposide, and / or a histone deacetylase (HDAC) inhibitor.

[0200] 18. Use according to embodiment 17, wherein the histone deacetylase (HDAC) inhibitor is Vorinostat, Romidepsin Resminostat, Panobinostat, or Belinostat.

[0201] 19. A method for blocking drug-resistance in primary malignant T-cells comprising contacting the malignant T-cells with an antibacterial enzyme effective against Staphylococcus aureus.

[0202] 20. A method according to embodiment 19, wherein the chemotherapeutic drug where the cancer is refractive to, is an anthracycline such as Doxorubin, a topoisomerase II inhibitor such as Etoposide, and / or a histone deacetylase (HDAC) inhibitor.

[0203] 21. A method according to embodiment 20, wherein the histone deacetylase (HDAC) inhibitor is Vorinostat, Romidepsin Resminostat, Panobinostat, or Belinostat.

[0204] Examples

[0205] Abstract

[0206] Patients with Sezary syndrome (SS), a leukemic variant of cutaneous T cell lymphoma (CTCL), are prone to infection with Staphylococcus aureus (S. aureus) and have a poor prognosis due to treatment-resistance. Here, we report that S. aureus and staphylococcal enterotoxins (SE) induce drug-resistance in malignant T-cells against therapeutics commonly used in CTCL. Supernatant from patient-derived, SE-producing S. aureus and recombinant SE significantly inhibit cell death induced by histone deacetylase (HDAC) inhibitor Romidepsin in primary malignant T-cells from SS patients. Bacterial killing by an engineered, bacteriophage-derived, S. aureus-specific endolysin (XZ.700; SEQ ID NO: 76 herein; see e.g. WO 2017 / 046021). abrogates the effect of S. aureus supernatant. Likewise, mutations in MHC Class II binding sites of SE type-A (SEA) and anti-SEA antibody block induction of resistance. Importantly, SE also triggers resistance to other HDAC inhibitors (Vorinostat and Resminostat) and chemotherapeutic drugs (doxorubicin and etoposide). Multimodal single-cell sequencing indicates TCR, NFkB, and JAK / STAT signaling pathways (previously associated with treatment-resistance) as putative mediators of SE-induced drugresistance. In support, inhibition of TCR-signaling and Protein Kinase C (upstream of NFkB) counteracts SE-induced rescue from drug-induced cell death. Inversely, SE cannot rescue from cell death induced by proteasome / NFkB inhibitor Bortezomib. Inhibition of JAK / STAT only blocks SE- induced rescue of malignant T-cells in some but not other patients suggesting two distinct ways that SE can induce drug-resistance.

[0207] In conclusion, we have demonstrated that S. aureus enterotoxins induce drug-resistance in primary malignant T-cells. These findings indicate that S. aureus enterotoxins cause clinical treatmentresistance in SS patients and that anti-bacterial measures directed against S. aureus, such as treatment with an endolysin will improve the outcome of cancer-directed therapy in patients harboring S. aureus.

[0208] Introduction

[0209] Sezary syndrome (SS) is a malignant disorder of T cells that belongs to the spectrum of Cutaneous T cell lymphoma (CTCL), a non-Hodgkin lymphoma (1-4). The etiology of CTCL still remains unresolved (5). Dysregulation of JAK / STAT, PLCg, and NFkB signalling has repeatedly been linked to malignant cell proliferation, drug resistance, and inflammation in CTCL (6-8).

[0210] An important feature of CTCL is an increased susceptibility to bacterial infections, which are also major causes of disease morbidity and mortality (9-15). Skin barrier defects induced by the malignant T cells are likely ports of entrance for bacterial infection (16-17).

[0211] Here, we have demonstrated for the first time that SE-producing S. aureus can induce drugresistance in malignant T cells and that anti-bacterial therapy specifically targeting S. aureus in CTCL patients may improve the outcome of cancer-directed therapy.

[0212] Materials and Methods

[0213] Patient Material

[0214] This study includes 17 SS patients. The patient characteristics are mentioned in Table 2. Blood samples from SS patients were collected after obtaining written consent from patients as well as institutional approvals from Denmark (Committee on Health Research Ethics, H-16025331) and Germany. Experiments were performed in accordance with the Declaration of Helsinki.

[0215] PBMC isolation, cell culture, inhibitors, and pulse experiments.

[0216] Peripheral blood mononuclear cells (PBMCs) from blood of SS patients (SS PBMCs) were isolated by density gradient centrifugation following manufacturer’s protocol (Stem Cell Technologies). PBMCs were cultured in RPMI 1640 media (Sigma) supplemented with 10% pooled human serum (Copenhagen Hospital Blood Bank) and 1 % antibiotics (Penicillin and Streptomycin) (Sigma). For SE stimulation, a pool of SE (SEA, SEB, SEC2, SED and SEI) (Toxin Technologies) at 50-100 ng / mL was added to the cells before treatment with CTCL drugs. For cytokine experiments, a pool of IL-2 family cytokines - IL-2 (2 x 103U / ml, Novartis), IL-4, IL-7, and IL-15 (10 ng / mL each, Peprotech) was added. Inhibitors / drugs: A-419259 and Oligomycin (Sigma-Aldrich): Romidepsin, Dasatinib, Tofacitinib, Vorinostat, Resminostat, Doxorubicin, Bortezomib, Etoposide (Selleckchem); Crystal Violet and Sotrastaurin (MedChemExpress). For Romidepsin pulse experiments, cells were treated with high concentration of Romidepsin for 6 hours. Cells were washed and kept in normal media for 16 hours followed by incubation with and without SE pool for three days. Patient bacterial isolation and culture

[0217] S. aureus from CTCL patients was isolated and validated for the expression. Supernatants from S. aureus cultures were prepared as previously described (18). Briefly, S. aureus was prepared from an overnight culture diluted to an OD600nm of 0.01 in Tryptic Soy Broth (TSB) and regrown for 4 hours with or without 1 pg / mL XZ.700 (SEQ ID NO: 76 herein; see e.g. WO 2017 / 046021). Bacterial supernatants were prepared by centrifugation (10,000g for 10 minutes) followed by sterile filtration (0.22 pm filter).

[0218] Flow cytometry and cell sorting

[0219] Primary conjugated monoclonal antibodies against CD3, CD4, CD7, CD8, CD14, CD26, CD45, TCRCpI , TCRVpl , TCRVp2, TCRVp8, and TCRVpl 8 were purchased from BD Biosciences, BioLegend, Miltenyi Biotec, Beckman Coulter, or R&D Systems. Post culture, cells were pelleted by centrifugation, washed using FACS-PBS buffer (PBS + 1 % FBS + 0.02% NaNs). Cell surface staining (30 minutes on ice) was performed in FACS-PBS or Brilliant Stain Buffer (BD Biosciences). Malignant cells were gated as CD3+CD4+TCRVp#+. In patients where the dominant TCRVp clone could not be targeted, malignant cells were gated as CD3+CD4+CD7|OWand / or CD26- or CD3+CD4+CD26-TCRCp1 + / TCRCpI- (depending on whether the TCR of the malignant clone utilizes the TRBC1 or TRBC2 gene segment in its TCRp chain). Flow cytometric analysis was conducted using a 5-laser BD LSR-Fortessa at the Core Facility for Flow Cytometry at the University of Copenhagen. Flow cytometry data were visualized and analyzed using FlowJo (TreeStar) software. PI was added to exclude dead cells prior to sorting. Cell sorting was conducted on a 3- laser BD FACS Aria-ll, using a 100-pm nozzle. Purity of sorted cells was consistently above 95%.

[0220] Apoptosis assay and Western Blotting

[0221] For apoptotic experiments, at the end of cell culture, cells were stained with Mitotracker Red CMXRos (Thermofisher) by adding it to the culture media and were incubated at 37°C for 30 minutes. Cells were then washed, and flow cytometry procedure was followed. After cell surface staining, cells were stained with Propidium Iodide (PI) and Annexin V suspended in Annexin V binding buffer (BD Biosciences). Apoptotic cells were identified from Live (PI-) malignant or nonmalignant cells as MitotrackerlowAnnexin V+ i.e., if a patient had dominant TCRVpl clone, then the apoptotic population of malignant cells would be gatedas Pl- CD3+CD4+TCRVp1 +MitotrackerlowAnnexin V+. Western blotting was performed as previously described (20) and analyzed using Image Lab software (BioRad).

[0222] Cellular Indexing of Transcriptomes and Epitopes by Seguencing (CITE-seg)

[0223] For CITE-seq experiment, patient PBMC was treated with Romidepsin in the presence or absence of SE for 36 hours. Each condition was stained with a unique hashtag antibody together with a cocktail of TotalSeq-C surface antibodies. CITE-seq staining protocol and antibodies were individually optimized and titrated as previously described (19). Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seg)

[0224] For CITE-seq experiment, patient PBMC was treated with Romidepsin in the presence or absence of SE for 36 hours. Each condition was stained with a unique hashtag antibody together with a cocktail of TotalSeq-C surface antibodies. CITE-seq staining protocol and antibodies were individually optimized and titrated as previously described (19).

[0225] Statistics

[0226] Statistical analyses were performed using GraphPad Prism. Graphs were also made using that software. Error bars represent the standard error of the mean, and the level of statistical significance was set at *P < .05.

[0227] Results

[0228] S. aureus culture supernatants and SE induce drug resistance in malignant T cells.

[0229] Eradication of S. aureus with antibiotics reduced disease activity in patients undergoing anti-cancer treatment with doxorubicin, vorinostat, and alitretinoin. This confirms and extends previous data that eliminating bacterial colonization increased the efficacy of different types of ongoing anti-cancer treatment33. This prompted us to hypothesize that S. aureus and its toxins may directly induce drugresistance in malignant T cells. To address this hypothesis, we initially focused on the effect of S. aureus on malignant T cell responses to romidepsin, which is a potent inducer of apoptosis in malignant cells but not in non-malignant CD4+ T cells (Figure 8A). Accordingly, we examined whether supernatant from SE-producing S. aureus (isolated from affected SS patient skin) could modulate romidepsin-induced apoptosis in primary malignant cells. Thus, we treated SS PBMCs with romidepsin for 72 hours in the presence or absence of supernatant followed by flow cytometric analysis of apoptosis in malignant cells. Supernatant from SE-producing S. aureus drastically reduced romidepsin-induced apoptosis in malignant cells, while supernatant from endolysin (XZ.700)-treated S. aureus did not have this effect (Figure 1A & Figure 8B). As endolysin abrogates SE production and kills S. aureus (18), we hypothesized that the anti-apoptotic effect of S. aureus supernatants was mediated by SE. To address this, we treated SS PBMCs with romidepsin in the presence or absence of supernatants from two patient-derived S. aureus isolates that either produced or did not produce SE (Figure 8C). Intriguingly, only the S. aureus supernatant that contained SE showed potent reduction in romidepsin-induced apoptosis. To test if SE was sufficient to counteract the romidepsin-induced apoptosis, we treated SS PBMCs with romidepsin in the presence or absence of a pool of SE and analyzed apoptosis in malignant cells by assessing PARP cleavage (Figure 1 B) and by flow cytometry (Figures 8D-8E). As hypothesized, romidepsin-induced apoptosis was markedly reduced in the presence of SE in a dose- and time-dependent manner (Figure 1 C). The anti-apoptotic effect of SE was not restricted to this treatment-regimen. Thus, even when added 16 hours after pulse-treatment with romidepsin, SE blocked the apoptotic effect of very high concentrations of romidepsin (25 to 100 nM) (Figure 1 D). Analysis of S. aureus supernatants identified SEA as the dominating type of SE produced by patient-derived S. aureus (18). Consistent with these results, recombinant SEA (SEAwt) drastically diminished romidepsin-induced apoptosis (Figure 1 E-F). In contrast, a mutated SEA (SEAF47A / D227A), that cannot bind to MHC II (21), had no effect (Figure 1 E-F). As expected, the effect of SEA was abrogated by addition of a blocking anti- SEA antibody (Figure 1 G-H). Together, this indicates that (i) SEA is sufficient to counteract treatment-induced apoptosis, (ii) this effect is dependent on the superantigenic properties of SEA, and (iii) the observation is not due to off-target effects or contaminants.

[0230] SE largely overrides the transcriptional effect of Romidepsin in malignant T cells.

[0231] In our SS cohort, malignant cells from nine out of the ten romidepsin-naive patients were sensitive to romidepsin as measured by a significant drop in cell survival following treatment (Figure 2A-B). Importantly, presence of SE drastically improved malignant cell survival following romidepsin treatment (Figure 2A-B). To explore this effect, we performed single-cell cellular indexing of transcriptomes and epitopes sequencing (CITE-seq) analysis of SS PBMCs treated with romidepsin in the presence or absence of SE (Figure 2C-H). Cell cultures were collected after 36 hours to capture the transcriptional profiles prior to the onset of cell death induced by romidepsin (sensitivity of the malignant cells to romidepsin was confirmed by flow cytometry after 72 hours culture. Cell types were identified by their surface protein expression and malignant cells were defined by their monoclonal TCR and expression of malignant-associated markers TOX (22) and KIR3DL2 (23) in concert with low surface levels of CD7 (24) and CD26 (25) (Figure 2C-D). While romidepsin and SE individually induced strong transcriptional responses in the malignant cells (Figure 2E-F), treatment with romidepsin in the presence of SE largely resembled the response to SE alone as evident by their differentially expressed genes and co-clustering in transcriptional space. This trend was also evident from gene-set enrichment analysis (GSEA) and transcription factor (TF) activity analysis (Figure 2G-H). These findings suggest that upon treatment with romidepsin in the presence of SE, the SE-induced gene-regulation largely overshadows the romidepsin-mediated changes in gene expression. Both GSEA and TF activity analysis identified induction of key signaling pathways including NFkB signaling, cytokine-mediated JAK / STAT signaling, and TCR / BCR signaling upon SE exposure, both in isolation and in combination with romidepsin treatment (Figure 2G-H).

[0232] SE-mediated drug resistance of malignant T cells is not limited to Romidepsin.

[0233] As SE significantly reduced romidepsin-induced apoptosis of malignant cells, we tested whether SE also counteracted the effects of other clinically used HDAC inhibitors (HDACi), vorinostat (26) and resminostat (27). Like romidepsin, treatment of SS PBMCs with either vorinostat or resminostat induced potent cell death and both were counteracted by the presence of SE, resulting in survival of malignant cells (Figure 3A-D). Our CITE-seq analysis indicated that SE modulated signaling pathways involved in the survival of malignant cells such as NFkB and JAK / STAT. Thus, we hypothesized that the pro-survival effect of SE on malignant cells may not be limited to HDACi. Doxorubicin and etoposide are two chemotherapeutic agents that induce DNA damage and have been used as a treatment for CTCL (28, 29). We selected PBMCs from SS patients that responded to doxorubicin or etoposide ex vivo and tested whether presence of SE also counteracted the cytotoxic effect on malignant cells of these chemotherapeutics. Indeed, we found that SE exposure induced marked resistance in malignant cells towards both doxorubicin and etoposide (Figure 3E- H). Furthermore, the effect of SE-induced drug-resistance was not limited solely to HDACi and drugs inducing DNA damage, as SE exposure also maintained malignant cell survival following treatment with the ATP synthase inhibitor Oligomycin. Together, this indicates that presence of SE does not directly interfere with the mode of action of these agents, but rather promote overall drug resistance and survival of the malignant cells.

[0234] As NFkB signaling is enhanced by SE-treatment (Figure 2G-H), we investigated whether using the clinically approved proteasome and NFkB inhibitor, bortezomib (7, 30), could abrogate SE-induced drug resistance. We found that bortezomib induced potent malignant cell death that could not be rescued by SE (Figure 3I-J). Likewise, SE did not confer resistance to crystal violet (gentian violet (31); which is shown to inhibit NFkB in CTCL (32). These findings suggests that SE-induced drug resistance may be driven by enhanced NFkB-mediated cell survival of malignant cells.

[0235] SE-induced drug resistance is mediated by TCR signaling via LCK-PKC-NFkB.

[0236] NFkB is known to be vital for the survival of malignant cells in CTCL and is directly induced by TCR activation (33, 34) Our CITE-seq analysis confirmed that SE triggered profound TCR mediated activation of malignant cells (as shown by pathway enrichment and induction of surface CD25, CD69 and CD71 expression; Figure 2G) suggesting that SE-induced drug-resistance could be mediated through TCR engagement. Thus, we examined whether blockage of TCR-signaling could abrogate SE-induced drug-resistance. TCR signaling is contingent upon early activation of protein tyrosine kinases like LCK (SRC-family of tyrosine kinases), a critical kinase in the initiation of TCR- signaling following antigen presentation (35). Accordingly, we tested whether SRC inhibitor A- 419259 would block SE-induced drug-resistance. A-419259 treatment effectively blocked SE abrogation of romidepsin-induced cell death (Figure 4A-B). Similar results were obtained with dasatinib, a clinically approved inhibitor used against different cancers. Dasatinib, known to inhibit LCK (36), had little effect on malignant cell survival by itself, but abrogated the SE-mediated resistance to romidepsin (Figure 4C-D). As TCR-induced NFkB signaling is downstream of Protein Kinase C (PKC)-0 activation, we blocked PKC- 0 with a clinical inhibitor, sotrastaurin (37). Sotrastaurin significantly inhibited SE-induced romidepsin resistance (Figure 4E-F). Next, we inhibited the MAP kinase cascade in TCR signaling as MAP kinases have previously been linked to HDACi resistance (39). Using two clinical MEK inhibitors, mirdametinib and selumetinib, we found that neither inhibitor abrogated SE-induced romidepsin resistance. Taken together, our findings support the hypothesis that SE-induced drug-resistance is induced by TCR-signaling through the PKC-0 pathway leading to NFkB activation.

[0237] SE-induced drug-resistance can be both direct and indirect through release of IL-2R cytokines.

[0238] In addition to NFkB signaling, our CITE-seq data indicated that JAK-STAT mediated interleukin signaling and STAT3 activity was induced in malignant cells in the presence of SE (Figure 2G-H). IL-2 and its family members stimulate JAK1- and JAK3- leading to STAT3 / 5 activation (39). To determine if indirect signaling through cytokines could facilitate SE-induced drug-resistance, we treated SS PBMCs ex vivo with romidepsin in the presence or absence of a cocktail of IL-2 family cytokines (IL-2, IL-4, IL-7, and IL-15). We observed that the IL-2 family cytokines were sufficient to protect malignant cells from cell death induced by romidepsin (Figure 5A-B). To determine if cytokine signaling is necessary for SE-induced drug resistance, we added either A-419259 (to block TCR signaling) or a clinically used JAK inhibitor, tofacitinib (known to block IL-2 family cytokine signaling (40)), while treating SS PBMCs with romidepsin in the presence or absence of either SE or IL-2 family cytokines. Consistent with the results above, both SE and cytokines induced romidepsin resistance in malignant T cells (Figure 5C). Importantly, A-419259 completely blocked SE-induced romidepsin resistance but did not affect cytokine-induced romidepsin resistance (Figure 5C). In contrast, while tofacitinib completely blocked cytokine-induced romidepsin resistance (Figure 5C), we found remarkable differences in the effect of tofacitinib on SE-induce romidepsin resistance between the patients (Figure 5C-F). In three of the five investigated patient PBMCs, we found only modest or no reduction in SE-induced romidepsin resistance in the presence of tofacitinib (JAK-independent; Figure 5C-D). In the remaining two patient PBMCs, SE-induced romidepsin resistance was almost completely abrogated by the presence of tofacitinib, suggesting that cytokine signaling was necessary for the effect in these patients (JAK-dependent; Figure 5E- F). These individual differences in the impact of JAK / STAT on SE-induced HDACi resistance were not surprising because SS is a highly heterogeneous disease (41 - 43) and SE can induce STAT3 activation indirectly in malignant cells through IL-2 family cytokines released by non-malignant, SE- responsive bystander CD4+ T cells. To determine if TCR signaling is sufficient to induce drugresistance in patients that respond to SE in a JAK-dependent fashion and to investigate the dependence of non-malignant cells, we sorted malignant T cells from the PBMCs of three SS patients (based on CD3, CD4, CD8, CD7, CD26 and TCR-C01 expression) (Figure 6A). Due to the lack of surface markers that define malignant T cells, sorted malignant cells will unavoidably contain a minute non-malignant CD4+ T cells. Nonetheless, even in these highly enriched conditions SE still induced potent drug-resistance in sorted malignant T cells. Fortwo patients, this resistance was abrogated by the presence of tofacitinib (JAK-dependent) whereas malignant cells from one patient showed JAK-independent resistance (Figure 6B-C).

[0239] Discussion

[0240] In this study, we have demonstrated that S. aureus and its toxins induce resistance to drug-induced cell-death in primary malignant T cells from SS patients. Thus, supernatants from SE-expressing S. aureus isolated from SS skin largely protected malignant cells from cell-death induced by romidepsin, whereas endolysin-treated SE-producing S. aureus and SE-negative S. aureus had no effect. As only live S. aureus express exoproteins such as SE, these findings imply that the effect was mediated by SE. In support, the inhibitory effect on romidepsin-induced apoptosis was fully replicated by highly purified SEA and abrogated by antibody-blocking and mutations in the MHC class II binding domain of SEA (SEAF47A / D227A), that prevent TCR-MHC class II crosslinking (a prerequisite for SEA-mediated, TCR activation (21)). For decades, it has been suspected - but never proven - that bacterial superantigens play a pathogenetic role in CTCL. The present findings indicate that SE-mediated induction of drug-resistance could be one of the missing links between S. aureus and disease aggravation. Importantly, induction of resistance was not confined to romidepsin. On the contrary, SE also induced malignant T cell resistance to other HDACi (vorinostat and resminostat), chemotherapeutic drugs (doxorubicin and etoposide) and metabolic inhibitors (oligomycin). However, the protective effect was not universal and unspecific, as SE could not rescue malignant cells from cell death induced by bortezomib and crystal violet, which have also been used for treatment of CTCL.

[0241] Short-term pulse-treatment of malignant cells with a high dose of romidepsin was equally potent at inducing malignant cell-death. Importantly, presence of SE sixteen hours after the removal of the romidepsin pulse was still sufficient to counteract the drug-induced cell-death, showing that SE- induced drug-resistance is mediated downstream of the drug entering and initiating its effect. Consequently, SE-induced drug-resistance was not mediated through direct interaction with the drug nor blocking entrance or facilitating its removal. In line with a broad resistance mechanism, SE-induced resistance appears to be mediated through the cellular response to SE rather than direct interference with the treatment. In support, our CITE-seq analysis identified three potentially important pathways - NFkB, TCR, and STAT3. NFkB activity has been implicated in resistance to HDACi and chemotherapeutic drugs. In vitro studies on different cancer cell lines have shown that inhibiting NFkB enhanced sensitivity to treatment with doxorubicin (44 - 45) and etoposide (46 - 47). Together with our present finding that SE activates NFkB signaling in malignant cells and overrides most of the transcriptional changes induced by romidepsin, it is likely that SE induce drug resistance - at least partly - through the NFkB pathway. This conclusion is consistent with our observations that SE could not rescue malignant cells from cell-death induced by bortezomib and crystal violet which has been shown to inhibit NFkB activity in malignant cells from CTCL patients (7, 32).

[0242] TCR-signaling depends on activation of the SRC tyrosine kinase, LCK, resulting in NFkB activation through a PKC-dependent pathway which promotes cell-survival by inhibiting pro-apoptotic proteins (48 - 49). Accordingly, blockage of both early TCR signaling (dasatinib) and PKC activity (sotrastaurin) inhibited SE-induced drug-resistance. The response was selective to the PKC pathway, as inhibition of MEK (which has been implicated in HDACi resistance (38) was not sufficient to counteract SE-induced drug-resistance. These differences between cancer- and cellmodels suggest that HDACi resistance may be dependent on both cell-intrinsic features and factors in the tumor microenvironment.

[0243] Our CITE-seq data showed that SE enhanced STAT3 activation in HDACi treated cultures. JAK / STAT signaling is also induced by cytokines including the IL-2 family, which stimulate JAK- dependent, STAT-mediated cell survival in malignant cells from SS patients (18, 50 - 51). Indeed, our results show that IL-2 family cytokines provided survival signals, which protected malignant cells from drug-induced apoptosis. These findings provide first evidence that cytokines - which are expressed in the tumor microenvironment - may play a role in drug-resistance in CTCL patients. Surprisingly, JAK-STAT activation was only necessary for SE-induced drug-resistance in some patients but not all. As illustrated in Figure 7, these findings suggested that two pathways are involved: (i) direct TCR-dependent, JAK-independent pathway mediated through PKC-dependent NFkB signaling (Figure 7, upper part) and (ii) an indirect JAK-dependent pathway which rely on SE- 5 induced cytokine production in bystander T cells or the malignant cells themselves in an auto- / paracrine fashion (Figure 7, lower part). Experiments with sorted malignant cells confirmed that SE could induce drug-resistance even in highly enriched cultures from both JAK-dependent and - independent malignant populations. At present, it is not known why different responses were observed in malignant T cells from different patients. As SE can either directly (via TCRVD 10 interaction) or indirectly (via bystander T cells) activate malignant cells, this may explain why induction of drug-resistance in malignant cells from some patients rely on cytokine signaling, which can be blocked by tofacitinib, while others do not. Interestingly, a study in Chronic lymphocytic leukemia showed IL-6 induced resistance to vorinostat through STAT3 activation and that inhibition of STAT3 reversed vorinostat resistance (52). Taken together, these findings support the 15 hypothesis that SE-producing S. aureus can induce STAT3-dependent resistance to drugs such as HDACi.

[0244] In conclusion, we demonstrate that S. aureus and its toxins induce drug resistance in malignant T cells. These findings provide one of the missing links between S. aureus and cancer and grant an explanation how skin colonization by SE-producing bacteria can fuel disease activity in CTCL. 20 Moreover, our results highlight the need to specifically eradicate and prevent S. aureus skin colonization in addition to targeting the malignant cells in CTCL patients. In the present study this has been performed using the XZ.700 endolysin. It is evident that treatment with other (native of chimeric) endolysins effective against S. aureus will provide comparable results as obtained with the XZ.700 endolysin.

[0245] 25

[0246] Table 2 Patient characteristics

[0247] SS - Sezary syndrome; MF - Mycosis fungoides; NA - Not available; ECP - Extracorporal photopheresis; IFN - Interferon; PUVA - Psoralen and ultraviolet A; UVB - Ultraviolet B; TSEI - Total skin electron irradiation. Treatment indicates the patient clinical treatments at the time point of blood collection where PBMCs were isolated and used for experiments.

[0248] References

[0249] 1 . Sezary A, Bouvrain Y. Erythrodermie avec presence de cellules monstrueuses dans le derme et le sang circulant. Bull Soc Fr Dermatol Syphiligr. 1938;45:254-260.

[0250] 2. Brouet JC, Flandrin G, Seligmann M. Indications of the thymus-derived nature of the proliferating cells in six patients with Sezary's syndrome. N Engl J Med. 1973;289(7):341-344.

[0251] 3. Lutzner M, Edelson R, Schein P, Green I, Kirkpatrick C, Ahmed A. Cutaneous T-cell lymphomas: the Sezary syndrome, mycosis fungoides, and related disorders. Ann Intern Med. 1975;83(4):534-552.

[0252] 4. Girardi M, Heald PW, Wilson LD. The Pathogenesis of Mycosis Fungoides. New England Journal of Medicine. 2004;350(19):1978-1988.

[0253] 5. Kim YH, Liu HL, Mraz-Gernhard S, Varghese A, Hoppe RT. Long-term outcome of 525 patients with mycosis fungoides and Sezary syndrome - Clinical prognostic factors and risk for disease progression. Archives of Dermatology. 2003;139(7):857-866.

[0254] 7. Sors A, Jean-Louis F, Pellet C, et al. Down-regulating constitutive activation of the NF- kappaB canonical pathway overcomes the resistance of cutaneous T-cell lymphoma to apoptosis. Blood. 2006;107(6):2354-2363.

[0255] 8. Netchiporouk E, Litvinov IV, Moreau L, Gilbert M, Sasseville D, Duvic M. Deregulation in STAT signaling is important for cutaneous T-cell lymphoma (CTCL) pathogenesis and cancer progression. Cell Cycle. 2014;13(21):3331-3335.

[0256] 9. Axelrod PI, Lorber B, Vonderheid EC. Infections complicating mycosis fungoides and Sezary syndrome. JAMA. 1992;267(10):1354-1358.

[0257] 10. Mirvish ED, Pomerantz RG, Geskin LJ. Infectious agents in cutaneous T-cell lymphoma. J Am Acad Dermatol. 2011 ;64(2):423-431 .

[0258] 11. Zhang Y, Seminario-Vidal L, Cohen L, et al. "Alterations in the Skin Microbiota Are Associated With Symptom Severity in Mycosis Fungoides". Front Cell Infect Microbiol. 2022;12:850509.

[0259] 12. Hooper MJ, LeWitt TM, Veon FL, et al. Nasal Dysbiosis in Cutaneous T-Cell Lymphoma Is Characterized by Shifts in Relative Abundances of Non-Staphylococcus Bacteria. JID Innov. 2022;2(5):100132.

[0260] 13. Allen PB, Switchenko J, Ayers A, Kim E, Lechowicz MJ. Risk of bacteremia in patients with cutaneous T-cell lymphoma (CTCL). Leukemia & Lymphoma. 2020;61 (11):2652-2658.

[0261] 14. Posner LE, Fossieck BE, Jr., Eddy JL, Bunn PA, Jr. Septicemic complications of the cutaneous T-cell lymphomas. Am J Med. 1981 ;71 (2):210-216.

[0262] 15. Bonin S, Tothova SM, Barbazza R, Brunetti D, Stanta G, Trevisan G. Evidence of multiple infectious agents in mycosis fungoides lesions. Exp Mol Pathol. 2010;89(1):46-50. 16. Gluud M, Pallesen EMH, Buus TB, et al. Malignant T cells induce skin barrier defects through cytokine-mediated JAK / STAT signaling in cutaneous T-cell lymphoma. Blood. 2023;141 (2):180-193.

[0263] 17. Thode C, Woetmann A, Wandall HH, et al. Malignant T Cells Secrete Galectins and Induce Epidermal Hyperproliferation and Disorganized Stratification in a Skin Model of Cutaneous T-Cell Lymphoma. Journal of Investigative Dermatology. 2015;135(1):238-246.

[0264] 18. Pallesen EMH, Gluud M, Vadivel CK, et al. Endolysin inhibits skin colonization by patient-derived Staphylococcus aureus and malignant T cell activation in cutaneous T cell lymphoma. J Invest Dermatol. 2023.

[0265] 19. Buus TB, Herrera A, Ivanova E, et al. Improving oligo-conjugated antibody signal in multimodal single-cell analysis. eLife. 2021 ;10:e61973.

[0266] 20. Vadivel CK, Gluud M, Torres-Rusillo S, et al. JAK3 Is Expressed in the Nucleus of Malignant T Cells in Cutaneous T Cell Lymphoma (CTCL). Cancers. 2021 ;13(2).

[0267] 21 . Mehindate K, Thibodeau J, Dohlsten M, Kalland T, Sekaly RP, Mourad W. Cross-linking of major histocompatibility complex class II molecules by staphylococcal enterotoxin A superantigen is a requirement for inflammatory cytokine gene expression. J Exp Med. 1995;182(5):1573-1577.

[0268] 22. Huang Y, Su MW, Jiang X, Zhou Y. Evidence of an oncogenic role of aberrant TOX activation in cutaneous T-cell lymphoma. Blood. 2015;125(9):1435-1443.

[0269] 23. Hurabielle C, Thonnart N, Ram-Wolff C, et al. Usefulness of KIR3DL2 to Diagnose, Follow-Up, and Manage the Treatment of Patients with Sezary Syndrome. Clin Cancer Res. 2017;23(14):3619-3627.

[0270] 24. Vonderheid EC, Bigler RD, Kotecha A, et al. Variable CD7 expression on T cells in the leukemic phase of cutaneous T cell lymphoma (Sezary syndrome). J Invest Dermatol. 2001 ;117(3):654-662.

[0271] 25. Jones D, Dang NH, Duvic M, Washington LT, Huh YO. Absence of CD26 expression is a useful marker for diagnosis of T-cell lymphoma in peripheral blood. Am J Clin Pathol. 2001 ;115(6):885-892.

[0272] 26. Olsen EA, Kim YH, Kuzel TM, et al. Phase lib multicenter trial of vorinostat in patients with persistent, progressive, or treatment refractory cutaneous T-cell lymphoma. J Clin Oncol. 2007;25(21):3109-3115.

[0273] 27. Resminostat for Maintenance Treatment of Patients With Advanced Stage Mycosis Fungoides (MF) or Sezary Syndrome (SS): https: / / ClinicalTrials.gov / show / NCT02953301.

[0274] 28. Wollina U, Dummer R, Brockmeyer NH, et al. Multicenter study of pegylated liposomal doxorubicin in patients with cutaneous T-cell lymphoma. Cancer. 2003;98(5):993-1001 .

[0275] 29. Purnak S, Azar J, Mark LA. Etoposide as a single agent in the treatment of mycosis fungoides: A retrospective analysis. Dermatologic Therapy. 2018;31 (2).

[0276] 30. Zinzani PL, Musuraca G, Tani M, et al. Phase II trial of proteasome inhibitor bortezomib in patients with relapsed or refractory cutaneous T-cell lymphoma. J Clin Oncol. 2007;25(27):4293- 31 . Westergaard SA, Lechowicz MJ, Harrington M, Elsey J, Arbiser JL, Khan MK. Induction of remission in a patient with end-stage cutaneous T-cell lymphoma by concurrent use of radiation therapy, gentian violet, and mogamulizumab. JAAD Case Rep. 2020;6(8):761-765.

[0277] 32. Wu J, Wood GS. Analysis of the Effect of Gentian Violet on Apoptosis and Proliferation in Cutaneous T-Cell Lymphoma in an In Vitro Study. JAMA Dermatology. 2018;154(10):1191-1198.

[0278] 33. Chang TP, Vancurova I. NFkappaB function and regulation in cutaneous T-cell lymphoma. Am J Cancer Res. 2013;3(5):433-445.

[0279] 34. Paul S, Schaefer BC. A new look at T cell receptor signaling to nuclear factor-KB. Trends Immunol. 2013;34(6):269-281 .

[0280] 35. Courtney AH, Lo WL, Weiss A. TCR Signaling: Mechanisms of Initiation and Propagation. Trends in Biochemical Sciences. 2018;43(2):108-123.

[0281] 36. Schade AE, Schieven GL, Townsend R, et al. Dasatinib, a small-molecule protein tyrosine kinase inhibitor, inhibits T-cell activation and proliferation. Blood. 2008;1 11 (3):1366-1377.

[0282] 37. A Dose Finding Study of AEB071 Assessing Psoriasis Area and Severity Index in Patients With Plaque Psoriasis: https: / / ClinicalTrials.gov / show / NCT00885196.

[0283] 38. Chakraborty AR, Robey RW, Luchenko VL, et al. MAPK pathway activation leads to Bim loss and histone deacetylase inhibitor resistance: rationale to combine romidepsin with an MEK inhibitor. Blood. 2013;121 (20):4115-4125.

[0284] 39. Hammaren HM, Virtanen AT, Raivola J, Silvennoinen O. The regulation of JAKs in cytokine signaling and its breakdown in disease. Cytokine. 2019;118:48-63.

[0285] 40. Meyer DM, Jesson Ml, Li X, et al. Anti-inflammatory activity and neutrophil reductions mediated by the JAK1 / JAK3 inhibitor, CP-690,550, in rat adjuvant-induced arthritis. J Inflamm (Lond). 2010;7:41.

[0286] 41. Borcherding N, Voigt AP, Liu V, Link BK, Zhang W, Jabbari A. Single-Cell Profiling of Cutaneous T-Cell Lymphoma Reveals Underlying Heterogeneity Associated with Disease Progression. Clin Cancer Res. 2019;25(10):2996-3005.

[0287] 42. Buus TB, Willerslev-Olsen A, Fredholm S, et al. Single-cell heterogeneity in Sezary syndrome. Blood Adv. 2018;2(16):2115-2126.

[0288] 43. Gaydosik AM, Tabib T, Geskin LJ, et al. Single-Cell Lymphocyte Heterogeneity in Advanced Cutaneous T-cell Lymphoma Skin Tumors. Clin Cancer Res. 2019;25(14):4443-4454.

[0289] 44. Chiao PJ, Na R, Niu J, Sclabas GM, Dong Q, Curley SA. Role of Rel / NF-kappaB transcription factors in apoptosis of human hepatocellular carcinoma cells. Cancer. 2002;95(8):1696-1705.

[0290] 45. Abdin SM, Tolba MF, Zaher DM, Omar HA. Nuclear factor-KB signaling inhibitors revert multidrug-resistance in breast cancer cells. Chemico-Biological Interactions. 2021 ;340:109450.

[0291] 46. Morotti A, Cilloni D, Pautasso M, et al. NF-kB inhibition as a strategy to enhance etoposide-induced apoptosis in K562 cell line. American Journal of Hematology. 2006;81 (12):938- 945. 47. Meley D, Spiller DG, White MR, McDowell H, Pizer B, See V. p53-mediated delayed NF- kappaB activity enhances etoposide-induced cell death in medulloblastoma. Cell Death Dis. 2010;1(5):e41.

[0292] 48. Bertolotto C, Maulon L, Filippa N, Baier G, Auberger P. Protein kinase C theta and epsilon promote T-cell survival by a rsk-dependent phosphorylation and inactivation of BAD. J Biol

[0293] Chem. 2000;275(47):37246-37250.

[0294] 49. Cheng J, Montecalvo A, Kane LP. Regulation of NF-kappaB induction by TCR / CD28. Immunol Res. 2011 ;50(2-3):113-117.

[0295] 50. Willerslev-Olsen A, Gjerdrum LMR, Lindahl LM, et al. Staphylococcus aureus Induces Signal Transducer and Activator of Transcription 5-Dependent miR-155 Expression in Cutaneous

[0296] T-Cell Lymphoma. J Invest Dermatol. 2021 ;141 (10):2449-2458.

[0297] 51 . Willerslev-Olsen A, Krejsgaard T, Lindahl LM, et al. Staphylococcal enterotoxin A (SEA) stimulates STAT3 activation and IL-17 expression in cutaneous T-cell lymphoma. Blood. 2016.

[0298] 52. Lu K, Fang XS, Feng LL, et al. The STAT3 inhibitor WP1066 reverses the resistance of chronic lymphocytic leukemia cells to histone deacetylase inhibitors induced by interleukin-6.

[0299] Cancer Letters. 2015;359(2):250-258.

Claims

Claims1 . An antibacterial enzyme effective against Staphylococcus aureus for use in the treatment of a subject suffering from cancer refractive to a histone deacetylase (HDAC) inhibitor, said treatment comprising administration of an effective amount of the antibacterial enzyme to the subject.

2. An antibacterial enzyme effective against Staphylococcus aureus for use according to claim 1 , wherein, further to the antibacterial enzyme, a chemotherapeutic drug is administered to the subject.

3. An antibacterial enzyme effective against Staphylococcus aureus for use according to claim 1 or 2, wherein the cancer is lymphoma, such as cutaneous T cell lymphoma (CTCL), such as the leukemic variant Sezary syndrome (SS).

4. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of claims 2 and 3, wherein the further administered chemotherapeutic drug is an anthracycline such as Doxorubin, a topoisomerase II inhibitor such as Etoposide, and / or a histone deacetylase (HDAC) inhibitor such as Vorinostat, Romidepsin, Resminostat, Panobinostat, and Belinostat.

5. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of the preceding claims, wherein the cancer is refractive due to a staphylococcal enterotoxin (SE), such as a SE produced by Staphylococcus aureus.

6. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of the preceding claims, 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.

7. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of the preceding claims, wherein the antibacterial enzyme is a chimeric lysin, preferably a recombinant chimeric endolysin comprising one or more heterologous domains8. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of the preceding claims, wherein the Staphylococcus aureus is an antibiotic-resistant Staphylococcus aureus, such as a methicillin resistant Staphylococcus aureus (MRSA).

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

10. An antibacterial enzyme effective against Staphylococcus aureus for use according to any one of the preceding claims, wherein the subject is a mammal, such as a live stock animal, a domestic animal or a human.11 . A method of treatment of a subject suffering from a cancer refractive to a histone deacetylase (HDAC) inhibitor, said treatment comprising administration of an effective amount of an antibacterial enzyme effective against Staphylococcus aureus to the subject.

12. Use of an antibacterial enzyme effective against Staphylococcus aureus for the manufacture of a medicament for the treatment of a cancer refractive to a histone deacetylase (HDAC) inhibitor, said treatment comprising administration of an effective amount of an antibacterial enzyme effective against Staphylococcus aureus to the subject.

13. A method for blocking histone deacetylase (HDAC) inhibitor-resistance in primary malignant T- cells comprising contacting the malignant T-cells with an antibacterial enzyme effective against Staphylococcus aureus.

Images