Clec3a-derived antimicrobial peptides

Abstract

The present invention generally relates to antimicrobial polypeptides, and more specifically to antimicrobial polypeptides derived from C-type lectin domain family 3-member A (CLEC3A) having improved properties. The present invention further relates to pharmaceutical compositions comprising said polypeptides. The antimicrobial polypeptides are particularly useful in the treatment of bacterial infections, especially those caused by drug resistant bacteria.

Description

[0001] CLEC3A-derived antimicrobial peptides

[0002] Field of the invention

[0003] The present invention generally relates to antimicrobial (poly)peptides, and more specifically to antimicrobial polypeptides derived from C-type lectin domain family 3-member A (CLEC3A) having improved properties. The present invention further relates to pharmaceutical compositions comprising said polypeptides. The antimicrobial polypeptides are particularly useful in the treatment of bacterial infections, especially those caused by drug resistant bacteria.

[0004] Background of the invention

[0005] The emergence of multi-drug-resistant bacteria poses a great threat to the health-care system1. To counteract the impact of antimicrobial resistance alternatives to common antibiotics like antimicrobial peptides (AMPs) are in dire need1. AMPs are short peptides of 10 to 50 amino acids and a highly positive net charge1. Besides p-sheet formations, loops and extended random coils, APMs often display amphipathic a-helices as a secondary structure2.

[0006] AMPs exhibit a potent antimicrobial activity and are commonly found as a component of immunity in various species34where they play an important role in pathogen defense. They provide innate immunity by binding to the membranes of microbes causing membrane disruption56. The molecular mechanism of action of AMPs often involves the incorporation of the peptide into the bacterial membrane or cell wall, leading to their disruption via destabilization and pore formation56. AMPs are usually synthesized as inactive precursors and the first step in activating AMPs is the processing via proteolytic cleavage78. Peptides derived from C-type lectin domain family 3 member A (CLEC3A)9, a cartilage-specific member of the C-type lectin superfamily8, have the potential to be used as alternatives to antibiotics. Besides its carbohydrate recognition domain (CRD) encoded by exon 3, CLEC3A contains an a-helical oligomerization domain encoded by exon 2 as well as a 16 amino acids long, positively charged region and a signal peptide at the N- terminus encoded by exon 1 , which contains eight positively charged residues9. CLEC3A shows great similarity to AMP precursors and in prior works, the CLEC3A-derived peptides HT-16, which includes the positively charged N-terminus, and HT-47, which additionally includes the oligomerization domain, have shown considerable antimicrobial activity on gram-positive and gram-negative bacteria10. The assessed antimicrobial activity of the peptides was comparable to the human cathelicidin LL-37, one of the best-studied AMPs10. However, in contrast to LL-37, HT-16 and HT-47 did not show any toxic effects on primary human cartilage cells within an incubation time of two hours10. Moreover, coating the commonly used prosthetic material titanium with the AMPs significantly reduced the number of bacteria that adhered to the titanium10

[0007] Modifications to naturally occurring AMPs are important tools to prepare them for clinical application. This is the case since natural AMPs most often have a disadvantage that makes them not suitable for clinical applications. In the case of LL-37, its disadvantage is its high cytotoxicity11-15. Since in the body it is only locally released by immune cells like neutrophils16, monocytes17, T-cells, B-cells and natural killer cells18it still has a major biological role. However, its cytotoxicity disables it for a systemic application as it is conventional in the treatment of infectious diseases. Another common disadvantage of natural AMPs is their relatively short-lived biostability as they are severely susceptible to proteases19 20. Systemically applied AMPs should be stable in physiological fluids like blood, or serum to be able to reach the site of infection. Additionally, they should not be susceptible to bacterial proteases.

[0008] Consequently, there remains a constant need for novel and improved AMPs.

[0009] Summary of the invention

[0010] The present inventors have addressed this need by developing novel CLEC3A-derived antimicrobial polypeptides having improved properties. Specifically, the polypeptides of the present invention have shown improved antimicrobial activity, especially against drug resistant bacteria such as MRSA, and thus are promising candidates as an antibiotic alternative for clinical applications.

[0011] Accordingly, its broadest aspect the present invention provides a polypeptide comprising, in order from N-terminus to C-terminus:

[0012] (a) optionally at least one tryptophan (W) residue;

[0013] (b) a peptide fragment A, which is positively charged and is composed of 4 to 18 amino acids;

[0014] (c) a peptide linker L, which is composed of 2 to 5 amino acids selected from the group consisting of glycine (G), serine (S) and threonine (T);

[0015] (d) a peptide fragment B, which exhibits an alpha-helical structure and is composed of 12 to 28 amino acids. In a further aspect, the present invention provides a pharmaceutical composition comprising a polypeptide according to the present invention.

[0016] In a further aspect, the present invention provides the medical use of the polypeptide or pharmaceutical composition according to the present invention, especially in the treatment of a bacterial infection.

[0017] The present invention may be summarized by the following items:

[0018] 1. A polypeptide comprising, in order from N-terminus to C-terminus:

[0019] (a) optionally at least one tryptophan (W) residue;

[0020] (b) a peptide fragment A, which is positively charged and is composed of 4 to 18 amino acids;

[0021] (c) a peptide linker L, which is composed of 2 to 5 amino acids selected from the group consisting of glycine (G), serine (S) and threonine (T);

[0022] (d) a peptide fragment B, which exhibits an alpha-helical structure and is composed of 12 to 28 amino acids.

[0023] 2. The polypeptide according to item 1, wherein at least 65% of the amino acids in peptide fragment A are positively charged amino acids.

[0024] 3. The polypeptide according to item 1, wherein at least 70% of the amino acids in peptide fragment A are positively charged amino acids.

[0025] 4. The polypeptide according to item 1, wherein at least 75% of the amino acids in peptide fragment A are positively charged amino acids.

[0026] 5. The polypeptide according to item 1, wherein at least 65% of the amino acids in peptide fragment A are positively charged amino acids selected from the group consisting of lysine (K) and arginine (R). The polypeptide according to item 1 , wherein at least 70% of the amino acids in peptide fragment A are positively charged amino acids selected from the group consisting of lysine (K) and arginine (R). The polypeptide according to item 1 , wherein at least 75% of the amino acids in peptide fragment A are positively charged amino acids selected from the group consisting of lysine (K) and arginine (R). The polypeptide according to any one of items 1 to 7, wherein the peptide fragment A comprises i-1) the amino acid sequence RKHSKRRVR (SEQ ID NO: 1), or i-2) a variant amino acid sequence of i-1) having 1 , 2, 3 or 4 modifications relative to the amino acid sequence of i-1), wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof. The polypeptide according to any one of items 1 to 8, wherein the peptide fragment A comprises i-1) the amino acid sequence RKHSKRRVR (SEQ ID NO: 1), or i-2) a variant amino acid sequence of i-1) in which 1 , 2, 3 or 4 amino acids are substituted relative to the amino acid sequence of i-1). The polypeptide according to item 8 or 9, where the substitution(s) in the variant amino acid sequence of i-2) is (are) a conservative substitution(s). The polypeptide according to any one of items 8 to 10, wherein in the variant amino acid sequence of i-2) at least six of the nine amino acids are positively charged amino acids selected from the group consisting of lysine (K) and arginine (R). The polypeptide according to any one of items 8 to 11 , wherein in the variant amino acid sequence of i-2) at least one lysine is substituted by arginine and / or at least one arginine is substitute by lysine relative to the amino acid sequence of i-1). The polypeptide according to any one of items 1 to 12, wherein the polypeptide fragment A comprises ii-1) the amino acid sequence HTSRLKARKHSKRRVR (SEQ ID NO: 2), or ii-2) a variant amino acid sequence of ii-1) having 1 , 2, 3, 4, 5, 6, 7 or 8 modifications relative to the amino acid sequence of ii-1), wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof. 14. The polypeptide according to any one of items 1 to 13, wherein the polypeptide fragment A comprises ii-1) the amino acid sequence HTSRLKARKHSKRRVR (SEQ ID NO: 2), or ii-2) a variant amino acid sequence of ii-1) in which 1 , 2, 3, 4, 5, 6, 7 or 8 amino acids are substituted relative to the amino acid sequence of ii-1).

[0027] 15. The polypeptide according to item 13 or 14, where the substitution(s) in the variant amino acid sequence of ii-2) is (are) a conservative substitution(s).

[0028] 16. The polypeptide according to any one of items 13 to 15, wherein the variant amino acid sequence of ii-2) comprises the amino acid sequence of i-1) or the variant amino acid sequence of i-2).

[0029] 17. The polypeptide according to any one of items 1 to 16, wherein peptide fragment A is composed of 5 to 18 amino acids.

[0030] The polypeptide according to any one of items 1 to 17, wherein peptide fragment A is composed of 5 to 15 amino acids.

[0031] The polypeptide according to any one of items 1 to 17, wherein peptide fragment A is composed of 5 to 12 amino acids.

[0032] 20. The polypeptide according to any one of items 1 to 17, wherein peptide fragment A is composed of 5 to 9 amino acids.

[0033] The polypeptide according to any one of items 1 to 17, wherein peptide fragment A is composed of 9 to 18 amino acids.

[0034] 22. The polypeptide according to any one of items 1 to 17, wherein peptide fragment A is composed of 9 to 15 amino acids.

[0035] The polypeptide according to any one of items 1 to 17, wherein peptide fragment A is composed of 9 to 12 amino acids.

[0036] 24. The polypeptide according to any one of items 1 to 23, wherein the peptide fragment A consists of i-1) the amino acid sequence RKHSKRRVR (SEQ ID NO: 1), or i-2) a variant amino acid sequence of i-1) having 1, 2, 3 or 4 modifications relative to the amino acid sequence of i-1), wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof. The polypeptide according to any one of items 1 to 24, wherein the peptide fragment A consists of i-1) the amino acid sequence RKHSKRRVR (SEQ ID NO: 1), or i-2) a variant amino acid sequence of i-1) in which 1 , 2, 3 or 4 amino acids are substituted relative to the amino acid sequence of i-1). The polypeptide according to item 24 or 25, where the substitution(s) in the variant amino acid sequence of i-2) is (are) a conservative substitution(s). The polypeptide according to any one of items 24 to 26, wherein in the variant amino acid sequence of i-2) at least six of the nine amino acids are positively charged amino acids selected from the group consisting of lysine (K) and arginine (R). The polypeptide according to any one of items 1 to 27, wherein the polypeptide fragment A consists of ii-1) the amino acid sequence HTSRLKARKHSKRRVR (SEQ ID NO: 2), or ii-2) a variant amino acid sequence of ii-1) having 1 , 2, 3, 4, 5, 6, 7 or 8 modifications relative to the amino acid sequence of ii-1), wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof. The polypeptide according to any one of items 1 to 28, wherein the polypeptide fragment A consists of ii-1) the amino acid sequence HTSRLKARKHSKRRVR (SEQ ID NO: 2), or ii-2) a variant amino acid sequence of ii-1) in which 1 , 2, 3, 4, 5, 6, 7 or 8 amino acids are substituted relative to the amino acid sequence of ii-1). The polypeptide according to item 27 or 28, where the substitution(s) in the variant amino acid sequence of ii-2) is (are) a conservative substitution(s). The polypeptide according to any one of items 27 to 30, wherein the variant amino acid sequence of ii-2) comprises the amino acid sequence of i-1) or the variant amino acid sequence of i-2). The polypeptide according to any one of items 1 to 23, wherein the peptide fragment A consists of iii-1) the amino acid sequence KRRVR (SEQ ID NO: 3), or iii-2) a variant amino acid sequence of iii-1) in which 1 , 2 or 3 amino acids are substituted relative to the amino acid sequence of iii-1). The polypeptide according to item 32, where the substitution(s) in the variant amino acid sequence of iii-2) is (are) a conservative substitution(s). The polypeptide according to item 32 or 33, wherein in the variant amino acid sequence of iii-2) at least one lysine is substituted by arginine and / or at least one arginine is substitute by lysine relative to the amino acid sequence of iii-1). The polypeptide according to item 1 , wherein the peptide fragment A consists of the amino acid sequence RKHSKRRVR (SEQ ID NO: 1). The polypeptide according to any one of items 1 to 35, wherein peptide fragment B comprises iv-1) the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4), or iv- 2) a variant amino acid sequence of iv-1) having 1 , 2, 3, 4, 5, 6, 7 or 8 modifications relative to the amino acid sequence of iv-1) and which is capable of forming an alpha-helical structure, wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof. The polypeptide according to any one of items 1 to 36, wherein peptide fragment B comprises iv-1) the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4), or iv- 2) a variant amino acid sequence in which 1 , 2, 3, 4, 5, 6, 7 or 8 amino acids are substituted relative to the amino acid sequence of iv-1) and which is capable of forming an alpha-helical structure. The polypeptide according to item 36 or 37, where the substitution(s) in the variant amino acid sequence of iv-2) is (are) a conservative substitution(s). The polypeptide according to any one of items 1 to 38, wherein peptide fragment B comprises v-1) the amino acid sequence LKTQIEKLWTEVNALKEIQALQTVCL (SEQ ID NO: 5), or v-2) a variant amino acid sequence of v-1) having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 modifications relative to the amino acid sequence of v-1) and which is capable of forming an alpha-helical structure, wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof. The polypeptide according to any one of items 1 to 39, wherein peptide fragment B comprises v-1) the amino acid sequence LKTQIEKLWTEVNALKEIQALQTVCL (SEQ ID NO: 5), or v-2) a variant amino acid sequence in which 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 amino acids are substituted relative to the amino acid sequence of v-1) and which is capable of forming an alpha-helical structure.

[0037] 41 . The polypeptide according to item 39 or 40, where the substitution(s) in the variant amino acid sequence of v-2) is (are) a conservative substitution(s).

[0038] 42. The polypeptide according to any one of items 39 to 41 , wherein the variant amino acid sequence of v-2) comprises the amino acid sequence of iv-1) or the variant amino acid sequence of iv-2).

[0039] 43. The polypeptide according to any one of items 1 to 42, wherein peptide fragment B is composed of 12 to 26 amino acids.

[0040] 44. The polypeptide according to any one of items 1 to 42, wherein peptide fragment B is composed of 12 to 20 amino acids.

[0041] 45. The polypeptide according to any one of items 1 to 42, wherein peptide fragment B is composed of 12 to 18 amino acids.

[0042] 46. The polypeptide according to any one of items 1 to 42, wherein peptide fragment B is composed of 15 to 26 amino acids.

[0043] 47. The polypeptide according to any one of items 1 to 42, wherein peptide fragment B is composed of 15 to 20 amino acids.

[0044] 48. The polypeptide according to any one of items 1 to 42, wherein peptide fragment B is composed of 15 to 18 amino acids.

[0045] 49. The polypeptide according to any one of items 1 to 48, wherein peptide fragment B consists of iv-1) the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4), or iv-2) a variant amino acid sequence of iii-1) having 1 , 2, 3, 4, 5, 6, 7 or 8 modifications relative to the amino acid sequence of iii-1) and which is capable of forming an alpha-helical structure, wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof.

[0046] 50. The polypeptide according to any one of items 1 to 49, wherein peptide fragment B consists of iv-1) the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4), or iv-2) a variant amino acid sequence of iv-1) in which 1, 2, 3, 4, 5, 6, 7 or 8 amino acids are substituted relative to the amino acid sequence of iv-1) and which is capable of forming an alpha-helical structure. The polypeptide according to item 49 or 50, where the substitution(s) in the variant amino acid sequence of iv-2) is (are) a conservative substitution(s). The polypeptide according to any one of items 1 to 51 , wherein peptide fragment B consists of v-1) the amino acid sequence LKTQIEKLWTEVNALKEIQALQTVCL (SEQ ID NO: 5), or v-2) a variant amino acid sequence of v-1) having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 modifications relative to the amino acid sequence of v-1) and which is capable of forming an alpha-helical structure, wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof. The polypeptide according to any one of items 1 to 52, wherein peptide fragment B consists of v-1) the amino acid sequence LKTQIEKLWTEVNALKEIQALQTVCL (SEQ ID NO: 5), or v-2) a variant amino acid sequence in which 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acids are substituted relative to the amino acid sequence of v-1) and which is capable of forming an alpha-helical structure. The polypeptide according to item 52 or 53, where the substitution(s) in the variant amino acid sequence of v-2) is (are) a conservative substitution(s). The polypeptide according to any one of items 52 to 54, wherein the variant amino acid sequence of v-2) comprises the amino acid sequence of iv-1) or the variant amino acid sequence of iv-2). The polypeptide according to any one of items 1 to 34, wherein peptide fragment B consists of the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4). The polypeptide according to any one of items 1 to 56, wherein the peptide linker L is composed of 3, 4 or 5 amino acids. The polypeptide according to any one of items 1 to 57, wherein the peptide linker L is composed of 3 amino acids. 59. The polypeptide according to any one of items 1 to 58, wherein the peptide linker L is exclusively composes of glycine (glycine-linker), is exclusively composed of glycine and serine (glycine-serine-linker), is exclusively composed of glycine and threonine (glycinethreonine linker), is exclusively composed of serine and threonine (serine-threonine linker) or is composed of glycine, serine and threonine (glycine-serine-threonine linker).

[0047] 60. The polypeptide according to any one of items 1 to 59, wherein the peptide linker L is exclusively composes of glycine (glycine-linker).

[0048] 61. The polypeptide according to any one of items 1 to 57, wherein the peptide linker L is selected from the group consisting of GG, GX, XG, GGG, XGG, GXG, GGX, GGGG (SEQ ID NO: 8), XGGG, GXGG, GGXG, GGGX, GGGGG (SEQ ID NO: 9), XGGGG (SEQ ID NO: 10), GXGGG (SEQ ID NO: 11), GGXGG (SEQ ID NO: 12), GGGXG (SEQ ID NO: 13) and GGGGX (SEQ ID NO: 14), wherein X is S or T, preferably S.

[0049] 62. The polypeptide according to any one of items 1 to 58, wherein the peptide linker L consists of the amino acid sequence GGG.

[0050] 63. The polypeptide according to any one of items 1 to 57, wherein the peptide linker L is selected from the group consisting of SS, SX, XS, SSS, XSS, SXS, SSX, SSSS (SEQ ID NO: 15), XSSS, SXSS, SSXS, SSSX, SSSSS (SEQ ID NO: 16), XSSSS (SEQ ID NO: 17), SXSSS (SEQ ID NO: 18), SSXSS (SEQ ID NO: 19), SSSXS (SEQ ID NO: 20) and SSSSX (SEQ ID NO: 21), wherein X is G or T, preferably G.

[0051] 64. The polypeptide according to any one of items 1 to 58, wherein the peptide linker L consists of the amino acid sequence SSS.

[0052] 65. The polypeptide according to any one of items 1 to 57, wherein the peptide linker L is selected from the group consisting of TT, TX, XT, TTT, XTT, TXT, TTX, TTTT (SEQ ID NO: 22), XTTT, TXTT, TTXT, TTTX, TTTTT (SEQ ID NO: 23), XTTTT (SEQ ID NO: 24), TXTTT (SEQ ID NO: 25), TTXTT (SEQ ID NO: 26), TTTXT (SEQ ID NO: 27) and TTTTX (SEQ ID NO: 28), wherein X is G or S, preferably G.

[0053] 66. The polypeptide according to any one of items 1 to 58, wherein the peptide linker L consists of the amino acid sequence TTT.

[0054] 67. The polypeptide according to any one of items 1 to 67, wherein (a) is present. 68. The polypeptide according to any one of items 1 to 67, wherein (a) is 1, 2, 3, 4 or 5 tryptophan (W) residues.

[0055] 69. The polypeptide according to any one of items 1 to 68, wherein (a) is 1 tryptophan (W) residue.

[0056] 70. The polypeptide according to any one of items 1 to 67, wherein (a) is 2 tryptophan (W) residues.

[0057] 71. The polypeptide according to any one of items 1 to 67, wherein (a) is 3 tryptophan (W) residues.

[0058] 72. The polypeptide according to any one of items 1 to 67, wherein (a) is 4 tryptophan (W) residues.

[0059] 73. The polypeptide according to any one of items 1 to 67, wherein (a) is 5 tryptophan (W) residues.

[0060] 74. The polypeptide according to any one of items 1 to 66 having the structure Fragment A- Linker-Fragment B.

[0061] 75. The polypeptide according to any one of items 1 to 66 having the structure Tryptophan (W)- Fragment A-Linker-Fragment B.

[0062] 76. The polypeptide according to item 1 , which comprises or consists of the amino acid sequence RKHSKRRVRGGGLKTQIEKLWTEVNALKEI (SEQ ID NO: 6).

[0063] 77. The polypeptide according to item 1 , which comprises or consists of the amino acid sequence WRKHSKRRVRGGGLKTQIEKLWTEVNALKEI (SEQ ID NO: 7).

[0064] 78. The polypeptide according to any one of items 1 to 77, which is composed of at most 50 amino acids.

[0065] 79. The polypeptide according to any one of items 1 to 77, which is composed of at most 40 amino acids. 80. The polypeptide according to any one of items 1 to 77, which is composed of at most 31 amino acids.

[0066] 81. The polypeptide according to any one of items 1 to 80, which is composed of L-amino acids, D-amino acids and combinations thereof.

[0067] 82. The polypeptide according to any one of items 1 to 81 , which is exclusively composed of L- amino acids.

[0068] 83. The polypeptide according to any one of items 1 to 81 , which is exclusively composed of D- amino acids.

[0069] 84. The polypeptide according to any one of items 1 to 83, wherein the C-terminal amino acid is amidated.

[0070] 85. The polypeptide according to any one of items 1 to 83, wherein the N-terminal amino acid is acetylated.

[0071] 86. The polypeptide according to any one of items 1 to 85 for use as an antimicrobial agent.

[0072] 87. The polypeptide according to any one of items 1 to 85 for use in medicine.

[0073] 88. The polypeptide according to any one of items 1 to 85 for use in the treatment of a microbial infection in a subject.

[0074] 89. The polypeptide for use according to item 88, wherein the microbial infection is a bacterial infection, a fungal infection or a viral infection.

[0075] 90. The polypeptide for use according to item 88 or 89, wherein the subject is a mammal.

[0076] 91. The polypeptide for use according to item 88 or 89, wherein the mammal is a human.

[0077] 92. The polypeptide for use according to item 88 or 91, wherein the subject is an non-human animal. The polypeptide for use according to item 92, wherein the non-human animal is selected from the group consisting of cat, dog, horse, cow, pig, sheep, goat, rabbit, guinea pig, monkey and avine including chicken, duck, goose and turkey. The polypeptide according to any one of items 88 to 93, wherein the microbial infection is a bacterial infection. The polypeptide for use according to item 94, wherein the bacterial infection is caused by a gram-positive bacterium. The polypeptide for use according to item 95, wherein the gram-positive bacterium is a coccus. The polypeptide for use according to item 95 or 96, wherein the gram-positive bacterium is of the genus Staphylococcus or Streptococcus. The polypeptide for use according to item 95 or 96, wherein the gram-positive bacterium is Staphylococcus aureus. The polypeptide for use according to any one of items 95 to 98, wherein the gram-positive bacterium is a drug resistant bacterium. The polypeptide for use according to item 99, wherein the drug resistant bacterium is methicillin resistant Staphylococcus aureus (MRSA). The polypeptide for use according to item 94, wherein the bacterial infection is caused by a gram-negative bacterium. The polypeptide for use according to item 101, wherein the gram-negative bacterium is of the genus Pseudomonas. The polypeptide for use according to item 102, wherein the gram-negative bacterium is Pseudomonas aeruginosa. Pharmaceutical composition comprising at least one polypeptide according to any one of items 1 to 83 and at least one pharmaceutically acceptable carrier and / or excipient. 105. llse of a polypeptide according to any one of items 1 to 85 in the manufacture of a medicament.

[0078] 106. Use of a polypeptide according to any one of items 1 to 85 in the manufacture of a medicament for the treatment of a microbial infection in a subject.

[0079] 107. Use of a polypeptide according to any one of items 1 to 85 in the manufacture of a medicament for the treatment of a bacterial infection in a subject.

[0080] 108. Method for the treatment of a bacterial infection in a subject, comprising administering to the subject a therapeutically effective amount of the polypeptide according to any one of items 1 to 85 or the pharmaceutical composition of item 104.

[0081] 109. Use of the polypeptide according to any one of items 1 to 85 for coating at least part of the surface of a solid material or for incorporating into at least a part of a solid material.

[0082] 110. Coating comprising the polypeptide according to any one of items 1 to 85, and optionally at least one further antimicrobial agent.

[0083] 111. A solid material, which is characterized in that at least part, preferably all, of its surface is covered by a coating comprising the polypeptide according to any one of items 1 to 85, and optionally at least one further antimicrobial agent.

[0084] 112. A solid material, which is characterized in that the polypeptide according to any one of items 1 to 85 and optionally at least one further antimicrobial agent is incorporated or partially incorporated.

[0085] 113. The solid material according to item 111 or 112, which is a medical device, preferably an implantable medical device.

[0086] 114. The solid material according to item 113, which is a prosthesis such as an endoprosthesis such as a total endoprosthesis or a partial endoprosthesis, a joint replacement, a partial prosthesis, a surface replacement prosthesis, a vascular prosthesis or a heart valve prosthesis, an implant such as a dental implant or a bioimplant, a cartilage substitute, a bone substitute, an arterial access or a stent.

[0087] 115. The use of a polypeptide according to any one of items 1 to 85 in a disinfectant or cleaning agent, as a preservative or in a packaging material. 116. A bacteriocidal composition which contains at least one polypeptide according to any one of items 1 to 85 and a suitable carrier.

[0088] Brief description of drawings

[0089] Figure 1 : Modified CLEC3A-derived peptides. Depicted are CLEC3A-derived peptides (ST-16, HT-16, DK-29, and HT-47) aligned to their origin in the CLEC3A protein. Furthermore, modified CLEC3A-derived peptides (RK-9, WRK-9, RK-31 , WRK-31 , RK-30, and WRK-30) are depicted underneath, with lines linking them to their original peptide. Dotted black framed boxes indicate abolishment of positive net-charge, grey regions in black framed boxes indicate truncations, grey striped areas indicate inserted linkers and W in front of the white regions in black framed boxes indicate added tryptophan residues.

[0090] Figure 2: Determining the antimicrobial activity of modified CLEC3A-derived peptides. (A) Viable Count Assay using LL-37 (* p=0.0279), HT-16, HT-47, ST-16 (Ctrl 1), DK-29 (Ctrl 2), RK- 9 (** p=0.0048), and WRK-9 in P. Aeruginosa. The bar chart shows the determined MIC50 of the peptides. (B) Viable Count Assay using LL-37 (** p=0.0094), HT-47, DK-29 (Ctrl 2), RK-31 , WRK- 31 , RK-30 and WRK-30 in P. Aeruginosa. The bar chart shows the determined MIC50 of the peptides. (C) Viable Count Assay using LL-37, HT-16, HT-47, ST-16 (Ctrl 1), DK-29 (Ctrl 2), RK- 9, and WRK-9 in S. Aureus. The bar chart shows the determined MIC50 of the peptides. (D) Viable Count Assay using LL-37, HT-47, DK-29 (Ctrl 2), RK-31 , WRK-31 (* p=0.0421), RK-30 and WRK-30 in S. Aureus. The bar chart shows the determined MIC50 of the peptides. (E) Viable Count Assay using LL-37, HT-16, HT-47, ST-16 (Ctrl 1), DK-29 (Ctrl 2), RK-9, and WRK-9 in MRSA. The bar chart shows the determined MIC50 of the peptides. (F) Viable Count Assay using LL-37 (** p=0.0029), HT-47, DK-29 (Ctrl 2), RK-31 , WRK-31 , RK-30 (“ p=0.0094) and WRK-30 (** p=0.0013) in MRSA. The bar chart shows the determined MIC50 of the peptides. Depicted are averages and standard deviations. Statistical significance was calculated using Prism 9 with paired Anova test followed by Dunett test. All experiments were repeated three times (n=3).

[0091] Figure 3: Cytotoxicity of modified CLEC3A-derived peptides. (A) Cytotoxicity Assay of NIH3T3 cells with LL-37 (30pM ** p=0.0027), HT-16, HT-47, ST-16 (Ctrl 1), DK-29 (Ctrl 2), RK-9, WRK-9, RK-30, WRK-30 in 0.3pM, 3pM, and 30pM over 24h. Vancomycin, Cholistin, Untreated and TritonX (**** p<0.0001) treated samples were used as additional controls. (B) Cytotoxicity Assay of NIH3T3 cells LL-37 (30 pM **** p<0.0001), HT-16 (0.3pM * p=0.0495), HT-47 (3pM * p=0.0439), ST-16 (Ctrl 1) (0.3pM * p=0.0324, 3pM * p=0.0397), DK-29 (Ctrl 2) (3pM * p=0.0246), RK-9 (0.3pM * p=0.0378, 3pM * p=0.0454), WRK-9 (0.3pM * p=0.0155, 3pM ** p=0.0095), RK- 30 (0.3pM * p=0.0106, 3 M * p=0.0456, 30pM * p=0.0349), WRK-30 (0.3pM ** p=0.0051 , 3pM ** p=0.0055, 30pM * p=0.0240) in 0.3pM, 3pM, and 30pM over 96h. Vancomycin (* p=0.0354), Cholistin, Untreated and TritonX (*** p=0.0002, **** p<0.0001) treated samples were used as additional controls. (C) Cytotoxicity Assay of NIH3T3 cells with LL-37 (**** p<0.0001), HT-47, DK- 29 (Ctrl 2), and WRK-30 in 40pM, 60pM, and 80pM over 24h. Vancomycin, Cholistin, Untreated and TritonX (**** p<0.0001) treated samples were used as additional controls. The number of viable cells was determined using MTS (CellTiter 96 Aqueous Solution Reagent from Promega). All values are percentages normed to the untreated controls (untreated controls were set as 100%). Depicted are averages and standard deviations. Statistical significance was calculated using Prism 9 with paired Anova test followed by Dunett test. All experiments were repeated three times (n=3). (D) Table of the therapeutic indices of HT-47 and WRK-30 against P. aeruginosa, S. aureus and MRSA, calculated using the equation TI=cytotox / MIC50.

[0092] Figure 4: Bacterial burden on the titanium-plate (Ti-plate) and in the wound tissue of infected mice. (A) Bacterial count in absolute numbers found on the Ti-plates. (B) Bacterial burden found on the Ti-plates normed to the controls in %. (C) Bacterial count in absolute numbers found in the wound tissue. TiBP-WRK-30 p < 0.0001. (D) Bacterial burden found in the wound tissue normed to the controls in %. TiBP-WRK-30 p < 0.0001. Depicted are averages with standard deviations. Statistical significance was calculated by comparing TiBP-WRK-30 to the uncoated control (Ctrl). Experiments were repeated four times

[0093] Detailed description of the invention

[0094] Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled person in the fields of biochemistry, molecular biology, genetics, and microbiology.

[0095] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified. Polypeptide of the present invention

[0096] As noted above, the present invention generally pertains to novel CLEC3A-derived antimicrobial (poly)peptides. The present invention is based in the surprising finding that introducing certain modifications to CLEC3A-derived antimicrobial polypeptides improves their properties, notably their antimicrobial activity. Particularly, the present inventors have shown that introducing a small and flexible peptide linker between the two peptide fragments primarily responsible for the antimicrobial activity of CLEC3A-derived antimicrobial polypeptides results in an increase of the antimicrobial activity. Further improvements are achieved if at least one tryptophan (W) residue are added to the N terminus as this increases the hydrophobicity of the modified polypeptide.

[0097] Thus, in its broadest aspect, the present invention provides a polypeptide comprising, in order from N-terminus to C-terminus:

[0098] (a) optionally at least one tryptophan (W) residue;

[0099] (b) a peptide fragment A, which is positively charged and is composed of 4 to 18 amino acids;

[0100] (c) a peptide linker L, which is composed of 2 to 5 amino acids selected from the group consisting of glycine (G), serine (S) and threonine (T);

[0101] (d) a peptide fragment B, which exhibits an alpha-helical structure and is composed of 12 to 28 amino acids.

[0102] Generally, the peptide linker L is composed of 2, 3, 4 or 5 amino acids selected from the group consisting of glycine (G), serine (S) and threonine (T). The peptide linker may be a glycine-linker, a glycine-serine-linker, a glycine-threonine linker, a serine-threonine linker or a glycine-serine- threonine linker. The

[0103] According to some embodiments, the peptide linker L is composed of 2 amino acids.

[0104] According to some embodiments, the peptide linker L is composed of 3 amino acids.

[0105] According to some embodiments, the peptide linker L is composed of 4 amino acids.

[0106] According to some embodiments, the peptide linker L is composed of 5 amino acids. According to some embodiments, the peptide linker L is exclusively composes of glycine (glycine- linker).

[0107] According to some embodiments, the peptide linker L is exclusively composed of glycine and serine (glycine-serine-linker).

[0108] According to some embodiments, the peptide linker L is exclusively composed of glycine and threonine (glycine-threonine linker).

[0109] According to some embodiments, the peptide linker L is serine and threonine (serine-threonine linker).

[0110] According to some embodiments, the peptide linker L is composed of glycine, serine and threonine (glycine-serine-threonine linker).

[0111] According to some embodiments, the peptide linker L is selected from the group consisting of GG, GX, XG, GGG, XGG, GXG, GGX, GGGG (SEQ ID NO: 8), XGGG, GXGG, GGXG, GGGX, GGGGG (SEQ ID NO: 9), XGGGG (SEQ ID NO: 10), GXGGG (SEQ ID NO: 11), GGXGG (SEQ ID NO: 12), GGGXG (SEQ ID NO: 13) and GGGGX (SEQ ID NO: 14), wherein X is S or T, preferably S.

[0112] According to some embodiments, the peptide linker L consists of the amino acid sequence GGG.

[0113] According to some embodiments, the peptide linker L is selected from the group consisting of SS, SX, XS, SSS, XSS, SXS, SSX, SSSS (SEQ ID NO: 15), XSSS, SXSS, SSXS, SSSX, SSSSS (SEQ ID NO: 16), XSSSS (SEQ ID NO: 17), SXSSS (SEQ ID NO: 18), SSXSS (SEQ ID NO: 19), SSSXS (SEQ ID NO: 20) and SSSSX (SEQ ID NO: 21), wherein X is G or T, preferably G.

[0114] According to some embodiments, the peptide linker L consists of the amino acid sequence SSS.

[0115] According to some embodiments, the peptide linker L is selected from the group consisting of TT, TX, XT, TTT, XTT, TXT, TTX, TTTT (SEQ ID NO: 22), XTTT, TXTT, TTXT, TTTX, TTTTT (SEQ ID NO: 23), XTTTT (SEQ ID NO: 24), TXTTT (SEQ ID NO: 25), TTXTT (SEQ ID NO: 26), TTTXT (SEQ ID NO: 27) and TTTTX (SEQ ID NO: 28), wherein X is G or S, preferably G.

[0116] According to some embodiments, the peptide linker L consists of the amino acid sequence TTT. Generally, the peptide fragment A has a positively charge which allows the binding of the polypeptide to the bacterial membrane and contributes to the antimicrobial activity of the polypeptide. Suitably, at least 50% of the amino acids in peptide fragment A should be positively charged amino acids. Preferably, at least 65% of the amino acids in peptide fragment A are positively charged amino acids.

[0117] Thus, According to some embodiments, at least 65% of the amino acids in peptide fragment A are positively charged amino acids. According to some embodiments, at least 70% of the amino acids in peptide fragment A are positively charged amino acids. According to some embodiments, at least 75% of the amino acids in peptide fragment A are positively charged amino acids.

[0118] According to some embodiments, at least 65% of the amino acids in peptide fragment A are positively charged amino acids selected from the group consisting of lysine (K) and arginine (R).

[0119] According to some embodiments, at least 70% of the amino acids in peptide fragment A are positively charged amino acids selected from the group consisting of lysine (K) and arginine (R).

[0120] According to some embodiments, at least 75% of the amino acids in peptide fragment A are positively charged amino acids selected from the group consisting of lysine (K) and arginine (R).

[0121] According to some embodiments, the peptide fragment A comprises i-1) the amino acid sequence RKHSKRRVR (SEQ ID NO: 1), or i-2) a variant amino acid sequence of i-1) having 1 , 2, 3 or 4 modifications relative to the amino acid sequence of i-1), wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof.

[0122] According to some embodiments, the peptide fragment A comprises i-1) the amino acid sequence RKHSKRRVR (SEQ ID NO: 1), or i-2) a variant amino acid sequence of i-1) in which 1 , 2, 3 or 4 amino acids are substituted relative to the amino acid sequence of i-1). Preferably, the substitution(s) in the variant amino acid sequence of i-2) is (are) a conservative substitution(s).

[0123] According to some embodiments, the peptide fragment A consists of i-1) the amino acid sequence RKHSKRRVR (SEQ ID NO: 1), or i-2) a variant amino acid sequence of i-1) having 1 , 2, 3 or 4 modifications relative to the amino acid sequence of i-1), wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof.

[0124] According to some embodiments, the peptide fragment A consists of i-1) the amino acid sequence RKHSKRRVR (SEQ ID NO: 1), or i-2) a variant amino acid sequence of i-1) in which 1 , 2, 3 or 4 amino acids are substituted relative to the amino acid sequence of i-1). Preferably, the substitution(s) in the variant amino acid sequence of i-2) is (are) a conservative substitution(s).

[0125] According to some embodiments, in the variant amino acid sequence of i-2) at least six of the nine amino acids are positively charged amino acids selected from the group consisting of lysine (K) and arginine (R).

[0126] According to some embodiments, in the variant amino acid sequence of i-2) at least one lysine is substituted by arginine and / or at least one arginine is substitute by lysine relative to the amino acid sequence of i-1).

[0127] According to some embodiments, the polypeptide fragment A comprises ii-1) the amino acid sequence HTSRLKARKHSKRRVR (SEQ ID NO: 2), or ii-2) a variant amino acid sequence of ii- 1) having 1 , 2, 3, 4, 5, 6, 7 or 8 modifications relative to the amino acid sequence of ii-1), wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof.

[0128] According to some embodiments, the polypeptide fragment A comprises ii-1) the amino acid sequence HTSRLKARKHSKRRVR (SEQ ID NO: 2), or ii-2) a variant amino acid sequence of ii- 1) in which 1 , 2, 3, 4, 5, 6, 7 or 8 amino acids are substituted relative to the amino acid sequence of ii-1). Preferably, the substitution(s) in the variant amino acid sequence of i-2) is (are) a conservative substitution(s).

[0129] According to some embodiments, the polypeptide fragment A consists of ii-1) the amino acid sequence HTSRLKARKHSKRRVR (SEQ ID NO: 2), or ii-2) a variant amino acid sequence of ii- 1) having 1 , 2, 3, 4, 5, 6, 7 or 8 modifications relative to the amino acid sequence of ii-1), wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof.

[0130] According to some embodiments, the polypeptide fragment A consists of ii-1) the amino acid sequence HTSRLKARKHSKRRVR (SEQ ID NO: 2), or ii-2) a variant amino acid sequence of ii- 1) in which 1 , 2, 3, 4, 5, 6, 7 or 8 amino acids are substituted relative to the amino acid sequence of ii-1). Preferably, the substitution(s) in the variant amino acid sequence of i-2) is (are) a conservative substitution(s).

[0131] According to some embodiments, the variant amino acid sequence of ii-2) comprises the amino acid sequence of i-1) or the variant amino acid sequence of i-2). According to some embodiments, the peptide fragment A consists of iii-1) the amino acid sequence KRRVR (SEQ ID NO: 3), or iii-2) a variant amino acid sequence of iii-1) in which 1 , 2 or 3 amino acids are substituted relative to the amino acid sequence of iii-1). Preferably, the substitution(s) in the variant amino acid sequence of i-2) is (are) a conservative substitution(s).

[0132] According to some embodiments, in the variant amino acid sequence of iii-2) at least one lysine is substituted by arginine and / or at least one arginine is substitute by lysine relative to the amino acid sequence of iii-1).

[0133] The peptide fragment A generally has a length from 4 to 18 amino acids, such as from 5 to 18 amino acids, from 5 to 15 amino acids, from 5 to 12 amino acids, from 5 to 9 amino acids, from 7 to 18 amino acids, from 7 to 15 amino acids, from 7 to 12 amino acids, from 9 to 18 amino acids, from 9 to 15 amino acids, from 9 to 12 amino acids, or from 8 to 10 amino acids.

[0134] According to some embodiments, the peptide fragment A is composed of 5 to 18 amino acids.

[0135] According to some embodiments, the peptide fragment A is composed of 5 to 15 amino acids.

[0136] According to some embodiments, the peptide fragment A is composed of 5 to 12 amino acids.

[0137] According to some embodiments, peptide fragment A is composed of 5 to 9 amino acids.

[0138] According to some embodiments, the peptide fragment A is composed of 7 to 18 amino acids.

[0139] According to some embodiments, the peptide fragment A is composed of 7 to 15 amino acids.

[0140] According to some embodiments, the peptide fragment A is composed of 7 to 12 amino acids.

[0141] According to some embodiments, peptide fragment A is composed of 7 to 9 amino acids.

[0142] According to some embodiments, the peptide fragment A is composed of 9 to 18 amino acids.

[0143] According to some embodiments, the peptide fragment A is composed of 9 to 15 amino acids.

[0144] According to some embodiments, the peptide fragment A is composed of 9 to 12 amino acids.

[0145] According to some embodiments, peptide fragment A is composed of 8 to 10 amino acids. According to some embodiments, peptide fragment A is composed of 9 amino acids.

[0146] According to some embodiments, the peptide fragment A consists of the amino acid sequence RKHSKRRVR (SEQ ID NO: 1).

[0147] Generally, the peptide fragment B exhibits an alpha-helical structure. As used herein “alpha helical” structure refers to a three dimensional structural conformation which is commonly found in naturally occurring proteins and polypeptides. The alpha helix conformation found in naturally occurring proteins and polypeptides has its side chains extending to the outside of the structure, has a complete turn every 3.6 amino acids, is right-handed and typically has hydrogen bonding between the carbonyl groups of the amide bond and an amide N — H group 4 amino acids further on in the sequence. Among the types of polypeptide structures, the alpha-helix is the most prevalent and easily predicted based on amino acid sequence. Principles for the design of polypeptides with a-helical structures are well-studied and established in the art (see e.g., Garner & Harding, "Design and synthesis of a- helical peptides and mimetics," Org. Biomol. Chem., 2007, 5, 3577-3585; and Doig, "Stability and Design of a-Helical Peptides," Progress in Molecular Biology and Translational Science, Vol. 83, pp. 1-52, Elsevier 2008). These general principles can be used to design a polypeptide fragment having an alpha helical structure that are useful in the present invention.

[0148] The propensity of an amino acid to form an a-helix structure can be estimated based on the difference in free energy, estimated in kcal / mol per residue when the amino acid is in an a-helix, relative to the amino acid alanine, which is arbitrarily set as zero. The alpha-helix forming propensity of amino acid residues have been estimated based on free energy differences (see e.g., Pace, et al. (1998), "A Helix Propensity Scale Based on Experimental Studies of Peptides and Proteins," Biophysical Journal 75: 422-427). Amino acid residues that are favorable to helix formation include, for example, alanine, arginine, lysine, methionine, leucine, glutamic acid, glutamine, cysteine, isoleucine, phenylalanine, tyrosine, tryptophan, histidine and aspartic acid, and especially alanine, arginine, lysine, methionine, leucine, glutamic acid and glutamine. Proline and glycine have poor helix-forming propensities. Proline is an amino acid residue that disrupts an amino acid sequence from forming a helical structure, such as an a-helix. Glycine also tends to disrupt helical structures. However, both can act as the first residue of a helix.

[0149] Methods for determining alpha-helix formation are well-known in the art and include, but are not limited to, circular dichroism spectroscopy (CD spectroscopy). According to some embodiments, peptide fragment B comprises iv-1) the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4), or iv-2) a variant amino acid sequence of iv-1) having 1 , 2, 3, 4, 5, 6, 7 or 8 modifications relative to the amino acid sequence of iv-1) and which is capable of forming an alpha-helical structure, wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof.

[0150] According to some embodiments, peptide fragment B comprises iv-1) the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4), or iv-2) a variant amino acid sequence in which 1 , 2, 3, 4, 5, 6, 7 or 8 amino acids are substituted relative to the amino acid sequence of iv-1) and which is capable of forming an alpha-helical structure. Preferably the substitution(s) in the variant amino acid sequence of iv-2) is (are) a conservative substitution(s).

[0151] According to some embodiments, peptide fragment B consists of iv-1) the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4), or iv-2) a variant amino acid sequence of iii-1) having 1 , 2, 3, 4, 5, 6, 7 or 8 modifications relative to the amino acid sequence of iii-1) and which is capable of forming an alpha-helical structure, wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof.

[0152] According to some embodiments, peptide fragment B consists of iv-1) the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4), or iv-2) a variant amino acid sequence of iv-1) in which 1 , 2, 3, 4, 5, 6, 7 or 8 amino acids are substituted relative to the amino acid sequence of iv-1) and which is capable of forming an alpha-helical structure. Preferably the substitution(s) in the variant amino acid sequence of iv-2) is (are) a conservative substitution(s).

[0153] According to some embodiments, peptide fragment B comprises v-1) the amino acid sequence LKTQIEKLWTEVNALKEIQALQTVCL (SEQ ID NO: 5), or v-2) a variant amino acid sequence of v-1) having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 modifications relative to the amino acid sequence of v-1) and which is capable of forming an alpha-helical structure, wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof.

[0154] According to some embodiments, peptide fragment B comprises v-1) the amino acid sequence LKTQIEKLWTEVNALKEIQALQTVCL (SEQ ID NO: 5), or v-2) a variant amino acid sequence in which 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 amino acids are substituted relative to the amino acid sequence of v-1) and which is capable of forming an alpha-helical structure. Preferably the substitution(s) in the variant amino acid sequence of iv-2) is (are) a conservative substitution(s). According to some embodiments, peptide fragment B consists of v-1) the amino acid sequence LKTQIEKLWTEVNALKEIQALQTVCL (SEQ ID NO: 5), or v-2) a variant amino acid sequence of v-1) having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 modifications relative to the amino acid sequence of v-1) and which is capable of forming an alpha-helical structure, wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof.

[0155] According to some embodiments, peptide fragment B consists of v-1) the amino acid sequence LKTQIEKLWTEVNALKEIQALQTVCL (SEQ ID NO: 5), or v-2) a variant amino acid sequence in which 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acids are substituted relative to the amino acid sequence of v-1) and which is capable of forming an alpha-helical structure. Preferably the substitution(s) in the variant amino acid sequence of iv-2) is (are) a conservative substitution(s).

[0156] According to some embodiments, the variant amino acid sequence of v-2) comprises the amino acid sequence of iv-1) or the variant amino acid sequence of iv-2).

[0157] The peptide fragment B generally has a length from 12 to 28 amino acids, such as from 12 to 26 amino acids, from 12 to 20 amino acids, from 12 to 18 amino acids, from 15 to 26 amino acids, from 15 to 20 amino acids, from 15 to 18 amino acids, from 18 to 26 amino acids, from 18 to 20 amino acids, or from 17 to 19 amino acids.

[0158] According to some embodiments, peptide fragment B is composed of 12 to 26 amino acids.

[0159] According to some embodiments, peptide fragment B is composed of 12 to 20 amino acids.

[0160] According to some embodiments, peptide fragment B is composed of 12 to 18 amino acids.

[0161] According to some embodiments, peptide fragment B is composed of 15 to 26 amino acids.

[0162] According to some embodiments, peptide fragment B is composed of 15 to 20 amino acids.

[0163] According to some embodiments, peptide fragment B is composed of 15 to 18 amino acids.

[0164] According to some embodiments, peptide fragment B is composed of 18 to 26 amino acids.

[0165] According to some embodiments, peptide fragment B is composed of 18 to 20 amino acids. According to some embodiments, peptide fragment B is composed of 17 to 19 amino acids.

[0166] According to some embodiments, peptide fragment B is composed of 18 amino acids.

[0167] According to some embodiments, peptide fragment B consists of the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4).

[0168] According to some embodiments, element (a) is present.

[0169] According to some embodiments, (a) is 1 , 2, 3, 4 or 5 tryptophan (W) residues.

[0170] According to some embodiments, (a) is 1 tryptophan (W) residue.

[0171] According to some embodiments, (a) is 2 tryptophan (W) residues.

[0172] According to some embodiments, (a) is 3 tryptophan (W) residues.

[0173] According to some embodiments, (a) is 4 tryptophan (W) residues.

[0174] According to some embodiments, (a) is 5 tryptophan (W) residues.

[0175] According to some embodiments, the polypeptide has the structure Fragment A-Linker- Fragment B.

[0176] According to some embodiments, the polypeptide has the structure Tryptophane (W)-Fragment A-Linker-Fragment B.

[0177] According to some embodiments, the polypeptide comprises, in order from N-terminus to C- terminus:

[0178] (a) optionally at least one tryptophan (W) residue;

[0179] (b) a peptide fragment A, which consists of i-1) the amino acid sequence RKHSKRRVR (SEQ ID NO: 1), or i-2) a variant amino acid sequence of i-1) in which 1 , 2, 3 or 4 amino acids are substituted relative to the amino acid sequence of i-1); preferably wherein the substitution(s) is (are) a conservative substitution(s); (c) a peptide linker L, which is composed of 2 to 5 amino acids selected from the group consisting of glycine (G), serine (S) and threonine (T);

[0180] (d) a peptide fragment B, which consists of iv-1) the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4), or iv-2) a variant amino acid sequence of iv-1) in which 1 , 2, 3, 4, 5, 6, 7 or 8 amino acids are substituted relative to the amino acid sequence of iv-1) and which is capable of forming an alpha-helical structure, preferably wherein the substitution(s) is (are) a conservative substitution(s).

[0181] According to some embodiments, the polypeptide comprises, in order from N-terminus to C- terminus:

[0182] (a) optionally at least one tryptophan (W) residue;

[0183] (b) a peptide fragment A, which consists of i-1) the amino acid sequence RKHSKRRVR (SEQ ID NO: 1), or i-2) a variant amino acid sequence of i-1) in which 1 , 2, 3 or 4 amino acids are substituted relative to the amino acid sequence of i-1); preferably wherein the substitution(s) is (are) a conservative substitution(s);

[0184] (c) a peptide linker L selected from the group consisting of GG, GX, XG, GGG, XGG, GXG, GGX, GGGG (SEQ ID NO: 8), XGGG, GXGG, GGXG, GGGX, GGGGG (SEQ ID NO: 9), XGGGG (SEQ ID NO: 10), GXGGG (SEQ ID NO: 11), GGXGG (SEQ ID NO: 12), GGGXG (SEQ ID NO: 13) and GGGGX (SEQ ID NO: 14), wherein X is S or T, preferably S;

[0185] (d) a peptide fragment B, which consists of iv-1) the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4), or iv-2) a variant amino acid sequence of iv-1) in which 1 , 2, 3, 4, 5, 6, 7 or 8 amino acids are substituted relative to the amino acid sequence of iv-1) and which is capable of forming an alpha-helical structure, preferably wherein the substitution(s) is (are) a conservative substitution(s).

[0186] According to some embodiments, the polypeptide comprises or consists of the amino acid sequence RKHSKRRVRGGGLKTQIEKLWTEVNALKEI (SEQ ID NO: 6).

[0187] According to some embodiments, the polypeptide comprises or consists of the amino acid sequence WRKHSKRRVRGGGLKTQIEKLWTEVNALKEI (SEQ ID NO: 7). According to some embodiments, the polypeptide is composed of at most 50 amino acids, such as at most at most 49 amino acids, at most 48 amino acids, at most 47 amino acids, at most 46 amino acids, at most 45 amino acids, at most 44 amino acids, at most 43 amino acids, at most 42 amino acids, at most 41 amino acids, at most 40 amino acids, at most 39 amino acids, at most 38 amino acids, at most 37 amino acids, at most 36 amino acids, at most 35 amino acids, at most 34 amino acids, at most 33 amino acids, at most 32 amino acids, at most 31 amino acids or at most 30 amino acids, at most 29 amino acids, at most 28 amino acids, at most 27 amino acids, at most 26 amino acids, at most 25 amino acids or at most 24 amino acids.

[0188] According to some embodiments, the polypeptide is composed of at most 40 amino acids.

[0189] According to some embodiments, the polypeptide is composed of at most 31 amino acids.

[0190] The polypeptide of the invention can be composed of L-amino acids, D-amino acids or combinations thereof. For example, the polypeptide of the invention can be exclusively composed of L-amino acids or can be exclusively composed of D-amino acids. Preferably, the polypeptide of the invention is exclusively composed of L-amino acids

[0191] The polypeptide of the invention may contain further modifications at the C-terminus and / or N- terminus selected from C-terminal amidation, C-terminal glycosylation, C-terminal lipidation, C- terminal methylation, N-terminal acetylation, N-terminal lipidation, and N-terminal methylation. Such modifications are well known to the skilled person and can be achieved by established procedures.

[0192] According to some embodiments, the C-terminal amino acid is amidated.

[0193] According to some embodiments, the N-terminal amino acid is acetylated.

[0194] A polypeptide of the present invention can be obtained through chemical synthesis based on the selected amino acid sequence. Examples of conventional peptide synthesis methods are well known to the skilled persons and include, e.g., those described in: (i) Peptide Synthesis, Interscience, New York, 1966; (ii) The Proteins, Vol. 2, Academic Press, New York, 1976; and (iii) Barany G. & Merrifield R. B., Peptides Vol. 2, Solid Phase Peptide Synthesis, Academic Press, New York, 1980, 100-18.

[0195] Alternatively, a polypeptide of the present invention can be obtained by known genetic engineering methods and recombinant technology. For example, a suitable vector comprising a polynucleotide encoding the polypeptide of the present invention in an expressible form (e.g., downstream of a regulatory sequence corresponding to a promoter sequence) can be prepared and transformed into a suitable host cell. The host cell will then be cultured to produce the polypeptide of the present invention. The polypeptide of the present invention can also be produced in vitro using an in vitro translation system.

[0196] The present invention thus also provides a polynucleotide encoding a polypeptide of the present invention, as well as a vector, such as an expression vector, comprising the polynucleotide.

[0197] In order to facilitate expression of a polypeptide in a host cell, the vector may comprise suitable regulatory elements such as a promoter that is functional in the host cell to cause the production of mRNA molecules and that is operably linked to the nucleotide sequence encoding the polypeptide.

[0198] Uses of the polypeptide of the invention

[0199] As noted above, the polypeptides of the present invention have shown improved antimicrobial activity, especially against drug resistant bacteria such as MRSA, and thus are promising candidates as an antibiotic alternative for clinical applications.

[0200] Thus, the present invention provides in a further aspect a polypeptide of the present invention for use as an antimicrobial agent.

[0201] The present invention further provides in a further aspect a polypeptide of the present invention for use in medicine. The term “medicine” includes both human medicine and veterinary medicine.

[0202] The present invention further provides in a further aspect a polypeptide of the present invention for use in the treatment of a microbial infection in a subject, such as bacterial infection, fungal infection or viral infection.

[0203] In a more specific aspect, the present invention provides a polypeptide of the present invention for use in the treatment of a bacterial infection in a subject.

[0204] The term "subject" includes any individual that has a microbial infection that can be treated or prevented by the polypeptide of the present invention. In particular, the subject may be a human, but may also be a non-human animal such as a non-human mammal, such as dog, cat, horse, cattle, rabbit, monkey, camel, deer, guinea pig, donkey, mouse, rat, etc., or other vertebrate such as avines, such as a chicken, duck, goose or turkey, reptiles, amphibians or fish. Thus, according to some embodiments, the subject is a mammal. According to some embodiments, the subject is a human. According to some embodiments, the subject is a non-human mammal, such as such as dog, cat, horse, cattle, rabbit, monkey, camel, deer, guinea pig, donkey, mouse, or rat.

[0205] The bacterium causing the infection may be a Gram-positive or Gram-negative bacterium, and especially may be a drug resistant Gram-positive or Gram-negative bacterium.

[0206] Non-limiting example of gram-positive and gram-negative bacteria include bacteria of the genera Staphylococcus, Streptococcus, Pseudomonas, Escherichia, Enterobacter, Erwinia, Klebsiella, Morganella, Salmonella, Shigella, Yersinia, Acinetobacter, Acinetobacter, Agrobacterium, Francisella, Legionella, Rhizobium, and Haemophilus. Preferred examples of bacteria include bacteria of the genera Staphylococcus, Streptococcus and Pseudomonas.

[0207] Specific, non-limiting examples gram-positive and gram-negative bacteria include Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Pseudomonas aeruginosa, Pseudomonas syringae, Escherichia coli, Enterobacter cloacae, Erwinia amylovora, Klebsiella pneumoniae, Morganella morganii, Salmonella typhimurium, Salmonella typhi, Shigella clysenteriae, Yersinia enterocolitica, Acinetobacter calcoaceticus, Acinetobacter baumannii, Agrobacterium tumefaciens, Francisella tularensis, Legionella pneumophila, Rhizobium meliloti, and Haemophilus influenzae. Preferred examples of bacteria include bacteria of the genera Staphylococcus aureus and Pseudomonas aeruginosa.

[0208] Thus, according to some embodiments, the bacterial infection is caused by a gram-positive bacterium.

[0209] According to some embodiments, the gram-positive bacterium is a coccus.

[0210] According to some embodiments, the gram-positive bacterium is of the genus Staphylococcus or Streptococcus.

[0211] According to some embodiments, the gram-positive bacterium is Staphylococcus aureus.

[0212] According to some embodiments, the gram-positive bacterium is a drug resistant bacterium.

[0213] According to some embodiments, the drug resistant bacterium is methicillin resistant Staphylococcus aureus (MRSA). According to some embodiments, the bacterial infection is caused by a gram-negative bacterium.

[0214] According to some embodiments, the gram-negative bacterium is of the genus Pseudomonas.

[0215] According to some embodiments, the gram-negative bacterium is Pseudomonas aeruginosa.

[0216] The present invention provides in a further aspect a pharmaceutical composition comprising at least one polypeptide of the present invention and at least one pharmaceutically acceptable carrier and / or excipient.

[0217] A pharmaceutical composition according to the invention may contain further pharmaceutically active substances, in particular an antibiotic (preferably vancomycin, streptomycin, tetracycline or penicillin).

[0218] A pharmaceutical composition of the present invention may contain one or more different polypeptides of the present invention.

[0219] Suitably, a pharmaceutical composition of the present invention will comprise a therapeutically effective amount of the at least one polypeptide of the present invention. The pharmaceutical compositions of the invention are designed to treat infections of a bacteria-infected animal, such as mammal, including human. Compositions according to the invention are effective in particular in infections with Gram-positive bacteria, in particular S. aureus.

[0220] The pharmaceutical composition of the invention may contain at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a biocompatible substance which does not induce the production of antibodies by itself which could be hazardous to the subject to whom the pharmaceutical composition of the invention is administered Suitable pharmaceutically acceptable carriers within the meaning of the invention are solvents (preferably water, buffered aqueous solutions, brine, glycerol and ethanol), diluents or other liquid binders (such as dispersion or suspension aids), surface active agents, isotonic agents, thickeners, emulsifiers, preservatives, encapsulating agents, solid binders or lubricants. Liposomes or carrier polymers to which the peptides or peptide multimers of the invention are preferably coupled (preferably biocompatible proteins or polyethylene glycol). Suitable carrier polymers are preferably slowly degradable macromolecules, especially b preferably proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids or inactivated viral components. The carriers are appropriately selected for their compatibility with the peptide and according to the particular application and dosage. Preferably, a pharmaceutical composition according to the invention may contain at least one excipient, such as humectant, emulsifier and / or pH-buffering substance.

[0221] The polypeptide of the present invention may be present in the form of a salt. For this, a salt of the polypeptide can be prepared by known methods, typically by mixing the polypeptide with a pharmaceutically acceptable acid to form an acid salt of the polypeptide, or with a pharmaceutically acceptable base, wherein a basic salt of the polypeptide is formed. Suitable pharmaceutically acceptable acids are selected from formic, acetic, propionic, lactic, glycolic, oxalic, pyruvic, succinic, maleic, malonic, cinnamic, sulfuric, hydrochloric, hydrobromic, nitric, perchloric, phosphoric and thiocyanic acid. These acids form ammonium salts with the free amino groups of the peptides or peptide multimers. Suitable pharmaceutically acceptable bases are selected from ethylamine, methylamine, dimethylamine, triethylamine, isopropylamine, diisopropylamine and other monoalkylamines, dialkylamines and trialkylamines and arylamines. These bases form carboxylates with the free carboxylic acid groups of the peptides or peptide multimers.

[0222] The pharmaceutical composition of the present invention may be in the form of a capsule, tablet, troche, dragee, drop, suppository, powder, spray, ointment, paste, cream, inhalant, patch or as an aerosol.

[0223] The pharmaceutical composition of the present invention may further comprise at least one further antimicrobial agent. A "further antimicrobial agent" may for example be at least one (e.g., 1 , 2, 3, or 4) agent directed against microorganisms other than the polypeptide of the present invention. Advantageously, the further antimicrobial agent is an antibiotic agent, such as one selected from the group consisting of aminoglycosides (e.g., gentamicin, tobramycin, streptomycin), penicillins (e.g., clavulanic acid, amoxicillin), cephalosporins (e.g., cefaclor, cefixime), lincosamides (e.g. clindamycin), macrolides (e.g. erythromycin, azithromycin, roxithromycin, clarithromycin), gyrase inhibitors, quinolones, tetracyclines (e.g. doxycycline), sulfonamides (e.g. cotrim oxazole), carbapenems (e.g. imipenem), and glycopeptides (e.g., vancomycin, teicoplanin), and combinations thereof. In other embodiments, the further antimicrobial agent may be an antifungal agent selected from the group of azole antifungal agents such as one selected from the group consisting of clotrimazole, bifonazole, econazole, isoconazole, ketoconazole, miconazole, sertaconazole, allylamine, amorolfine, caspofungin, ciclopirox, flucytosine, thiocarbamate such as tolnaftate, and polyene antibiotics such as nystatin and amphotericin B, and combinations thereof. For treating a microbial infection, such as a bacterial infection, a subject is administered a therapeutically effective amount of the polypeptide of the present invention or pharmaceutical composition of the present invention. The direct administration of the polypeptide or pharmaceutical composition may be carried out locally or systemically, preferably orally, parenterally, intraperitoneally, intravenously, intramuscularly, cutaneous, pulmonarily or interstitially.

[0224] The present invention also provides a method for the treatment of a microbial infection, such as a bacterial infection, in a subject. For this purpose, a subject is administered a therapeutically effective amount of the polypeptide or pharmaceutical composition according to the invention.

[0225] The term "therapeutically effective amount" as used herein refers to the amount of the polypeptide of the present invention which is capable of reducing or completely preventing the proliferation and / or colonization of the microbial agent, such as a bacterium. The precise effective amount for a subject will depend on its size and condition , the type and extent of the disease and the therapeutics or the combination of several therapeutics selected for treatment.

[0226] The amount of the polypeptide according to the present invention, which is necessary for an antimicrobial effective dose, is determined taking into account the infection-causing pathogen, the severity of the infection and the age, weight, sex of the patient (and possibly further patient parameters). The frequency of dosages depends on the factors mentioned above and is preferably between one and six doses per day over a treatment period of about three days to a maximum of one week. Appropriate dosing regimens may range from 0.1 to 25 mg / kg body weight such as from 0.1 to 20 mg / kg body weight, from 0.1 to 15 mg / kg body weight, from 0.1 to 10 mg / kg body weight, from 0.1 to 5 mg / kg body weight or from 0.1 to 2.5 mg / kg body weight, e.g., from 0.5 to 25 mg / kg body weight, from 0.5 to 20 mg / kg body weight, from 0.5 to 15 mg / kg body weight, from 0.5 to 10 mg / kg body weight, from 0.5 to 5 mg / kg body weight, from 0.5 to 2.5 mg / kg body weight, from 1 to 25 mg / kg body weight, from 1 to 20 mg / kg body weight, from 1 to 15 mg / kg body weight, from 1 to 10 mg / kg body weight, from 1 to 5 mg / kg body weight, from 1 to 2.5 mg / kg body weight, from 2.5 to 25 mg / kg body weight, from 2.5 to 20 mg / kg body weight, from 2.5 to 15 mg / kg body weight, from 2.5 to 10 mg / kg body weight, from 2.5 to 5 mg / kg body weight, from 5 to 25 mg / kg body weight, from 5 to 20 mg / kg body weight, from 5 to 15 mg / kg body weight, or from 5 to 10 mg / kg body weight. Administration may be 1 to 3 times per day. However, the concentration and dosage regimens should not be construed as limited to the ranges and values indicated. Any effective concentration and dosing regimen are included within the scope of the invention. The present invention provides in a further aspect the use of a polypeptide of the present invention in the manufacture of a medicament. The medicament may be for use in the treatment of a microbial infection, such as bacterial infection, in a subject.

[0227] The polypeptide of the present invention may also be used as a coating or as part of a coating, such as a coating on a medical device.

[0228] The present invention thus provides in a further aspect the use of the polypeptide of the present invention for coating at least part of the surface of a solid material or for incorporating into at least a part of a solid material. Preferably, the solid material is covered completely by a coating comprising the polypeptide of the present invention.

[0229] The coating may comprise at least one further antimicrobial agent. A "further antimicrobial agent" may for example be one as detailed above.

[0230] In the context of the present invention, "incorporation into a solid material" means that the polypeptide of the present invention and, optionally, the at least one further antimicrobial agent are within the solid material. Incorporation into the solid material is carried out by methods known in the prior art. Incorporation may be performed during manufacture of the solid material, wherein polypeptide of the present invention, and optionally the at least one further antimicrobial agent, is admixed with the materials comprising the solid material prior to manufacture of the solid material. Alternatively, the incorporation may be performed after the solid material is manufactured, wherein the solid material is treated, e.g., impregnated, with the polypeptide of the present invention and optionally the at least one further antimicrobial agent, such as a plaster, a wound dressing or a tamponade, so that the active agent is distributed or partially distributed in the solid material. The skilled person knows that the incorporation is carried out depending on the type of solid material and which methods are to be used.

[0231] The coating or incorporation of the polypeptide of the present invention, and optionally the at least one further antimicrobial agent, is preferably performed outside the subject (ex vivo). However, the coating may also occur when the solid material is inserted into a subject, e.g., by sprinkling the solid material with the polypeptide of the present invention and optionally the at least one further antimicrobial agent. The coating or incorporation of the polypeptide of the present invention, and optionally the at least one further antimicrobial agent, serves to inhibit microbial adhesion to or microbial penetration of the solid material before, during, or after the solid material is introduced into or applied to a subject. This is particularly important when the solid is introduced into a subject and remains there for a period of time or forever.

[0232] For coating or incorporation, the solid materials are treated with solutions containing the polypeptide of the present invention at concentrations of 0.1 pM up to the solubility limit of the polypeptide, such as in the range of 0.1 to 100 pM, such as 1 to 20, 25, 30, 35, 40, 45 or 50 pM or 2 to 20, 25, 30, 35, 40, 45 or 50 pM. Preferably, the effective range is from 2.5 to 20 pM, such as 2.5 pM, 5 pM, 10 pM or 20 pM treated. At these concentrations, the solid materials are coated with effective concentrations or effective concentrations are incorporated into the solid materials that result in killing and / or preventing adhesion of microorganisms. However, the concentration specifications should not be understood to be limited to the ranges and values indicated. Any concentration effective for coating or incorporating into solid materials and killing and / or preventing adhesion of microorganisms is included within the scope of the invention.

[0233] The present invention provides in a further aspect a coating comprising the polypeptide of the present invention, and optionally at least one further antimicrobial agent. A coating of the present invention may contain one or more different polypeptides of the present invention.

[0234] The present invention also provides in a further aspect a solid material, which is characterized in that at least part, preferably all, of its surface is covered by a coating comprising the polypeptide of the present invention, and optionally at least one further antimicrobial agent, or which is characterized in that the polypeptide of the present invention and optionally at least one further antimicrobial agent is incorporated or partially incorporated. A solid material of the present invention may contain one or more different polypeptides of the present invention. Preferably, the coated solid material or solid material into which the polypeptide of the present invention and optionally the at least one further antimicrobial agent is incorporated is for treating an indication as defined above.

[0235] As used herein, “solid material” means any solid material that can be coated with or into which the polypeptide of the present invention, and optionally at least one other antimicrobial agent, can be incorporated. The solid material may comprise or consist of a substance that is tolerated and not rejected or otherwise antagonized by the subject into which the solid material is introduced or to which the solid material is applied. The solid material may, for example, be a medical device. For purposes of the present invention, a medical device is a solid that is inserted into or applied to a subject to perform multiple tasks, such as a replacement for a body part such as a prosthesis, a replacement for a physiological function such as a catheter, a stabilization of a body part such as in the context of a fractured joint or bone, an ornament such as a piercing, or an overlay to treat or prevent a microbial infection of the skin or a wound. Preferably, the medical device is an implantable device, more preferably a prosthesis such as an endoprosthesis such as a total endoprosthesis or a partial endoprosthesis, a joint replacement, a partial prosthesis, a surface replacement prosthesis, a vascular prosthesis or a heart valve prosthesis, an implant such as a dental implant or a bioimplant, a cartilage substitute, a bone substitute, an arterial access or a stent, or preferably the solid is a piercing, screw, wire or plate such as in the context of a joint fracture or bone fracture, an indwelling venous cannula, a bladder catheter, a cerebrospinal fluid catheter, a broviac catheter or a tamponade.

[0236] The solid material may comprise or consist of one or more materials known to be included in medical devices such as those mentioned above. The solid material may comprise or consist of one or more materials and / or alloys selected from metal such as titanium, iron, cobalt, chromium, aluminum and molybdenum, plastic, ceramic and bone cement.

[0237] Optionally, one or more coupling agents may be employed to facilitate binding of the polypeptide of the present invention, and optionally at least one further antimicrobial agent, to the solid material. The one or more coupling agents may thus form part of the coating. Suitable coupling agents and techniques for protein immobilization are well known to the skilled person. The type of the coupling agent may vary depending on the specific solid material to be coated by the polypeptide of the present invention, and optionally the at least one further antimicrobial agent. Non-limiting examples of coupling agents include bifunctional linkers, i.e. molecules having with two different reactive ends, one end binding to the surface, and the other to the polypeptide, aldehyde-containing agents, such as glutaraldehyde, and peptides, that can be added, directly or indirectly, to the N-terminus of the polypeptide of the present invention and have a high affinity for the solid material in question. A non-limiting example of such peptides has the sequence RPRENRGRERGL (SEQ ID NO: 35), which has a high affinity for titanium and can be used as a biological coupling agent to immobilize the polypeptide of the present invention onto titanium surfaces.

[0238] Generally, it may be advantageous to include a flexible linker, such as a flexible peptide linker, between the coupling agent and the polypeptide of the present invention, to allow sufficient flexibility of the polypeptide. Such peptide linker may, for example, be directly fused to the N- terminus of the polypeptide of the present invention. A suitable flexible peptide linker may, for example, be any one of linkers L detailed above with respect to the polypeptide of the present invention. For example, the flexible linker may be a peptide linker selected from the group consisting of GG, GX, XG, GGG, XGG, GXG, GGX, GGGG (SEQ ID NO: 8), XGGG, GXGG, GGXG, GGGX, GGGGG (SEQ ID NO: 9), XGGGG (SEQ ID NO: 10), GXGGG (SEQ ID NO: 11), GGXGG (SEQ ID NO: 12), GGGXG (SEQ ID NO: 13) and GGGGX (SEQ ID NO: 14), wherein X is S or T, preferably S. A preferred peptide linker is GGG. In case of, e.g., a thiol-reactive agent being used as coupling agent, the peptide linker may include at its N-terminus a cysteine or any other amino acid containing a thiol group, such as homocysteine, which allows the formation of a disulfide bond.

[0239] It is understood that the details given in connection with the polypeptide for use equally applies to all other therapeutic aspects as detailed above and vice versa.

[0240] In addition to the therapeutic use of the polypeptide according to the present invention for the treatment of infections, they are also advantageously applicable in disinfectants or cleaning agents.

[0241] The present invention therefore also provides the use of a polypeptide according to the present invention in a disinfectant or cleaning agent, as a preservative or in a packaging material.

[0242] The present invention further provides a bacteriocidal composition which contains at least one polypeptide according to the invention and a suitable carrier. Suitable carriers are liquids or binders which are commonly used in bactericidal compositions, preferably ethanol. A bactericidal composition according to the present invention may preferably be used for disinfecting or cleaning surfaces or objects, in particular for avoiding or removing biofilms.

[0243] Certain definitions

[0244] “Polypeptide," or "protein" are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids.

[0245] "Reference sequence" or “reference polypeptide sequence” refers to a defined sequence to which another sequence is compared. In the context of the present invention a reference amino acid sequence may be any amino acid sequence set forth in SEQ ID NO: 1 to 7. “Substitution” or “substituted” refers to a modification of the reference polypeptide sequence by replacing one amino acid residue with another, for instance, the replacement of an arginine residue with a lysine residue in a polypeptide sequence is an amino acid substitution.

[0246] "Conservative substitution" refers to a substitution of an amino acid residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar class of amino acids. By way of example and not limitation, an amino acid with a basic side chain may be substituted with another amino acid with a basic side chain, e.g., lysine and arginine; an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain, e.g., aspartic acid or glutamic acid; an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid, e.g., alanine, valine, leucine, and isoleucine; an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain, e.g., serine and threonine; an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, and histidine; and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0247] "Non-conservative substitution" refers to a substitution of an amino acid in a reference polypeptide sequence with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0248] "Deletion" or “deleted” refers to a modification of the refence polypeptide sequence by removal of one or more amino acids in the reference polypeptide sequence. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, four or more amino acids or 5 or more amino acids, or up to 10% of the total number of amino acids or up to 20% of the total number of amino acids making up the reference polypeptide sequence while retaining function or activity and / or retaining the improved properties of the reference polypeptide. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide sequence. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. "Insertion" or “inserted” refers to a modification of the reference polypeptide sequence by insertion of one or more amino acids within the reference polypeptide. Insertions can comprise insertion of 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids or 5 or more amino acids while retaining function or activity and / or retaining the improved properties of the reference polypeptide. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the reference polypeptide.

[0249] “Addition” or “added” refers to a modification of the reference polypeptide sequence by addition of one or more amino acids to the carboxy or amino terminus of the reference polypeptide, or both. Additions can comprise addition of 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids or 5 or more amino acids while retaining function or activity and / or retaining the improved properties of the reference polypeptide.

[0250] It is understood that the indicated number of modifications generally define the total number of modifications to the reference polypeptide sequence. The modifications may solely be amino acid substitutions, be it conservative or non-conservative substitutions, or both, preferably however conservative substitution. They may solely be amino acid deletions. They may solely be amino acid insertions. The modifications may be a mix of these specific modifications, such as amino acid substitutions and amino acid deletions.

[0251] It is further understood that in case of one or more amino acid insertions, and hence a shift in the position of an amino acid originally present in the reference polypeptide, the original amino acid in its new position is not to be construed as being a “substituted” amino acid. Similarly, in case of one or more amino acid deletions within the reference polypeptide, and hence a shift in the position of an amino acid originally present in the reference polypeptide, the original amino acid in its new position is not to be construed as being a “substituted” amino acid.

[0252] Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and sub ranges within a numerical limit or range are specifically included as if explicitly written out.

[0253] Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Examples

[0254] Example 1

[0255] 1. Peptide design / modification

[0256] To enhance the antimicrobial efficacy of CLEC3A-derived AMPs we decided to use 3 different modification approaches in the first round of modification. These approaches resulted in a total of 6 different modified CLEC3A-derived peptides (Fig. 1).

[0257] First, we truncated the CLEC3A-derived peptides HT-16 and HT-47 to reduce them to their core sequence needed for their antimicrobial activity (Fig. 1). From Elezagic et. al. (2019) we learned that the positively charged N-terminus of CLEC3A encoded for by exon 1 , which is contained in HT-16 and HT-47, is essential for the antimicrobial activity of CLEC3A-derived peptides. Therefore, we discarded the parts of the amino acid sequence of the N-terminus that showed only a low positive charge density, leading to the fragment RK-9 (Fig. 1). In the case of HT-47 we also truncated the a-helical part encoded for by exon 2 (Fig. 1). To determine the actual sequence needed for the a-helix we used prediction tools from Network Protein Sequence Analysis tool from prabi21and verified the amphipathicity visually with Helical Wheel Projections from NetWheels (http: / / lbqp.unb.br / NetWheels). Combined this led to the modified peptide RK-31 (Fig. 1).

[0258] Second, HT-47 exhibits direct bacterial killing through binding to LPS, or LTA and permeabilizing the bacterial membrane, which leads to a leakage of cell content10. For its molecular mechanism of action, HT-47 binds the bacterial membrane through its positively charged N-terminus and then the amphipathic a-helix is inserted into the membrane10. This mechanism requires certain flexibility between the positively charged domain and the a-helix. Sequence shortening can cause steric tension and peptides can lose their flexibility and activity. Therefore, a small, flexible Glycine-linker between the positively charged region and the a-helix was introduced, resulting in the peptide RK-30 (Fig. 1).

[0259] Lastly, we added a tryptophan residue to the N-Terminus of RK-9, RK-31 , and RK-30 to enhance the antimicrobial effect of the CLEC3A-derived AMPs and raise their specificity against bacteria (Fig. 1). The antimicrobial effect is enhanced through the tryptophan residue since it raises the hydrophobicity of the peptide, facilitating more easy incorporation of the peptide into the bacterial membrane22. Since we added the tryptophan residue to the positively charged N-terminus we hypothesize that it will anchor the peptide, bound through electrostatic interaction to the bacterial membrane, further. Adding a tryptophan residue to the modified peptides resulted in the new peptides WRK-9, WRK-31 , and WRK-30 (Fig. 1).

[0260] Table 1 : Overview of native and modified CLEC3A-derived peptides. Table comparing the amino acid sequences of CLEC3A-derived peptides (ST-16, HT-16, DK-29, and HT-47) and modified CLEC3A-derived peptides (RK-9, WRK-9, RK-31 , WRK-31 , RK-30, and WRK-30). All sequences are shown from N- to C-Terminus. Size (Da) and netcharge were calculated using the peptide property calculator from Innovagen (http: / / www.pepcalc.com / ). The hydrophobicity was calculated using the peptide analyzing tool from Thermo Fischer Scientific and the hydrophobic moment was calculated using heliQuest (https: / / heliquest.ipmc.cnrs.fr / cgi- bin / ComputParams.py). ST-16 = SEQ ID NO: 29; HT-16 = SEQ ID NO: 2; DK-29 = SEQ ID NO: 30; HT-47 = SEQ ID NO: 31 ; RK-9 = SEQ ID NO: 1 ; WRK-9 = SEQ ID NO: 32; RK-31 = SEQ ID NO: 33; WRK-31 = SEQ ID NO: 34; RK-30 = SEQ ID NO: 6; WRK-30 = SEQ ID NO: 7.

[0261] 2. Peptide synthesis

[0262] Peptides were synthesized on Fmoc-Wang resin beads to receive peptides with a free C-Terminus. The first amino acid was already coupled to the resin with a loading of 0.4 - 0.8 mmol / g. Further, amino acids were coupled using an automated peptide synthesizer from MultiSynTech by double coupling steps with 8 eq. Fmoc-aa-OH, Oxyma pure®, and DIC. DG-motives were coupled manually as Fmoc-Asp(OtBu)-(Dmb)Gly-OH dipeptide using HATLI (2 eq.) and DI PEA (2 eq.) in DMF, afterwards, peptide synthesis was finalized using the automated peptide synthesizer. Peptides HT-16, RK-9, and WRK-9 were removed from the resin using TFA / TIS / H2O (95:2.5:2.5 v / v / v) for 3 h and precipitated in ice-cold diethyl ether. The other peptides were removed from the resin using TFA / Ethandithiol / Thioanisol (90:3:7) and afterward precipitated in ice-cold diethyl ether. Purification of the peptides was achieved by RP-HPLC with a C18 column using acetonitrile in water (0.1 % TFA), and fractions were analyzed by analytical LCMS. The final purity of all peptides was 99% (except HT-47 with 91 %). Peptides LL-37, ST-16 (Ctrl 1), HT-47, and dWRK-30 were custom ordered from Genosphere (Paris, France) with a purity of >95%. Peptide DK-29 (Ctrl 2) was custom ordered from Biomatik (Wilmington, USA) with a purity of >95%.

[0263] 3. Antimicrobial activity assay

[0264] 3.1 Bacterial strains

[0265] Pseudomonas aeruginosa (Psae-27853), Staphylococcus aureus (ATCC-29213), and Methicillin-resistant Staphylococcus Aureus (MRSA-43300) were used. Bacteria were cultivated in tryptic soy broth (TSB) (Merck, Darmstadt, Germany) at 37°C and 225rpm or on TSB-agarose plates at 37°C.

[0266] 3.2 Assay conditions

[0267] Bacteria were grown to an OD620 of 0.5 and harvested by centrifugation and washed with tris-glucose buffer (TG buffer: 10 mM tris, 5 mM glucose; pH=7.4) (P. aeruginosa and S. aureus) or phosphate-glucose (PG buffer: 10 mM K2HPO4, 5 mM glucose; pH=7.4) (MRSA). Bacteria were adjusted to 2x106CFU / ml and were incubated in 1 :2 dilutions for 2 h at 37°C with CLEC3A-derived and modified CLEC3A-derived peptides in TG buffer or PG buffer. The known AMP LL-37 was used as a positive control, while the CLEC3A-derived peptides ST-16 (Ctrl 1) and DK-29 (Ctrl 2) as well as untreated bacteria were used as negative controls. Afterward, the bacteria were plated out onto TSB-agarose plates in previously determined dilutions on TSB-agarose plates and cultured overnight at 37°C. The number of grown colonies was determined with the cell counter plugin of Imaged and is presented as a percentage of the number of grown colonies from the untreated control. The MIC50 was defined as the concentration of antimicrobial peptide where only 50% of bacteria survived. Peptides were used in the following concentrations (Tab. 2). Table 2: Peptide concentrations used in the antimicrobial activity assay.

[0268] 3.3 Results

[0269] The most important characteristic of AMPs is their antimicrobial activity. Therefore, we first assessed the antimicrobial activity of the modified CLEC3A-derived peptides in an antimicrobial activity assay and determined their MICso (Fig. 2).

[0270] First, we investigated the antimicrobial activity of the peptides modified from the native CLEC3A-derived peptides HT-16 and HT-47 against the gram-negative bacterial strain of P. aeruginosa. RK-9 and WRK-9 modified from HT-16 exhibited higher MICso than the native peptide (Fig. 2A). In the case of RK-9, the MICso was increased by 8-fold, whereas for WRK-9 it was only increased by 1.9-fold, meaning that the antimicrobial activity of these peptides was decreased compared to the native peptide HT-16. RK-31 and WRK-31 , in their used, maximal concentration of 5 pM, did not achieve a bacterial killing of 50% therefore no MICso could be determined (Fig. 2B). However, RK-30 showed only a slight increase by 1.3-fold in its MICso compared to the native HT-47 and WRK-30 showed an MICso in the same range as HT-47 (Fig. 2B). Second, we assessed the antimicrobial activity of the modified peptides against the gram-positive bacterial strain of S. aureus. Here, similar to the results with P. aeruginosa the MICso of RK-9 and WRK-9 were, compared to the native HT-16, increased by 8-fold and 2.4-fold, respectively (Fig. 2C). However, for RK-31 , and WRK-31 , an MICso could be determined. The MICso of RK-30 showed a stark increase of 16-fold, while WRK-31 exhibited an increase of the MICso by 3-fold, compared to the native HT-47 (Fig. 2D). RK-30 showed only an increase in MICso by 2-fold, compared to HT-47. In contrast, WRK-30 showed a decrease in MICso by 0.7-fold compared to the native HT-47 (Fig. 2D). Lastly, we determined the antimicrobial activity of the modified peptides against a drug-resistant strain of S. aureus, namely methicillin-resistant S. aureus (MRSA). Interestingly, MRSA compared to the not resistant strains, showed increased MICso for all peptides even the control LL-37 (Fig. 2). This increase is caused by the different buffer used for this strain, which was a necessity since MRSA curiously did not survive in the Tris buffer used for the other strains. Therefore, we increased the used concentrations of the peptides to a maximum of 10 pM. RK-9 was not able to kill at least 50% of the bacteria within the limit of the concentration maximum of 10 pM, while WRK-9 only showed an increase in the MICso, compared to the native HT-16, by 1.2-fold (Fig. 2E). Just as in the case of RK-9, RK-31 and WRK-31 did not achieve a bacterial killing of 50%, therefore no MICso could be determined (Fig. 2F). It is notable that not only WRK-30 showed a decrease in MICso by 3.7-fold, compared to the native HT-47, but also RK-30 showed a decrease in MICso by 2.4-fold (Fig. 2F). Thus, the MICso of RK-30 and WRK-30 against MRSA are closer to the MICso of the positive control LL-37, than to the MICso of HT-47 from which they were modified.

[0271] Our above-described findings show that the addition of a single tryptophan residue to the N-terminus enhances the antimicrobial activity of a peptide greatly and reduces the MICso by at least 30% (supplementary table 1). We successfully modified the peptide HT-47 to WRK-30, which exhibited only a comparable antimicrobial activity against gram-negative P. aeruginosa, but a strongly enhanced activity against gram-positive S. aureus and MRSA. Additionally, RK-30 also showed a strongly enhanced activity against gram-positive S. aureus and MRSA. As the modified CLEC3A-derived peptides RK-31 and WRK-31 only showed a slight antimicrobial activity with a high MIC50 against S. aureus and no activity against P. aeruginosa and MRSA, we did not include them in further experiments. 4. Cytotoxicity assay

[0272] 4.1 Assay conditions

[0273] NIH3T3 cells were seeded and precultured overnight. For 24 h incubation with peptides a density of 10x103cells / well and for 96 a density of 5x103cells / well. After preculturing the seeded cells, the culture medium was replaced with a peptide-containing medium. Cells were incubated with a peptide-containing medium for 24 h and 96 h. Afterward, the peptide containing medium was removed and cells were washed with PBS before adding MTS to the culture medium. Cells were incubated with MTS medium for 1 h at 37°C for cells incubated with peptides for 24 h and absorbance was measured at 490 nm. Cells incubated with peptides for 96 h were incubated with MTS medium for 2 h at 37°C before measuring the absorbance. Samples were measured in triplicates and the cell viability of three individual experiments is presented as a percentage of the measured OD from the untreated control. As further controls vancomycin (final concentration 7.35 pg / ml), colistin (final concentration 147 pg / ml), and 1% TritronX in culture medium were used. Peptides were used in the following concentrations. Table 3: Peptide concentrations used in the cytotoxicity assay.

[0274] The therapeutic index of the peptides was calculated using the following equation: TI=ccytotox / MIC50 4.2 Results

[0275] An AMP does not only need to exhibit a potent antimicrobial activity to find use in clinical applications, but it also has to show low cytotoxicity towards mammalian cells, thus granting a wide therapeutic window. Therefore, we next determined the cytotoxicity of the modified CLEC3A-derived peptides against murine fibroblasts of the NIH3T3 cell line (Fig. 3).

[0276] First, we tested the modified CLEC3A-derived AMPs in rather low concentrations corresponding to the MICso of HT-47 against S. aureus, as well as 10-fold and 100-fold of the MIC50 of HT-47 against S. aureus. Incubation of NIH3T3 cells with HT-16 (Ctrl 1), the modified CLEC3A-derived peptides or conventional antibiotics over 24 h did not impair the cell survival or growth (Fig. 3A). However, HT-47 and DK-29 (Ctrl 2), which contains the same amphipathic a-helix as HT-47, showed a concentration-dependent tendency for cytotoxicity (Fig. 3A). This indicates that the amphipathic a-helix of HT-47 interacts with the murine cells, probably their membranes, and impairs their normal metabolism. Additionally, we could verify the stark cytotoxic effect of LL-37, which obliterated the cell population when used with a concentration of 30 pM (Fig. 3A). This is comparable to the use of 1% TritronX in the culture medium. Incubating the murine fibroblasts for 96 h with the peptides showed similar results as the incubation over 24 h. Neither HT 16, Ctrl 1 , the modified CLEC3A-derived peptides, nor the conventional antibiotics showed any cytotoxic effects (Fig. 3B). On the contrary, cells incubated with these peptides grew and survived better (in most cases statistically significant) than the untreated control. The cytotoxic tendency of HT-47 deepened a bit but did still not reach statistical significance (Fig. 3B). While LL-37 left few surviving cells at 30 pM in the 24 h incubation, no surviving cells can be found after an incubation time of 96 h (Fig. 3B).

[0277] Finally, to investigate the therapeutic window of the most promising, novel, modified CLEC3A-derived AMP WRK-30 and the native CLEC3A-derived AMP HT-47 we decided to use even higher concentrations of 40 mM, 60 pM, and 80 pM over an incubation time of 24 h. Again HT-47 showed a strong, concentration-dependent tendency towards cytotoxicity, which still did not reach statistical significance (Fig. 3C). Since a concentration of 80 pM HT-47 for an incubation time of 24 h led to a decrease in cell survival of 50% (Fig. 3C) we deemed 60 pM as the maximal concentration of HT-47 suitable for in vivo experiments and thus as the upper limit of its therapeutic window. In contrast, a concentration of 80 pM of WRK-30 did not show cytotoxic effects (Fig. 3C). Therefore, we defined the maximal concentration of WRK-30, suitable for in vivo experiments and thus as the upper limit of its therapeutic window as 80 pM. The resulting therapeutic index, which is defined by the ratio of the maximal peptide concentration that does not show cytotoxicity and the MICso, is therefore overall smaller in the case of HT-47 compared to WRK-30 (Fig 3D).

[0278] We showed that the modifications to turn HT-47 into WRK-30 did not only enhance the antimicrobial activity of the modified WRK-30 but also decrease the cytotoxic potential, and raise the maximal concentration suitable for in vivo experiments of WRK-30. Thus, the therapeutic window of WRK-30 was greatly widened by the modifications.

[0279] 5. Statistical analysis

[0280] Statistical analysis of results was performed with Prism 9.3.1 (471) (GraphPad, San Diego CA, USA). Shown are calculated averages and standard deviations obtained from three individual experiments. Statistical significance was determined with a paired ANOVA followed by a Dunnett-test and multiple comparisons.

[0281] Example 2

[0282] Titanium is a frequently used biomaterial for arthroplasties and also generally to insert into the patient’s body in the form of screws and the like. However, bacteria are able to adhere to titanium and possibly form biofilms which are tough to treat. An antimicrobial coat on the titanium using TiBP- WRK-30 could help to reduce infections after surgeries. To test this, we performed surgeries on mice, inserted AMP coated titanium plates (Ti plates) subcutaneously (s.c.), and infected the surgery wound with S. aureus by administering bacteria into it before wound closure. As control uncoated Ti plates were used.

[0283] 1. Mice

[0284] Female C57BL / 6N mice were purchased from Charles River Laboratories Germany (Sulzfeld, Germany). Animals were housed in a specific pathogen-free S1 animal facility, kept under temperature and humidity-controlled conditions, and a 12 hours light and 12 hours dark schedule in IVC cages. Food and water were provided ad libidum and all animals were euthanized humanely using CO2. Experiments were performed in accordance with German law and the procedure was approved by the local government authority LANUV under permit no. SI- 02.04.2019. A458. Before including shipped mice in the experiments, they were given an equilibration time of 1 week to settle into their new environment and reduce stress. Mice were subjected to the surgery when they reached an age of 7 to 8 weeks. Animals were only taken out of their cages and manipulated under class II laminar flow biological safety cabinets. 2. Bacterial strains

[0285] Staphylococcus aureus (ATCC-29213) was used and cultivated in tryptic soy broth (TSB) (Merck, Darmstadt, Germany) liquid culture as well as on plates TSB-agarose plates.

[0286] Bacteria were grown at 37°C under constant agitation in TSB to an OD (620 nm) of 0.5. For further use, the bacterial solution was adjusted to 103CFU in 20pl of sterile 0.9% NaCI.

[0287] 3. Peptides

[0288] Chimeric peptide TiBP-WRK-30, in which a titanium binding protein is fused to the N-terminus of the AMP via a flexible glycine linker, were custom-ordered from Genosphere Biotechnologies (Paris, France) with a purity of >95% and a salt exchange from TFE to acetate.

[0289] Table 4: Amino acid sequence of the used peptide TiBP-WRK-30.

[0290] The titanium binding protein (TiBP) including a shirt GGG linker is presented in italic.

[0291] 4. Peptide-coating of titanium plates

[0292] Titanium plates (Ti-plates) with a thickness of 0.5 mm, a diameter of 4 mm, and a purity of +99.6% were purchased from Goodfellow / Merck (Darmstadt, Germany). Ti-plates were soaked in 70% ethanol for 72 hours and afterward sonicated for 15 min at 37 Hz in 1 :1 acetone:methanol, isopropyl alcohol, and deionized water. Afterward, Ti-plates were transferred into a sterile 96-well plate and sterilized under UV-light for 15 min on each side. To coat the washed and sterilized Ti-plates, they were incubated with 60 pM of the chimeric peptide in PBS at 37°C under constant agitation overnight. Control Ti-plates were incubated in PBS only. After incubation, the coated Ti-plates were transferred into sterile reaction tubes and washed with 1 ml of PBS twice. 5. Biomaterial-associated infection mouse model

[0293] Anesthesia of mice was achieved by Ketamine (100mg / kg) and Xylazine (10mg / kg) in 0,9% NaCI to a maximal concentration of 0.05ml / 10g. Repeated anesthesia was done with 1 / 2 of the initial dose. The depth of the anesthesia was checked by intertoe reflex. The animals were placed on a heating pad and bepanthen eye and nose ointment was administered to their eyes. The surgical site (neck) was shaved, and washed with 70% of ethanol, an incision was made and a subcutaneous (s.c.) pocked was opened. A chimeric peptide-coated Ti-plate was placed inside the s.c. pocket and 103CFU bacteria in 20pl of sterile 0.9% NaCI were pipetted into the pocket. The skin of the animals was closed with VetBond closure glue and animals received 0.1 mg / kg (max 0.1 ml / 10g) buprenorphine in 0.9% NaCI s.c. for pain management. Control animals were subjected to the same procedure, however receiving an uncoated Ti-plate. The mice woke under supervision and after the surgery, they were transferred into separate cages. 6 h and 21 h after the surgery the mice get another dose of buprenorphine intraperitoneal and the water is supplemented with 0.009mg / ml buprenorphine for access during the whole monitoring phase of 24 h. After 24 h the mice were euthanized humanely using CO2.

[0294] Blood was drawn via cardiac puncture into EDTA-filled vials, the Ti-plate was taken out and the tissue surrounding the Ti-plate (excluding the skin) was taken. The blood was centrifuged for 10 min at maximum speed and the obtained serum was frozen at -80°C for later use. Ti-plates were taken up in 100pl of 0,9% NaCI, sonicated for 15 min at 37 Hz and the resulting solution was streaked out directly as well as in serial dilutions onto pre-warmed TSB-agar plates. The dissected tissue was taken up in 0,9% NaCI in 5-times the volume of its weight and homogenized for 1 min at medium speed with an Omni TH Homogenizer. Again, the resulting solution was streaked out directly as well as in serial dilutions onto pre-warmed TSB-agar plates. Streaked-out agar plates were incubated for 20 h at 37°C before pictures were taken and colonies were counted.

[0295] 6. Statistical analysis

[0296] Prism 9.3.1 (471) (GraphPad, San Diego CA, USA) was used for statistical analysis of the results. Graphs and tables show calculated averages and standard deviations obtained from four individual experiments. Statistical significance was determined with a paired ANOVA test followed by multiple comparisons comparing TiBP-WRK-30 to the uncoated control (Ctrl) and lastly a Dunnett test. 7. Results

[0297] We conducted a study to determine the number of bacteria on implanted Tiplates. Tiplates coated with TiBP-WRK-30 showed a significantly lower number of viable colonies than the uncoated control plates (Fig. 4A and 4B). Notably, TiBP-WRK-30 was able to eliminate adherent bacteria (Fig. 4A). On average, a coat with TiBP-WRK-30 resulted in a 99.9% reduction in bacterial count (confidence interval ±0.19) (Fig. 4B). Therefore, TiBP-WRK-30 not only retains its antimicrobial activity when immobilized on a surface but also retains its efficacy in an in vivo setting, making it suitable for therapeutic applications.

[0298] Infections after implantations can happen as biofilms on the implanted biomaterial or spread into the surrounding tissue. Therefore, we also investigated the bacterial burden in the wound tissue surrounding the implanted Ti-plate. Similar to the bacterial count on the Ti-plate, we observed a reduction in viable bacteria in the wound tissue (Fig. 4C and 4D). Again, in some cases, TiBP-WRK-30 was able to reduce the number of bacteria to zero (Fig. 4C). On average TiBP-WRK-30 achieved a reduction of 99,9% (confidence interval ±0.16) (Fig. 4D). This shows that TiBP-WRK-30 not only reduces the bacterial count on the biomaterial it is bound to but also in the surrounding wound tissue.

[0299] Cited References

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[0301] 2 Powers, J. P. & Hancock, R. E. The relationship between peptide structure and antibacterial activity. Peptides 24, 1681-1691 (2003). https: / / doi.org: 10.1016 / j. peptides.2003.08.023

[0302] 3 Zasloff, M. Antimicrobial Peptides of Multicellular Organisms: My Perspective. Adv Exp Med Biol 1117, 3-6 (2019). https: / / doi.org: 10.1007 / 978-981 -13-3588-4_1

[0303] 4 Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 415, 389-395 (2002). https: / / doi.org: 10.1038 / 415389a

[0304] 5 Park, Y. & Hahm, K. S. Antimicrobial peptides (AMPs): peptide structure and mode of action. J Biochem Mol Biol 38, 507-516 (2005). https: / / doi.org: 10.5483 / bmbrep.2005.38.5.507

[0305] 6 Chan, D. I., Prenner, E. J. & Vogel, H. J. Tryptophan- and arginine-rich antimicrobial peptides: structures and mechanisms of action. Biochim Biophys Acta 1758, 1184-1202 (2006). https: / / doi. org: 10.1016 / j.bbamem.2006.04.006

[0306] 7 Tsunezumi, J., Higashi, S. & Miyazaki, K. Matrilysin (MMP-7) cleaves C-type lectin domain family 3 member A (CLEC3A) on tumor cell surface and modulates its cell adhesion activity. J Cell Biochem 106, 693-702 (2009). https: / / doi.org: 10.1002 / jcb.22062

[0307] 8 Shinnar, A. E., Butler, K. L. & Park, H. J. Cathelicidin family of antimicrobial peptides: proteolytic processing and protease resistance. Bioorg Chem 31 , 425-436 (2003). https: / / doi.org: 10.1016 / s0045-2068(03)00080-4

[0308] 9 Neame, P. J., Tapp, H. & Grimm, D. R. The cartilage-derived, C-type lectin (CLECSF1): structure of the gene and chromosomal location. Biochim Biophys Acta 1446, 193-202 (1999). https : / / doi . org : 10.1016 / s0167-4781 (99)00087- 1

[0309] 10 Elezagic, D. et al. Antimicrobial peptides derived from the cartilage. -specific C-type Lectin Domain Family 3 Member A (CLEC3A) - potential in the prevention and treatment of septic arthritis. Osteoarthritis Cartilage 27, 1564-1573 (2019). https: / / doi.org: 10.1016 / j.joca.2019.06.007

[0310] 11 Sall, J. et al. The antimicrobial peptide LL-37 alters human osteoblast Ca2+ handling and induces Ca2+-independent apoptosis. J Innate Immun 5, 290-300 (2013). https: / / doi.org: 10.1159 / 000346587

[0311] 12 Oren, Z., Lerman, J. C., Gudmundsson, G. H., Agerberth, B. & Shai, Y. Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity. Biochem J 341 ( Pt 3), 501-513 (1999).

[0312] 13 Johansson, J., Gudmundsson, G. H., Rottenberg, M. E., Berndt, K. D. & Agerberth, B. Conformation-dependent antibacterial activity of the naturally occurring human peptide LL- 37. J Biol Chem 273, 3718-3724 (1998). https: / / doi.org: 10.1074 / jbc.273.6.3718

[0313] 14 Frasca, L. & Lande, R. Role of defensins and cathelicidin LL37 in auto-immune and auto- inflammatory diseases. Curr Pharm Biotechnol 13, 1882-1897 (2012). https: / / doi.org: 10.2174 / 138920112802273155

[0314] 15 Barlow, P. G. et al. The human cathelicidin LL-37 preferentially promotes apoptosis of infected airway epithelium. Am J Respir Cell Mol Biol 43, 692-702 (2010). https: / / doi.org: 10.1165 / rcmb.2009-02500C

[0315] 16 Turner, J., Cho, Y., Dinh, N. N., Waring, A. J. & Lehrer, R. I. Activities of LL-37, a cathelin- associated antimicrobial peptide of human neutrophils. Antimicrob Agents Chemother 42, 2206-2214 (1998). https: / / doi.org: 10.1128 / AAC.42.9.2206

[0316] 17 Bowdish, D. M., Davidson, D. J., Speert, D. P. & Hancock, R. E. The human cationic peptide LL-37 induces activation of the extracellular signal-regulated kinase and p38 kinase pathways in primary human monocytes. J Immunol 172, 3758-3765 (2004). https: / / doi.org: 10.4049 / jimmunol.172.6.3758

[0317] 18 Agerberth, B. et al. The human antimicrobial and chemotactic peptides LL-37 and alpha- defensins are expressed by specific lymphocyte and monocyte populations. Blood 96, 3086-3093 (2000). 19 Vlieghe, P., Lisowski, V., Martinez, J. & Khrestchatisky, M. Synthetic therapeutic peptides: science and market. Drug Discov Today 15, 40-56 (2010). https: / / doi.org: 10.1016 / j.drudis.2009.10.009

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Claims

Claims1. A polypeptide comprising, in order from N-terminus to C-terminus:(a) optionally at least one tryptophan (W) residue;(b) a peptide fragment A, which is positively charged and is composed of 4 to 18 amino acids;(c) a peptide linker L, which is composed of 2 to 5 amino acids selected from the group consisting of glycine (G), serine (S) and threonine (T);(d) a peptide fragment B, which exhibits an alpha-helical structure and is composed of 12 to 28 amino acids.

2. The polypeptide according to claim 1 , wherein at least 70% of the amino acids in peptide fragment A are positively charged amino acids selected from the group consisting of lysine (K) and arginine (R).

3. The polypeptide according to claim 1 or 2, wherein the peptide fragment A comprises i-1) the amino acid sequence RKHSKRRVR (SEQ ID NO: 1), or i-2) a variant amino acid sequence of i-1) having 1 , 2, 3 or 4 modifications relative to the amino acid sequence of i- 1), wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof.

4. The polypeptide according to any one of claims 1 to 3, wherein the peptide fragment A comprises i-1) the amino acid sequence RKHSKRRVR (SEQ ID NO: 1), or i-2) a variant amino acid sequence of i-1) in which 1 , 2, 3 or 4 amino acids are substituted relative to the amino acid sequence of i-1).

5. The polypeptide according to any one of claims 1 to 4, wherein the peptide fragment A consists of i-1) the amino acid sequence RKHSKRRVR (SEQ ID NO: 1), or i-2) a variant amino acid sequence of i-1) having 1 , 2, 3 or 4 modifications relative to the amino acid sequence of i-1), wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof.

6. The polypeptide according to any one of claims 1 to 5, wherein the peptide fragment A consists of i-1) the amino acid sequence RKHSKRRVR (SEQ ID NO: 1), or i-2) a variant amino acid sequence of i-1) in which 1 , 2, 3 or 4 amino acids are substituted relative to the amino acid sequence of i-1).

7. The polypeptide according to any one of claims 1 to 6, wherein peptide fragment B comprises iv-1) the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4), or iv- 2) a variant amino acid sequence of iv-1) having 1 , 2, 3, 4, 5, 6, 7 or 8 modifications relative to the amino acid sequence of iv-1) and which is capable of forming an alpha-helical structure, wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof.

8. The polypeptide according to any one of claims 1 to 7, wherein peptide fragment B comprises iv-1) the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4), or iv- 2) a variant amino acid sequence in which 1 , 2, 3, 4, 5, 6, 7 or 8 amino acids are substituted relative to the amino acid sequence of iv-1) and which is capable of forming an alpha-helical structure.

9. The polypeptide according to any one of claims 1 to 8, wherein peptide fragment B consists of iv-1) the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4), or iv-2) a variant amino acid sequence of iii-1) having 1 , 2, 3, 4, 5, 6, 7 or 8 modifications relative to the amino acid sequence of iii-1) and which is capable of forming an alpha-helical structure, wherein the modifications are selected from substitutions, deletions, insertions, additions and combinations thereof.

10. The polypeptide according to any one of claims 1 to 9, wherein peptide fragment B consists of iv-1) the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4), or iv-2) a variant amino acid sequence of iv-1) in which 1 , 2, 3, 4, 5, 6, 7 or 8 amino acids are substituted relative to the amino acid sequence of iv-1) and which is capable of forming an alpha-helical structure.11 . The polypeptide according to any one of claims 1 to 10, wherein fragment A consists of the amino acid sequence RKHSKRRVR (SEQ ID NO: 1) and peptide fragment B consists of the amino acid sequence LKTQIEKLWTEVNALKEI (SEQ ID NO: 4).

12. The polypeptide according to any one of claims 1 to 11 , wherein the peptide linker L is selected from the group consisting of GG, GX, XG, GGG, XGG, GXG, GGX, GGGG (SEQID NO: 8), XGGG, GXGG, GGXG, GGGX, GGGGG (SEQ ID NO: 9), XGGGG (SEQ ID NO: 10), GXGGG (SEQ ID NO: 11), GGXGG (SEQ ID NO: 12), GGGXG (SEQ ID NO: 13) and GGGGX (SEQ ID NO: 14), wherein X is S or T, preferably S.

13. The polypeptide according to any one of claims 1 to 12, wherein the peptide linker L is GGG.

14. The polypeptide according to any one of claims 1 to 13, wherein (a) is present.

15. The polypeptide according to any one of claims 1 to 14, wherein (a) is 1 tryptophan (W) residue.

16. The polypeptide according to claim 1 , which comprises or consists of the amino acid sequence RKHSKRRVRGGGLKTQIEKLWTEVNALKEI (SEQ ID NO: 6).

17. The polypeptide according to claim 1 , which comprises or consists of the amino acid sequence WRKHSKRRVRGGGLKTQIEKLWTEVNALKEI (SEQ ID NO: 7).

18. The polypeptide according to any one of claims 1 to 17 for use as an antimicrobial agent.

19. The polypeptide according to any one of claims 1 to 17 for use in the treatment of a microbial infection in a subject.

20. Pharmaceutical composition comprising at least one polypeptide according to any one of claims 1 to 17 and at least one pharmaceutically acceptable carrier and / or excipient.21 . Use of the polypeptide according to any one of claims 1 to 17 for coating at least part of the surface of a solid material or for incorporating into at least a part of a solid material.

22. A solid material, which is characterized in that at least part, preferably all, of its surface is covered by a coating comprising the polypeptide according to any one of claims 1 and 17 and optionally at least one further antimicrobial agent.

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