Treatment of lymphoma

Abstract

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

Description

Technical Field

[0001] This invention relates to the medical field, and more specifically to the treatment of lymphoma. Background Technology

[0002] Cezari syndrome (SS) is a malignant disorder of T cells belonging to the cutaneous T-cell lymphoma (CTCL) (non-Hodgkin lymphoma) spectrum. SS is a leukemic variant of cutaneous T-cell lymphoma (1-4). Patients with SS are susceptible to Staphylococcus aureus (S. aureus) infection and have a poor prognosis due to treatment resistance.

[0003] Therefore, there is a need to improve the treatment and treatment outcomes of Cezari syndrome. Detailed Implementation

[0004] The inventors have determined that Staphylococcus aureus and its toxins (staphylococcal enterotoxin (SE)) induce drug resistance in malignant T cells, particularly resistance to histone deacetylase (HDAC) inhibitors. These findings provide a missing link between Staphylococcus aureus and cancer and explain how skin colonization of SE-producing bacteria can contribute to disease activity in CTCL.

[0005] Therefore, in a first aspect, an antibacterial enzyme effective against Staphylococcus aureus is provided for use in treating a subject with cancer refractory to chemotherapy drugs, preferably histone deacetylase (HDAC) inhibitors, said treatment comprising administering an effective amount of the antibacterial enzyme to the subject.

[0006] In the embodiments herein, an antibacterial enzyme effective against Staphylococcus aureus for use is referred to as "an" or "an effective antibacterial enzyme against Staphylococcus aureus for use according to the invention", "an" or "an antibacterial enzyme as disclosed herein" or explicitly "an" or "the antibacterial enzyme".

[0007] In the embodiments described herein, the effective amount of antibacterial enzyme can be interpreted as a therapeutically effective amount, meaning an amount of antibacterial enzyme sufficient to treat, delay, or improve the symptoms of the subject's condition (preferably SS) when administered to the subject. Preferably, the effective amount eliminates resistance of cancer cells to chemotherapeutic agents, preferably histone deacetylase (HDAC) inhibitors, such that the effectiveness of the chemotherapeutic agent is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, at least 2, 3, 4, 5, 6, 7, 8, 9 times, 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 compared to when the antibacterial enzyme is not administered to the subject.

[0008] In the embodiments herein, the term "chemotherapy-resistant cancer" means that cancer cells are less sensitive to chemotherapy drugs, preferably histone deacetylase (HDAC) inhibitors, due to SE interference. For the hypothesized mechanism, see [link to relevant documentation]. Figure 7 In this article, "less sensitive" means that, compared with the absence of SE, cancer cells are less sensitive to chemotherapy drugs, preferably histone deacetylase (HDAC) inhibitors, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, at least 2, 3, 4, 5, 6, 7, 8, 9, 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.

[0009] In the embodiments described herein, administration of antibacterial enzymes or compositions containing antibacterial enzymes to a subject may be performed in any manner known to those skilled in the art, such as systemic, local, intravenous, topical, oral, via dressings (e.g., wound dressings), via bandages, rectum, vagina, intramuscular, via artery, intraperitoneum, intranasal, subcutaneous, endoscopic, percutaneous, mucosal, and intrathecal administration.

[0010] In the embodiments described herein, the chemotherapy drugs for cancer that are refractory to treatment may be anthracyclines, such as doxorubicin; topoisomerase II inhibitors, such as etoposide; and / or preferably histone deacetylase (HDAC) inhibitors, such as vorinostat, romidesin, remixstat, pabisostat, and benoristat. In one embodiment, the histone deacetylase (HDAC) inhibitor is vorinostat; in another embodiment, the histone deacetylase (HDAC) inhibitor is romidesin; in yet another embodiment, the histone deacetylase (HDAC) inhibitor is remixstat; in yet another embodiment, the histone deacetylase (HDAC) inhibitor is pabisostat; and in yet another embodiment, the histone deacetylase (HDAC) inhibitor is benoristat.

[0011] In the embodiments described herein, treatment including administering an effective amount of an antibacterial enzyme to a subject may include administering a chemotherapeutic agent to the subject in addition to the antibacterial enzyme.

[0012] In the embodiments described herein, cancer can be any type of cancer refractory to chemotherapy, preferably a histone deacetylase (HDAC) inhibitor, such as, but not limited to, lymphoma, such as cutaneous T-cell lymphoma (CTCL), such as leukodermal Cezari syndrome (SS), which is widely described elsewhere herein. Therefore, in the embodiments described herein, cancer refractory to chemotherapy, preferably a histone deacetylase (HDAC) inhibitor, can be leukodermal Cezari syndrome of CTCL.

[0013] In the embodiments described herein, the further administered chemotherapy drug can be any chemotherapy drug that the cancer may be refractory to. Therefore, in the embodiments described herein, the chemotherapy drug can be anthracyclines, such as doxorubicin; topoisomerase II inhibitors, such as etoposide; and / or histone deacetylase (HDAC) inhibitors, such as vorinostat, romidesin, remixstat, pabilistat, and benoristat. In the embodiments described herein, the further administered chemotherapy drug is preferably a histone deacetylase (HDAC) inhibitor, and can be vorinostat. In the embodiments described herein, the further administered chemotherapy drug can be romidesin. In the embodiments described herein, the further administered chemotherapy drug can be remixstat. In the embodiments described herein, the further administered chemotherapy drug can be pabilistat. In the embodiments described herein, the further administered chemotherapy drug can be benoristat.

[0014] In the embodiments described herein, cancer may be refractory to chemotherapy-preferred histone deacetylase (HDAC) inhibitors due to staphylococcal enterotoxins (SEs) such as those produced by Staphylococcus aureus. The hypothetical mechanisms by which staphylococcal enterotoxins (SEs) such as those produced by Staphylococcus aureus are involved in the refractory nature of cancer to chemotherapy-preferred histone deacetylase (HDAC) inhibitors are extensively described in the examples herein. In this document, SE can be any staphylococcal enterotoxin. In this document, SE can 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). In this document, SE can be staphylococcal enterotoxin (SE) type A.

[0015] In the embodiments described herein, the antibacterial enzyme is preferably selected from the group consisting of: bacteriocins or their functional portions, bacterial lysins or autolysins or their functional portions, phage lysins or their functional portions, and chimeric lysins or their functional portions. In the embodiments described herein, the antibacterial enzyme has peptidoglycan hydrolase activity; hydrolysis of peptidoglycan leads to bacterial lysis. In the embodiments described herein, the antibacterial enzyme may have more than one type of peptidoglycan hydrolase activity. In the embodiments described herein, the antibacterial enzyme may be a chimeric lysin, such as a recombinant chimeric endosomal containing one or more heterologous domains.

[0016] In the embodiments described herein, the functional portion of an antibacterial enzyme means that a portion of the functional antibacterial enzyme is still capable of cleaving its natural target bacteria through the hydrolysis of peptidoglycan. Preferably, the activity of the functional portion is at least 20%, 30%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of that of a fully functional antibacterial enzyme.

[0017] In the embodiments described herein, the bacteriocin may be any bacteriocin with peptidoglycan hydrolase activity, such as class IIIa bacteriocins. Preferred bacteriocins herein are lysostaphin, mutanolysin, and enterolysin.

[0018] In the embodiments described herein, bacterial lysins or autolysins can 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).

[0019] In the embodiments described herein, phageendolysins are also referred to as endosynovins and can be any phageendolysin with peptidoglycan hydrolase activity. Phageendolysins are known to those skilled in the art; see, for example, Bacteriophageendolysins as novel antimicrobials. Schmelcher M, Donovan DM, Loessner MJ. Future Microbiol. 2012 Oct;7(10): l 147-7.

[0020] In the embodiments described herein, the endosomal can 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 [Escherichia coli 0157:H7 phage φVIO and identification of a phage-encoded immunity protein that modifies the 0157 antigen]. Perry LL, SanMiguel P, Minocha U, Terekhov AI, Shroyer ML, Farris LA, Bright N, Reuhs BL, Applegate BM. FEMS Microbiol Lett. [FEMS Microbiol Letters] 2009 Mar;292(2):182-6); STM0907 of phage FELS-1. FelsO (Nature [Nature]). October 25, 2001; 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); phage ε15 εl5p25 (Virology. December 20, 2007; 369(2):234-44. Epub September 7, 2007.The genome of epsilon15, a serotype-converting, Group E1 Salmonella enterica-specific bacteriophage [ε15 (serotype-converting Group E1 Salmonella enterica-specific bacteriophage) genome]. 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 Sequences: YP 001595885.1 and J Bacteriol. [Journal of Bacteriology] February 2008; 190(4): 1429-35. The genome and structural proteome of YuA, a new Pseudomonas aeruginosa phage resembling M6 [YuA (a new Pseudomonas aeruginosa phage similar to M6) genome and structural proteome]. 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 [the complete genome sequence of phage B3 (a Mu-like phage of Pseudomonas aeruginosa)]. Braid MD, Silhavy JL, Kitts CL, Cano RJ, Howe MM. J Bacteriol. [Journal of Bacteriology] October 2004; 186(19):6560-74); BcepMu22 of phage BcepMu (J Mol Biol. [Journal of Molecular Biology] June 25, 2004; 340(1):49-65.Burkholderiacenocepacia phage BcepMu and a family of Mu-like phages encoding potential pathogenesis factors. Summer EJ1, Gonzalez CF, Carlisle T, Mebane LM, Cass AM, SawaCG, LiPuma J, Young R); Phage F116p62 of Fl 16 (Gene. Feb. 14, 2005;346:187-94. The genome of the Pseudomonas aeruginosa generalized transducing bacteriophage Fl 16. Byrne Ml, Kropinski AM.); Phage Fels2 STM2715.S.Fels2 (Nature). October 25, 2001; 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, SR, Gilcrease, EB, Winn-Stapley, DA, Schicklmaier, P., Schmieger, H., Pedulla, ML, Ford, ME, Houtz, J.M., Hatfull, GF and Hendrix, R.W. The generalized transducing Salmonellabacteriophage ESI 8: Complete genome sequence and DNA package strategy [Salmonella spp. generalized transducing phage ESI 8: Complete genome sequence and DNA package strategy] J. Bacteriol. [Journal of Bacteriology] 187 (3), 1091-1104 (2005)); SPSV3_gp23 of phage SETP3 (J Med Microbiol. [Journal of Medical Microbiology] 2009 Jan; 58 (Part 1): 86-93. Characterization of bacteria used in the Salmonella enterica serovar Enteritidis phage-typing scheme [Characterization of bacteria used in the Salmonella enterica serovar Enteritidis phage-typing scheme]. De Lappe Nl, Doran G, O'Connor J, O'Hare C, Cormican M); φ32_17 of phage OEC032 (Genomic and Proteomic analysis of phiEco32, a novel Escherichia colibacteriophage [φEco32 (a novel Escherichia colibacteriophage) genomic and proteomic analysis]. 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. [Journal of Molecular Biology] 28 March 2008; 377(3):774-89); HK022p54 of phage HK022 (J Mol Biol. [Journal of Molecular Biology] 26 May 2000; 299(1):27-51.Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoid bacteriophages [J Mol Biol. [Molecular Biology Journal] May 26, 2000; 299(1): 27-51. Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoid bacteriophages [J Mol Biol. [Molecular Biology Journal] May 26, 2000; 299(1): 27-51. Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoid bacteriophages [J Mol Biol. [Molecular Biology Journal] May 26, 2000; 299(1): 27-51. Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoid bacteriophages [J Mol Biol. [Molecular Biology Journal] May 26, 2000; 299(1): 27-51. Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoid bacteriophages [J Mol Biol. [Molecular Biology Journal] May 26, 2000; 299(1): 27-51. RW); HK620p36 of phage HK620 (Nucleotide sequence of coliphage HK620 and the evolution of lambdoid phages [Escherichia coli phage HK620 nucleotide sequence and the evolution of lambdoid phages]. Clark AJ, Inwood W, Cloutier T, Dhillon TS. J MolBiol. [Journal of Molecular Biology] Aug 24, 2001; 311(4):657-79); VIP0007 of phage El (Molecular characterization of the Salmonella enterica serovar Typhi Vi-typing bacteriophage El [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.[Journal of Bacteriology] April 2008; 190(7):2580-7); Sf6p62 of phage SF6 (J Mol Biol. [Journal of Molecular Biology] May 28, 2004; 339(2):379-94. The chromosome of Shigella flexneri bacteriophage Sf6:complete nucleotide sequence, genetic mosaicism, and DNA packaging [Chromosome of Shigella flexneri phage 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. [Journal of Bacteriology] April 2002; 184(7): 1974-87. Complete genomic sequence of SfV, A serotype-converting temperate bacteriophage of Shigella flexneri [complete genome sequence of SfV (serotype-converting temperate phage of Shigella flexneri)]. Allison GE1, Angeles D, Tran-Dinh N, Verma NK); gp22 of phage BCEPC6B (Summer, EJ, Christian, BN, Collins, J., Morrison, W., Patel, P., Wells, W., Mebane, L., Gonzalez, CF and Young, RF GenBank: AAT38381.1); Nazgul38 of phage BCEPNAZGUL (Summer, EJ, Peek, ML, Haliburton J.R., Hall, E., Heusinkveld, K., Simser J., No, EG, Gonzalez, CF and Young, RF NCBI reference sequence: NP918971.2); K of phage P2 (P2p09) (Christie, GE, Haggard-Ljungquist, E. and Calendar, R. NCBI reference sequence: NP 046765.)1) Phage WO of K (Wφ09) (Esposito, D., Schmidt, BJ, Bloom, FR and Christie, GEGenBank: AAN28227.1); Phage RV5 of rv5_gp085 (Virol J. [Journal of Virology] 2013 Mar 6; 10:76. The host-range, genomics and proteomics of Escherichia coli O157:H7 bacteriophage rV5 [Escherichia coli O157:H7 phage rV5 host range, genomics and proteomics]. Kropinski AMI, Waddell T, Meng J, Franklin K, Ackermann HW, Ahmed R, Mazzocco A, Yates J 3, Lingohr EJ, Johnson RP); Phage JS98 of EpJS98_gpl 16 (Zuber, S., Ngom-Bru, C, Barretto, C, Bruttin, A., Brussow, H., and Denou, E. Genome analysis of phage JS98 defines a fourteenth 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, I. NCBI reference sequence: YP 002003950.1); gp3.5 of phage BA14 (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); CKVlF_gpl6 of phage K1F (Scholl, D. and Merril, C.).The Genome of Bacteriophage K1F, a T7-Like Phage That Has Acquired the Ability To Replicate on Kl Strains of Escherichia coli [J. Bacterid. 187 (24), 8499-8503 (2005)]; T3pl8 of Phage T3 (Pajunen, ML, Elizondo, MR, Skurnik, M., Kieleczawa J. and Molineux, IJ Complete nucleotide sequence and likely recombinatorial origin of bacteriophage T3 [J. Mol. Biol. 319 (5)] 1115-1132); gh-lpl2 of phage GH-1 (Kovalyovai.V. and Kropinski, AM. The complete genomic sequence of lytic bacteriophage gh-1 infecting Pseudomonas putida—evidence for close relationship to the T7 group [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 Kl l (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, acytotoxin-converting phage of Pseudomonas aeruginosa: implications for phage evolution and horizontal gene transfer via bacteriophages [φCTX (cytotoxin-converting phage of Pseudomonas aeruginosa): implications for phage evolution and horizontal gene transfer via bacteriophages] Mol. Microbiol. [Molecular Microbiology] 31 (2), 399-419 (1999); Bcep43-27 of phage BCEP43 (Summer, EJ, Gonzalez, CF, Bomer, M., Carlile, T., Embry, A., Kucherka, AM, Lee, J., Mebane, L., Morrison, WC, Mark, L., King, MD, LiPuma, J.J., Vidaver, AK and Young, R. Divergence and mosaicism among virulentsoil phages of the Differences and mosaicism among virulent soil phages of the Burkholderia cepacia complex [Journal of Bacteriol.] 188 (1), 255-268 (2006); Bcep781-27 of phage BCEP781 (Summer, EJ, Gonzalez, CF, Bomer, M., Carlile, T., Embry, A., Kucherka, AM, Lee, J., Mebane, L., Morrison, WC, Mark, L., King, MD, LiPuma, J.J., Vidaver, AK and Young, R. Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex [Journal of Bacteriol.] 188 (1), 255-268 (2006)); ...[Journal of Bacteriology] 188(1), 255-268 (2006)); BcepNY3 gene 26 of phage BCEP1 (Summer, EJ, Gonzalez, CF, Bomer, M., Carlile, T., Embry, A., Kucherka, AM, Lee, J., Mebane, L., Morrison, WC, Mark, L., King, MD, LiPuma, J.J., Vidaver, AK and Young, R. Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex [Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex] J. Bacteriol. [Journal of Bacteriology] 188(1), 255-268 (2006)); BcepNY3 gene 26 of phage BCEPNY3 (GenBank: ABR10561.1 Summer, EJ, Orchard, RC, Attenhofer, K., Coffey, A., Gill, J.J., Gonzalez, CF, and Young, R.); gp45 of phage ΦE12-2 (NCBI reference sequence: YP 001111195.1 DeShazer, D., Ronning, CM, Brinkac, LM, and Merman, WC); gp28 of phage Φ52237 (DeShazer, D, and Merman, WC NCBI reference sequence: YP 293741.1 DeShazer, D., Ronning, CM, Brinkac, LM, and Merman, WC); p27p30 of phage ΦP27 (Recktenwald, J., 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 [The nucleotide sequence of phage φP27, which encodes Shiga toxin (Stx) 2e, is not related to the genomes of other Stx phages, but its modular genetic structure is conserved.] Infect. Immun.[Infection and Immunity] 70 (4), 1896-1908 (2002)); RB49pl02 of phage RB49 (Monod, C, Repoila, F., Kutateladze, M., Tetart, F. and Krisch, HM The genome of the pseudo T-even bacteriophages, adiverse group that resembles T4 [The genome of the pseudo T-even bacteriophages, adiverse group that resembles T4] J. Mol. Biol. [Journal of Molecular Biology] 267 (2), 237-249 (1997)); φl-pl02 of phage Φ1 (Arbiol, C, Comeau, AM, Kutateladze, M., Adamia, R. and Krisch, HM Mobileregulatory cassettes mediate modular shuffling in t4-type phage genomes [Mobile regulatory cassettes mediate modular shuffling in t4-type phage genomes] Genome Biol Evol [Genomics, Biology & Evolution] 2010, 140-152 (2010)); lys (T5.040) of phage T5 (NCBI reference sequence: YP 006868.1 Ksenzenko, VN, Kaliman, AV, Krutilina, AI and Shlyapnikov, MG); YP 001956952.1 of phage 201φ2-l (Thomas J.A., Rolando, MR, Carroll, CA, Shen, PS, Belnap, DM, Weintraub, ST, Serwer, P. and Hardies, SC Characterization of Pseudomonas chlororaphis myovirus 201 varphi2-l via genomic sequencing, mass spectrometry, and electron microscopy) Virology [Virology] 376 (2), 330-338 (2008)); Aehlp339 of phage Aehl (NCBI reference sequence: NP 944217.1 Petrov, V., Nolan J.).Bertrand, C., Letarov, AV., Krisch, HM. and Karam, J.D.); YYZgp45 of phage YYZ-2008 (GenBank: ACI32381.1 Zhang, Y., Laing, CR., Kropinski, A. and Gannon, VJP); endosomalin of Pseudomonas aeruginosa phage φKZ, g l44 (J Biol Chem. [Journal of Biochemistry] 2008 Mar 14;283(11):7242-50. Structure of the bacteriophage phi KZ lytic transglycosylasegpl44 [Structure of phage φKZ lytic transglycosylate gpl44]. Fokine A, Miroshnikov KA, Shneider MM, Mesyanzhinov VV, Rossmann MG.) and EL, El.1 88 (Mol Microbiol. [Molecular Microbiology] September 2007; 65(5): 1334-44. Mura lytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages phiKZ and EL [Mura lytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages phiKZ and EL]. BriersYl, Volckaert G, Cornelissen A, Lagaert S, Michiels CW, Hertveldt K, LavigneR), endolysin of phage LUZ24 (NCBI reference sequence: YP 001671940.1) and Escherichia coli phage N4gp61 (JMol Biol. [Journal of Molecular Biology] February 16, 2007; 366(2):406-19. Coliphage N4 N-acetylmuramidase defines a new family of murein hydrolases [E. coli phage N4N-acetylmellarinase defines a new family of mural hydrolases]. Stojkovic EA1, Rothman-Denes LB), STM0016 endolysin (NCBI reference sequence: NP 459021.1), PSP3 endolysin (NP 958065).1) and the endolysin of Salmonella enteritidis phage PVPSE1 (PVP-SElgpl46 (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. [Molecular Biology Journal] 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 [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 φ11 endolysin (Lytic activity of recombinantbacteriophage phi 11 and phi 12 endolysins on whole cells and biofilms of Staphylococcus aureus [Recombinant phage φ11 and φ12 endolysins on whole cells and biofilms of Staphylococcus aureus]. Sass P, Bierbaum G. Appl Environ Microbiol. [Applied Environmental Microbiology] January 2007; 73(l):347-52), φMRU 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 [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. [Journal of Bacteriology] October 2005; 187(20):7161-4), Clostridium perfringens PlyS9 (WO 2010003943 A1; Bacteriophage [phage]. April 2012; 2(2):89-97. Inducible Clostridium perfringens bacteriophages OS9 and OS63: Different genome structures and a fully functional sigK intervening element [Inducible Clostridium perfringens phage OS9 and OS63: Different genome structures and a fully functional sigK intervening element]. KimKP1, Born Y, Lurz R, Eichenseher F, Zimmer M, Loessner MJ, Klumpp J.), Ply3626 (Zimmer, M., Scherer, S. and Loessner, MJ Genomic analysis of Clostridium perfringens bacteriophage phi3626, which integrates into guaA and possibly affects sporulation [Genomic analysis of Clostridium perfringens phage φ3626 (which integrates into guaA and may affect sporulation)] J. Bacteriol. [Journal of Bacteriology] 184 (16), 4359-4368 (2002)), Clostridium difficile: CD27L endosynovin (J Bacteriol. [Journal of Bacteriology] October 2008; 190(20):6734-40.Molecular characterization of a Clostridium difficile bacteriophage and its cloned biologically active endolysin [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 [Bifunctional peptidoglycan lysin of Streptococcus agalactiae bacteriophage B30]. Pritchard DG, Dong S, Baker JR, Engler JA. Microbiology. July 2004; 150(Part 7):2079-87), Palamidinase encoded by phage Dp-1 (J Biol Chem. October 2004; 279(42):43697-707. Structural and Thermodynamic characterization of Pal, a phage natural chimeric lysin active against pneumococci [Pal (a phage natural chimeric lysin active against pneumococci) structure and thermodynamic characterization]. Varea Jl, Monterroso B, Saiz JL, Lopez-Zumel C, Garcia JL, Laynez J, Garcia P, Menendez M.), CI endolysin PlyC (PlyC: a multimericbacteriophage lysin [PlyC: a multimeric phage lysin]. Nelson D, Schuch R, Chahales P, Zhu S, Fischetti VA. Proc Natl Acad Sci US A. [Proceedings of the National Academy of Sciences of the United States of America] July 11, 2006; 103(28): 10765-70), Cpl-1 endolysin (Gene [Gene]. January 31, 1990; 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). January 2005; 49(l): 11-7. Removal of Group B Streptococcus colonized in the vagina and oropharynx of mice by phage lysis enzymes. Cheng Ql, Nelson D, Zhu S, Fischetti VA.), Enterococcus: PlyV12 (J Bacteriol. [Journal of Bacteriology] July 2004; 186(14): 4808-12. Identification of a broadly active phage lytic enzyme with lethal activity against antibiotic-resistant Enterococcus faecalis and Enterococcus [Identification of broadly active bacteriophage lysins with lethal activity against antibiotic-resistant Enterococcus faecalis]. Yoong PI, Schuch R, Nelson D, Fischetti VA.

[0021] Many antibacterial enzymes are composed of different domains, such as cell wall-binding domains and one or more cleavage domains exhibiting peptidoglycan hydrolase activity, such as amidase domains, peptidase_M23 domains, and CHAP (cysteine-, histidine-dependent amidase / peptidase) domains. These domains can be used to design chimeric endosomals that do not exist in nature. Such endosomals are clearly within the scope of the antibacterial enzymes described in this embodiment.

[0022] In the embodiments described herein, the chimeric endosomal can be a chimeric endosomal comprising, for example, WO2012 / 150858, WO 2013 / 169104, WO 2016 / 142445, WO 2017 / 046021, WO 2015155244, WO2015005787, WO 2011 / 023702, WO 2012 / 146738, WO 2003 / 082184, WO 2010 / 011960, WO2010 / 149795, WO 2010 / 149792, WO 2012 / 094004, WO 2011 / 023702, WO 2011 / 065854, WO2011 / 076432, WO The endolysins proposed in WO 2011 / 134998, WO 2012 / 059545, WO 2012 / 085259, WO 2012146738, and WO2018 / 091707 are polypeptides or composed thereof having at least about 70% sequence identity. Further preferred endolysins are those comprising or composed of polypeptides having at least about 70% sequence identity with endolysins or their functional portions represented by one of the following sequences: SEQ ID NOs: 1 to 81 in Table 1.

[0023] Those skilled in the art will understand that a single domain of the endolysin disclosed and / or referenced herein can be combined with other domains to produce chimeric endolysins. Such chimeric endolysins are clearly within the scope of antibacterial enzymes described in the embodiments herein, including, but not limited to, domains represented by one of the following sequences: SEQ ID NO: 82 to 95 or their functional portions.

[0024] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented as SEQ ID NO: 1.

[0025] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 2.

[0026] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 3.

[0027] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 4.

[0028] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 5.

[0029] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 6.

[0030] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 7.

[0031] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 8.

[0032] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 9.

[0033] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 10.

[0034] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented by SEQ ID NO: 11.

[0035] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 12.

[0036] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 13.

[0037] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented by SEQ ID NO: 14.

[0038] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 15.

[0039] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 16.

[0040] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented as SEQ ID NO: 17.

[0041] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 18.

[0042] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented as SEQ ID NO: 19.

[0043] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 20.

[0044] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented by SEQ ID NO: 21.

[0045] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 22.

[0046] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented as SEQ ID NO: 23.

[0047] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented by SEQ ID NO: 24.

[0048] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented as SEQ ID NO: 25.

[0049] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 26.

[0050] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 27.

[0051] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 28.

[0052] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 29.

[0053] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 30.

[0054] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented by SEQ ID NO: 31.

[0055] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented as SEQ ID NO: 32.

[0056] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented as SEQ ID NO: 33.

[0057] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 34.

[0058] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 35.

[0059] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented by SEQ ID NO: 36.

[0060] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented by SEQ ID NO: 37.

[0061] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented by SEQ ID NO: 38.

[0062] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 39.

[0063] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 40.

[0064] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented by SEQ ID NO: 41.

[0065] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 42.

[0066] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 43.

[0067] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 44.

[0068] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented as SEQ ID NO: 45.

[0069] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 46.

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

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

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

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

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

[0075] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion 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 portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 53.

[0077] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion 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 portion thereof having at least about 70% sequence identity with an endolysin or its functional portion 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 portion thereof having at least about 70% sequence identity with an endolysin or its functional portion 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 portion thereof having at least about 70% sequence identity with an endolysin or its functional portion 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 portion thereof having at least about 70% sequence identity with an endolysin or its functional portion 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 portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 59.

[0083] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 60.

[0084] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 61.

[0085] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 62.

[0086] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 63.

[0087] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 64.

[0088] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 65.

[0089] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 66.

[0090] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 67.

[0091] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 68.

[0092] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 69.

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

[0094] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented by SEQ ID NO: 71.

[0095] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 72.

[0096] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 73.

[0097] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented as SEQ ID NO: 74.

[0098] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 75.

[0099] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 76.

[0100] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with the endolysin or its functional portion represented by SEQ ID NO: 77.

[0101] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 78.

[0102] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of the following: a polypeptide or functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion represented as SEQ ID NO: 79.

[0103] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 80.

[0104] In the embodiments herein, the endolysin or chimeric endolysin may comprise or consist of a polypeptide or a functional portion thereof having at least about 70% sequence identity with an endolysin or its functional portion as represented by SEQ ID NO: 81.

[0105] In the embodiments described herein, the antibacterial enzyme may or may not have a tag attached to it, such as a peptide tag that can be used to promote enzyme expression and / or affinity purification, to immobilize the enzyme to a surface, or to serve as a marker or labeled moiety for detection of the enzyme in various ELISA assays, for example, by antibody binding. Such a tag may be a His tag, such as a C-terminal 6x His tag. Those skilled in the art know how to incorporate a His tag into the mRNA encoding the antibacterial enzyme.

[0106] In the embodiments described herein, the antibacterial enzyme may or may not have an antibacterial peptide attached to it, such as cationic or polycationic peptides, amphiphilic peptides, sushi peptides, defensins, and hydrophobic peptides. Examples of such antibacterial peptides are those proposed in WO 2015 / 155244 and WO 2016 / 142445.

[0107] In the embodiments described herein, the domains of antibacterial enzymes may or may not be separated by a linker. Such a linker may be native to the domain or exogenous to the domain.

[0108] Those skilled in the art will understand that a polypeptide may lack one or more encoded amino acids, particularly at the C-terminus or N-terminus of the polypeptide. Therefore, in the embodiments described herein, the antibacterial enzyme may lack one or more amino acids at the C-terminus or N-terminus, such as M-terminal methionine.

[0109] In the embodiments described herein, Staphylococcus aureus may be antibiotic-resistant Staphylococcus aureus, such as methicillin-resistant Staphylococcus aureus (MRSA).

[0110] In the embodiments described herein, the antibacterial enzyme may be present or may be present as a source of the antibacterial enzyme (such as a polynucleotide encoding the antibacterial enzyme). Such a polynucleotide encoding the antibacterial enzyme may conveniently be mRNA. The mRNA may be present in a delivery medium. Such mRNA constructs containing such mRNA encoding the antibacterial enzyme and delivery media are broadly described in EP 23190916.9, which is incorporated herein by reference. The use of the constructs described in EP 23190916.9 is explicitly included in the embodiments herein.

[0111] In the embodiments described herein, antibacterial enzymes may be present in compositions such as pharmaceutical compositions, wherein the compositions further comprise pharmaceutically acceptable excipients or carriers. Such pharmaceutically acceptable carriers, or interchangeably described herein as pharmaceutically acceptable excipients, may be any such carrier known to those skilled in the art. Pharmaceutically acceptable excipients used to manufacture pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Such excipients may optionally be included in pharmaceutical formulations. Based on the formulator's judgment, excipients such as cocoa butter and suppository wax, colorants, coating agents, sweeteners, flavoring agents, and / or performance agents may be present in the composition. Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dicalcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and / or combinations thereof.

[0112] Exemplary granulating agents and / or dispersants include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginate, guar gum, citrus pomace, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose, methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate, sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0113] Exemplary surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, astragalus gum, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate and magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, glyceryl triacetate monostearate, ethylene distearate, glyceryl monostearate and propylene monostearate, polyvinyl alcohol), and ear Homers (e.g., carboxylated polymethylene, polyacrylic acid, acrylic polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan, polyoxyethylene sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monostearate, sorbitan monostearate, etc.). Sorbitol 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), polyvinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl lauryl ester, sodium lauryl sulfate, Poloxamer 188. Cetrimonium bromide, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0114] Exemplary adhesives include, but are not limited to, starches (e.g., corn starch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., gum arabic, sodium alginate, Irish moss extract, solanum, psyllium husk gum, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), magnesium aluminum silicate, and larch arabinogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts: silica; polymethyl methacrylate; waxes; water; alcohols; etc.; and combinations thereof.

[0115] 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, α-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citrate monohydrate, disodium edetate, dipotassium edetate, edetate, 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, benzyl alcohol bromnitrile glycol, bromophthalic acid trimethylammonium chloride, hexadecylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexacodyl, imidazolidinyl urea, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal.

[0116] Exemplary antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Exemplary alcoholic preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, parabens, 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, tocopheryl acetate, deferoxamine mesylate, bromobrown trimethylammonium, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, and methylparaben.

[0117] Exemplary buffers 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, propionic acid, calcium levulinate, pentadienoic acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginate, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and / or combinations thereof.

[0118] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0119] Examples of oils include, but are not limited to, almond oil, avocado oil, babassu oil, bergamot oil, blackcurrant seed oil, borage oil, juniper oil, chamomile oil, canola oil, amaranth oil, Brazilian palm oil, castor oil, cinnamon oil, cocoa butter, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, flaxseed oil, geraniol oil, gourd oil, grapeseed oil, hazelnut oil, hyssop oil, isopropyl myristate, jojoba oil, kukui oil, mixed lavender oil, lavender oil, lemon oil, litsea cubeba oil, macadamia nut oil, mallow oil, mango seed oil, meadowfoam seed oil, mink oil, nutmeg oil, olive oil, orange oil, and orange salmon oil. Roughie oil, palm oil, palm kernel oil, peach kernel oil, peanut oil, poppy seed oil, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, shaquana oil, peppermint oil, sea buckthorn oil, sesame oil, shea butter, silicone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, ailanthus oil, vetiver oil, walnut oil, and wheat germ oil. Exemplary oils include, but are not limited to, butyl stearate, caprylic / capric triglyceride, caprylic / capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyl dodecyl alcohol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0120] In the embodiments described herein, the composition may be a solid composition such as a dry powder composition, or a liquid composition, such as an aqueous composition that may be a solution and / or suspension. Liquid dosage forms for, for example, oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art (e.g., water or other solvents), solubilizers and emulsifiers (such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide), oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters and mixtures thereof. In addition to inert diluents, oral compositions may include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and / or aromatizers. In the embodiments described herein for parenteral administration, the composition may be mixed with a solubilizer such as Cremophor / alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer and / or combinations thereof.

[0121] Injectable formulations (e.g., sterile injectable aqueous or oily suspensions) can be formulated using suitable dispersants, wetting agents, and / or suspending agents according to known techniques. Sterile injectable formulations can be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenteral-acceptable diluents and / or solvents, for example, as a solution in 1,3-butanediol. Acceptable media and solvents that can be used include water, USP Ringer's solution, and isotonic sodium chloride solution. Sterile, non-volatile oils are commonly used as solvents or suspension media. For this purpose, any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectable formulations.

[0122] Injectable preparations can be sterilized, for example, by filtering through a bacterial retention filter and / or incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0123] Similar types of solid compositions can be used as fillers in soft-filled and hard-filled gelatin capsules that use such excipients as lactose (or milk sugar) and high molecular weight polyethylene glycol.

[0124] Solid dosage forms such as tablets, sugar-coated pills, capsules, pellets, and granules can be prepared using coatings and shells (such as enteric coatings and other coatings well known in the field of pharmaceutical formulation). They may optionally contain light-blocking agents and may have compositions that, optionally in a delayed manner, release only or preferentially one or more active ingredients in a portion of the intestine. Examples of encapsulation compositions that can be used include polymeric substances and waxes. Similar types of solid compositions can be used as fillers in soft-filled and hard-filled gelatin capsules that use such excipients as lactose (or milksugar) and high molecular weight polyethylene glycol.

[0125] Topical and / or transdermal dosage forms of the composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, and / or patches. Generally, the active ingredient is blended under sterile conditions with pharmaceutically acceptable excipients and / or any desired preservatives and / or buffers, as needed. Additionally, within the scope of this invention is the use of transdermal patches, which typically offer the added advantage of controlled delivery of the compound to the body. Such dosage forms can be prepared, for example, by dissolving and / or dispersing the compound in a suitable medium. Alternatively or additionally, the rate can be controlled by providing a rate-controlled membrane and / or by dispersing the compound in a polymer matrix and / or gel.

[0126] In the embodiments described herein, the subject can be any animal subject, preferably a mammal, livestock (such as a cow), domestic animal (like a dog or cat), or human subject.

[0127] The medical uses disclosed in this embodiment can be defined as products used as medicines for treating the conditions, as well as methods of using the product to treat the conditions, products used in the preparation of medicines for treating the conditions, and the use of the product to treat the conditions. These medical uses are all contemplated by this invention.

[0128] Therefore, in the embodiments herein, a method is provided for treating a subject with cancer refractory to chemotherapy drugs (preferably histone deacetylase (HDAC) inhibitors, more preferably histone deacetylase (HDAC) inhibitors as described above), said treatment comprising administering to the subject an effective amount of an antibacterial enzyme effective against Staphylococcus aureus.

[0129] Therefore, in the embodiments herein, the use of an antibacterial enzyme effective against Staphylococcus aureus for the manufacture of a medicament for treating cancer refractory to chemotherapy drugs (preferably histone deacetylase (HDAC) inhibitors, more preferably histone deacetylase (HDAC) inhibitors as described above) is provided, said treatment comprising administering to a subject an effective amount of the antibacterial enzyme effective against Staphylococcus aureus.

[0130] In a second aspect, a method for blocking drug resistance in primary malignant T cells is provided, the method comprising contacting these malignant T cells with an antibacterial enzyme effective against Staphylococcus aureus. Features of this aspect are preferably those of embodiments of the first aspect. The malignant T cells can be derived from any subject suffering from cancer refractory to chemotherapy. Chemotherapy drugs refractory to the cancer can be anthracyclines, such as doxorubicin; topoisomerase II inhibitors, such as etoposide; and / or preferably histone deacetylase (HDAC) inhibitors, such as vorinostat, romidesin, remidesat, pabisostat, and benoristat. In embodiments, the histone deacetylase (HDAC) inhibitor is vorinostat; in one embodiment, the histone deacetylase (HDAC) inhibitor is romidesin; in embodiments, the histone deacetylase (HDAC) inhibitor is remidesat; in embodiments, the histone deacetylase (HDAC) inhibitor is pabisostat; in embodiments, the histone deacetylase (HDAC) inhibitor is benoristat. Preferably, the malignant T cells are malignant T cells derived from SS patients.

[0131] Table 1: Sequence Overview

[0132] Attached Figure Description

[0133] Figure 1. SE rescues malignant cells from romidesin-induced apoptosis, while endolysin eliminates this rescue.

[0134] A. Flow cytometry plot depicting apoptotic population of phylogenetic malignancies from PBMCs of SS patients after 72 h of treatment with supernatants of romidesin and Staphylococcus aureus cultures isolated from different CTCL patients (treated alone or with endolysin).

[0135] B. Western blot analysis depicting PARP expression in PBMCs of SS patients after treatment with SE and / or romidesin for 24 h and 48 h. GAPDH was used as a loading control.

[0136] C. A graph depicts the apoptotic populations of phage malignant cells from PBMCs of SS patients (n = 5) after treatment with SE (PBS) and SE stimulation followed by different concentrations of romidesin for 72 h and 144 h. Two-way ANOVA was performed, followed by Cidal multiple comparison test. P < 0.01, P < 0.0005.

[0137] D. A diagram depicting romidesin-induced apoptosis in SE-rescued malignant cells from high concentrations of romidesin treatment (as mentioned in the romidesin pulse experiment in Materials and Methods).

[0138] E. Representative flow cytometry plot from PBMCs of SS patients, showing the results using SEA or mutant SEA (SEA). F47A / D227A Apoptotic population of phylogenetic malignant cells after treatment with romidesin for 72 h.

[0139] F. Figure 1E The bar graph shows that in SS patients (n = 5), SEA, rather than mutant SEA, rescued malignant cells from romidesin-induced apoptosis.

[0140] G. A representative flow cytometry plot from PBMCs of SS patients, showing an apoptotic population of phylogenetic malignancies after treatment with SEA or SEA and anti-SEA antibodies and romidesin for 72 h.

[0141] H. Figure 1G The bar graph shows that in SS patients (n = 4), SEA rescued malignant cells, but SEA blocking antibodies inhibited the rescue of malignant cells, making them susceptible to romidesin-induced apoptosis.

[0142] For F. and H., a one-way ANOVA was performed using the R.M. method, followed by a Tukey multiple comparison test. P < 0.05.

[0143] Figure 2. SE largely counteracts the effect of romidesin in malignant T cells.

[0144] A. A representative flow cytometry image of phylogenetic malignant cells from PBMCs of SS patients, showing SE-induced cell survival after 72 h of treatment with 2 nM romidasine.

[0145] B. Figure 2AThe bar chart depicts the significant survival of SE-induced malignant cells after romidesin treatment in SS patients (n = 11). Red dots represent PBMCs from SS patients who did not respond to romidesin treatment. For statistical analysis, ordinary one-way ANOVA was performed, followed by Tukey's multiple comparison test. P < 0.01, P < 0.0005, P < 0.0001.

[0146] C. - H. Single-cell transcriptome and epitope sequencing cell index (CITE-Seq) analysis of SS PBMCs treated with romedixin in the presence or absence of SE.

[0147] Figure 3. SE rescue of malignant cells from cell death induced by different drugs used in CTCL treatment.

[0148] Representative flow cytometry images of phylogenetic malignancies from PBMCs of SS patients after the following treatments with and without SE stimulation, and bar graphs depicting viable malignant cell populations in PBMCs of more SS patients:

[0149] A. - B. Vorinostat (n = 5)

[0150] C. - D. Remistatin (n = 3)

[0151] E.-F. Doxorubicin (n = 4) (using Mitotracker instead of PI, and the gating strategy for flow cytometry is similar to that for other drug treatments).

[0152] G.-H.etoposide (n = 5)

[0153] I. - J. Bortezomib (n = 4).

[0154] For statistical analysis, a simple one-way ANOVA was performed, followed by a Dunnett multiple comparison test. P < 0.05, P < 0.005, P < 0.0005.

[0155] Figure 4. SE-mediated rescue from romidesin can be blocked by targeting LCK and PKC kinases involved in TCR signaling.

[0156] Representative flow cytometry plots of phylogenetic malignancies from PBMCs of SS patients after treatment with and without SE stimulation and romidexin, and bar graphs depicting viable malignant cell populations in PBMCs of more SS patients:

[0157] A. - BA-419259 (Src inhibitor) (n = 8)

[0158] C. - D. Dasatinib (n = 6)

[0159] E.-F. Sotrastulin (PKC inhibitor) (n = 5).

[0160] For statistical analysis, one-way ANOVA with Reynolds analysis was performed, followed by Tukey multiple comparison test. P < 0.05, P < 0.01, P < 0.0005, P < 0.0001.

[0161] Figure 5. IL-2 family cytokines rescue malignant cells from romidesin-mediated cell death.

[0162] A. A representative flow cytometry image of phylogenetic malignant cells from PBMCs of SS patients, depicting the survival of malignant cells induced by treatment with cytokines (IL2, IL4, IL7, IL15) after 72 h of romidesin treatment.

[0163] B. Figure 5A The bar chart represents the percentage of viable malignant cell populations after different treatments, with cytokine treatment inducing significant cell survival after romidesin treatment (n = 7). For statistical analysis, ordinary one-way ANOVA was performed, followed by Tukey multiple comparison tests. P < 0.0001.

[0164] C. Representative flow cytometry plots from SS patients treated with romidasin after a 72-hour study, depicting malignant cells with SRC inhibitor (A-419259) and JAK inhibitor (tofacitinib) that blocked SE stimulation alone or cytokine treatment alone. Controls show an untreated viable cell population after 72 hours.

[0165] E. A representative flow cytometry plot from JAK-dependent SS patients, depicting how tofacitinib inhibits SE-induced malignant cell survival after romidisin treatment.

[0166] D and F bar graphs depict the percentage of malignant cells that survived 72 h after SE stimulation and treatment with romidasin alone or with tofacitinib. Figure 5D JAK dependence = Tofacitinib + Romidesin blocking SE-mediated survival in SS patients (n = 2), and Figure 5F JAK non-dependency = Tofacitinib cannot eliminate SE-mediated survival in SS patients after romidesin treatment (n = 3)).

[0167] Figure 6. IL-2 family cytokines rescue malignant cells from romidesin-mediated cell death.

[0168] A. Representative flow cytometry plots depicting the percentage of malignant cell populations in PBMCs of SS patients before and after sorting.

[0169] B. Representative flow cytometry plots depicting the survival of sorted malignant cells after 72 h of treatment with SE-stimulated and romedixin-treated cells, and showing that the JAK inhibitor (tofacitinib) could not block SE-mediated survival (JAK-independent).

[0170] C. Representative flow cytometry plot depicting the JAK inhibitor (tofacitinib) blocking SE-mediated survival of sorted malignant cells co-treated with romidasin (JAK-dependent).

[0171] Figure 7 SE activates rescue malignant T cells via direct and indirect TCR.

[0172] Elucidation of the putative mechanism by which Staphylococcus aureus and its SEs can induce drug resistance in malignant T cells - a cytokine-independent pathway driven by direct TCR ( Figure 7 (the upper part) and the indirect cytokine-driven pathway that may involve neighboring CD4 T cells ( Figure 7 (Lower part). SRC inhibitors block SE-mediated TCR-dependent survival (both direct and indirect), while tofacitinib only blocks cytokine-dependent survival mediated by indirect activation.

[0173] Figure 8. SE rescues malignant cells from romidesin-induced apoptosis, while endolysin eliminates this rescue.

[0174] A. A representative flow cytometry image from PBMCs of SS patients, depicting the apoptosis of malignant T cells induced by different concentrations of romidesin after 72 h, but not by non-malignant T cells.

[0175] B. Representative flow cytometry plot depicting the apoptotic population of phylogenetic malignancies from PBMCs of additional SS patients after 72 h of treatment with supernatants of romidesin and Staphylococcus aureus cultures (alone or with endolysin) isolated from different CTCL patients.

[0176] C. Representative flow cytometry plot depicting the apoptotic population of phylogenetic malignancies from PBMCs of additional SS patients after 72 h of treatment with supernatants from two different cultures: one producing (Staphylococcus aureus supernatant (SEA)) and the other not producing (Staphylococcus aureus supernatant (SEA-free)).

[0177] D - E. Representative flow cytometry images depicting apoptotic populations of phylogenetic malignancies from PBMCs (left and right) of two SS patients after 24, 48, and 72 h of romidesin treatment.

[0178] definition

[0179] "Sequence identity" is defined herein as the relationship between two or more amino acid (peptide, polypeptide, or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by sequence comparison. In the art, "identity" also refers to the degree of sequence relevance between amino acid or nucleotide sequences, as determined, such as by matching strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one peptide or polypeptide and its conserved amino acid substitutions with the sequence of a second peptide or polypeptide. In a preferred embodiment, identity or similarity is calculated over the entire SEQ ID NO as identified herein. "Identity" and "similarity" can be readily calculated using known methods, including but not limited to those described in: *Computational Molecular Biology*, edited by Lesk, AM, Oxford University Press, New York, 1988; *Biocomputing: Informatics and Genome Projects*, edited by Smith, DW, Academic Press, New York, 1993; *Computer Analysis of Sequence Data*, Part I, edited by Griffin, AM and HG, Humana Press, New Jersey, 1994; *Sequence Analysis in Molecular Biology*, von Heine, G., Academic Press, 1987; and *Sequence Analysis Primer*, edited by Gribskov, M. and Devereux, J., M. Stockton Press, New York. 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math. 48:1073 (1988).

[0180] Preferred methods for determining identity were designed to obtain the maximum match between test sequences. Methods for determining identity and similarity were incorporated into publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, for example, the GCG package (Devereux, J. et al., Nucleic AcidsResearch 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, SF et al., J. Mol. Biol. 215:403-410 (1990)). The BLAST X procedure is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, MD [Bethesda, MD] 20894; Altschul, S. et al., J. Mol. Biol. [Journal of Molecular Biology] 215:403-410 (1990)). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0181] Preferred parameters for peptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. [Journal of Molecular Biology] 48:443-453 (1970); Comparison matrix: BLOSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. [Proceedings of the National Academy of Sciences] 89:10915-10919 (1992); Vacancy penalty: 12; and Vacancy length penalty: 4. A program that can be used with these parameters is publicly available as the “Ogap” program from the Genetics Computer Group in Madison, Wisconsin. The aforementioned parameters are the default parameters for amino acid comparison (no penalty for terminal vacancies).

[0182] Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. [Journal of Molecular Biology] 48:443-453 (1970); Comparison matrix: Match = +10, Mismatch = 0; Vacancy penalty: 50; Vacancy length penalty: 3. These are available as a gap program from the Genetics Computer Group in Madison, Wisconsin. The default parameters for nucleic acid comparison are given above.

[0183] Optionally, in determining the degree of amino acid similarity, those skilled in the art may also consider so-called “conservative” amino acid substitutions, as will be apparent to them. Conservative amino acid substitution refers to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy side chains includes serine and threonine; a group of amino acids with amide-containing side chains includes asparagine and glutamine; a group of amino acids with aromatic side chains includes phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains includes lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains includes cysteine ​​and methionine. Preferred groups of conserved amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitution variants of the amino acid sequences disclosed herein are those in which at least one residue has been removed from the disclosed sequence and a different residue has been inserted at its position. Preferably, the amino acid changes are conserved. Preferred conservative substitutions for each naturally occurring amino acid are as follows: Ala to ser; Arg to lys; Asn to gln or his; Asp to glu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; His to asn or gln; Ile to leu or val; Leu to ile or val; Lys to arg; gln 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.

[0184] A “nucleic acid molecule” or “polynucleotide” (these 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 isolated from a naturally occurring gene, or a nucleic acid molecule modified to contain nucleic acid segments combined or juxtaposed in a manner not found in nature. Nucleic acid molecules are represented by nucleotide sequences. Optionally, the nucleotide sequences present in the nucleic acid construct are operatively linked to one or more control sequences that direct the production or expression of the peptide or polypeptide in a cell or subject.

[0185] "Operationally linked" is defined herein as a configuration in which a control sequence is suitably placed relative to a nucleotide sequence encoding the polypeptide of the present invention, such that the control sequence directs the production / expression of the peptide or polypeptide of the present invention in cells and / or subjects. "Operationally linked" can also be used to define a configuration in which a sequence is suitably placed relative to another sequence encoding a functional domain, such that the chimeric polypeptide is encoded in cells and / or subjects.

[0186] "Expression" is interpreted to include any step involved in the production of peptides or polypeptides, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0187] “Control sequence” is defined herein as comprising all components that are essential or advantageous for peptide expression. At a minimum, the control sequence comprises a promoter as well as transcription and translation termination signals. Optionally, the promoter, represented by a nucleotide sequence present in the nucleic acid construct, is operatively linked to another nucleotide sequence encoding a peptide or polypeptide as identified herein.

[0188] The term "transformation" refers to a permanent or transient genetic change induced by the incorporation of new DNA (i.e., exogenous DNA) into a cell. When the cell is a bacterial cell, as contemplated in this invention, the term generally refers to an extrachromosomal self-replicating vector with selective antibiotic resistance.

[0189] "Expression vector" can be any vector that allows for convenient recombinant DNA procedures and can result in the expression of a nucleotide sequence encoding the polypeptide of the present invention in cells and / or subjects. As used herein, the term "promoter" refers to a nucleic acid fragment for controlling the transcription of one or more genes or nucleic acids, located upstream of the transcription start site of the gene in the transcriptional direction. It involves binding sites identified by the presence of binding sites for DNA-dependent RNA polymerases, transcription start sites, and any other DNA sequences, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art that directly or indirectly function to regulate the amount of transcription of the promoter. In the context of the present invention, the promoter preferably terminates at nucleotide -1 of the transcription start site (TSS).

[0190] As used herein, "polypeptide" refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. Polypeptides are composed of a continuous sequence of amino acids. The term "polypeptide" encompasses both naturally occurring and synthetic molecules.

[0191] The sequence identity of the polynucleotide, polynucleotide construct, or polypeptide described herein 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 the case of 100% sequence identity, the polynucleotide or polypeptide is completely identical to the sequence of the described SEQ ID NO:. Sequence identity is preferably determined over the entire length of the target sequence.

[0192] The sequence information provided herein should not be narrowly interpreted as needing to include incorrectly identified bases. Those skilled in the art can identify such incorrectly identified bases and know how to correct such errors.

[0193] In this document and its claims, the verb “comprising” and its inflections are used in a non-limiting sense to mean including the items following the word, but not excluding items not specifically mentioned. Furthermore, the verb “consisting of” can be replaced by “consisting substantially of”, meaning that a product or composition, nucleic acid molecule, peptide, polypeptide, nucleic acid construct, carrier, or cell as defined herein may contain additional components besides those specifically identified; said additional components do not alter the distinctive features of the invention. Additionally, reference to an element by the indefinite article “a” or “an” does not exclude the possibility of more than one element unless the context explicitly requires the presence of one and only one element. Therefore, the indefinite article “a” or “an” generally means “at least one”. The word “about” or “approximately” when used in conjunction with a numerical value (e.g., about 10) preferably means that the value can be a given value (10) plus or minus 10% of that value.

[0194] All patents and references cited in this specification are incorporated herein by reference in their entirety.

[0195] Unless otherwise stated, each embodiment described herein may be combined with another embodiment described herein.

[0196] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0197] Other embodiments

[0198] Further embodiments of the invention are listed below.

[0199] 1. An antibacterial enzyme effective against Staphylococcus aureus for use in treating a subject with cancer refractory to chemotherapy drugs, said treatment comprising administering an effective amount of the antibacterial enzyme to the subject.

[0200] 2. The effective antibacterial enzyme against Staphylococcus aureus as described in Example 1, wherein the chemotherapeutic agent for which the cancer is refractory is an anthracycline, such as doxorubicin; a topoisomerase II inhibitor, such as etoposide; and / or a histone deacetylase (HDAC) inhibitor.

[0201] 3. An effective antibacterial enzyme against Staphylococcus aureus as described in Example 1 or 2, wherein the histone deacetylase (HDAC) inhibitor is vorinostat, romidesin, remistat, parbisitat, or benostat.

[0202] 4. An effective antibacterial enzyme against Staphylococcus aureus as described in any one of Examples 1 to 3, wherein, in addition to the antibacterial enzyme, a chemotherapeutic agent is administered to the subject.

[0203] 5. An effective antibacterial enzyme against Staphylococcus aureus as described in any one of Examples 1 to 4, wherein the cancer is a lymphoma, such as cutaneous T-cell lymphoma (CTCL), such as leukemia-related Cezari syndrome (SS).

[0204] 6. An effective antibacterial enzyme against Staphylococcus aureus as described in any of Examples 4 and 5, wherein the further administered chemotherapeutic agent is an anthracycline, such as doxorubicin; a topoisomerase II inhibitor, such as etoposide; and / or a histone deacetylase (HDAC) inhibitor, such as vorinostat, romidazine, remistat, parbistat, and benostat.

[0205] 7. An effective antibacterial enzyme against Staphylococcus aureus as described in any of the foregoing embodiments, wherein the cancer is refractory due to staphylococcal enterotoxins (SE), such as SE produced by Staphylococcus aureus.

[0206] 8. An effective antibacterial enzyme against Staphylococcus aureus according to any of the foregoing embodiments, wherein the antibacterial enzyme is selected from the group consisting of: bacteriocins or their functional portions, bacteriosins or autolysins or their functional portions, phage lysins or their functional portions, and chimeric lysins or their functional portions.

[0207] 9. An effective antibacterial enzyme against Staphylococcus aureus according to any of the foregoing embodiments, wherein the antibacterial enzyme is a chimeric endosomal, preferably a recombinant chimeric endosomal comprising one or more heterologous domains.

[0208] 10. An effective antibacterial enzyme against Staphylococcus aureus according to any of the foregoing embodiments, wherein the Staphylococcus aureus is an antibiotic-resistant Staphylococcus aureus, such as methicillin-resistant Staphylococcus aureus (MRSA).

[0209] 11. An effective antibacterial enzyme against Staphylococcus aureus according to any of the foregoing 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.

[0210] 12. An effective antibacterial enzyme against Staphylococcus aureus as described in any of the foregoing embodiments, wherein the subject is a mammal, such as livestock, domestic animals, or a human.

[0211] 13. A method of treating a subject with cancer refractory to chemotherapy, said treatment comprising administering to the subject an effective amount of an antibacterial enzyme effective against Staphylococcus aureus.

[0212] 14. The treatment method according to Example 13, wherein the chemotherapy drug that is refractory to the cancer is an anthracycline, such as doxorubicin; a topoisomerase II inhibitor, such as etoposide; and / or a histone deacetylase (HDAC) inhibitor.

[0213] 15. The treatment method according to Example 14, wherein the histone deacetylase (HDAC) inhibitor is vorinostat, romidesin, remistat, pabistat, or benostat.

[0214] 16. The use of an antibacterial enzyme effective against Staphylococcus aureus in the manufacture of a medicament for treating cancers refractory to chemotherapy, said treatment comprising administering to the subject an effective amount of an antibacterial enzyme effective against Staphylococcus aureus.

[0215] 17. The use according to Example 16, wherein the chemotherapeutic agent for which the cancer is refractory is an anthracycline, such as doxorubicin; a topoisomerase II inhibitor, such as etoposide; and / or a histone deacetylase (HDAC) inhibitor.

[0216] 18. The use as described in Example 17, wherein the histone deacetylase (HDAC) inhibitor is vorinostat, romidesin, remistat, pabistat, or benostat.

[0217] 19. A method for blocking drug resistance in primary malignant T cells, the method comprising contacting the malignant T cells with an antibacterial enzyme effective against Staphylococcus aureus.

[0218] 20. The method according to Example 19, wherein the chemotherapy drug that is refractory to the cancer is an anthracycline, such as doxorubicin; a topoisomerase II inhibitor, such as etoposide; and / or a histone deacetylase (HDAC) inhibitor.

[0219] 21. The method according to Example 20, wherein the histone deacetylase (HDAC) inhibitor is vorinostat, romidesin, remistat, pabistat, or benostat. Example

[0220] summary

[0221] Patients with Cézari syndrome (SS) (a leukemic variant of cutaneous T-cell lymphoma (CTCL)) are susceptible to Staphylococcus aureus (S. aureus) infection and have a poor prognosis due to treatment resistance. Here, we report on drug resistance inducible by Staphylococcus aureus and staphylococcal enterotoxin (SE) in malignant T cells to commonly used therapeutic agents in CTCL. Supernatant of patient-derived SE-producing Staphylococcus aureus and recombinant SE significantly inhibited cell death induced by the histone deacetylase (HDAC) inhibitor romidesin in primary malignant T cells from SS patients. The effect of Staphylococcus aureus supernatant was eliminated by bacteriocytosis with engineered phage-derived Staphylococcus aureus-specific endolysin (XZ.700; SEQ ID NO: 76 in this paper; see, for example, WO 2017 / 046021). Similarly, mutations in the MHC class II binding site of SEA type (SEA) and anti-SEA antibodies blocked the induction of resistance. Importantly, SE also triggers resistance to other HDAC inhibitors (vorinostat and remixstat) and chemotherapeutic agents (doxorubicin and etoposide). Multimodal single-cell sequencing indicated that the TCR, NFkB, and JAK / STAT signaling pathways (previously associated with treatment resistance) are putative mediators of SE-induced drug resistance. Supportingly, inhibition of TCR signaling and protein kinase C (upstream of NFkB) counteracted SE-induced rescue from drug-induced cell death. In contrast, SE failed to rescue cell death induced by the proteasome / NFkB inhibitor bortezomib. Inhibition of JAK / STAT blocked SE-induced rescue against malignant T cells in some patients but not in others, suggesting that SE can induce drug resistance in two distinct ways.

[0222] In summary, we have demonstrated that Staphylococcus aureus enterotoxin induces drug resistance in primary malignant T cells. These findings suggest that Staphylococcus aureus enterotoxin leads to clinical treatment resistance in patients with SS, and that antibacterial measures against Staphylococcus aureus (such as treatment with endolysin) will improve cancer-targeted therapy outcomes in patients with Staphylococcus aureus.

[0223] introduction

[0224] Cezari syndrome (SS) is a malignant disorder of T cells belonging to the cutaneous T-cell lymphoma (CTCL) (non-Hodgkin lymphoma) spectrum (1-4). The etiology of CTCL remains unresolved (5). Dysregulation of JAK / STAT, PLCg, and NFkB signaling has been repeatedly associated with malignant cell proliferation, drug resistance, and inflammation in CTCL (6-8).

[0225] A key characteristic of CTCL is increased susceptibility to bacterial infections, which are a major cause of disease morbidity and mortality (9-15). Skin barrier defects induced by malignant T cells may be an entry point for bacterial infection (16-17).

[0226] Here, we demonstrate for the first time that SE-producing Staphylococcus aureus can induce drug resistance in malignant T cells, and that antibacterial therapy specifically targeting Staphylococcus aureus can improve outcomes of cancer-targeted therapy in CTCL patients.

[0227] Materials and methods

[0228] Patient materials

[0229] This study included 17 SS patients. Patient characteristics are described in Table 2. Blood samples were collected from SS patients after obtaining their written consent and approval from institutions in Denmark (Committee on Health Research Ethics, H-16025331) and Germany. The experiment was conducted in accordance with the Declaration of Helsinki.

[0230] PBMC isolation, cell culture, inhibitor and pulse experiments.

[0231] Peripheral blood mononuclear cells (PBMCs) from SS patients were isolated by density gradient centrifugation, following the manufacturer's protocol (Stem Cell Technologies). PBMCs were cultured in RPMI 1640 medium (Sigma) supplemented with 10% conjugated human serum (Copenhagen Hospital Blood Bank) and 1% antibiotics (penicillin and streptomycin) (Sigma). For SE stimulation, a pool of 50–100 ng / mL of SE (SEA, SEB, SEC2, SED, and SEI) (Toxin Technologies) was added to the cells prior to treatment with CTCL. For cytokine assays, an IL-2 family cytokine pool—IL-2 (2 × 10⁻⁶)—was added. 3 U / ml, Novartis), IL-4, IL-7, and IL-15 (10 ng / mL each, Peprotech). Inhibitors / drugs: A-419259 and oligomycins (Sigma-Aldrich); romidesin, dasatinib, tofacitinib, vorinostat, remixstat, doxorubicin, bortezomib, etoposide (Selleckchem); crystal violet and sotrastolin (MedChemExpress). For romidesin pulse assays, cells were treated with high concentrations of romidesin for 6 hours. Cells were washed and held in normal medium for 16 hours, followed by incubation for three days with and without SE pools.

[0232] Patient bacterial isolation and culture

[0233] Staphylococcus aureus was isolated from CTCL patients and its expression was verified. Supernatants from Staphylococcus aureus cultures were prepared as previously described (18). Briefly, Staphylococcus aureus was prepared from overnight cultures diluted to 0.01 OD600 nm in tryptone soybean broth (TSB) and allowed to regrow for 4 hours with or without 1 μg / mL XZ.700 (SEQ ID NO: 76; see, for example, WO 2017 / 046021). Bacterial supernatants were prepared by centrifugation (10,000 g, 10 min) followed by sterile filtration (0.22 μm filter).

[0234] Flow cytometry and cell sorting

[0235] Primary conjugated monoclonal antibodies targeting CD3, CD4, CD7, CD8, CD14, CD26, CD45, TCRCβ1, TCRVβ1, TCRVβ2, TCRVβ8, and TCRVβ18 were purchased from BD Biosciences, BioLegend, Miltenyi Biotec, Beckman Coulter, or R&D Systems. After culture, cells were centrifuged and washed with FACS-PBS buffer (PBS + 1% FBS + 0.02% NaN3). Cell surface staining was performed in FACS-PBS or Brilliant staining buffer (BD Biosciences) (on ice for 30 minutes). Malignant cells were gated as CD3+CD4+TCRVβ#+. In patients where the dominant TCRVβ clone could not be targeted, malignant cells were gated as CD3+CD4+CD7. 低 And / or CD26- or CD3+CD4+CD26-TCRCβ1+ / TCRCβ1- (depending on whether the TCR of a malignant clone utilizes the TRBC1 or TRBC2 gene segment in its TCRβ chain). Flow cytometry analysis was performed using a 5-laser BD LSR-Fortessa at the core flow cytometry facility of the University of Copenhagen. Flow cytometry data were visualized and analyzed using FlowJo (TreeStar) software. PI was added before sorting to exclude dead cells. Cell sorting was performed using a 100-µm nozzle on a three-laser BD FACS Aria-II. The purity of sorted cells was consistently above 95%.

[0236] Apoptosis assay and Western blot

[0237] For apoptosis assays, at the end of cell culture, cells were stained with Mitotracker Red CMXRos (Thermofisher) (by adding it to the culture medium) and incubated at 37°C for 30 minutes. Cells were then washed and subjected to flow cytometry. After staining the cell surface, cells were stained with propidium iodide (PI) and annexin V (suspended in annexin V binding buffer (BD Biosciences)). Apoptotic cells from live (PI-) malignant or non-malignant cells were identified as Mitotracker cells. 低Annexin V+, meaning that if a patient has a dominant TCRVβ1 clone, the apoptotic population of malignant cells will be gated to PI-CD3+CD4+TCRVβ1+Mitotracker. 低 Annexin V+. As previously described (20), Western blotting was performed and analyzed using Image Lab software (BioRad).

[0238] CITE-Seq (Cellular Transcriptome and Epitope Sequencing Index)

[0239] For CITE-seq experiments, patient PBMCs were treated with romidesin for 36 hours in the presence or absence of SE. Each condition was stained with a unique subject-labeled antibody along with a mixture of TotalSeq-C surface antibodies. CITE-seq staining protocols and antibodies were individually optimized and stepwise as previously described (19).

[0240] CITE-Seq (Cellular Transcriptome and Epitope Sequencing Index)

[0241] For CITE-seq experiments, patient PBMCs were treated with romidesin for 36 hours in the presence or absence of SE. Each condition was stained with a unique subject-labeled antibody along with a mixture of TotalSeq-C surface antibodies. CITE-seq staining protocols and antibodies were individually optimized and stepwise as previously described (19).

[0242] statistics

[0243] Statistical analysis was performed using GraphPad Prism. The software was also used to create graphs. Error bars represent the standard error of the mean, and the statistical significance level was set to [value missing]. P < 0.05.

[0244] result

[0245] Staphylococcus aureus culture supernatant and SE induce drug resistance in malignant T cells.

[0246] Eradication of Staphylococcus aureus with antibiotics reduced disease activity in patients undergoing anticancer therapy with doxorubicin, vorinostat, and iveretinoin. This confirms and expands previous data that eliminating bacterial colonization increases the efficacy of different types of ongoing anticancer treatments. 33 This led us to hypothesize that Staphylococcus aureus and its toxins could directly induce drug resistance in malignant T cells. To address this hypothesis, we initially focused on the effect of Staphylococcus aureus on the response of malignant T cells to romidesin, a potent inducer of apoptosis in malignant cells but not in non-malignant CD4+ T cells. Figure 8A Therefore, we examined whether the supernatant from SE-producing Staphylococcus aureus (isolated from the skin of affected SS patients) could modulate romidesin-induced apoptosis in primary malignant cells. We treated SS PBMCs with romidesin for 72 h with or without supernatant, followed by flow cytometry analysis of malignant cell apoptosis. The supernatant from SE-producing Staphylococcus aureus significantly reduced romidesin-induced apoptosis in malignant cells, while the supernatant from Staphylococcus aureus treated with endolysin (XZ.700) did not have this effect. Figure 1A and Figure 8B Since endosomalin eliminates SE production and kills Staphylococcus aureus (18), we hypothesized that the anti-apoptotic effect of Staphylococcus aureus supernatant is SE-mediated. To address this, we treated SS PBMCs with romidesin in the presence or absence of supernatant from two patient-derived Staphylococcus aureus isolates that produced or did not produce SE. Figure 8C Interestingly, only Staphylococcus aureus supernatant containing SE showed a significant reduction in romidesin-induced apoptosis. To test whether SE was sufficient to counteract romidesin-induced apoptosis, we treated SS PBMCs with romidesin in the presence or absence of an SE pool and evaluated PARP cleavage (…). Figure 1B ) and flow cytometry ( Figures 8D-8E The study analyzed apoptosis in malignant cells. As hypothesized, romidesin-induced apoptosis was significantly reduced in a dose- and time-dependent manner in the presence of SE. Figure 1C SE's anti-apoptotic effect is not limited to this treatment regimen. Therefore, even when added 16 hours after pulsed treatment with romidesin, SE blocked the apoptotic effects of very high concentrations of romidesin (25 to 100 nM). Figure 1D Analysis of the Staphylococcus aureus supernatant identified SEA as the major type of SE produced by patient-derived Staphylococcus aureus (18). Consistent with these results, recombinant SEA (SEA) was identified as a major type of SE produced by Staphylococcus aureus from patients. wt It significantly reduced romidesin-induced apoptosis. Figures 1E-1F In contrast, the mutant SEA (SEA) cannot bind to MHC II. F47A / D227A (21) No effect ( Figures 1E-1F As expected, the effect of SEA was eliminated by adding a blocking anti-SEA antibody. Figures 1G-1H In summary, this indicates that (i) SEA is sufficient to counteract treatment-induced apoptosis, (ii) this effect depends on the superantigenic properties of SEA, and (iii) the observations are not due to off-target effects or contaminants.

[0247] SE largely counteracted the transcriptional effects of romidesin in malignant T cells.

[0248] In our SS cohort, malignant cells from nine out of ten romidesin-naïve patients were sensitive to romidesin, as measured by a significant decrease in cell survival after treatment. Figures 2A-2B Importantly, the presence of SE significantly improved the survival of malignant cells after romidesin treatment. Figures 2A-2B To explore this effect, we performed single-cell transcriptome and epitope sequencing-based cell indexing (CITE-Seq) analysis on SS PBMCs treated with romidecin in the presence and absence of SE. Figures 2C-2H Cell cultures were collected after 36 hours to capture the transcriptional profile prior to the onset of romidesin-induced cell death, and the sensitivity of malignant cells to romidesin was confirmed by flow cytometry after 72 hours of culture. Cell types were identified by their surface protein expression, and malignant cells were defined by the expression of their monoclonal TCRs and malignancy-associated markers TOX (22) and KIR3DL2 (23), as well as by the binding of low surface levels of CD7 (24) and CD26 (25). Figures 2C-2D Although romidesin and SE alone induce strong transcriptional responses in malignant cells ( Figures 2E-2F However, treatment with romidesin in the presence of SEs largely mirrors the response to individual SEs, as evidenced by co-clustering in differentially expressed genes and transcriptional spaces. This trend is also supported by gene set enrichment analysis (GSEA) and transcription factor (TF) activity analysis. Figures 2G-2H These findings suggest that SE-induced gene regulation in the presence of SE largely masks romidexin-mediated changes in gene expression. Both GSEA and TF activity assays, both alone and in combination with romidexin treatment, identified the induction of key signaling pathways, including NF-κB signaling, cytokine-mediated JAK / STAT signaling, and TCR / BCR signaling following SE exposure. Figures 2G-2H ).

[0249] SE-mediated drug resistance in malignant T cells is not limited to romidesin.

[0250] Since SE significantly reduced romidesin-induced apoptosis in malignant cells, we tested whether SE also counteracted the effects of other clinically used HDAC inhibitors (HDACi), vorinostat (26), and remixstat (27). Similar to romidesin, treatment of SS PBMCs with vorinostat or remixstat induced effective cell death, and both were counteracted by the presence of SE, resulting in the survival of malignant cells ( Figures 3A-3D Our CITE-seq analysis showed that SE-regulated signaling pathways are involved in the survival of malignant cells (such as NFkB and JAK / STAT). Therefore, 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 to treat CTCL (28, 29). We selected ex vivo PBMCs from SS patients who responded to doxorubicin or etoposide and tested whether the presence of SE also counteracted the cytotoxic effects of these chemotherapeutic agents on malignant cells. Indeed, we found that SE exposure induced significant resistance in malignant cells to both doxorubicin and etoposide. Figures 3E-3H Furthermore, the role of SE-induced drug resistance is not limited to HDACi and DNA-damaging drugs, as SE exposure maintained malignant cell survival even after treatment with the ATP synthase inhibitor oligomycin. In summary, this suggests that the presence of SE does not directly interfere with the mode of action of these agents, but rather promotes overall drug resistance and survival of malignant cells.

[0251] Due to the enhancement of NFkB signal conduction through SE processing ( Figures 2G-2H We investigated whether the clinically approved proteasome and NF-κB inhibitor bortezomib (7, 30) could eliminate SE-induced drug resistance. We found that bortezomib induced potent malignant cell death that SE could not rescue ( Figures 3I-3J Similarly, SE did not confer resistance to crystal violet (gentian violet) (31); crystal violet showed inhibition of NFkB in CTCL (32). These findings suggest that SE-induced drug resistance may be driven by enhanced NFkB-mediated cell survival in malignant cells.

[0252] SE-induced drug resistance is caused by LCK-PKC-N F TCR signal transduction is mediated by kB.

[0253] It is known that NFkB is essential for the survival of malignant cells in CTCL and is directly induced by TCR activation (33,34). Our CITE-seq analysis confirmed that SE triggers strong TCR-mediated activation of malignant cells (as shown by pathway enrichment and induction by expression of surface CD25, CD69 and CD71). Figure 2GThis indicates that SE-induced drug resistance can be mediated by TCR conjugation. Therefore, we examined whether blocking TCR signaling could eliminate SE-induced drug resistance. TCR signaling depends on the early activation of protein tyrosine kinases like LCK (the SRC family of tyrosine kinases), which are key kinases in the initiation of TCR signaling after antigen presentation (35). Therefore, we tested whether the SRC inhibitor A-419259 would block SE-induced drug resistance. A-419259 treatment effectively blocked the elimination of SE from romidesin-induced cell death (SE). Figures 4A-4B Similar results were obtained with dasatinib (a clinically approved inhibitor for treating various cancers). Dasatinib (known to inhibit LCK (36)) itself has little effect on malignant cell survival, but it eliminated SE-mediated resistance to romidesin ( Figures 4C-4D Since TCR-induced NFkB signaling is downstream of protein kinase C (PKC)-θ activation, we used the clinical inhibitor sotrastuxin to block PKC-θ (37). Sotrastuxin significantly inhibited SE-induced romidixin resistance ( Figures 4E-4F Next, we inhibited the MAP kinase cascade in TCR signaling, as MAP kinase has previously been associated with HDACi resistance (39). Using two clinical MEK inhibitors (midatinib and selumetinib), we found that neither inhibitor eliminated SE-induced romidisin resistance. In summary, our findings support the hypothesis that SE-induced drug resistance is induced by TCR signaling via the PKC-θ pathway leading to NFkB activation.

[0254] SE-induced drug resistance can be either direct or indirect, through the release of IL-2R cytokines.

[0255] In addition to NFkB signaling, our CITE-seq data indicate that, in the presence of SE, JAK-STAT-mediated interleukin signaling and STAT3 activity were induced in malignant cells. Figures 2G-2H IL-2 and its family members stimulate JAK1 and JAK3, leading to STAT3 / 5 activation (39). To determine whether indirect signaling via cytokines can promote SE-induced drug resistance, we treated SS PBMCs in vitro with romidesin in the presence or absence of a mixture of IL-2 family cytokines (IL-2, IL-4, IL-7, and IL-15). We observed that IL-2 family cytokines were sufficient to protect malignant cells from romidesin-induced cell death ( Figures 5A-5BTo determine whether cytokine signaling is essential for SE-induced drug resistance, we added A-419259 (to block TCR signaling) or the clinically used JAK inhibitor tofacitinib (known to block IL-2 family cytokine signaling (40)) to romidesin-treated SS PBMCs in the presence or absence of SE or IL-2 family cytokines. Consistent with the above results, both SE and cytokines induce romidesin resistance in malignant T cells (40). Figure 5C Importantly, A-419259 completely blocks SE-induced romidasine resistance, but does not affect cytokine-induced romidasine resistance. Figure 5C In contrast, although tofacitinib completely blocked cytokine-induced romidixin resistance ( Figure 5C However, we found that the effect of tofacitinib on SE-induced romidixine resistance varied significantly among patients. Figures 5C-5F In three of the patients' PBMCs across five studies, we found only a modest or no reduction in SE-induced romidixine resistance in the presence of tofacitinib (JAK-independent). Figures 5C-5D In the remaining two patient PBMCs, SE-induced romidesin resistance was almost completely eliminated by the presence of tofacitinib, indicating that cytokine signaling is essential for this effect in these patients (JAK-dependent). Figures 5E-5F These individual differences in the effect of JAK / STAT on SE-induced HDACi resistance are not unexpected, given that SS is a highly heterogeneous disease (41–43) and that SE can indirectly induce STAT3 activation in malignant cells via IL-2 family cytokines released by non-malignant SE-responsive neighboring CD4+ T cells. To determine whether TCR signaling is sufficient to induce drug resistance in patients responding to SE in a JAK-dependent manner and to investigate non-malignant cell dependence, we sorted malignant T cells (based on CD3, CD4, CD8, CD7, CD26, and TCR-Cβ1 expression) from PBMCs of three SS patients. Figure 6A Due to the lack of surface markers defining malignant T cells, sorted malignant cells will inevitably contain trace amounts of non-malignant CD4+ T cells. Nevertheless, even under these highly enriched conditions, SE still induces effective drug resistance in sorted malignant T cells. This resistance was eliminated by the presence of tofacitinib in two patients (JAK-dependent), while malignant cells from one patient showed JAK-independent resistance. Figures 6B-6C ).

[0256] discuss

[0257] In this study, we have demonstrated that Staphylococcus aureus and its toxins induce drug-induced cell death in primary malignant T cells from SS patients. Thus, the supernatant from SE-expressing Staphylococcus aureus isolated from SS skin largely protected malignant cells from romidesin-induced cell death, while endolysin-treated SE-producing and SE-negative Staphylococcus aureus did not have this effect. Since only live Staphylococcus aureus expresses extracellular proteins such as SE, these findings suggest that the effect is SE-mediated. As a supporting measure, the inhibitory effect on romidesin-induced apoptosis was fully reproduced by highly purified SEA, and by antibody blocking and mutation of the MHC class II binding domain of SEA (preventing TCR-MHC class II crosslinking (a prerequisite for SEA-mediated TCR activation (21))) (SEA) F47A / D227A The inhibitory effect on romidesin-induced apoptosis was eliminated. For decades, the pathogenic role of bacterial superantigens in CTCL has been suspected but never proven. The findings of this invention suggest that SE-mediated induction of drug resistance may be a missing link between Staphylococcus aureus and disease exacerbation. Importantly, the induction of resistance is not limited to romidesin. In contrast, SE also induces resistance in malignant T cells to other HDACi (vorinostat and remixstat), chemotherapy drugs (doxorubicin and etoposide), and metabolic inhibitors (oligomycin). However, the protective effect is not universal and nonspecific, as SE cannot rescue malignant cells from cell death induced by bortezomib and crystal violet, which have also been used to treat CTCL.

[0258] Treatment of malignant cells with short-term pulses of high-dose romidesin was equally effective in inducing malignant cell death. Importantly, the presence of SE sixteen hours after the removal of the romidesin pulse was still sufficient to counteract drug-induced cell death, indicating that SE-induced drug resistance is mediated downstream of drug entry and the initiation of its action. Therefore, SE-induced drug resistance is not mediated by direct interaction with the drug, nor by blocking entry or promoting its removal. Consistent with a wide range of resistance mechanisms, the manifestation of SE-induced resistance is mediated by cellular responses to SE rather than by direct interference with treatment. As support, our CITE-seq analysis identified three potentially important pathways—NFkB, TCR, and STAT3. NFkB activity is associated with resistance to HDACi and chemotherapy drugs. In vitro studies on different cancer cell lines showed that inhibition of NFkB enhanced sensitivity to treatment with doxorubicin (44–45) and etoposide (46–47). Combined with our current findings that SE activates NFkB signaling in malignant cells and counteracts most of the transcriptional changes induced by romedixin, it is possible that SE induces drug resistance at least partially through the NFkB pathway. This conclusion is consistent with our observation that SE cannot rescue malignant cells from cell death induced by bortezomib and crystal violet, which have been shown to inhibit NFkB activity in malignant cells from CTCL patients (7, 32).

[0259] TCR signaling depends on the activation of SRC tyrosine kinase LCK, which leads to NFkB activation via a PKC-dependent pathway that promotes cell survival by inhibiting pro-apoptotic proteins (48–49). Therefore, blocking both early TCR signaling (dasatinib) and PKC activity (sotretinoin) inhibits SE-induced drug resistance. This response is selective for the PKC pathway, as inhibition of MEK (as associated with HDACi resistance (38)) is insufficient to counteract SE-induced drug resistance. These differences between cancer and cellular models suggest that HDACi resistance may depend on intrinsic cellular characteristics and factors within the tumor microenvironment.

[0260] Our CITE-seq data showed that SE enhanced STAT3 activation in HDACi-treated cultures. JAK / STAT signaling was also induced by cytokines, including the IL-2 family, which stimulated JAK-dependent STAT-mediated cell survival in malignant cells from SS patients (18, 50–51). Indeed, our results indicate that IL-2 family cytokines provide survival signals that protect malignant cells from drug-induced apoptosis. These findings are the first to demonstrate that cytokines expressed in the tumor microenvironment may play a role in drug resistance in CTCL patients. Surprisingly, JAK-STAT activation is required for SE-induced drug resistance only in some, not all, patients. Figure 7 As shown, these findings indicate the involvement of two pathways: (i) a direct TCR-dependent JAK-independent pathway, mediated by PKC-dependent NFkB signaling ( Figure 7 (ii) the indirect JAK-dependent pathway, which depends on SE-induced cytokine production in a self-secreting / paracrine manner in neighboring T cells or malignant cells themselves. Figure 7 (Lower part). Experiments using sorted malignant cells confirmed that SE can induce drug resistance even in cultures highly enriched from both JAK-dependent and non-JAK-dependent malignant populations. Currently, it is unclear why different responses are observed in malignant T cells from different patients. Because SE can directly (via TCRV) The activation of malignant cells (either through interaction or indirectly via neighboring T cells) could explain why drug resistance is induced in malignant cells from some patients in a manner dependent on cytokine signaling (which can be blocked by tofacitinib), but not in malignant cells from other patients. Interestingly, studies in chronic lymphocytic leukemia have shown that IL-6 induces resistance to vorinostat via STAT3 activation, and that inhibition of STAT3 reverses vorinostat resistance (52). In summary, these findings support the hypothesis that SE-producing Staphylococcus aureus can induce STAT3-dependent resistance to drugs such as HDACi.

[0261] In summary, we demonstrate that Staphylococcus aureus and its toxins induce drug resistance in malignant T cells. These findings provide a missing link between Staphylococcus aureus and cancer and explain how skin colonization by SE-producing bacteria contributes to disease activity in CTCL. Furthermore, our results highlight the need for specific eradication and prevention of Staphylococcus aureus skin colonization in CTCL patients, in addition to targeting malignant cells. In this study, this was performed using XZ.700 endolysin. Treatment with other (natural or chimeric) endolysins effective against Staphylococcus aureus has been demonstrated to provide results comparable to those obtained with XZ.700 endolysin.

[0262] Table 2 Patient Characteristics

[0263]

[0264] SS - Cézari syndrome; MF - mycosis fungoides; NA - not available; ECP - extracorporeal photodissociation; IFN - interferon; PUVA - psoralen and ultraviolet A; UVB - ultraviolet B; TSEI - total body electron beam irradiation. Treatment instructions are given to patients at the time point of blood collection, at which time PBMCs are isolated and used for experiments. References

[0265] 1.Sézary A, Bouvrain Y. Erythrodermie avec de cellulosemonstrueuses dans le derme et le sang circulant. Bull Soc Fr DermatolSyphiligr. 1938;45:254-260.

[0266] 2.Brouet JC, Flandrin G, Seligmann M. Indications of the thymus-derived nature of the proliferating cells in six patients with Sezary'ssyndrome. N Engl J Med. 1973;289(7):341-344.

[0267] 3.Lutzner M, Edelson R, Schein P, Green I, Kirkpatrick C, Ahmed A.Cutaneous T-cell lymphomas: the Sezary syndrome, mycosis fungoides, andrelated disorders. Ann Intern Med. 1975;83(4):534-552.

[0268] 4.Girardi M, Heald PW, Wilson LD. The Pathogenesis of MycosisFungoides. New England Journal of Medicine. 2004;350(19):1978-1988.

[0269] 5.Kim YH, Liu HL, Mraz-Gernhard S, Varghese A, Hoppe RT. Long-termoutcome of 525 patients with mycosis fungoides and Sezary syndrome - Clinicalprognostic factors and risk for disease progression. Archives of Dermatology.2003;139(7):857-866.

[0270] 7.Sors A, Jean-Louis F, Pellet C, et al. Down-regulating constitutiveactivation of the NF-kappaB canonical pathway overcomes the resistance ofcutaneous T-cell lymphoma to apoptosis. Blood. 2006;107(6):2354-2363.

[0271] 8.Netchiporouk E, Litvinov IV, Moreau L, Gilbert M, Sasseville D,Duvic M. Deregulation in STAT signaling is important for cutaneous T-celllymphoma (CTCL) pathogenesis and cancer progression. Cell Cycle. 2014;13(21):3331-3335.

[0272] 9.Axelrod PI, Lorber B, Vonderheid EC. Infections complicatingmycosis fungoides and Sezary syndrome. JAMA. 1992;267(10):1354-1358.

[0273] 10.Mirvish ED, Pomerantz RG, Geskin LJ. Infectious agents incutaneous T-cell lymphoma. J Am Acad Dermatol. 2011;64(2):423-431.

[0274] 11.Zhang Y, Seminario-Vidal L, Cohen L, et al. "Alterations in theSkin Microbiota Are Associated With Symptom Severity in Mycosis Fungoides".Front Cell Infect Microbiol. 2022;12:850509.

[0275] 12.Hooper MJ, LeWitt TM, Veon FL, et al. Nasal Dysbiosis in CutaneousT-Cell Lymphoma Is Characterized by Shifts in Relative Abundances of Non-Staphylococcus Bacteria. JID Innov. 2022;2(5):100132.

[0276] 13.Allen PB, Switchenko J, Ayers A, Kim E, Lechowicz MJ. Risk ofbacteremia in patients with cutaneous T-cell lymphoma (CTCL). Leukemia &Lymphoma. 2020;61(11):2652-2658.

[0277] 14.Posner LE, Fossieck BE, Jr., Eddy JL, Bunn PA, Jr. Septicemiccomplications of the cutaneous T-cell lymphomas. Am J Med. 1981;71(2):210-216.

[0278] 15.Bonin S, Tothova SM, Barbazza R, Brunetti D, Stanta G, Trevisan G.Evidence of multiple infectious agents in mycosis fungoides lesions. Exp MolPathol. 2010;89(1):46-50.

[0279] 16.Gluud M, Pallesen EMH, Buus TB, et al. Malignant T cells induceskin barrier defects through cytokine-mediated JAK / STAT signaling incutaneous T-cell lymphoma. Blood. 2023;141(2):180-193.

[0280] 17.Thode C, Woetmann A, Wandall HH, et al. Malignant T Cells SecreteGalectins and Induce Epidermal Hyperproliferation and DisorganizedStratification in a Skin Model of Cutaneous T-Cell Lymphoma. Journal ofInvestigative Dermatology. 2015;135(1):238-246.

[0281] 18.Pallesen EMH, Gluud M, Vadivel CK, et al. Endolysin inhibits skincolonization by patient-derived Staphylococcus aureus and malignant T cellactivation in cutaneous T cell lymphoma. J Invest Dermatol. 2023.

[0282] 19.Buus TB, Herrera A, Ivanova E, et al. Improving oligo-conjugatedantibody signal in multimodal single-cell analysis. eLife. 2021;10:e61973.

[0283] 20.Vadivel CK, Gluud M, Torres-Rusillo S, et al. JAK3 Is Expressed inthe Nucleus of Malignant T Cells in Cutaneous T Cell Lymphoma (CTCL).Cancers. 2021;13(2).

[0284] 21.Mehindate K, Thibodeau J, Dohlsten M, Kalland T, Sékaly RP, MouradW. Cross-linking of major histocompatibility complex class II molecules bystaphylococcal enterotoxin A superantigen is a requirement for inflammatorycytokine gene expression. J Exp Med. 1995;182(5):1573-1577.

[0285] 22.Huang Y, Su MW, Jiang X, Zhou Y. Evidence of an oncogenic role ofaberrant TOX activation in cutaneous T-cell lymphoma. Blood. 2015;125(9):1435-1443.

[0286] 23.Hurabielle C, Thonnart N, Ram-Wolff C, et al. Usefulness ofKIR3DL2 to Diagnose, Follow-Up, and Manage the Treatment of Patients withSezary Syndrome. Clin Cancer Res. 2017;23(14):3619-3627.

[0287] 24.Vonderheid EC, Bigler RD, Kotecha A, et al. Variable CD7expression on T cells in the leukemic phase of cutaneous T cell lymphoma(Sezary syndrome). J Invest Dermatol. 2001;117(3):654-662.

[0288] 25.Jones D, Dang NH, Duvic M, Washington LT, Huh YO. Absence of CD26expression is a useful marker for diagnosis of T-cell lymphoma in peripheralblood. Am J Clin Pathol. 2001;115(6):885-892.

[0289] 26.Olsen EA, Kim YH, Kuzel TM, et al. Phase IIb multicenter trial ofvorinostat in patients with persistent, progressive, or treatment refractorycutaneous T-cell lymphoma. J Clin Oncol. 2007;25(21):3109-3115.

[0290] 27.Resminostat for Maintenance Treatment of Patients With AdvancedStage Mycosis Fungoides (MF) or Sézary Syndrome (SS): https: / / ClinicalTrials.gov / show / NCT02953301.

[0291] 28.Wollina U, Dummer R, Brockmeyer NH, et al. Multicenter study ofpegylated liposomal doxorubicin in patients with cutaneous T-cell lymphoma.Cancer. 2003;98(5):993-1001.

[0292] 29.Purnak S, Azar J, Mark LA. Etoposide as a single agent in thetreatment of mycosis fungoides: A retrospective analysis. DermatologicTherapy. 2018;31(2).

[0293] 30.Zinzani PL, Musuraca G, Tani M, et al. Phase II trial ofproteasome inhibitor bortezomib in patients with relapsed or refractorycutaneous T-cell lymphoma. J Clin Oncol. 2007;25(27):4293-4297.

[0294] 31.Westergaard SA, Lechowicz MJ, Harrington M, Elsey J, Arbiser JL,Khan MK. Induction of remission in a patient with end-stage cutaneous T-celllymphoma by concurrent use of radiation therapy, gentian violet, andmogamulizumab. JAAD Case Rep. 2020;6(8):761-765.

[0295] 32.Wu J, Wood GS. Analysis of the Effect of Gentian Violet onApoptosis and Proliferation in Cutaneous T-Cell Lymphoma in an In VitroStudy. JAMA Dermatology. 2018;154(10):1191-1198.

[0296] 33.Chang TP, Vancurova I. NFkappaB function and regulation incutaneous T-cell lymphoma. Am J Cancer Res. 2013;3(5):433-445.

[0297] 34.Paul S, Schaefer BC. A new look at T cell receptor signaling tonuclear factor-κB. Trends Immunol. 2013;34(6):269-281.

[0298] 35.Courtney AH, Lo WL, Weiss A. TCR Signaling: Mechanisms ofInitiation and Propagation. Trends in Biochemical Sciences. 2018;43(2):108-123.

[0299] 36.Schade AE, Schieven GL, Townsend R, et al. Dasatinib, a small-molecule protein tyrosine kinase inhibitor, inhibits T-cell activation andproliferation. Blood. 2008;111(3):1366-1377.

[0300] 37.A Dose Finding Study of AEB071 Assessing Psoriasis Area andSeverity Index in Patients With Plaque Psoriasis: https: / / ClinicalTrials.gov / show / NCT00885196.

[0301] 38.Chakraborty AR, Robey RW, Luchenko VL, et al. MAPK pathwayactivation leads to Bim loss and histone deacetylase inhibitor resistance:rationale to combine romidepsin with an MEK inhibitor. Blood. 2013;121(20):4115-4125.

[0302] 39.Hammarén HM, Virtanen AT, Raivola J, Silvennoinen O. Theregulation of JAKs in cytokine signaling and its breakdown in disease.Cytokine. 2019;118:48-63.

[0303] 40.Meyer DM, Jesson MI, Li X, et al. Anti-inflammatory activity andneutrophil reductions mediated by the JAK1 / JAK3 inhibitor, CP-690,550, in ratadjuvant-induced arthritis. J Inflamm (Lond). 2010;7:41.

[0304] 41.Borcherding N, Voigt AP, Liu V, Link BK, Zhang W, Jabbari A.Single-Cell Profiling of Cutaneous T-Cell Lymphoma Reveals UnderlyingHeterogeneity Associated with Disease Progression. Clin Cancer Res. 2019;25(10):2996-3005.

[0305] 42.Buus TB, Willerslev-Olsen A, Fredholm S, et al. Single-cellheterogeneity in Sézary syndrome. Blood Adv. 2018;2(16):2115-2126.

[0306] 43.Gaydosik AM, Tabib T, Geskin LJ, et al. Single-Cell LymphocyteHeterogeneity in Advanced Cutaneous T-cell Lymphoma Skin Tumors. Clin CancerRes. 2019;25(14):4443-4454.

[0307] 44.Chiao PJ, Na R, Niu J, Sclabas GM, Dong Q, Curley SA. Role of Rel / NF-kappaB transcription factors in apoptosis of human hepatocellularcarcinoma cells. Cancer. 2002;95(8):1696-1705.

[0308] 45.Abdin SM, Tolba MF, Zaher DM, Omar HA. Nuclear factor-κB signalinginhibitors revert multidrug-resistance in breast cancer cells. Chemico-Biological Interactions. 2021;340:109450.

[0309] 46.Morotti A, Cilloni D, Pautasso M, et al. NF-kB inhibition as astrategy to enhance etoposide-induced apoptosis in K562 cell line. AmericanJournal of Hematology. 2006;81(12):938-945.

[0310] 47.Meley D, Spiller DG, White MR, McDowell H, Pizer B, See V. p53-mediated delayed NF-kappaB activity enhances etoposide-induced cell death inmedulloblastoma. Cell Death Dis. 2010;1(5):e41.

[0311] [ PMC free article ] [ PubMed ] 48.Bertolotto C, Maulon L, Filippa N, Baier G, Auberger P. Protein kinase C theta and epsilon promote T-cell survival by a rsk-dependentphosphorylation and inactivation of BAD. J Biol Chem. 2000;275(47):37246–37250.

[0312] 49.Cheng J, Montecalvo A, Kane LP. Regulation of NF-kappaB induction by TCR / CD28. Immunol Res. 2011;50(2-3):113-117.

[0313] 50.Willerslev-Olsen A, Gjerdrum LMR, Lindahl LM, et al.Staphylococcus aureus Induces Signal Transducer and Activator ofTranscription 5-Dependent miR-155 Expression in Cutaneous T-Cell Lymphoma. JInvest Dermatol. 2021;141(10):2449–2

[0314] 51.Willerslev-Olsen A, Krejsgaard T, Lindahl LM, et al.Staphylococcal enterotoxin A (SEA) stimulates STAT3 activation and IL-17expression in cutaneous T-cell lymphoma. Blood. 2016.

[0315] 52.Lu K, Fang XS, Feng LL, et al. The STAT3 inhibitor WP1066 reversesthe resistance of chronic lymphocytic leukemia cells to histone deacetylaseinhibitors induced by interleukin-6. Cancer Letters. 2015;359(2):250-258.

Claims

1. An antibacterial enzyme effective against Staphylococcus aureus, the antibacterial enzyme being used in the treatment of a subject with cancer refractory to histone deacetylase (HDAC) inhibitors, said treatment comprising administering an effective amount of the antibacterial enzyme to the subject.

2. The antibacterial enzyme according to claim 1 for use against Staphylococcus aureus, wherein, In addition to the antibacterial enzyme, the subject was also given chemotherapy drugs.

3. The antibacterial enzyme effective against Staphylococcus aureus as described in claim 1 or 2, wherein the cancer is a lymphoma, such as cutaneous T-cell lymphoma (CTCL), such as leukemia-related Cezari syndrome (SS).

4. The effective antibacterial enzyme against Staphylococcus aureus according to any one of claims 2 and 3, wherein the further administered chemotherapeutic agent is an anthracycline, such as doxorubicin; a topoisomerase II inhibitor, such as etoposide; and / or a histone deacetylase (HDAC) inhibitor, such as vorinostat, romidazine, remistat, parbisitat, and benostat.

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

6. The effective antibacterial enzyme against Staphylococcus aureus according to any one of the preceding claims, wherein the antibacterial enzyme is selected from the group consisting of: bacteriocins or their functional portions, bacteriosins or autolysins or their functional portions, phage lysins or their functional portions, and chimeric lysins or their functional portions.

7. An effective antibacterial enzyme against Staphylococcus aureus according to any one of the preceding claims, wherein the antibacterial enzyme is a chimeric endosomal, preferably a recombinant chimeric endosomal comprising one or more heterologous domains.

8. The effective antibacterial enzyme against Staphylococcus aureus according to any one of the preceding claims, wherein the Staphylococcus aureus is an antibiotic-resistant Staphylococcus aureus, such as methicillin-resistant Staphylococcus aureus (MRSA).

9. An effective antibacterial enzyme against Staphylococcus aureus 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 effective antibacterial enzyme against Staphylococcus aureus according to any one of the preceding claims, wherein the subject is a mammal, such as livestock, domestic animals, or a human.

11. A method of treating a subject with cancer refractory to histone deacetylase (HDAC) inhibitors, said treatment comprising administering to the subject an effective amount of an antibacterial enzyme effective against Staphylococcus aureus.

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

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

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