Description

Novel proteins specific for pyoverdine and pyochelin

I. BACKGROUND

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 α-ketoglutaryl, Pvd type II succinyl, Pvd type II succinamid, Pvd type II α-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 expressesType II Pvd and P.

aeruginosa R and pa6 expressesType III Pvd, whereby each Pvd type includs all three members of the respective type, and each said strain also expresses pyochelin.

In this regard, we identified the pyoverdins and pyochelin as targets which are crucial for P. aeruginosa's pathogenicity and developed specific inhibitors for such targets, as disclosed here, 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. In addition, we selected inhibitors that distinguish free and iron-loaded pyochelin.

The present invention was made as a result of activities undertaken on behalf of Pieris AG, Sanofi-Aventis and Sanofi-Pasteur Inc., which are parties to an existing joint research agreement, and was made within the scope of the joint research agreement.

II. DEFINITIONS

The following list defines terms, phrases, and abbreviations used throughout the instant specification. All terms listed and defined herein are intended to encompass all grammatical forms.

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).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 (type l-lll, Figure 1 ) and a cognate FpvA receptor.

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 (see Figure 1 .)

As used herein, "detectable affinity" means the ability to bind to a selected target with an affinity constant of generally at least about 10"5 M or below. Lower affinities 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) or a fusion polypeptide thereof 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."

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" scaffold. Said term also includes fragments of a mutein and variants as described herein. Muteins of the present disclosure, fragments or variants thereof preferably retain the function of binding to pyoverdine or pyochelin as described herein.

The term "fragment" as used herein in connection with the muteins of the disclosure relates to proteins or peptides derived from full-length mature human lipocalin 2 that are N-terminally and/or C-terminally shortened, i.e. lacking at least one of the N-terminal and/or C-terminal amino acids. Such fragments may include at least 10, more such as 20 or 30 or more consecutive amino acids of the primary sequence of the mature human lipocalin 2 and are usually detectable in an immunoassay of the mature human lipocalin 2. In general, the term "fragment", as used herein with respect to the corresponding protein ligand of a mutein of the disclosure or of the combination according to the disclosure or of a fusion protein described herein, relates to N-terminally and/or C-terminally shortened protein or peptide ligands, which retain the capability of the full length ligand to be recognized and/or bound by a mutein according to the disclosure.

The term "mutagenesis" as used herein means that the experimental conditions are chosen such that the amino acid naturally occurring at a given sequence position of the mature human lipocalin 2 can be substituted by at least one amino acid that is not present at this specific position in the respective natural polypeptide sequence. The term "mutagenesis" also includes the (additional) modification of the length of sequence segments by deletion or insertion of one or more amino acids. Thus, it is within the scope of the disclosure that, for example, one amino acid at a chosen sequence position is replaced by a stretch of three random mutations, leading to an insertion of two amino acid residues compared to the length of the respective segment of the wild type protein. Such an insertion or deletion may be introduced independently from each other in any of the peptide segments that can be subjected to mutagenesis in the disclosure.

The term "random mutagenesis" means that no predetermined single amino acid (mutation) is present at a certain sequence position but that at least two amino acids can be incorporated with a certain probability at a predefined sequence position during mutagenesis.

"Identity" is a property of sequences that measures their similarity or relationship. The term "sequence identity" or "identity" as used in the present disclosure means the percentage of pair-wise identical residues - following (homologous) alignment of a sequence of a polypeptide of the disclosure with a sequence in question - with respect to the number of residues in the longer of these two sequences. Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the product by 100.

The term "homology" is used herein in its usual meaning and includes identical amino acids as well as amino acids which are regarded to be conservative substitutions (for example, exchange of a glutamate residue by an aspartate residue) at equivalent positions in the linear amino acid sequence of a polypeptide of the disclosure (e.g., any mutein of the disclosure).

The percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version blastp 2.2.5 (November 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res. 25, 3389-3402). In this

embodiment the percentage of homology is based on the alignment of the entire

polypeptide sequences (matrix: BLOSUM 62; gap costs: 11 .1 ; cutoff value set to 10"3) including the propeptide sequences, preferably using the wild type protein scaffold as reference in a pairwise comparison. It is calculated as the percentage of numbers of "positives" (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

Specifically, in order to determine whether an amino acid residue of the amino acid sequence of a mutein different from the wild-type human lipocalin 2 corresponds to a certain position in the amino acid sequence of the wild-type human lipocalin 2, a skilled artisan can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs such as BLAST2.0, which stands for Basic Local Alignment Search Tool or ClustalW or any other suitable program which is suitable to generate sequence alignments. Accordingly, the wild-type human lipocalin 2 can serve as "subject sequence" or "reference sequence", while the amino acid sequence of a mutein different from the wild-type human lipocalin 2 described herein serves as "query sequence". The terms "reference sequence" and "wild type sequence" are used interchangeably herein.

"Gaps" are spaces in an alignment that are the result of additions or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or

replacements, may have a lower degree of sequence identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity using standard parameters, for example Blast (Altschul, et al. (1997) Nucleic Acids Res. 25, 3389-3402), Blast2 (Altschul, et al. (1990) J. Mol. Biol. 215, 403-410), and Smith-Waterman (Smith, et al. (1981 ) J. Mol. Biol. 147, 195-197).

The term "variant" as used in the present disclosure relates to derivatives of a protein or peptide that include modifications of the amino acid sequence, for example by substitution, deletion, insertion or chemical modification. Such modifications do in some embodiments not reduce the functionality of the protein or peptide. Such variants include proteins, wherein one or more amino acids have been replaced by their respective D-stereoisomers or by amino acids other than the naturally occurring 20 amino acids, such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline. However, such substitutions may also be conservative, i.e. an amino acid residue is replaced with a chemically similar amino acid residue. 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.

By a "native sequence" human lipocalin 2 is meant human lipocalin 2 that has the same amino acid sequence as the corresponding polypeptide derived from nature. Thus, a native sequence human lipocalin 2 can have the amino acid sequence of the respective naturally-occurring human lipocalin 2. Such native sequence polypeptide can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence" polypeptide specifically encompasses naturally-occurring truncated or secreted forms of the human lipocalin 2, naturally-occurring variant forms such as alternatively spliced forms and naturally-occurring allelic variants of human lipocalin 2. A polypeptide "variant" means a biologically active polypeptide having at least about 50%, 60%, 70%, 80% or at least about 85% amino acid sequence identity with the native sequence polypeptide. Such variants include, for instance, polypeptides in which one or more amino acid residues are added or deleted at the N- or C- terminus of the polypeptide. Generally a variant has at least about 70%, including at least about 80%, such as at least about 85% amino acid sequence identity, including at least about 90% amino acid sequence identity or at least about 95% amino acid sequence identity with the native sequence polypeptide.

The term "position" when used in accordance with the disclosure means the position of either an amino acid within an amino acid sequence depicted herein or the position of a nucleotide within a nucleic acid sequence depicted herein. To understand the term " correspond" or "corresponding" as used herein in the context of the amino acid sueqnece positions of one or more muteins, a corresponding position is not only determined by the number of the preceding nucleotides/amino acids. Accordingly, the position of a given amino acid in accordance with the disclosure which may be substituted may vary due to deletion or addition of amino acids elsewhere in a (mutant or wild-type) human lipocalin 2. Similarly, the position of a given nucleotide in accordance with the present disclosure which may be substituted may vary due to deletions or additional nucleotides elsewhere in a mutein or wild type human lipocalin 2 5'-untranslated region (UTR) including the promoter and/or any other regulatory sequences or gene (including exons and introns).

Thus, for a corresponding position in accordance with the disclosure, it is preferably to be understood that the positions of nucleotides/amino acids may differ in the indicated number than similar neighbouring nucleotides/amino acids, but said neighbouring nucleotides/amino acids, which may be exchanged, deleted, or added, are also comprised by the one or more corresponding positions.

In addition, for a corresponding position in a mutein based on a reference scaffold in accordance with the disclosure, it is preferably to be understood that the positions of nucleotides/amino acids are structurally corresponding to the positions elsewhere in a mutein or wild-type human lipocalin 2, even if they may differ in the indicated number.

The term "organic molecule" or "small organic molecule" as used herein for the non-natural target denotes 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, preferably between 100 and 1000 Dalton, and optionally including one or two metal atoms.

The word "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.

A "subject" is a vertebrate, preferably a mammal, more preferably a human. 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 cynomolgous monkeys and etc., to name only a few illustrative examples. Preferably, the mammal herein is human.

An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations.

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.

III. DESCRIPTIONS OF FIGURES

Figure 1 : shows the structure of P. aeruginosa siderophores. Fig. 1 A-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 chormophore part can be a succinyl, succinamid or a-ketoglutaryl side chain; and Fig.l 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 (Fig.2A), Pvd II s (+Fe) binding to the lipocalin mutein SEQ ID NO: 36 (Fig.2B), Pvd III (+Fe) binding to the lipocalin mutein SEQ ID NO: 53 (Fig.2C) and Pyochelin (+Fe) binding to SEQ ID NO: 62 (Fig.2D). In addition, absence of binding of the respective siderophores at 1200 nM (200 nM for Pyochelin) to the negative control lipocalin SEQ ID NO: 64 is shown in Fig.2E-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 Pyoverdin 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-induced 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

200pg/mouse, with a maximal effect at 2000 g/mouse.

Figure 6: shows the amino acid sequence expressed for crystallisation including a start methionine at position 1 , a lysine at postion 2, a hexahistidine tag at postion 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.

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.

IV. DETAILED DESCRIPTION OF THE DISCLOSURE

The current disclosure provides a polypeptide having binding specificity for pyoverdine type I, II, III or pyochelin, wherein the polypeptide comprises an hNGAL mutein that binds pyoverdine type I, II, III or pyochelin with detectable affinity.

The term "human lipocalin 2" or "human Lcn 2" or "human NGAL" or "hNGAL" 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 lipocalin 2 mutein of the disclosure may also be designated herein as "an hNGAL mutein". The amino acid sequence shown in SWISS-PROT/UniProt Data Bank

Accession Number P80188 may be used as a preferred "reference sequence", more preferably the amino acid sequence shown in SEQ ID NO: 1 is used as reference sequence.

In some embodiments, an hNGAL mutein binding pyoverdine (type I, II or III) or pyochelin with detectable affinity may include at least one amino acid substitution of a native cysteine residue by another amino acid, for example, a serine residue. In some other embodiments, a mutein binding pyoverdine or pyochelin with detectable affinity 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 briges. 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 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, 11 , 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.

Further, 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 seqeunce 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 ).

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 the 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, 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, with the proviso that the altered position or

sequence is retained and that one or more gaps are possible.

As used herein, a mutein of the disclosure "specifically binds" a target (for example, pyoverdine or pyochelin) if it is able to discriminate between that target 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.

In one embodiment, the muteins of the disclosure are fused at its N-terminus and/or its C-terminus to a fusion partner which is a protein domain that extends the serum half-life of the mutein. In further particular embodiments, the protein domain is a 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 muteins of the disclosure are conjugated to a compound that extends the serum half-life of the mutein. More preferably, the mutein is conjugated to a compound selected from the group consisting of a polyalkylene glycol molecule, a hydroethylstarch, an Fc part of an immunoglobulin, a CH3 domain of an immoglobulin, a CH4 domain of an immunoglobulin, an albumin binding peptide, and an albumin binding protein.

In yet another embodiment, the current disclosure relates to a nucleic acid molecule comprising a nucleotide sequence encoding a mutein disclosed herein. The disclosure encompasses a host cell containing said nucleic acid molecule.

Muteins specific for pyoverdine.

Study of the P. aeruginosa isolates so far helped classify pyoverdine into three different types (Meyer et al., Use of Siderophores to Type Pseudomonads: The Three

Pseudomonas Aeruginosa Pyoverdine Systems, Microbiology, 1997; vol. 143 no. 1 35-43). Roughly 42% of the P. aeruginosa isolates have a pyoverdine system identical to that of Pvd type I, 42% of the P. aeruginosa isolates behave like Pvd type II, while 16% of the P. aeruginosa isolates belong to Pvd type III, respectively (Cornells et al., 1989a; Table 4). Each type has three members (subtypes) differening 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.

To tackle P. aeruginosa producing different types of pyoverdine, the present disclosure provides hNGAL muteins directed against different types of pyoverdine. The disclosure also provides useful applications for such muteins, methods of making pyoverdine-binding hNGAL muteins described herein as well as compositions comprising such muteins. Pyoverdine-binding hNGAL muteins of the disclosure as well as compositions thereof may be used in methods of detecting pyoverdine in a sample or in methods of binding of pyoverdine in a subject. No such hNGAL muteins having these features attendant to the uses provided by present disclosure have been previously described.

Pyoverdine did not bind to the natural wild-type hNGAL, while hNGAL's natural ligand, enterobactin, docks into the calyx of hNGAL with high affinity. Pyoverdine, therefore, is a virulence factor and a stealth siderophore that evades hNGAL recognition, allowing P. aeruginosa to establish infection (Peek et al., Pyoverdine, the Major Siderophore in Pseudomonas aeruginosa, Evades NGAL Recognition, Interdisciplinary Perspectives on Infectious Diseases, 2012).

Accordingly, it is an object of the present disclosure to provide muteins derived from human neutrophil gelatinase associated lipocalin (NGAL), also termed as human lipocalin 2, which muteins, in contrast to nature wild-type hNGAL, have high specificity for pyoverdine.

Exemplary muteins specfic for pyoverdine.

In one aspect, the present disclosure relates to novel, 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 with detectable affinity, such as an affinity measured by a KQ 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 KQ 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 disdsore 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 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 in an assay essentially described in Example 8.

In this regard, the disclosure relates to a polypeptide, wherein said polypeptide inlcudes an hNGAL mutein, and said hNGAL 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, Via, Ala or Pro; Tyr 52→ Met, Trp or Pro; Ser 68→ Asp, Via 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 the following substitution in comparison with the linear polypeptide sequence of the mature hNGAL: Gin 28→ His; Lys 46→ Glu; Thr 54→ Via or Ala; lie 55→ Via; 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:

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;

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;

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;

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;

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;

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;

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;

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;

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;

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→ Via; Tyr 132→ Gly; Lys 134→ Asn;

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;

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;

Gin 28→ His; Ala 40→Gly; lie 41→ Trp; Lys 46→ Glu; Gin 49→ Leu; Tyr 52→ Met; Thr 54→ Ala; lie 55→ Via; 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;

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;

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;

Gin 28→ His; Ala 40→Gly; lie 41→ Trp; Lys 46→ Glu; Gin 49→ Leu; Tyr 52→ Met; Thr 54→ Ala; lie 55→ Via; 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

Gin 28→ His; Ala 40→Gly; lie 41→ Trp; Lys 46→ Glu; Gin 49→ Leu; Tyr 52→ Met; Thr 54→ Ala; lie 55→ Via; 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 sequnce 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 sequnce 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%, preferably more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 92% and most preferably 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 KQ 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 disclsore 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 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 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 in an assay essentially described in Example 10.

In this regard, the disclosure relates to a polypeptide, wherein said polypeptide inlcudes an hNGAL mutein, and said hNGAL 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 orThr; 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, 11 , 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 the following substitution 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:

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;

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;

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;

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;

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;

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;

Gin 28→ His; Leu 36→ Via; 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;

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;

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;

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;

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;

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;

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→ 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;

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;

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;

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;

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;

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

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 sequnce 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 sequnce 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%, preferably more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 92% and most preferably 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 KQ 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 disclsore 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 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 in an assay essentially described in Example 8.

In this regard, the disclosure relatesi to a polypeptide, wherein said polypeptide inlcudes an hNGAL mutein, and said hNGAL 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-l I 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→ Phe or Glu; Ala 40→ Trp, Leu or Arg; lie 41→ Met, Arg, Ala, Leu orTrp; 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, 11 , 12, or even more or all mutated amino acid residues at these sequence positions of the mature hNGAL.

Additionally, a Pvd-type-l I l-binding hNGAL mutein according to the disclosure may also comprise the following substitution 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:

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→ Gly; Tyr 106→ Glu; Lys 125→ Trp; Ser 127→ His; Tyr 132→ Phe; Lys 134→ Gin;

Gin 28→ His; Leu 36→ Phe; Ala 40→ Arg; lie 41→ Trp; Gin 49→ lie; Tyr 52→ Tyr;

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

Gin 28→ His; Leu 36→ Phe; Ala 40→ Leu; lie 41→ Leu; Gin 49→ Arg; Tyr 52→ Arg; Ser 68→ Asp; Leu 70→ Arg; Arg 72→ Leu; Lys 73→ Tyr; Asp 77→ lie; Trp 79 → Ser; Arg 81→ Ala; Cys 87→ Ser; Asn 96→ Gly; Tyr 100→ Ala; Leu 103→ Phe; Tyr 106→ Glu; Lys 125→ Trp; Ser 127→ Ala; Lys 134→ Glu;

Gin 28→ His; Leu 36→ Phe; Ala 40→ Trp; lie 41→ Arg; Gin 49→ Pro; Tyr 52→ Ser; Ser 68→ Asn; Leu 70→ Arg; Arg 72→ Trp; Lys 73→ Pro; Asp 77→ Arg; Trp 79→ Ser; Arg 81→ Ser; Cys 87→ Ser; Asn 96→ Met; Tyr 100→ Pro; Leu 103→ Gly; Tyr 106→ Glu; Lys 125→ Trp; Ser 127→ Phe; Tyr 132→ Phe; Lys 134→ Glu;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Gin 49→ Lys; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Gin; Trp 79→ Asp; Arg 81→ Ala; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Gin 49→ Met; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ Gin; Asp 77→ Lys; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Thr; Gin 49→ Met; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ Arg; Asp 77→ Lys; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Via; Tyr 100→ Asp; Leu 103→ Gin; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Gin 49→ Met; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Gin 49→ Lys; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ Tyr; Asp 77→ Gin; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ -; Tyr 100→ Glu; Leu 103→ Gin; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Leu 42→ Arg; Gin 49→ Met; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79 → Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Ser 105→ Pro; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Asp 47→ Asn; Gin 49→ Met; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79 → Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Ser 105→ Pro; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp; Thr 145→ Pro;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Asp 45→ Gly; Lys 46→ Arg; Gin 49→ Met; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Ser 105→ Pro; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Leu 42→ Arg; Gin 49→ Met; Tyr 52→ Met; Asn 65→ Asp; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Ser 105→ Pro; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Asp 47→ Asn; Gin 49→ Met; Tyr 52→ Met; Asn 65→ Asp; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Ser 105→ Pro; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp; Thr 145→ Pro;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Asp 45→ Gly; Lys 46→ Arg; Gin 49→ Met; Tyr 52→ Met; Asn 65→ Asp; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100 → Asp; Leu 103→ Gin; Ser 105→ Pro; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp; or

Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Leu 42→ Arg; Gin 49→ Met; Tyr 52→ Met; Asn 65→ Asp; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79 → Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin;

Ser 105→ Pro; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp.

In the residual region, i.e. the region differing from 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, 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: 38-53 or a fragment or variant thereof.

The amino acid sequence of a Pvd-type-l I 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 sequnce selected from the group consisting of SEQ ID NOs: 38-53.

The disclosure also includes structural homologues of an hNGAL mutein having an amino acid sequnce selected from the group consisting of SEQ ID NOs: 38-53, which structural homologues have an amino acid sequence homology or sequence identity of more than about 60%, preferably more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 92% and most preferably more than 95% in relation to said hNGAL mutein.

A Pvd-type-l I 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).

Applications of muteins specific for pyoverdine

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.

Numerous possible applications for the pyoverdine-binding muteins of the disclosure, therefore, exist in medicine. In one further aspect, the disclosure relates to the use of a pyoverdine-binding mutein disclosed herein for detecting pyoverdine (type I, II or III) in a sample as well as a respective method of diagnosis.

The present disclosure also involves the use of one or more pyoverdine-binding muteins as described for complex formation with pyoverdine (type I, II or III).

Therefore, in another aspect of the disclosure, the disclosed muteins are used for the detection of pyoverdine (type I, II or III). Such use may include the steps of contacting one or more said muteins, under suitable conditions, with a sample suspected of containing pyoverdine, thereby allowing formation of a complex between the muteins and pyoverdine (type I, II or III), and detecting the complex by a suitable signal.

The detectable 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 pyoverdine-binding muteins disclosed herein may also be used for the separation of pyoverdine (type I, II or III). Such use may include the steps of contacting one or more said muteins, under suitable conditions, with a sample supposed to contain pyoverdine (type I, II and/or III), thereby allowing formation of a complex between the muteins and pyoverdine (type I, II or III), and separating the complex from the sample.

In the use of the disclosed muteins for the detection of pyoverdine as well as the separation of pyoverdine (type I, II or III), the muteins and/or pyoverdine or a domain or fragment thereof may be immobilized on a suitable solid phase.

In still another aspect, the present disclosure features a diagnostic or analytical kit comprising a pyoverdine-binding mutein according to the disclosure.

In addition to their use in diagnostics, in yet another aspect, the disclosure

encompasses the use of a pyoverdine-binding mutein of the disclosure or a composition comprising such mutein for the binding of pyoverdine (type I, II or III) in a subject and/or inhibiting or lessening growth of P. aeruginosa in a subject.

In still another aspect, the present disclosure features a method of binding pyoverdine (type I, II or III) in a subject, comprising administering to said subject an effective amount of one or more pyoverdine-binding muteins of the disclosure or of one or more compositions comprising such muteins.

In still another aspect, the present disclosure involves a method for inhibiting or lessening growth of P. aeruginosa in a subject, comprising administering to said subject an effective amount of one or more pyoverdine-binding muteins of the disclosure or of one or more compositions comprising such muteins.

Muteins specific for pyochelin

In addition, the present disclosure fulfills the need for alternative inhibitors of pyochelin by providing hNGAL muteins that bind pyochelin and useful applications therefor.

Accordingly, the disclosure also provides methods of making and using the pyochelin-binding muteins described herein as well as compositions that may be used in methods of detecting pyochelin in a sample or in methods of binding of pyochelin in a subject. No such hNGAL muteins having these features attendant to the uses provided by present disclosure have been previously described.

Exemplary muteins specific for pyochelin

In one aspect, the present disclosure relates to an hNGAL mutein that binds pyochelin complexed with iron with a KQ of about 20 nM or lower, such as 1 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 disclsore are capable of binding pyochelin with complexed iron, with an affinity measured by an IC50 value of about 500 nM or lower, for example, when measured in an ELISA assay essentially described in Example 5.

In some still further embodiments, one or more hNGAL muteins of this disclsore are capable of binding pyochelin 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 still further embodiments, one or more hNGAL muteins of this disclsore are capable of binding pyochelin 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 pyochelin with an IC50 value of about 150 nM or lower in a competition ELISA format essentially described in Example 7.

In some embodiments, the mutein is capable of inhibiting bacterial growth of Pvd I knock-out (ApvdA) in an assay essentially described in Example 8.

In this regard, the disclosure relates to a polypeptide, wherein said polypeptide inlcudes an hNGAL mutein, and said hNGAL 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, 34, 36, 40-41 , 44-46, 49, 52, 54, 65, 68, 70, 72-74, 77, 79-81 , 87, 96, 100, 103, 106, 108, 123, 125, 127, 132, 134 and 141 , and wherein said polypeptide binds pyochelin.

Table 6: Stability after 3 freeze / thaw cycles (F/T); 1 week storage in PBS at 37°C and 1 week storage in human (hu), mouse (mu) or rat plasma assessed by recovery of activity in qELISA and monomer content in analytical SEC: stable in qELISA = 100 +/-15 %; stable in aSEC = 100 +/- 5% (recovery of monomer peak area compared to non-stressed reference sample); for all samples including references a monomer content of 100 area percent has been detected.

1 week hu 1 week mu 1 week rat

3xF/T, -20°C 1mg/ml 1 week PBS, 37°C, 1mg/ml

plasma, 37°C plasma, 37°C plasma, 37°C sidero- % recovery of % monomer in % recovery of % monomer

Mutein % recovery of activity in qELISA

phore activity in qELISA aSEC activity in qELISA in aSEC

SEQ ID N0:16 Pvd I 102 98 86 98 86 100 100

SEQ ID NO: 36 Pvd II 99 101 104 98 93 91 110

SEQ ID NO: 53 Pvd III 98 99 107 102 92 83 101

SEQ ID NO: 62 pch 107 100 95 104 97 102 95

Example 10: In vivo potency of lipocalin muteins in mouse model

The prophylactic effect of SEQ ID NO: 19 following intravenous (i.v.) administration in a P. aeruginosa-induced pulmonary infection in mice was studied.

SEQ ID NO: 19 was administered 1 hour before infection and at time of infection. Lung bacteria load was evaluated 24h after infection.

The strain used in this study was P. aeruginosa (ATCC27853). Starting from P.

aeruginosa stored at -80°C in PBS/ 15% Glycerol, an overnight culture was conducted at 37°C under shaking in Mueller-Hinton broth, and followed by additional subculture (1 ΟΟμΙ overnight culture + 100ml of MHB) until end of logarithmic phase of growth. The culture was washed twice and resuspended in phosphate-buffer saline before to be frozen at 1 E+09 CFU/ml. For each experiment a fresh vial was thawed and inoculum verified by viable counts.

7 to 8 weeks-old Male Swiss mice (5 animals/group) purchased from Janvier

laboratories, (Route des chenes sees, 53940 Le Genest Saint lie, France), were allowed at least 5 days acclimatization prior to use. Animals were maintained at temperature of 22±2°C with relative humidity of 40 - 70 % and 12-15 air fresh changes/hour. Light cycle 12/12 hours: light 7 a.m. to 7 p.m. (normal cycle).

Temperature and relative humidity derivations are recorded continuously. Animals were housing 5 per cages and they allowed access to water and standard diet (AO4 C standard diet (SAFE)) ad libitum. All experiments were performed with approval of the ethic committee of Sanofi R&D (CEPAL).

Lung infection was induced by intranasal challenge of male Swiss mice with 1 .E+07 CFU/mouse of P. aeruginosa in 50μΙ NaCI 0.9%.

SEQ ID NO: 19 at concentrations of 200, 400, 1000 or 2000Mg/mouse was administered 1 h before infection and at time of infection, with i.v. bolus.

Twenty four hours after infection, animals were euthanized and bacterial count from lung homogenates were determined and expressed in Iog10 CFU/ml as mean ± sem.

Statistical analysis was performed using SAS v9.2. The Excel software 2003 was used for figure presentations. Comparisons on SEQ ID NO: 19 doses versus vehicle were evaluated with a one-way analysis of variance followed by Dunnett's test (ZAR J.H., « Biostatistical Analysis », Prentice Hall International Editions, 4eme edition, 1999.; C.W. Dunnett, "A multiple comparison procedure for comparing several treatments with a control", J. Amer. Statist. Assoc., 50 (1955), pp. 1096-1 121 , 1955).

In a P.aeruginosa-induced lung infection model in mice, SEQ ID NO: 19 was

administrated 1 hour before and at time of bacteria challenge and SEQ ID NO: 19 prevented the development of infection in mice in a dose-dependent manner. A significant prevention effect was observed for SEQ ID NO: 19 starting at 200 g/mouse, with a maximal effect at 2000 g/mouse.

Example 1 1 : Crystallisation

To determine the three dimensional structure of SEQ ID NO: 31 protein in complex with Pvd-Fe the following procedure was applied.

The protein sequence depicted in Figure 6 was cloned in the pET-24a plasmid and expressed as N-terminally tagged 6His-TEV protease recognition site construct.

The plasmid was used to transform BL21 (DE3) Star E.coli cells and the resulted clones were inoculated in Overnight Express Instant TB Medium (Novagen) and the cells were harvested after 47 hours of incubation at 18°C with 200 RPM agitation at final OD600 4.7. The cell pellet was resuspended in buffer containing 500mM NaCI, 10mM

Imidazole, 1 mM MgCI2, 1 mM TCEP, 5 % glycerol and 20mM Tris pH 7.4 and lysed by standard ultra-sonication procedure. The resulted extract was cleared by low speed centrifugation and supernatant was filtered throw 22 nm membrane before loading to Ni NTA (Qiagen) 5 ml column pre-equilibrated with 100 mM NaCI, 10mM Imidazole, 100 mM HEPES pH 8 buffer. The protein was eluted by linear gradient of imidazole 10mM to 300mM and further dialyzed overnight to 100 mM NaCI, 10mM Imidazole, 100 mM HEPES pH 8 buffer. The protein was concentrated to 20 mg/ml and loaded to Gel filtration Superdex 75 column (GE). The resulted protein was dialyzed to 100 mM NaCI, 10 mM HEPES pH 8 buffer overnight in the presence of TEV protease (1/50 ratio) to remove 6His N-terminal tag following by negative Ni NTA purification step as described above to separate the cleaved protein. Final protein was concentrated to 12 mg/ml in 100 mM NaCI, 50 mM HEPES pH 7.5 aliquoted, snap frozen in liquid nitrogen and stored at -80°C for further use.

For crystallization the protein was incubated with 10 x times higher molar concentration of Pvd-Fe overnight and plated for crystallization screening carried out in SBS format plates where 100 nl_ protein drops were mixed with 100nl_ of crystallization screening solution in vapor diffusion sitting drops format experiments at 20°C and 4°C. A number of crystallization hits were detected and crystallization conditions were further optimized in order to obtain well diffracting x-ray quality crystals.

The crystals diffraction quality was assessed using synchrotron x-ray source and the best diffracting crystals were obtained under 20% PEG3350 and 0.2M LiSO4 conditions at 20°C. The best crystals were cryoprotected by increasing PEG3350 concentration to 35% than snap frozen in liquid nitrogen and 1 .8A data set was collected at 100K temperature.

X-ray data were processed by MOSFLM and the protein structure was determined by molecular replacement method using pdb 1 LKE as a search model and the structural model was further refined to Rfree=0.233 - R=0.200 quality in P41212 with 2 ternary protein complexes per asymmetric unit.

The protein structure presents classical lipocalin scaffold with Pvd-Fe bound to both mutein proteins present in the asymmetric unit, Figure 7. The amino acid residues involved in the Pvd-Fe binding analysed and presented on Figure 8. The oxygens of the Pvd directly binding Fe are identified and presented on Figure 9.

The present invention pertains to a polypeptide having binding specificity for pyoverdine type I, II, III or pyochelin, wherein the polypeptide comprises an hNGAL mutein that binds pyoverdine type I, II, III or pyochelin with detectable affinity.

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 another embodiment said mutein is capable of binding pyoverdine type I complexed with iron with a KQ of about 20 nM or lower when measured by Biacore T200 instrument

in an assay essentially described in Example 6.

In another embodiment said hNGAL mutein is 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, when measured in an ELISA assay essentially described in Example 5.

In another embodiment the hNGAL mutein is capable of inhibiting iron uptake mediated by pyoverdine type I with an IC50 value of about 150 nM or lower in a competition ELISA format essentially described in Example 7.

In another embodiment the hNGAL mutein is capable of inhibiting bacterial growth of Pvd I strain in an assay essentially described in Example 8.

In another embodiment the hNGAL mutein comprises a mutated amino acid residue at one or more positions corresponding to 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 of the linear polypeptide sequence of the mature hNGAL (SEQ ID NO: 1 ).

In another embodiment the amino acid sequence of the hNGAL mutein comprises at least one of the following mutated amino acid residues in comparison with the linear polypeptide sequence of the mature hNGAL: 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, Via, Ala or Pro; Tyr 52→ Met, Trp or Pro; Ser 68→ Asp, Via 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 another embodiment the amino acid sequence of the hNGAL mutein comprises the following substitution in comparison with the linear polypeptide sequence of the mature hNGAL: Gin 28→ His; Lys 46→ Glu; Thr 54→ Via or Ala; lie 55→ Via; Lys 59→ Arg; Asn 65→ Asp or Gin; lie 80→ Thr; Cys 87→ Ser or Asn; and Thr 136→ Ala.

In another embodiment the hNGAL mutein comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 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 of the linear polypeptide sequence of the mature human NGAL (SEQ ID NO: 1 ).

In another embodiment the hNGAL mutein comprises one of the following sets of amino acid substitutions in comparison with the linear polypeptide sequence of the mature hNGAL:

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;

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;

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;

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;

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;

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;

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;

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;

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;

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→ Via; Tyr 132→ Gly; Lys 134→ Asn;

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;

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;

Gin 28→ His; Ala 40→Gly; lie 41→ Trp; Lys 46→ Glu; Gin 49→ Leu; Tyr 52→ Met; Thr 54→ Ala; lie 55→ Via; 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;

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;

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;

Gin 28→ His; Ala 40→Gly; lie 41→ Trp; Lys 46→ Glu; Gin 49→ Leu; Tyr 52→ Met; Thr 54→ Ala; lie 55→ Via; 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

Gin 28→ His; Ala 40→Gly; lie 41→ Trp; Lys 46→ Glu; Gin 49→ Leu; Tyr 52→ Met; Thr 54→ Ala; lie 55→ Via; 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 another embodiment the hNGAL mutein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-18 or a fragment or variant thereof.

In another embodiment said hNGAL mutein is capable of binding pyoverdine type II complexed with iron with a KQ of about 20 nM or lower when measured by Biacore

T200 instrument in an assay essentially described in Example 6.

In another embodiment said hNGAL mutein is 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, when measured in an ELISA assay essentially described in Example 5.

In another embodiment the hNGAL mutein is capable of inhibiting iron uptake mediated by pyoverdine type II with an IC50 value of about 150 nM or lower in a competition ELISA format essentially described in Example 7.

In another embodiment the hNGAL mutein is capable of inhibiting bacterial growth of Pvd II strain in an assay essentially described in Example 8.

In another embodiment said hNGAL mutein is capable of inhibiting growth of P.

aeruginosa stains expressing pyoverdine type II in an assay essentially described in Example 9.

In another embodiment the hNGAL mutein comprises a mutated amino acid residue at one or more positions corresponding to 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 of the linear

polypeptide sequence of the mature hNGAL (SEQ ID NO: 1 ).

In another embodiment the amino acid sequence of the hNGAL mutein comprises at least one of the following mutated amino acid residues in comparison with the linear polypeptide sequence of the mature hNGAL: Leu 36→ Asn, lie or Val; Ala 40→ Glu, Gly, Asn, Thr or His; lie 41→ Arg, Val orThr; 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 another embodiment the amino acid sequence of the hNGAL mutein comprises the following substitution 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 another embodiment the hNGAL mutein comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 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 of the linear polypeptide sequence of the mature human NGAL (SEQ ID NO: 1 ).

In another embodiment the hNGAL mutein comprises one of the following sets of amino acid substitutions in comparison with the linear polypeptide sequence of the natural wildtype hNGAL:

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;

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;

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;

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;

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;

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;

Gin 28→ His; Leu 36→ Via; 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;

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;

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;

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;

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;

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;

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→ 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;

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;

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;

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;

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;

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

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.

In another embodiment the hNGAL mutein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-37 or a fragment or variant thereof.

In another embodiment said mutein is capable of binding pyoverdine type III complexed with iron with a KQ of about 20 nM or lower when measured by Biacore T200 instrument in an assay essentially described in Example 6.

In another embodiment said hNGAL mutein is capable of binding Pvd type III succinyl, Pvd type III succinamid and Pvd type III a-ketoglutaryl with and without complexed iron, with an affinity measured by an IC50 value of about 200 nM or lower, when measured in an assay essentially described in Example 5.

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

In another embodiment the hNGAL mutein is capable of inhibiting bacterial growth of Pvd III strain in an assay essentially described in Example 8.

In another embodiment the hNGAL mutein comprises a mutated amino acid residue at one or more positions corresponding to 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 of the linear polypeptide sequence of the mature hNGAL (SEQ ID NO: 1 ).

In another embodiment the amino acid sequence of the hNGAL mutein comprises at least one of the following mutated amino acid residues in comparison with the linear polypeptide sequence of the mature hNGAL: Leu 36→ Phe or Glu; Ala 40→ Trp, Leu or Arg; lie 41→ Met, Arg, Ala, Leu orTrp; 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 another embodiment the amino acid sequence of the hNGAL mutein comprises the following substitution 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 another embodiment the hNGAL mutein comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 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 of the linear polypeptide sequence of the mature human NGAL (SEQ ID NO: 1 )

In another embodiment the hNGAL mutein comprises one of the following sets of amino acid substitutions in comparison with the linear polypeptide sequence of the mature hNGAL:

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→ Gly; Tyr 106→ Glu; Lys 125→ Trp; Ser 127→ His; Tyr 132→ Phe; Lys 134→ Gin;

Gin 28→ His; Leu 36→ Phe; Ala 40→ Arg; lie 41→ Trp; Gin 49→ lie; Tyr 52→ Tyr; Ser 68→ Gin; Leu 70→ Asn; Arg 72→ Trp; Lys 73→ Leu; Asp 77→ Ala; Trp 79→ Ser; Arg 81→ Ser; Cys 87→ Ser; Asn 96→ Arg; Tyr 100→ lie; Leu 103→ Pro; Tyr 106→ Glu; Lys 125→ Thr; Ser 127→ lie; Tyr 132→ Phe; Lys 134→ Glu;

Gin 28→ His; Leu 36→ Phe; Ala 40→ Leu; lie 41→ Leu; Gin 49→ Arg; Tyr 52→ Arg; Ser 68→ Asp; Leu 70→ Arg; Arg 72→ Leu; Lys 73→ Tyr; Asp 77→ lie; Trp 79 → Ser; Arg 81→ Ala; Cys 87→ Ser; Asn 96→ Gly; Tyr 100→ Ala; Leu 103→ Phe; Tyr 106→ Glu; Lys 125→ Trp; Ser 127→ Ala; Lys 134→ Glu;

Gin 28→ His; Leu 36→ Phe; Ala 40→ Trp; lie 41→ Arg; Gin 49→ Pro; Tyr 52→ Ser; Ser 68→ Asn; Leu 70→ Arg; Arg 72→ Trp; Lys 73→ Pro; Asp 77→ Arg; Trp 79→ Ser; Arg 81→ Ser; Cys 87→ Ser; Asn 96→ Met; Tyr 100→ Pro; Leu 103→ Gly; Tyr 106→ Glu; Lys 125→ Trp; Ser 127→ Phe; Tyr 132→ Phe; Lys 134→ Glu;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Gin 49→ Lys; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Gin; Trp 79→ Asp; Arg 81→ Ala; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Gin 49→ Met; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ Gin; Asp 77→ Lys; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Thr; Gin 49→ Met; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ Arg; Asp 77→ Lys; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Via; Tyr 100→ Asp; Leu 103→ Gin; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Gin 49→ Met; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Gin 49→ Lys; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ Tyr; Asp 77→ Gin; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ -; Tyr 100→ Glu; Leu 103→ Gin; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Leu 42→ Arg; Gin 49→ Met; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79 → Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Ser 105→ Pro; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Asp 47→ Asn; Gin 49→ Met; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79 → Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Ser 105→ Pro; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp; Thr 145→ Pro;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Asp 45→ Gly; Lys 46→ Arg; Gin 49→ Met; Tyr 52→ Met; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Ser 105→ Pro; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Leu 42→ Arg; Gin 49→ Met; Tyr 52→ Met; Asn 65→ Asp; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Ser 105→ Pro; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Asp 47→ Asn; Gin 49→ Met; Tyr 52→ Met; Asn 65→ Asp; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79→ Asp; Arg 81→ Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Ser 105→ Pro; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp; Thr 145→ Pro;

Gin 28→ His; Leu 36→ Glu; Ala 40→ Leu; lie 41 → Ala; Asp 45→ Gly; Lys 46→ Arg; Gin 49→ Met; Tyr 52→ Met; Asn 65→ Asp; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79→ Asp; Arg 81 → Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100 → Asp; Leu 103→ Gin; Ser 105→ Pro; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp; or

Leu 36→ Glu; Ala 40→ Leu; lie 41→ Ala; Leu 42→ Arg; Gin 49→ Met; Tyr 52→ Met; Asn 65→ Asp; Ser 68→ Glu; Leu 70→ Arg; Lys 73→ His; Asp 77→ Lys; Trp 79 → Asp; Arg 81 → Via; Cys 87→ Ser; Asn 96→ Leu; Tyr 100→ Asp; Leu 103→ Gin; Ser 105→ Pro; Tyr 106→ Glu; Ser 127→ Val; Tyr 132→ Phe; Lys 134→ Trp.

In another embodiment the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38-53 or a fragment or variant thereof.

In another embodiment said hNGAL mutein is capable of binding pyochelin complexed with iron with a KQ of about 20 nM or lower when measured by Biacore T200 instrument in an assay essentially described in Example 6.

In another embodiment said hNGAL mutein is capable of binding pyochelin with complexed iron, with an affinity measured by an IC50 value of about 500 nM or lower, when measured in an assay essentially described in Example 5.

In another embodiment said hNGAL mutein is capable of binding pyochelin without complexed iron, with an affinity measured by an IC50 value of about 200 nM or lower, when measured in an assay essentially described in Example 5.

In another embodiment said hNGAL mutein is capable of binding pyochelin with and without complexed iron, with an affinity measured by an IC50 value of about 200 nM or lower, when measured in an assay essentially described in Example 5.

In another embodiment the hNGAL mutein is capable of inhibiting iron uptake mediated by pyochelin with an IC50 value of about 150 nM or lower in a competition ELISA format essentially described in Example 7.

In another embodiment the hNGAL mutein is capable of inhibiting bacterial growth of Pvd I knock-out (ApvdA) in an assay essentially described in Example 8

In another embodiment the hNGAL mutein comprises a mutated amino acid residue at one or more positions corresponding to positions 28, 34, 36, 40-41 , 44-46, 49, 52, 54, 65, 68, 70, 72-74, 77, 79-81 , 87, 96, 100, 103, 106, 108, 123, 125, 127, 132, 134 and 141 of the linear polypeptide sequence of the mature hNGAL (SEQ ID NO: 1 ).

In another embodiment the amino acid sequence of the hNGAL mutein comprises at least one of the following mutated amino acid residues in comparison with the linear polypeptide sequence of the mature hNGAL: Leu 36→ His, Met or Val; Ala 40→ lie, Gin, Tyr or Phe; lie 41→ Leu, His orTrp; Gin 49→ His, Arg, Ser or Ala; Tyr 52→ Leu, Trp or Pro; Ser 68→ Asp or His; Leu 70→ Arg or Trp; Arg 72→ His, lie, Ala, Ser or Gly; Lys 73→ Asn, Met, Pro, Phe, Gin or Arg; Asp 77→ Arg, Thr, Pro or Asp; Trp 79→ Ala, Arg, Lys or Asp; Arg 81→ Thr, lie or Trp; Asn 96→ Met, Asn, Pro or Ala; Tyr 100 → Gly, His or Glu; Leu 103→ Gly, Met, His or Gin; Tyr 106→ Met, Gly, Arg or Trp; Lys 125→ Trp, Phe, Gly or Leu; Ser 127→ Arg, Trp, Asp or lie; Tyr 132→ Ala, Glu or Thr; and Lys 134→ Leu, Val, Asn or Phe.

In another embodiment the amino acid sequence of the hNGAL mutein comprises the following substitution in comparison with the linear polypeptide sequence of the mature hNGAL: Gin 28→ His; Val 34→ Leu; Glu 44→ Gly; Asp 45→ Gly; Lys→ Arg or Tyr; Asn 65→ Asp; lie 80→ Thr; Cys 87→ Ser; Leu 94→ Phe; Val 108→ Ala; Phe 123→ Ser and Thr 141→Ala.

In another embodiment the hNGAL mutein comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 mutated amino acid residues at the sequence positions 28, 34, 36, 40-41 , 44-46, 49, 52, 54, 65, 68, 70, 72-74, 77, 79-81 , 87, 96, 100, 103, 106, 108, 123, 125, 127, 132, 134 and 141 of the linear polypeptide sequence of the mature human NGAL (SEQ ID NO: 1 ).

In another embodiment the hNGAL mutein comprises one of the following sets of amino acid substitutions in comparison with the linear polypeptide sequence of the mature hNGAL:

Gin 28→ His; Ala 40→ lie; lie 41→ Leu; Gin 49→ His; Tyr 52→ Leu; Ser 68→ His; Leu 70→ Thr; Arg 72→ Lys; Lys 73→ Trp; Asp 77→ lie; Trp 79→ Ser; Arg 81→ His; Cys 87→ Ser; Asn 96→ Met; Tyr 100→ Asn; Leu 103→ His; Tyr 106→ Met; Lys 125→ Trp; Ser 127→ Asp; Tyr 132→ Glu; Lys 134→ Leu;

Gin 28→ His; Leu 36→ His; Ala 40→ Gin; lie 41→ Trp; Gin 49→ Arg; Tyr 52→ Trp; Ser 68→ Asp; Leu 70→ Asp; Arg 72→ Ala; Lys 73→ lie; Asp 77→ His; Trp 79→ Arg; Arg 81→ Thr; Cys 87→ Ser; Tyr 100→ His; Leu 103→ Gly; Tyr 106→ Gly; Lys

125→ Phe; Ser 127→ lie; Tyr 132→ Ala; Lys 134→ Phe;

Gin 28→ His; Leu 36→ Met; Ala 40→ Phe; lie 41→ His; Gin 49→ Ser; Tyr 52→ Pro; Ser 68→ His; Leu 70→ Pro; Arg 72→ Trp; Lys 73→ Ala; Asp 77→ Ala; Trp 79→ Lys; Arg 81→ lie; Cys 87→ Ser; Asn 96→ Ala; Tyr 100→ Gly; Leu 103→ Met; Tyr 106→ Trp; Lys 125→ Gly; Ser 127→ Trp; Tyr 132→ Thru; Lys 134→ Val;

Gin 28→ His; Leu 36→ Val; Ala 40→ Tyr; lie 41→ Trp; Gin 49→ Ala; Ser 68→ Asp; Leu 70→ Arg; Arg 72→Trp; Lys 73→ Arg; Asp 77→ Arg; Trp 79→ Asp; Arg 81→ Trp; Cys 87→ Ser; Asn 96→ Pro; Tyr 100→ Glu; Leu 103→ Gin; Tyr 106→ Arg; Lys 125→ Leu; Ser 127→ Arg; Tyr 132→ Ala; Lys 134→ Asn;

Gin 28→ His; Via 34→ Leu; Leu 36→ Met; Ala 40→ Phe; lie 41→ His; Gin 49→ Ser; Tyr 52→ Pro; Ser 68→ His; Leu 70→ Pro; Arg 72→ Trp; Lys 73→ Ala; Asp 77→ Ala; Trp 79→ Lys; lie 80→ Thr; Arg 81→ lie; Cys 87→ Ser; Asn 96→ Ala; Tyr 100→ Gly; Leu 103→ Met; Tyr 106→ Trp; Phe 123→ Ser; Lys 125→ Gly; Ser 127→ Trp; Tyr 132→ Thru; Lys 134→ Val; Thr 141→ Ala;

Gin 28→ His; Leu 36→ Met; Ala 40→ Phe; lie 41→ His; Gin 49→ Ser; Tyr 52→ Pro; Ser 68→ His; Leu 70→ Pro; Arg 72→ Trp; Lys 73→ Ala; Asp 77→ Ala; Trp 79→ Lys; lie 80→ Thr; Arg 81→ lie; Cys 87→ Ser; Asn 96→ Ala; Tyr 100→ Gly; Leu 103→ Met; Tyr 106→ Trp; Phe 123→ Ser; Lys 125→ Gly; Ser 127→ Trp; Tyr 132→ Thru; Lys 134→ Val;

Gin 28→ His; Leu 36→ His; Ala 40→ Gin; lie 41→ Trp; Asp 45→ Gly; Lys 46→ Arg; Gin 49→ Arg; Tyr 52→ Trp; Ser 68→ Asp; Leu 70→ Asp; Arg 72→ Ala; Lys 73→ lie; Asp 77→ Leu; Trp 79→ Arg; Arg 81→ Thr; Cys 87→ Ser; Tyr 100→ His; Leu 103 → Gly; Tyr 106→ Gly; Lys 125→ Phe; Ser 127→ lie; Tyr 132→ Ala; Lys 134→ Phe;

Gin 28→ His; Leu 36→ His; Ala 40→ Gin; lie 41→ Trp; Glu 44→ Gly; Lys 46→ Tyr; Gin 49→ Arg; Tyr 52→ Trp; Ser 68→ Asp; Leu 70→ Asp; Arg 72→ Ala; Lys 73→ lie; Lys 74→ Glu; Asp 77→ His; Trp 79→ Arg; Arg 81→ Thr; Cys 87→ Ser; Leu 94 → Phe; Tyr 100→ His; Leu 103→ Gly; Tyr 106→ Gly; Val 108→ Ala; Lys 125→ Phe; Ser 127→ lie; Tyr 132→ Ala; Lys 134→ Phe; or

Leu 36→ His; Ala 40→ Gin; lie 41→ Trp; Asp 45→ Gly; Lys 46→ Arg; Gin 49→ Arg; Tyr 52→ Trp; Asn 65→ Asp; Ser 68→ Asp; Leu 70→ Asp; Arg 72→ Ala; Lys 73→ lie; Asp 77→ Leu; Trp 79→ Arg; Arg 81→ Thr; Cys 87→ Ser; Tyr 100→ His; Leu 103 → Gly; Tyr 106→ Gly; Lys 125→ Phe; Ser 127→ lie; Tyr 132→ Ala; Lys 134→ Phe.

In another embodiment the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 54-63 or a fragment or variant thereof.

In another embodiment the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-63 or a fragment or variant thereof.

In another embodiment said hNGAL mutein comprises one or more non-native cysteine residues substituting one or more amino acids of a wild type hNGAL.

In another embodiment said hNGAL mutein comprises at least one amino acid substitution of a native cysteine residue by another amino acid.

In another embodiment said another amino acid is a serine residue.

In another embodiment the hNGAL mutein is conjugated to a compound selected from the group consisting of an organic molecule, an enzyme label, a radioactive label, a colored label, a fluorescent label, a chromogenic label, a luminescent label, a hapten, digoxigenin, biotin, a cytostatic agent, a toxins, a metal complex, a metal, and colloidal gold.

In another embodiment the hNGAL mutein is fused at its N-terminus and/or its C-terminus to a fusion partner which is a protein, or a protein domain or a peptide.

In another embodiment the hNGAL mutein is conjugated to a compound that extends the serum half-life of the polypeptide.

In another embodiment the polypeptide comprises a compound that extends the serum half-life is selected from the group consisting of a polyalkylene glycol molecule, hydroethylstarch, a 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 another embodiment the polyalkylene glycol is polyethylene (PEG) or an activated derivative thereof.

In another embodiment a nucleic acid molecule is encompassed comprising a nucleotide sequence encoding any of the polypeptides mentioned herein.

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

In another embodiment the nucleic acid molecule is comprised in a vector or in a phagemid vector.

In another embodiment a host cell is encompassed containing a nucleic acid molecule of any one of the ones mentioned herein.

In another embodiment a method of producing any of the polypeptide described herein is encompassed, wherein the polypeptide is produced starting from the nucleic acid coding for the polypeptide by means of genetic engineering methods.

In another embodiment the polypeptide is produced in a bacterial or eucaryotic host organism and is isolated from this host organism or its culture.

In another embodiment a composition is encompassed comprising one or more polypeptides selected from the group consisting of (i) a polypeptide specific for pyoverdine type I, (ii) a polypeptide specific for pyoverdine type II, (iii) a polypeptide specific for pyoverdine type III and (iv) a polypeptide specific for pyochelin.

In another embodiment the composition comprises two or more polypeptides selected from the group consisting of (i) a polypeptide specific for pyoverdine type I, (ii) a polypeptide specific for pyoverdine type II, (iii) a polypeptide specific for pyoverdine type III and (iv) a polypeptide specific for pyochelin.

In another embodiment the composition comprises three or four polypeptides selected from the group consisting of (i) a polypeptide specific for pyoverdine type I, (ii) a polypeptide specific for pyoverdine type II, (iii) a polypeptide specific for pyoverdine type III and (iv) a polypeptidespecific for pyochelin.

In another embodiment the composition comprises the polypeptide specific for pyoverdine type I.

In another embodiment the composition comprises the polypeptide specific for pyoverdine type II.

In another embodiment the composition comprises the polypeptide specific for pyoverdine type III.

In another embodiment the composition comprises the polypeptide specific for pyochelin.

In another embodiment said composition further includes at least one pharmaceutically acceptable adjuvant, diluent or carrier.

In another embodiment the method of binding pyoverdine type I, II, III and/or pyochelin in a subject comprises administering to said subject an effective amount of any of the compositions mentioned herein.

In another embodiment a method is encompassed for inhibiting or lessening growth of P. aeruginosa in a subject, comprising administering to said subject an effective amount of the composition of of any of the ones mentioned herein.

In another embodiment a kit is encompassed comprising one or more containers, separately or in admixture, and the composition of any of the ones mentioned herein.

In another embodiment the use of (i) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyoverdine type I, (ii) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyoverdine type II, (iii) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyoverdine type III and/or (iv) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyochelin is encompassed, for the binding of pyoverdine type I, II, III and/or pyochelin in a subject.

In another embodiment the use of (i) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyoverdine type I, (ii) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyoverdine type II, (iii) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyoverdine type III and/or (iv) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyochelin is encompassed, for preventing or reducing iron-uptake by P. aeruginosa through pyochelin and/or pyoverdine in a subject.

In another embodiment the use of (i) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyoverdine type I, (ii) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyoverdine type II, (iii) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyoverdine type III and/or (iv) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyochelin is encompassed, for the treatment or alleviation of P. aeruginosa biofilm infection in a subejct.

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

In another embodiment said first, second, third and/or fourth polypeptides are administered in combination, including concurrently, concomitantly or in series.

In another embodiment said first, second, third and/or fourth polypeptides are administered independent from each other, including at individual intervals at

independent points of time.

In another embodiment a combination comprising (i) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyoverdine type I, (ii) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyoverdine type II, (iii) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyoverdine type III and/or (iv) a polypeptide according to any polypeptide mentioned herein cabable of binding to pyochelin.

Embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present embodiments have been specifically disclosed by preferred embodiments and optional features, modification and variations thereof may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention. All patents, patent applications, textbooks and peer-reviewed publications described herein are hereby incorporated by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further embodiments will become apparent from the following claims.

CLAIMS

1 . A polypeptide having binding specificity for pyoverdine type I, II, III or pyochelin, wherein the polypeptide comprises an hNGAL mutein that binds pyoverdine type I, II, III or pyochelin with detectable affinity.

2. The polypeptide 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. A nucleic acid molecule comprising a nucleotide sequence encoding the

polypeptide of any one of claims 1 or 29.

4. A host cell containing a nucleic acid molecule of claim 3.

5. A method of producing the polypeptide according to any one of claims 1 or 2 , wherein the polypeptide is produced starting from the nucleic acid coding for the polypeptide by means of genetic engineering methods.

6. A composition comprising one or more polypeptides selected from the group consisting of (i) a polypeptide specific for pyoverdine type I, (ii) a polypeptide specific for pyoverdine type II, (iii) a polypeptide specific for pyoverdine type III and (iv) a polypeptide specific for pyochelin.

7. A method of binding pyoverdine type I, II, III and/or pyochelin in a subject

comprising administering to said subject an effective amount of the composition of claim 13.

8. A kit comprising in one or more containers, separately or in admixture, and the composition of claim 6.

9. Use of (i) a polypeptide according to claim 1 cabable of binding to pyoverdine type I, (ii) a polypeptide according to claim 1 cabable of binding to pyoverdine type II, (iii) a polypeptide according to claim 1 cabable of binding to pyoverdine type III and/or (iv) a polypeptide according to claim 1 cabable of binding to pyochelin, for the binding of pyoverdine type I, II, III and/or pyochelin in a subject.

10. Use of (i) a polypeptide according to claim 1 cabable of binding to pyoverdine type I, (ii) a polypeptide according to claim 1 cabable of binding to pyoverdine type II, (iii) a polypeptide according to claim 1 cabable of binding to pyoverdine type III and/or (iv) a polypeptide according to claim 1 cabable of binding to pyochelin, for the binding of pyoverdine type I, II, III and/or pyochelin in a subject, for preventing or reducing iron-uptake by P. aeruginosa through pyochelin and/or pyoverdine in a subject.

1 1 . Use of (i) a polypeptide according to claim 1 cabable of binding to pyoverdine type I, (ii) a polypeptide according to claim 1 cabable of binding to pyoverdine type II, (iii) a polypeptide according to claim 1 cabable of binding to pyoverdine type III and/or (iv) a polypeptide according to claim 1 cabable of binding to pyochelin, for the binding of pyoverdine type I, II, III and/or pyochelin in a subject, for the treatment or alleviation of P. aeruginosa biofilm infection in a subejct.

12. A combination of two or more polypeptides according to any one of the

claims 1 or 2.