Antibacterial peptides

Abstract

The present invention relates to polypeptides comprising a sequence at least 85% identical to a sequence of SEQ ID NO: 2 or to a sequence of SEQ ID NO: 3. The invention further relates to fusion proteins containing the polypeptides, and to uses thereof in medicine and in the treatment and / or prevention of bacterial infections.

Description

[0001] Antibacterial peptides

[0002] This application claims the priority of the European Patent Application 24383460.3 filed on December 24th, 2024.

[0003] Technical Field

[0004] The present invention relates to the field of clinical and veterinary microbiology. In particular, it relates to a new polypeptide and its use as antibacterial agent. The polypeptide of the invention is particularly useful for eliminating antibiotic resistant Gram-negative bacteria and minimizing the emergence of new resistances. Background Art

[0005] Antimicrobial Resistance (AMR) represents a critical challenge in contemporary medicine, characterized by the ability of bacteria to withstand previously effective antimicrobial treatments. This phenomenon, while naturally occurring as a bacterial survival mechanism, has seen a marked escalation in recent decades, with the proliferation of multidrug-resistant (MDR) and pan drug-resistant (PDR) strains, colloquially termed "superbugs”. These developments have rendered conventional antimicrobial therapies increasingly ineffective, posing a significant threat to global health.

[0006] In response, innovative non-antibiotic treatment options are under exploration, including phage therapies, and phage-derived enzyme treatments (known as enzybiotics), which present expansive research opportunities. Promising enzybiotics under development are based in, for example, lysins, which are phage enzymes, targeting specific bacterial species. The main drawback of lysins is that the outer membrane of Gram-negative bacteria hinders their action when added from the outside since they cannot reach the peptidoglycan layer. Protein engineering has attempted to improve the anti-bacterial efficiency of lysins by integrating an outer membrane-permeating polypeptide into lysins or by co-administering them with an outer membranepermeating polypeptide. However, lysin-based enzybiotics often present from low stability levels and their anti-bacterial activity is limited.

[0007] There is, therefore, the need of developing new enzybiotics with enhanced efficacy and stability against Gram-negative bacteria.

[0008] Summary of Invention

[0009] The present invention provides polypeptides with LysM domain structure that have a surprisingly high cell wall-permeating activity and a surprisingly high stability. The polypeptides of the invention allow engineering enzybiotics with a remarkable efficacity, particularly against Gram-negative bacteria.

[0010] As shown in the examples below, the present inventors used the polypeptides of the invention for generating various proteins with augmented stability, and augmented activity against Gram-negative pathogens,characterized by low minimum inhibitory concentration (MIC) values against most tested bacteria, a rapid bactericidal action within one hour of treatment, and antibiofilm capabilities, both in preventing and eradicating biofilms. Furthermore, these proteins achieved 100% survival in pulmonary in vivo models when used in conjunction with standard antibiotic therapies, with no observed toxicity at the tested protein concentrations. The invention provides six polypeptides, called cell-wall binding (CWB) polypeptides (CWB30-CWB35) comprising at least one Lysine motif (LysM) domain structure. These polypeptides containing LysM domains showed a surprisingly enhanced cell-permeating activity and stability, as shown in examples below. Stability was in fact one of the main drawbacks of outer membrane-disrupting polypeptides.

[0011] Thus, in a first aspect, the invention provides a polypeptide comprising a sequence at least 70 % identical to a sequence of SEQ ID NO: 2 or to a sequence of SEQ ID NO: 3, optionally at least 70 % identical to a sequence of SEQ ID NO: 2 or to a sequence of SEQ ID NO: 3.

[0012] In a second aspect, the invention provides a fusion protein comprising the polypeptide as defined in the first aspect.

[0013] In a third aspect, the invention provides a polynucleotide that encodes the polypeptide as defined in the first aspect or the fusion protein as defined in the second aspect.

[0014] In a fourth aspect, the invention provides a gene construct comprising a polynucleotide as defined in the third aspect operatively linked to an expression promoter.

[0015] In a fifth aspect, the invention provides an expression vector comprising the polynucleotide of the third aspect or the gene construct as defined in the fourth aspect.

[0016] In a sixth aspect, the invention provides a host cell comprising the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth aspect, or the expression vector as defined in the fifth aspect.

[0017] In a seventh aspect, the invention provides a method for producing the polypeptide as defined in the first aspect, or the fusion protein as defined in the second aspect; wherein the method comprises the steps of: (I) culturing a host cell as defined in the sixth aspect under conditions suitable for the production of the polypeptide or the fusion protein; or, alternatively, (I') in vitro transcription and / or translation of the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth, and / or the vector as defined in the fifth aspect; (ii) recovering the polypeptide or the fusion protein produced in step (I) or (I'); and (ill) optionally, isolating and / or purifying the polypeptide or the fusion protein.

[0018] In an eighth aspect, the invention provides a polypeptide or a fusion protein obtainable by the method defined in the seventh aspect.

[0019] In a nineth aspect, the invention provides a composition comprising the polypeptide as defined in the first aspect, the fusion protein as defined in the second aspect, the polynucleotide as defined in the third aspect,the gene construct as defined in the fourth aspect, the vector as defined in the fifth aspect, the host cell as defined in the sixth aspect, and / or the polypeptide or fusion protein as defined in the eighth aspect.

[0020] In an tenth aspect, the invention provides the polypeptide as defined in the first aspect, the fusion protein as defined in the second aspect, the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth aspect, the vector as defined in the fifth aspect, the host cell as defined in the sixth aspect, the polypeptide or fusion protein as defined in the eighth aspect, and / or the composition as defined in the nineth aspect; for use in medicine.

[0021] In a eleventh aspect, the invention provides the polypeptide as defined in the first aspect, the fusion protein as defined in the second aspect, the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth aspect, the vector as defined in the fifth aspect, the host cell as defined in the sixth aspect, the polypeptide or fusion protein as defined in the eighth aspect, and / or the composition as defined in the nineth aspect; for use in the treatment and / or prevention of a bacterial infection.

[0022] This aspect may also be formulated as the use of the polypeptide as defined in the first aspect, the fusion protein as defined in the second aspect, the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth aspect, the vector as defined in the fifth aspect, the host cell as defined in the sixth aspect, the polypeptide or fusion protein as defined in the eighth aspect, or the composition as defined in the nineth aspect; for the manufacture of a medicament for the treatment and / or prevention of a bacterial infection.

[0023] The present invention also relates to a method for the treatment and / or prevention of a bacterial infection, the method comprising administering a pharmaceutically effective amount of the polypeptide as defined in the first aspect, the fusion protein as defined in the second aspect, the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth aspect, the vector as defined in the fifth aspect, the host cell as defined in the sixth aspect, the polypeptide or fusion protein as defined in the eighth aspect, and / or the composition as defined in the nineth aspect, together with at least one pharmaceutically acceptable excipient, diluent, or carrier; in a subject in need thereof, including a human.

[0024] In a twelfth aspect, the invention provides a kit of parts comprising the polypeptide as defined in the first aspect, the fusion protein as defined in the second aspect, the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth aspect, the vector as defined in the fifth aspect, the host cell as defined in the sixth aspect, the polypeptide or fusion protein as defined in the eighth aspect, and / or the composition as defined in the nineth aspect; and, optionally, instructions for its use.

[0025] In a thirteen aspect, the invention provides an in vitro method of inhibiting the growth, or reducing the population, or killing of Gram-negative bacteria; wherein the method comprises the step of contacting the bacteria with the polypeptide as defined in the first aspect, the fusion protein as defined in the second aspect, the host cell as defined in the sixth aspect, the polypeptide or fusion protein as defined in the eighth aspect, and / or the composition as defined in the nineth aspect.In a further aspect, the invention provides the use of a polypeptide as defined in the first aspect and / or the fusion protein as defined in the second aspect, in a method of inhibiting the growth, or reducing the population, or killing of Gram-negative bacteria.

[0026] In a fourteenth aspect, the invention relates to the use of a polypeptide as defined in the first aspect and / or the fusion protein as defined in the second aspect, as disinfectant for materials and / or surfaces.

[0027] In a further aspect, the invention provides an in vitro method for disinfecting materials and / or surfaces.

[0028] In a fifteenth aspect, the invention provides an antibody that specifically binds to a polypeptide comprising or consisting of a sequence selected from the group consisting of a sequence of SEQ ID NO: 2, a sequence of SEQ ID NO: 3, SEQ ID NO: 6 and a sequence of SEQ ID NO: 16; and / or a fusion protein comprising or consisting of a sequence selected from the group consisting of a sequence of SEQ ID NO: 32, a sequence of SEQ ID NO: 33, a sequence of SEQ ID NO: 36, a sequence of SEQ ID NO: 39, and a sequence of SEQ ID NO: 45.

[0029] Brief Description of Drawings

[0030] Fig. 1 shows Exponential growing cultures of E. colih CC 25922 exposed to 10 piM of the indicated proteins in human serum for 24 h. Graph represent the mean ± standard deviation of 3 biological replicates. “T” stands for "Time”.

[0031] Fig. 2. (A) shows Time-kill assay conducted on clinical strains exposed to equimolar concentrations (1 piM) of EPLETT200 (SEQ ID NO: 39) for durations of 1 and 3 hours. The outcomes, expressed as the mean ± standard deviation from three replicates, indicate bacterial reduction in Iog10 units compared to the untreated control. Statistically significant differences from the control (p<0.05) were identified using the Student's t-test, as denoted by asterisks on the bars. Black columns for each tested strain refer to "Log reduction 1 h”. White columns for each tested strain, refer to "Log reduction 3h”. "Log R” stands for "Log Reduction”. (B) shows Time-kill assay conducted on clinical strains exposed to equimolar concentrations (1 piM) of EPLETT207 (SEQ ID NO: 45) for durations of 1 and 3 hours. The outcomes, expressed as the mean ± standard deviation from three replicates, indicate bacterial reduction in Iog10 units compared to the untreated control. Statistically significant differences from the control (p<0.05) were identified using the Student's t-test, as denoted by asterisks on the bars. Black columns for each tested strain refer to "Log reduction 1 h”. White columns for each tested strain, refer to "Log reduction 3h”. "Log R” stands for "Log Reduction”.

[0032] Fig. 3 shows Differential Scanning Fluorimetry (DSF) of EPLETT200 (SEQ ID NO: 39) with varying NaCI concentrations. The data illustrates the mean ± standard deviation from nine replicates. An ANOVA test indicated no statistically significant differences in the melting temperature (Tm) when salt concentrations ranged from 100 to 500 mM (p > 0.05). "MT” in the figure stands for "melting temperature”.

[0033] Fig 4. Shows confocal microscopy images of A. baumannii treated with EPLETT200 (SEQ ID NO: 39).

[0034] Representative time-lapse images (at 0 minutes, 3 minutes, 6 minutes, and 9 minutes) showing morphologicalchanges in A. baumannii cells after treatment with EPLETT200 (SEQ ID NO: 39). Pore formation (indicated by white arrows) is observed as early as 3 minutes post-treatment, followed by progressive cellular disruption up to 9 minutes. Scale bar: 5 m. "min” stands for minutes.

[0035] Detailed description of the invention

[0036] All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.

[0037] As used herein, the indefinite articles "a” and "an” are synonymous with "at least one” or "one or more”.

[0038] Unless indicated otherwise, definite articles used herein, such as "the” also include the plural of the noun. As explained above, in a first aspect the invention provides a polypeptide comprising a sequence at least 70% identical to a sequence of SEQ ID NO: 2 or to a sequence of SEQ ID NO: 3.

[0039] A "polypeptide” is a chain of amino acids linked by a peptide bond (i.e., amide bonds) including more than 10 amino acids. The terms polypeptide and protein are used here interchangeably. As the skilled person will know, polypeptides can be produced by synthetic routes that belong to the common general knowledge, for example by solid-phase peptide synthesis on a rink amide resin using Fmoc-o-amine-protected amino acid. They can also be produced biologically, for example, using recombinant bacteria.

[0040] In the present invention the term "identical" or "identity" refers to the percentage of positions that are identical in the two sequences when the sequences are optimally aligned. If, in the optimal alignment, a position in a first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, the sequences exhibit identity with respect to that position. The percentage of identity determines the number of identical nucleotides or amino acids over a defined length in a given alignment. Thus, the level of identity between two sequences or ("percent sequence identity") is measured as a ratio of the number of identical positions shared by the sequences with respect to the number of positions compared (i.e., percent sequence identity = (number of identical positions / total number of positions compared) x 100). A gap, i.e., a position in an alignment where a nucleotide or amino acid is present in one sequence but not in the other, is regarded as a position with non-identical nucleotide or amino acid and is counted as a compared position. A number of mathematical algorithms for rapidly obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. For purposes of the present invention, the sequence identity between two nucleic acid or amino acid sequences is preferably determined using algorithms based on global alignment, such as the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), preferably implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277); or the BLAST Global Alignment tool (Altschul et al., "Basic local alignment search tool”, 1990, J. Mol. Biol, v. 215, pages 403-410), using default settings. Local alignment alsocan be used when the sequences being compared are substantially the same length.

[0041] The terms "Lysin motif domain”, "LysM domain”, "LysinM domain” or "Lysin domain” are used interchangeably in the present disclosure, and refer to a small globular domain which is found in a wide range of microbial proteins. They interact with substrates containing N-acetylglucosamine such as the peptidoglycan. The domain reference in InterPro (http: / / www.ebi.ac.uk / interpro / ) database is IPR018392. Structurally, it is composed of two alpha helices interconnected by two intersecting beta sheets. The sequences SEQ ID NO: 2 and the sequence of SEQ ID NO: 3 are each Lysine Motif domains.

[0042] Thus, the invention provides polypeptides with at least one LysM domain.

[0043] Therefore, in particular embodiments, the polypeptide comprises peptidoglycan binding activity. In another particular embodiment, the polypeptide comprises cell-wall binding activity.

[0044] The term "peptidoglycan binding activity” refers to the ability of a polypeptide to specifically recognize and bind to peptidoglycan, which is a major structural component of bacterial cell walls. Such binding can facilitate enzymatic degradation of the cell-wall.

[0045] The skilled person will know how to determine presence of peptidoglycan binding activity, following routine methods known in the art, for example by an affinity-based binding assay or a pull-down assay, without the need of inventive skill.

[0046] As indicated above, the polypeptides of the first aspect surprisingly presented cell-permeating activity. In particular they confer cell-permeating activity to the fusion proteins in which they are present.

[0047] Therefore, in particular embodiments, the polypeptide comprises cell-permeating activity. In particular, outermembrane permeating activity. In particular embodiments, Gram-negative permeating activity. In particular, Gram-negative outer membrane permeating activity.

[0048] In particular embodiments, the Gram-negative bacteria are from a genus selected from the group consisting of Acinetobacter, Pseudomonas, Escherichia, Klebsiella, and combinations thereof. In a more particular embodiment, the Gram-negative bacteria are from a species selected from the group consisting of A. baumannii, E. coli, K. pneumoniae, P. aeruginosa, and combinations thereof.

[0049] The term "bacteria” in the present disclosure refers to one or more bacterium.

[0050] The term "cell-permeating activity” or "cell-wall permeating activity” in the present disclosure refers to the ability of a polypeptide to disrupt or destabilize the cell-wall and thus allowing the polypeptide to cross and / or reach a target within or beyond the periplasmatic space, for example the peptidoglycan layer. The term "outermembrane permeating activity” refers to the particular ability of a polypeptide to disrupt or destabilize the outer-membrane of the cell-wall of Gram-negative bacteria. When the polypeptide of the invention forms part of a fusion protein, the fusion protein also comprises cell-permeating activity or outer-membrane permeating activity, by which the fusion protein can access the periplasmic space of the Gram-negative bacteria.A person skilled in the art will know how to determine the presence of cell-permeating activity in a polypeptide. For instance, the ability of a polypeptide to permeabilize the outer membrane of a Gram-negative bacteria can be assessed by examining the release of the periplasmic b-lactamase of Pseudomonas aeruginosa PA01 after the treatment of said bacterium with the polypeptide to be studied, as reproduced herein below for illustrative purposes.

[0051] An overnight culture of P. aeruginosa PA01 (500 pi) is diluted with 30 ml of pre-warmed (37°C) LB medium and incubated with shaking (150 rpm) for 2 h at 37°C. Following the addition of 0.25 mg 11 imipenem to induce b-lactamase production, the cultures are incubated with shaking for an additional 3 h and harvested by centrifugation at 5,000 g for 10 min at room temperature. Cell pellets are washed once with PBS and resuspended PBS to a final volume of 10 ml. The polypeptide is added to the cell suspensions (25 pi) at final concentrations of 40 pg ml-1 and 200 pg ml-1 (total volume, 50 ml). The mixture is incubated at 37°C for 10 min and centrifuged in an Eppendorf tube at 15,000 g for 30 min at room temperature. The control experiment condition can be performed using the supernatant of sonicated P. aeruginosa PA01 cells. The supernatants are collected and b-lactamase activity is assayed using 100 mM chromogenic cephalothin (CENTA) as substrate. The hydrolysis of CENTA can be monitored by continuously recording the absorbance at 405 nm. Release of b-lactamase (percent) is defined as the ratio of the b-lactamase activity under the control condition to that under the test condition. If the tested polypeptide shows b-lactamase releasing activity similar to or higher than the control condition, it means that it comprises cell-permeating activity.

[0052] As shown in the examples below, the LysM domains of the invention surprisingly exhibited greater stability in serum, as predicted through in silico models and confirmed in vitro. More in particular, the LysM domain confers greater stability to the polypeptides and / or fusion proteins containing them.

[0053] Therefore, in particular embodiments, the polypeptide comprises high stability. More in particular, the polypeptide has a half-life of at least 1.5, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 hours in human serum; particularly at 37°C. In particular, the polypeptide has a half-life of at least around 30 hours in human serum; particularly at 37°C.

[0054] The skilled person will know how to determine the shelf life and stability, in particular the half-life in a particular medium and condition, of the proteins, following routine methods known in the art, for example in silico analysis for example using ProtParam, without the need of inventive skill.

[0055] In particular embodiments, the polypeptide comprises a sequence at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 2 or to a sequence of SEQ ID NO: 3; particularly at least 95%; more particularly at least 99%; even more particularly 100%, identical to a sequence of SEQ ID NO: 2 or to a sequence of SEQ IDNO: 3.

[0056] Polypeptides may comprise repetitions of LysM domains. In fact, as shown in the examples below, adding additional LysM domains enhances both stability and activity of polypeptides and fusion proteins.

[0057] Therefore, in particular embodiments, the polypeptide comprises at least two sequences independently selected from the group consisting of a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, identical to a sequence of SEQ ID NO: 2; and a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 3. In particular, the polypeptide comprises at least two sequences independently selected from the group consisting of a sequence at least 90% identical to a sequence of SEQ ID NO: 2; and a sequence at least 90% identical to a sequence of SEQ ID NO: 3. In particular, the polypeptide comprises at least two sequences independently selected from the group consisting of a sequence at least 95% identical to a sequence of SEQ ID NO: 2; and a sequence at least 95% identical to a sequence of SEQ ID NO: 3. In particular, the polypeptide comprises at least two sequences independently selected from the group consisting of a sequence at least 96% identical to a sequence of SEQ ID NO: 2; and a sequence at least 96% identical to a sequence of SEQ ID NO: 3. In particular, the polypeptide comprises at least two sequences independently selected from the group consisting of a sequence at least 97% identical to a sequence of SEQ ID NO: 2; and a sequence at least 97% identical to a sequence of SEQ ID NO: 3. In particular, the polypeptide comprises at least two sequences independently selected from the group consisting of a sequence at least 98% identical to a sequence of SEQ ID NO: 2; and a sequence at least 98% identical to a sequence of SEQ ID NO: 3. In particular, the polypeptide comprises at least two sequences independently selected from the group consisting of a sequence at least 99% identical to a sequence of SEQ ID NO: 2; and a sequence at least 99% identical to a sequence of SEQ ID NO: 3. In particular, the polypeptide comprises at least two sequences independently selected from the group consisting of a sequence 100% identical to a sequence of SEQ ID NO: 2; and a sequence 100% identical to a sequence of SEQ ID NO: 3. This embodiment is meant to encompass polypeptides where the at least two sequences are the same or different.

[0058] In one embodiment, the polypeptide of the invention further comprises a spacer; particularly between LysM domains.

[0059] The terms "linker” and "spacer” are used herein interchangeably, and refer to an amino acid sequence acting as a hinge region between polypeptide domains or between fusion protein polypeptides, allowing them to move independently from one another while maintaining the three-dimensional form of the individual domainsor polypeptides. In this sense, linkers can exhibit structural properties that influence inter-domain mobility. Non-limiting examples of linkers, each serving to optimize the structural and functional integration of polypeptide domains or fusion protein polypeptides, are:

[0060] "Flexible linkers”: characterized by structural ductility or flexibility, enabling free movement between domains; typically, a flexible linking peptide comprises 2 or more amino acids selected from glycine, serine, alanine, and threonine. In particular, at least 65%, more specifically 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acids in the flexible linker are selected from this group. Such linkers may also contain repeats of residues, particularly Gly and Ser. The length of the flexible linker can vary, particularly from 2 to 30 amino acids, more specifically 5 to 25, and even more specifically 10 to 20 amino acids.

[0061] "Rigid linkers”: Designed to maintain a fixed spatial orientation between domains, minimizing their relative movement. These linkers often include proline-rich sequences or amino acids prone to forming stable, extended structures. "Helix-forming linkers”: Linkers that adopt a helical conformation, which can stabilize the relative positioning of domains while allowing controlled rotation or alignment. Such linkers may include amino acid sequences with a propensity for o-helix formation, like alanine or leucine-rich motifs.

[0062] Thus, in one embodiment, the polypeptide comprises or consists of the formula (I): (S1)n-LysM1-(S2)m-LysM2-(S3)p; wherein n, m and p are integers independently selected from 0 or 1; LysM1 and LysM2 are independently selected from a sequence at least 70% identical to a sequence of SEQ ID NO: 2 and to a sequence of SEQ ID NO: 3; S1 is a sequence at least 70% identical to a sequence of SEQ ID NO: 4; S3 is a sequence at least 70% identical to a sequence of SEQ ID NO: 4; and S2 is a sequence at least 70% identical to a sequence of SEQ ID NO: 5. In particular embodiments, m is 1, n is selected from 0 or 1, and p is 0. In more particular embodiments, m is 1, n is 1, and p is 0. In particular embodiments, m is 1, p is selected from 0 or 1, and n is 0. In more particular embodiments, m is 1, p is 1, and n is 0.

[0063] In particular embodiments, the polypeptide comprises or consists of formula (II): (L1)z-(S1)n-LysM1-(S2)m-LysM2; wherein z, n, and m, are integers independently selected from 0 or 1; LysM1 and LysM2 are independently selected from a sequence at least 70% identical to a sequence of SEQ ID NO: 2 and to a sequence of SEQ ID NO: 3; S1 is a sequence at least 70% identical to a sequence of SEQ ID NO: 4; S2 is a sequence at least 70% identical to a sequence of SEQ ID NO: 5; and L1 is a linker. In particular embodiments, m is 1, n is 0 and z is 0. In particular embodiments, m is 0, n is 1, and z is 0. In particular embodiments, m is 1 , n is 1 , and z is 1.

[0064] In formula (II), the (L1)z module is at the N-terminal end and the LysM2 module at the C-terminal end of the polypeptide. However, the polypeptide can also be in the opposite order. I.e. (L1)z can be at the C-terminal end of the polypeptide and the LysM2 at the N-terminal end of the polypeptide. Therefore, in particular embodiments, the polypeptide comprises or consists of formula (III): LysM1-(S2)m-LysM2-(S1)n-(L1)z; wherein z, n and m, are integers independently selected from 0 or 1; L1 is a linker; and LysM1 and LysM2 are independently selected from a sequence at least 70% identical to a sequence of SEQ ID NO: 2 and to a sequence of SEQ ID NO: 3; S1 is a sequence at least 70% identical to a sequence of SEQ ID NO: 4; S2 is asequence at least 70% identical to a sequence of SEQ ID NO: 5.

[0065] In particular embodiments, the polypeptide comprises a sequence at least 70% identical to a sequence of SEQ ID NO: 6. In particular embodiments, the polypeptide comprises a sequence at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 6; particularly at least 95%; more particularly at least 99%; even more particularly 100%, identical to a sequence of SEQ ID NO: 6.

[0066] In particular embodiments, the polypeptide comprises or consists of formula (IV): (L1)z-2xLysM; wherein z is an integer selected from 0 or 1; L1 is a linker; and 2xLysM comprises or consists of a sequence at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 6; particularly at least 95%; more particularly at least 99%; even more particularly 100%, identical to a sequence of SEQ ID NO: 6.

[0067] In particular embodiments, in any one formulas (l)-(lll), S1 consists of a sequence at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 4; particularly at least 95%; more particularly at least 99%; even more particularly 100%, identical to a sequence of SEQ ID NO: 4.

[0068] In particular embodiments, in formula (I), S3 consists of a sequence at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 4; particularly at least 95%; more particularly at least 99%; even more particularly 100%, identical to a sequence of SEQ ID NO: 4.

[0069] In particular embodiments, in any one formulas (l)-(lll), S2 consists of a sequence at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 5; particularly at least 95%; more particularly at least 99%; even more particularly 100%, identical to a sequence of SEQ ID NO: 5.

[0070] In particular embodiments, in any one formulas (l)-(lll), LysM1 consists of a sequence at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 2; particularly at least 95%; more particularly at least 99%; even more particularly 100%, identical to a sequence of SEQ ID NO: 2; and LysM2 consists of a sequence at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%,or 100% identical to a sequence of SEQ ID NO: 3; particularly at least 95%; more particularly at least 99%; even more particularly 100%, identical to a sequence of SEQ ID NO: 3.

[0071] In particular embodiments, in any one formulas (l)-(lll), LysM1 consists of a sequence at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 3; particularly at least 95%; more particularly at least 99%; even more particularly 100%, identical to a sequence of SEQ ID NO: 3; and LysM2 consists of a sequence at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 2; particularly at least 95%; more particularly at least 99%; even more particularly 100%, identical to a sequence of SEQ ID NO: 2.

[0072] In particular embodiments, in any one formulas (I l)-(IV), L1 is a linker selected from the group consisting of a rigid linker, a flexible linker, an helix-forming linker, and a linker of sequence at least 50% identical to sequence selected from the group consisting of a sequence of SEQ ID NO: 10, a sequence of SEQ ID NO: 11, a sequence of SEQ ID NO: 12, a sequence of SEQ ID NO: 13, a sequence of SEQ ID NO: 14, and a sequence of SEQ ID NO: 15.

[0073] In more particular embodiments, in any one formulas (ll)-(IV), L1 is a rigid linker comprising or consisting of a sequence at least 50% identical to a sequence of SEQ ID NO: 7. In particular, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 7; particularly at least 80%; more particularly at least 90%; even more particularly 100%, identical to a sequence of SEQ ID NO: 7. In more particular embodiments, in any one formulas (ll)-(IV), L1 is a flexible linker comprising or consisting of a sequence at least 50% identical to a sequence of SEQ ID NO: 8. In particular, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 8; particularly at least 80%; more particularly at least 90%; even more particularly 100%, identical to a sequence of SEQ ID NO: 8. In more particular embodiments, in any one formulas (ll)-(IV), L1 is a helix-forming linker comprising or consisting of a sequence at least 50% identical to a sequence of SEQ ID NO: 9. In particular, the helix-forming linker comprises or consists of a sequence at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 9; particularly at least 80%; more particularly at least 90%; even more particularly 100%, identical to a sequence of SEQ ID NO: 9.In more particular embodiments, in any one formulas (I l)-(IV), L1 is a linker comprising or consisting of a sequence at least 55%, at least 60%, at least 65%, or at least 70% identical to a sequence selected from the group consisting of a sequence of SEQ ID NO: 10, a sequence of SEQ ID NO: 11, a sequence of SEQ ID NO: 12, of sequence of SEQ ID NO: 13, a sequence of SEQ ID NO: 14, and a sequence of SEQ ID NO: 15. In particular, L1 is a linker comprising or consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of a sequence of SEQ ID NO: 10, a sequence of SEQ ID NO: 11, a sequence of SEQ ID NO: 12, of sequence of SEQ ID NO: 13, a sequence of SEQ ID NO: 14, and a sequence of SEQ ID NO: 15.

[0074] In particular embodiments, in any one formulas (I l)-(IV), L1 is a linker comprising or consisting of a sequence at least 50% identical to a sequence of SEQ ID NO: 15. In particular, at least 55%, 60%, 65%, or 70% identical to a sequence of SEQ ID NO: 15; particularly at least 60%; more particularly at least 65%; even more particularly 70%, identical to a sequence of SEQ ID NO: 15. More in particular, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 15; particularly at least 90%; more particularly at least 95%; even more particularly 100%, identical to a sequence of SEQ ID NO: 15.

[0075] Table 1. List of linkers, characteristics and DNA and protein sequences:

[0076]

[0077] In particular embodiments, the polypeptide comprises a sequence at least 70% identical to a sequence of SEQ ID NO: 16. In particular, the polypeptide comprises a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16; particularly at least 95%; more particularly at least 99%; even moreparticularly 100%, identical to a sequence of SEQ ID NO: 16.

[0078] In particular embodiments, the polypeptide consists of a sequence at least 70% identical to a sequence of SEQ ID NO: 16. In particular, the polypeptide consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16; particularly at least 95%; more particularly at least 99%; even more particularly 100%, identical to a sequence of SEQ ID NO: 16.

[0079] In particular embodiments, the polypeptide comprises:

[0080] - a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 2; and a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 3; - a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 6; or, alternatively,

[0081] - a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16.

[0082] As explained above and as shown in examples, the fusion or combination of the polypeptides of the invention, in particular CWB30 (SEQ ID NO: 16), with polypeptides comprising peptidoglycan degrading activity enhances the efficacy against Gram-negative bacteria, notably including A. baumannii, E. coli, and P. aeruginosa. Also, as shown in the examples, CWB30 (SEQ ID NO: 16) is stable at high salt concentrations. CWB30 (SEQ ID NO: 16) also enhances stability in salt to fusion proteins that comprise this polypeptide. In particular embodiments, the polypeptide is an isolated, synthetic, or recombinant polypeptide.

[0083] In particular embodiments, the polypeptide is positively charged at pH 7. More in particular, the polypeptide comprises a positive charge at pH 7 of at least 1.5, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15. In particular, the polypeptide comprises a positive charge at pH 7 of at least 10. In particular, the polypeptide comprises a positive charge at pH 7 of around 15.

[0084] Thus, in a second aspect the invention provides a fusion protein comprising the polypeptide as defined in the first aspect.

[0085] As used herein, "fusion protein" refers to an amino acid sequence which comprises at least two polypeptide sequences that are never found together in nature. Therefore, the fusion protein as such is never found in nature as such. Fusion proteins of the present invention are also referred to as engineered phage lytic enzymes (EPLEs).

[0086] It should be appreciated that the fusion proteins provided herein may be arranged in any configuration. Theorder of linkage of the polypeptides is not meant to be particularly limiting so long as the particular arrangement of the polypeptides produces a functional fusion protein. The polypeptides may be fused directly or through a linker.

[0087] In particular embodiments, the fusion protein further comprises a polypeptide comprising peptidoglycandegrading activity. More in particular, the fusion protein comprises a polypeptide with activity selected from the group consisting of peptidoglycan hydrolase activity, peptidoglycan lyase activity, oxidative cleavage activity, transglycosylase activity, carboxypeptidase activity, endopeptidase activity, amidase activity, and a combination thereof. More in particular, the fusion protein comprises a polypeptide comprising peptidoglycan hydrolase activity.

[0088] In other words, in particular embodiments, the fusion protein further comprises a polypeptide which acts in the periplasmic space to decrease the rate of growth of the Gram-negative bacterium. In particular, by degrading the peptidoglycan layer.

[0089] Non-limiting examples of polypeptides with peptidoglycan degrading activity include: EAD2 (SEQ ID NO: 17), EAD3 (SEQ ID NO: 18), EAD4 (SEQ ID NO: 19), and EAD7 (SEQ ID NO: 20). These are also known as lysozyme domains. More in particular phage lysozyme domains.

[0090] A person skilled in the art will know how to determine presence of peptidoglycan degrading activity. For instance, peptidoglycan hydrolase activity can be determined by zymography or by turbidometric assays which follow the activity of the enzyme in Micrococcus lysodeikticus cells. The skilled person would know how to determine peptidoglycan hydrolase activity by this or other conventionally used methods in molecular biology. For example, fluorescence assays where the peptidoglycan is labelled with fluorescence and is detected once the enzyme has acted (Invitrogen EnzChek® lysozyme Assay kit). The skilled person will know methods for determining presence of other peptidoglycan degrading activities.

[0091] In particular embodiments, the polypeptide comprising peptidoglycan-degrading activity comprises a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence selected from the group consisting of a sequence of SEQ ID NO: 17, a sequence of SEQ ID NO: 18, a sequence of SEQ ID NO: 19 and a sequence of SEQ ID NO: 20.

[0092] In particular embodiments, the polypeptide comprising peptidoglycan degrading activity comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, at least 99% or 100% of the PG hydrolase activity of a sequence selected from the group consisting of a sequence of SEQ ID NO: 17, a sequence of SEQ ID NO: 18, a sequence of SEQ ID NO: 19 and a sequence of SEQ ID NO: 20.

[0093] A particular example of a polypeptide comprising peptidoglycan degrading activity is EAD7 which is a polypeptide obtained from the phage Arya. In particular it comprises peptidoglycan hydrolase activity.

[0094] In particular embodiments, the polypeptide comprising peptidoglycan degrading activity comprises asequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 20.

[0095] The fusion protein may, apart from the polypeptide as defined in the first aspect, comprise a second polypeptide having at least one activity from said polypeptide as defined in the first aspect.

[0096] Thus, in particular embodiments, the fusion protein further comprises a second polypeptide comprising peptidoglycan binding activity. In another particular embodiment, the polypeptide comprises cell-wall binding activity.

[0097] In particular embodiments, the fusion protein further comprises a second polypeptide comprising cellpermeating activity. In particular, outer-membrane permeating activity. In particular embodiments, Gramnegative permeating activity. In particular, Gram-negative outer membrane permeating activity.

[0098] In particular embodiments, the fusion protein further comprises a second polypeptide.

[0099] In particular embodiments, the fusion protein further comprises a second polypeptide comprising or consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence selected from the group consisting of a sequence of SEQ ID NO: 21, a sequence of SEQ ID NO: 16, a sequence of SEQ ID NO: 22, a sequence of SEQ ID NO: 23, a sequence of SEQ ID NO: 24, a sequence of SEQ ID NO: 25, a sequence of SEQ ID NO: 26, a sequence of SEQ ID NO: 27, a sequence of SEQ ID NO: 28, a sequence of SEQ ID NO: 29, a sequence of SEQ ID NO: 30, and a sequence of SEQ ID NO: 31.

[0100] In particular embodiments, the fusion protein further comprises a second polypeptide comprising peptidoglycan-binding activity and / or cell-permeating activity. In particular, comprising peptidoglycan-binding activity and cell-permeating activity.

[0101] In particular embodiments, the fusion protein further comprises a second polypeptide comprising peptidoglycan binding activity and / or cell-permeating activity; particularly wherein the second polypeptide comprising peptidoglycan binding activity and / or cell-permeating activity comprises or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence selected from the group consisting of a sequence of SEQ ID NO: 21, a sequence of SEQ ID NO: 16, a sequence of SEQ ID NO: 22, a sequence of SEQ ID NO: 23, a sequence of SEQ ID NO: 24, a sequence of SEQ ID NO: 25, a sequence of SEQ ID NO: 26, a sequence of SEQ ID NO: 27, a sequence of SEQ ID NO: 28, a sequence of SEQ ID NO: 29, a sequence of SEQ ID NO: 30, and a sequence of SEQ ID NO: 31.

[0102] In particular embodiments, the fusion protein comprises high stability. More in particular, the fusion protein has a half-life of at least 1.5, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, atleast 25, at least 26, at least 27, at least 28, at least 29, or at least 30 hours in human serum. In particular, the fusion protein has a half-life of at least around 30 hours in human serum.

[0103] In particular embodiments, the fusion protein comprises:

[0104] - a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16; and a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 20;

[0105] - a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16; a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 20; and a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 29;

[0106] - a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16; a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 20; a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 29; and a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 21; or, alternatively,

[0107] - a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16; a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 20; and a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 21.

[0108] For example, the fusion proteins EPLETT44 of sequence of SEQ ID NO: 32, EPLETT45 of sequence of SEQ ID NO: 140, EPLETT46 of sequence of SEQ ID NO: 143 and EPLETT48 of sequence of SEQ ID NO: 149, which comprise the polypeptides as shown in Table 3A and Table 3B, showed high potency (Figure 1). In particular, EPLETT44 (SEQ ID NO: 32) which comprises and EAD7 (SEQ ID NO: 20) polypeptide and a CWB30 (SEQ ID NO: 16) polypeptide, surprisingly exhibited the highest potency as shown in the examples. In particular embodiments, the fusion protein comprises or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 32.It was also demonstrated that engineering variants adding LysM domains to the C-terminus of proteins enhanced both stability and activity. EPLETT50 (SEQ ID NO: 33) was developed, featuring EAD7 (SEQ ID NO: 20) followed by a repeated CWB30 (SEQ ID NO: 16), thus containing four LysM domains. As shown in the examples, it presented surprisingly high efficacy against E. coli.

[0109] In particular embodiments, the fusion protein comprises or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 33.

[0110] Further fusion proteins, in particular EPLETT195 to EPLETT209, as shown in the examples, also demonstrated remarkable activity against A. baumannii.

[0111] Thus, in particular embodiments, the fusion protein comprises or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence selected from the group consisting of a sequence of SEQ ID NO: 34, a sequence of SEQ ID NO: 35, a sequence of SEQ ID NO: 36, a sequence of SEQ ID NO: 37, a sequence of SEQ ID NO: 38, a sequence of SEQ ID NO: 39, a sequence of SEQ ID NO: 40, a sequence of SEQ ID NO: 41, a sequence of SEQ ID NO: 42, a sequence of SEQ ID NO: 43, a sequence of SEQ ID NO: 44, a sequence of SEQ ID NO: 45, a sequence of SEQ ID NO: 46, and a sequence of SEQ ID NO: 47.

[0112] In this regard, the fusion proteins of the invention EPLETT197 of sequence of SEQ ID NO: 36, EPLETT200 of sequence of SEQ ID NO: 39, and EPLETT207 of sequence of SEQ ID NO: 45 were in particular identified as the surprisingly most active and stable candidates. Furthermore, EPLETT197 (SEQ ID NO: 36), EPLETT200 (SEQ ID NO: 39), and EPLETT207 (SEQ ID NO: 45), showed activity against clinically significant pathogens including strains of A. baumannii, E. coli, and P. aeruginosa, as shown in the examples.

[0113] In particular embodiments, the fusion protein comprises or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence selected from the group consisting of a sequence of SEQ ID NO: 36, a sequence of SEQ ID NO: 39, and a sequence of SEQ ID NO: 45.

[0114] Notably, EPLETT200 (SEQ ID NO: 39) and EPLETT207 (SEQ ID NO: 45) exhibited the most potent activity. In vivo models also demonstrated that EPLETT200 (SEQ ID NO: 39) and EPLETT207 (SEQ ID NO: 45), which carry CWB30 (SEQ ID NO: 16), did not show cumulative toxicity. In vivo pneumonia models of A. baumannii indicated that EPLETT200 (SEQ ID NO: 39) and EPLETT207 (SEQ ID NO: 45) are capable of protecting mice. EPLETT207 (SEQ ID NO: 45) and EPLETT200 (SEQ ID NO: 39) also prevented and eliminated pre-formed biofilms. In particular, pre-formed biofilms of A. baumannii, that could not be eliminated by antibiotics. Also, EPLETT200 (SEQ ID NO: 39) showed synergism with different antibiotic such as colistin, gentamicin, aztreonam, meropenem and / or sulbactam.

[0115] In particular embodiments, the fusion protein comprises or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQID NO: 39.

[0116] In particular embodiments, the fusion protein comprises or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 45.

[0117] Notably, EPLETT200 (SEQ ID NO: 39) achieved the highest survival rates in protecting mice in pneumonia models, as shown in the examples. Also, EPLETT200 (SEQ ID NO: 39) did not generate bacterial resistance. In particular embodiments, the fusion protein comprises or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence selected from the group consisting of a sequence of SEQ ID NO: 32, a sequence of SEQ ID NO: 33, a sequence of SEQ ID NO: 36, a sequence of SEQ ID NO: 39, and a sequence of SEQ ID NO: 45.

[0118] The combination of peptidoglycan degradation activity and cell permeation activity results in antibacterial activity. This can be achieved by the integration in a same fusion protein of a polypeptide comprising cellpermeating activity (e.g. the polypeptide of the first aspect) and a polypeptide comprising peptidoglycan degrading activity. Or it can be also achieved by the separate administration of a polypeptide or a fusion protein comprising cell-permeating activity in combination with a polypeptide or fusion protein comprising peptidoglycan degrading activity.

[0119] Thus, in particular embodiments, the fusion protein comprises an activity selected from the group consisting of peptidoglycan binding activity, cell-permeating activity, peptidoglycan-degrading activity, antibacterial activity, and a combination thereof. More in particular, the fusion protein comprises an activity selected from the group consisting of cell-permeating activity and / or peptidoglycan-degrading activity.

[0120] In particular embodiments, the antibacterial activity is selected from bacteriostatic activity and / or bactericidal activity.

[0121] The term "bactericidal activity" in the present disclosure refers to the ability of causing, or being able to cause, the death of bacteria to an extent of at least a 3-log (99.9%) reduction among an initial population of bacteria. The term "bacteriostatic activity" in the present disclosure refers to the ability of inhibiting, or being able to inhibit, bacterial growth, including inhibiting growing bacterial cells, thus causing an at least 2-log (99%) and up to just under a 3-log (99.9%) reduction among an initial population of bacteria.

[0122] The term "antibacterial activity” is used generically to refer to both bacteriostatic and bactericidal activities. In particular embodiments, the antibacterial activity, bacteriostatic activity and / or bactericidal activity is against Gram-negative bacteria.

[0123] In particular embodiments, the Gram-negative bacteria are from a genus selected from the group consisting of Acinetobacter, Pseudomonas, Escherichia, Klebsiella, and combinations thereof; particularly, the Gramnegative bacteria are from a species selected from the group consisting of A. baumannii, E. coli, K.pneumoniae, P. aeruginosa, and combinations thereof.

[0124] The polypeptide of the first aspect and the fusion protein of the second aspect may be produced by any method known in the art. For example, they may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a linker. Methods for recombinant protein expression and purification are well known in the art, and belong to the common general knowledge of the skilled person.

[0125] In particular embodiments, the fusion protein further comprises further polypeptides comprising antibacterial activity. More in particular, bacteriostatic activity and / or bactericidal activity.

[0126] In particular embodiments, the fusion protein is an isolated, synthetic, or recombinant fusion protein.

[0127] In particular embodiments, the polypeptide of the first aspect and / or the fusion protein of the second aspect may comprise a specific affinity purification tag. Illustrative, non-limiting examples of affinity tags include glutathione S-transferase (GST), maltose E binding protein, protein A, FLAG tag, hexa-histidine, myc tag or the influenza HA tag. Said affinity purification tag can be fused directly in-line or, alternatively, fused to the polypeptide via a cleavable linker, i.e., a peptide segment containing an amino acid sequence that is specifically cleavable by enzymatic or chemical means (i.e., a recognition / cleavage site).

[0128] In particular embodiments, the polypeptide of the first aspect and / or the fusion protein of the second aspect may be chemically modified. A chemical modification includes but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties. Chemical modifications can occur anywhere in the polypeptide or the fusion protein, including the amino acid side chains, as well as the amino or carboxyl termini. Such modification can be present at more than one site in the polypeptide or the fusion protein. Furthermore, one or more side groups, or terminal groups of the polypeptide or fusion protein may be protected by protective groups known to the person skilled in the art.

[0129] It is well-known in the state of the art how to modify (i.e., derivatize) the terminal ends of a polypeptide or a protein, for example, to improve its stability or efficacy. It should be appreciated that any of the polypeptide of the first aspect and / or the fusion protein of the second aspect are provided with or without any N-terminal and / or C-terminal modification.

[0130] As used herein, "N-terminal modification" refers to the modification of the NH2 of the N-terminal residue of the polypeptide or protein, resulting in a compound being linked to the NH moiety. Examples of N-terminal modifications include, but are not limited to acetylation. As used herein, "C-terminal modification" refers to the modification of the carboxyl moiety of the C-terminal residue of the polypeptide or protein. Examples of C-terminal modifications include, but are not limited to amidation, esterification and reduction.

[0131] Thus, in particular embodiments, the N-terminal and / or C-terminal modification comprises or consists of a compound selected from the group consisting of phosphoryl group, glycosyl group, acyl group, alkyl group, carboxyl group, hydroxyl group, biotinyl group, ubiquitinyl group, and amido group; particularly, wherein theacyl group is selected from the group consisting of acetyl group, lauroyl group, myristoyl group, and palmitoyl group.

[0132] In another embodiment, the N-terminal modification comprises or consists of a compound selected from the group consisting of acetyl group, urea group, formyl group, carbamate group, pyroglutamyl group, and sulfonamide group, and / or the C-terminal modification comprises or consists of a compound selected from the group consisting of amido group, N-alkyl amido group, or aldehyde group.

[0133] In particular embodiments, the polypeptide and / or the fusion protein of the invention is a linear polypeptide or fusion protein or a circularized polypeptide or fusion protein (i.e., cyclic). There are several methods in the art to circularize polypeptides or proteins known to the skilled person.

[0134] In particular embodiments, the polypeptide of the first aspect and / or the fusion protein of the second aspect is conjugated to a further agent. Non-limiting examples of further agents to be conjugated with the polypeptide and / or the fusion protein, include nanoparticles, antibiotics, cell-penetrating peptides, among other.

[0135] In particular embodiments, the polypeptide of the first aspect and the fusion protein of the second aspect may contain an attachment of duration enhancing moieties. A non-limiting example of duration enhancing moiety is polyethylene glycol. Polyethylene glycol ("PEG”) has been used in the art to obtain therapeutic polypeptides or fusion proteins of enhanced duration. The PEG backbone (CH2CH2— 0— )n, wherein n is the number of repeating monomers, is flexible and amphiphilic. When attached to another chemical entity, such as the polypeptide or the fusion protein of the invention, PEG polymer chains can protect such polypeptide or fusion protein from immune response and other clearance mechanisms. As a result, pegylation can lead to improved efficacy and safety by optimizing pharmacokinetics, increasing bioavailability, and decreasing immunogenicity and dosing amount and / or frequency.

[0136] The polypeptide and / or the fusion protein may be encoded in nucleic acids for their expression and production. Also, the polypeptide of the first aspect and / or the fusion protein of second aspect may be administered directly, or they can be expressed inside target cells of interest by means of gene therapy. To these aims the invention also provides, in a third aspect, a polynucleotide that encodes the polypeptide as defined in the first aspect and / or the fusion protein as defined in the second aspect.

[0137] The term "polynucleotide” refers to a molecule comprising two or more individual nucleotide residues linked to each other via a phosphodiester linkage. In particular, deoxyribonucleotides or ribonucleotides.

[0138] Polynucleotides can be single or double stranded, and it includes, but it is not limited to, nucleotide sequences coding for polypeptides. In this description, polynucleotides used for obtaining the polypeptides by recombinant technology have a varying length depending on the construct.

[0139] In particular embodiments, the polynucleotide is DNA (single or double stranded).

[0140] In particular embodiments, the polynucleotide comprises or consists of a sequence selected from the group consisting of a sequence of SEQ ID NO: 48, a sequence of SEQ ID NO: 49, a sequence of SEQ ID NO: 52, asequence of SEQ ID NO: 62, and a combination thereof.

[0141] In particular embodiments, the polynucleotide comprises or consists of a sequence selected from the group consisting of a sequence of SEQ ID NO: 48 and a sequence of SEQ ID NO: 49; and further comprises a sequence of SEQ ID NO: 50 and / or a sequence of SEQ ID NO: 51.

[0142] In particular embodiments, the polynucleotide comprises or consists of a sequence selected from the group consisting of a sequence of SEQ ID NO: 48, a sequence of SEQ ID NO: 49, and a sequence of SEQ ID NO: 52; and further comprises a sequence selected from the group consisting of a sequence of SEQ ID NO: 53, a sequence of SEQ ID NO: 54, a sequence of SEQ ID NO: 55, a sequence of SEQ ID NO: 56, a sequence of SEQ ID NO: 57, a sequence of SEQ ID NO: 58, a sequence of SEQ ID NO: 59, a sequence of SEQ ID NO: 60, and a sequence of SEQ ID NO: 61.

[0143] In particular embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NO: 62.

[0144] In particular embodiments, the polynucleotide further comprises a sequence selected from the group consisting of a sequence of SEQ ID NO: 67, a sequence of SEQ ID NO: 62, a sequence of SEQ ID NO: 68, a sequence of SEQ ID NO: 69, a sequence of SEQ ID NO: 70, a sequence of SEQ ID NO: 71, a sequence of SEQ ID NO: 72, a sequence of SEQ ID NO: 73, a sequence of SEQ ID NO: 74, a sequence of SEQ ID NO: 75, a sequence of SEQ ID NO: 76, a sequence of SEQ ID NO: 77, a sequence of SEQ ID NO: 63, a sequence of SEQ ID NO: 64, a sequence of SEQ ID NO: 65, a sequence of SEQ ID NO: 66, and combinations thereof. In particular embodiments, the polynucleotide comprises or consists of a sequence selected from the group consisting of a sequence of SEQ ID NO: 78, a sequence of SEQ ID NO: 79, a sequence of SEQ ID NO: 80, a sequence of SEQ ID NO: 81, a sequence of SEQ ID NO: 82, a sequence of SEQ ID NO: 83, a sequence of SEQ ID NO: 84, a sequence of SEQ ID NO: 85, a sequence of SEQ ID NO: 86, a sequence of SEQ ID NO: 87, a sequence of SEQ ID NO: 88, a sequence of SEQ ID NO: 89, a sequence of SEQ ID NO: 90, a sequence of SEQ ID NO: 91, a sequence of SEQ ID NO: 92, and a sequence of SEQ ID NO: 93. In particular embodiments, the polynucleotide comprises or consists of a sequence selected from the group consisting of a sequence of SEQ ID NO: 78, a sequence of SEQ ID NO: 79, a sequence of SEQ ID NO: 82, a sequence of SEQ ID NO: 85, and a sequence of SEQ ID NO: 91. In particular embodiments, the polynucleotide comprises or consists of a sequence selected from the group consisting of a sequence of SEQ ID NO: 78 and a sequence of SEQ ID NO: 79. In particular embodiments, the polynucleotide comprises or consists of a sequence selected from the group consisting of a sequence of SEQ ID NO: 82, a sequence of SEQ ID NO: 85, and a sequence of SEQ ID NO: 91.

[0145] In particular embodiments, the polynucleotide is RNA.

[0146] In particular embodiments, the polynucleotide comprises or consists of a sequence selected from the group consisting of a sequence of SEQ ID NO: 94, a sequence of SEQ ID NO: 95, a sequence of SEQ ID NO: 98, a sequence of SEQ ID NO: 108, and a combination thereof.In particular embodiments, the polynucleotide comprises or consists of a sequence selected from the group consisting of a sequence of SEQ ID NO: 94 and a sequence of SEQ ID NO: 95; and further comprises a sequence of SEQ ID NO: 96 and / or a sequence of SEQ ID NO: 97.

[0147] In particular embodiments, the polynucleotide comprises or consists of a sequence selected from the group consisting of a sequence of SEQ ID NO: 94, a sequence of SEQ ID NO: 95, and a sequence of SEQ ID NO: 98; and further comprises a sequence selected from the group consisting of a sequence of SEQ ID NO: 99, a sequence of SEQ ID NO: 104, a sequence of SEQ ID NO: 105, a sequence of SEQ ID NO: 106, a sequence of SEQ ID NO: 100, a sequence of SEQ ID NO: 101, a sequence of SEQ ID NO: 102, a sequence of SEQ ID NO: 103, and a sequence of SEQ ID NO: 107.

[0148] In particular embodiments, the polynucleotide comprises or consists of a sequence of SEQ ID NO: 108. In particular embodiments, the polynucleotide further comprises a sequence of SEQ ID NO: 113, a sequence of SEQ ID NO: 108, a sequence of SEQ ID NO: 114, a sequence of SEQ ID NO: 115, a sequence of SEQ ID NO: 116, a sequence of SEQ ID NO: 117, a sequence of SEQ ID NO: 118, a sequence of SEQ ID NO: 119, a sequence of SEQ ID NO: 120, a sequence of SEQ ID NO: 121, a sequence of SEQ ID NO: 122, a sequence of SEQ ID NO: 123, a sequence of SEQ ID NO: 109, a sequence of SEQ ID NO: 110, a sequence of SEQ ID NO: 111, a sequence of SEQ ID NO: 112, and combinations thereof.

[0149] In particular embodiments, the polynucleotide comprises or consists of a sequence selected from the group consisting of a sequence of SEQ ID NO: 124, a sequence of SEQ ID NO: 125, a sequence of SEQ ID NO: 126, a sequence of SEQ ID NO: 127, a sequence of SEQ ID NO: 128, a sequence of SEQ ID NO: 129, a sequence of SEQ ID NO: 130, a sequence of SEQ ID NO: 131, a sequence of SEQ ID NO: 132, a sequence of SEQ ID NO: 133, a sequence of SEQ ID NO: 134, a sequence of SEQ ID NO: 135, a sequence of SEQ ID NO: 136, a sequence of SEQ ID NO: 137, a sequence of SEQ ID NO: 138, and a sequence of SEQ ID NO: 139. In particular embodiments, the polynucleotide comprises or consists of a sequence selected from the group consisting of a sequence of SEQ ID NO: 124, a sequence of SEQ ID NO: 125, a sequence of SEQ ID NO: 128, a sequence of SEQ ID NO: 131, and a sequence of SEQ ID NO: 138. In particular embodiments, the polynucleotide comprises or consists of a sequence selected from the group consisting of a sequence of SEQ ID NO: 124 and a sequence of SEQ ID NO: 125. In particular embodiments, the polynucleotide comprises or consists of a sequence selected from the group consisting of a sequence of SEQ ID NO: 128, a sequence of SEQ ID NO: 131, and a sequence of SEQ ID NO: 138.

[0150] In particular embodiments, the polynucleotide is an isolated, synthetic, or recombinant polynucleotide.

[0151] In particular embodiments, the polynucleotide comprises a sequence optimized for expression in one or more cell types. For example, the polynucleotide sequence may be optimized for expression in a mammalian cell (e.g., a HEK 293T cell). The polynucleotide may be codon optimized for expressing in a mammalian cell using Integrated DNA Technologies (IDT), GeneArt, CoIler, and GenScript. Alternatively, in particular embodiments, the polynucleotide comprises a sequence optimized for expression in a bacterial expression system.Polynucleotides may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a polynucleotide may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA / RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the term "polynucleotide" includes nucleic acid analogues, e.g., analogues having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogues such as analogues having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogues (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); modified base analogues of DNA or RNA (e.g. 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1 -methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5' -methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0152] In a fourth aspect, the invention provides a gene construct comprising a polynucleotide as defined in the third aspect operatively linked to an expression promoter.

[0153] A "gene construct” according to the invention can also be named as an "expression cassette”. It refers to a polynucleotide sequence which is operatively linked to an expression promoter, said promoter controlling expression of the sequence coding for the polynucleotide and / or the fusion protein. For "operatively linked” is to be understood that the sequence coding for the fusion protein is disposed after the sequence of the promoter (in the 5'-3' direction), or near the promoter in case restriction sites are included, or other stabilizing elements of the gene construction are present. The gene constructs (expression cassette) may also comprisesmall fragments with useful sequences to adapt it to more complex expression systems (vectors, plasmids), or a polyadenylation tail disposed after the sequence coding for the polypeptide and / or fusion protein of interest. The expression cassette itself is also an expression system, being vectors or plasmids further used to protect the gene construct, or to promote entrance to cells in case of viral vectors. The "promoters” are of DNA regions that initiate transcription of a particular genes. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5' region of the sense strand).

[0154] Promoters can be about 100-1000 base pairs long. A "constitutive promoter” is a promoter that is active in all circumstances in the cell, contrary to others that are regulated, becoming active in the cell only in response to specific stimuli, such as "inducible promoters”.

[0155] In any case, examples of suitable expression promoters include those conventionally used in molecular biology and known to the skilled person.

[0156] As above explained, in a fifth aspect, the invention provides an expression vector comprising the polynucleotide of the third aspect and / or the gene construct as defined in the fourth aspect.

[0157] For the efficient expression of the polypeptide and / or fusion protein codified by the polynucleotide and / or the gene construct, the polynucleotide and / or the gene construct are inserted in a vector. A "vector” is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and / or expressed (e.g., plasmid, cosmid, Lambda phages). A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. These vectors may carry elements (sequences) the skilled person will know aiding in the expression and stability of the expressed sequence, as well as tags or labels to purify the recombinant expressed polypeptide.

[0158] Examples of suitable expression vectors include those conventionally used in biomedicine and known to the skilled person.

[0159] Vectors can be designed for expression in prokaryotic or eukaryotic cells. For example, vectors can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Alternatively, expression vectors can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0160] In any case, examples of suitable expression vectors include those conventionally used in molecular biology and known to the skilled person.

[0161] In a sixth aspect, the invention provides a host cell comprising the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth aspect, and / or the expression vector as defined in the fifth aspect.

[0162] A polynucleotide, a gene construct, and / or a vector is transfected to an appropriate cell system (host cells), which by means of their replicative and expression biochemical machinery will allow the obtaining of thepolypeptide and / or fusion protein.

[0163] Examples of suitable host cells and culture conditions include those conventionally used in cell biology and known to the skilled person.

[0164] In particular embodiments, the host cell is a eukaryotic host cell. In alternative embodiments, the host cell is a prokaryotic host cell. Host cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC)).

[0165] In a seventh aspect, the invention provides a method for producing the polypeptide as defined in the first aspect and / or the fusion protein as defined in the second aspect; wherein the method comprises the steps of: (I) culturing a host cell as defined in the sixth aspect under conditions suitable for the production of the polypeptide and / or the fusion protein; or, alternatively, (”) in vitro transcription and / or translation of the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth, and / or the vector as defined in the fifth aspect; (II) recovering the polypeptide and / or the fusion protein produced in step (I) or (I'); and (ill) optionally, isolating and / or purifying the polypeptide and / or the fusion protein.

[0166] In particular embodiments, the method further comprises a first step of transforming of a host cell with a vector as defined in the fifth aspect comprising the polynucleotide and / or gene construct as defined in sixth aspect, to obtain a host cell as defined in the sixth aspect.

[0167] In particular embodiments, the step (ill) is carried out by enzymatic and / or mechanical disruption of the cell host, optionally in presence of a one or more of solvents, detergents, and surfactants, in particular non-ionic surfactants, and protease inhibitors.

[0168] The skilled person in the art will know which enzymes, reagents and / or physical means are currently employed for the purification of a compound of interest (i.e., a polypeptide and / or fusion protein) from a host cell culture.

[0169] Examples of enzymes include the lysozyme. Examples of physical or mechanical means include centrifugation steps, frozen and thaw cycles.

[0170] Examples of non-ionic surfactants include Triton X-100, polyglycerol alkyl ethers, glucosyl dialkyl ethers, crownethers, ester-linked surfactants, polyoxyethylene alkyl ethers, Brij, Spans (sorbitan esters) and Tweens (Polysorbates).

[0171] In an eighth aspect, the invention provides a polypeptide and / or a fusion protein obtainable by the method defined in the seventh aspect. All the particular embodiments of the method are applicable to the polypeptide and / or fusion protein obtainable by means of the same.

[0172] In a nineth aspect, the invention provides a composition comprising the polypeptide as defined in the first aspect, the fusion protein as defined in the second aspect, the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth aspect, the vector as defined in the fifth aspect, the host cell asdefined in the sixth aspect, and / or the polypeptide or fusion protein as defined in the eighth aspect.

[0173] In particular embodiments of the nineth aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the polypeptide as defined in the first aspect, the fusion protein as defined in the second aspect, the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth aspect, the vector as defined in the fifth aspect, the host cell as defined in the sixth aspect, and / or the polypeptide or fusion protein as defined in the eighth aspect; with at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0174] The expression "pharmaceutical composition” encompasses both compositions intended for human as well as for non-human animals. The skilled in the art understands that a pharmaceutical composition must comprise a therapeutically effective amount of the active compound. The expression "therapeutically effective amount" as used herein, refers to the amount of polypeptide, fusion protein, polynucleotide, gene construct, vector, and / or host cell that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed. The particular dose of compound administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations.

[0175] The expression "pharmaceutically acceptable excipient, diluent or carrier" refers to pharmaceutically acceptable materials, compositions or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and non-human animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0176] A composition that includes the polypeptide, the fusion protein, the polynucleotide, the gene construct, the vector, and / or the host cell, can be delivered to a subject by a variety of routes including, without limitation, by local (e.g. topical, rectal, ocular, etc.) or systemic administration. Systemic administration, may include oral or parental (e.g., intravenous, subcutaneous, intramuscular and intraperitoneal) administration. Additionally, it is also possible to administer the composition comprising the agent of the invention I ntranasally or sublingually which allows systemic administration by a non-aggressive mode of administration.

[0177] In particular embodiments, the pharmaceutical composition is for being administered to the patient via mucosa (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenterally (e.g., subcutaneous, intravenous, intramuscular, or intraarterial injection, either bolus or infusion), orally, transdermally or via inhalation by means e.g. of an aerosol.

[0178] In particular embodiments, the pharmaceutical composition is administered by injection e.g subcutaneous, intraperitoneal, intravesically, intravenous, intracerebroventricular, by infusion, e.g., using a reservoir or osmotic minipump or intramuscular.In particular embodiments, the pharmaceutical composition is for intraventricular administration. In particular embodiments, the route of delivery is intravascular (e.g. intraarterial or intravenous) or subcutaneous injection. More in particular, for systemic intravascular administration.

[0179] Typically, compositions for intravenous, intramuscular, subcutaneous, intraperitoneal or intraventricular administration are solutions in sterile isotonic aqueous buffer. In particular embodiments, the composition can contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Illustrative non-limiting examples of pH buffering agents include Tris-HCI buffer, acetate buffer, citrate and phosphate buffer or combinations thereof. The term "acetate buffer”, "citrate buffer” and "phosphate buffer” as used herein can refer to a buffer system comprising an organic acid (acetic acid, citric acid and phosphoric acid, respectively) and a salt thereof. Each of them can be added in a sufficient amount. In particular embodiments, the pH of the composition according may be in the range from about 4 to about 8. In particular, from about 5 to about 7. In particular pH 5, pH 5.5, pH 6, pH 6.5 and pH 7.

[0180] The polypeptide, the fusion protein, the polynucleotide, the gene construct, and the vector can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids and the like, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, thriethylamine, 2-ethylamino ethanol, histidine, procaine or similar.

[0181] Examples of suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

[0182] The relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered.

[0183] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Excipients such as coloring agents, coating agents, sweetening, and flavoring agents can be present in the composition, according to the judgment of the formulator.

[0184] The pharmaceutical compositions of the invention can be presented in any dosage form, for example, solid or liquid.

[0185] Solid dosage forms for oral administration may include conventional capsules, sustained release capsules,conventional tablets, sustained-release tablets, chewable tablets, sublingual tablets, effervescent tablets, pills, suspensions, powders, granules and gels. Formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate, etc. Such dosage forms can also comprise, as in normal practice, additional substances other than inert diluents, such as dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. In the case of capsules, tablets, effervescent tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can be prepared with enteric coatings.

[0186] Where necessary, the polypeptide, fusion protein, polynucleotide, gene construct, vector and / or host cell are comprised in a composition also including a solubilizing agent and a local anesthetic to ameliorate any pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The formulation can be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials.

[0187] The effective quantity of the polypeptide, the fusion protein, the polynucleotide, the gene construct, the vector and / or of the host cell, can vary within a wide range and, in general, will vary depending on the particular circumstances of application, duration of the exposure and other considerations. In particular embodiments, the dose ranges from 0.01 mg / kg to 20 mg / kg; in particular from 0.05 mg / kg to 10 mg / kg; in particular such as from 0.1 mg / kg to 5 mg / kg; or more in particular from 1 mg / kg to 2 mg / kg.

[0188] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and combinations thereof.

[0189] Exemplary granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and combinations thereof.

[0190] Exemplary binding agents include, but are not limited to, starch (e.g., corn-starch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and syntheticgums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone), magnesium aluminium silicate (Veegum), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; and combinations thereof. Exemplary preservatives may include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, ascorbyl stearate, ascorbyl oleate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate.

[0191] Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and combinations thereof.

[0192] Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0193] Various delivery systems are known in the art, including encapsulation in liposomes, microbubbles, emulsions, microparticles, microcapsules and the like.

[0194] In particular embodiments, the pharmaceutical composition further comprises a liposome, a microbubble, an emulsion, a microparticle, a microcapsule and / or a nanoparticle encapsulating the polypeptide, the fusion protein, the polynucleotide, gene construct, the vector, and / or the host cell. In particular embodiments, the nanoparticle is a lipid nanoparticle. The skilled person would appreciate that the nanoparticle is to be biocompatible and protect the active ingredient from degradation. The encapsulation in the nanoparticle can be performed using well-known methods in the state of the art.In an tenth aspect, the invention provides the polypeptide as defined in the first aspect, the fusion protein as defined in the second aspect, the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth aspect, the vector as defined in the fifth aspect, the host cell as defined in the sixth aspect, the polypeptide or fusion protein as defined in the eighth aspect, and / or the composition as defined in the nineth aspect; for use in medicine. In other words, they are provided for use as a medicament, and more particularly, for use in therapy.

[0195] In a eleventh aspect, the invention provides the polypeptide as defined in the first aspect, the fusion protein as defined in the second aspect, the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth aspect, the vector as defined in the fifth aspect, the host cell as defined in the sixth aspect, the polypeptide or fusion protein as defined in the eighth aspect, and / or the composition as defined in the nineth aspect; for use in the treatment and / or prevention of a bacterial infection. In other words, they are for use in the prophylactic and / or therapeutic treatment of a bacterial infection.

[0196] In one embodiment of the eleventh aspect, the treatment and / or prevention comprises the administration of the polypeptide, the fusion protein, the polynucleotide, the gene construct, the vector, the host cell, and / or the composition in combination with another drug; particularly an antibiotic.

[0197] In particular embodiments, the bacterial infection is caused by Gram-negative bacteria. In particular the Gramnegative bacteria are selected from the group consisting of bacteria of the genus Acinetobacter, Bacteroides, Campylobacter, Fusobacterium, Haemophilus, Helicobacter, Mobiluncus, Porphyromonas, Prevotella, Pseudomonas and Veillonella, and the Enterobacteriaceae family (also referred herein as Enterobacteria). Some illustrative but not limiting examples of Enterobacteria include bacteria of the genus Citrobacter, Enterobacter, Escherichia, Klebsiella, Proteus, Salmonella, Serratia, Shigella and Yersinia.

[0198] In particular embodiments, the Gram-negative bacteria are selected from the group consisting of the genus Acinetobacter, Pseudomonas, Escherichia, Klebsiella, Serratia and Citrobacter. In particular embodiments, from the group consisting of Acinetobacter, Pseudomonas, Escherichia and Klebsiella. In particular embodiments, the Gram-negative bacteria are selected from the group consisting of E. coli, K. pneumoniae, A. baumannii, P. aeruginosa, S. marcescens and C. freundii. More in particular, the Gram-negative bacteria are selected from the group consisting of E. coli, K. pneumoniae, A. baumannii and P. aeruginosa.

[0199] In particular embodiments, the Gram-negative bacteria are a drug-resistant strain. More in particular, a multidrug-resistant (MDR) strain.

[0200] The term "drug-resistant” in a context of a pathogen and more specifically a bacterium, generally refers to a bacterium that is resistant to the antimicrobial activity of a drug. When used in a more particular way, drugresistance specifically refers to antibiotic resistance. In some cases, a bacterium that is generally susceptible to a particular antibiotic can develop resistance to the antibiotic, thereby becoming a drug-resistant microbe or strain. A “multidrug-resistant” pathogen is one that has developed resistance to at least two classes of antimicrobial drugs, each used as monotherapy. For example, certain strains of E. coli are capable ofproducing the so-called extended-spectrum beta-lactamases (ESBLs). ESBLs are enzymes that degrade certain antibiotics such as penicillin or cephalosporin, so the strains that produce these ESBLs are resistant to these antibiotics. In addition, strains of ESBLs-producing E. coli resistant to carbapenems have already been found, one of the few antibiotics effective against this to ESBLs-producing E. coli.

[0201] One skilled in the art can readily determine if a bacterium is drug-resistant using routine laboratory techniques that determine the susceptibility or resistance of a bacterium to a drug or antibiotic.

[0202] In particular embodiments, the Gram-negative bacteria are of the Enterobacteriaceae family. In particular embodiments, the infection is caused by Escherichia coli. The E. coli strain is not particularly limited to any serotype. Illustrative non-limiting examples include E. co / / bacteria from serotypes 01 (e.g., 01 A), 02, 06, (e.g., 06A), 025 (e.g. O25B) or 0157 serotypes, which are serotypes frequently involved in urinary tract infections. In particular embodiments, the E.coli is a serotype 06 strain (such as E. coli ATCC 25922), or a serotype 0157 strain (such as E. coli O157:H7). In particular embodiments, the E. coli strain is a drugresistant strain. More in particular, a multidrug-resistant (MDR) strain.

[0203] These MDR strains can be resistant to one, two, three, four, five, six, seven or more antibiotics, such as for example sulphonamides (e.g., sulfamethoxaxole and trimethoprim-sulfamethoxaxole), penicillins (e.g. ticarcillin, ticarcillin-clavulanate, piperacillin, piperacillin-tazobactam, amoxicillin, amoxicillin-clavulanate), cephalosporins (e.g., ceftazidime, cefepime, cefoperazone), monobactams (e.g., aztreonam), lincosamides (e.g., lincomycin), fluoroquinolones (e.g., ciprofloxacin, levofloxacin, norfloxacin), carbapenems (e.g., imipenem, meropenem, ertapenem, doripenem), aminoglycosides (e.g., gentamicin, tobramycin, amikacin), and polymyxins (e.g., colistin, polymyxin B). In particular embodiments, the MDR strain is resistant to one or more antibiotics selected from the list consisting of sulphonamides, penicillins, lincosamides, fluoroquinolones, aminoglycosides and tetracyclines. In particular to all listed antibiotics. In particular embodiments, the MDR strain is resistant to one or more, including all, of the antibiotics selected from the list consisting of sulfonamide, amoxicillin, lincomycin, linco-spectin® (lincomycin + spectinomycin), enrofloxacin, neomycin and doxycycline.

[0204] In particular embodiments, the Gram-negative bacteria are carbapenem-resistant.

[0205] In particular embodiments, the Gram-negative bacteria are Escherichia coli. In particular embodiments, Escherichia coli resistant to one or more of sulfamide, amoxicillin, lincomycin, lincospectin, enrofloxacin, neomycin, and doxycycline.

[0206] In particular embodiments, the Gram-negative bacteria are Klebsiella pneumoniae. In particular embodiments, Klebsiella pneumonia resistant to one or more of beta lactams (e.g., carbapenems), fluoroquinolones and trimethoprim / sulfamethoxazole.

[0207] In particular embodiments, the Gram-negative bacteria are Acinetobacter baumannii. In particular embodiments, Acinetobacter baumannii resistant to one or more of aminoglycosides and

[0208] trimethoprim / sulfamethoxazole.In particular embodiments, the Gram-negative bacteria are Pseudomonas aeruginosa. In particular embodiments, Pseudomonas aeruginosa resistant to beta lactams.

[0209] In particular embodiments, the Gram-negative bacteria are a clinical strain.

[0210] In particular embodiments, the bacterial infection is an hospital-acquired bacterial infection.

[0211] In particular embodiments, the bacterial infection is a device-associated bacterial infection.

[0212] In particular embodiments, the bacterial infection is a nosocomial infection.

[0213] In particular embodiments, the bacterial infection is a biofilm-associated bacterial infection. More in particular, it is for use in the prevention and / or treatment of a biofilm-associated bacterial infection in combination with at least one other drug; particularly an antibiotic. Even more in particular, wherein the other drug is ciprofloxacin and / or meropenem.

[0214] In particular embodiments, the polypeptide as defined in the first aspect, the fusion protein as defined in the second aspect, the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth aspect, the vector as defined in the fifth aspect, the host cell as defined in the sixth aspect, the polypeptide or fusion protein as defined in the eighth aspect, and / or the composition as defined in the nineth aspect; for use in the treatment and / or prevention of pneumonia. More in particular, wherein the pneumonia is selected from the group consisting of hospital-acquired bacterial pneumonia (HABP), ventilator-associated bacterial pneumonia (VABP), and carbapenem resistant (CRAB)-related pneumonia. In particular wherein the pneumonia is caused by A. baumannii strains exhibiting varying levels of antibiotic resistance. More in particular, wherein the A. baumannii strains are multidrug-resistant and / or carbapenem-resistant strains. As indicated above and in the examples, certain fusion proteins of the invention (EPLEs) performed specially well for certain bacterial species.

[0215] In particular embodiments, the fusion protein consisting of or comprising a sequence selected from the group consisting of a sequence of SEQ ID NO: 32 and a sequence of SEQ ID NO: 33, is for use in the treatment of a bacterial infection caused by Escherichia coli.

[0216] In particular embodiments, the fusion protein consisting of or comprising a sequence selected from the group consisting of a sequence of SEQ ID NO: 34, a sequence of SEQ ID NO: 35, a sequence of SEQ ID NO: 36, a sequence of SEQ ID NO: 37, a sequence of SEQ ID NO: 38, a sequence of SEQ ID NO: 39, a sequence of SEQ ID NO: 40, a sequence of SEQ ID NO: 41, a sequence of SEQ ID NO: 42, a sequence of SEQ ID NO: 43, a sequence of SEQ ID NO: 44, a sequence of SEQ ID NO: 45, a sequence of SEQ ID NO: 46, and a sequence of SEQ ID NO: 47, is for use in the treatment of a bacterial infection caused by Acinetobacter baumannii.

[0217] In particular embodiments, the fusion protein consisting of or comprising a sequence selected from the group consisting of a sequence of SEQ ID NO: 36, a sequence of SEQ ID NO: 39, and a sequence of SEQ ID NO:45, is for use in the treatment of a bacterial infection caused by a bacterial species selected from the group consisting of Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa.

[0218] In particular embodiments, the fusion protein consisting of or comprising a sequence of SEQ ID NO: 39, is for use in the treatment of a bacterial infection. In particular, a bacterial infection caused by a Gram-negative bacterium. More in particular, a gram-negative bacteria selected from the group consisting of Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and a combination thereof. Even more in particular, the fusion protein consisting of or comprising a sequence of SEQ ID NO: 39, is for use in the treatment of a bacterial infection caused by A. baumannii,' particularly multidrug-resistant A. baumannii,' more particularly carbapenem-resistant A. baumannii.

[0219] The infection caused by Gram-negative bacteria may occur in any organ or tissue of the subject. In particular embodiments, the infection caused by Gram-negative bacteria occurs in blood, gastrointestinal tract, heart, cardiovascular system, liver, lung, respiratory tract, kidney, urinary tract, nervous central system, skin, subcutaneous tissues or surgical wounds. In particular embodiments, the infection caused by Gram-negative bacteria occurs in the urinary tract. In particular embodiments, the infection occurs in blood. In particular embodiments, the infection is caused by one or more of the genus or species described herein above. The Gram-negative bacteria causing the infection to be treated can be human or veterinary pathogenic bacteria or strains. In particular embodiments, Gram-negative bacteria are human pathogenic bacteria or strains. In particular embodiments, Gram-negative bacteria are veterinary pathogenic bacteria or strains, such as avian or non-human mammals pathogenic bacteria or strains.

[0220] As indicated above, in particular embodiments, the polypeptide, the fusion protein, the nucleic acid, the gene construct, the vector, the host cell, or the composition is administered in combination with another drug. In particular embodiments, the other drug is selected from the group consisting of permeabilizing agents of the outer membrane of the Gram-negative bacteria, antiseptic reagents, lantibiotics, bacteriocins, other endolysins, antibiotics, and combinations thereof.

[0221] The term "suitable” in the context of an antibiotic being suitable for use against certain bacteria refers to an antibiotic that was found to be effective against those bacteria even if resistance subsequently developed. Permeabilizing agents of the outer membrane of the Gram-negative bacteria, including, but not limited to metal chelators as e.g. EDTA, TRIS, lactic acid, lactoferrin, polymyxins, citric acid. This may be part of the same or separate compositions.

[0222] Therapeutic agents, including antiseptic reagents, lantibiotics, bacteriocins, other endolysins or antibiotics. Antiseptic reagents include, but are not limited to Daquin's solution, sodium or potassium hypochlorite solution, solution of sodium benzenesulfochloramide, certain iodine preparations, such as iodopovidone, peroxides as urea perhydrate solutions and pH-buffered peracetic acid solutions, alcohols with or without antiseptic additives, weak organic acids such as sorbic acid, benzoic acid, lactic acid and salicylic acid, some phenolic compounds, such as hexachlorophene, triclosan and Dibromol, and cation-active compounds, suchas benzalkonium, Chlorhexidine, methylisothiazolinone, a-terpineol, thymol, chloroxylenol octenidine solutions.

[0223] In particular embodiments, the other drug is an antibiotic. Traditional antibiotics used against Gram-negative bacteria which may be used in the present invention include, but are not limited to, sulphonamides (e.g., sulfamethoxazole and trimethoprim-sulfamethoxazole), penicillins (e.g., ticarcillin, piperacillin, amoxicillin, including ureidopenicillins [e.g. azlocillin, piperacillin, mezlocillin]), cephalosporins (e.g., ceftazidime, cefepime, cefoperazone), monobactams (e.g., aztreonam), lincosamides (e.g., lincomycin), fluoroquinolones (e.g., ciprofloxacin, levofloxacin, norfloxacin), carbapenems (e.g., imipenem, meropenem, ertapenem, doripenem), aminoglycosides (e.g., gentamicin, tobramycin, amikacin), and polymyxins (e.g., colistin, polymyxin B).

[0224] In particular embodiments, the other drug is selected from the group consisting of a sulphonamide, a penicillin, a monobactam, a lincosamide, a fluoroquinolone, a carbapenem, an aminoglycoside, a polymyxin, and a combination thereof.

[0225] In particular embodiments, the other drug is colistin, gentamicin, aztreonam, meropenem, sulbactam, and a combination thereof. More in particular, the other drug is colistin.

[0226] Dosage and administration regimens for the polypeptide, fusion protein, the polynucleotide, the gene construct, the vector and / or the host cell of the invention have been described herein. The other drug may be administered at a dose and / or on a time schedule generally used for that other drug as single agent or in combination therapies.

[0227] In particular embodiments, administration of the polypeptide, fusion protein, the polynucleotide, the gene construct, the vector and / or the host cell of the invention as described herein is simultaneous to the administration of said other drug, as part of the same or separate compositions. In particular embodiments, administration as described herein is sequential (prior to or subsequent) to the administration of said another drug. In a twelfth aspect, the invention provides a kit of parts comprising the polypeptide as defined in the first aspect, the fusion protein as defined in the second aspect, the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth aspect, the vector as defined in the fifth aspect, the host cell as defined in the sixth aspect, the polypeptide or fusion protein as defined in the eighth aspect, and / or the composition as defined in the nineth aspect; and, optionally, instructions for its use.

[0228] In a thirteen aspect, the invention provides an in vitro method of inhibiting the growth, or reducing the population, or killing of Gram-negative bacteria; wherein the method comprises the step of contacting the bacteria with the polypeptide as defined in the first aspect, the fusion protein as defined in the second aspect, the polynucleotide as defined in the third aspect, the gene construct as defined in the fourth aspect, the vector as defined in the fifth aspect, the host cell as defined in the sixth aspect, the polypeptide or fusion protein as defined in the eighth aspect, and / or the composition as defined in the nineth aspect.

[0229] Reagents, tools, and / or instructions for performing the methods described herein can be provided in a kit. Forexample, the kit can contain reagents, tools, and instructions for use in the prophylactic and / or therapeutic treatment of an infection caused by Gram-negative bacteria or for conducting an in vitro method of inhibiting the growth, or reducing the population, or killing of Gram-negative bacteria. For instance, said Gram-negative bacteria are of one or more of the genus or species described herein above.

[0230] In a fourteenth aspect, the invention relates to the use of a polypeptide as defined in the first aspect, and / or the fusion protein as defined in the second aspect, as disinfectant for materials and / or surfaces; in particular, for abiotic materials and / or abiotic surfaces.

[0231] "Abiotic” refers to non-living materials, surfaces, or substances that are inherently devoid of life and are not derived from biological processes. The term does not preclude the presence of living organisms, such as microorganisms, on or in contact with these materials or surfaces; rather, it distinguishes the material or surface itself as non-living. Non limiting examples include synthetic polymers, metals, ceramics, and minerals. More in particular, in hospitals as well as in private households. These materials and / or surfaces include but are not limited to medical devices such as joint replacements and other types of orthopaedic instrumentation, prosthetic heart valves, pacemakers, implantable defibrillators, urinary catheters and stents, peritoneal dialysis catheters, intravascular catheters, cerebrospinal fluid shunts, breast implants, and vascular grafts and stents. A composition for this use may comprise the polypeptide and / or the fusion protein, and optionally further comprise other disinfectants and / or surfactants.

[0232] Also as shown in the examples, in addition to its preventive effects, the fusion protein of the disclosure demonstrated the capacity to eliminate pre-formed, 24-hour mature biofilms. In particular, biofilms of A. baumannii.

[0233] Thus, the disclosure also relates to the use of a polypeptide as defined in the first aspect, and / or the fusion protein as defined in the second aspect, in preventing the formation of biofilms and / or eliminating biofilms in materials and / or surfaces; in particular for abotic materials and / or abiotic surfaces. And in particular, of A. baumannii biofilms.

[0234] In a fifteenth aspect, the invention provides an antibody that specifically binds to a polypeptide comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 16; and / or a fusion protein comprising or consisting of a sequence selected from the group consisting of a sequence of SEQ ID NO: 32, a sequence of SEQ ID NO: 33, a sequence of SEQ ID NO: 36, a sequence of SEQ ID NO: 39, and a sequence of SEQ ID NO: 45.

[0235] Examples of antibodies include, but are not limited to monoclonal antibodies, polyclonal antibodies, antibody fragments, antibody derivatives, Fab fragments, Fab' fragments, F(ab)2 fragments, Fd fragments, Fv fragments, single-chain Fv fragments (scFv), diabodies, tribodies, tetrabodies, dimers, trimers, and minibodies. In particular embodiments of the fifteenth aspect, the antibody is a monoclonal antibody. The antibodies can be produced by any method known in the art for the generation of antibodies, in particular, by chemical synthesis or by recombinant expression techniques. Monoclonal antibodies can be prepared using awide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in W02009100309.

[0236] In further aspects, the invention provides:

[0237] - a polypeptide comprising a sequence of SEQ ID NO: 1 (Y-T-V-K-X1-G-D-T-L-S-X2-I-A-X3-K-Y-X4-T-X5-X6-X7-K-J8-X9-X10-X11-N-G-I-X12-N-X13-N-K-I-X14-V-X15-Q-X16-J17-R-X18-X19);

[0238] wherein Xi is selected from K and R; X2 is selected from K and G; X3 is selected from A and R; X4 is selected from G and B; X5 is selected from T and S; Xe is selected from Y and V; X7 is selected from Q and A; Js is selected from I and L; X9 is selected from A and V; X10 is selected from A and M; Xu is selected from D and L; X12 is selected from S and K; X13 is selected from P and N; X14 is selected from S and Y; X15 is selected from G and N; X is selected from V and S; J17 is selected from L and I; X is selected from I and V; and X19 is selected from T and K.

[0239] - a polypeptide comprising a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 21. Also, the invention provides a fusion protein comprising this polypeptide, in particular a fusion protein that comprises or consists of a sequence of SEQ ID NO: 155. The polypeptide and / or the fusion protein of this further aspect may be codified by a polynucleotide comprising a sequence selected from the group consisting of a sequence of SEQ ID NO: 67, a sequence of SEQ ID NO: 113, a sequence of SEQ ID NO: 156, and a sequence of SEQ ID NO: 157. - a polypeptide comprising a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 22. Also, the invention provides a fusion protein comprising this polypeptide, in particular a fusion protein that comprises or consists of a sequence of SEQ ID NO: 140. The polypeptide and / or the fusion protein of this further aspect may be codified by a polynucleotide comprising a sequence selected from the group consisting of a sequence of SEQ ID NO: 68, a sequence of SEQ ID NO: 114, a sequence of SEQ ID NO: 141, and a sequence of SEQ ID NO: 142. - a polypeptide comprising a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 23. Also, the invention provides a fusion protein comprising this polypeptide, in particular a fusion protein that comprises or consists of a sequence of SEQ ID NO: 143. The polypeptide and / or fusion protein of this further aspect may be codified by a polynucleotide comprising a sequence selected from the group consisting of a sequence of SEQ ID NO: 69, a sequence of SEQ ID NO: 115, a sequence of SEQ ID NO: 144, and a sequence of SEQ ID NO: 145.

[0240] - a polypeptide comprising a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 24. Also, the invention provides a fusion protein comprising this polypeptide, in particular a fusion protein that comprises or consists of a sequence of SEQ ID NO: 146. The polypeptide and / or fusion protein of this further aspect may be codified by a polynucleotide comprising a sequence selected from the group consisting of a sequence of SEQ ID NO: 70,a sequence of SEQ ID NO: 116, a sequence of SEQ ID NO: 147, and a sequence of SEQ ID NO: 148.

[0241] - a polypeptide comprising a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 25. Also, the invention provides a fusion protein comprising this polypeptide, in particular a fusion protein that comprises or consists of a sequence of SEQ ID NO: 149. The polypeptide and / or the fusion protein of this further aspect may be codified by a polynucleotide comprising a sequence selected from the group consisting of a sequence of SEQ ID NO: 71, a sequence of SEQ ID NO: 117, a sequence of SEQ ID NO: 150, and a sequence of SEQ ID NO: 151. - a polypeptide comprising a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 26. Also, the invention provides a fusion protein comprising this polypeptide, in particular a fusion protein that comprises or consists of a sequence of SEQ ID NO: 152. The polypeptide and / or the fusion protein of this further aspect may be codified by a polynucleotide comprising a sequence selected from the group consisting of a sequence of SEQ ID NO: 72, a sequence of SEQ ID NO: 118, a sequence of SEQ ID NO: 153, and a sequence of SEQ ID NO: 154. It should be understood that the particular embodiments described for the first aspect, as well as the aspects comprising or relating to that first aspect (i.e. aspects 2-15) and their embodiments, are equally applicable to and intended to be encompassed within the scope of these further aspects of the invention.

[0242] It is contemplated that any features described herein can optionally be combined with any of the embodiments of the polypeptide, fusion protein, polynucleotide, gene construct, vector, host cell, composition, kit, any use, medical use, method of treatment, method of manufacturing a medicament and combination therapies of the invention; and any embodiment discussed in this specification can be implemented with respect to any of these. It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention.

[0243] The term "or combinations thereof' as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof' is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAG, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled person will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0244] As used herein, words of approximation such as, without limitation, "about", "around”, "approximately” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion,a numerical value herein that is modified by a word of approximation such as "about" may vary from the stated value by plus / minus 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. Accordingly, the term "about” may mean the indicated value plus / minus 10% of its value, particularly the indicated value plus / minus 5% of its value, more particularly the indicated value plus / minus 2% of its value.

[0245] Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise” encompasses the case of "consisting of'. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

[0246] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses: 1. A polypeptide comprising a sequence at least 70% identical to a sequence of SEQ ID NO: 2 or to a sequence of SEQ ID NO: 3.

[0247] 2. The polypeptide according to clause 1, wherein the polypeptide comprises cell-permeating activity.

[0248] 3. The polypeptide according to any one of clauses 1-2, wherein the polypeptide comprises a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 2 or to a sequence of SEQ ID NO: 3.

[0249] 4. The polypeptide according to any one of clauses 1-2, wherein the polypeptide comprises at least two sequences independently selected from the group consisting of:

[0250] - a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 2; and

[0251] - a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 3.

[0252] 5. The polypeptide according to any one of clauses 1-2, wherein the polypeptide comprises:

[0253] - a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 2; and a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 3; - a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 6; or, alternatively,

[0254] - a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16.6. A fusion protein comprising the polypeptide as defined in any one of clauses 1-5.

[0255] 7. The fusion protein according to clause 6, further comprising a polypeptide comprising peptidoglycandegrading activity.

[0256] 8. The fusion protein according to any one of clauses 6-7, wherein the polypeptide comprising peptidoglycan degrading activity comprises a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence selected from the group consisting of a sequence of SEQ ID NO: 17, a sequence of SEQ ID NO: 18, a sequence of SEQ ID NO: 19, and a sequence of SEQ ID NO: 20.

[0257] 9. The fusion protein according to any one of clauses 6-8, further comprising a second polypeptide comprising cell-permeating activity; particularly wherein the second polypeptide comprising cell-permeating activity comprises a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence selected from the group consisting of a sequence of SEQ ID NO: 21, a sequence of SEQ ID NO: 16, a sequence of SEQ ID NO: 22, a sequence of SEQ ID NO: 23, a sequence of SEQ ID NO: 24, a sequence of SEQ ID NO: 25, a sequence of SEQ ID NO: 26, a sequence of SEQ ID NO: 27, a sequence of SEQ ID NO: 28, a sequence of SEQ ID NO: 29, a sequence of SEQ ID NO: 30, and a sequence of SEQ ID NO: 31.

[0258] 10. The fusion protein according to any one of clauses 6-9, wherein the fusion protein comprises:

[0259] - a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16; and a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 20;

[0260] - a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16; a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 20; and a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 29;

[0261] - a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16; a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 20; a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 29; and a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 21; or, alternatively,- a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16; a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 20; and a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 21.

[0262] 11. The fusion protein according to any one of clauses 6-10, wherein the fusion protein comprises or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence selected from the group consisting of a sequence of SEQ ID NO: 32, a sequence of SEQ ID NO: 33, a sequence of SEQ ID NO: 36, a sequence of SEQ ID NO: 39, and a sequence of SEQ ID NO: 45.

[0263] 12. The fusion protein according to any one of clauses 7-11, wherein the fusion protein comprises an activity selected from the group consisting of peptidoglycan binding activity, cell-permeating activity, peptidoglycandegrading activity, antibacterial activity, and combinations thereof.

[0264] 13. The fusion protein according to clause 12, wherein the antibacterial activity is against Gram-negative bacteria; particularly wherein the Gram-negative bacteria are from a genus selected from the group consisting of Acinetobacter, Pseudomonas, Escherichia, Klebsiella, and combinations thereof.

[0265] 14. A polypeptide as defined in any one of clauses 1-5 or a fusion protein as defined in any one of clauses 6-13 for use in medicine; particularly for use in the treatment and / or prevention of a bacterial infection caused by Gram-negative bacteria.

[0266] 15. An in vitro method of inhibiting the growth or reducing the population of Gram-negative bacteria, comprising the step of contacting the bacteria with the polypeptide as defined in any one of clauses 1-5 and / or the fusion protein as defined in any one of clauses 6-13.

[0267] Examples

[0268] Example 1. Materials and methods

[0269] Example 1.1. Bacterial strains and culture media

[0270] All the bacterial strains tested are detailed in Table 2. A. baumannii strains used in this study were routinely grown in tryptic soy broth (TSB; Condalab, Madrid, Spain) at 37°C with shaking at 200 rpm or on TSB plates containing 2% (wt / vol) bacteriological agar (TSA). Escherichia coli and P. aeruginosa strains were grown at 37°C with shaking in lennox broth (LB; Condalab, Madrid, Spain) or on plates of LB supplemented with 2% (w / v) agar. Particularly the E. coli BL21 (DE3) (Fisher, Spain) transformed with the plasmid pET29b(+) and that were used for protein cloning and expression, were grown at 37°C with shaking in LB medium or on plates of LB supplemented with 2% (w / v) agar and with the antibiotic for selection of the clones, 50 pg / mL of kanamycin (Fisher, Spain). All media were sterilized by autoclave at 120°C for 15 min or by filtration usingsterile 0.2 pm cellulose acetate filters.

[0271] Table 2:

[0272]

[0273]

[0274] >

[0275] >

[0276]

[0277] >

[0278] >

[0279] >

[0280] >

[0281] >

[0282] >

[0283] >

[0284]

[0285] >

[0286] >

[0287] >

[0288]

[0289] Example 1.2. Physicochemical and functional bioinformatics analysis of the proteins

[0290] The main physicochemical properties of polypeptides, fusion proteins and domains were calculated using the bioinformatic software Geneious Prime® 2022.1.1 and further versions up to Geneious Prime® 2025.0.2. (the main characteristics of polypeptides and fusion proteins are found at Table 3(A-H)). More specifically, the protein sequences of polypeptides, domains and EPLEs were analyzed using Interproscan from Geneious Prime and the percentage of pairwise similarity was calculated by BLASTP. The protein sequences were compared by CLUSTAL W alignment. Half-life stability of the proteins was determined using ProtParam. The 3D structure of polypeptides, fusion proteins and domains was predicted using Alphafold2 through Colabfold. The skilled person knows alternatives methodologies to the software and tools cited herein.

[0291] Table 3A:

[0292]

[0293]

[0294] Table 3B

[0295]

[0296] Table 3C

[0297]

[0298]

[0299] Table 3D

[0300]

[0301] Table 3E

[0302]

[0303]

[0304] Table 3F

[0305]

[0306] Table 3G

[0307]

[0308] Table 3H

[0309]

[0310]

[0311] Example 1.3. Plasmid construction and DNA manipulation

[0312] The genes encoding the polypeptides of interest were optimized based on E. coli codon usage using Geneious Prime software and synthetized at Twist Bioscience (San Fracisco, CA, USA). The EPLEs and polypeptides for expression were construed by known cloning techniques. This result in the cloning of the gene of interest into the pET29b(+) vector that introduces a C-terminal His6-tag and carries an ampicillin resistance gene the vectors where then transformed into chemically competent E. coli BL21 cells following suppliers' instructions by incubating the cells with the plasmid of interest for 30 min and performing a heat shock of 30 s at 42°C. Then cells are incubated in SOC medium for 1 h and plated onto LB agar supplemented with kanamycin as described above. Once transformed, the plasmid was extracted by miniprep using the NZYMiniprep kit (Lisboa, Portugal) and then sequenced by LGC genomics (Berlin, Germany) to ensure the correct sequence of the insert into the pET29b(+). The sequences were checked using Geneious Prime software.

[0313] Example 1.4. Protein expression and purification

[0314] Several colonies of E. coli BL21(DE3) transformed with the pET29b(+) vector containing the desired gene were harvested and incubated at 37°C shaking 200 rpm in 10 mL of LB supplemented with 50 pg / mL of kanamycin for 16-24 h. Then the culture is diluted in 1 L of prewarmed LB and incubated at 37°C shaking until the exponential growth phase (GD6000.5-0.6) was reached. Protein expression was then induced by addition of 0.5 mM IPTG (isopropyl-B-D-thiogalactopyranoside) and further incubation for 16-20 h at 16°C. Cells were collected after centrifugation (10,000 rpm, 4°C, 30 min) and suspended in 3 mL / g of cells of lysis buffer (20 mM TpNa, 500 mM NaCI, 10 mM imidazole, pH 7.5 supplemented with 0.1 mM PMSF, 1 mM benzamidine, and 1 mg / mL lysozyme). Pellets were freeze / thawed three times at -80°C. Sonication was carried out afterwards (10 x 30 s pulses with 30 s recovery on ice, 70% of amplitude) using the Branson Sonifier Power Supply (Branson, Danbury). Then, the suspension was centrifuged at 10,000 rpm, 4°C for 30 min and filtered using 0.45 pm PMSF membrane filters before purification. Proteins were purified using manifold coupled with the His GraviTrap™ Columns (Cytiva) and following the supplier's recommendations. Briefly, the clear lysate is applied to the column, followed by a washing with 20 mL of lysis buffer and 50 mL of washing buffer (20 mM TpNa, 500 mM NaCI, 50 mM imidazole, pH 7.5); the purified proteins were recovered with 3 mL of elution buffer (20 mM TpNa, 500 mM NaCI, 500 mM imidazole, pH 7.5). Subsequently, proteins were desalinized using ZebaTM Spin Desalting Columns 7K MWCO (Fisher, USA) and 20 mM TpNa, 75 mM NaCI, pH 7.5. Protein purity was evaluated in 8-16% SurePage™ gels (GenScript, Nanjing) with a constant voltage (200 V) in Tris-MOPS-SDS electrophoresis buffer (GenScript, Nanjing) and further revealed via conventional Coomassie staining. Proteins were sterilized by filtration using PMSF 0.2 pm membrane filters (VWR, Spain).The quantification of purified proteins was performed by NanoPhotometer® (Implen GmbH).

[0315] Example 1.5. Minimum inhibitory concentration (MIC) assay

[0316] The MIC of the fusion proteins was determined in triplicate using a standard broth microdilution method in casamino acid medium against a group of clinical strains (Table 1). To do that, two-fold dilutions of each protein were added to a microtiter plate containing 105-106CFU / mL. The MIC is herein defined as the lowest protein concentration that inhibited visible bacterial growth after incubation at 37°C. The final MIC values for each fusion protein and strain correspond to the mode of three independent biological repeats. Growth and sterility controls were included. The MIC was calculated as an average of 3 replicates. The MIC is expressed in piM or pig / mL for comparison of the activity of the proteins since the proteins tested do not have the same molecular weight. To make this transformation the online calculator from Bioline was used, although alternative calculator tools or methods are known by the skilled person. The MIC50 and MIC90 of the proteins were calculated as the lowest concentration of an antimicrobial capable to inhibit 50% (percentile 0.5) and 90% (percentile 0.9) of bacterial isolates, respectively.

[0317] Example 1.6. Time-killing assays

[0318] Time-killing assays were assessed, according to the common general knowledge, in microwells. Briefly, freshly grown colonies were picked from a plate and resuspended in the corresponding media of growth for each bacterium until getting an ODeoo=0.3 then an intermediate dilution is performed in CAA or in heat inactivated human serum so the final bacteria concentration in 200 piL well is 105-106CFU / mL. The proteins were added to an equimolar amount of 1 piM or 10 piM for the experiment with human serum. The mixture was incubated from 1 to 24 h and then cell counts were performed by plating 1:10 dilution of the bacteria onto media agar plates specific for each bacterium. The experiments were performed using 3 biological replicates. For combination studies, time-kill assays were also conducted using EPLETT200 (SEQ ID NO: 39) together with a last resource antibiotic, colistin at subinhibitory concentrations (typically 0.1x-0.25xMIC for the antibiotic and 0.25X-1XMIC for EPLETT200 (SEQ ID NO: 39)) to evaluate potential synergistic effects. Bacterial counts were determined as described above, and synergism was defined as a >2 logiOCFU / mL reduction for the combination compared with the most active single agent after 24 h of incubation.

[0319] Example 1.7. Animal Ethics Issues

[0320] C57BL / 6J female mice, aged seven weeks, were sourced from Charles River Laboratories. Throughout the experimental procedures, the animals were housed in compliance with regulatory standards, with unrestricted access to food and water. The care and use of the animals adhered to the European Directive on the protection of animals used for scientific purposes. Ethical approval for the study was granted by the Ethics and Clinical Research Committee of the University Hospital Virgen del Rocio, Seville, Spain, and the Consejeria De Agricultura, Pesca, Agua Y Desarrollo Rural, Junta de Andalucia . The number of animals utilized was minimized to achieve scientifically valid results.Example 1.8. Cumulative toxicity of the proteins

[0321] Three cohorts of n=6, 7-week-old healthy C57BL / 6 J female mice were administered intraperitoneally with a single daily dose over a period of three days, at concentrations of 1, 5 and 10 mg / kg for EPLETT200 (SEQ ID NO: 39) and EPLETT207 (SEQ ID NO: 45). Post-administration, over a seven-day observation period, the mice were monitored for indicative signs of pain, including reduced water and food intake (indicative of dehydration), social isolation, self-mutilation, tremors or spasms, dyspnea, alterations in physical activity levels, chromodacryorrhoea, muscle stiffness, piloerection, teeth grinding, and weight loss.

[0322] Additionally, for each protein, a separate group of n=6, 7-week-old healthy C57BL / 6 J female mice received the same dosage as those in the cumulative toxicity studies. Twenty-four hours following each treatment, two animals per protein per day were randomly selected and sacrificed for serum extraction. The serum samples were subsequently frozen at -80°C prior to being dispatched for further analysis.

[0323] Example 1.9. Detection of inflammatory response, hepatotoxicity and nephrotoxicity

[0324] Hepatotoxicity and nephrotoxicity assessments involved the collection of serum samples after mice dead or after euthanasia from the cumulative toxicity assay. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were quantified using the Mouse Alanine Aminotransferase ELISA Kit and Mouse Aspartate Aminotransferase Kit, respectively, from Innovative Research. Serum creatinine and blood urea nitrogen (BUN) concentrations were measured with the Mouse Creatinine Assay Kit from Crystal Chem and the Urea Nitrogen (BUN) Colorimetric Detection Kit from Invitrogen, respectively. Inflammatory cytokines, including TNF-o and IL-6, were analyzed using enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems, adhering to the manufacturer's protocols.

[0325] Example 1.10. Confirmation of the minimum lethal dose (MLP) of carbapenem resistant A. baumannii R&25 in the murine pneumonia model

[0326] To validate the minimum lethal dose (MLD) of 8.64 Iog10 CFU / mL in the murine pneumonia model as previously characterized using the A. baumannii R625 strain, a cohort of eight mice was inoculated with this concentration. Mice, anesthetized with ketamine and xylazine intraperitoneally, received an intratracheal inoculation of 50 pl of the 8.64 Iog10 CFU / mL solution. Mortality was recorded in six subjects over a seven-day period or until death occurred. Additionally, two mice were euthanized for two hours post-inoculation to verify bacterial load in the lungs and bloodstream. Lung and blood samples were collected and processed immediately post-mortem or post-euthanasia (sodium thiopental, intraperitoneally). Following euthanasia or death, aseptic thoracotomies were conducted, and blood samples were obtained aseptically for quantitative culture analysis (Iog10 CFU / mL). Subsequently, lungs were aseptically excised, weighed, and homogenized in sterile saline using a Stomacher 80 (Tekmar Co., OH, USA) for quantitative culture assessment (Iog10 CFU / g).

[0327] Example 1.11. Efficacy of protein in experimental carbapenem-resistant A. baumannii pneumonia murine model.Upon verification of the inoculum responsible for the infection using the selected strain, anaesthetized mice from the designated treatment groups were administered an intratracheal inoculation of 50 pl containing an 8.64 Iog10 CFU / mL concentration. The therapeutic groups were evaluated in separate weekly experiments, with each group consisting of five animals per week, to minimize potential errors.

[0328] The study involved the random assignment of animals into distinct treatment cohorts (n=15):

[0329] 1. Control Group: Infected, untreated animals.

[0330] 2. CMS Group: Infected animals treated with a suboptimal dose of colistimethate sodium (2.5 mg / kg, intraperitoneally, every 24 hours for 72 hours).

[0331] 3. EPLETT200 (SEQ ID NO: 39) Group: Infected animals treated with EPLETT200 (SEQ ID NO: 39) protein (5 mg / kg, every 24 hours, intraperitoneally).

[0332] 4. CMS + EPLETT200 (SEQ ID NO: 39) Combination Group: Infected animals treated with a combination of CMS and EPLETT200 (SEQ ID NO: 39) protein.

[0333] 5. EPLETT207 (SEQ ID NO: 45) Group: Infected animals treated with EPLETT207 (SEQ ID NO: 45) protein (5 mg / kg, every 24 hours, intraperitoneally).

[0334] 6. CMS + EPLETT207 (SEQ ID NO: 45) Combination Group: Infected animals treated with a combination of CMS and EPLETT207 (SEQ ID NO: 45) protein.

[0335] Three therapeutic interventions were initiated 2 hours post-infection and administered over a 72-hour period, with subsequent treatments occurring every 24 hours following the initial dose. Mortality was assessed over the 72-hour treatment period. Upon the conclusion of the study, or upon animal demise or euthanasia (via intraperitoneal administration of sodium thiopental), lung and blood samples were aseptically collected for quantitative culture analysis, reported as Iog10 CFU / g for lung tissue and Iog10 CFU / mL for blood. Blood samples also underwent qualitative culture analysis.

[0336] Example 1.12. Statistical analyses

[0337] In accordance with the characteristics of the in vitro data, such as normality and homoscedasticity, the Kruskal-Wallis test was employed alongside the U-Mann Whitney test. Additionally, Student's t-test was conducted for evaluating in vitro bactericidal activity assays. Statistical analyses were performed using GraphPad InStat version 3.0 (GraphPad Software, San Diego, CA) and Stata 15.0 (StataCorp LLC, College Station, TX).

[0338] Mortality rates and the incidence of positive qualitative blood cultures are presented as percentages.

[0339] Intergroup differences were assessed using the two-tailed Fisher's exact test. Quantitative bacterial cultures in pulmonary and blood samples, reported as log 10 CFU / g and log 10 CFU / mL, respectively, with means ±(plus / minus) standard deviation, were analyzed using Tukey's post hoc tests following confirmation of variance homogeneity via Levene's test. A p-value of less than 0.05 was deemed statistically significant. Dataanalysis was conducted using SPSS software, version 25.0 (SPSS Inc.). Toxicity data were expressed as mean ±(plus / minus) standard error of the mean (SEM). Statistical comparisons between toxicity groups were performed using non-parametric Kruskal-Wallis test and non-parametric Mann-Whitney u test. A p-value < 0.05 was considered statistically significant.

[0340] Example 1.13. Differential Scanning Fluorimetry (DSF)

[0341] The experiment was conducted using a CFX Duet Real-Time PCR System (BIO-RAD, Hercules, USA). The reaction mixture contained SYPRO Orange (Invitrogen, Spain) diluted in milliQ water to 500x, ensuring final concentrations at 5x along with 1% DMSO (Labkem, Spain) and the protein of interest. From this mixture, 20 pl were added per well into a qPCR plate before the protein. To determine best protein concentration, an initial titration assay was conducted, ideally between 1-20 pM, in buffer 20 mM TpNa, 75 mM NaCI, pH 7.5. Following protein concentration determination, various buffers ranging from 0 to 500 mM of NaCI were tested. The PCR System run included a 2-minute incubation at 25°C and a melting curve analysis from 25°C to 99°C with 0.2°C increments. All assays were performed in triplicate, including blanks for data normalization. For data analysis, raw data files were converted to a TSA-CRAFT compatible format, with data representation accomplished using a Python-based script.

[0342] Example 1.14. Confocal microscopy

[0343] Live-cell confocal microscopy was used to visualize the membrane-disruptive activity of EPLETT200 (SEQ ID NO: 39) against A. baumannii. Bacteria were grown overnight in tryptic soy broth (TSB) at 37°C with shaking and then diluted 1:10 in fresh medium and incubated for 2 h to reach exponential phase. For imaging, 10 pL of the culture were placed on a microscope slide and overlaid with a 1% low-melting agarose pad (1 x 1 cm) prepared in sterile water. The agarose pad was covered with a coverslip to form a sealed "sandwich” chamber, maintaining the bacteria in a hydrated environment.

[0344] Then 500 pg / mL of EPLETT200 (SEQ ID NO: 39) were added between the coverslips at the highest soluble concentration immediately before imaging. Samples were observed on an inverted confocal scanning laser microscope (DMI8; Leica Microsystems) equipped with a Leica DFC365FX digital camera, maintained at 37°C. Time-lapse imaging was performed for up to 60 min, acquiring frames every minute to monitor real-time morphological changes, pore formation, and cell lysis events.

[0345] Example 1.15. Minimum bactericidal concentration (MBC) assay

[0346] The MBC of the proteins was determined following standard procedures adapted from the CLSI guideline M26-A. After MIC determination, 10 pL from wells showing no visible growth were plated in duplicate onto blood agar and incubated at 35 ± 2 °C for 24-72 h depending on the bacterial species. The MBC is herein defined as the lowest protein concentration producing a > 99.9% (3-logiO) reduction in viable counts relative to the initial inoculum (2-8 x 105CFU / mL). All assays were performed in triplicate, and results are expressed as mean MBC values in pg / mL.Example 1.16. Turbidity Reduction Assay (TRA)

[0347] Peptidoglycan from A. baumannii was obtained from exponential-phase cultures (0D600= 0.3-0.4) grown in tryptic soy broth (TSB) at 37 °C with shaking. Cells from 1 L culture were harvested by centrifugation (10,000 x g, 10 min, 4 °C) and resuspended in 25 mL of chloroform-saturated 20 mM Tris-Na buffer (pH 7.4). The suspension was gently mixed for 45 min at 50-60 rpm to remove outer membrane components. After centrifugation (10,000 x g, 10 min, 4 °C), pellets were washed twice with 20 mM Tris-Na (pH 7.4) and resuspended to reach an optical density of 1.0 at 600 nm when diluted 1:1 with buffer. The resulting peptidoglycan preparation, a crude sacculi suspension containing residual cell wall fragments, was stored at-20 °C until use in muralytic activity assays. Protein activity was assayed in 96-well plates containing peptidoglycan adjusted to 0D600= 1 ■ Reactions were incubated at 37°C, with 0D600recorded every 2 min for 30 min using a Tecan plate reader. Specific activity was calculated as A0D / (min'pM), using the values obtained within the linear region of the reaction.

[0348] Example 1.17. Checkerboard assay and calculation of fractional inhibitory concentration index (FICI) The interaction between EPLETT200 (SEQ ID NO: 39) and antibiotics was assessed by the broth microdilution checkerboard method in cation-adjusted Mueller-Hinton broth. Two-fold serial dilutions of EPLETT200 (SEQ ID NO: 39) and each antibiotic were combined in 96-well plates to obtain concentration matrices from 4xMIC to 1 / 16xMIC. After inoculation with 5x10sCFU / mL and incubation at 37°C for 18-20 h, the MIC of each compound alone and in combination was determined. The fractional inhibitory concentration index (FICI) was calculated as FIC_A = A / MIC_A and FIC_B = B / MIC_B, where A and B represent the concentrations of each agent in combination and MIC_A and MIC_B their MICs when tested alone. The fractional inhibitory concentration index (FICI) was defined as FICI = FIC_A + FIC_B. Interactions were interpreted as synergistic (FICI < 0.5), indifferent (0.5 < FICI < 4), or antagonistic (FICI > 4).

[0349] Example 1.18. / n silico prediction of mechanism of action

[0350] Protein structure and function were analyzed using AlphaFold2 (https: / / www.deepmind.com / research / open-source / alphafold) through ColabFold (https: / / colabfold.mmseqs.com / ) for 3D prediction, and InterProScan (https: / / www.ebi.ac.uk / interpro / search / sequence / ) for domain annotation. Ligand- and ion-binding sites were identified with P2Rank (https: / / github.com / rdk / p2rank), ProBIS (https: / / probis.cmm.ki.si / ), MIB2 (https: / / bioinfo.gmu.edu / MIB2 / ), and lonCom (https: / / zhanggroup.org / lonCom / ). Sequence alignment and electrostatic surface mapping were performed using Geneious Prime (https: / / www.geneious.com / ) and PyMOL (https: / / pymol.Org / 2 / ).

[0351] Example 1.19. Biofilm elimination assay

[0352] Biofilms of Acinetobacter baumannii were formed on cellulose-coated pegs (MBEC Assay®; Innovotech Inc., Edmonton, AB, Canada) by incubating bacterial cultures in suitable growth medium for 24 h at 37 °C under static conditions. After biofilm maturation, the peg lids were transferred to fresh plates containing 200 pL of EPLETT200 (SEQ ID NO: 39) (200 pg / mL), ciprofloxacin (80 pg / mL), meropenem (128 pg / mL), or theircombinations. T reatments were applied in a double-dose format (3 + 3 h incubation at 37 °C). Following exposure, pegs were rinsed in PBS, stained with 0.1% crystal violet for 10 min, and destained with 30% ethanol. The remaining biofilm biomass was quantified by measuring absorbance at 595 nm, and results were expressed as the percentage of biofilm removal relative to untreated controls.

[0353] Example 1.20. Bacterial resistance and mutagenesis assays

[0354] To evaluate the potential for resistance development against EPLETT200 (SEQ ID NO: 39), A. baumannii, P. aeruginosa and E. coli cultures were subjected to serial passages under sublethal exposure. Briefly, bacteria were grown in CAA broth and exposed daily to 0.5xMIC concentrations for up to 25 consecutive rounds, followed by five additional passages in the absence of the compound. At each round, the minimum inhibitory concentration (MIC) was determined by broth microdilution, and resistance was defined as an eightfold or higher increase compared with the initial MIC. Three independent biological replicates were performed for each strain tested, with appropriate growth and sterility controls included. Cefiderocol, meropenem, and colistin were included against A. baumannii as positive controls.

[0355] The occurrence of spontaneous resistant mutants was assessed by plating 107- 108CFU of overnight cultures onto agar media containing EPLETT200 (SEC ID NO: 39) at 2x to 4xMIC. Plates were incubated for 48 h at 37 °C, and emerging colonies were purified and confirmed for resistance by MIC determination. Mutants exhibiting MICs > 8 times higher than the parental strain were classified as resistant.

[0356] For chemical mutagenesis, mid-exponential cultures (CD600= 0.5-0.6) were treated with 5 pig / mL N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 30 min at 37 °C with agitation. Cells were subsequently washed, resuspended in fresh medium, and incubated overnight to allow recovery. The resulting populations were plated on selective agar containing EPLETT200 (SEC ID NO: 39) at 2x to 4xMIC, and resistant colonies were isolated and confirmed by MIC testing. All confirmed mutants were preserved in glycerol stocks at -80 °C for further analysis.

[0357] Example 1.21. Cytotoxicity assay

[0358] The cytotoxicity of EPLETT200 (SEC ID NO: 39) was evaluated in human hepatic (HepG2) and lung fibroblast (MRC-5) cell lines cultured in standard conditions (37 °C, 5 % CO2, humidified atmosphere). Cells were seeded in 96-well plates and exposed for 24 h to serial concentrations of EPLETT200 (SEC ID NO: 39) ranging from 0.02 to 200 pig / mL Cell viability was quantified spectrophotometrically using the MTT colorimetric assay (570 nm). A 10 % DMSO solution was used as positive control for cytotoxicity, and untreated cells served as the negative control. Each condition was tested in triplicate, and results were expressed as percentage of proliferation relative to untreated control (mean ± SD).

[0359] Example 1.23. Biodistribution study

[0360] Biodistribution and organ targeting of EPLETT200 (SEC ID NO: 39) were assessed after intravenous administration of technetium-99m-labeled protein in healthy BALB / c male mice (n = 4). Radiolabeling wasperformed via an indirect tricarbonyl-mediated method using ["mTc][Tc(CO)3(H2O)3]+, achieving > 97 % radiochemical purity. Each animal received 5.3 MBq (« 100 pig protein) in 110 piL intravenously.

[0361] In vivo imaging was conducted using a U-SPECT6 / E-class SPECT / CT system (Ml Labs, Netherlands) equipped with a UHR-RM-1 mm collimator. Whole-body scans were acquired at 15 min, 30 min, 1 h, 4 h, and 6 h post-injection under continuous isoflurane anesthesia (2 % in O2). Images were reconstructed using the "mTc photopeak (145 keV ± 20 %) and analyzed with PMOD software (PMOD Technologies, Switzerland) to obtain standardized uptake values (SUVmean).

[0362] At 6 h post-administration, animals were euthanized, and organs (brain, eyes, heart, kidneys, liver, testicles), blood, and urine were collected. Samples were weighed and analyzed using a calibrated gamma counter (Hidex Automatic Gamma Counter, Finland). Radioactivity was expressed as the percentage of injected dose per gram of tissue (%ID / g), corrected for decay.

[0363] Example 2. CWBs of the invention from polypeptides containing LysM domains exhibit enhanced predicted stability in human serum.

[0364] The half-life of the PCP8 (SEQ ID NO: 30) in mammalian reticulocytes is estimated to be relatively short at 1.4 hours. In contrast, four out of six polypeptides are predicted to exhibit extended stability, with a half-life of 30 hours in human serum. As shown in Table 4.

[0365] Table 4: CWB conserved domains, homology, estimated half-life in serum

[0366]

[0367] Example 3. CWB30 (SEQ ID NO: 16) enhanced the activity against E. coli of the proteins when combined with an enzymatic activity polypeptide

[0368] To evaluate the potential enhancement of activity through the incorporation of the new cell-wall binding polypeptides (CWB) into a fusion protein (EPLE). Specifically, six EPLE variants were engineered (EPLETT44-EPLETT49), each featuring the EAD7 (SEQ ID NO: 20) polypeptide in conjunction with one of the six distinct CWB polypeptides of the invention (CWB30-CWB35). The corresponding genes were cloned into the expression vector pET29b(+) by techniques know by the skilled person, and subsequently purified via nickel immobilized metal affinity chromatography (IMAC). All constructs demonstrated successful expression and purification through nickel IMAC except for EPLETT47 (SEC ID NO: 146) and EPLETT49 (SEC ID NO: 152) (data not shown), these two were discarded for further experiments.

[0369] The bactericidal efficacy of EPLEs was evaluated against E. coli ATCC 25922 and compared against TT206 (SEC ID NO: 158) (which includes an enzymatic activity polypeptide (EAD7 (SEC ID NO: 20)) and a reference cell-wall binding polypeptide (PCP8 (SEQ ID NO: 30)) in human serum (Figure 1). Exponentially growingbacterial cultures were treated with 10 pM of the proteins in human serum. All the EPLEs tested (EPLETT44 (SEQ ID NO: 32), EPLETT45 (SEQ ID NO: 140), EPLETT46 (SEQ ID NO: 146) and EPLETT48 (SEQ ID NO: 149)) exhibited improved reduction in bacterial count compared to TT206 (SEQ ID NO: 158). Among them, EPLETT44 (SEQ ID NO: 32) (with a similar structure that the TT206 (SEQ ID NO: 158) but changing the cellwall binding polypeptide: EAD7 +CWB30) emerged as the most potent protein, achieving a 1 Log unit reduction in bacterial count. Proteins' activity was further assessed using a MIC assay in CAA media and the same strain, which confirmed the serum results. EPLETT44 (SEQ ID NO: 32) exhibited the lowest MIC at 0.3 pM, whereas TT206 (SEQ ID NO: 158) had a MIC of 2.5 pM, and the remaining proteins had MIC values exceeding 2.5 pM (Figure 1).

[0370] The findings demonstrate that the incorporation of CWB30 (SEQ ID NO: 16) to EAD7 (SEQ ID NO: 20) enhances the protein's activity against E. coli, even in the presence of human serum. Among all the polypeptides evaluated, CWB30 (SEQ ID NO: 16) exhibited the most promising in vitro results.

[0371] It was also demonstrated that engineering variants adding LysM domains to the C-terminus of proteins enhanced both stability and activity. EPLETT50 (SEQ ID NO: 33) was developed, featuring EAD7 (SEQ ID NO: 20) followed by a repeated CWB30 (SEQ ID NO: 16), thus containing four LysM domains (two LysM1 (SEQ ID NO: 2) and two LysM2 (SEQ ID NO: 3); or two 2xLysM (SEQ ID NO: 6). The MIC against E. coli ATC 25922 matched that of EPLETT44 (SEQ ID NO: 32) at 0.3 pM, with reduced aggregation observed in SDS-PAGE analysis (data not shown). EPLETT50 (SEQ ID NO: 33) demonstrated efficacy against E. coli clinical strains, with a median MIC of 0.17 pM across all strains (Table 5).

[0372] Table 5. MIC values of EPLETT50 (SEQ ID NO: 33) (pM) against clinically relevant E. coli strains including carbapenem resistant strains. Values represent mean ±standard deviation of 3 biological replicates.

[0373]

[0374] Example 4. The incorporation / fusion of CWB30 (SEQ ID NO: 16) into / with various activity polypeptides enhanced the protein's efficacy against Gram-negative bacteria, notably including A. baumannii, E. coli, and P. aeruginosa.

[0375] EPLETT 117 (SEQ ID NO: 155) is characterized for its outer membrane-degrading activity, exhibiting a minimum inhibitory concentration (MIC) of 0.8 pM against A. baumannii DSM 25645. EPLETT117 (SEQ ID NO: 155) consists of CWB28 (SEQ ID NO: 21), a PG_binding 1 (IPR002477) type polypeptide, and a POP3(SEQ ID NO: 29), a positively charged peptide derived from a lysin. To enhance protein activity, various polypeptide combinations of CWB28 (SEQ ID NO: 21), POP3 (SEQ ID NO: 29), EAD7 (SEQ ID NO: 20), and CWB30 (SEQ ID NO: 16) were explored. The resulting proteins, EPLETT195 to EPLETT209, demonstrated activity against A baumannii, with EPLETT197 (SEQ ID NO: 36), EPLETT200 (SEQ ID NO: 39), and EPLETT207 (SEQ ID NO: 45) identified as the most active and stable candidates.

[0376] Table 6. MIC values of EPLETT195-EPLETT209 (pM) against A baumannii DSM 25645. Expression and purity are checked by SDS-PAGE after IMAC purification, considering "Ok” when purity was higher or equal than 85% (i.e. >85%). Stability was tested by frozen the proteins with 20% glycerol and performing SDS-PAGE.

[0377] >

[0378]

[0379] *ND: not determined

[0380] The minimum inhibitory concentration (MIC) values for (EPLETT197 SEQ ID NO: 36), EPLETT200 (SEQ ID NO: 39), and EPLETT207 (SEQ ID NO: 45) were determined against clinically significant pathogens, including strains of A. baumannii, E. coli, and P. aeruginosa (Table 7 and Table 1). Notably, EPLETT200 (SEQ ID NO: 39) and EPLETT207 (SEQ ID NO: 45) exhibited the most potent activity, with M IC50 and MIC90 values approximately at 0.3 M and 1 pM, respectively, across all tested strains.

[0381] Table 7. MIC values of EPLETT197 (SEQ ID NO: 36), EPLETT200 (SEQ ID NO: 39) and EPLETT207 (SEQ ID NO: 45) (pi M) against 50 clinical MDR or carbapenem resistant clinical strains of relevant A. baumannii, E. coli and P. aeruginosa. MIC50 and MIC90 are calculated as percentile 0.5 and 0.9 respectively.

[0382]

[0383] |

[0384]

[0385] The experimental findings indicate that EPLETT200 (SEQ ID NO: 39) and EPLETT207 (SEQ ID NO: 45), when tested at a concentration equivalent to MIC90 (1 pM) in a time-kill assay, demonstrate potent bactericidal activity. The proteins effectively eradicate the majority of bacterial cells within 1 hour, with no observable regrowth after 3 hours of treatment, as illustrated in Figure 2A and Figure 2B.

[0386] Example 5. In vivo models demonstrate that EPLETT200 (SEQ ID NO: 39) and EPLETT207 (SEQ ID NO: 45), which carry CWB30 (SEQ ID NO: 16), do not show cumulative toxicity.

[0387] The conducted toxicity assessments used three concentrations of EPLETT200 (SEQ ID NO: 39) and EPLETT207 (SEQ ID NO: 45), specifically 1, 5, and 10 mg / kg. Throughout the 7-day study duration, none of the evaluated treatments exhibited any of the 11 symptoms indicative of pain or toxicity. Additionally, the weights of individual animals were monitored, revealing no significant differences (data not shown).

[0388] Hepatic (ALT and AST) and renal (serum creatinine and BUN) toxicity markers, along with inflammatory markers (TNF-a, IL-6), were assessed in blood samples from mice exposed to the proteins for 72 hours at the 3 concentrations mentioned above to evaluate acute toxicity. The findings indicated no significant differences compared to serum baseline values (Table 8).

[0389] Table 8. Levels of inflammatory, hepatotoxicity, and nephrotoxicity markers following a 72-hour treatment regimen consisting of three doses administered every 24 hours. Values represent expressed as the mean ± standard deviation of 2 biological replicates.

[0390]

[0391]

[0392] Example 6. In vivo pneumonia models of A. baumannii indicate that EPLETT200 (SEQ ID NO: 39) and EPLETT207 (SEQ ID NO: 45) are capable of protecting mice as stand alone and achieving complete survival when used in conjunction with suboptimal antibiotic doses.

[0393] To validate the appropriate pneumonia model for A. baumannii, eight anesthetized mice underwent intratracheal inoculation with 50 pl of an 8.64 Iog10 cfu / mL suspension. Over a seven-day observation period, mortality was recorded in six subjects, with all succumbing between 46 and 68 hours post-infection. Postmortem, aseptic collection and guantitative analysis of lung and blood samples revealed 100% bacteremia, with bacterial loads of 11.22 ± 0.28 Iog10 CFU / g in lung tissue and 9.39 ± 0.56 Iog10 CFU / ml in blood, thereby confirming successful infection establishment. Moreover, the bacterial counts after 2 h of infection were determined to be 8.94 ± 0.00 and 4.43 ± 0.36 in lungs and blood, respectively.

[0394] The study demonstrated that among the various combinations evaluated, EPLETT200 (SEQ ID NO: 16) in conjunction with colistin resulted in complete survival. It is important to highlight that the colistin doses administered were suboptimal, thereby reducing its potential toxicity in humans (Table 9). Notably, both EPLETT200 (SEQ ID NO: 39) (10 mg / kg)and EPLETT207 (SEQ ID NO: 45) yield favourable survival outcomes. Notably, EPLE achieved survival rates comparable to those observed with colistin. Survival was in both cases enhanced when the EPLE was used in combination with colistin.

[0395] Table 9. In vivo evaluation of the therapeutic efficacy of EPLETT200 (SEQ ID NO: 39), EPLETT207 (SEQ ID NO: 45), and CMS, both individually and in combination, within an experimental pneumonia model. Statistical analyses were conducted using Fisher's exact test, Tukey's post hoc test, and Levene's test. Significance was determined as follows: a: p<0.05 compared to the control group; b: p<0.05 compared to the protein group; c: D<0.05 compared to the CMS group.

[0396] <

[0397] <

[0398]

[0399] ip: intraperitoneal; CMS: colistimethate sodium.Further evaluation of EPLETT200 (SEQ ID NO: 39) activity, intravenous (IV) administration of EPLETT200 (SEQ ID NO: 39) as a monotherapy achieved 100% survival, clearly superior to the 0% observed in untreated controls and to the 73% survival with 2.5 mg / kg colistimethate sodium (CMS) alone. Interestingly, combining EPLETT200 (SEQ ID NO: 39) with suboptimal CMS dosing did not further improve survival outcomes or bacterial clearance, which remained comparable to EPLETT200 (SEQ ID NO: 39) alone. EPLETT200 (SEQ ID NO: 39) resulted in >3 log reduction in the bacterial counts in lung and blood compared to untreated controls (Table 11). These results establish a robust in vivo proof of concept for systemic antibacterial activity and support the potential of EPLETT200 (SEQ ID NO: 39) in treating multidrug-resistant A. baumannii infections.

[0400] Table 11. In vivo evaluation of the therapeutic efficacy of EPLETT200 (SEQ ID NO: 39), EPLETT207 (SEQ ID NO: 45), and CMS, both individually and in combination, within an experimental pneumonia model. Statistical analyses were conducted using Fisher's exact test, Tukey's post hoc test, and Levene's test. Significance was determined as follows: a: p<0.05 compared to the control group; b: p<0.05 compared to the protein group; c: p<0.05 compared to the CMS group, in: intraperitoneal; CMS: colistimethate sodium.

[0401] <

[0402]

[0403] Example 7. CWB30 (SEQ ID NO: 16) is stable at high salt concentrations and confers stability in salt to proteins that carry this polypeptide

[0404] Protein stability for CWB30 (SEQ ID NO: 16) and PCP8 (SEQ ID NO: 30) was assessed via Differential Scanning Fluorimetry (DSF). This technique employs real-time PCR to observe thermally induced protein denaturation by detecting fluorescence changes in SYPRO Orange dye, which preferentially binds to unfolded proteins. The resulting fluorescence data facilitates the visualization of thermostability curves and the determination of the melting temperature (Tm), defined as the temperature at which fluorescence intensity reaches 50% of its maximum. In the absence of protein or when a denatured protein is present, the Tm corresponds to the initial temperature (Table 10). The data revealed that CWB30 (SEQ ID NO: 16) demonstrates superior stability, evidenced by a thermal shift ATm of 2.6 °C.

[0405] Table 10. Tm of CWB30 (SEQ ID NO: 16) and PCP8 (SEQ ID NO: 30) measured by DSF in a standard 20 mM sodium phosphate buffer, 75 mM NaCI, pH 7.4. Values represent media ± standard deviation of three biological replicates.

[0406]

[0407] The differential scanning fluorimetry (DSF) analysis was also conducted on EPLETT200 (SEQ ID NO: 39), which incorporates CWB30 (SEQ ID NO: 16), utilizing buffers with increasing salt concentrations (Figure 3). The findings indicated that EPLETT200 (SEQ ID NO: 39) exhibits a high tolerance to elevated salt concentrations, with optimal thermal stability observed at NaCI concentrations of 100 mM or higher (Figure 3).

[0408] Example 8. EPLETT200 (SEQ ID NO: 39) exhibits a dual bactericidal mechanism combining enzymatic peptidoglycan degradation and outer membrane permeabilization

[0409] The predicted three-dimensional structure of EPLETT200 (SEQ ID NO: 39), obtained through AlphaFold2 modelling, revealed a modular four-domain architecture with well-defined folding and high structural confidence. Ligand-binding predictions using P2Rank and ProBIS identified conserved cavities within the protein consistent with peptidoglycan recognition, while MIB2 and lonCom analyses revealed potential ionbinding pockets (Ca2+, Mg2+, Zn2+, Fe2+), which without being bound to any theory, would indicate that EPLETT200 (SEQ ID NO: 39) chelates divalent cations and destabilize the outer membrane. The combination of these findings, supported by structural overlays and electrostatic mapping, indicates a dual mechanism of action involving enzymatic muralysis and membrane permeabilization.

[0410] A single LyM domain is capable of providing binding strength to a lysin, and multiple LysM domains in a lysin contribute primarily in an additive manner to carbohydrate and / or peptidoglycan binding affinity. This already indicates that the cell-permeating activity of a polypeptide comprising multiple LysM domains occurs due to the added effects of each LysM domain. Therefore, it is plausible that a polypeptide comprising one of said multiple LysM domain does also have cell-permeating activity by itself. Moreover, the in silico mechanism of action reported for EPLETT200 (SEQ ID NO: 39) shows that the CWB30 (SEQ ID NO: 16) region itself contains predicted ligand- and ion-binding pockets formed by residues located within each LysM domain (for example, a Ca2+-binding pocket involving residues 304K / 308D / 309N within LysM1, and Na+-binding pockets involving residues 276-280 in LysM1 or 345-381 in LysM2). This indicates that each LysM1 and LysM2 independently present a structurally complete binding surface for substrate / ion interaction. On this basis, it is further corroborated that a construct comprising only one first LysM1 (SEQ ID NO: 2) or LysM2 (SEQ ID NO: 3) would still plausibly exhibit the same underlying cell-permeating mechanism; and also that it would be plausibly useful in medicine and in particular for use in the treatment and / or prevention of a bacterial infection caused by Gram-negative bacteria.

[0411] In vitro evaluation of antimicrobial activity confirmed the bioinformatic predictions. The inhibitory potency of EPLETT200 (SEQ ID NO: 39) was determined by minimum inhibitory concentration (MIC) ranging from 4 to 64 pig / mL across A. baumannii strains.

[0412] Consistent with these results, the enzymatic (muralytic) activity of EPLETT200 (SEQ ID NO: 39) was quantified using a kinetic turbidity reduction assay (TRA) against A. baumannii peptidoglycan. The decrease in optical density at 600 nm was recorded over 30 minutes at 37°C, and specific activity was calculated as AOD / (min'piM), using the values obtained within the linear region of the reaction. EPLETT200 (SEQ ID NO: 39) exhibited specific activities ranging from 0.775 to 1.475 AOD'min“1'piM“1, confirming its ability toenzymatically degrade bacterial cell walls through peptidoglycan hydrolysis mediated by the EAD7 (SEQ ID NO: 20) catalytic domain.

[0413] Complementary confocal microscopy experiments provided direct visual evidence supporting the proposed dual mechanism of outer membrane permeabilization and lytic activity. A. baumannii cells treated with EPLETT200 (SEQ ID NO: 39) displayed rapid morphological alterations and pore formation within minutes of exposure, followed by progressive cell disruption and lysis. The images (Fig. 4) acquired using an inverted confocal scanning laser microscope revealed cell wall alteration consistent with enzymatic degradation and outer membrane destabilization.

[0414] Example 9. Broad and consistent bactericidal activity of EPLETT200 (SEQ ID NO: 39) against clinical Gramnegative isolates

[0415] The distribution of EPLETT200 (SEQ ID NO: 39) MICs across Gram-negative pathogens showed a predominance of values between 4 - 16 pig / mL (Table 12).

[0416] Overall, MIC and MBC values of EPLETT200 (SEQ ID NO: 39) against A. baumannii, E. coli and P. aeruginosa were generally close, most often within one or two dilution steps, supporting a predominantly bactericidal profile. In A. baumannii, MIC values were commonly 16 pig / mL, with MBCs ranging from 16 to 64 pig / mL Several isolates presented identical MIC and MBC values (e.g., 16 / 16 pig / mL), while in others the MBC was higher (e.g., 16 / 64 pig / mL or 32 / 64 pig / mL), indicating that a higher concentration was required for bactericidal effect (Table 12). In summary, EPLETT200 (SEQ ID NO: 39) showed MIC and MBC values that were generally consistent across the tested Gram-negative pathogens, with most isolates displaying similar or closely related values.

[0417] Table 12. Antimicrobial in vitro MIC and MBC data for EPLETT200 against a group of clinical bacteria.

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425] Example 10. EPLETT200 (SEQ ID NO: 39) displays rapid, MIC-dependent bactericidal activity and synergism with antibiotics against A. baumannii

[0426] To further investigate the combinatorial potential of EPLETT200 (SEQ ID NO: 39) with clinically relevant antibiotics, checkerboard assays were performed against several A. baumannii strains (Table 13). The results indicated colistin consistently showing favourable interactions across all isolates tested. Notably, for strain CRAB-3, synergy was observed at a low combination ratio of 4 pg / mL EPLETT200 (SEQ ID NO: 39) and 0.25 pg / mL colistin. For A. baumannii MDR-1, the EPLETT200 (SEQ ID NO: 39) showed synergism with colistin (3:0.25), gentamicin (6:40), and aztreonam (6:20), while for A. baumannii CRAB-2 synergism was exhibited with gentamicin and sulbactam. Although the effect with colistin was classified as indifferent in A. baumannii CRAB-1, two out of three replicates showed clear synergy at low concentrations (0.06 pg / mL EPLETT200 (SEQ ID NO: 39) +0.5 pg / mL colistin), suggesting a concentration-dependent interaction.

[0427] Table 13. Checkerboard assay results showing the interaction between EPLETT200 (SEQ ID NO: 39)_and selected antibiotics against A. baumannii strains. Ratios are presented as pg / mL of protein : antibiotic. A: antagonism; I: indifferent; S: synergism. I*: In the case of A. baumannii CRAB-1, although the overall outcome was classified as indifferent, synergy was observed in two out of three replicates at a concentration of 0.06 pg / mL EPLETT200 and 0.5 pg / mL colistin. ATM: aztreonam; GEN: gentamycin; AMI: amikacin; CIP: ciprofloxacin; COL: colistin; IMI: imipenem; MEM: meropenem; S: sulbactam.

[0428]

[0429] Checkerboard synergy data were validated by time-kill experiments using the A. baumannii CRAB-1 strain. Treatment with subinhibitory concentrations of colistin (O.IxMlC) or EPLETT200 (SEQ ID NO: 39) (0.25xMIC) alone led to limited bacterial reduction, with surviving cells detectable after 24 h. However, the combination of both agents resulted in complete killing within 24 h (Table 14). Importantly, colonies recovered after 24 h exposure to either agent alone did not exhibit increased MIC values, confirming that no resistance had emerged during the experiment.

[0430] Together, these findings support the clinical development of EPLETT200 (SEQ ID NO: 39) in combination with antibiotics, particularly colistin, for the treatment of resistant A. baumannii infections.Table 14. In vitro synergy of EPLETT200 (SEQ ID NO: 39) with colistin against A. baumannii CRAB-1. Timekill curves of CRAB-1 exposed to subinhibitory concentrations of colistin (0.1 xMIC; 4 pg / mL) or EPLETT200 (SEC ID NO: 39) (0.25xMIC; 4 pg / mL). Bacterial counts (logiOCFU / mL) were monitored over 24 h. Survivors recovered after 24 h of treatment were re-tested by MIC assays to confirm that no resistance to either colistin

[0431]

[0432] Example 11. Evaluation of the antibiofilm properties of EPLETT200 (SEC ID NO: 39)

[0433] The antibiofilm properties of EPLETT200 (SEC ID NO: 39) were comprehensively evaluated in both biofilm prevention and elimination models using A. baumannii, a clinically relevant pathogen associated with persistent hospital-acquired infections.

[0434] When administered at sub micromolar concentrations, EPLETT200 (SEQ ID NO: 39) effectively prevented the formation of A. baumannii biofilms. The minimum biofilm prevention concentration required to achieve maximal inhibition was 25 pg / mL (approximately 0.5 pM), resulting in a robust average reduction of 51.5 ± 7.0% in biofilm formation. Notably, this inhibitory effect was achieved with 100 to 1000-fold lower concentrations than those required by conventional antibiotics tested in parallel, such as meropenem, ciprofloxacin, and nitrofurantoin. These findings underscore the potent prophylactic efficacy of EPLETT200 (SEQ ID NO: 39) and support its application in the prevention of device-associated or nosocomial infections where biofilm formation is a critical challenge.

[0435] In addition to its preventive effects, EPLETT200 (SEQ ID NO: 39) demonstrated the capacity to eliminate preformed, 24-hour mature biofilms of A. baumannii. A double-dose treatment regimen (3 + 3 h exposure) led to an average biofilm biomass reduction of approximately 30%. By contrast, treatment with ciprofloxacin or meropenem alone at clinically relevant concentrations produced minimal to no measurable biofilm removal under the same conditions (Table 15).

[0436] Importantly, when EPLETT200 (SEQ ID NO: 39) was combined with either ciprofloxacin or meropenem, the level of biofilm elimination significantly increased, reaching -40% biomass reduction. This synergistic effect highlights the potential utility of EPLETT200 as an adjuvant in combination therapies targeting resilient biofilm-associated infections (Table 15).

[0437] Table 15. In vitro elimination of pre-formed biofilms of A. baumannii b EPLETT200 (SEQ ID NO: 39) and its combinations with antibiotics. Percentage of biofilm removal following treatment with EPLETT200 (SEQ ID NO: 39) (200 pg / mL), ciprofloxacin (80 pg / mL), meropenem (128 pg / mL), or their combinations. Treatments were administered in a double-dose format (3 +3 h). Statistical analysis: a p < 0.05 vs correspondingantibiotic alone; b p < 0.05 vs EPLETT200 alone.

[0438]

[0439] Example 12. No resistance or mutagenic variants emerge after prolonged exposure to EPLETT200 (SEQ ID NO: 39)

[0440] Resistance studies demonstrated that A. baumannii, P. aeruginosa, and E. coli did not develop resistance to EPLETT200 (SEQ ID NO: 39), even after 25 consecutive passages under subinhibitory pressure. In contrast, conventional antibiotics such as meropenem, colistin, and cefiderocol exhibited progressive increases in MIC values, particularly against A baumannii (Table 16). The stability of EPLETT200 (SEQ ID NO: 39) susceptibility was further confirmed through five additional passages performed in the absence of selective pressure, which maintained unaltered MIC values across all bacterial species. In comparison, the elevated MICs observed for the antibiotic-treated strains remained stable, confirming the persistence of resistance (Table 17). Moreover, neither spontaneous nor chemically induced resistant mutants could be obtained for EPLETT200 (SEQ ID NO: 39), with a resistance freguency below 10“8, highlighting its low mutagenic potential and strong barrier to resistance development.

[0441] Table 16. Fold-change in MIC values over 25 consecutive passages of A. baumannii, E. coli and P. aeruginosa exposed daily to subinhibitory concentrations (0.5xMIC) of EPLETT200 (SEQ ID NO: 39) compared with conventional antibiotics (meropenem, cefiderocol, and colistin).

[0442]

[0443]

[0444] Table 17. Stability of MIC values after withdrawal of antimicrobial pressure. MICs of A. baumannii, E. coli, and P. aeruginosa after 25 serial passages under subinhibitory concentrations followed by five additional passages in the absence of antimicrobial exposure.

[0445] >

[0446]

[0447] Example 13. EPLETT200 (SEQ ID NO: 39) shows no cytotoxic effects on human hepatic and fibroblast cells The cytotoxic potential of EPLETT200 (SEQ ID NO: 39) was assessed in human hepatic (HepG2) and lung fibroblast (MRC-5) cell lines. Cells were exposed for 24 h to serial concentrations ranging from 0.02 to 200 pig / mL No cytotoxic effects were observed at any concentration tested (Table 18).

[0448] In HepG2 cells, relative proliferation ranged from 105% at 200 pig / mL to 22% at 2 pig / mL, compared with the untreated control (100 %), indicating that EPLETT200 (SEQ ID NO: 39) did not impair cell growth. In MRC-5 fibroblasts, proliferation values remained between 73 % and 109 % of control levels, with no statistically significant differences observed. The positive control (10 % DMSO) reduced viability to below 15 %, confirming assay validity.

[0449] Overall, these results indicate that EPLETT200 (SEQ ID NO: 39) does not exert cytotoxic or antiproliferative effects on hepatic or fibroblast cell lines under the tested conditions (Table 18).

[0450] Table 18. In vitro cytotoxicity of EPLETT200 (SEQ ID NO: 39) in human hepatic (HepG2) and fibroblast (MRC-5) cell lines. Cell proliferation after 24 h exposure to increasing concentrations of EPLETT200 (SEQ ID NO: 39), expressed as mean ± standard deviation (n = 3) and percentage of proliferation relative to untreated controls. DMSO (10 %) was used as a positive control for cytotoxicity. No reduction in cell viability was observed in either cell line across the tested concentration range.

[0451]

[0452] |

[0453]

[0454] Example 14. EPLETT200 (SEQ ID NO: 39) displays selective pulmonary retention and renal clearance after intravenous administration

[0455] Biodistribution analysis was conducted to evaluate the systemic disposition and tissue targeting of EPLETT200 (SEQ ID NO: 39) following intravenous (IV) administration in healthy mice. The protein was radiolabeled with technetium-99m, and its in vivo distribution was monitored using SPECT / CT imaging complemented with ex vivo gamma counting.

[0456] Imaging data revealed rapid systemic distribution, with predominant renal clearance and detectable accumulation in the renal cortex, consistent with elimination through the urinary pathway. Low hepatic retention was observed, and no radioactivity was detected in the brain, confirming that EPLETT200 (SEQ ID NO: 39) does not cross the blood-brain barrier. Notably, pulmonary uptake was evident as early as 15 minutes post-injection and persisted up to 6 hours, indicating selective retention in the lungs. This illustrates the capacity of EPLETT200 (SEQ ID NO: 39) to penetrate tissues and target specific organs, such as the lungs, within the initial hours of treatment. Imaging data not shown.

[0457] Ex vivo quantification at 6 hours confirmed these findings, showing the highest radioactivity levels in urine, kidneys, liver, and lungs, while negligible amounts were found in other organs, including heart, eyes, testicles, and brain (Table 19). This biodistribution pattern supports a favourable pharmacological profile, with targeted pulmonary exposure and minimal off-target accumulation.

[0458] Table 19. Ex vivo biodistribution of EPLETT200 (SEQ ID NO: 39) at 6 hours post-intravenous administration.

[0459]

[0460] Tissue-associated radioactivity is expressed as the percentage of injected dose per gram of tissue (%ID / g). The highest accumulation was observed in the urine, liver, kidneys, and lungs. Notably, EPLETT200 (SEQ ID NO: 39) remained detectable in the lungs 6 hours post-administration, consistent with its targeted pulmonary activity. Distribution to other organs such as eyes, heart, testicles, and brain was minimal, supporting a favourable tissue specificity profile. Detection in the lungs was confirmed as early as 15 minutes postadministration (data not shown).

[0461] Citation List

[0462] Patent Literature

[0463] WQ2009100309

[0464] Non Patent Literature

[0465] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277

[0466] Altschul et al., "Basic local alignment search tool”, 1990, J. Mol. Biol, v. 215, pages 403-410

[0467] Clinical and Laboratory Standards Institute (CLSI): M26-A - Methods for Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline

Claims

Claims1. A polypeptide comprising a sequence at least 85 % identical to a sequence of SEQ ID NO: 2 or to a sequence of SEQ ID NO: 3.

2. The polypeptide according to claim 1, wherein the polypeptide comprises cell-permeating activity.

3. The polypeptide according to any one of claims 1-2, wherein the polypeptide comprises a sequence at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 2 or to a sequence of SEQ ID NO: 3.

4. The polypeptide according to any one of claims 1-2, wherein the polypeptide comprises at least two sequences independently selected from the group consisting of:- a sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 2; and- a sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 3.

5. The polypeptide according to any one of claims 1-2, wherein the polypeptide comprises:- a sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 2; and a sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 3;- a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 6; or, alternatively,- a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16.

6. A fusion protein comprising the polypeptide as defined in any one of claims 1-5.

7. The fusion protein according to claim 6, further comprising a polypeptide comprising peptidoglycandegrading activity.

8. The fusion protein according to claim 7, wherein the polypeptide comprising peptidoglycan degrading activity comprises a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence selected from the group consisting of a sequence of SEQ ID NO: 17, a sequence of SEQ ID NO: 18, a sequence of SEQ ID NO: 19, and a sequence of SEQ ID NO: 20.

9. The fusion protein according to any one of claims 6-8, further comprising a second polypeptide comprising cell-permeating activity; particularly wherein the second polypeptide comprising cell-permeating activity comprises a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 99%, or 100% identical to a sequence selected from the group consisting of a sequence of SEQ ID NO: 21, a sequence of SEQ ID NO: 16, a sequence of SEQ ID NO: 22, a sequence of SEQ ID NO: 23, a sequence of SEQ ID NO: 24, a sequence of SEQ ID NO: 25, a sequence of SEQ ID NO: 26, a sequence of SEQ ID NO: 27, a sequence of SEQ ID NO: 28, a sequence of SEQ ID NO: 29, a sequence of SEQ ID NO: 30, and a sequence of SEQ ID NO: 31.

10. The fusion protein according to any one of claims 6-9, wherein the fusion protein comprises:- a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16; and a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 20;- a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16; a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 20; and a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 29;- a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16; a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 20; a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 29; and a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 21; or, alternatively,- a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 16; a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 20; and a polypeptide consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence of SEQ ID NO: 21.

11. The fusion protein according to any one of claims 6-10, wherein the fusion protein comprises or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a sequence selected from the group consisting of a sequence of SEQ ID NO: 32, a sequence of SEQ ID NO: 33, a sequence of SEQ ID NO: 36, a sequence of SEQ ID NO: 39, and a sequence of SEQ ID NO: 45.

12. The fusion protein according to any one of claims 7-11, wherein the fusion protein comprises an activity selected from the group consisting of peptidoglycan binding activity, cell-permeating activity, peptidoglycandegrading activity, antibacterial activity, and combinations thereof.

13. The fusion protein according to claim 12, wherein the antibacterial activity is against Gram-negative bacteria; particularly wherein the Gram-negative bacteria are from a genus selected from the group consisting of Acinetobacter, Pseudomonas, Escherichia, Klebsiella, and combinations thereof.

14. A polypeptide as defined in any one of claims 1-5 or a fusion protein as defined in any one of claims 6-13 for use in medicine; particularly for use in the treatment and / or prevention of a bacterial infection caused by Gram-negative bacteria.

15. An in vitro method of inhibiting the growth or reducing the population of Gram-negative bacteria, comprising the step of contacting the bacteria with the polypeptide as defined in any one of claims 1-5 and / or the fusion protein as defined in any one of claims 6-13.

Images