FUSION MOLECULES

I. TECHNICAL FIELD OF THE INVENTION

The present invention relates to fusion molecules that have binding specificity for pyoverdine type I, II and III and pyochelin and can be used in various applications, including diagnostic and/or therapeutic applications, for example, to inhibit or reduce growth of P. aeruginosa and/or to prevent or treat P. aeruginosa biofilm infection as well as diseases or disorders associated with P. aeruginosa biofilm infection. The present invention also concerns methods of producing the fusion molecules described herein as well as compositions and kits comprising such fusion molecules. The present invention further relates to nucleic acid molecules encoding the fusion molecules described herein.

II. BACKGROUND OF THE INVENTION

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that causes acute infections, primarily in association with tissue injuries. P. aeruginosa forms biofilms on indwelling devices and on the pulmonary tissues of patients with the genetic disorder, cystic fibrosis. Biofilm infections are difficult to treat with conventional antibiotic therapies. However, research has demonstrated that iron is essential for proper biofilm formation by P. aeruginosa, and therefore iron-uptake systems are potential targets for anti-Pseudomonas therapies.

P. aeruginosa is able to scavenge iron from the host environment by using the secreted iron-binding siderophores, pyochelin and pyoverdine. Pyoverdine (Pvd) is a peptide-linked hydroxamate- and catecholate-type ligand, and pyochelin (Pch) a derivatized conjugate of salicylate and two molecules of cysteine and having phenol, carboxylate, and amine ligand functionalities. Both Pvd and Pch have demonstrated roles in P. aeruginosa virulence with some indication of synergism. Double-deficient mutants unable to make either siderophore are much more attenuated in virulence than either single-deficient mutant unable to make just one of the two siderophores (Takase et al., Infection and immunity, Apr.2000, p.1834-1839). Furthermore, pyoverdine acts as a signalling molecule to control production of several virulence factors as well as pyoverdine itself; while it has been proposed that pyochelin may be part of a system for obtaining divalent metals such as ferrous iron and zinc for P. aeruginosa' s pathogenicity, in addition to ferric iron (Visca et al., 1992).

Three structurally different pyoverdine types or groups have been identified from several P. aeruginosa strains: from P. aeruginosa ATCC 15692 (Briskot et al., 1989, Liebigs Ann Chem,

p.375-384), from P. aeruginosa ATCC 27853 (Tappe et al., 1993, J.Prakt-Chem., 335, p.83-87) and from a natural isolate, P. aeruginosa R (Gipp et al., 1991 , Z. Naturforsch, 46c, p.534-541 ). Moreover, comparative biological investigations on 88 clinical isolates and the two collection strains mentioned above revealed three different strain-specific pyoverdine-mediated iron uptake systems (Cornells et al., 1989, Infect Immun., 57, p.3491 -3497; Meyer et al., 1997, Microbiology, 143, p.35-43) according to the reference strains: P. aeruginosa ATCC 15692 (Type I Pvd or Pvd I), P. aeruginosa ATCC 27853 (Type II Pvd or Pvd II) and the clinical isolates P. aeruginosa R and pa6 (Type III Pvd or Pvd III).

Each pyoverdine type has three members (subtypes) differing in the side chain which is succinyl, succinamid or a-ketoglutaryl, namely, Pvd type I succinyl, Pvd type I succinamid, Pvd type I a-ketoglutaryl, Pvd type II succinyl, Pvd type II succinamid, Pvd type II a-ketoglutaryl, Pvd type III succinyl, Pvd type III succinamid and Pvd type III a-ketoglutaryl.

Each P. aeruginosa strain expresses one Pvd type i.e. P. aeruginosa ATCC 15692 expresses Type I Pvd, P. aeruginosa ATCC 27853 expresses Type II Pvd and P. aeruginosa R and pa6 expresses Type III Pvd, whereby each Pvd type includes all three members of the respective type, and each said strain also expresses pyochelin.

The inventors have identified the pyoverdines and pyochelin as targets which are crucial for P. aeruginosa's pathogenicity and developed specific inhibitors for such targets, i.e. for each type of Pvd including for every type the three members (subtypes) differing in the side chain (Pvd I s, Pvd I sa, Pvd I aKG, Pvd II s, Pvd II sa, Pvd II aKG, Pvd III s, Pvd III sa, Pvd III aKG) as well as for Pch, and in every case to the free siderophore as well as to the siderophore with bound iron without creating the strong selective pressure imposed by conventional antibiotics (see also EP 15 305 242.8).

The biofilm mode of growth is believed to be critical for persistent P. aeruginosa infections (Costerton et al., 1999; Singh et al., 2000) and the dual expression of Pvd and Pch genes is necessary for normal biofilm development (Banin et al., 2005). Given that P. aeruginosa produces an impressive array of virulence factors, all playing a role in its pathogenicity, a preferred strategy to efficiently inhibit P. aeruginosa virulence is to target several virulence factors.

III. SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a fusion molecule having binding specificity for pyoverdine type I, II and III and pyochelin, comprising

(a) a first polypeptide comprising or consisting of a human neutrophil gelatinase-associated lipocalin (hNGAL) mutein that binds pyoverdine type I;

(b) a second polypeptide comprising or consisting of an hNGAL mutein that binds pyoverdine type II;

(c) a third polypeptide comprising or consisting of an hNGAL mutein that binds pyoverdine type III; and

(d) a fourth polypeptide comprising or consisting of an hNGAL mutein that binds pyochelin; wherein the first, second, third and fourth polypeptides are covalently linked.

In one embodiment, the hNGAL mutein comprises a mutated amino acid residue at one or more positions corresponding to positions 28, 34, 36, 39-42, 44-47, 49, 52, 54-55, 65, 68, 70, 72-75, 77, 79-81 , 87, 96, 100, 103, 106, 108, 123, 125, 127, 132, 134, 141 and 145 of the linear polypeptide sequence of the mature hNGAL (SEQ ID NO: 1 ).

In one embodiment, the hNGAL mutein binds pyoverdine type I, pyoverdine type II, pyoverdine type III and pyochelin, respectively, with a dissociation constant KD of 200 nM or lower.

In one embodiment, the first, second, third and fourth polypeptides are covalently linked via linker molecules.

In one embodiment, the linker molecules are peptide linkers.

In one embodiment, the fusion molecule further comprises a multimerization domain allowing the multimerization of the fusion molecule.

In one embodiment, the multimerization domain is a dimerization domain allowing the dimerization of the fusion molecule.

In one embodiment, the dimerization domain is selected from the group consisting of an Fc domain, an IgE heavy-chain domain 2 (EHD2), an IgM heavy-chain domain 2 (MHD2), an IgG heavy-chain domain 3 (GHD3), an IgA heavy-chain domain 3 (AHD2), an IgD heavy-chain domain 3 (DHD3), an IgE heavy-chain domain 4 (EHD4), an IgM heavy-chain domain 4 (MHD4), an uteroglobin dimerization domain and variants or fragments of any one of the foregoing.

In one embodiment, the Fc domain is a human lgG4-Fc domain.

In one embodiment, the fusion molecule has a general formula selected from the group consisting of

/V'- X1 - L1 - X2 - L2 - X3 - L3 - X4 -C' (I),

ΛΓ- Xi - U - X2 - L2 - X3 - L3 - X4 - L4 - MD -C (II),

/V'- MD - L1 - X1 - L2 - X2 - L3 - X3 - L4 - X4 -C' (II I),

N'- Xi - Li - X2 - L2 - MD - L3 - X3 - L4 - X4 -C (IV), N'- Xi - Li - MD - L2 - X2 - L3 - X3 - L4 - X4 -C (V), and

ΛΓ- Xi - Li - X2 - L2 - X3 - L3 - MD - L4 - X4 -C (VI) wherein

Xi , X2, X3 and X are, at each occurrence, selected from the group consisting of the first, second, third and fourth polypeptides, with the proviso that the fusion molecule comprises each of the first, second, third and fourth polypeptides;

MD comprises a multimerization domain; and

Li , L2, L3 and L4 are, at each occurrence, independently selected from a covalent bond and a linker molecule.

In one embodiment, the fusion molecule is present as a multimeric (e.g., dimeric) complex.

In one embodiment, the fusion molecule further comprises at least one label or tag allowing the detection and/or isolation of the fusion molecule.

In one embodiment, the fusion molecule further comprises one or more modifications increasing the stability of the fusion molecule and/or extending the serum half-life of the fusion molecule.

In one embodiment, the fusion molecule inhibits or reduces iron-uptake by P. aeruginosa through pyochelin and/or pyoverdine.

In one embodiment, the fusion molecule inhibits or reduces virulence factor expression by P. aeruginosa.

In one embodiment, the fusion molecule inhibits or reduces pyochelin- and/or pyoverdine-mediated signaling.

In one embodiment, the fusion molecule inhibits or reduces P. aeruginosa bacterial growth.

In one embodiment, the fusion molecule is associated with or conjugated/fused to a pharmaceutically active agent.

In one embodiment, the pharmaceutically active agent is selected from the group consisting of an antibiotic, a cytostatic agent, a toxin, a metal or metal compound/complex, a chelating agent, a hapten and an antibody.

In a further aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding the fusion molecule as defined above.

In one embodiment, the nucleic acid molecule is operably linked to a regulatory sequence to allow expression of the nucleic acid molecule.

In one embodiment, the nucleic acid molecule is comprised in a vector.

In another aspect, the present invention relates to a host cell containing a nucleic acid molecule as defined above.

In yet another aspect, the present invention relates to a method of producing the fusion molecule as defined above, wherein the fusion molecule is produced (i) by culturing a host cell as defined above under conditions that allow the expression of the fusion molecule and by isolating the fusion molecule from the host cell or its culture medium, or (ii) by in vitro translating a nucleic acid molecule as defined above.

In a further aspect, the present invention relates to a pharmaceutical composition comprising a fusion molecule as defined above, a nucleic acid molecule as defined above, or a cell as defined above.

In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

In one embodiment, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

In one embodiment, the additional pharmaceutically active agent is selected from the group consisting of an antibiotic, a cytostatic agent, a toxin, a metal or metal compound/complex, a chelating agent, a hapten and an antibody.

In yet another aspect, the present invention relates to a kit comprising a fusion molecule as defined above, a nucleic acid molecule as defined above, a cell as defined above, or a pharmaceutical composition as defined above.

In yet another aspect, the present invention relates to a fusion molecule as defined above, a nucleic acid molecule as defined above, a cell as defined above, or a pharmaceutical composition as defined above for use as a medicament.

In yet another aspect, the present invention relates to a fusion molecule as defined above, a nucleic acid molecule as defined above, a cell as defined above, or a pharmaceutical composition as defined above for use in the prevention or treatment of P. aeruginosa biofilm infection in a subject.

In yet another aspect, the present invention relates to a fusion molecule as defined above, a nucleic acid molecule as defined above, a cell as defined above, or a pharmaceutical composition as defined above for use in the prevention or treatment of a disease or disorder associated with or caused by P. aeruginosa biofilm infection in a subject.

In yet another aspect, the present invention relates to a method of preventing or treating P. aeruginosa biofilm infection in a subject, comprising administering an effective amount of a fusion molecule as defined above, a nucleic acid molecule as defined above, a cell as defined above, or a pharmaceutical composition as defined above to the subject.

In yet another aspect, the present invention relates to a method of preventing or treating a disease or disorder associated with or caused by P. aeruginosa biofilm infection in a subject, comprising administering an effective amount of a fusion molecule as defined above, a nucleic acid molecule as defined above, a cell as defined above, or a pharmaceutical composition as defined above to the subject.

In one embodiment, the P. aeruginosa biofilm infection is acute or chronic infection.

IV. DESCRIPTION OF THE FIGURES

Figure 1 : shows the structure of P. aeruginosa siderophores. Fig. 1A-C show the structures of the three P. aeruginosa pyoverdines. Fig.lA: Structure of Pvd type I (see Birskot et al., 1989); Fig.l B: Structure of Pvd type II (see Birskot et al., 1989); Fig.l C: Structure of Pvd type III (Gipp et al., 1991 ); Fig.l D: R attached to the chromophore part can be a succinyl, succinamid or a-ketoglutaryl side chain; and Fig.1 E: Structure of pyochelin (Brandel et al., 201 1 ).

Figure 2: provides typical measurements of on-rate and off-rate by surface plasmon resonance for Pvd I s (+Fe) binding to the lipocalin mutein SEQ ID NO: 16 (A), Pvd II s (+Fe) binding to the lipocalin mutein SEQ ID NO: 36 (B), Pvd III (+Fe) binding to the lipocalin mutein SEQ ID NO: 53

(C) and pyochelin (+Fe) binding to SEQ ID NO: 62 (D). In addition, absence of binding of the respective siderophores at 1200 nM (200 nM for pyochelin) to the negative control SEQ ID NO: 64 is shown in Fig.1 E-H.

Figure 3: shows an exemplary specificity and crossreactivity profile for the lipocalin mutein SEQ ID NO: 35 as determined by surface plasmon resonance. Specific binding to pyoverdine II succinyl, succinamid and a-ketoglutaryl is demonstrated, while absence of binding to pyoverdines of type I and type III, pyochelin, enterobactin and desferoxamin is shown. High concentrations of 2 μΜ are used for all analytes.

Figure 4: shows exemplary data from growth inhibition assay. Fig. 4A: Pvd I specific mutein SEQ ID NO: 16 shows growth inhibition of a Pvd I specific P. aeruginosa strain (ATCC27853) compared to the control culture growing without mutein. Fig. 4B: Pvd II specific muteins SEQ ID NOs: 19 and 36 show growth inhibition of a Pvd II specific P. aeruginosa strain (ATCC15692) compared to the control culture growing without mutein. SEQ ID NO: 36 has a higher binding affinity compared to SEQ ID NO: 19 and shows a greater growth inhibition. Fig. 4C: Pvd III specific mutein SEQ ID NO: 53 shows growth inhibition of a Pvd III specific P. aeruginosa strain (ATCC33360) compared to the control culture growing without mutein. Fig. 4D: Pch specific muteins SEQ ID NO: 62 shows growth inhibition of a Pvd I knock out P. aeruginosa strain (ATCC15692 ApvdA) relying on Pch for iron uptake compared to the control culture growing without mutein. 10μΜ lipocalin muteins were applied in the assay.

Figure 5: shows in a P. aeruginosa-'mduced lung infection model in mice that administration of SEQ ID NO: 19, 1 hour before and at time of bacteria challenge, prevents the development of infection in mice in a dose-dependent manner. A significant prevention effect was observed starting from SEQ ID NO: 19 at 200μg mouse, with a maximal effect at 2000μg mouse.

Figure 6: shows the amino acid sequence expressed for crystallisation (SEQ ID NO: 129), including a start methionine at position 1 , a lysine at position 2, a hexahistidine tag at position 3 - 8, a linker region of amino acids DYDIPTT at postion 9 - 15 (SEQ ID NO: 132), the tobacco etch viral (TEV) protease cleavage site ENLYFQG at position 16 - 22 (SEQ ID NO: 133) followed by the amino acid sequence of the mutein of interest from position 23 onwards.

Figure 7: shows the SEQ ID NO: 31 - Pvd-Fe complex structure. An overlay of two SEQ ID NO: 31 molecules i.e. chain A and chain B from an asymmetric unit is shown.

Figure 8: shows SEQ ID NO: 31 and Pvd-Fe interactions. Two molecules from asymmetric unit are overlaid. Side chains interacting with Pvd-Fe are depicted.

Figure 9: shows the Pvd composition. Oxygen atoms involved in iron binding are boxed.

Figure 10A: shows the structure of a fusion molecule according to the present invention. Lipocalin muteins one, two, three and four (LM 1 , 2, 3, 4) were genetically fused via linker molecules (here: peptide linkers of the sequence (G4S)2; see SEQ ID NO: 141 ), wherein LM 1 (SEQ ID NO: 34 or 36) bound to the pyoverdine II group siderophores with bound iron ion and without complexed iron ion, LM 2 (SEQ ID NO: 16 or 17) bound to the pyoverdine I group siderophores with bound iron ion and without complexed iron ion, LM 3 (SEQ I D NO: 50 or 53) bound to the pyoverdine III group siderophores with bound iron ion and without complexed iron ion and LM 4 (SEQ ID NO: 62 or 63) bound to pyochelin siderophore with bound iron ion and without complexed iron ion. The fusion molecule construct (SEQ ID NO: 134 or 135) comprised binding capacity to all P. aeruginosa siderophores in one molecule and had one binding moiety for each Pvd group and one for pyochelin.

Figure 10B: shows the structure of an Fc-fusion molecule construct according to the present invention. Lipocalin muteins one, two, three and four (LM 1 , 2, 3, 4) were genetically fused via linker molecules (here: peptide linkers of the sequence (G4S)2; see SEQ ID NO: 141 ) to the N-terminus of the human lgG4-Fc domain (hu lgG4-Fc; SEQ ID NO: 140), wherein LM 1 (SEQ ID NO: 34 or 36) bound to the pyoverdine II group siderophores with bound iron ion and without complexed iron ion, LM 2 (SEQ ID NO: 16 or 17) bound to the pyoverdine I group siderophores with bound iron ion and without complexed iron ion, LM 3 (SEQ I D NO: 50 or 53) bound to the pyoverdine III group siderophores with bound iron ion and without complexed iron ion and LM 4 (SEQ ID NO: 62 or 63) bound to pyochelin siderophore with bound iron ion and without complexed iron ion. The Fc-fusion molecule construct (SEQ ID NO: 136 or 137) comprised binding capacity to all P. aeruginosa siderophores in one molecule and had two binding moieties for each Pvd group and for pyochelin.

Figure 10C: shows the structure of an Fc-fusion molecule construct according to the present invention. Lipocalin muteins one, two, three and four (LM 1 , 2, 3, 4) were genetically fused via linker molecules (here: peptide linkers of the sequence (G4S)2; see SEQ ID NO: 141 ) to the C-terminus of the human lgG4-Fc domain (hu lgG4-Fc; SEQ ID NO: 140), wherein LM 1 (SEQ ID NO: 34) bound to the pyoverdine II group siderophores with bound iron ion and without complexed iron ion, LM 2 (SEQ ID NO: 17) bound to the pyoverdine I group siderophores with bound iron ion and without complexed iron ion, LM 3 (SEQ ID NO: 50) bound to the pyoverdine III group siderophores with bound iron ion and without complexed iron ion and LM 4 (SEQ ID NO: 63) bound to pyochelin siderophore with bound iron ion and without complexed iron ion. The Fc-fusion molecule construct (SEQ ID NO: 138) comprised binding capacity to all P.

aeruginosa siderophores in one molecule and had two binding moieties for each Pvd group and for pyochelin.

Figure 10D: shows the structure of an Fc-fusion molecule construct according to the present invention. Lipocalin muteins one and two and three and four (LM 1 , 2, 3, 4) were genetically fused via linker molecules (here: peptide linkers of the sequence (G4S)2; see SEQ ID NO: 141 ). LM 1 and LM 2 were genetically fused to the N-terminus of the human lgG4-Fc domain (hu lgG4-Fc; SEQ ID NO: 140), and LM 3 and LM 4 were genetically fused to the C-terminus of the human lgG4-Fc domain (hu lgG4-Fc; SEQ ID NO: 140), wherein LM 1 (SEQ ID NO: 34) bound to the pyoverdine II group siderophores with bound iron ion and without complexed iron ion, LM 2 (SEQ ID NO: 17) bound to the pyoverdine I group siderophores with bound iron ion and without complexed iron ion, LM 3 (SEQ ID NO: 50) bound to the pyoverdine III group siderophores with bound iron ion and without complexed iron ion and LM 4 (SEQ ID NO: 63) bound to pyochelin siderophore with bound iron ion and without complexed iron ion. The Fc-fusion molecule construct (SEQ ID NO: 139) comprised binding capacity to all P. aeruginosa siderophores in one molecule and had two binding moieties for each Pvd group and for pyochelin.

Figure 1 1 : shows a Coomassie-stained SDS-PAGE gel of a purified fusion molecule (here: SEQ ID NO: 134).

Figure 12: shows binding of the different Fc-fusion molecule constructs of SEQ ID NOs: 137, 138 and 139 and of the respective single lipocalin muteins of SEQ ID NOs: 17, 34, 50 and 63 to the iron loaded succinyl variants of the different pyoverdine groups and to iron loaded pyochelin in solution, as determined in an ELISA based assay. All Fc-fusion molecule formats were active in binding to the single targets and had IC50 values comparable to those of single lipocalin muteins.

Figure 13: shows that fusion molecules (here: SEQ ID NO: 134) are capable of simultaneous engagement of pyoverdine and pyochelin. Biotinylated pyoverdine from each of the respective groups (I, II and III) with a succinyl residue was captured on neutravidin coated MSD plates, and the fusion molecule was allowed to bind. Bound fusion molecule was detected via Sulfo-tag labeled pyochelin.

Figure 14: shows that fusion molecules (here: SEQ ID NO: 134) are capable of inhibiting growth of either a strain producing Pvd I (A), Pvd II (B) or Pvd III (C), or a ApvdA Pvdll knock out strain (D) in iron limited medium.

Figure 15: shows that fusion molecules (here: SEQ ID NO: 134, referred to as RA10680550) significantly reduce P. aeruginosa lung burden (CFU/gram of lung tissue) in a chronic model of infection after a single dose at day 3 and that no relapse occurs up to day 15. It further shows that the fusion molecules do not antagonize Tobramycin activity, and that, in a combination therapy, the two activities synergize. The geometric mean burden of each treatment is indicated by the horizontal bar. Above each column the Log-io reduction (LogR) of that treatment group compared to the vehicle is recorded (LOD = limit of detection).

Figure 16: shows that fusion molecules (here: SEQ ID NO: 134, also referred to as RA10680550) block the increase of total white cell count in the lung of infected rats and thereby increases the clearance of infection. The Tobramycin / fusion molecule combination shows a clear benefit versus each of the single treatments. Absolute BAL white blood cell (monocytes and neutrophils) counts mean + SD (cells per ml of BAL) at days 3, 5, 7, 9 and 15 post infection (Log scale).

Figure 17: shows that fusion molecules decrease virulence factor expression during the early phase of colonization (A) or established colonization (B). The tested fusion molecule of SEQ ID NO: 134 shows a significant effect on the virulence gene expression of rhll of P. aeruginosa, rhll is involved in quorum sensing by producing N-butanoyl homoserine lactone (BHL). BHL binds to its receptor RhIR, thereby inducing expression of a complement of genes, including their own loci (completing the autoinducing circuits).

Figure 18: shows that the binding capacity of fusion molecules is not influenced by the length of the linker molecule separating the four lipocalin muteins. The tested linker molecules were glycine, GSGGSG (SEQ ID NO: 142) and (G4S)2 (SEQ ID NO: 141 ).

Figure 19: shows that Fc-fusion molecules prevent the production of pyoverdine by exponentially growing bacteria in an iron starved medium. The means and s.e.m. of cfu/ml and fluorescence arbitrary unit values upon treatment with an Fc-fusion molecule (SEQ ID NO: 136) or a control Fc-fusion molecule are shown for P. aeruginosa strain ATCC27853 (A and B), strain ATCC15692 (C and D) and strain ATCC33360 (E and F).

Figure 20: shows that Fc-fusion molecules prevent the production of pyoverdine by exponentially growing bacteria in an iron starved medium (see also Figure 20). The normalized relative amounts of pyoverdine fluorescence upon treatment with an Fc-fusion molecule (SEQ ID NO: 136) or a control Fc-fusion molecule are shown for strains ATCC27853 (A), ATCC15692 (B) and ATCC33360 (C).

Figure 21 : shows that the effects shown in Figures 19 and 20 are pyoverdine-specific. The mean values of fluorescence in cultures of strain ATCC27853 and in a pvdA/pchD double mutant strain of ATCC27853 show that the mutant strain unable to synthesize these siderophores does not exhibit any fluorescence signal.

Figure 22: shows that the effects shown in Figures 19 and 20 are pyoverdine-specific (see also Figure 21 ). To normalize the fluorescence signal, purified pyoverdine II obtained from the supernatant of wild-type ATCC27853 was used to spike the cultures prior to fluorescence signal acquisition, and a calibration curve was obtained.

Figure 23: shows that the drop in fluorescence signal in the presence of an Fc-fusion molecule was not due to fluorescence quenching upon pyoverdine binding by the compound. The Fc-fusion molecule (SEQ ID NO: 136) was added extemporaneously to a culture supernatant containing pyoverdine (obtained with a filtered 24 hours-culture of ATCC27853), and fluorescence was read immediately and after 24h. As shown by the mean values of fluorescence observed in this experiment, no fluorescence quenching was detectable.

Figure 24: shows that Fc-fusion molecules influence the kinetics of bacterial growth. The impact of 1 mg/mL treatment with an Fc-fusion molecule (SEQ ID NO: 136) on strains ATCC15692 and ATCC33360 are shown as means and s.e.m. of cfu/mL in (A) and (B), respectively.

Figure 25: shows the impact of iron concentration on Ppchd transcriptional activity. The means and s.e.m. of the observed luminescence show that pchD promoter expression was induced in the C-MS medium without addition of iron and repressed by iron addition, thereby validating the functionality of the reporter fusion.

Figure 26: shows the impact of treatment with an Fc-fusion molecule (SEQ ID NO: 136) on Ppchd transcriptional activity in the presence of various amounts of supplementary iron. The means and s.e.m. of the observed luminescence show that promoter activity was abolished in the presence of the Fc-fusion molecule regardless of the amount of supplemented iron, whereas the control Fc-fusion molecule had no effect.

Figure 27: shows the impact of a range of doses of an Fc-fusion molecule (SEQ ID NO: 136) on PpchD transcriptional activity tested with no iron supplement. The points of means and s.e.m. of the observed luminescence were fitted to a standard dose-response curve and yielded an IC50 value of 0.03 mg/mL (95% CI: 0.02 - 0.05) for the Fc-fusion molecule, whereas it was undefined for the isotypic control Fc-fusion molecule.

V. DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (lUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (Sambrook, J. et al. (2001 ) Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps although in some embodiments such other member, integer or step or group of members, integers or steps may be excluded, i.e. the subject-matter consists in the inclusion of a stated member, integer or step or group of members, integers or steps. The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

A. Fusion molecules

The present invention provides fusion molecules having binding specificity for pyoverdine type I, II and III and pyochelin, comprising

(a) a first polypeptide comprising or consisting of a human neutrophil gelatinase-associated lipocalin (hNGAL) mutein that binds pyoverdine type I;

(b) a second polypeptide comprising or consisting of an hNGAL mutein that binds pyoverdine type II;

(c) a third polypeptide comprising or consisting of an hNGAL mutein that binds pyoverdine type III; and

(d) a fourth polypeptide comprising or consisting of an hNGAL mutein that binds pyochelin; wherein the first, second, third and fourth polypeptides are covalently linked.

The term "fusion molecule" generally refers to molecules created by joining two or more distinct molecules (e.g., (poly-)peptides or proteins), particularly head-to-tail (e.g., N-terminus to C-terminus or vice versa), resulting in a single molecule with functional properties derived from each of the original molecules (e.g., (poly-)peptides or proteins). In one embodiment, the fusion molecule is a fusion protein.

The term "binding specificity", as used herein, refers to the ability of a ligand (e.g., the fusion molecule of the invention) to discriminate between the target(s) (e.g., pyoverdine type I, II and III and pyochelin) and one or more reference targets, since binding specificity is not an absolute, but a relative property. Specific binding can be determined, for example, in accordance with Western blots, ELISA-, RIA-, ECL-, IRMA-tests, FACS, IHC and peptide scans.

As used herein, "pyoverdine" means a fluorescent siderophore that is produced by the gram negative bacterium Pseudomonas aeruginosa under iron-deficient growth conditions and has high affinity for iron. Pyoverdines are composed of three structural parts: a dihydroxyquinoline chromophore, a side chain and a variable peptidic chain. The peptide chain moiety is involved in receptor recognition and binding. Three different Pvds, differing in their peptide chain, have been identified (types l-lll; Figure 1A-D).The size and amino acid composition of pyoverdine types are unique to each species, as well as the pyoverdine recognition specificity. Three P. aeruginosa strains can be distinguished, each producing a different pyoverdine type and a cognate FpvA receptor. Each type has three members (subtypes) differing in the side chain which is succinyl, succinamid or a-ketoglutaryl, namely, Pvd type I succinyl, Pvd type I succinamid, Pvd type I a-ketoglutaryl, Pvd type II succinyl, Pvd type II succinamid, Pvd type II a-ketoglutaryl, Pvd type III succinyl, Pvd type III succinamid and Pvd type III a-ketoglutaryl.

As used herein, "pyochelin" means a thiazoline derivatized conjugate of salicylate and two molecules of cysteine and having phenol, carboxylate, and amine ligand functionalities, produced by P. aeruginosa and solubilizing ferric iron. Pyochelin is a structurally unique siderophore possessing phenolate, but neither a hydroxamate nor a catecholate moiety (Figure 1 E).

The term "neutrophil gelatinase-associated lipocalin (hNGAL)" (also referred to as "human lipocalin 2" or "human Lcn 2" or "human NGAL" or simply "lipocalin"), as used herein, refers to the mature human neutrophil gelatinase-associated lipocalin (NGAL) with the SWISS-PROT/UniProt Data Bank Accession Number P80188. A human NGAL (hNGAL) mutein may also be designated herein as "lipocalin mutein" or simply "mutein". The amino acid sequence shown in SWISS-PROT/UniProt Data Bank Accession Number P80188 may be used as a particular "reference sequence". In one embodiment, the amino acid sequence shown in SEQ ID NO: 1 is used as reference sequence. Wild-type hNGAL does not bind to pyoverdines or pyochelin. The natural ligand of wild-type hNGAL is enterobactin, which docks into the calyx of hNGAL with high affinity.

As used herein, a "mutein," a "mutated" entity (whether protein or nucleic acid), or "mutant" refers to the exchange, deletion, or insertion of one or more nucleotides or amino acids,

compared to the naturally occurring (wild-type) nucleic acid or protein reference sequence/scaffold. Said term also includes fragments of a mutein and variants as described herein. Muteins for use in the fusion molecules of the invention, fragments or variants thereof preferably retain the function of binding to pyoverdine or pyochelin as described herein.

In one embodiment, the hNGAL muteins comprised by the first, second, third and fourth polypeptides are specific for or specifically bind to pyoverdine type I, II and III and pyochelin, respectively.

When used herein in the context of hNGAL muteins that bind to pyoverdine or pyochelin, the term "specific for" includes that the mutein is directed against, binds to, or reacts with pyoverdine or pyochelin, respectively. Thus, being directed to, binding to or reacting with includes that the mutein specifically binds to pyoverdine or pyochelin, respectively. The term "specifically" in this context means that the mutein reacts with a pyoverdine protein or a pyochelin protein, as described herein, but essentially not with another protein. The term "another protein" includes any non-pyoverdine or non-pyochelin protein, respectively, including proteins closely related to or being homologous to pyoverdine or pyochelin against which the muteins disclosed herein are directed to. However, pyoverdine or pyochelin proteins, fragments and/or variants from species other than human such as those described in the context of the definition "subject" are not excluded by the term "another protein". The term "does not essentially bind" means that the mutein of the present disclosure does not bind another protein, i.e., shows a cross-reactivity of less than 30%, particularly 20%, more particularly 10%, even more particularly less than 9, 8, 7, 6 or 5%. Whether the mutein specifically reacts as defined herein above can easily be tested, inter alia, by comparing the reaction of a lipocalin mutein of the present disclosure with pyoverdine or pyochelin and the reaction of said mutein with (an) other protein(s). "Specific binding" can also be determined, for example, in accordance with Western blots, ELISA-, RIA-, ECL-, IRMA-tests, FACS, IHC and peptide scans.

In one embodiment, the hNGAL muteins comprised by the first, second, third and fourth polypeptides bind to pyoverdine type I, II and III and pyochelin, respectively, with detectable affinity.

As used herein, "detectable affinity" means the ability to bind to a selected target with a dissociation constant KD of generally about 10"5 M or lower, e.g., about 10"6 M or lower, or about 10"7 M or lower. Lower affinities (i.e., higher KD values) are generally no longer measurable with common methods such as ELISA and therefore of secondary importance.

As used herein, "binding affinity" of a protein of the disclosure (e.g. a mutein of human lipocalin 2) to a selected target (in the present case, pyoverdine or pyochelin), can be measured (and thereby KD values of a mutein-ligand complex be determined) by a multitude of methods known to those skilled in the art. Such methods include, but are not limited to, fluorescence titration, direct ELISA, competition ELISA, calorimetric methods, such as isothermal titration calorimetry (ITC), and surface plasmon resonance (Biacore). Such methods are well established in the art and examples thereof are also detailed below.

It is also noted that the complex formation between the respective binder and its ligand is influenced by many different factors such as the concentrations of the respective binding partners, the presence of competitors, pH and the ionic strength of the buffer system used, and the experimental method used for determination of the dissociation constant KD (for example fluorescence titration, direct ELISA, competition ELISA or surface plasmon resonance, just to name a few) or even the mathematical algorithm which is used for evaluation of the experimental data.

Therefore, it is also clear to the skilled person that the KD values (dissociation constant of the complex formed between the respective binder and its target ligand) may vary within a certain experimental range, depending on the method and experimental setup that is used for determining the affinity of a particular mutein for a given ligand. This means that there may be a slight deviation in the measured KD values or a tolerance range depending, for example, on whether the KD value was determined by surface plasmon resonance (Biacore), by competition ELISA, or by "direct ELISA".

In some embodiments, the mutein specific for pyoverdine (type I, II or III) as used in the fusion molecule of the invention is able to bind pyoverdine (type I, II or III, respectively) with a dissociation constant KD of 200 nM or lower, or about 100 nM or lower, or about 50 nM or lower, or about 25 nM or lower, or about 15 nM or lower. In some embodiments, the mutein specific for pyochelin as used in the fusion molecule of the invention is able to bind pyochelin with a dissociation constant KD of 200 nM or lower, or about 100 nM or lower, or about 50 nM or lower, or about 25 nM or lower, or about 15 nM or lower. In some further particular embodiments, a mutein of the fusion molecule according to the present invention binds pyoverdine (type I, II or III) or pyochelin, respectively, with a dissociation constant for pyoverdine (type I, II or III, respectively) or pyochelin of about 10 nM or lower, or about 5 nM or lower, or about 2 nM or lower, or about 1 nM or lower, or about 0.1 nM or lower, or about 10 pM or lower.

In particular embodiments, the fusion molecule of the invention binds pyoverdine (type I, II and III) and pyochelin with a respective dissociation constant KD of 200 nM or lower, or about 100 nM or lower, or about 50 nM or lower, or about 25 nM or lower, or about 15 nM or lower, or about 10 nM or lower, or about 5 nM or lower, or about 2 nM or lower, or about 1 nM or lower, or about 0.1 nM or lower, or about 10 pM or lower.

In one embodiment, the KD value is determined by surface plasmon resonance (Biacore), by competition ELISA, or by "direct ELISA".

hNGAL muteins for use in the fusion molecules of the present invention as well as methods for their generation and identification are disclosed/described in detail in EP 15 305 242.8 and PCT/EP2016/053226 as well as in the section "hNGAL muteins" below.

In a particular embodiment, an hNGAL mutein that is specific for pyoverdine type I is shown in any one of SEQ ID NOs: 2-18 (e.g., SEQ ID NO: 16). In a particular embodiment, an hNGAL mutein that is specific for pyoverdine type II is shown in any one of SEQ ID NOs: 19-37 (e.g., SEQ ID NO: 36). In a particular embodiment, an hNGAL mutein that is specific for pyoverdine type III is shown in any one of SEQ ID NOs: 38-53 (e.g., SEQ ID NO: 53). In a particular embodiment, an hNGAL mutein that is specific for pyochelin is shown in any one of SEQ ID NOs: 54-63 (e.g., SEQ ID NO: 62).

The term "covalently linked", as used herein, refers to linkage via a covalent bond or via a covalent linker molecule. In one embodiment, the first, second, third and fourth polypeptides are covalently linked via linker molecules.

The term "linker molecules", as used herein, refers to molecules adapted to connect/link protein moieties, e.g., the first, second, third and fourth polypeptides as defined herein. A linker molecule in accordance with the present invention may have any size/length. However, it is preferably long enough to provide an adequate degree of flexibility to prevent the connected/linked moieties from interfering with each other's activity, for example by steric hindrance, and to allow for proper protein folding; yet it is preferably short enough to provide stability (e.g., proteolytic stability) in the cell. Suitable linker molecules are known to a person skilled in the art and include, for example, peptides as well as non-peptidic molecules, e.g., non-peptidic oligomers and polymers of suitable lengths. According to the present invention, the various linker molecules within a single fusion molecule described herein may be the same or different.

In one embodiment, the linker molecules are peptide linkers. A peptide linker in accordance with the present invention may have any length, i.e., it may comprise any number of amino acid residues. However, it is preferably long enough to provide an adequate degree of flexibility to prevent the connected/linked moieties from interfering with each other's activity, for example by steric hindrance, and to allow for proper protein folding; yet it is preferably short enough to provide stability (e.g., proteolytic stability) in the cell. In some embodiments, the peptide linkers have a length of 1 to 30 amino acids, or a length of 1 to 25 amino acids, or a length of 1 to 20 amino acids, or a length of 1 to 15 amino acids, or a length of 1 to 12 amino acids or a length of 1 to 10 amino acids. Thus, according to the present invention, a peptide linker may be composed of a single amino acid residue, e.g., glycine (Gly, G).

The amino acids of a peptide linker in accordance with the present invention may be selected from all naturally or non-naturally occurring amino acids, in particular from the amino acids glycine (Gly, G), serine (Ser, S) and threonine (Thr, T). In one embodiment, the peptide linker is a glycine-serine-threonine-rich linker or glycine-serine-rich linker, wherein at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the amino acids are a glycine or serine or threonine residue or a glycine or serine residue, respectively. In another embodiment, the amino acids are selected from glycine, serine and threonine, i.e., the peptide linker is exclusively composed of glycine, serine and threonine residues (referred to as a glycine-serine-threonine linker). In yet another embodiment, the peptide linker is exclusively composed of glycine and serine residues (referred to as a glycine-serine linker).

In one embodiment, the peptide linker is a glycine-serine linker and has a length of 1 to 30 amino acids, or a length of 1 to 25 amino acids, or a length of 1 to 20 amino acids, or a length of 1 to 15 amino acids, or a length of 1 to 12 amino acids or a length of 1 to 10 amino acids. Particular peptide linkers in accordance with the present invention have the general formula (GGGGX)n, wherein X is, at each occurrence, independently selected from S and T, and n is an integer selected from 1 to 5, or 1 to 4, or 1 to 3, or 1 and 2. In one embodiment, the peptide linker has the amino acid sequence of SEQ ID NO: 141 . In another embodiment, the peptide linker has the amino acid sequence of SEQ ID NO: 142.

In one embodiment, the fusion molecule further comprises a multimerization domain allowing the multimerization of the fusion molecule. Multimerization may occur by non-covalent interaction and/or covalent interaction, in particular via one or more disulfide bonds, between multiple (e.g., 2, 3 or 4, particularly 2 or 3, more particularly 2) multimerization domains.

Suitable multimerization domains are known to a person skilled in the art and include, for example, trimerization domains, such as a tenascin trimerization motif, a collectin trimerization domain and streptavidin, and dimerization domains, such as an Fc domain, an IgE heavy-chain domain 2 (EHD2), an IgM heavy-chain domain 2 (MHD2), an IgG heavy-chain domain 3 (GHD3), an IgA heavy-chain domain 3 (AHD2), an IgD heavy-chain domain 3 (DHD3), an IgE heavy-chain domain 4 (EHD4), an IgM heavy-chain domain 4 (MHD4), an uteroglobin

dimerization domain. In one embodiment, the Fc domain is a human lgG4-Fc domain, which, in a particular embodiment, comprises or consists of the amino acid sequence of SEQ ID NO: 140. Also included are variants or fragments of any one of the foregoing domains, e.g., domains that have been modified so as to extend their half-life and/or increase their efficiency, as long as they still allow multimerization (e.g., dimerization) of the fusion molecule, i.e. are functional. Suitable modifications are known to a person skilled in the art and include, but are not limited to, modifications of the Fc domain which increase its affinity for FcRn, as described, for example, in Zalevsky, J. et al. (2010), Nature Biotechnology, 28(2):157-9 (e.g., N434S, V259IA 308F, M252Y/S254T/T256E, M428L/N434S, and V259IA 308F/M428L).

In one embodiment, the fusion molecule has a general formula selected from the group consisting of

/V'- X1 - L1 - X2 - L2 - X3 - L3 - X4 -C' (I),

ΛΓ- Xi - Li - X2 - L2 - X3 - L3 - X4 - L4 - MD -C (II),

/V'- MD - L1 - X1 - L2 - X2 - L3 - X3 - L4 - X4 -C' (III),

ΛΓ- Xi - U - X2 - L2 - MD - L3 - X3 - L4 - X4 -C (IV), ΛΓ- Xi - U - MD - L2 - X2 - L3 - X3 - L4 - X4 -C (V), and

ΛΓ- Xi - Li - X2 - L2 - X3 - L3 - MD - L4 - X4 -C (VI) wherein

X-i, X2, X3 and X are, at each occurrence, selected from the group consisting of the first, second, third and fourth polypeptides, with the proviso that the fusion molecule comprises each of the first, second, third and fourth polypeptides;

MD comprises a multimerization domain; and

U, L2, L3 and L4 are, at each occurrence, independently selected from a covalent bond and a linker molecule.

According to the present invention, L-i , L2, L3 and L4 may be the same or different.

In one embodiment, the fusion molecule is present as a multimeric (e.g., dimeric) complex.

Therefore, in one aspect, the present invention also provides a multimere (or multimeric complex) comprising two or more fusion molecules described herein (e.g., a dimer or dimeric complex). Such complex may also be referred to as fusion molecule complex. In one embodiment, the two or more fusion molecules non-covalently or covalently (e.g., via disulfide bonds) associate to form the fusion molecule complex.

In one embodiment, the fusion molecule further comprises at least one label or tag allowing the detection and/or isolation of the fusion molecule.

A "label or tag allowing the detection and/or isolation of the fusion molecule" is meant to include any labels/tags known in the art for these purposes. They include affinity tags such as the Strep-tag® or Strep-tag® II (Schmidt, T.G.M. et al. (1996) J. Mol. Biol. 255, 753-766), the myc-tag, the FLAG-tag, the His6-tag or the HA-tag or proteins such as maltose binding protein (MBP) and glutathione-S-transferase (GST) as well as combinations thereof (e.g., Strep-tag® or Strep-tag® II and His-6-tag). Proteins with chromogenic or fluorescent properties such as the green fluorescent protein (GFP) or the yellow fluorescent protein (YFP) are suitable labels as well.

The amino acid sequence of a (poly)peptide label or tag may be introduced at any position within the amino acid sequence of the fusion molecule, and may, for example, take the shape of a loop within the encoded protein structure (e.g., within any of the peptide linkers described herein or even within the muteins as long as the label/tag does not interfere with their function), or it may be N-terminally or C-terminally fused. The label or tag may further contain a cleavage site that allows a removal of the label or tag from the fusion molecule. Similarly, non-peptidic labels or tags, e.g., fluorescent dyes, may be conjugated to the fusion molecule at any suitable site.

In general, it is possible to label the fusion molecules of the invention with any appropriate chemical substance or enzyme, which directly or indirectly generates a detectable compound or signal in a chemical, physical, optical, or enzymatic reaction. An example for a physical reaction and at the same time optical reaction/marker is the emission of fluorescence upon irradiation or the emission of X-rays when using a radioactive label. Alkaline phosphatase, horseradish peroxidase and β-galactosidase are examples of enzyme labels (and at the same time optical labels) which catalyze the formation of chromogenic reaction products.

The fusion molecule may also comprise an amino acid sequence for facilitating secretion of the molecule, such as an N-terminal secretion signal, or a signal sequence. The fusion molecule of the invention may further comprise a binding domain which serves, e.g., to enhance selectivity for a specific cell type. This can be achieved, e.g., by providing a binding domain that binds to a specific antigen expressed on the surface of said cell type.

In one embodiment, the fusion molecule further comprises one or more modifications increasing the stability of the fusion molecule and/or extending the serum half-life of the fusion molecule.

In one embodiment, "stability" of the fusion molecule relates to the "half-life" of the fusion molecule, e.g., in vivo. "Half-life" relates to the period of time which is needed to eliminate half of the activity, amount, or number of molecules.

The fusion molecule may in some embodiments be conjugated to a moiety that extends the serum half-life of the fusion molecule (in this regard see also PCT publication WO 2006/56464 A2 where such conjugation strategies are described with references to muteins of human neutrophil gelatinase-associated lipocalin with binding affinity for CTLA-4). The moiety that extends the serum half-life may be a polyalkylene glycol molecule, hydroxyethyl starch, polysialic acid, fatty acid molecules, such as palmitic acid (Vajo & Duckworth 2000, Pharmacol. Rev. 52, 1-9), an Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, an albumin binding peptide, an albumin binding protein, or transferrin to name only a few. The albumin binding protein may be a bacterial albumin binding protein, an antibody, an antibody fragment including domain antibodies (see US patent 6,696,245, for example), or a mutein with binding activity for albumin. Accordingly, suitable conjugation partners for extending the half-life of a fusion molecule of the invention include an albumin binding protein, for example, a bacterial albumin binding domain, such as the one of streptococcal protein G (Konig, T, & Skerra, A. (1998) J. Immunol. Methods 218, 73-83). Other examples of albumin binding peptides that can be used as conjugation partner are, for instance, those having a Cys-Xaai-Xaa2-Xaa3-Xaa4-Cys consensus sequence, wherein Xaai is Asp, Asn, Ser, Thr, or Trp; Xaa2 is Asn, Gin, His, lie, Leu, or Lys; Xaa3 is Ala, Asp, Phe, Trp, or Tyr; and Xaa4 is Asp, Gly, Leu, Phe, Ser, or Thr as described in US patent application 2003/0069395 or Dennis et al. (Dennis, M. S., Zhang, M., Meng, Y. G., Kadkhodayan, M., Kirchhofer, D., Combs, D. & Damico, L. A. (2002) J Biol Chem 277, 35035-35043).

In other embodiments, albumin itself (Osborn, B.L. et al., 2002, J. Pharmacol. Exp. Ther. 303, 540-548), or a biological active fragment of albumin can be used as conjugation partner of a fusion molecule of the invention. The term "albumin" includes all mammal albumins such as human serum albumin or bovine serum albumin or rat albumin. The albumin or fragment thereof can be recombinantly produced as described in US patent 5,728,553 or European patent applications EP 0 330 451 and EP 0 361 991. Recombinant human albumin (Recombumin®) Novozymes Delta Ltd. (Nottingham, UK) can be conjugated or fused to a fusion molecule of the invention in order to extend the half-life of the fusion molecule.

If the albumin-binding protein is an antibody fragment it may be a domain antibody. Domain antibodies (dAbs) are engineered to allow precise control over biophysical properties and in vivo half-life to create the optimal safety and efficacy product profile. Domain antibodies are for example commercially available from Domantis Ltd. (Cambridge, UK and MA, USA).

Using transferrin as a moiety to extend the serum half-life of the fusion molecules of the invention, the fusion molecules can be genetically fused to the N- or C-terminus, or both, of non-glycosylated transferrin. Non-glycosylated transferrin has a half-life of 14-17 days, and a transferrin fusion protein will similarly have an extended half-life. The transferrin carrier also provides high bioavailability, biodistribution and circulating stability. This technology is commercially available from BioRexis (BioRexis Pharmaceutical Corporation, PA, USA). Recombinant human transferrin (DeltaFerrin™) for use as a protein stabilizer/half-life extension partner is also commercially available from Novozymes Delta Ltd. (Nottingham, UK).

If an Fc part of an immunoglobulin is used for the purpose to prolong the serum half-life of the muteins of the invention, the SynFusion™ technology, commercially available from Syntonix Pharmaceuticals, Inc (MA, USA), may be used. The use of this Fc-fusion technology allows the creation of longer-acting biopharmaceuticals and may for example consist of two copies of each of the at least four muteins linked to the Fc region of an antibody to improve pharmacokinetics, solubility, and production efficiency.

Yet another alternative to prolong the half-life of the fusion molecules of the invention is to fuse to the N-or C-terminus of the fusion molecules long, unstructured, flexible glycine-rich sequences (for example poly-glycine with about 20 to 80 consecutive glycine residues). This approach disclosed in WO 2007/038619, for example, has also been term "rPEG" (recombinant PEG).

If polyalkylene glycol is used as conjugation partner, the polyalkylene glycol can be substituted, unsubstituted, linear or branched. It can also be an activated polyalkylene derivative. Examples of suitable compounds are polyethylene glycol (PEG) molecules as described in WO 99/64016, in US Patent 6,177,074 or in US Patent 6,403,564 in relation to interferon, or as described for other proteins such as PEG-modified asparaginase, PEG-adenosine deaminase (PEG-ADA) or PEG-superoxide dismutase (see for example, Fuertges et al. (1990) The Clinical Efficacy of Poly(Ethylene Glycol)-Modified Proteins J. Control. Release 11 , 139-148). The molecular weight of such a polymer, such as polyethylene glycol, may range from about 300 to about 70.000 Dalton, including, for example, polyethylene glycol with a molecular weight of about 10.000, of about 20.000, of about 30.000 or of about 40.000 Dalton. Moreover, as, e.g., described in US patents 6,500,930 or 6,620,413, carbohydrate oligo- and polymers such as starch or hydroxyethyl starch (HES) can be conjugated to a fusion molecule of the invention for the purpose of serum half-life extension.

In one embodiment, the fusion molecules of the invention are fused at their N-terminus and/or their C-terminus to a fusion partner which is a protein domain that extends the serum half-life of the fusion molecules. In further particular embodiments, the protein domain is an Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, an albumin binding peptide, or an albumin binding protein.

In another embodiment, the fusion molecules of the invention are conjugated to a compound that extends the serum half-life of the fusion molecules. More particularly, the mutein is conjugated to a compound selected from the group consisting of a polyalkylene glycol molecule, a hydroxyethyl starch, an Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, an albumin binding peptide, and an albumin binding protein.

In one embodiment, the fusion molecule inhibits or reduces iron-uptake by P. aeruginosa through pyochelin and/or pyoverdine.

In one embodiment, the fusion molecule reduces virulence factor expression by P. aeruginosa.

In one embodiment, the fusion molecule inhibits or reduces P. aeruginosa bacterial growth.

In one embodiment, the fusion molecule is associated with or conjugated/fused to a pharmaceutically active agent. Such conjugates can be produced by methods well known in the art.

In one embodiment, the pharmaceutically active agent is selected from the group consisting of an antibiotic, a cytostatic agent, a toxin, an enzyme, a metal or metal compound/complex, a chelating agent, a radionuclide, a small organic molecule, a therapeutically active peptide, a therapeutically active nucleic acid molecule, a hapten and an antibody.

Suitable antibiotics include Tobramycin, Azithromycin, Aztreonam, Colistin and carbapenems.

Suitable metals or metal compounds/complexes are gallium, gallium compounds and gallium-based complexes.

The term "small organic molecule", as used herein, refers to an organic molecule comprising at least two carbon atoms, but preferably not more than 7 or 12 rotatable carbon bonds, having a molecular weight in the range between 100 and 2000 Dalton, particularly between 100 and 1000 Dalton, or < 900 Dalton, and optionally including one or two metal atoms.

The term "therapeutically active peptide" refers, e.g., to peptides acting as agonists/antagonists of a cell surface receptor or peptides competing for a protein binding site on a given cellular target.

The term "therapeutically active nucleic acid molecule" refers, e.g., to antisense nucleic acid molecules, small interfering RNAs, micro RNAs or ribozymes.

Particular pharmaceutically active agents include Dornase alfa, corticosteroids, leukotriene modifiers, N-acetylcysteine, inhaled glutathione, anticholinergics, ibuprofen and p2-adrenergic receptor agonists.

In one embodiment, the fusion molecule comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 134 to 139 and SEQ ID NOs: 143 to 186, wherein, in particular embodiments, the amino acid sequences of SEQ ID NOs: 144 to 186 lack the (C-terminal) Strep-tag® II and His6 tags. In one embodiment, the fusion molecule comprises or consists of the amino acid sequence of SEQ ID NO: 134 or SEQ ID NO: 136.

B. hNGAL muteins for use in the fusion molecules of the present invention

In some embodiments, an hNGAL mutein binding pyoverdine (type I, II or III) or pyochelin may include at least one amino acid substitution of a native cysteine residue (e.g., the cysteine residue at position 87 of the linear polypeptide sequence of human NGAL, SEQ ID NO: 1 ) by another amino acid, for example, a serine residue. In some other embodiments, a mutein binding pyoverdine or pyochelin may include one or more non-native cysteine residues substituting one or more amino acids of wild-type hNGAL. In a further particular embodiment, an hNGAL mutein according to the disclosure includes at least two amino acid substitutions of a native amino acid by a cysteine residue, hereby to form one or more cysteine bridges. In some embodiments, said cysteine bridge may connect at least two loop regions. The definition of these regions is used herein in accordance with Flower (Flower, 1996, supra, Flower, et al., 2000, supra) and Breustedt et al. (2005, supra).

In some embodiments, an hNGAL mutein of the disclosure does not bind to enterobactin.

In one aspect, the present disclosure includes various hNGAL muteins that bind pyoverdine or pyochelin, e.g., with at least detectable affinity. In this sense, pyoverdine or pyochelin is regarded as a non-natural ligand of the reference wild-type hNGAL, where "non-natural ligand" refers to a compound that does not bind to wild-type human lipocalin 2 under physiological conditions. By engineering wild-type hNGAL with one or more mutations at certain sequence positions, the present inventors have demonstrated that high affinity and high specificity for the non-natural ligand, pyoverdine or pyochelin, is possible. In some embodiments, at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or even more nucleotide triplet(s) encoding certain sequence positions on wild-type I human lipocalin 2, a random mutagenesis may be carried out through substitution at these positions by a subset of nucleotide triplets.

The muteins of the disclosure may have a mutated amino acid residue at any one or more, including at least at any one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve, of the sequence positions corresponding to certain sequence positions of the linear polypeptide sequence of hNGAL, such as sequence positions 28, 34, 36, 39-42, 44-47, 49, 52, 54-55, 65, 68, 70, 72-75, 77, 79-81 , 87, 96, 100, 103, 106, 108, 123, 125, 127, 132, 134, 141 and 145 of the linear polypeptide sequence of human NGAL (SEQ ID NO: 1 ). In some embodiments, the mutated amino acid residue represents a conservative or a non-conservative substitution. In some embodiments, the hNGAL mutein comprises a mutated amino acid residue at one or more positions corresponding to positions 28, 54, 65 and 87 of the linear polypeptide sequence of the mature hNGAL (SEQ ID NO: 1 ).

A mutein of the disclosure may include the wild type (natural) amino acid sequence of the "parental" protein scaffold (such as hNGAL) outside the mutated amino acid sequence positions. In some embodiments, an hNGAL mutein according to the disclosure may also carry one or more amino acid mutations at a sequence position/ positions as long as such a mutation does, at least essentially not hamper or not interfere with the binding activity and the folding of the mutein. Such mutations can be accomplished very easily on DNA level using established standard methods (Sambrook, J. et al. (2001 ) Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Illustrative examples of alterations of the amino acid sequence are insertions or deletions as well as amino acid substitutions. Such substitutions may be conservative, i.e. an amino acid residue is replaced with an amino acid residue of chemically similar properties, in particular with regard to polarity as well as size. Examples of conservative substitutions are the replacements among the members of the following groups: 1 ) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan. On the other hand, it is also possible to introduce non-conservative alterations in the amino acid sequence. In addition, instead of replacing single amino acid residues, it is also possible to either insert or delete one or more continuous amino acids of the primary structure of the human lipocalin 2 as long as these deletions or insertion result in a stable folded/functional mutein (for example, hNGAL muteins with truncated N- and C-terminus). In such mutein, for instance, one or more amino acid residues are added or deleted at the N- or C- terminus of the polypeptide. Generally such a mutein may have about at least 70%, including at least about 80%, such as at least about 85%

amino acid sequence identity, with the amino acid sequence of the mature hNGAL. As an illustrative example, the present disclosure also encompasses hNGAL muteins as defined above, in which four amino acid residues (G-N-l-K; positions 95-98; SEQ ID NO: 130) of the linear polypeptide sequence of the mature hNGAL have been deleted (e.g. SEQ ID NO: 46).

The amino acid sequence of an hNGAL mutein disclosed herein has a high sequence identity to the mature hNGAL (SEQ ID NO: 1 ) when compared to sequence identities with other lipocalins. In this general context, the amino acid sequence of a mutein of the disclosure is at least substantially similar to the amino acid sequence of the natural wild-type hNGAL, e.g., with the proviso that possibly there are gaps (as defined below) in an alignment that are the result of additions or deletions of amino acids. A respective sequence of a mutein of the disclosure, being substantially similar to the sequences of the mature hNGAL, has, in some embodiments, at least 70% identity or sequence homology, at least 75% identity or sequence homology, at least 80% identity or sequence homology, at least 82% identity or sequence homology, at least 85% identity or sequence homology, at least 87% identity or sequence homology, or at least 90% identity or sequence homology including at least 95% identity or sequence homology, to the sequence of the mature hNGAL, e.g., with the proviso that the altered position or sequence is retained and that one or more gaps are possible.

a) hNGAL muteins specific for pyoverdine

In one aspect, the present disclosure relates to specific-binding human lipocalin 2 (human Lcn2 or hNGAL) muteins specific for one type of pyoverdine, such as Pvd type I, Pvd type II or Pvd type III.

One embodiment of the current disclosure relates to a mutein that is capable of binding one type of pyoverdine, e.g., with detectable affinity, such as an affinity measured by a KD of about 200 nM or lower, such as about 150 nM or lower.

In one aspect, the current disclosure provides an hNGAL mutein that is capable of binding Pvd type I complexed with iron with a KD of about 20 nM or lower, such as 15 nM or lower, for example,

when measured by Biacore T200 instrument in an assay essentially described in Example 6.

In some further embodiments, one or more hNGAL muteins of this disclosure are capable of binding Pvd type I succinyl, Pvd type I succinamid and Pvd type I a-ketoglutaryl with and without complexed iron, with an affinity measured by an IC50 value of about 200 nM or lower, for example, when measured in an ELISA assay essentially described in Example 5.

In some embodiments, the mutein is capable of inhibiting iron uptake mediated by pyoverdine type I succinyl with an IC50 value of about 150 nM or lower, for example, in a competition ELISA format essentially described in Example 7.

In some embodiments, the mutein is capable of inhibiting bacterial growth of Pvd I strain, for example, in an assay essentially described in Example 8.

In this regard, the disclosure relates to a polypeptide (e.g., a first polypeptide as referred to herein), wherein said polypeptide comprises or consists of an hNGAL mutein, and said hNGAL mutein in comparison with the linear polypeptide sequence of the mature hNGAL, comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or even more, mutated amino acid residues at the sequence positions 28, 36, 39-41 , 46, 49, 52, 54-55, 59, 65, 68, 70, 72-75, 77, 79-81 , 87, 96, 100, 103, 106, 125, 127, 132, 134 and 136, and wherein said polypeptide binds Pvd type I, including Pvd type I succinyl, Pvd type I succinamid and Pvd type I a-ketoglutaryl.

In some embodiments, a Pvd-type-l-binding hNGAL mutein of the disclosure includes, at any one or more of the sequence positions 36, 40-41 , 49, 52, 68, 70, 72-73, 77, 79, 81 , 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of the mature hNGAL (SEQ ID NO: 1 ), one or more of the following mutated amino acid residues: Leu 36→ Asn, Thr, Val, Trp or Phe; Ala 40→ Gly, Asn, Thr or Phe; lie 41→ Arg, Ala, Thr, Phe or Trp; Gin 49→ lie, Leu, Val, Ala or Pro; Tyr 52→ Met, Trp or Pro; Ser 68→ Asp, Val or Glu; Leu 70→ Gin, Trp, Asp or Thr; Arg 72→ Trp, Ala, Ser, Leu, Pro or Glu; Lys 73→ Asp, Leu, Ala, Glu or Asn; Asp 77→ Arg, Leu, Tyr, Ser, Gin, Thr, lie or Asn; Trp 79→ Gin, Asp, Ser, Arg, Met or Glu; Arg 81→ Gin, Gly, lie, Glu, His or Asp; Asn 96→ His, lie, Gly, Tyr or Asp; Tyr 100→ Lys, Glu, Asn, Ser, Phe or Tyr; Leu 103→ Lys, Pro, Gin, His, Asp, Tyr, Glu, Trp or Asn; Tyr 106→ His, Gin or Phe; Lys 125→ Arg, Ser, Trp, Tyr, Val or Gly; Ser 127→ Trp, Asn, Ala, Thr, Tyr, His, lie, Val or Asp; Tyr 132 → Trp, Asn, Gly or Lys; and Lys 134 → Asn, His, Trp, Gly, Gin or Asp. In some embodiments, an hNGAL mutein of the disclosure includes two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or even more or all mutated amino acid residues at these sequence positions of the mature hNGAL.

Additionally, a Pvd-type-l-binding hNGAL mutein according to the disclosure may also comprise one or more or all of the following substitutions in comparison with the linear polypeptide sequence of the mature hNGAL: Gin 28→ His; Lys 46→ Glu; Thr 54→ Val or Ala; lie 55→ Val; Lys 59→ Arg; Asn 65→ Asp or Gin; lie 80→ Thr; Cys 87→ Ser or Asn; and Thr 136→ Ala.

In some additional embodiments, an hNGAL mutein of the disclosure, which binds to Pvd type I, includes the following amino acid replacements in comparison with the linear polypeptide sequence of the mature hNGAL:

(a) Gin 28→ His; Leu 36→ Asn; Ala 40→Gly; lie 41 → Trp; Gin 49→ lie; Tyr 52→ Met;

Ser 68→ Val; Leu 70→ Gin; Arg 72→ Trp; Lys 73→ Asp; Asp 77→ Leu; Trp 79→ Gin; Arg 81→ Gin; Cys 87→ Ser; Asn 96→ His; Tyr 100→ Lys; Leu 103→ His; Tyr 106→ His; Lys 125→ Arg; Ser 127→ Trp; Tyr 132→ Trp; Lys 134→ Asp;

(b) Gin 28→ His; Leu 36→ Thr; Ala 40→Gly; lie 41→ Phe; Gin 49→ Leu; Tyr 52→ Trp;

Leu 70→ Trp; Arg 72→ Ala; Lys 73→ Leu; Asp 77→ Tyr; Trp 79→ Asp; Arg 81→ Gly;

Cys 87→ Ser; Asn 96→ lie; Tyr 100→ Glu; Leu 103→ His; Tyr 106→ Gin; Lys 125→ Trp; Ser 127→Asn; Tyr 132→ Asn; Lys 134→ Gin;

(c) Gin 28→ His; Leu 36→ Trp; Ala 40→Thr; lie 41 → Thr; Gin 49→ Pro; Tyr 52→ Pro;

Ser 68→ Asp; Leu 70→ Gin; Arg 72→ Ser; Lys 73→ Glu; Asp 77→ Ser; Trp 79→ Ser; Arg 81 → lie; Cys 87→ Ser; Asn 96→ Gly; Tyr 100→ Asn; Leu 103→ Lys; Tyr

106→ His; Lys 125→ Tyr; Ser 127→ Ala; Tyr 132→ Gly; Lys 134→ Asn;

(d) Gin 28→ His; Leu 36→ Phe; Ala 40→ Asn; lie 41→ Arg; Gin 49→ Pro; Tyr 52→ Met;

Ser 68→ Asp; Leu 70→ Thr; Arg 72→ Glu; Lys 73→ Ala; Asp 77→ Arg; Trp 79→ Arg; Arg 81→ lie; Cys 87→ Ser; Asn 96→ Tyr; Tyr 100→ Lys; Leu 103→ Pro; Tyr 106 → Phe; Lys 125→ Ser; Ser 127→ Thr; Tyr 132→ Trp; Lys 134→ Gly;

(e) Gin 28→ His; Ala 40→Gly; lie 41 → Trp; Gin 49→ Val; Tyr 52→ Met; Ser 68→ Val;

Leu 70→ Asp; Arg 72→ Glu; Lys 73→ Leu; Asp 77→ Arg; Trp 79→ Met; Arg 81 → Glu; Cys 87→ Ser; Asn 96→Asp; Tyr 100→ Phe; Leu 103→ Trp; Tyr 106→ Gin; Lys 125→ Gly; Ser 127→ Tyr; Tyr 132→ Trp; Lys 134→ His;

(f) Gin 28→ His; Leu 36→ Val; Ala 40→ Phe; lie 41→ Phe; Gin 49→ Ala; Tyr 52→ Pro;

Ser 68→ Glu; Leu 70→ Trp; Arg 72→ Leu; Lys 73→ Asn; Asp 77→ Gin; Trp 79→ Glu; Arg 81 → His; Cys 87→ Ser; Asn 96→ Tyr; Leu 103→ Tyr; Tyr 106→ His; Lys 125→ Val; Ser 127→ His; Tyr 132→ Lys; Lys 134→ Trp;

(g) Gin 28→ His; Leu 36→ Trp; Ala 40→Thr; lie 41 → Thr; Gin 49→ Pro; Tyr 52→ Pro;

Ser 68→ Asp; Leu 70→ Gin; Arg 72→ Ser; Lys 73→ Glu; Asp 77→ Ser; Trp 79→

Ser; lie 80→ Thr; Arg 81 → lie; Cys 87→ Ser; Asn 96→ Gly; Tyr 100→ Ser; Leu 103 → Gin; Tyr 106→ His; Lys 125→ Tyr; Ser 127→ lie; Tyr 132→ Gly; Lys 134→ Asn;

(h) Gin 28→ His; Leu 36→ Trp; Ala 40→Thr; lie 41 → Thr; Gin 49→ Pro; Tyr 52→ Pro;

Ser 68→ Asp; Leu 70→ Gin; Arg 72→ Ser; Lys 73→ Asp; Asp 77→ Ser; Trp 79→ Ser; Arg 81 → lie; Cys 87→ Ser; Asn 96→ Gly; Tyr 100→ Asn; Leu 103→ Asp; Tyr

106→ His; Lys 125→ Tyr; Ser 127→ Val; Tyr 132→ Gly; Lys 134→ Asn;

(i) Gin 28→ His; Leu 36→ Trp; Ala 40→Thr; lie 41 → Thr; Gin 49→ Pro; Tyr 52→ Pro;

Ser 68→ Asp; Leu 70→ Gin; Arg 72→ Ser; Lys 73→ Glu; Asp 77→ Thr; Trp 79→

Ser; Arg 81 → lie; Cys 87→ Ser; Asn 96→ Asp; Tyr 100→ Asn; Leu 103→ Glu; Tyr 106→ His; Lys 125→ Tyr; Ser 127→ Asp; Tyr 132→ Gly; Lys 134→ Asn;

G) Gin 28→ His; Leu 36→ Trp; Ala 40→Thr; lie 41 → Thr; Gin 49→ Pro; Tyr 52→ Pro;

Ser 68→ Asp; Leu 70→ Gin; Arg 72→ Ser; Lys 73→ Asp; Asp 77→ Val; Trp 79→ Ser; Arg 81 → lie; Cys 87→ Ser; Asn 96→ Gly; Tyr 100→ Asn; Leu 103→ Asn; Tyr

106→ His; Lys 125→ Tyr; Ser 127→ Val; Tyr 132→ Gly; Lys 134→ Asn;

(k) Gin 28→ His; Ala 40→Gly; lie 41 → Trp; Gin 49→ Leu; Tyr 52→ Met; Ser 68→ Val;

Leu 70→ Asp; Arg 72→ Glu; Lys 73→ Leu; Asp 77→ Arg; Trp 79→ Met; Arg 81 → Glu; Cys 87→ Ser; Asn 96→ Asp; Tyr 100→ Ser; Leu 103→ Trp; Tyr 106→ Gin; Lys 125→ Gly; Ser 127→ Tyr; Tyr 132→ Trp; Lys 134→ His;

(I) Gin 28→ His; Leu 36→ Trp; Ala 40→Thr; lie 41 → Thr; Gin 49→ Pro; Tyr 52→ Pro;

Thr 54→ Val; Ser 68→ Asp; Leu 70→ Gin; Arg 72→ Ser; Lys 73→ Glu; Lys 75→ Glu; Asp 77→ Ser; Trp 79→ Ser; lie 80→ Thr; Arg 81→ lie; Cys 87→ Ser; Asn 96→ Gly; Tyr 100→ Ser; Leu 103→ Gin; Tyr 106→ His; Lys 125→ Tyr; Ser 127→ Thr; Tyr 132→ Gly; Lys 134→ Asn;

(m) Gln 28→ His; Ala 40→Gly; lie 41→ Trp; Lys 46→ Glu; Gin 49→ Leu; Tyr 52→ Met;

Thr 54→ Ala; lie 55→ Val; Lys 59→ Arg; Ser 68→ Val; Leu 70→ Asp; Arg 72→ Glu; Lys 73→ Leu; Lys 74→ Glu; Lys 75→ Glu; Asp 77→ Arg; Trp 79→ Met; lie 80→ Thr; Arg 81→ Glu; Ser 87→ Asn; Asn 96→ Asp; Tyr 100→ Ser; Leu 103→ Trp; Tyr 106→ Gin; Lys 125→ Gly; Ser 127→ Tyr; Tyr 132→ Trp; Lys 134→ His;

(n) Leu 36→ Trp; Asn 39→ Asp; Ala 40→Thr; lie 41 → Thr; Gin 49→ Pro; Tyr 52→ Pro;

Thr 54→ Val; Asn 65→ Asp; Ser 68→ Asp; Leu 70→ Gin; Arg 72→ Ser; Lys 73→ Glu; Lys 75→ Glu; Asp 77→ Ser; Trp 79→ Ser; lie 80→ Thr; Arg 81→ lie; Cys 87→ Ser; Asn 96→ Gly; Tyr 100→ Ser; Leu 103→ Gin; Tyr 106→ His; Lys 125→ Tyr; Ser 127→ Thr; Tyr 132→ Gly; Lys 134→ Asn; Thr 136→ Ala;

(o) Leu 36→ Trp; Ala 40→Thr; lie 41 → Ala; Gin 49→ Pro; Tyr 52→ Pro; Thr 54→ Val;

Asn 65→ Asp; Ser 68→ Asp; Leu 70→ Gin; Arg 72→ Ser; Lys 73→ Glu; Lys 75→ Glu; Asp 77→ Ser; Trp 79→ Ser; lie 80→ Thr; Arg 81→ lie; Cys 87→ Ser; Asn 96→ Gly; Tyr 100→ Ser; Leu 103→ Gin; Tyr 106→ His; Lys 125→ Tyr; Ser 127→ Thr; Tyr 132→ Gly; Lys 134→ Asn; Thr 136→ Ala;

(p) Gin 28→ His; Ala 40→Gly; lie 41→ Trp; Lys 46→ Glu; Gin 49→ Leu; Tyr 52→ Met;

Thr 54→ Ala; lie 55→ Val; Lys 59→ Arg; Asn 65→ Asp; Ser 68→ Val; Leu 70→ Asp; Arg 72→ Glu; Lys 73→ Leu; Lys 74→ Glu; Lys 75→ Glu; Asp 77→ Arg; Trp 79→ Met; lie 80→ Thr; Arg 81→ Glu; Ser 87→ Asn; Asn 96→ Asp; Tyr 100→ Ser; Leu 103 → Trp; Tyr 106→ Gin; Lys 125→ Gly; Ser 127→ Tyr; Tyr 132→ Trp; Lys 134→ His; or

(q) Gin 28→ His; Ala 40→Gly; lie 41→ Trp; Lys 46→ Glu; Gin 49→ Leu; Tyr 52→ Met;

Thr 54→ Ala; lie 55→ Val; Lys 59→ Arg; Asn 65→ Gin; Ser 68→ Val; Leu 70→ Asp; Arg 72→ Glu; Lys 73→ Leu; Lys 74→ Glu; Lys 75→ Glu; Asp 77→ Arg; Trp 79→

Met; lie 80→ Thr; Arg 81→ Glu; Ser 87→Asn; Asn 96→ Asp; Tyr 100→ Ser; Leu 103 → Trp; Tyr 106→ Gin; Lys 125→ Gly; Ser 127→ Tyr; Tyr 132→ Trp; Lys 134→ His.

In the residual region, i.e. the region differing from sequence positions 28, 36, 39-41 , 46, 49, 52, 54-55, 59, 65, 68, 70, 72-75, 77, 79-81 , 87, 96, 100, 103, 106, 125, 127, 132, 134 and 136, an hNGAL mutein of the disclosure may include the wild type (natural) amino acid sequence outside the mutated amino acid sequence positions.

In further particular embodiments, a mutein according to the current disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-18 or a fragment or variant thereof.

The amino acid sequence of a Pvd-type-l-binding hNGAL mutein of the disclosure may have a high sequence identity, such as at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90% identity, including at least 95% identity, to a sequence selected from the group consisting of SEQ ID NOs: 2-18.

The disclosure also includes structural homologues of an hNGAL mutein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-18, which structural homologues have an amino acid sequence homology or sequence identity of more than about 60%, particularly more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 92% and most particularly more than 95% in relation to said hNGAL mutein.

A Pvd-type-l-binding hNGAL mutein according to the present disclosure can be obtained by means of mutagenesis of a naturally occurring form of human lipocalin 2. In some embodiments of the mutagenesis, a substitution (or replacement) is a conservative substitution. Nevertheless, any substitution - including non-conservative substitution or one or more from the exemplary substitutions below - is envisaged as long as the mutein retains its capability to bind to Pvd type I, and/or it has an identity to the then substituted sequence in that it is at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or higher identity to the amino acid sequence of the mature human lipocalin 2 (SWISS-PROT Data Bank Accession Number P80188).

In another aspect, the current disclosure provides an hNGAL mutein that binds Pvd type II complexed with iron with a KD of about 20 nM or lower, such as 5 nM or lower, for example, when measured by Biacore T200 instrument in an assay essentially described in Example 6.

In some still further embodiments, one or more hNGAL muteins of this disclosure are capable of binding Pvd type II succinyl, Pvd type II succinamid and Pvd type II a-ketoglutaryl with and without complexed iron, with an affinity measured by an IC50 value of about 200 nM or lower, for example, when measured in an ELISA assay essentially described in Example 5.

In some embodiments, the mutein is capable of inhibiting iron uptake mediated by pyoverdine type II succinyl with an IC50 value of about 150 nM or lower, for example, in a competition ELISA format essentially described in Example 7.

In some embodiments, the mutein is capable of inhibiting bacterial growth of Pvd II strain, for example, in an assay essentially described in Example 8.

In some other embodiments, the mutein is capable of inhibiting or lessening growth of P. aeruginosa stains expressing pyoverdine type II, for example, in an assay essentially described in Example 10.

In this regard, the disclosure relates to a polypeptide (e.g., a second polypeptide as referred to herein), wherein said polypeptide comprises or consists of an hNGAL mutein, and said hNGAL mutein in comparison with the linear polypeptide sequence of the mature hNGAL, comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or even more, mutated amino acid residues at the sequence positions 28, 36, 40-41 , 49, 52, 54, 65, 68, 70, 72-75, 77, 79, 81 , 87, 96, 100, 103, 106, 125, 127, 132 and 134, and wherein said polypeptide binds Pvd type II.

In some embodiments, a Pvd-type-ll-binding hNGAL mutein of the disclosure includes, at any one or more of the sequence positions 36, 40-41 , 49, 52, 68, 70, 72-73, 77, 79, 81 , 87, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of the mature hNGAL (SEQ ID NO: 1 ), one or more of the following mutated amino acid residues: Leu 36→ Asn, lie or Val; Ala 40→ Glu, Gly, Asn, Thr or His; lie 41→ Arg, Val or Thr; Gin 49→ Gly, Ala or Pro; Tyr 52→ Asn, Gly, Trp or Pro; Ser 68→ Asp, Arg or Glu; Leu 70→ Arg or Trp; Arg 72→ His, lie, Ala, Ser or Gly; Lys 73→ Asn, Met, Pro, Phe, Gin or Arg; Asp 77→ His, lie, Met, Lys, Gly or Asn; Trp 79→ Ser, Tyr, Ala, Asp, Phe or Trp; Arg 81→ Glu, Ser, Tyr or Asp; Asn 96→ Met, lie, Arg, Asp, Lys, Asn or Ala; Tyr 100→ Lys, Glu, Asn, Ser, Phe or Tyr; Leu 103→ Thr, lie, Gin, Gly, Met, His, Trp or Val; Tyr 106→ Met, Gin, Ala, lie, Asn, Gly, Met or Phe; Lys 125→ Ala, lie or Asn; Ser 127→ Lys, Arg, Ser, Met, Asp or Asn; Tyr 132→ Met, Phe, Asn, Ala, lie, Gly or Val; and Lys 134→ Trp or Tyr. In some embodiments, an hNGAL mutein of the disclosure includes two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, or even more or all mutated amino acid residues at these sequence positions of the mature hNGAL.

Additionally, a Pvd-type-ll-binding hNGAL mutein according to the disclosure may also comprise one or more or all of the following substitutions in comparison with the linear polypeptide sequence of the mature hNGAL: Gin 28→ His; Thr 54→ Ala; Asn 65→ Asp or Gin and Cys 87→ Ser.

In some additional embodiments, an hNGAL mutein of the disclosure, which binds to Pvd type II, includes the following amino acid replacements in comparison with the linear polypeptide sequence of the mature hNGAL:

(a) Gin 28→ His; Leu 36→ Val; Ala 40→ Glu; lie 41→ Val; Gin 49→ Gly; Tyr 52→ Pro;

Ser 68→ Glu; Leu 70→ Arg; Arg 72→ His; Lys 73→ Asn; Asp 77→ Asn; Trp 79→ Ser; Arg 81→ Glu; Cys 87→ Ser; Tyr 100→ Asn; Leu 103→ Gin; Tyr 106→ Met; Ser 127→ Lys; Tyr 132→ Gly; Lys 134→ Trp;

(b) Gin 28→ His; Ala 40→ Thr; lie 41 → lie; Gin 49→ Gly; Tyr 52→ Asn; Ser 68→ Asp;

Leu 70→ Arg; Arg 72→ lie; Lys 73→ Met; Asp 77→ His; Trp 79→ Tyr; Arg 81→ Glu;

Cys 87→ Ser; Asn 96→ lie; Tyr 100→ Asn; Leu 103→ Thr; Tyr 106→ Gin; Lys 125 → lie; Ser 127→ Arg; Tyr 132→ Met; Lys 134→ Trp;

(c) Gin 28→ His; Leu 36→ lie; Ala 40→Thr; lie 41 → Val; Gin 49→ Gly; Tyr 52→ Pro;

Ser 68→ Glu; Leu 70→ Arg; Arg 72→ Ala; Lys 73→ Pro; Asp 77→ lie; Trp 79→ Ser; Arg 81→ Ser; Cys 87→ Ser; Asn 96→ Met; Tyr 100→ Ser; Leu 103→ Gly; Tyr 106→

Ala; Lys 125→ Lys; Tyr 132→ Val; Lys 134→ Trp;

(d) Gin 28→ His; Ala 40→ Asn; Gin 49→ Ala; Tyr 52→ Pro; Ser 68→ Glu; Leu 70→ Arg;

Arg 72→ Ser; Lys 73→ Gin; Asp 77→ Met; Trp 79→ Ala; Arg 81 → Tyr; Cys 87→ Ser; Asn 96→ Arg; Tyr 100→ Pro; Leu 103→ Thr; Tyr 106→ lie; Lys 125→ Lys; Ser 127→ Met; Tyr 132→ Phe; Lys 134→ Trp;

(e) Gin 28→ His; Ala 40→ His; Gin 49→ Ala; Tyr 52→ Pro; Ser 68→ Glu; Leu 70→ Asp;

Arg 72→ Gly; Lys 73→ Arg; Asp 77→ His; Trp 79→ Trp; Arg 81 → Glu; Cys 87→ Ser; Asn 96→ Arg; Tyr 100→ Asp; Leu 103→ Met; Tyr 106→ Phe; Lys 125→ Ala; Ser 127→ Asp; Tyr 132→ Asn; Lys 134→ Trp;

(f) Gin 28→ His; Leu 36→ Asn; Ala 40→ Gly; lie 41→ Arg; Gin 49→ Pro; Tyr 52→ Trp;

Ser 68→ Arg; Leu 70→ Trp; Arg 72→ Asn; Lys 73→ Gin; Asp 77→ Lys; Trp 79→ Asp; Arg 81 → Glu; Cys 87→ Ser; Asn 96→ Asp; Tyr 100→ Thr; Leu 103→ Trp; Tyr 106→ Asn; Lys 125→ Asn; Ser 127→ Met; Tyr 132→ lie; Lys 134→ Tyr;

(g) Gin 28→ His; Leu 36→ Val; Ala 40→Thr; lie 41 → Thr; Gin 49→ Gly; Tyr 52→ Gly;

Ser 68→ Glu; Leu 70→ Arg; Arg 72→ Gly; Lys 73→ Arg; Asp 77→ Gly; Trp 79→

Trp; Arg 81 → Glu; Cys 87→ Ser; Asn 96→ Ala; Tyr 100→ Trp; Leu 103→ lie; Tyr 106→ Gly; Lys 125→ Lys; Ser 127→ Asn; Tyr 132→ Val; Lys 134→ Trp;

(h) Gin 28→ His; Leu 36→ Val; Ala 40→ Glu; lie 41→ Val; Gin 49→ Gly; Tyr 52→ Pro;

Ser 68→ Glu; Leu 70→ Arg; Arg 72→ His; Lys 73→ Asn; Asp 77→ Asn; Trp 79→

Ser; Arg 81 → Glu; Cys 87→ Ser; Asn 96→ Lys; Tyr 100→ Asn; Leu 103→ Val; Tyr

106→ Met; Lys 125→ Asn; Ser 127→ Lys; Tyr 132→ Gly; Lys 134→ Trp;

(i) Gin 28→ His; Leu 36→ Val; Ala 40→ Thr; lie 41→ Val; Gin 49→ Gly; Tyr 52→ Pro;

Ser 68→ Glu; Leu 70→ Arg; Arg 72→ His; Lys 73→ Asn; Asp 77→ Asn; Trp 79→

Ser; Arg 81→ Glu; Cys 87→ Ser; Leu 103→ Gin; Tyr 106→ Met; Ser 127→ Lys; Tyr

132→ Val; Lys 134→ Trp;

G) Gin 28→ His; Leu 36→ Val; Ala 40→ Thr; lie 41→ Val; Gin 49→ Gly; Tyr 52→ Pro;

Ser 68→ Glu; Leu 70→ Arg; Arg 72→ His; Asp 77→ Asn; Trp 79→ Phe; Arg 81 →

Glu; Cys 87→ Ser; Asn 96→ Lys; Tyr 100→ His; Leu 103→ Gin; Tyr 106→ Met; Ser

127→ Lys; Tyr 132→ Ala; Lys 134→ Trp;

(k) Gin 28→ His; Leu 36→ Val; Ala 40→ Gly; lie 41→ Val; Gin 49→ Gly; Tyr 52→ Pro;

Ser 68→ Glu; Leu 70→ Arg; Arg 72→ His; Lys 73→ Asn; Asp 77→ Asn; Trp 79→ Trp; Arg 81→ Glu; Cys 87→ Ser; Tyr 100→ Asn; Leu 103→ His; Tyr 106→ Met; Ser

127→ Lys; Tyr 132→ Gly; Lys 134→ Trp;

(I) Gin 28→ His; Leu 36→ Val; Ala 40→ Thr; lie 41 → lie; Gin 49→ Gly; Tyr 52→ Asn;

Ser 68→ Asp; Leu 70→ Arg; Arg 72→ lie; Lys 73→ Phe; Asp 77→ His; Trp 79→ Tyr;

Arg 81 → Asp; Cys 87→ Ser; Leu 103→ Met; Tyr 106→ Gin; Lys 125→ lie; Ser 127 → Arg; Tyr 132→ lie; Lys 134→ Trp;

(m) Gln 28→ His; Leu 36→ Val; Ala 40→ Thr; lie 41 → lie; Gin 49→ Gly; Tyr 52→ Asn;

Ser 68→ Asp; Leu 70→ Arg; Arg 72→ lie; Lys 73→ Arg; Asp 77→ His; Trp 79→ Tyr;

Arg 81 → Asp; Cys 87→ Ser; Leu 103→ Thr; Tyr 106→ Gin; Lys 125→ lie; Ser 127

→ Arg; Tyr 132→ lie; Lys 134→ Trp;

(n) Gin 28→ His; Leu 36→ Val; Ala 40→ Glu; lie 41→ Val; Gin 49→ Gly; Tyr 52→ Pro;

Asn 65→ Asp; Ser 68→ Glu; Leu 70→ Arg; Arg 72→ His; Lys 73→ Asn; Asp 77→

Asn; Trp 79→ Phe; Arg 81→ Glu; Cys 87→ Ser; Asn 96→ Lys; Tyr 100→ Asn; Leu

103→ Val; Tyr 106→ Met; Lys 125→ Asn; Ser 127→ Lys; Tyr 132→ Gly; Lys 134→

Trp;

(o) Gin 28→ His; Leu 36→ Val; Ala 40→ Glu; lie 41→ Val; Gin 49→ Gly; Tyr 52→ Pro;

Asn 65→ Gin; Ser 68→ Glu; Leu 70→ Arg; Arg 72→ His; Lys 73→ Asn; Asp 77→ Asn; Trp 79→ Phe; Arg 81→ Glu; Cys 87→ Ser; Asn 96→ Lys; Tyr 100→ Asn; Leu 103→ Val; Tyr 106→ Met; Lys 125→ Asn; Ser 127→ Lys; Tyr 132→ Gly; Lys 134→ Trp;

(p) Gin 28→ His; Leu 36→ Val; Ala 40→ Thr; lie 41 → lie; Gin 49→ Gly; Tyr 52→ Asn;

Thr 54→ Ala; Asn 65→ Asp; Ser 68→ Asp; Leu 70→ Arg; Arg 72→ lie; Lys 73→ Arg; Asp 77→ His; Trp 79→ Tyr; Arg 81→ Asp; Cys 87→ Ser; Leu 103→ Thr; Tyr 106 → Gin; Lys 125→ lie; Ser 127→ Arg; Tyr 132→ lie; Lys 134→ Trp;

(q) Gin 28→ His; Leu 36→ Val; Ala 40→ Thr; lie 41 → lie; Gin 49→ Gly; Tyr 52→ Asn;

Thr 54→ Ala; Asn 65→ Gin; Ser 68→ Asp; Leu 70→ Arg; Arg 72→ lie; Lys 73→ Arg;

Asp 77→ His; Trp 79→ Tyr; Arg 81 → Asp; Cys 87→ Ser; Leu 103→ Thr; Tyr 106→

Gin; Lys 125→ lie; Ser 127→ Arg; Tyr 132→ lie; Lys 134→ Trp;

(r) Leu 36→ Val; Ala 40→ Thr; lie 41→ lie; Gin 49→ Gly; Tyr 52→ Asn; Thr 54→ Ala;

Asn 65→ Asp; Ser 68→ Asp; Leu 70→ Arg; Arg 72→ lie; Lys 73→ Arg; Asp 77→

His; Trp 79→ Tyr; Arg 81 → Asp; Cys 87→ Ser; Leu 103→ Thr; Tyr 106→ Gin; Lys

125→ lie; Ser 127→ Arg; Tyr 132→ lie; Lys 134→ Trp; or

(s) Gin 28→ His; Leu 36→ Val; Ala 40→ Thr; lie 41 → lie; Gin 49→ Gly; Tyr 52→ Asn;

Asn 65→ Gin; Ser 68→ Asp; Leu 70→ Arg; Arg 72→ lie; Lys 73→ Arg; Asp 77→ His;

Trp 79→ Tyr; Arg 81→ Asp; Cys 87→ Ser; Leu 103→ Thr; Tyr 106→ Gin; Lys 125→ lie; Ser 127→ Arg; Tyr 132→ lie; Lys 134→ Trp.

In the residual region, i.e. the region differing from sequence positions 28, 36, 40-41 , 49, 52, 54, 65, 68, 70, 72-75, 77, 79, 81 , 87, 96, 100, 103, 106, 125, 127, 132 and 134, an hNGAL mutein of the disclosure may include the wild type (natural) amino acid sequence outside the mutated amino acid sequence positions.

In further particular embodiments, a mutein according to the current disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-37 or a fragment or variant thereof.

The amino acid sequence of a Pvd-type-ll-binding hNGAL mutein of the disclosure may have a high sequence identity, such as at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90% identity, including at least 95% identity, to a sequence selected from the group consisting of SEQ ID NOs: 19-37.

The disclosure also includes structural homologues of an hNGAL mutein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-37, which structural homologues have an amino acid sequence homology or sequence identity of more than about 60%, particularly more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 92% and most particularly more than 95% in relation to said hNGAL mutein.

A Pvd-type-ll-binding hNGAL mutein according to the present disclosure can be obtained by means of mutagenesis of a naturally occurring form of human lipocalin 2. In some embodiments of the mutagenesis, a substitution (or replacement) is a conservative substitution. Nevertheless, any substitution - including non-conservative substitution or one or more from the exemplary substitutions below - is envisaged as long as the mutein retains its capability to bind to Pvd type I, and/or it has an identity to the then substituted sequence in that it is at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or higher identity to the amino acid sequence of the mature human lipocalin 2 (SWISS-PROT Data Bank Accession Number P80188).

In still another aspect, the current disclosure provides an hNGAL mutein that binds Pvd type III complexed with iron with a KD of about 20 nM or lower, such as 10 nM or lower, for example, when measured by Biacore T200 instrument in an assay essentially described in Example 6.

In some still further embodiments, one or more hNGAL muteins of this disclosure are capable of binding Pvd type III succinyl, Pvd type III succinamid and Pvd type II a-ketoglutaryl with and without complexed iron, with an affinity measured by an IC50 value of about 200 nM or lower, for example, when measured in an ELISA assay essentially described in Example 5.

In some embodiments, the mutein is capable of inhibiting iron uptake mediated by pyoverdine type III with an IC50 value of about 150 nM or lower, for example, in a competition ELISA format essentially described in Example 7.

In some embodiments, the mutein is capable of inhibiting bacterial growth of Pvd III strain, for example, in an assay essentially described in Example 8.

In this regard, the disclosure relates to a polypeptide (e.g., a third polypeptide as referred to herein), wherein said polypeptide comprises or consists of an hNGAL mutein, and said hNGAL mutein in comparison with the linear polypeptide sequence of the mature hNGAL, comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or even more, mutated amino acid residues at the sequence positions 28, 36, 40-42, 45-47, 49, 52, 65, 68, 70, 72-73, 77, 79, 81 , 87, 96, 100, 103, 105-106, 125, 127, 132, 134 and 145, and wherein said polypeptide binds Pvd type III.

In some embodiments, a Pvd-type-lll-binding hNGAL mutein of the disclosure includes, at any one or more of the sequence positions 36, 40-41 , 49, 52, 68, 70, 72-73, 77, 79, 81 , 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of the mature hNGAL (SEQ ID NO: 1 ), one or more of the following mutated amino acid residues: Leu 36→ Phe or Glu; Ala 40→ Trp, Leu or Arg; lie 41 → Met, Arg, Ala, Leu or Trp; Gin 49→ His, lie, Arg, Lys, Met or Pro; Tyr 52→ Asn, Tyr, Arg, Ser or Met; Ser 68→ Asp, Asn, Glu or Gin; Leu 70→ Lys, Asn or Arg; Arg 72→ Leu, Arg, Gin or Tyr; Lys 73→ His, Leu, Ala, Pro, Gin or Tyr; Asp 77→ Ala, lie, Lys, Gin or Arg; Trp 79→ Ser or Asp; Arg 81 → His, Ala, Ser or Val; Asn 96→ Met, lie, Arg,

Gly, Leu or Val; Tyr 100→ Ala, lie, Asn, Pro or Asp; Leu 103→ Gin, Gly, Phe or Pro; Tyr 106 → Glu; Lys 125→ Trp or Thr; Ser 127→ Val, His, lie, Phe or Ala; Tyr 132→ Phe; and Lys 134 → Trp, Gin or Glu. In some embodiments, an hNGAL mutein of the disclosure includes two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, or even more or all mutated amino acid residues at these sequence positions of the mature hNGAL.

Additionally, a Pvd-type-lll-binding hNGAL mutein according to the disclosure may also comprise one or more or all of the following substitutions in comparison with the linear polypeptide sequence of the mature hNGAL: Gin 28→ His; Leu 42→ Arg; Asp 45→ Gly; Lys 46→ Arg; Asp 47→ Asn; Asn 65→ Asp; Cys 87→ Ser; Ser 105→ Pro and Thr 145→ Pro.

In some additional embodiments, an hNGAL mutein of the disclosure, which binds to Pvd type III, includes the following amino acid replacements in comparison with the linear polypeptide sequence of the mature hNGAL:

(a) Gin 28→ His; Leu 36→ Phe; Ala 40→ Trp; lie 41→ Met; Gin 49→ His; Tyr 52→ Asn;

Ser 68→ Glu; Leu 70→ Lys; Arg 72→ Gin; Lys 73→ Ala; Asp 77→ lie; Trp 79→ Ser; Arg 81→ His; Cys 87→ Ser; Asn 96→ lie; Tyr 100→ Asn; Leu 103→ may thus allow the formation of disulfide bonds in the cytosol (Venturi et al. (2002) J. Mol. Biol. 315, 1 -8.).

However, the fusion molecule as described herein may not necessarily be generated or produced only by use of genetic engineering. Rather, such fusion molecule can also be obtained by chemical synthesis such as Merrifield solid phase polypeptide synthesis or by in vitro transcription and translation. Methods for the solid phase and/or solution phase synthesis of proteins are well known in the art (see e.g. Bruckdorfer, T. et al. (2004) Curr. Pharm. Biotechnol. 5, 29-43), and so are methods for in vitro transcription/translation. It is also possible that promising mutations are identified using molecular modeling and then to synthesize the wanted (designed) polypeptide in vitro and investigate the binding activity for pyoverdine type I, II, III and/or pyochelin. Methods for the solid phase and/or solution phase synthesis of proteins are well known in the art (see e.g. Bruckdorfer, T. et al. (2004) Curr. Pharm. Biotechnol. 5, 29-43).

The skilled worker will appreciate methods useful to prepare fusion molecules contemplated by the present invention but whose protein or nucleic acid sequences are not explicitly disclosed herein. As an overview, such modifications of the amino acid sequence include, e.g., directed mutagenesis of single amino acid positions in order to simplify sub-cloning of an hNGAL mutein gene or its parts by incorporating cleavage sites for certain restriction enzymes. In addition, these mutations can also be incorporated to further improve the affinity of a mutein for its target (e.g. pyoverdine or pyochelin, respectively). Furthermore, mutations can be introduced to modulate certain characteristics of the muteins and fusion molecules, such as to improve folding stability, serum stability, protein resistance or water solubility or to reduce aggregation tendency, if necessary. For example, naturally occurring cysteine residues may be mutated to other amino acids to prevent disulphide bridge formation.

The current disclosure also relates to a nucleic acid molecule comprising a nucleotide sequence encoding a mutein disclosed herein. In this regard, the present disclosure provides nucleotide sequences encoding some muteins of the disclosure as shown in SEQ ID NOs: 65-126. Since the degeneracy of the genetic code permits substitutions of certain codons by other codons specifying the same amino acid, the disclosure is not limited to a specific nucleic acid molecule encoding a mutein as described herein but encompasses all nucleic acid molecules that include nucleotide sequences encoding a functional mutein. The disclosure further encompasses a host cell containing said nucleic acid molecule. All aspects and embodiments relating to nucleic acid molecules and (host) cells disclosed in connection with the fusion molecules of the present

invention are also applicable to single muteins and their combinations. The same applies to methods of their production.

D. Compositions and kits

In a further aspect, the present invention relates to a pharmaceutical composition comprising a fusion molecule as defined herein, a nucleic acid molecule as defined herein, or a cell as defined herein.

In some embodiments, the pharmaceutical compositions of the invention are sterile and/or contain an effective amount of the fusion molecules, nucleic acid molecules or cells described herein to generate the desired reaction or the desired effect.

The term "effective amount", as used throughout the description, refers to an amount that is sufficient to effect beneficial or desired results, wherein an effective amount can be administered in one or more administrations. In the case of treatment of a particular disease or of a particular condition, the beneficial or desired result particularly relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The beneficial or desired result in a treatment of a disease or of a condition may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of an agent or composition described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the subject, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the agents described herein may depend on various of such parameters. In the case that a reaction in a subject is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

Pharmaceutical compositions are usually provided in a uniform dosage form and may be prepared in a manner known per se. A pharmaceutical composition may, e.g., be in the form of a solution or suspension.

A pharmaceutical composition may further comprise one or more carriers and/or excipients, all of which are, in particular embodiments, pharmaceutically acceptable. The term "pharmaceutically acceptable", as used herein, refers to the non-toxicity of a material which, in particular embodiments, does not interact with the action of the active agent of the pharmaceutical composition.

The term "carrier" refers to an organic or inorganic component, of a natural or synthetic nature, in which the active component is combined in order to facilitate, enhance or enable application. According to the invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a subject.

Pharmaceutical compositions suitable for parenteral administration usually comprise a sterile aqueous or non-aqueous preparation of the active compound, which, in a particular embodiment, is isotonic to the blood of the recipient. Examples of compatible carriers/solvents/diluents for parenteral administration are, e.g., sterile water, Ringer solution, Ringer lactate, isotonic sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers. In addition, usually sterile, fixed oils are used as solution or suspension medium.

The term "excipient", as used herein, is intended to include all substances which may be present in a pharmaceutical composition and which are not active ingredients, such as salts, binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffer substances, flavouring agents, or colorants.

Salts, which are not pharmaceutically acceptable, may be used for preparing pharmaceutically acceptable salts and are included in the invention. Pharmaceutically acceptable salts of this kind comprise in a non limiting way those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic acids, and the like. Pharmaceutically acceptable salts may also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.

Suitable preservatives for use in a pharmaceutical composition include benzalkonium chloride, chlorobutanol, paraben and thimerosal.

Suitable buffer substances for use in a pharmaceutical composition include acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt.

The pharmaceutical composition may also comprise one or more pharmaceutically acceptable adjuvants.

In one embodiment, the pharmaceutical composition is formulated for systemic administration, in particular parenteral (e.g., subcutaneous) administration. In another embodiment, the pharmaceutical composition is formulated for inhalation.

In one embodiment, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

In one embodiment, the additional pharmaceutically active agent is selected from the group consisting of an antibiotic, a cytostatic agent, a toxin, an enzyme, a metal or metal compound/complex, a chelating agent, a radionuclide, a small organic molecule, a therapeutically active peptide, a therapeutically active nucleic acid molecule, a hapten and an antibody. Suitable antibiotics include Tobramycin, Azithromycin, Aztreonam, Colistin and carbapenems.

Suitable metals or metal compounds/complexes are gallium, gallium compounds and gallium-based complexes.

The term "small organic molecule", as used herein, refers to an organic molecule comprising at least two carbon atoms, but preferably not more than 7 or 12 rotatable carbon bonds, having a molecular weight in the range between 100 and 2000 Dalton, particularly between 100 and 1000 Dalton, or < 900 Dalton, and optionally including one or two metal atoms.

The term "therapeutically active peptide" refers, e.g., to peptides acting as agonists/antagonists of a cell surface receptor or peptides competing for a protein binding site on a given cellular target.

The term "therapeutically active nucleic acid molecule" refers, e.g., to antisense nucleic acid molecules, small interfering RNAs, micro RNAs or ribozymes.

Particular additional pharmaceutically active agents, with which the fusion molecules of the present invention can be combined, include Dornase alfa, corticosteroids, leukotriene modifiers, N-acetylcysteine, inhaled glutathione, anticholinergics, ibuprofen and p2-adrenergic receptor agonists.

In yet another aspect, the present invention relates to a kit comprising a fusion molecule as defined herein, a nucleic acid molecule as defined herein, a cell as defined herein, or a pharmaceutical composition as defined herein. The kit may be a diagnostic or analytical kit.

As used herein, the term "kit of parts (in short: kit)" refers to an article of manufacture comprising one or more containers and, optionally, a data carrier. Said one or more containers may be filled with one or more of the means or reagents disclosed herein. Additional containers may be included in the kit that contain, e.g., diluents, buffers and further reagents. Said data carrier may be a non-electronic data carrier, e.g., a graphical data carrier such as an information leaflet, an information sheet, a bar code or an access code, or an electronic data carrier such as a compact disk (CD), a digital versatile disk (DVD), a microchip or another semiconductor-based electronic data carrier. The access code may allow the access to a database, e.g., an internet database, a centralized, or a decentralized database. Said data carrier may comprise instructions for the use of the fusion molecule, nucleic acid molecule, cell and/or pharmaceutical composition of the present invention.

All aspects and embodiments relating to compositions and kits disclosed in connection with the fusion molecules of the present invention are also applicable to single muteins and their combinations.

E. Diagnostic and therapeutic applications

Pyoverdines are the main siderophores of pseudomonads such as P. aeruginosa. In vitro experiments indicated a potential role of the P. aeruginosa pyoverdine in iron release from ferritransferrin but the ability of pyoverdine to compete for iron in vivo has only recently been demonstrated (Meyer et al., 1996, Infection and Immunity, 64, p.518-523). It was observed using a burned-mouse model that the absence of pyoverdine production in mutants raised from a virulent parental strain correlated with a loss of virulence of these mutants and that virulence was restored when the homologous pyoverdine originating from the wild-type strain was supplemented. Furthermore, supplementation with a heterologous pyoverdine did not restore the virulence of the latter mutants. Thus, a precise knowledge of the pyoverdine-mediated iron uptake system used by a given P. aeruginosa isolate during infection appears a prerequisite for developing new ways of treatment of P. aeruginosa infections via bacterial iron metabolism, e.g., by blocking the pyoverdine biosynthesis or the pyoverdine-mediated iron transport.

Pyochelin (Pch) is one of the two major siderophores produced and secreted by Pseudomonas aeruginosa to assimilate iron. It chelates iron in the extracellular medium and transports it into the cell via a specific outer membrane transporter, FptA. Pch strongly chelates divalent metals such as Zn(ll) (pZn = 1 1 .8 at p[H] 7.4) and Cu(ll) (pCu = 14.9 at p[H] 7.4) and forms predominantly 1 : 2 (M2+/Pch) complexes. Siderophores are not only devoted to iron(lll) shuttling but most likely display other specific biological roles in the subtle metals homeostasis in microorganisms.

Therefore, numerous possible applications for the pyoverdine/pyochelin-binding fusion molecules of the invention exist in medicine.

In one aspect, the present invention relates to a fusion molecule as defined herein, a nucleic acid molecule as defined herein, a cell as defined herein, or a pharmaceutical composition as defined herein for use as a medicament.

The term "medicament", as used herein, refers to a substance/composition used in therapy, i.e., in the treatment of a disease.

The term "treatment of a disease", as used herein, includes curing, shortening the duration, ameliorating, preventing, slowing down or inhibiting progression or worsening, or preventing or delaying the onset of a disease or the symptoms thereof.

According to the invention, the term "disease" refers to any pathological state, in particular a disease or disorder associated with or caused by P. aeruginosa biofilm infection in a subject.

In another aspect, the present invention relates to a fusion molecule as defined herein, a nucleic acid molecule as defined herein, a cell as defined herein, or a pharmaceutical composition as defined herein for use in the prevention or treatment of P. aeruginosa biofilm infection in a subject.

The term "subject", as used throughout the description, means according to the invention a subject for treatment or diagnosis, in particular a diseased subject (also referred to as "patient") or a subject expected/assumed/at risk to be diseased. A "subject" is a vertebrate, particularly a mammal. The term "mammal" is used herein to refer to any animal classified as a mammal, including, without limitation, humans, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, rats, pigs, apes such as cynomolgus monkeys etc., to name only a few illustrative examples. In a particular embodiment, the mammal is a human.

In yet another aspect, the present invention relates to a fusion molecule as defined herein, a nucleic acid molecule as defined herein, a cell as defined herein, or a pharmaceutical composition as defined herein for use in the prevention or treatment of a disease or disorder associated with or caused by P. aeruginosa biofilm infection in a subject.

In yet another aspect, the present invention relates to a method of preventing or treating P. aeruginosa biofilm infection in a subject, comprising administering an effective amount of a

fusion molecule as defined herein, a nucleic acid molecule as defined herein, a cell as defined herein, or a pharmaceutical composition as defined herein to the subject.

In yet another aspect, the present invention relates to a method of preventing or treating a disease or disorder associated with or caused by P. aeruginosa biofilm infection in a subject, comprising administering an effective amount of a fusion molecule as defined herein, a nucleic acid molecule as defined herein, a cell as defined herein, or a pharmaceutical composition as defined herein to the subject.

In one embodiment, the P. aeruginosa biofilm infection is acute or chronic infection.

In one embodiment, the disease or disorder associated with or caused by P. aeruginosa biofilm infection is selected from the group consisting of cystic fibrosis, hospital acquired pneumonia, ventilator-associated pneumonia, urinary tract infection, eye infection, ear infection and burn wound infection.

In yet another aspect, the present invention relates to a fusion molecule as defined herein, a nucleic acid molecule as defined herein, a cell as defined herein, or a pharmaceutical composition as defined herein for use in binding of pyoverdine (type I, II and/or III) and/or pyochelin in a subject and/or inhibiting or lessening growth of P. aeruginosa in a subject.

In still another aspect, the present invention features a method of binding pyoverdine (type I, II and/or III) and/or pyochelin in a subject, comprising administering to said subject an effective amount of a fusion molecule as defined herein, a nucleic acid molecule as defined herein, a cell as defined herein, or a pharmaceutical composition as defined herein.

In still another aspect, the present invention features a method for inhibiting or lessening growth of P. aeruginosa in a subject, comprising administering to said subject an effective amount of a fusion molecule as defined herein, a nucleic acid molecule as defined herein, a cell as defined herein, or a pharmaceutical composition as defined herein.

In yet a further aspect, the present invention relates to the use of a fusion molecule as defined herein, a nucleic acid molecule as defined herein, a cell as defined herein, or a pharmaceutical composition as defined herein in the manufacture of a medicament for (i) the prevention or treatment of P. aeruginosa biofilm infection in a subject, (ii) the prevention or treatment of a disease or disorder associated with or caused by P. aeruginosa biofilm infection in a subject, or (iii) inhibiting or lessening growth of P. aeruginosa in a subject.

The agents and compositions described herein may be administered via any conventional route, such as by topical or systemic (i.e., enteral or parenteral) administration. Parenteral administration includes injection or infusion, e.g., intravenously, intraarterially, subcutaneously, intradermally or intramuscularly. Topical administration includes inhalation as well as eye and ear drops. In particular embodiments, the fusion molecules of the invention are administered by inhalation (fusion molecules) or systemically, e.g., subcutaneously (Fc-fusion molecule constructs).

The present invention further relates to the use of a fusion molecule disclosed herein for detecting pyoverdine (type I, II and/or III) and/or pyochelin in a sample as well as a respective method of diagnosis.

The term "detect", "detection", "detectable" or "detecting" as used herein is understood both on a quantitative and a qualitative level, as well as a combination thereof. It thus includes quantitative, semi-quantitative and qualitative measurements of a molecule of interest.

Such use may include the steps of contacting one or more fusion molecules, under suitable conditions, with a sample suspected of containing pyoverdine and/or pyochelin, thereby allowing formation of a complex between the fusion molecule and pyoverdine (type I, II and/or III) and/or pyochelin, and detecting the complex by a suitable signal. The signal can be caused by a label, as explained above, or by a change of physical properties due to the binding, i.e. the complex formation, itself. One example is surface plasmon resonance, the value of which is changed during binding of binding partners from which one is immobilized on a surface such as a gold foil.

The fusion molecules disclosed herein may also be used for the separation of pyoverdine and/or pyochelin. Such use may include the steps of contacting such fusion molecule, under suitable conditions, with a sample supposed to contain pyoverdine and/or pyochelin, thereby allowing formation of a complex between the fusion molecule and pyoverdine and/or between the fusion molecule and pyochelin, respectively, and separating the complex from the sample.

In the use of the disclosed fusion molecules for the detection of pyoverdine and/or pyochelin as well as the separation of pyoverdine and/or pyochelin, the fusion molecules and/or pyoverdine and/or pyochelin or a domain or fragment thereof may be immobilized on a suitable solid phase.

Accordingly, the presence or absence of pyoverdine and/or pyochelin, e.g., in a sample, as well as its concentration or level may be determined.

A "sample" is defined as a biological sample taken from any subject. Biological samples include, but are not limited to, blood, serum, urine, feces, semen, or tissue.

The fusion molecules disclosed herein can be used in many fields similar to antibodies or fragments thereof. For example, the fusion molecules can be used for labeling with an enzyme, an antibody, a radioactive substance or any other group having biochemical activity or defined binding characteristics. By doing so, their respective targets can be detected or brought in contact with them. In addition, fusion molecules of the invention can serve to detect chemical structures by means of established analytical methods (e.g., ELISA or Western Blot) or by microscopy or immunosensorics. In this regard, the detection signal can either be generated directly by use of a suitable fusion molecule conjugate or indirectly by immunochemical detection of the bound fusion molecule via an antibody.

All aspects and embodiments relating to diagnostic and therapeutic applications disclosed in connection with the fusion molecules of the present invention are also applicable to single muteins and their combinations.

Additional objects, advantages, and features of this invention will become apparent to those skilled in the art upon examination of the following Examples and the attached Figures thereof, which are not intended to be limiting. Thus, it should be understood that although the present invention is specifically disclosed by exemplary embodiments and optional features, modifications and variations of the disclosures embodied herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. In particular, all aspects and embodiments disclosed in connection with the fusion molecules of the invention are also applicable to single muteins disclosed herein as well as to their combinations, and vice versa.

VI. EXAMPLES

Example 1 : Purification and biotinylation of Pseudomonas aeruginosa siderophores

P. aeruginosa produces three groups of pyoverdines i.e. pyoverdine type I, pyoverdine type II & pyoverdine type III. Each group has three forms differing in the side chain which is succinyl, succinamid or a-ketoglutaryl. In addition P. aeruginosa produces pyochelin. All ten siderophores can complex iron as Fe3+.

For selection and screening of muteins of interest, the siderophores may be biotinylated. Biotinylation was performed for pyoverdine I succinyl variant at the succinyl side chain, for pyoverdine II succinyl variant at the L-ornithine side chain and for pyoverdine III succinyl variant mainly at the glycine side chain. Pyochelin was biotinylated at the phenol ring.

Example 2: Selection of muteins specifically binding to P. aeruginosa siderophores

hNGAL-based libraries, generated by random mutagenesis of mature hNGAL, were used for

CLAIMS

1 . A fusion molecule having binding specificity for pyoverdine type I, II and III and pyochelin, comprising

(a) a first polypeptide comprising a human neutrophil gelatinase-associated lipocalin (hNGAL) mutein that binds pyoverdine type I;

(b) a second polypeptide comprising an hNGAL mutein that binds pyoverdine type II;

(c) a third polypeptide comprising an hNGAL mutein that binds pyoverdine type III; and

(d) a fourth polypeptide comprising an hNGAL mutein that binds pyochelin;

wherein the first, second, third and fourth polypeptides are covalently linked.

2. The fusion molecule of claim 1 , wherein the hNGAL mutein comprises a mutated amino acid residue at one or more positions corresponding to positions 28, 34, 36, 39-42, 44-47, 49, 52, 54-55, 65, 68, 70, 72-75, 77, 79-81 , 87, 96, 100, 103, 106, 108, 123, 125, 127, 132, 134, 141 and 145 of the linear polypeptide sequence of the mature hNGAL (SEQ ID NO: 1 ).

3. The fusion molecule of claim 1 or 2, wherein the hNGAL mutein binds pyoverdine type I, pyoverdine type II, pyoverdine type III and pyochelin, respectively, with a dissociation constant KD of 200 nM or lower.

4. The fusion molecule of any one of claims 1 to 3, wherein the first, second, third and fourth polypeptides are covalently linked via linker molecules, in particular peptide linkers.

5. The fusion molecule of any one of claims 1 to 4, further comprising a multimerization domain allowing the multimerization of the fusion molecule, in particular a dimerization domain allowing the dimerization of the fusion molecule.

6. The fusion molecule of claim 5, wherein the dimerization domain is selected from the group consisting of an Fc domain, an IgE heavy-chain domain 2 (EHD2), an IgM heavy-chain domain 2 (MHD2), an IgG heavy-chain domain 3 (GHD3), an IgA heavy-chain domain 3 (AHD2), an IgD heavy-chain domain 3 (DHD3), an IgE heavy-chain domain 4 (EHD4), an IgM heavy-chain domain 4 (MHD4), an uteroglobin dimerization domain and variants or fragments of any one of the foregoing, wherein, in particular, the Fc domain is a human lgG4-Fc domain.

7. The fusion molecule of any one of claims 1 to 6, having a general formula selected from the group consisting of

/V'- X1 - L1 - X2 - L2 - X3 - L3 - X4 -C' (I),

ΛΓ- Xi - Li - X2 - L2 - X3 - L3 - X4 - - MD -C (II),

ΛΓ- MD - Li - Xi - L2 - X2 - L3 - X3 - - X4 -C (II I), ΛΓ- Xi - Li - X2 - L2 - MD - L3 - X3 - L4 - X4 -C (IV), W- X1 - L1 - MD - L2 - X2 - L3 - X3 - L4 - X4 -C' (V), and

N'- Xi - Li - X2 - L2 - X3 - L3 - MD - L4 - X4 -C (VI) wherein

Xi . 2, 3 and X4 are, at each occurrence, selected from the group consisting of the first, second, third and fourth polypeptides, with the proviso that the fusion molecule comprises each of the first, second, third and fourth polypeptides;

MD comprises a multimerization domain; and

Li , L2, L3 and L4 are, at each occurrence, independently selected from a covalent bond and a linker molecule.

8. The fusion molecule of any one of claims 1 to 7, being present as a multimeric (e.g., dimeric) complex.

9. The fusion molecule of any one of claims 1 to 8, wherein the fusion molecule has one or more of the following properties:

(i) it further comprises at least one label or tag allowing the detection and/or isolation of the fusion molecule;

(ii) it further comprises one or more modifications increasing the stability of the fusion molecule and/or extending the serum half-life of the fusion molecule;

(iii) it inhibits or reduces iron-uptake by P. aeruginosa through pyochelin and/or pyoverdine; (iv) it inhibits or reduces virulence factor expression by P. aeruginosa;

(v) it inhibits or reduces pyochelin- and/or pyoverdine-mediated signalling;

(vi) it inhibits or reduces P. aeruginosa bacterial growth; and

(vii) it is associated with or conjugated/fused to a pharmaceutically active agent, wherein, in particular, the pharmaceutically active agent is selected from the group consisting of an antibiotic, a cytostatic agent, a toxin, a metal or metal compound/complex, a chelating agent, a hapten and an antibody.

10. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion molecule of any one of claims 1 to 9.

1 1 . A host cell containing a nucleic acid molecule of claim 10.

12. A pharmaceutical composition comprising a fusion molecule of any one of claims 1 to 9, a nucleic acid molecule of claim 10, or a cell of claim 1 1 .

13. A kit comprising a fusion molecule of any one of claims 1 to 9, a nucleic acid molecule of claims 10, a cell of claim 1 1 , or a pharmaceutical composition of claim 12.

14. A fusion molecule of any one of claims 1 to 9, a nucleic acid molecule of claim 10, a cell of claim 1 1 , or a pharmaceutical composition of claim 12 for use as a medicament.

15. A fusion molecule of any one of claims 1 to 9, a nucleic acid molecule of claim 10, a cell of claim 1 1 , or a pharmaceutical composition of claim 12 for use in the prevention or treatment of P. aeruginosa biofilm infection in a subject.

16. A fusion molecule of any one of claims 1 to 9, a nucleic acid molecule of claim 10, a cell of claim 1 1 , or a pharmaceutical composition of claim 12 for use in the prevention or treatment of a disease or disorder associated with or caused by P. aeruginosa biofilm infection in a subject.

17. The fusion molecule, nucleic acid molecule, cell or pharmaceutical composition for use of claim 15 or 16, wherein the P. aeruginosa biofilm infection is acute or chronic infection.