The present invention relates to a pharmaceutical composition comprising an endolysin or an artilysin and an anti-cancer agent. In addition, the present invention relates to a pharmaceutical composition according to this invention for use as a medicament and for use in the treatment of a proliferative disease or disorder.

Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020. According to the World Health Organization the most common in 2020 (in terms of new cases of cancer) were: breast cancer (2.26 million cases), lung cancer (2.21 million cases), colon and rectum cancer (1.93 million cases), prostate cancer (1.41 million cases), skin cancer (non-melanoma) (1.20 million cases) and stomach cancer (1.09 million cases). Therefor, investigation of new strategies of cancer diagnosis and therapeutics is needed.

Traditional treatments of cancer inaccessible to surgery or as adjuvant to surgery are multimodal comprising chemotherapy, immunotherapy, hormonal therapy and radiotherapy. The application of chemotherapeutic s has led to improved survival rates; however, this treatment is accompanied by non-specific cytotoxicity to normal cells and appearance of cancer drug resistance. Thus, another approach the active targeting, i.e. the targeting of nanomedicines with various cancer- specific ligands is being extensively explored. The active targeting is driven by specific binding of a ligand attached to a nanomedicine to a tumor cell receptor (Haley and Frenkel, 2008). In this connection anticancer peptides (ACP) have been designed. However, the use of peptides in clinical treatments has many disadvantages such as the low selectivity of some of the ACPs molecules, the high cost of production in large scale, and their low resistance to proteolytic cleavage (Hu et al., 2011). There are also some concerns related to the use of ACPs whose sequences are close to human and natural antimicrobial peptides (AMPs) due to a possible compromise of the human natural defense and consequently threat to public health.

Thus, there is a need for novel vehicles targeting cancer cells, wherein these vehicles may provide an anti-cancer agent. This object is solved by the subject matter defined in the claims and set forth below.

The term "protein" as used herein refers synonymously to the term "polypeptide". The term “protein” as used herein refers to a linear polymer of amino acid residues linked by peptide bonds in a specific sequence. The amino-acid residues of a protein may be modified by e.g. covalent attachments of various groups such as carbohydrates and phosphate. Other substances may be more loosely associated with the polypeptide chains, such as heme or lipid, giving rise to the conjugated proteins which are also comprised by the term “protein” as used herein. There are various ways in which the polypeptide chains fold have been elucidated, in particular with regard to the presence of alpha helices and beta-pleated sheets. The term “protein” as used herein refers to all four classes of proteins being all-alpha, all-beta, alpha/beta and alpha plus beta.

The term "fusion protein" as used herein refers to an expression product resulting from the fusion of two nucleic acid sequences. Such a protein may be produced, e.g., in recombinant DNA expression systems. Moreover, the term “fusion protein” as used herein refers to a fusion of a first amino acid sequence, in particular an endolysin, autolysin and/or other peptidoglycan hydrolase, with a second or further amino acid sequence. The second or further amino acid sequence is preferably a peptide stretch, in particular a cationic and/or polycationic peptide. Preferably, said second and/or further amino acid sequence is foreign to and not substantially homologous with any domain of the first amino acid sequence.

The term “artilysin” as used herein refers to a fusion protein comprising an endolysin and a peptide stretch, in particular a cationic and/or polycationic peptide.

The term “peptide” as used herein refers to short peptides consisting of from about 2 to about 100 amino acid residues, more preferably from about 4 to about 50 amino acid residues, more preferably to about 5 to 30 amino acid residues, wherein the amino group of one amino acid residue is linked to the carboxyl group of another amino acid residue by a peptide bond. A peptide may have a specific function. A peptide can be a naturally occurring peptide or a synthetically designed and produced peptide. The peptide can be, for example, derived or removed from a native protein by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid phase synthesis) or molecular biology techniques (see Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). Preferred synthetically produced peptides are e.g. cationic, polycationic, amphipathic or hydrophobic peptides. Preferred naturally occurring peptides are e.g. antimicrobial peptides.

As used herein, the term "cationic peptide" refers to a peptide having positively charged amino acid residues. Preferably a cationic peptide has a pKa-value of 9.0 or greater. Typically, at least four of the amino acid residues of the cationic peptide can be positively charged, for example, lysine or arginine. "Positively charged" refers to the side chains of the amino acid residues which have a net positive charge at about physiological conditions. The term “cationic peptide” as used herein refers also to polycationic peptides.

The term “polycationic peptide” as used herein refers to a synthetically designed and produced peptide composed of mostly positively charged amino acid residues, in particular lysine, arginine and/or histidine residues, more preferably lysine and/or arginine residues. A peptide is composed of mostly positively charged amino acid residues if at least about 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95 or about 100 % of the amino acid residues are positively charged amino acid residues, in particular lysine and/or arginine residues. The amino acid residues being not positively charged amino acid residues can be neutrally charged amino acid residues and/or negatively charged amino acid residues and/or hydrophobic amino acid residues. Preferably the amino acid residues being not positively charged amino acid residues are neutrally charged amino acid residues, in particular serine and/or glycine.

The term, "antimicrobial peptide" (AMP) as used herein refers to any naturally occurring peptide that has microbicidal and/or microbistatic activity on for example bacteria, viruses, fungi, yeasts, mycoplasma and protozoa. Thus, the term “antimicrobial peptide” as used herein refers in particular to any peptide having anti-bacterial, anti-fungal, anti-mycotic, anti- parasitic, anti-protozoal, anti-viral, anti-infectious, anti-infective and/or germicidal, algicidal, amoebicidal, microbicidal, bactericidal, fungicidal, parasiticidal, protozoacidal, protozoicidal properties, in particular sushi peptides and defensin. The antimicrobial peptide may be a member of the RNAse A super family, a defensin, cathelicidin, granulysin, histatin, psoriasin, dermicidine or hepcidin. The antimicrobial peptide may be naturally occurring in insects, fish, plants, arachnids, vertebrates or mammals.

The term “sushi peptide” as used herein refers to complement control proteins (CCP) having short consensus repeats. The sushi module of sushi peptides functions as a protein -protein interaction domain in many different proteins. Peptides containing a Sushi domain have been shown to have antimicrobial activities. Preferably, sushi peptides are naturally occurring antimicrobial peptides.

The term "amphiphatic peptide" as used herein refers to synthetic peptides having both hydrophilic and hydrophobic functional groups. Preferably, the term “amphiphatic peptide” as used herein refers to a peptide having a defined arrangement of hydrophilic and hydrophobic groups e.g. amphiphatic peptides may be e.g. alpha helical, having predominantly non polar side chains along one side of the helix and polar residues along the remainder of its surface.

The term "hydrophobic group" as used herein refers to chemical groups such as amino acid side chains which are substantially water insoluble, but soluble in an oil phase, with the solubility in the oil phase being higher than that in water or in an aqueous phase. In water, amino acid residues having a hydrophobic side chain interact with one another to generate a nonaqueous environment. Examples of amino acid residues with hydrophobic side chains are valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and glycine residues.

The term “endolysin” as used herein refers to an enzyme which is suitable to hydrolyse bacterial cell walls. The term “endolysin” as used herein comprise naturally occurring endolysins, e.g. encoded by bacteriophages or bacterial viruses as well as recombinant endolysins. The recombinant endolysins may be shuffled, i.e. composed of heterologous domains like CBDs or EADs derived from different endolysins. “Endolysins” comprise at least one “enzymatically active domain” (EAD) having at least one of the following activities: endopeptidase, N-acetyl-muramoyl-L-alanine-amidase (amidase), N-acetyl-muramidase, N- acetyl-glucosaminidase (lysozyme) or transglycosylases. In addition, the endolysins may contain also regions which are enzymatically inactive, and bind to the cell wall of the host bacteria, the so-called CBDs (cell wall binding domains). The endolysin may contain one, two or more CBDs. However, the term “endolysin” as used herein refers also to enzymes having at least one EAD but no CBDs. Generally, the cell wall binding domain is able to bind different components on the surface of bacteria. Preferably, the cell wall binding domain is a peptidoglycan binding domain and binds to the bacteria’s peptidoglycan.

The term “EAD” as used herein refers to the enzymatically active domain of an endolysin. The EAD is responsible for hydrolysing bacterial peptidoglycans. It exhibits at least one enzymatic activity of an endolysin. The EAD can also be composed of more than one enzymatically active module. The term “EAD” is used herein synonymously with the term “catalytic domain”.

Recent studies have shown that all cancer cells exhibit negative surface charges that are directly proportional to the secreted lactic acid, which is due to a unique cancer metabolic characteristic, namely a high rate of glycolysis (Le, Wenjun et al., Biophysics Report, February 2019, Vol. 5, Issue 1). Meanwhile, several normal cells were found charge-neutral. The most typical feature of cancer cells is their abnormal glucose metabolism pathway (Chaffer and Weinberg 2011; You and Jones 2012; Warburg 1956). Cancer cells use the glycolytic pathway that subsequently leads to secretion of lactic acid (Hanahan and Weinberg 2011). In normal tissues the so-called Pasteur effect can be observed (Fu et al. 2013; Warburg 1924), i.e. 90% of ATP is derived from oxidative phosphorylation, and only 10% is derived from aerobic glycolysis. In contrast to that, about 80% of glucose is used in cancer cells to produce ATP in the glycolytic pathway, but not through the oxidative phosphorylation pathway, what is known as the Warburg effect (Schell et al. 2014; Copeland and Turner 1980). The highly active glycolysis of cancer cells requires the rapid and large intake of high levels of glucose to generate energy in order to meet the rapid growth requirements. In addition, cancer cells secrete a large quantity of lactic acid, while normal cells produce carbon dioxide and water. Almost all metabolically active cancer cells, both in vivo and in vitro, secrete large amounts of lactate ions as mobile anions (Gadsby 2009). This is the result of active glycolysis, in which glucose uptake and lactate secretion can be up to 30 times higher than in normal cells (Hanahan and Weinberg 2011). A large number of cancer cells secrete lactate ions outside the cell across the plasma membrane. These ions pass through the plasma membrane and will change the surface charge. Thus, it is assumed that the charge on the surface of cancer cells is mainly due to the secretion of lactate across the plasma membrane. It should be noted that cancer cells may have a slightly elevated surface content of negatively- charged immobilized molecules (e.g., sialic acid), which is 30%-50% more than normal cells, but this is hardly comparable to the elevated levels of glycolysis and lactate secretion, that is 30 times higher than normal cell levels (Dubyak 2004; Gadsby et al. 2009). Therefore, the negative charge generated on the cancer cells is due to different sugar metabolism compared to normal cells.

Like cancer cells, Gram-positive and Gram-negative bacteria cells exhibit a negative charge of the cell surface. Bacterial membranes are negatively charged with lipids such as phosphatidylglycerol, cardiolipin, or phosphatidyl serine. The outer membrane of a Gramnegative bacteria is also negatively charged as it contains anionic lipopolysaccharides. On the other hand, mammalian cell membranes consist largely of zwitterionic phospholipids (neutral in net charge) such as phosphatidylethanolamine, phosphatidylcholine or sphingomyelin.

Antimicrobials against Gram-positive and Gram-negative bacteria cells like endolysins and artilysins are positively charged and thus, the electrostatic interaction between these components plays a great role in the lysis process of bacteria. Such electrostatic interaction may be used between negatively charged cancer cells and positively charged antimicrobials, like endolysins and artilysins.

Endolysins are peptidoglycan hydrolases encoded by bacteriophages (or bacterial viruses), however there are also recombinant endolysins which are engineered dependent on the desired functions. They are synthesized during late gene expression in the lytic cycle of phage multiplication and mediate the release of progeny virions from infected cells through degradation of the bacterial peptidoglycan. They are either B(l,4)-glycosylases (lysozymes), transglycosylases, amidases or endopeptidases. Antimicrobial application of endolysins was already suggested in 1991 by Gasson (GB2243611). Although the killing capacity of endolysins has been known for a long time, the use of these enzymes as antibacterials was ignored due to the success and dominance of antibiotics. Only after the appearance of multiple antibiotic resistant bacteria this simple concept of combating human pathogens with endolysins received interest. A compelling need to develop totally new classes of antibacterial agents emerged and endolysins used as 'enzybiotics' - a hybrid term of 'enzymes' and 'antibiotics' - perfectly met this need. In 2001, Fischetti and coworkers demonstrated for the first time the therapeutic potential of bacteriophage Cl endolysin towards group A streptococci (Nelson et al., 2001). Since then many publications have established endolysins as an attractive and complementary alternative to control bacterial infections, particularly by Gram-positive bacteria. Subsequently different endolysins against other Gram-positive pathogens such as Streptococcus pneumoniae (Loeffler et al., 2001), Bacillus anthracis (Schuch et al., 2002), S. agalactiae (Cheng et al., 2005) and Staphylococcus aureus (Rashel et al, 2007) have proven their efficacy as enzybiotics. In general, endolysins are built up from of at least one “enzymatically active domain” (EAD) having at least one of the following activities: endopeptidase, N-acetyl-muramoyl-L-alanine-amidase (amidase), N-acetyl- muramidase, N-acetyl-glucosaminidase (lysozyme) or transglycosylases. In addition, the endolysins may contain also regions which are enzymatically inactive, and bind to the cell wall of the host bacteria, the so-called CBDs (cell wall binding domains).

Artilysins comprise an endolysin fused to a peptide with lipopolysachharide (LPS) or in general membrane disrupting activity. LPS is a major component of the outer membrane of Gram-negative bacteria. It increases the negative charge of the cell membrane and protects the membrane from certain kinds of chemical attack. To a certain degree said LPS protects the membrane of Gram-negative bacteria also from endolysins added from outside of the bacteria. However, the LPS can be disrupted by peptide stretches having a LPS disrupting activity as e.g. positively charged peptides. Moreover, said peptide stretches may be involved in the outer membrane protein transport mechanism, a destabilisation of structural outer membrane proteins and/or in lipid-dependent destabilisation. It was found that a peptide stretch having LPS disrupting activity or in general membrane disrupting activity promotes the passage of an endolysin fused to said peptide stretch through the outer membrane of Gram-negative bacteria. After the promoted pass of the endolysin through the outer membrane of Gramnegative bacteria, the cell wall of the Gram-negative bacterium can be more easily be disrupted or desintegrated by the endolysin due to degradation of the peptidoglycan layer followed by osmotic lysis when the internal cell pressure of the bacterium cannot longer be resisted.

In contrast to Gram-negative bacteria, Gram-positive bacteria do not possess an outer membrane. The cytoplasmic membrane is surrounded by an up to 25 nm thick layer of peptidoglycan (which is only up to 5 nm for Gram-negative bacteria) which forms the cell wall. Main purpose of the cell wall of Gram-positives is to maintain bacterial shape and to counteract the internal bacterial cell pressure. Peptidoglycan, or murein, is a polymer consisting of sugars and amino acids. The sugar component consists of alternating residues of P-(l,4) linked N-acetylglucosamine and N-acetylmuramic acid residues compose the sugar components. A peptide chain of three to five amino acids is attached to the N-acetylmuramic acid. The peptide chain can be cross-linked to the peptide chain of another strand forming a 3D mesh-like layer. The peptide chain may contain D- and L- amino acid residues and the composition may vary for different bacteria. Thus, the cell wall of Gram-positive bacteria composed of peptidoglycan may be disrupted by enzymes like endolysins and the EAD of artilysins representing peptidoglycan hydrolases.

Consequently, depending on the bacteria type -Gram-positive or Gram-negative - and the resulting specific structure of the bacteria type an endolysin or artilysin is engineered, i.e. the specific EAD and CBD as well as a peptide with LPS disrupting or membrane disrupting activity with the desired activity is chosen and combined. This specificity of endolysins and artilysins for bacteria may be transferred to the different cancer cell types and thus, enables an engineering of endolysins or artilysins with specific targeting activity for specific cancer cell types.

The selectivity of the endolysins and artilysins for cancer cells is based on the opposite charge, i.e. a negativ charge of cancer cells compared to healthy mammalian cells consisting largely of zwitterionic phospholipids (neutral in net charge).

Endolysins and artilysins are thus selected or engineered to bind and lysate Gram-negative or Gram-positive bacteria cells. According to this, also the artilysins according to the present invention may be engineered to target different, specific cancer cells and thus, function as vehicle for anti-cancer agents which may be delivered to the specific target site

The present invention relates to a pharmaceutical composition comprising an endolysin or an artilysin and an anti-cancer agent.

The endolysin or the endolysin part of the artilysin according to the present invention is preferably encoded by bacteriophages specific for Gram-negative bacteria such as Gramnegative bacteria of bacterial groups, families, genera or species comprising strains pathogenic for humans or animals like Enterobacteriaceae (Escherichia, especially E. coli, Salmonella, Shigella, Citrobacter, Edwardsiella, Enterobacter, Elafnia, Klebsiella, especially K. pneumoniae, Morganella, Proteus, Providencia, Serratia, Yersinia), P seudomonadaceae Pseudomonas, especially P. aeruginosa, Burkholderia, Stenotrophomonas, Shewanella, Sphingomonas, Comamonas), Neisseria, Moraxella, Vibrio, Aeromonas, Brucella, Francisella, Bordetella, Legionella, Bartonella, Coxiella, Haemophilus, Pasteurella, Mannheimia, Actinobacillus, Gardnerella, Spirochaetaceae (Treponema and Borrelia), Leptospiraceae, Campylobacter, Helicobacter, Spirillum, Streptobacillus, Bacteroidaceae (Bacteroides, Fusobacterium, Prevotella, Porphyromonas), Acinetobacter, especially A. baumanii.

In another preferred embodiment, the endolysin or the endolysin part of the artilysin is encoded by bacteriophages specific for Gram-positive bacteria such as Gram-positive bacteria of bacterial groups, families, genera or species comprising strains pathogenic for humans or animals, in particular of the phylum Actinobacteria, in particular of the class Actinobacteridae, in particular of the order Actinomycetales, in particular of the families Actinomycineae: Actinomycetaceae (Actinomyces, Mobiluncus), Corynebacterineae : Mycobacteriaceae (Mycobacterium), Nocardiaceae, Corynebacteriaceae, Frankineae: Frankiaceae, Micrococcineae: Brevibacteriaceae and Propionibacteriaceae

(Propionibacterium) and of the order Bifidobacteriales, in particular of the families Bifidobacteriaceae (Bifidobacterium, Falcivibrio, Gardnerella) and other subclasses: Acidimicrobidae, Coriobacteridae, Rubrobacteridae, Sphaerobacteridae; and of the phylum Firmicutes, in particular of the class Bacilli, in particular of the order Bacillales, in particular of the families: Bacillaceae (Bacillus), Listeriaceae (Listeria), Staphylococcaceae (Staphylococcus, Gemella, Jeotgalicoccus) and of the order Lactobacillales, in particular of the families: Enterococcaceae (Enterococcus), Lactobacillaceae (Lactobacillus, Pediococcus), Leuconostocaceae (Leuconostoc), Streptococcaceae (Lactococcus, Streptococcus) and of the class Clostridia, in particular of the order: Clostridiales (Clostridium, Peptostreptococcus, Selenomonas), Halanaerobiales and Thermoanaerobacterales, and of the class Tenericutes/Mollicutes, in particular of the order: Mycoplasmatales (Mycoplasma, Ureaplasma), Entomoplasmatales (Spiroplasma), Anaeroplasmatales (Erysipelothrix), Acholeplasmatales (Acholeplasma), Haloplasmatales ( Haloplasma ).

In another preferred embodiment of the present invention the endolysins or the artilysin according to the present invention comprise modifications and/or alterations of the amino acid sequences. Such alterations and/or modifications may comprise mutations such as deletions, insertions and additions, substitutions or combinations thereof and/or chemical changes of the amino acid residues, e.g. biotinylation, acetylation, PEGylation, chemical changes of the amino-, SH- or carboxyl-groups.

The peptide of the artilysin according to the present invention may be linked to the endolysin part by additional amino acid residues e.g. due to cloning reasons. Preferably, said additional amino acid residues may be not recognized and/or cleaved by proteases. Preferably said peptide may be linked to the enzyme by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid residues. Preferably, the peptide fused on the N-terminus of the endolysin part of the artilysin according to the invention further comprises additional amino acids on its N- terminus. Preferably the peptide comprises the amino acid methionine (Met), or methionine, glycine and serine (Met-Gly-Ser) or alanine, methionine and glycine (Ala-Met-Gly). In another preferred embodiment the peptide is linked to the N-terminus of the endolysin part by the additional amino acid residues, in particular glycine and serine (Gly-Ser). In another preferred embodiment the peptide is linked to the C-terminus of the endolysin part by the additional amino acid residues, in particular glycine and serine (Gly-Ser).

In one aspect of the invention the peptide with membrane and/or LPS disrupting activity comprises a positively charged peptide, which comprises one or more of the positively charged amino acids being lysine, arginine and/or histidine. Preferably, more than 80%, preferably more than 90%, preferably 100% of the amino acids in said peptide are positively charged amino acids. Advantageously, the cationic peptide is fused at the N-terminal and/or the C-terminal end of the artilysin, thus enhancing the cationicity of the latter proteins.

In a preferred embodiment the artilysin comprises an endolysin and a peptide fused thereto said peptide comprising about 3 to about 50, more preferably about 5 to about 20, for instance about 5 to about 15 amino acid residues and at least 20, 30, 40, 50, 60 or 70%, more preferably at least 80%, for instance at least 90% of the said amino acid residues are either arginine or lysine residues.

Preferably, the peptide of the artilysin is fused to the N-terminus and/or to the C-terminus of the endolysin. In a particular preferred embodiment said peptide is only fused to the N- terminus of the endolysin. However, also preferred are artilysins having a peptide both on the N-terminus and on the C-terminus. Said peptides on the N-terminus and on the C-terminus can be the same or distinct peptides.

The peptide of the artilysin according to the present invention is preferably covalently bound to the enzyme. Preferably, said peptide consists of at least 5, more preferably at least of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or at least 100 amino acid residues. Especially preferred is a peptide comprising about 5 to about 100 amino acid residues, about 5 to about 50 or about 5 to about 30 amino acid residues. In one aspect of the present invention the peptide is selected from the group of cationic peptides, polycationic peptides, hydrophobic peptides, antimicrobial peptides and amphiphatic peptides.

In one aspect of the present invention the fused peptide is a cationic and/or polycationic peptide, which comprises one or more of the positively charged amino acid residues of lysine, arginine and/or histidine, in particular of lysine and/or arginine. Preferably, more than about 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95 or 99 % of the amino acid residues in said peptide stretch are positively charged amino acid residues, in particular lysine and/or arginine residues. Especially preferred are peptides consisting of about 100 % positively charged amino acid residues, in particular arginine and/or lysine residues, wherein preferably about 60 % to about 70 % of said positively charged amino acid residues are lysine residues and about 30% to about 40 % of said positively charged amino acid residues are arginine residues. Peptides consisting of either only arginine or only lysine are also preferred.

In another preferred embodiment of the present invention the cationic peptide comprises beside the positively charged amino acid residues, in particular lysine and/or arginine residues, neutrally charged amino acid residues, in particular glycine and/or serine residues. Preferred are cationic peptides consisting of about 70 % to about 100 %, or about 80 % to about 95 %, or about 85 % to about 90 % positively charged amino acid residues, in particular lysine, arginine and/or histidine residues, more preferably lysine and/or arginine residues and of about 0 % to about 30 %, or about 5 % to about 20 %, or about 10 % to about 20 % neutrally charged amino acid residues, in particular glycine and/or serine residues.

In another preferred embodiment of the present invention the cationic peptides comprise beside the positively charged amino acid residues, in particular lysine and/or arginine residues, hydrophobic amino acid residues, in particular valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and glycine residues, more preferably alanine, valine, leucine, isoleucine, phenylalanine, and/or tryptophan residues. Preferred are cationic peptides consisting of about 70 % to about 100 %, or about 80 % to about 95 %, or about 85 % to about 90 % positively charged amino acid residues, in particular lysine and/or arginine residues and of about 0 % to about 30 %, or about 5 % to about 20 %, or about 10 % to about 20 % hydrophobic amino acid residues, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and glycine residues, more preferably alanine, valine, leucine, isoleucine, phenylalanine, and/or tryptophan residues.

In a further embodiment of the present invention the peptide is an antimicrobial peptide comprising a positive net charge and around 50% hydrophobic amino acids. The antimicrobial peptides are amphiphatic, with a length of about 12 to about 50 amino acid residues. The antimicrobial peptides are naturally occurring in insects, fish, plants, arachnids, vertebrates or mammals. Preferably the antimicrobial peptide may be naturally occurring in radish, silk moth, wolf spider, frog, preferably in Xenopus laevis, Rana frogs, more preferably in Rana catesbeiana, toad, preferably Asian toad Bufo bufo gargarizans, fly, preferably in Drosophila, more preferably in Drosophila melanogaster, in Aedes aegypti, in honey bee, bumblebee, preferably in Bombus pascuorum, flesh fly, preferably in Sarcophaga peregrine, scorpion, horseshoe crab, catfish, preferably in Parasilurus asotus, cow, pig, sheep, porcine, bovine, monkey and human.

In a further embodiment of the present invention the peptide is a sushi peptide which is described by Ding JL, Li P, Ho B Cell Mol Life Sci. 2008 Apr;65(7-8): 1202-19. The Sushi peptides: structural characterization and mode of action against Gram- negative bacteria. Preferred sushi peptides of the fusion protein are sushi peptides SI and S3 and multiples thereof; FASEB J. 2000 Sep; 14(12): 1801- 13.

In a further embodiment of the present invention the peptide is a hydrophobic peptide, which comprises at least 90 % of the hydrophobic amino acid residues of valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and/or glycine. In another preferred embodiment the hydrophobic peptide of the fusion protein consist of about 90 % to about 95 %, or of about 90 to about 100%, or of about 95 % to about 100 % of the hydrophobic amino acid residues of valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and/or glycine.

In a further embodiment of the present invention the peptide is an amphiphatic peptide, which comprises one or more of the positively charged amino acid residues of lysine, arginine and/or histidine, combined to one or more of the hydrophobic amino acid residues of valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and/or glycine. Side chains of the amino acid residues are oriented in order that cationic and hydrophobic surfaces are clustered at opposite sides of the peptide. Preferably, more than about 30, 40, 50, 60 or 70% of the amino acids in said peptide are positively charged amino acids. Preferably, more than about 30, 40, 50, 60 or 70%, of the amino acid residues in said peptide are hydrophobic amino acid residues. Advantageously, the amphiphatic peptide is fused at the N-terminal and/or the C-terminal end of the enzyme having cell wall degrading activity, thus enhancing the amphiphaticity of the latter proteins.

In another embodiment of the present invention the peptide is an amphiphatic peptide consisting of at least 5, more preferably at least of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues. In a preferred embodiment at least about 30, 40, 50, 60 or 70% of the said amino acid residues of the amphiphatic peptide are either arginine or lysine residues and/or at least about 30, 40, 50, 60 or 70% of the said amino acid residues of the amphiphatic peptide are of the hydrophobic amino acids valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and/or glycine. In another preferred embodiment of the present invention the peptide is an amphiphatic peptide comprising beside the positively charged amino acid residues, in particular lysine and/or arginine residues, hydrophobic amino acid residues, in particular valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and glycine residues, more preferably alanine, valine, leucine, isoleucine, phenylalanine, and/or tryptophan residues. Preferred are amphiphatic peptides consisting of about 10 % to about 50 %, or about 20 % to about 50 %, or about 30 % to about 45 % or about 5 % to about 30 % positively charged amino acid residues, in particular lysine and/or arginine residues and of about 50 % to about 85 %, or about 50 % to about 90 %, or about 55 % to about 90 %, or about 60 % to about 90 %, or about 65 % to about 90 % hydrophobic amino acid residues, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and glycine residues, more preferably alanine, valine, leucine, isoleucine, phenylalanine, and/or tryptophan residues

Preferably, the peptide is no tag such as a His-tag, Strep-tag, Avi-tag, Myc-tag, Gst-tag, JS- tag, cystein-tag, FLAG-tag or other tags known in the art and no thioredoxin or maltose binding proteins (MBP). However, the artilysin or endolysin according to the present invention may comprise in addition such tag or tags.

The artilysins according to the present inventions are fusion proteins. Fusion proteins are constructed by linking at least two nucleic acid sequences using standard cloning techniques as described e.g. by Sambrook et al. 2001, Molecular Cloning: A Laboratory Manual. Such a protein may be produced, e.g., in recombinant DNA expression systems. Such fusion proteins according to the present invention can be obtained by fusing the nucleic acids for endolysin and the respective peptide stretch.

In another preferred embodiment of the present invention the peptides of the artilysin according to the present invention comprise modifications and/or alterations of the amino acid sequences. Such alterations and/or modifications may comprise mutations such as deletions, insertions and additions, substitutions or combinations thereof and/or chemical changes of the amino acid residues, e.g. biotinylation, acetylation, PEGylation, chemical changes of the amino-, SH- or carboxyl- groups. In a preferred embodiment of the present invention the anti-cancer agent according to the present invention refers to any agent suitable in the treatment of any cancer. In another embodiment the anti-cancer agent according to the present invention refers to a chemotherapeutic agent, a radiotherapy agent, an anti-cancer vaccine, an antibody, an immunotherapy agent and/or a cell therapy agent. In another embodiment the anti-cancer agent according to the present invention refers to alkylating agents, antimetabolites, natural products, and hormones. In another embodiment the anti-cancer agent according to the present invention refers to RNA, DNA, protein, peptide, immune cell, viral vector and antibodies. More preferably the RNA refers to mRNA, self-amlifying mRNA, siRNA, nonreplicating unmodified and modified mRNA and modified mRNA.

In another preferred embodiment the anti-cancer agent is coupled to the artilysin as vehicle via covalent binding, electrostatic interactions, hydrogen bonding, hydrophobic interactions and base stacking in a manner similar to protein-DNA interactions. In another preferred embodiment the artilysin and/or anti-cancer agent may be provided in the pharmaceutical composition according to the present invention included in lipid nanoparticles, solid lipid nanoparticles, polyplexes or cationic nanoemulsions.

A further aspect of the present invention is related to a pharmaceutical composition according to the present invention comprising an endolysin or an artilysin and an anti-cancer agent for use in the treatment of a proliferative disease. The term "proliferative disease" characterized by an abnormal proliferation of cells is used herein in a broad sense to include any disorder that requires control of the cell cycle. In a preferred embodiment, the proliferative disease refers to cancers, leukaemias, cardiovascular disorders such as restenosis and cardiomyopathy, auto-immune disorders such as glomerulonephritis and rheumatoid arthritis, dermatological disorders such as psoriasis, anti-inflammatory, anti-fungal, antiparasitic disorders such as malaria, emphysema and alopecia.

For this, the pharmaceutical composition of the present invention may be administered directly to a tumor or to a wide variety of locations including, for example, into sites such as the cerebral spinal fluid, bone marrow, joints, arterial endothelial cells, rectum, buccal/sublingual, vagina, the lymph system, to an organ selected from the group consisting of lung, liver, pancreas, spleen, skin, blood and brain, or to a site selected from the group consisting of tumors and interstitial spaces. Within other embodiments, the composition may be administered intraocularly, intranasally, sublingually, orally, topically, intravesically, intrathecal, intravenously, intraarterially e.g. into the intrahepatic artery, intraperitoneally, intracranially, intramuscularly, intraarticularily or subcutaneously. Other representative routes of administration include gastroscopy, ECRP and colonoscopy, which do not require full operating procedures and hospitalization, but may require the presence of medical personnel.

In a further aspect the present invention relates to a method of treating a disorder, disease or condition in a subject in need of treatment and/or prevention, which method comprises administering to said subject an effective amount of a pharmaceutical composition according to the present invention. The subject may be a human or an animal.

The dosage and route of administration used in a method of treatment (or prophylaxis) according to the present invention depends on the specific disease or target site to be treated. The route of administration may be for example oral, topical, nasopharyngeal, parenteral, inhalational, intravenous, intramuscular, intrathecal, intraspinal, endobronchial, intrapulmonal, intraosseous, intracardial, intraarticular, rectal, vaginal or any other route of administration.

For application of a pharmaceutical composition according to the present invention a formulation may be used that protects the active compounds from environmental influences such as proteases, oxidation, immune response etc., until it reaches the target site. Therefore, the formulation may be capsule, dragee, pill, powder, suppository, emulsion, suspension, gel, lotion, cream, salve, injectable solution, syrup, spray, inhalant or any other medical reasonable galenic formulation. Preferably, the galenic formulation may comprise suitable carriers, stabilizers, flavourings, buffers or other suitable reagents. For example, for topical application the formulation may be a lotion, cream, gel, salve or plaster, for nasopharyngeal application the formulation may be saline solution to be applied via a spray to the nose. For oral administration in case of the treatment of a specific target site e.g. in the intestine, it can be necessary to protect an artilysin according to the present invention from the harsh digestive environment of the gastrointestinal tract until the target site is reached. The present invention also relates to a pharmaceutical pack comprising one or more compartments, wherein at least one compartment comprises one or more artilysins according to the present invention and one or more anti-cancer agents or a composition according to the present invention.

It is to be understood that the foregoing general description are exemplary and explanatory only and are not restrictive of the invention, as claimed.