HK1232549B - Tear lipocalin muteins binding il-4 r alpha - Google Patents

Description

Tear lipocalin mutant protein that binds to IL-4 receptor α

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/61/352,461, filed with the USPTO on June 8, 2010, the entire contents of which are incorporated herein by reference.

Field of the Invention

The present invention relates to a mutein of human tear lipocalin that binds to IL-4 receptor alpha. The present invention also relates to corresponding nucleic acid molecules encoding such muteins and methods for their production. The present invention further relates to methods for producing such muteins. Finally, the present invention relates to pharmaceutical compositions comprising such muteins and various uses of the muteins.

background

Proteins that selectively bind to selected targets through non-covalent interactions generally play a key role in biotechnology, medicine, bioanalysis, and as reagents in the biological and life sciences. Antibodies, also known as immunoglobulins, are a prominent example of this type of protein. Despite their use in ligand/target recognition, binding, and/or separation, immunoglobulins are currently used almost exclusively. The use of other proteins with well-defined ligand-binding properties, such as lectins, remains limited to specialized cases.

Other proteinaceous binding molecules with antibody-like functions are members of the lipocalin family, which have naturally evolved to bind ligands. Lipocalins are produced in many organisms, including vertebrates, insects, plants, and bacteria. Members of the lipocalin family (Pervaiz, S. and Brew, K. (1987) FASEB J. 1, 209-214) are typically small, secreted proteins with a single polypeptide chain. They are characterized by a range of different molecular recognition properties: their ability to bind a variety of molecules (primarily hydrophobic molecules such as retinoids, fatty acids, cholesterol, prostaglandins, biliverdin, pheromones, tastants, and flavor enhancers), and their ability to bind to specific cell surface receptors and form macromolecular complexes. Although they were previously classified primarily as transport proteins, it is now clear that lipocalins perform a variety of physiological functions. These functions include roles in retinol transport, olfaction, pheromone signaling, and prostaglandin biosynthesis. Lipocalins have also been shown to be involved in the regulation of immune responses and in the mediation of cellular homeostasis (reviewed in, for example, Flower, D.R. (1996) Biochem. J. 318, 1-14 and Flower, D.R. et al. (2000) Biochim. Biophys. Acta 1482, 9-24).

The level of global sequence conservation among lipocalins is very low, often with less than 20% sequence identity. In stark contrast, their overall folding pattern is highly conserved. The central portion of the lipocalin structure consists of a single, eight-stranded, antiparallel β-sheet that closes onto itself to form a continuous, hydrogen-bonded β-barrel. This β-barrel forms a central cavity. One end of the barrel is sterically closed by an N-terminal peptide segment that passes through its base and three peptide loops connecting the β-sheet strands. The other end of the β-barrel is open to the solvent and contains a target binding site formed by four flexible peptide loops. It is this diversity of loops within the otherwise rigid lipocalin backbone that generates a variety of different binding modes, each capable of accommodating targets of varying size, shape, and chemical characteristics (e.g., reviewed in Flower, D.R. (1996), Flower, D.R. et al. (2000), supra, or Skerra, A. (2000) Biochim. Biophys. Acta 1482, 337-350).

International patent application WO 99/16873 discloses polypeptides of the lipocalin family having mutated amino acid positions in four peptide loop regions arranged at the ends of the cylindrical β-barrel structure surrounding the binding pocket and corresponding to those segments in the linear polypeptide sequence comprising amino acid positions 28 to 45, 58 to 69, 86 to 99, and 114 to 129 of the Pieris brassicae meta-bile pigment-binding protein. Members of the lipocalin family have been reported to be post-translationally modified, such as phosphorylation and glycosylation of tear lipocalin (e.g., You, J., et al. (2010) Electrophoresis 31, 1853-1861). However, post-translational modification is not required for their molecular recognition properties.

International patent application WO 00/75308 discloses muteins of post-bile pigment binding protein that specifically bind to digoxin, while international patent applications WO 03/029463 and WO 03/029471 relate to muteins of human neutrophil gelatinase-binding lipocalin (hNGAL) and apolipoprotein D, respectively. To further improve and optimize the ligand affinity, specificity and folding stability of lipocalin variants, various approaches have been proposed using different members of the lipocalin family (Skerra, A. (2001) Rev. Mol. Biotechnol. 74, 257-275; Schlehuber, S., and Skerra, A. (2002) Biophys. Chem. 96, 213-228), for example by replacing other amino acid residues. PCT Publication WO 2006/56464 discloses human neutrophil gelatinase-bound lipocalin muteins that have binding affinity for CTLA-4 in the low nanomolar range.

International Patent Application WO 2005/19256 discloses tear lipocalin mutants that have at least one binding site for different or identical target ligands, and provides methods for generating such human tear lipocalin mutants. According to this PCT patent application, certain stretches of amino acids in the primary sequence of tear lipocalin (particularly the loop region encompassing amino acids 7-14, 24-36, 41-49, 53-66, 69-77, 79-84, 87-98, and 103-110 of mature human tear lipocalin) are mutagenized to generate mutants with binding affinities. The resulting mutants have binding affinities for selected ligands ( _KD ) in the nanomolar range, often >100 nM. International Patent Application WO 2008/015239 discloses tear lipocalin mutants that bind to a given non-natural ligand, including IL-4 receptor α. In surface plasmon resonance experiments, the binding affinity was in the nanomolar range, as low as nearly ^1x10-10 M.

Human tear lipocalin (TLPC or Tlc), also known as lipocalin 1, lacrimal prealbumin, or Ebner's gland protein, was originally described as the major protein in human tears (approximately one-third of the total protein content), but is also found in several other secretory tissues, including the prostate, adrenal glands, thymus, mammary gland, testis, nasal and tracheal mucosa, and adrenocorticotroph cells of the pituitary gland. Homologous proteins have been found in macaques, chimpanzees, rats, mice, pigs, hamsters, cattle, dogs, and horses. Tear lipocalin is an unusual member of the lipocalin family because it exhibits an unusually broad ligand specificity compared to other lipocalins and a high affinity for relatively insoluble lipids (see Redl, B. (2000) Biochim. Biophys. Acta 1482, 241–248). This property of tear lipocalin has been attributed to its function in inhibiting the growth of corneal bacteria and fungi. A significant number of lipophilic compounds of different chemical classes, such as fatty acids, fatty alcohols, phospholipids, glycolipids, and cholesterol, are endogenous ligands for this protein. Interestingly, in contrast to other lipocalins, the strength of ligand (target) binding corresponds to the length of the hydrocarbon tails of the alkylamides and fatty acids. Thus, tear lipocalin binds most strongly to the least soluble lipids (Glasgow, B.J. et al. (1995) Curr. Eye Res. 14, 363-372; Gasymov, O.K. et al. (1999) Biochim. Biophys. Acta 1433, 307-320). The crystal structure of tear lipocalin reveals a particularly large cavity within its β-barrel (Breustedt, D.A. et al. (2005) J. Biol. Chem. 280, 1, 484-493).

Despite the progress made, it remains desirable to obtain human tear lipocalin muteins with improved binding properties (especially higher binding affinity) to IL-4 receptor alpha, simply for the purpose of further improving the suitability of human tear lipocalin muteins for diagnostic and therapeutic applications.

SUMMARY OF THE INVENTION

Therefore, one object of the present invention is to provide an improved human tear lipocalin mutein having high binding affinity to IL-4 receptor alpha.

This object is achieved by a human tear lipocalin mutein having the features stated in the claims, in particular claim 1 .

In a first aspect, the present invention provides a mutant protein of human tear lipocalin. The mutant protein binds to IL-4 receptor α. The mutant protein has a mutated amino acid residue at any one or more of sequence positions 27, 28, 30, 31, 33, 53, 57, 61, 64, 66, 80, 83, 104-106, and 108 of the linear polypeptide sequence of mature human tear lipocalin. This mutant protein further has a mutated amino acid residue at any two or more of sequence positions 26, 32, 34, 55, 56, 58, and 63 of the linear polypeptide sequence of mature human tear lipocalin. The amino acid sequence of the human tear lipocalin mutant protein comprises one of the following amino acid combinations: (1) Ser 26, Glu 34, Leu 55, Lys 58, (2) Ser 26, Asn 34, Ala 55, Lys 58, (3) Ser 26, Val 34, (4) Pro 26, Ser 34, (5) Pro 26, Ala55, (6) Leu 26, Trp 34, Ala 55, (7) Leu 26, Trp 34, Ile 58, (8) Asn 26, Asp 34, (9) Asn 26, Ala 55, (10) Tyr 26, His 34, Ala 55, (11) Tyr 26, His 34, Ala 58, (12) Lys 26, Arg 34, Ala 55, (13) Lys 26, Arg 34, Asn 58, (14) Glu 26, Gly 34, Ala 55, and (15) Glu 26, Gly 34, Leu 58.

As used herein, the term "position" refers to the position of an amino acid in an amino acid sequence described herein or the position of a nucleotide in a nucleic acid sequence described herein. The term "corresponding" as used herein also includes positions that are not determined solely by the number of the aforementioned nucleotides/amino acids. Thus, the position of a given amino acid according to the present invention that can be substituted can be altered by deletion or addition of an amino acid elsewhere in the (variant or wild-type) lipocalin. Similarly, the position of a given nucleotide according to the present invention that can be substituted can be altered by deletion or addition of a nucleotide elsewhere in the mutant protein or the 5'-untranslated region (UTR) of the wild-type lipocalin containing the promoter and/or any other regulatory sequences or genes (including exons and introns).

Therefore, according to the present invention, under "corresponding positions", it is preferably understood that the nucleotides/amino acids may differ in the numbers shown, but may still have similar adjacent nucleotides/amino acids. Said nucleotides/amino acids that may be exchanged, deleted or added are also encompassed by the term "corresponding positions".

In particular, to determine whether a nucleotide residue or amino acid residue in the amino acid sequence of a lipocalin different from that of the Tlc lipocalin mutein of the present invention aligns with a certain position in the nucleotide sequence or amino acid sequence of said Tlc lipocalin mutein (in particular any one of SEQ ID NOs: 2 to 11 or a sequence having one or more amino acid substitutions at positions 27, 28, 30, 31, 33, 53, 57, 61, 64, 66, 80, 83, 104 to 106 and 108 of the linear polypeptide sequence of Tcl (SEQ ID NO: 20), the skilled person can use means and methods well known in the art, such as alignment maps, manually or by using computer programs such as BLAST 2.0, which stands for Basic Local Alignment Search Tool or ClustalW or any other suitable program suitable for generating sequence alignments. Thus, a lipocalin mutein having any one of SEQ ID Nos: 2-11 or a sequence having one or more amino acid substitutions at positions 27, 28, 30, 31, 33, 53, 57, 61, 64, 66, 80, 83, 104-106 and 108 of the linear polypeptide sequence of Tcl (SEQ ID NO: 20) can serve as a "subject sequence" and an amino acid sequence of a lipocalin other than Tlc as a "query sequence."

In a second aspect, the present invention provides a method for producing a human tear lipocalin mutant. This mutant protein binds to IL-4 receptor α. The method comprises mutagenizing a nucleic acid molecule encoding human tear lipocalin at any one or more of amino acid sequence positions 27, 28, 30, 31, 33, 53, 57, 61, 64, 66, 80, 83, 104-106, and 108 of the mature human tear lipocalin linear polypeptide sequence. Furthermore, the method comprises mutagenizing a nucleic acid molecule encoding human tear lipocalin at any two or more of amino acid sequence positions 26, 32, 34, 55, 56, 58, and 63 of the mature human tear lipocalin linear polypeptide sequence. The result is one or more nucleic acids encoding human tear lipocalin mutants. The amino acid sequence of the encoded mutant protein comprises one of the following amino acid combination sets: (1) Ser 26, Glu 34, Leu 55, Lys 58, (2) Ser 26, Asn 34, Ala 55, Lys 58, (3) Ser 26, Val 34, (4) Pro 26, Ser 34, (5) Pro 26, Ala 55, (6) Leu 26, Trp 34, Ala 55, (7) Leu 26, Trp 34, Ile 58, (8) Asn 26, Asp 34, (9) Asn 26, Ala 55, (10) Tyr 26, His 34, Ala 55, (11) Tyr 26, His 34, Ala 58, (12) Lys 26, Arg (1) Lys 26, Arg 34, Asn 58, (1) Glu 26, Gly 34, Ala 55, and (1) Glu 26, Gly 34, Leu 58. The method further comprises expressing one or more nucleic acid molecules encoding the mutant proteins obtained thereby in an expression system. Further, the method comprises enriching one or more mutant proteins obtained thereby that bind to IL-4 receptor alpha by screening and/or separation.

In a third aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the mutant protein according to the first aspect.

In a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecule according to the third aspect.

In a fifth aspect, the present invention provides a pharmaceutical composition comprising a mutant protein of human tear lipocalin according to the first aspect, and further comprising a pharmaceutically acceptable excipient.

The invention is better understood with reference to the detailed description when considered in conjunction with the non-limiting embodiments and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the polypeptide sequence of S191.4-B24, a mutant protein of human tear lipocalin having binding affinity for IL-4 receptor alpha.

FIG2 shows the polypeptide sequences of exemplary muteins with high affinity for IL-4 receptor α (SEQ ID NO: 2-11).

Figure 3 shows the inhibition of TF-1 cell proliferation by increasing amounts of the muteins of the invention in the presence of IL-4 (A) and IL-13 (B).

FIG4 shows the _IC50 values of FIG3 and Biacore measurement data of the binding of the human tear lipocalin mutein of the present invention to IL-4 receptor α, such as human IL-4 receptor α.

Detailed description

The present invention provides a mutant protein of human tear lipocalin that has a particularly high affinity for IL-4 receptor α. The IL-4 receptor α, which is the target of the mutant protein of the present invention, is a typical mammalian protein, such as a human protein. In vivo, IL-4 receptor α can bind to interleukin-4 and interleukin-13 to regulate the production of IgE antibodies in B cells.

The binding affinity of the muteins according to the present invention has been found to be generally below 0.1 _nM , and in some embodiments, about 1 picomolar (pM) (see FIG. 4 ). The lipocalin muteins of the present invention are therefore capable of binding to IL-4 receptor α with a detectable affinity, i.e., a dissociation constant of at least 200 nM. In some embodiments, the lipocalin muteins of the present invention bind to IL-4 receptor α with a dissociation constant of at least about 10 nM, about 1 nM, about 0.1 nM, about 10 pM, or even less for IL-4 receptor α. The binding affinity of the muteins to a selected target (in this case, IL-4 receptor α) can be measured, and the _K value of the mutein-ligand complex can be determined by a variety of methods known to those skilled in the art. These methods include, but are not limited to, fluorescence titration, competitive ELISA, calorimetric methods such as isothermal titration calorimetry (ITC), and surface plasmon resonance (BIAcore). Examples of these methods are described in detail below (e.g., see Example 2).

The human interleukin-4 receptor α chain may have the amino acid sequence of SWISS PROT database accession number P24394 (SEQ ID NO: 18) or a fragment thereof. Illustrative examples of human interleukin-4 receptor α chain fragments include amino acids 26 to 232 of IL-4 receptor α. The amino acid sequence of human IL-13 receptor α1 is shown in SEQ ID NO: 19.

In general, the term "fragment" as used herein with respect to the protein ligand of the tear lipocalin mutein of the present invention relates to an N-terminally and/or C-terminally shortened protein or peptide ligand which retains the ability of the full-length ligand to be recognized and/or bound by the mutein according to the present invention.

Human tear lipocalin muteins that bind to IL-4 receptor α can be used as IL-4 antagonists and/or IL-13 antagonists or as inverse IL-4 agonists and/or inverse IL-13 agonists. Inverse agonists bind to the same binding site as agonists for a specific receptor and reverse the constitutive activity of the corresponding receptor. To date, the IL-4 receptor has not been reported to have endogenous kinase activity, so the muteins of the present invention can typically be used as IL-4 antagonists and/or IL-13 antagonists. In one embodiment, the human tear lipocalin muteins are used as antagonists of human IL-4 and/or human IL-13. In some embodiments, the muteins cross-react with macaque IL-4 and IL-4 receptor α and thus are used as antagonists of macaque ligands, such as IL-4 and/or IL-13. In some embodiments, the muteins cross-react with marmoset IL-4 and IL-4 receptor α and thus are used as antagonists of marmoset ligands, such as IL-4 and/or IL-13.

IL-4 receptor α can be used to define non-natural ligands for human tear lipocalin. The term "non-natural ligand" refers to a compound that does not bind to native mature human tear lipocalin under physiological conditions. As used herein, the term "human tear lipocalin" refers to the mature human tear lipocalin corresponding to the protein in the SWISS-PROT database accession number P31025. Mature human tear lipocalin does not contain the N-terminal signal peptide contained in the sequence of SWISS-PROT accession number P31025 (see Figure 2).

Compared to the sequence identity with other lipocalins (see above), the amino acid sequences of the muteins of the present invention have a high sequence identity with mature human tear lipocalin. In this general context, the amino acid sequences of the muteins of the present invention are at least substantially similar to the amino acid sequence of mature human tear lipocalin. The respective sequences of the muteins of the present invention are substantially similar to the sequence of mature human tear lipocalin, and in some embodiments have 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 the sequence of mature human tear lipocalin, provided that the mutation position or sequence is retained.

"Identity" refers to a property of sequences that measures their similarity or relatedness. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the result by 100. As two exemplary embodiments, the mutant protein of SEQ ID NO:3 has an 83.3% sequence identity with the amino acid sequence of mature human tear lipocalin, and the mutant protein of SEQ ID NO:7 has an 82.0% amino acid sequence identity with mature human tear lipocalin.

A "gap" is a space in an alignment caused by the addition or deletion of an amino acid. Thus, two identical sequences have 100% identity, but sequences that are less highly conserved and have additions, deletions, or substitutions may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity using standard parameters, such as 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 "mutated" or "mutant" with respect to a nucleic acid or polypeptide refers to an exchange, deletion or insertion of one or more nucleotides or amino acids, respectively, compared to a naturally occurring nucleic acid or polypeptide. Compared to the corresponding native human tear lipocalin, the mutein of the present invention comprises at least three substitutions.

In some embodiments, the mutant protein according to the present invention comprises at least two amino acid substitutions, including 2, 3, 4, 5 or more amino acid substitutions of natural amino acids with arginine residues. In some embodiments, the substituted amino acid can be located at any one of positions 27, 30, 57 and 83 relative to the amino acid sequence of mature human tear lipocalin.

In some embodiments, the muteins according to the present invention comprise amino acid substitutions of the native cysteine residues at positions 61 and/or 153 with serine residues. In this context, it is noted that removal of the structural disulfide bonds formed by cysteine residues 61 and 153 (see Breustedt et al., 2005, supra) of wild-type tear lipocalin (at the level of the respective native nucleic acid library) has been found to provide tear lipocalin muteins that are not only stably folded but also capable of binding a given non-native ligand with high affinity. Without wishing to be bound by theory, it is also believed that the elimination of structural disulfide bonds provides the further advantage of allowing the (spontaneous) generation or deliberate introduction of non-native, artificial disulfide bonds into the muteins of the present invention (see the Examples), for example, thereby increasing the stability of the muteins. In some embodiments, the muteins according to the present invention comprise amino acid substitutions of the native cysteine residue at position 101 with a serine residue. Furthermore, in some embodiments, the muteins according to the present invention comprise amino acid substitutions of the native arginine residue at position 111 with a proline residue. In some embodiments, a mutein according to the present invention comprises an amino acid substitution replacing the native lysine residue at position 114 with a tryptophan residue.

Human tear lipocalin muteins according to the present invention typically have one of asparagine, glutamic acid, proline, leucine, lysine, serine, and tyrosine at the position corresponding to amino acid position 26 of mature human tear lipocalin. In some embodiments, the muteins of the present invention have a sequence in which amino acid position 34 is unchanged relative to mature human tear lipocalin, and the sequence of the mutein comprises the amino acid substitutions Arg 26→Ser, Met 55→Leu, and Ser 58→Lys. In some embodiments, the muteins of the present invention have a sequence comprising the amino acid substitutions Arg 26→Pro and Glu 34→Ser. In some embodiments, the muteins of the present invention have a sequence comprising the amino acid substitutions Arg 26→Pro and Met 55→Ala. In some embodiments, the muteins of the present invention have a sequence comprising the amino acid substitutions Arg 26→Ser and Glu 34→Val. In some embodiments, the muteins of the present invention have a sequence comprising the amino acid substitutions Arg 26→Leu, Glu 34→Trp, and Met 55→Ala. In some embodiments, the mutant proteins of the present invention have a sequence comprising the amino acid substitutions Arg 26→Leu, Glu 34→Trp, and Ser 58→Ile. In some embodiments, the mutant proteins of the present invention have a sequence comprising the amino acid substitutions Arg 26→Ser, Glu 34→Asn, Met 55→Ala, and Ser 58→Lys. In some embodiments, the mutant proteins of the present invention have a sequence comprising the amino acid substitutions Arg 26→Asn and Glu 34→Asp. In some embodiments, the mutant proteins of the present invention have a sequence comprising the amino acid substitutions Arg 26→Asn and Met 55→Ala. In some embodiments, the mutant proteins of the present invention have a sequence comprising the amino acid substitutions Arg 26→Tyr, Glu 34→His, and Met 55→Ala. In some embodiments, the mutant proteins of the present invention have a sequence comprising the amino acid substitutions Arg 26→Tyr, Glu 34→His, and Ser 58→Ala. In some embodiments, the mutant protein of the present invention has a sequence comprising the amino acid substitutions Arg 26→Lys, Glu 34→Arg, and Met 55→Ala. In some embodiments, the mutant protein of the present invention has a sequence comprising the amino acid substitutions Arg 26→Lys, Glu 34→Arg, and Ser 58→Asn. In some embodiments, the mutant protein of the present invention has a sequence comprising the amino acid substitutions Arg 26→Glu, Glu 34→Arg, and Met 55→Ala. In some embodiments, the mutant protein of the present invention has a sequence comprising the amino acid substitutions Arg 26→Glu, Glu 34→Arg, and Ser 58→Leu.

In some embodiments, compared to the amino acid sequence of mature human tear lipocalin, the IL-4 receptor alpha binding human tear lipocalin muteins of the present invention have a mutated amino acid residue at sequence position 58 or sequence position 63. In some embodiments, the sequence of the muteins of the present invention is selected such that serine is not present at both amino acid positions 26 and 34, compared to the amino acid sequence of mature human tear lipocalin.

The lipocalin mutein may further comprise one or more (including at least two, at least three, or at least four) amino acid substitutions of a native amino acid residue with a cysteine residue at any one of positions 26-28, 30-34, 53, 55-58, 61, 63, 64, 66, 80, 83, 104-106, and 108 of the native mature human tear lipocalin relative to the amino acid sequence of the mature human tear lipocalin. In some embodiments, the mutein according to the present invention comprises an amino acid substitution of a native amino acid with a cysteine residue at position 28 or 105 relative to the amino acid sequence of the mature human tear lipocalin. In some embodiments, the mutein according to the present invention comprises an amino acid substitution of a native amino acid with a cysteine residue at position 28 or 105 relative to the amino acid sequence of the mature human tear lipocalin.

In some embodiments, the mutant protein according to the present invention has mutated amino acid residues at any three or more (including 3, 4, 5, 6 or 7) of sequence positions 26, 32, 34, 55, 56, 58 and 63 of the mature human tear lipocalin linear polypeptide sequence.

In some embodiments, the lipocalin muteins of the present invention have a mutated amino acid residue at position 26, wherein the mutated amino acid residue is one of asparagine, glutamine, proline, leucine, lysine, serine, and tyrosine. In some embodiments, the lipocalin muteins of the present invention have a mutated amino acid residue at position 32, wherein the mutated amino acid residue is one of histidine, lysine, tyrosine, and valine. In some embodiments, the lipocalin muteins of the present invention have a mutated amino acid residue at position 34, wherein the mutated amino acid residue is one of arginine, aspartic acid, asparagine, histidine, serine, tryptophan, and valine. In some embodiments, the lipocalin muteins of the present invention have a mutated amino acid residue at position 55, wherein the mutated amino acid residue is one of alanine and leucine. In some embodiments, the lipocalin muteins of the present invention have a mutated amino acid residue at position 56, wherein the mutated amino acid residue is one of alanine, glutamine, histidine, methionine, leucine, and lysine. In some embodiments, the lipocalin muteins of the present invention have a mutated amino acid residue at position 58, wherein the mutated amino acid residue is one of alanine, arginine, asparagine, histidine, isoleucine, and lysine. In some embodiments, the lipocalin muteins of the present invention have a mutated amino acid residue at position 63, wherein the mutated amino acid residue is one of glutamine, lysine, proline, and serine.

In some embodiments, the mutant protein according to the present invention comprises at least one of the substitutions Met 31→Ala, Leu 33→Tyr, Ser 61→Trp, Asp 80→Ser, Glu 104→Leu, His 106→Pro, and Lys 108→Gln. In some embodiments, the mutant protein according to the present invention comprises two or more, for example, 3, 4, 5, 6, or all, of the substitutions Met 31→Ala, Leu 33→Tyr, Ser 61→Trp, Asp 80→Ser, Glu 104→Leu, His 106→Pro, and Lys 108→Gln. In some embodiments, the mutant protein according to the present invention comprises the substitution Val 53→Ph or Val 53→Leu. The mutated amino acid residue may also comprise the substitution Val 64→Tyr or Val 64→Met. It may also comprise the substitution Ala 66→Leu or Ala 66→Asp.

In some embodiments, a human tear lipocalin mutein according to the present invention comprises a replacement amino acid for at least one or both cysteine residues present at each of sequence positions 61 and 153, as well as mutations in at least three amino acid residues at any of sequence positions 26-28, 30-34, 53, 55-58, 63, 64, 66, 80, 83, 104-106, and 108 of the mature human tear lipocalin linear polypeptide sequence. Positions 26-28 and 30-34 are contained within the AB loop, positions 53 and 55 are located at the extreme ends of the β-sheet, and positions 56-58 are subsequently contained within the CD loop. Surprisingly, positions 63, 64, and 66 are contained within the β-sheet (βD), and position 80 is located within the α-helical region. Position 83 is an amino acid that defines a single loop between this α-helical region and the β-sheet (βF). Positions 104-106 and 108 are contained in the GH loop within the binding site at the open end of the tear lipocalin β-barrel structure. Definitions of these regions, as used herein, are based on those of Flower (Flower, 1996, Flower et al., 2000, supra) and Breustedt et al. (2005, supra). Such mutant proteins can comprise at least two (including three, four, five, six, eight, ten, twelve, fourteen, fifteen, sixteen, seventeen, or eighteen) mutated amino acid residues at sequence positions 26-34, 55-58, sixty-three, sixty-four, eighty-seven, eighty-three, eighty-eight, ten, twelve, fourteen, fifteen, sixteen, seventeen, or eighteen) of the mature human tear lipocalin linear polypeptide sequence. In some embodiments, the mutant protein comprises the amino acid substitutions Cys 61→Ala, Phe, Lys, Arg, Thr, Asn, Tyr, Met, Ser, Pro, or Trp and Cys 153→Ser or Ala. This substitution proved effective in preventing the formation of the naturally occurring disulfide bond linking Cys 61 and Cys 153, thereby facilitating the processing of the mutein. However, tear lipocalin muteins that bind to IL-4 receptor alpha and form a disulfide bond between Cys 61 and Cys 153 are also part of the present invention.

In some embodiments, the mutein comprises at least one amino acid substitution, which can be another amino acid substitution selected from Arg 111→Pro and Lys 114→Trp. The mutein of the present invention can further comprise a cysteine substitution at position 101 of the native mature human tear lipocalin sequence with another amino acid. This substitution can be, for example, a mutation of Cys 101→Ser or Cys 101→Thr.

As described above, the mutant protein of the present invention comprises at least one amino acid substitution at position 27, 28, 30, 31, 33, 53, 57, 61, 64, 66, 80, 83, 104-106, and 108 of the mature human tear lipocalin linear polypeptide sequence. In some embodiments, the mutant protein of the present invention comprises two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1, 15, or 16) amino acid substitutions at these sequence positions of mature human tear lipocalin. In one embodiment, the mutant protein has a mutated amino acid residue at each of position 27, 28, 30, 31, 33, 53, 57, 61, 64, 66, 80, 83, 104-106, and 108 of the mature human tear lipocalin linear polypeptide sequence.

In some embodiments, the mutated amino acid residue at any one or more of sequence positions 27, 28, 30, 31, 33, 53, 57, 61, 64, 66, 80, 83, 104-106, and 108 of the mature human tear lipocalin linear polypeptide sequence relative to the mature human tear lipocalin amino acid sequence comprises one or more of the substitutions Met31→Ala, Leu33→Tyr, Ser61→Trp, Asp80→Ser, Glu104→Leu, His106→Pro, and Lys108→Gln. In some embodiments, the mutant protein of the present invention comprises two or more (e.g., 3, 4, 5, 6, or 7) amino acid substitutions at these sequence positions of mature human tear lipocalin.

In the remaining regions, i.e., regions other than sequence positions 26-28, 30-34, 53, 55-58, 63, 64, 66, 80, 83, 104-106, and 108, the lipocalin muteins of the present invention may comprise the wild-type (native) amino acid sequence, except for the mutated amino acid sequence positions. In some embodiments, the lipocalin muteins according to the present invention may also have one or more amino acid mutations at one or more sequence positions, provided that such mutations do not, at least substantially, impair or interfere with the binding activity and folding of the mutein. Such mutations can be readily accomplished at the DNA level using established standard methods. Illustrative examples of amino acid sequence alterations are insertions or deletions and amino acid substitutions. Such substitutions may be conservative, i.e., amino acid residues are replaced with amino acid residues of similar chemical properties, particularly with respect to polarity and size. Examples of conservative substitutions are substitutions between 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, non-conservative changes can also be introduced into the amino acid sequence. Furthermore, in addition to replacing single amino acid residues, one or more consecutive amino acids in the primary structure of tear lipocalin can be inserted or deleted, as long as these deletions or insertions result in a stable folded/functional mutant protein (see, for example, the experimental section where N- and C-terminally truncated mutant proteins are generated).

Such modifications of the amino acid sequence include site-directed mutagenesis of single amino acid positions to simplify subcloning of mutant lipocalin genes or portions thereof by incorporating cleavage sites for specific restriction enzymes. In addition, these mutations can be incorporated to further improve the affinity of the lipocalin mutein for a given target. Furthermore, mutations can be introduced to modulate certain characteristics of the mutein, for example to improve folding stability, serum stability, protein resistance or water solubility, or, if necessary, to reduce aggregation tendency. For example, naturally occurring cysteine residues can be mutated to other amino acids to prevent disulfide bond formation. Other amino acid sequence positions can also be intentionally mutated to cysteine to introduce new reactive groups, for example for conjugation to other compounds, such as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins, or for forming non-naturally occurring disulfide bonds. Exemplary possibilities for introducing a cysteine residue into the amino acid sequence of a human tear lipocalin mutein include substitutions Thr 40→Cys, Glu 73→Cys, Arg 90→Cys, Asp 95→Cys, and Glu 13→Cys. The thiol moiety generated on either side of amino acid positions 40, 73, 90, 95, and/or 131 can be used to pegylate or hesylate the mutein, for example, to increase the serum half-life of the respective tear lipocalin mutein.

The present invention also includes mutant proteins as described above, wherein the first four N-terminal amino acid residues (His-His-Leu-Leu; positions 1-4) of the mature human tear lipocalin sequence and/or the last two C-terminal amino acid residues (Ser-Asp; positions 157-158) of the mature human tear lipocalin sequence are deleted (see the Examples and the accompanying sequence listing). Another possible mutation of the wild-type sequence is to change the amino acid sequence at positions 5 to 7 (Ala Ser Asp) to Gly Gly Asp, as described in PCT application WO2005/019256.

In some embodiments, compared to mature human tear lipocalin, tear lipocalin muteins according to the present invention have one or more (including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of the following amino acid substitutions: Glu 27→Arg, Phe 28→Cys, Glu 30→Arg, Met 31→Ala, Leu 33→Tyr, Ile 57→Arg, Ser 61→Trp, Asp 80→Ser, Lys 83→Arg, Glu 104→Leu, Leu 105→Cys, His 106→Pro, Lys 108→Gln. In some embodiments, the muteins comprise all of these amino acid substitutions. In some embodiments, the muteins further comprise the amino acid substitutions Val 53→Phe, Val 64→Tyr, and Ala 66→Leu. In other embodiments, the mutant protein further comprises the set of amino acid substitutions Val 53→Leu, Val 64→Met, Ala 66→Asp.

In some embodiments, compared to mature human tear lipocalin, the tear lipocalin muteins according to the present invention comprise a combination of amino acid substitutions: Arg 26→Ser, Asn 32→Tyr, Met 55→Leu, Leu 56→Gln, and Ser58→Lys. In some embodiments, compared to mature human tear lipocalin, the tear lipocalin muteins according to the present invention comprise a combination of amino acid substitutions: Arg 26→Pro, Asn 32→Tyr, Glu 34→Ser, Met 55→Ala, Leu 56→Gln, and Glu 63→Lys. In some embodiments, compared to mature human tear lipocalin, the tear lipocalin muteins according to the present invention comprise a combination of amino acid substitutions: Arg 26→Leu, Asn 32→Phe, Glu 34→Trp, Met 55→Ala, Ser 58→Ile, and Glu 63→Ser. In some embodiments, compared to mature human tear lipocalin, the tear lipocalin mutein according to the present invention comprises a combination of amino acid substitutions Arg 26→Ser, Asn 32→Tyr, Glu 34→Val, Met 55→Ala, Leu 56→Ala, Ser 58→Ile, and Glu 63→Ser. In some embodiments, compared to mature human tear lipocalin, the tear lipocalin mutein according to the present invention comprises a combination of amino acid substitutions Arg 26→Ser, Asn 32→Val, Glu 34→Asn, Met 55→Ala, Leu 56→Gln, Ser 58→Lys, and Glu 63→Lys. In some embodiments, compared to mature human tear lipocalin, the tear lipocalin mutein according to the present invention comprises a combination of amino acid substitutions Arg 26→Tyr, Asn 32→Tyr, Glu 34→His, Met 55→Ala, Leu 56→His, Ser 58→Ala, and Glu 63→Lys. In some embodiments, compared to mature human tear lipocalin, the tear lipocalin mutein according to the present invention comprises a combination of amino acid substitutions Arg 26→Lys, Asn 32→Tyr, Glu 34→Arg, Met 55→Ala, Leu 56→Lys, Ser 58→Asn, and Glu 63→Pro. In some embodiments, the tear lipocalin mutein according to the present invention comprises a combination of amino acid substitutions Arg 26→Glu, Asn 32→His, Glu 34→Gly, Met 55→Ala, Leu 56→Met, Ser 58→Leu, Glu 63→Lys compared to mature human tear lipocalin.

In some embodiments, the mutant protein of the present invention comprises at least 6, 8, 10, 12, 14 or 16 amino acid substitutions selected from the following relative to the amino acid sequence of mature human tear lipocalin: Arg 26→Ser, Pro; Glu 27→Arg; Phe 28→Cys; Glu 30→Arg; Met 31→Ala; Asn 32→Tyr, His; Leu 33→Tyr, Glu 34→Gly, Ser, Ala, Asp, Lys, Asn, Thr, Arg; Leu 56→Gln; Ile 57→Arg; Ser 58→Ile, Ala, Arg, Val, Thr, Asn, Lys, Tyr, Leu, Met; Asp 80→Ser; Lys 83→Arg; Glu 104→Leu; Leu 105→Cys; His106→Pro and Lys 108→Gln.

In addition, the mutant protein may further comprise at least one amino acid substitution selected from the group consisting of: Met 39 → Val; Thr 42 → Met, Ala; Thr 43 → Ile, Pro, Ala; Glu 45 → Lys, Gly; Asn 48 → Asp, His, Ser, Thr; Val 53 → Leu, Phe, Ile, Ala, Gly, Ser; Thr 54 → Ala, Leu; Met 55 → Leu, Ala, Ile, Val, Phe, Gly, Thr, Tyr; Glu 63 → Lys, Gln, Ala, Gly, Arg; Val 64 → Gly, Tyr, Met, Ser, Ala, Lys, Arg, Leu, Asn, His, Thr, Ile; Ala 66 → Ile, Leu, Val, Thr, Met; Glu 69→Lys, Gly; Lys70→Arg, Gln, Glu; Thr 78→Ala; Ile 89→Val; Asp 95→Asn, Ala, Gly; and Tyr 100→His.

In one embodiment, the human tear lipocalin mutein that binds to IL-4 receptor α comprises the amino acid substitutions: Arg 26→Ser, Glu 27→Arg, Phe 28→Cys, Glu 30→Arg; Met31→Ala, Leu 33→Tyr, Leu56→Gln, Ile 57→Arg, Asp8 0→Ser, Lys 83→Arg, Glu 104→Leu, Leu 105→Cys, His 106→Pro, and Lys1 08→Gln.

In some embodiments, the human tear lipocalin mutein that binds to IL-4 receptor alpha comprises one of the following sets of amino acid substitutions:

(1)Arg 26→Ser, Glu 27→Arg, Phe 28→Cys, Glu 30→Arg, Met 31→Ala, Asn 32→Tyr, Leu 33→Tyr, Glu 34→Gly, Leu 56→Gln, Ile 57→Arg, Ser 58→Ile, Asp 80→Ser, Lys 83→Arg, Glu 104→Leu, Leu 105→Cys, His 106→Pro, Lys 108→Gln;

(2)Arg 26→Ser, Glu 27→Arg, Phe 28→Cys, Glu 30→Arg, Met 31→Ala, Asn 32→Tyr, Leu 33→Tyr, Glu 34→Lys, Leu 56→Gln, Ile 57→Arg, Ser 58→Asn, Asp 80→Ser, Lys 83→Arg, Glu 104→Leu, Leu 105→Cys, His 106→Pro, Lys 108→Gln;

(3)Arg 26→Ser, Glu 27→Arg, Phe 28→Cys, Glu 30→Arg, Met 31→Ala, Asn 32→Tyr, Leu 33→Tyr, Leu 56→Gln, Ile 57→Arg, Ser 58→Arg, Asp 80→Ser, Lys 83→Arg, Glu 104→Leu, Leu 105→Cys, His 106→Pro, Lys 108→Gln;

(4)Arg 26→Ser, Glu 27→Arg, Phe 28→Cys, Glu 30→Arg, Met 31→Ala, Asn 32→Tyr, Leu 33→Tyr, Glu 34→Ser, Leu 56→Gln, Ile 57→Arg, Asp 80→Ser, Lys 83→Arg, Glu 104→Leu, Leu 105→Cys, His 106→Pro, Lys 108→Gln;

(5)Arg 26→Ser, Glu 27→Arg, Phe 28→Cys, Glu 30→Arg, Met 31→Ala, Asn 32→His, Leu 33→Tyr, Glu 34→Ser, Leu 56→Gln, Ile 57→Arg, Ser 58→Ala, Asp 80→Ser, Lys 83→Arg, Glu 104→Leu, Leu 105→Cys, His 106→Pro, Lys 108→Gln;

(6) Arg 26→Ser, Glu 27→Arg, Phe 28→Cys, Glu 30→Arg, Met 31→Ala, Asn 32→Tyr, Leu 33→Tyr, Glu 34→Asp, Leu 56→Gln, Ile 57→Arg, Ser 58→Lys, Asp 80→Ser, Lys 83→Arg, Glu 104→Leu, Leu 105→Cys, His 106→Pro, Lys 108→Gln; and

(7)Arg 26→Ser, Glu 27→Arg, Phe 28→Cys, Glu 30→Arg, Met 31→Ala, Asn 32→Tyr, Leu 33→Tyr, Glu 34→Gly, Leu 56→Gln, Ile 57→Arg, Asp 80→Ser, Lys 83→Arg, Glu 104→Leu, Leu 105→Cys, His 106→Pro, Lys 108→Gln.

The human tear lipocalin muteins of the present invention may comprise, consist essentially of, or consist of any of the amino acid sequences shown in SEQ ID NOs: 3-11, or fragments or variants thereof. The term "fragment," as used herein with respect to the muteins of the present invention, refers to proteins or peptides derived from full-length mature human tear lipocalin that are N-terminally and/or C-terminally truncated, i.e., lack at least one of the N-terminal and/or C-terminal amino acids. Such fragments may comprise at least 10, or more (e.g., 20 or 30 or more), contiguous amino acids of the primary sequence of mature human tear lipocalin and are typically detectable in immunoassays for mature human tear lipocalin.

As used herein, the term "variant" refers to a derivative of a protein or peptide comprising a modification of the amino acid sequence, such as by substitution, deletion, insertion, or chemical modification. In some embodiments, such modification does not substantially reduce the function of the protein or peptide. Such variants include proteins in which one or more amino acids are replaced by their respective D stereoisomers or by amino acids other than the 20 naturally occurring amino acids (such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, valeric acid). However, such replacements can also be conservative, i.e., the amino acid residue is replaced by an amino acid residue with similar chemical properties. Examples of conservative replacements are replacements between 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.

Such a mutant protein may comprise at least 6, 8, 10, 12, 14 or 16 amino acid substitutions selected from the group consisting of Arg 26→Ser; Glu 27→Ile; Glu 30→Ser; Met 31→Gly; Asn 32→Arg; Leu 33→Ile; Glu 34→Tyr; Leu 56→Lys, Glu, Ala, Met; Ile 57→Phe; Ser 58→Arg; Asp 80→Ser, Pro; Lys 83→Glu, Gly; Glu 104→Leu; Leu 105→Ala; His 106→Val; and Lys 108→Thr relative to the amino acid sequence of mature human tear lipocalin, and may further comprise at least one amino acid substitution selected from the group consisting of Leu 4→Phe; Glu 63→Lys; Val 64→Met; Asp 72→Gly; Lys 76→Arg, Glu; Ile 88→Val, Thr; Ile89→Thr; Arg 90→Lys; Asp 95→Gly; Phe 99→Leu; and Gly 107→Arg, Lys, Glu.

In one embodiment, the mutant protein comprises the amino acid substitutions: Arg 26→Ser, Glu 27→Ile, Glu 30→Ser, Met 31→Gly, Asn 32→Arg, Leu 33→Ile, Glu 34→Tyr, Ile 57→Phe, Ser 58→Arg, Lys 83→Glu, Glu 104→Leu, Leu 105→Ala, His 106→Val, and Lys 108→Thr.

The tear lipocalin muteins of the present invention may exist as monomeric proteins. In some embodiments, the lipocalin muteins according to the present invention may be capable of spontaneous dimerization or oligomerization. The use of lipocalin muteins that form stable monomers may be advantageous in some applications, for example, due to faster diffusion and better tissue penetration. In other embodiments, the use of lipocalin muteins that spontaneously form homodimers or multimers may be advantageous because such multimers may provide (further) increased affinity and/or avidity for a given target. In addition, oligomeric forms of lipocalin muteins may have a lower dissociation rate or an extended serum half-life. If dimerization or multimerization of the muteins to form stable monomers is desired, this can be achieved, for example, by fusing respective oligomerization domains, such as the Jun-Fos domain or the leucine zipper, to the muteins of the present invention or by using "duocalins" (see below).

Lipocalin muteins according to the present invention can be obtained by mutagenesis of a naturally occurring form of human tear lipocalin. As used herein, the term "mutagenesis" refers to the selection of experimental conditions such that the naturally occurring amino acid at a given sequence position in human tear lipocalin (Swiss-Prot database entry P31025) is replaced by at least one amino acid not present at that specific position in the respective native polypeptide sequence. The term "mutagenesis" also encompasses (additional) modification of the length of a sequence segment by deletion or insertion of one or more amino acids. Thus, within the scope of the present invention, for example, an amino acid at a selected sequence position is replaced by three random mutations, resulting in the insertion of two amino acid residues compared to the length of each segment of the wild-type protein. Such deletions or insertions can be introduced independently of one another into any peptide segment that can be mutated in the present invention. In one exemplary embodiment of the present invention, several mutation insertions can be introduced into the AB loop of a selected lipocalin scaffold (see International Patent Application WO 2005/019256, incorporated herein by reference in its entirety). The term "random mutagenesis" means that no single amino acid (mutation) is predetermined to be present at a certain sequence position, but rather at least two amino acids may be incorporated at predefined sequence positions with a certain probability during mutagenesis.

The coding sequence of human tear lipocalin (Redl, B. et al. (1992) J. Biol. Chem. 267, 20282-20287) is used in the present invention as a starting point for mutagenesis of selected peptide segments. To mutagenesis the amino acid positions, one skilled in the art can choose to perform site-directed mutagenesis using various established standard methods. One commonly used technique is to introduce mutations by PCR (polymerase chain reaction) using a mixture of synthetic oligonucleotides with a degenerate base composition at the desired sequence position. For example, the use of the codons NNK or NNS (where N = adenine, cytosine, guanine or thymine; K = guanine or thymine; S = adenine or cytosine) during mutagenesis allows the incorporation of all 20 amino acids plus the amber stop codon, while the codon VVS limits the number of amino acids that can be incorporated to 12 because it excludes the amino acids Cys, Ile, Leu, Met, Phe, Trp, Tyr, Val from incorporation into the selected position of the polypeptide sequence; the use of the codon NMS (where M = adenine or cytosine) limits the number of possible amino acids at the selected sequence position to 11 because it excludes the amino acids Arg, Cys, Gly, Ile, Leu, Met, Phe, Trp, Val from incorporation into the selected sequence position. In this regard, it is noted that codons for other amino acids (in addition to the conventional 20 naturally occurring amino acids) such as selenocysteine or pyrrolysine can also be incorporated into the nucleic acid of the mutant protein. It is also possible to use "artificial" codons such as UAG, which are usually considered to be stop codons, in order to insert other unusual amino acids, such as o-methyl-L-tyrosine or p-aminophenylalanine, as described by Wang, L. et al. (2001) Science 292, 498-500 or Wang, L. and Schultz, P.G. (2002) Chem. Comm. 1, 1-11.

The use of nucleotide building blocks with reduced base pair specificity, such as inosine, 8-oxo-2'deoxyguanosine or 6(2-deoxy-b-D-furoxolosourea)-3,4-dihydro-8H-pyrimido-1,2-oxazin-7-one (Zaccolo et al. (1996) J. Mol. Biol. 255, 589-603), is another option for introducing mutations into selected sequence segments.

A further possibility is so-called triplet mutagenesis. This method uses a mixture of different nucleotide triplets, each encoding an amino acid, for incorporation into the coding sequence (B et al., 1994 Nucleic Acids Res 22, 5600-5607).

One possible strategy for introducing mutations into selected regions of each peptide is based on the use of four oligonucleotides, each of which is derived in part from one of the corresponding sequence segments to be mutated. When synthesizing these oligonucleotides, one skilled in the art can use a mixture of nucleic acid building blocks to synthesize nucleotide triplets corresponding to the amino acid positions to be mutated, so that the codons encoding all natural amino acids appear randomly, which ultimately leads to the generation of a library of lipocalin peptides. For example, the first oligonucleotide corresponds in its sequence (except for the position of the mutation) to the coding strand of the peptide segment to be mutated (at the most N-terminal position of the lipocalin polypeptide). Thus, the second oligonucleotide corresponds to the non-coding strand of the second sequence segment later in the polypeptide sequence. The third oligonucleotide, in turn, corresponds to the coding strand of the corresponding third sequence segment. Finally, the fourth oligonucleotide corresponds to the non-coding strand of the fourth sequence segment. A polymerase chain reaction is performed using each of the first and second oligonucleotides and, if necessary, each of the third and fourth oligonucleotides.

The amplified production of these two reactions can be merged into single nucleic acid by various known methods, and said single nucleic acid comprises the sequence from first to the 4th sequence section, and wherein sudden change is introduced into selected position.For this reason, two kinds of products for example can use flanking oligonucleotide and one or more intermediary nucleic acid molecules to carry out new polymerase chain reaction, and said intermediary nucleic acid molecule provides the sequence between second and the 3rd sequence section.When selecting number and arrangement in the oligonucleotide sequence that is used for mutagenesis, those skilled in the art have many alternative selections in his processing.

The nucleic acid molecules defined above can be ligated by ligating the nucleic acid encoding the lipocalin polypeptide and/or the vector with missing 5' and 3' sequences and cloned into known host organisms. Numerous established experimental procedures are available for ligation and cloning. For example, the recognition sequences for restriction endonucleases that are also present in the cloning vector sequence can be engineered into the sequences of the synthetic oligonucleotides. Thus, after amplification of the respective PCR products and enzymatic cleavage, the resulting fragments can be readily cloned using the corresponding recognition sequences.

Longer sequence segments within the gene encoding the protein selected for mutagenesis can also be subjected to random mutagenesis by known methods, for example, by utilizing the polymerase chain reaction under conditions that increase the error rate, by chemical mutagenesis, or by using bacterial mutant strains. These methods can also be used to further optimize the target affinity or specificity of the lipocalin mutein. Mutations that may occur outside the experimentally mutagenized segment are generally acceptable or may even prove to be advantageous, for example, if they contribute to improving the folding efficiency or folding stability of the lipocalin mutein.

In the method according to the present invention, a nucleic acid molecule encoding human tear lipocalin is first mutagenized at one or more of amino acid sequence positions 27, 28, 30, 31, 33, 53, 57, 61, 64, 66, 80, 83, 104-106, and 108 of the mature human tear lipocalin linear polypeptide sequence. Secondly, a nucleic acid molecule encoding human tear lipocalin can also be mutagenized at two or more of amino acid sequence positions 26, 32, 34, 55, 56, 58, and 63 of the mature human tear lipocalin linear polypeptide sequence. Of these latter amino acid sequence positions, at least one position to be mutated is selected from amino acid sequence position 58 and amino acid sequence position 63.

In one embodiment of the present invention, a method for producing a human tear lipocalin mutant protein comprises mutating at least 2, 3, 4, 5, 6, 8, 10, 12, 14, 15, 16, or 17 of the codons at any one of amino acid sequence positions 26-28, 30-34, 53, 55-58, 63, 64, 66, 80, 83, 104-106, and 108 of the mature human tear lipocalin linear polypeptide sequence. In one embodiment, all 22 of the codons at amino acid sequence positions 26, 27, 28, 30, 31, 32, 33, 34, 53, 55, 56, 57, 58, 63, 64, 66, 80, 83, 104, 105, 106, and 108 of the mature human tear lipocalin linear polypeptide sequence are mutated.

In one embodiment of the aforementioned method, at least 2, 3, 4, 5, 6, 8, 10, 12, 14, or 15 of the codons at any one of amino acid sequence positions 26-28, 30-34, 53, 55-58, 63, 64, 66, 80, 83, 104-106, and 108 of the mature human tear lipocalin linear polypeptide sequence are additionally mutated.

In a further embodiment of the present invention, the methods according to the present invention comprise mutation of two codons encoding cysteine at positions 61 and 153 of the linear polypeptide sequence of mature human tear lipocalin. In one embodiment, position 61 is mutated to encode an alanine, phenylalanine, lysine, arginine, threonine, asparagine, tyrosine, methionine, serine, proline, or tryptophan residue, to name a few possible examples. In an embodiment, where position 153 is mutated, an amino acid such as serine or alanine may be introduced at position 153.

In other embodiments of the invention as described herein, the codons at amino acid positions 111 and/or 114 of the amino acid sequence encoding the mature human tear lipocalin linear polypeptide sequence are mutated to encode, for example, arginine at position 111 and tryptophan at position 114.

Another embodiment of the methods of the present invention involves mutagenizing the codon encoding cysteine at position 101 of the mature human tear lipocalin linear polypeptide sequence so that this codon encodes any other amino acid. In one embodiment, the codon encoding the mutation at position 102 encodes serine. Thus, in some embodiments, two or all three cysteine codons at positions 61, 101, and 153 are replaced with codons for other amino acids.

According to the method of the present invention, starting with a nucleic acid encoding a human tear lipocalin, a mutant protein is obtained. This nucleic acid is mutated and introduced into a suitable bacterial or fungal host organism by means of recombinant DNA technology. The tear lipocalin nucleic acid library can be obtained using any suitable technique known in the art for generating mutant proteins with antibody-like properties (i.e., with affinity for a given target). Examples of such combinatorial approaches are described in detail in international patent applications such as WO 99/16873, WO 00/75308, WO 03/029471, WO 03/029462, WO 03/029463, WO 2005/019254, WO 2005/019255, WO 2005/019256, or WO 2006/56464. The contents of each of these patent applications are incorporated herein by reference in their entirety. After expressing the mutagenized amino acid sequence in a suitable host, clones carrying the genetic information for a plurality of individual lipocalin muteins that bind to a given target can be selected from the resulting library. These clones can be screened using well-known techniques, such as phage display (reviewed in Kay, B.K. et al. (1996), supra; Lowman, H.B. (1997), supra, or Rodi, D.J. and Makowski, L. (1999), supra), colony screening (reviewed in Pini, A. et al. (2002) Comb. Chem. High Throughput Screen. 5, 503-510), ribosome display (reviewed in Amstutz, P. et al. (2001) Curr. Opin. Biotechnol. 12, 400-405), or mRNA display as reported in Wilson, D.S. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 3750-3755, or in WO 99/16873, WO 00/75308, WO 03/029471, WO 03/029462, WO 03/029463, WO 2005/019254, WO 2005/019255, WO 2005/019256, or WO 2006/56464.

The nucleic acid molecule encoding the mutant protein is expressed using any suitable expression system. The obtained mutant protein is enriched by screening or separation methods. Screening can be performed, for example, under competitive conditions. The competitive conditions used herein refer to screening of the mutant protein comprising at least one of the following steps: in the step, the mutant protein and a given non-natural ligand of human tear lipocalin (i.e., IL-4 receptor α) are contacted in the presence of an additional ligand, and the additional ligand competes with the binding of the mutant protein to the IL-4 receptor α. The additional ligand can be a physiological ligand of the target (e.g., IL-4), an excess of the target itself, or any other non-physiological ligand of the target, which binds to at least one overlapping epitope to the epitope recognized by the mutant protein of the present invention and thereby interferes with the target binding of the mutant protein. Alternatively, the additional ligand complexes an epitope different from the binding site of the mutant protein to the target through an allosteric effect, thereby competing with the binding of the mutant protein.

Embodiments of phage display technology using the temperate M13 phage (reviewed in Kay, B.K. et al. (1996), supra; Lowman, H.B. (1997) or Rodi, D.J., & Makowski, L. (1999), supra) are provided as examples of screening methods that can be used in the present invention. Another embodiment of phage display technology that can be used to screen for mutant proteins of the present invention is the hyperphage phage technology described by Broders et al. (Broders et al. (2003) "Hyperphage. Improving antibody presentation in phage display." Methods Mol. Biol. 205: 295-302). Other temperate phages such as f1 or lytic phages such as T7 can also be used. For an exemplary screening method, an M13 phagemid is generated that allows for the expression of a mutant lipocalin amino acid sequence as a fusion protein having a signal sequence (e.g., the OmpA signal sequence) at the N-terminus and the capsid protein pill of bacteriophage M13, or a fragment thereof that can be incorporated into the phage capsid, at the C-terminus. A C-terminal fragment of the phage capsid protein, ΔpIII, comprising amino acids 217 to 406 of the wild-type sequence, can be used to generate the fusion protein. In one embodiment, a C-terminal fragment of pill is used in which the cysteine residue at position 201 is deleted or substituted with another amino acid.

Therefore, a further embodiment of the method of the present invention involves operably fusing a nucleic acid encoding one or more human tear lipocalin muteins to a gene encoding the coat protein pill of a fibrous bacteriophage of the M13 family or a fragment of such a coat protein at the 3' end by mutagenesis, so as to screen for at least one mutein that binds to a given ligand.

The fusion protein may comprise other components, such as affinity tags, which allow for the immobilization, detection and/or purification of the fusion protein or a portion thereof. In addition, a stop codon may be located between the sequence region encoding the lipocalin or a mutein thereof and the phage capsid gene or a fragment thereof, wherein said stop codon, such as an amber stop codon, is at least partially translated into amino acids during translation in a suitable suppressor strain.

For example, the phagemid vector pTLPC27, now also referred to as pTlc27, described herein, can be used to prepare a phagemid library encoding human tear lipocalin muteins. Nucleic acid molecules of the present invention encoding tear lipocalin muteins are inserted into the vector using two BstXI restriction sites. After ligation, the resulting nucleic acid mixture is used to transform a suitable host strain, such as E. coli XL1-Blue, to generate a large number of independent clones. If desired, individual vectors can be generated for use in preparing hyperphagemid libraries.

The resulting library is then superinfected in liquid culture with a suitable M13 helper phage or superphage to produce functional phagemids. The recombinant phagemid displays the lipocalin mutein on its surface as a fusion protein with the capsid protein pill or a fragment thereof, typically with the N-terminal signal sequence cleaved. On the other hand, it also possesses one or more copies of the native capsid protein pill provided by the helper phage, enabling infection of a recipient, typically a bacterial strain carrying an F- or F'-plasmid. In the case of superphage display, the superphage displays the lipocalin mutein on its surface as a fusion protein with the infectious capsid protein pill but without the native capsid protein. During or after infection with a helper phage or superphage, gene expression of the fusion protein between the lipocalin mutein and the capsid protein pill can be induced, for example, by the addition of anhydrotetracycline. Induction conditions are selected so that the majority of the phagemids obtained display at least one lipocalin mutein on their surface. In the case of superphage display, induction conditions produce a population of superphagemids carrying three to five fusion proteins consisting of a lipocalin mutein and the capsid protein pill. Various methods are known for isolating phagemids, such as precipitation with polyethylene glycol. Isolation typically occurs after an incubation period of 6-8 hours.

The isolated phagemids can then be screened by incubating with the desired target, wherein the target exists in a form that allows at least temporary fixation of these phagemids, and the phagemids carry mutant proteins with the same desired binding activity as the fusion protein in their capsids. In different embodiments known to those skilled in the art, the target can, for example, be conjugated to a carrier protein such as serum albumin and bound to a protein binding surface, such as polystyrene, through this carrier protein. Microtiter plates or so-called "immunobands" suitable for ELISA technology can, for example, be used for such target fixation. Alternatively, conjugates of the target with other binding groups such as biotin can be used. The target can then be fixed on a surface that selectively binds to this group, such as a microtiter plate or paramagnetic particles coated with streptavidin, neutravidin or avidin. If the target is fused to the Fc portion of an immunoglobulin, fixation can also be achieved using a surface, such as a microtiter plate or paramagnetic particles, which are coated with protein A or protein G.

The non-specific phagemid binding sites present on the surface can be saturated with blocking solutions, as they are known for ELISA methods. The phagemid is then contacted with the target fixed on the surface, usually in the presence of a physiological buffer. Unbound phagemid is removed by multiple washings. The phagemid particles retained on the surface are then washed off. For elution, several methods are feasible. For example, the phagemid can be eluted by adding a protease or in the presence of an acid, alkali, detergent or chaotropic salt or under appropriate denaturing conditions. Such a method is to use a buffer elution at pH 2.2, wherein the eluent is subsequently neutralized. Alternatively, a solution of free target can be added to compete with the fixed target for binding to the phagemid or target-specific phagemid, which can be eluted by competing with an immunoglobulin or a natural ligand protein that specifically binds to the target of interest.

Afterwards, the eluted phagemid is used to infect E. coli cells. Alternatively, nucleic acid can be extracted from the eluted phagemid and then used for sequence analysis, amplification, or transformation of cells in another way. Starting from the E. coli clones obtained in this way, fresh phagemids or superphagemids are again generated by superinfection with M13 helper phage or superphage according to the above method, and the phagemids amplified in this way are then screened again on the fixed target. Multiple screening cycles are generally necessary in order to obtain phagemids with mutant proteins of the present invention in a sufficiently enriched form. In some embodiments, the number of screening cycles is selected so that in subsequent functional analyses, at least 0.1% of the clones under investigation produce mutant proteins with detectable affinity for the target. Depending on the size, that is, the complexity of the library used, 2 to 8 cycles are generally required for this purpose.

For functional analysis of selected muteins, phagemids obtained from the screening cycle are used to infect E. coli strains, and the corresponding double-stranded plasmid DNA is then isolated. Starting from this phagemid DNA, or alternatively from single-stranded DNA extracted from the phagemid, the amino acid sequence of the selected mutein of the present invention can be determined by methods known in the art, from which the amino acid sequence can then be deduced. The mutated region or sequence of the entire tear lipocalin mutein can be subcloned into another expression vector and expressed in a suitable host organism. For example, the vector pTLPC26, now also referred to as pTlc26, can be used for expression in E. coli strains such as E. coli TG1. The tear lipocalin muteins thus produced can be purified by various biochemical methods. For example, tear lipocalin muteins generated using pTlc26 can carry an affinity peptide, a so-called affinity tag, at their C-terminus, allowing purification by affinity chromatography. Examples of affinity tags include, but are not limited to, biotin, Strep-tag, Strep-tag II (Schmidt et al., supra), oligohistidine, polyhistidine, an immunoglobulin domain, maltose binding protein, glutathione-S-transferase (GST), or calmodulin binding peptide (CBP).

Some affinity tags are haptens, such as, but not limited to, dinitrophenol and digoxin. Some affinity tags are epitope tags, such as -peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-Gly), T7 epitope (Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly), maltose binding protein (MBP), herpes simplex virus glycoprotein D sequence Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp HSV epitope The epitope of VSV-G is the sequence of Tyr-Pro-Tyr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Lys, the epitope of E is the sequence of Gly-Ala-Pro-Val-Pro-Tyr-Pro-Asp-Pro-Leu-Glu-Pro-Arg. , E2 epitope tag of the sequence Gly-Val-Ser-Ser-Thr-Ser-Ser-Asp-Phe-Arg-Asp-Arg, mammalian MAPK/ERK kinase C-terminal tag of the sequence Glu-Glu-Thr-Ala-Arg-Phe-Gln-Pro-Gly-Tyr-Arg-Ser-100 epitope tag, sequence Lys-Glu-Thr-Ala-Ala-Ala-Lys-Phe-Glu- The S-tag of Arg-Gln-His-Met-Asp-Ser, the "myc" epitope of the transcription factor c-myc of the sequence Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, and the small V5 epitope present on the P and V proteins of the paramyxovirus simian virus 5 (Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr). In addition, but generally not as a single tag, solubility-enhancing tags such as NusA, thioredoxin (TRX), small ubiquitin-like modifier (SUMO), and ubiquitin (Ub) can be used. Hapten and epitope tags can be used in conjunction with corresponding antibodies or antibody-like proteinaceous molecules as binding partners. The S-peptide epitope of the sequence Lys-Glu-Thr-Ala-Ala-Ala-Lys-Phe-Glu-Arg-Gln-His-Met-Asp-Ser can be used as an epitope tag to be linked to the respective antibody or to be bound to the S-protein as a binding partner (Hackbarth, JS et al., BioTechniques (2004) 37, 5, 835-839).

Screening can also be performed by other methods. Many corresponding embodiments are known to those skilled in the art or are described in the literature. In addition, a combination of methods can be used. For example, clones screened or at least enriched by "phage display" can also be subjected to "colony screening." The advantage of this procedure is that individual clones can be directly isolated for producing tear lipocalin mutants with detectable affinity for the target.

In addition to using E. coli as a host organism in the "phage display" technique or "colony screening" method, other bacterial strains, yeast or insect cells or mammalian cells can also be used for this purpose. In addition to screening for tear lipocalin muteins from random libraries as described above, evolutionary methods, including restriction-dependent mutagenesis, can also be used to optimize muteins that already have a certain binding activity for the target with respect to affinity or specificity for the target after repeated screening cycles.

It will be apparent to the skilled artisan that complex formation depends on many factors, such as the concentration of the binding partner, the presence of competitors, the ionic strength of the buffer system, and the like. Screening and enrichment are typically performed under conditions that allow for the isolation of lipocalin muteins that, when complexed with the desired target, have a dissociation constant of at least 200 nM. However, the washing and elution steps can be performed at varying degrees of stringency. Screening for kinetic characteristics is also feasible. For example, screening can be performed under conditions that favor complex formation between the target and the mutein (which exhibits slow dissociation from the target), or in other words, a low K _off rate. Alternatively, screening can be performed under conditions that favor rapid complex formation between the mutein and the target, or in other words, a high K _on rate. As a further exemplary option, screening can be performed under conditions that select for muteins with improved thermal stability (compared to wild-type tear lipocalin or to muteins that already have affinity for a previously screened target).

Once a mutant protein with affinity for a given target has been screened, it is also possible to subject this mutant protein to further mutagenesis in order to subsequently screen for variants with even higher affinity or variants with improved properties, such as higher thermal stability, improved serum stability, thermodynamic stability, improved solubility, improved monomeric behavior, improved resistance to thermal denaturation, chemical denaturation, proteolysis or detergents, etc. This further mutagenesis, which can be considered as in vitro "affinity maturation" for higher affinity, can be achieved by site-specific mutagenesis based on rational design or random mutagenesis. Another feasible method for obtaining higher affinity or improved properties is to use error-prone PCR, which results in point mutations within a selected range of positions in the lipocalin mutant protein sequence. Error-prone PCR can be performed according to any known protocol, such as the one described by Zaccolo et al. (1996) J. Mol. Biol. 255, 589-603. Other random mutagenesis methods suitable for this purpose include random insertion/deletion (RID) mutagenesis as described by Murakami, H et al. (2002) Nat. Biotechnol. 20, 76-81, or nonhomologous random recombination (NRR) as described by Bittker, J.A et al. (2002) Nat. Biotechnol. 20, 1024-1029. If desired, affinity maturation can also be performed according to the procedures described in WO 00/75308 or Schlehuber, S. et al. (2000) J. Mol. Biol. 297, 1105-1120, in which mutants of the post-bile pigment binding protein with high affinity for digoxin were obtained.

The tear lipocalin muteins of the present invention can be used to form complexes with IL-4 receptor alpha. The muteins can also bind to immunogenic fragments of IL-4 receptor alpha. Immunogenic fragments of IL-4 receptor alpha are fragments that possess one or more epitopes, mimotopes, or other antigenic determinants and are therefore capable of inducing an immune response or generating antibodies against them. Immunogenic fragments can include a single epitope or can have multiple epitopes. Because antigen presentation systems, such as carrier proteins, can be used to provide the desired size for recognition by the immune system, no specific size restrictions apply to immunogenic fragments. Thus, immunogenic fragments can also be "haptens," i.e., fragments that need not be antigenic themselves or can have low immunogenicity, particularly due to their low molecular weight and corresponding size. Generally, immunogenic fragments, alone or when presented on a carrier, can be bound by immunoglobulins, or, if presented via MHC molecules, by T cell receptors (TCRs). Generally, immunogenic fragments, alone or when presented in the form of an antigen presentation system, can elicit a humoral and/or cellular immune response, such as activation of B and/or T lymphocytes.

The target of the muteins of the present invention is the α chain of the transmembrane protein interleukin-4 receptor, which comprises a 207-amino acid extracellular domain. The secreted form of this extracellular domain, sIL-4 receptor α, also known as CD124, blocks IL-4 activity. The muteins of the present invention can bind to sIL-4 receptor α as well as any portion of the extracellular domain of IL-4 receptor α.

In this context, it is also pointed out that the formation of the complex between the respective mutein and its ligand is influenced by many different factors, such as the concentration of the respective binding partner, the presence of competitors, the pH and the ionic strength of the buffer system used, as well as the experimental method used to determine the dissociation constant _K (e.g. fluorescence titration, competition ELISA or surface plasmon resonance, etc.) or even the mathematical algorithm used to evaluate the experimental data.

Therefore, it is also obvious to the skilled person that the _K values given here (dissociation constant of the complex formed between the respective mutein and its ligand) may vary within a certain experimental range, depending on the method and experimental circumstances used to determine the affinity of a particular lipocalin mutein for a given ligand. This means that the measured K _values may have slight deviations or tolerance ranges, depending on, for example, whether the _K values are determined by surface plasmon resonance (Biacore) or by competitive ELISA.

Also included within the scope of the present invention are forms of the above-mentioned muteins, wherein the respective mutein has been altered or modified with respect to its immunogenic potential.

Cytotoxic T cells recognize peptide antigens on the cell surface of antigen presenting cells associated with class I major histocompatibility complex (MHC) molecules. The ability of peptides to bind to MHC molecules has allele specificity and is related to their immunogenicity. In order to reduce the immunogenicity of a given protein, it is very valuable to predict which peptides in the protein have the potential to be bound to the ability of a given MHC molecule. The method of using computational threading methods to identify potential T cell epitopes has been described before, to predict the combination of a given peptide sequence with MHC class I molecules (Altuvia et al. (1995) J. Mol. Biol. 249, 244-250).

This method can also be used to identify the potential t cell epitopes in mutein of the present invention, and depends on its intended use, carries out the selection of specific mutein based on the immunogenicity of its prediction. Further possible is, make the peptide region that has predicted to comprise t cell epitope carry out other mutagenesis, to reduce or eliminate these t cell epitopes and therefore minimize immunogenicity. Described and removed amphoteric epitopes (people such as Mateo (2000) Hybridoma 19,6,463-471) from genetically engineered antibodies, and can be suitable for mutein of the present invention.

The muteins thus obtained may possess minimized immunogenicity, which is ideal for their use in therapeutic and diagnostic applications, such as those described below.

For certain applications, it may also be beneficial to utilize the muteins of the present invention in a labeled form. Thus, the present invention also relates to lipocalin muteins conjugated to a label selected from the group consisting of an enzyme label, a radioactive label, a colored label, a fluorescent label, a chromogenic label, a luminescent label, a hapten, digoxigenin, biotin, a metal complex, a metal, and colloidal gold. The muteins may also be conjugated to a low molecular weight organic compound. As used herein, the term "low molecular weight organic compound" refers to a monomeric carbon-based compound that may have aliphatic, alicyclic, and/or aromatic moieties. In typical embodiments, a low molecular weight organic compound is an organic compound having a backbone of at least two carbon atoms, and in some embodiments, having no more than 7 or 12 rotatable carbon bonds. Such compounds have a molecular weight ranging from 100 to about 2000 Daltons, such as from about 100 to about 1000 Daltons. They may optionally contain one or two metal atoms.

In general, the lipocalin muteins can be labeled with any suitable chemical or enzyme that directly or indirectly generates a compound or signal detectable in a chemical, physical, optical, or enzymatic reaction. Examples of physical reactions (also optical reactions/labels) are the emission of fluorescence upon irradiation or the emission of X-rays when a radioactive label is used. Alkaline phosphatase, horseradish peroxidase, or β-galactosidase are examples of enzyme labels (also optical labels) that catalyze the formation of a chromogenic reaction product. In general, all labels commonly used for antibodies (except those that can only be used with carbohydrate moieties in the Fc portion of immunoglobulins) can also be conjugated to the muteins of the present invention. The muteins of the present invention can also be conjugated to any suitable therapeutic agent, for example, for targeted delivery of such agents to a given cell, tissue, or organ or for selective targeting of cells, such as tumor cells, without affecting surrounding normal cells. Examples of such therapeutic agents include radionuclides, toxins, small organic molecules, and therapeutic peptides (e.g., peptides that act as agonists/antagonists of cell surface receptors or peptides that compete for protein binding sites on a given cellular target). However, the lipocalin muteins of the present invention can also be conjugated to therapeutically active nucleic acids, such as antisense nucleic acid molecules, small interfering RNA, microRNA or ribozymes. Such conjugates can be produced by methods well known in the art.

In one embodiment, the mutein of the present invention can also be coupled to the targeting moiety of the specific human body region of the target, so that the mutein of the present invention is delivered to the desired region or range in vivo. An example of such modification may be required for passing through the blood-brain barrier. In order to pass through the blood-brain barrier, the mutein of the present invention can be coupled to the part that promotes active transport through this barrier (see Gaillard PJ et al., Diphtheria-toxin receptor-targeted brain drug delivery.International Congress Series, 2005 1277,185-198 or Gaillard PJ et al. .Targeted delivery across the blood-brain barrier.Expert Opin Drug Deliv.2005 2,2,299-309. This part can, for example, be obtained with trademark 2B-Trans ^TM (to-BBB technologies BV, Leiden, NL).

As mentioned above, the muteins of the present invention can, in some embodiments, be conjugated to a moiety that extends the serum half-life of the mutein (see also PCT Publication WO 2006/56464, which describes this conjugation strategy with reference to a human neutrophil gelatinase-associated lipocalin mutein with binding affinity for CTLA-4). The serum half-life-extending moiety can be a polyethylene glycol molecule, hydroxyethyl starch, a fatty acid molecule such as palmitic acid (Vajo & Duckworth 2000, Pharmacol. Rev. 52, 1-9), an Fc portion of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, albumin or a fragment thereof, an albumin-binding peptide or protein, transferrin, to name a few. The albumin-binding protein can be a bacterial albumin-binding protein, an antibody, an antibody fragment (including a domain antibody, see, for example, U.S. Patent No. 6,696,245), or a lipocalin mutein with binding activity for albumin. Accordingly, suitable conjugation partners for extending the half-life of the lipocalin muteins of the invention include albumin (Osborn, BL et al., 2002, J. Pharmacol. Exp. Ther. 303, 540-548), or albumin binding proteins, such as bacterial albumin binding domains, for example a streptococcal protein G (T. and Skerra, A. (1998) J. Immunol. Methods 218, 73-83). Other examples of albumin binding peptides that can be used as conjugation partners are those having the consensus sequence Cys-Xaa ₁ -Xaa ₂ -Xaa ₃ -Xaa ₄ -Cys, wherein Xaa ₁ is Asp, Asn, Ser, Thr or Trp; Xaa ₂ is Asn, Gln, His, Ile, Leu or Lys; Xaa ₃ is Ala, Asp, Phe, Trp or Tyr; and Xaa ₄ is Asp, Gly, Leu, Phe, Ser or Thr, as described in U.S. Patent Application 2003/0069395 or in Dennis et al. (Dennis, MS, Zhang, M., Meng, YG, Kadkhodayan, M., Kirchhofer, D., Combs, D. & Damico, LA (2002) J Biol Chem 277,35035-35043).

In other embodiments, albumin itself or biologically active fragments of albumin can be used as conjugation partners for the lipocalin muteins of the present invention. The term "albumin" includes all mammalian albumins, such as human serum albumin, bovine serum albumin, or rat albumin. Albumin or fragments thereof can be produced recombinantly, as described in U.S. Patent No. 5,728,553 or European Patent Applications EP 0 330 451 and EP 0 361 991. Recombinant human albumin (Novozymes Delta Ltd., Nottingham, UK) can be conjugated or fused to lipocalin muteins to extend the half-life of the muteins.

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

Transferrins, iron, and ferritin are used as the part of extending the serum half-life of mutein of the present invention, and described mutein can be fused to the N or C end or both of non-glycosylated transferrins, iron, and ferritin in a genetically engineered manner. The half-life of non-glycosylated transferrins, iron, and ferritin is 14-17 days, and transferrin fusion protein will similarly have the half-life of extension. Transferrins, iron, and ferritin carriers also provide high bioavailability, biodistribution and circulatory stability. This technology is commercially available from BioRexis (BioRexis Pharmaceutical Corporation, PA, USA). Recombinant human transferrins, iron, and ferritin (DeltaFerrin ^™ ) as a protein stabilizer/half-life extension partner is also commercially available from Novozymes Delta Ltd. (Nottingham, UK).

If the Fc portion of an immunoglobulin is used for the purpose of extending the serum half-life of a mutein of the present invention, SynFusion ^™ technology commercially available from Syntonix Pharmaceuticals, Inc. (MA, USA) can be used. The use of this Fc-fusion technology allows the production of longer-acting biopharmaceuticals and can, for example, consist of two copies of a mutein linked to the Fc region of an antibody to improve pharmacokinetics, solubility, and production efficiency.

Another option for extending the half-life of a mutant protein of the present invention is to fuse a long, unstructured, flexible, glycine-rich sequence (e.g., a polyglycine sequence having about 20 to 80 consecutive glycine residues) to the N or C-terminus of a mutant protein of the present invention. This approach is, for example, disclosed in WO 2007/038619 and is also referred to as "rPEG" (recombinant PEG).

In some embodiments, the polyalkylene glycol is a substituted, unsubstituted, linear or branched polyalkylene glycol. The polyalkylene glycol can be a substituted, unsubstituted, linear or branched polyalkylene glycol. It can also be an activated polyalkylene glycol derivative. Examples of suitable compounds include polyethylene glycol (PEG) molecules, such as those described in WO 99/64016, United States Patent (USP) 6,177,074 or United States Patent (USP) 6,403,564 relating to interferon, or asparaginase, PEG-adenosine deaminase (PEG-ADA) or PEG-superoxide dismutase modified for other proteins such as PEG- (e.g., see Fuertges et al. (1990) The Clinical Efficacy of Poly (Ethylene Glycol) -Modified Proteins J. Control. Release 11, 139-148). The molecular weight of such polymers, such as polyethylene glycol, can vary within the range of from about 300 to about 70,000 daltons, including, for example, polyethylene glycol having a molecular weight of about 10,000, about 20,000, about 30,000, or about 40,000 daltons. In addition, carbohydrate oligosaccharides and polymers such as starch or hydroxyethyl starch (HES) can be conjugated to the muteins of the invention to extend serum half-life, for example as described in U.S. Patent Nos. 6,500,930 or 6,620,413.

If one of the above-mentioned moieties is conjugated to the human tear lipocalin mutein of the present invention, conjugation to an amino acid side chain may be advantageous. Suitable amino acid side chains may be naturally present in the amino acid sequence of human tear lipocalin or may be introduced by mutagenesis. In the case of introducing a suitable binding site via mutagenesis, one feasible approach is to replace the amino acid with a cysteine residue at the appropriate position. In one embodiment, the mutation comprises at least one of the following: Thr 40→Cys, Glu 73→Cys, Arg 90→Cys, Asp 95→Cys, or Glu 131→Cys substitutions. The newly created cysteine residue at any of these positions can then be used to conjugate the mutein to a moiety that extends the serum half-life of the mutein, such as PEG or an activated derivative thereof.

In another embodiment, in order to provide for one of the above-mentioned parts being conjugated to the suitable amino acid side chain of the mutant protein of the present invention, artificial amino acid can be introduced by mutagenesis. Generally, such artificial amino acid can be designed to be more reactive, thus promoting conjugation with the desired part. An example of such artificial amino acid that can be introduced via artificial tRNA is p-acetyl-phenylalanine.

For several applications of the muteins disclosed herein, it may be beneficial to use them in the form of fusion proteins. In some embodiments, the human tear lipocalin muteins of the present invention are fused at their N-termini and their C-termini to a protein, protein domain or peptide, such as a signal sequence and/or affinity tag.

For pharmaceutical applications, the muteins of the invention can be fused to a fusion partner that increases the serum half-life of the mutein in vivo (see again PCT application WO 2006/56464, which describes suitable fusion partners with reference to human neutrophil gelatinase-associated lipocalin muteins that have binding affinity for CTLA-4). Similar to the conjugates described above, the fusion partner can be an Fc portion of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, albumin, an albumin-binding peptide or protein, transferrin, to name a few. Again, the albumin-binding protein can be a bacterial albumin-binding protein or a lipocalin mutein that has binding activity for albumin. Thus, suitable fusion partners for extending the half-life of the lipocalin muteins of the invention include albumin (Osborn, BL et al. (2002) J. Pharmacol. Exp. Ther. 303, 540-548, supra), or an albumin binding protein, e.g., a bacterial albumin binding domain, such as a streptococcal protein G (Osborn, BL et al. (2002) J. Pharmacol. Exp. Ther. 303, 540-548, supra), or an albumin binding protein, e.g., a bacterial albumin binding domain, e.g., a streptococcal protein G (Osborn, BL et al. (1998) J. Immunol. Methods 218, 73-83). Albumin-binding peptides described in Dennis et al. (2002) or U.S. Patent Application 2003/0069395 above, having a consensus sequence of Cys- _Xaa1 - _Xaa2 - _Xaa3 - _Xaa4 -Cys, wherein _Xaa1 is Asp, Asn, Ser, Thr, or Trp; _Xaa2 is Asn, Gln, His, Ile, Leu, or Lys; _Xaa3 is Ala, Asp, Phe, Trp, or Tyr; and _Xaa4 is Asp, Gly, Leu, Phe, Ser, or Thr. Albumin itself or biologically active fragments of albumin can also be used as fusion partners for the lipocalin muteins of the present invention. The term "albumin" includes all mammalian albumins, such as human serum albumin, bovine serum albumin, or rat serum albumin. The recombinant production of albumin or fragments thereof is well known in the art and is described, for example, in US Pat. No. 5,728,553, European Patent Applications EP 0 330 451 and EP 0 361 991.

Fusion partners can impart novel properties to the lipocalin muteins of the invention, such as enzymatic activity or binding affinity for other molecules. Examples of suitable fusion proteins are alkaline phosphatase, horseradish peroxidase, glutathione-S-transferase, the albumin binding domain of protein G, protein A, antibody fragments, oligomerization domains, lipocalin muteins with the same or different binding specificities (this results in the formation of "duocalins", see Schlehuber, S. and Skerra, A. (2001), Duocalins, engineered ligand-binding proteins with dual specificity derived from the lipocalin fold. Biol. Chem. 382, 1335-1342) or toxins.

In particular, the lipocalin muteins of the present invention can be fused to separate enzyme active sites so that the two "parts" of the resulting fusion protein act together on a given therapeutic target. The binding domain of the lipocalin mutein is linked to the pathogenic target, thereby allowing the enzyme domain to prevent the biological function of the target.

Affinity tags such as FLAG or FLAG II (Schmidt, TGM et al. (1996) J. Mol. Biol. 255, 753-766), myc-tags, FLAG-tags, His ₆ -tags or HA-tags or proteins such as glutathione-S-transferase, which also allow easy detection and/or purification of recombinant proteins, are further examples of suitable fusion partners. Finally, proteins with chromogenic or fluorescent properties such as green fluorescent protein (GFP) or yellow fluorescent protein (YFP) are also suitable fusion partners for the lipocalin muteins according to the invention.

As used herein, the term "fusion protein" also includes lipocalin muteins according to the present invention that contain a signal sequence. The signal sequence at the N-terminus of the polypeptide directs the polypeptide to a specific cellular compartment, such as the periplasm of E. coli or the endoplasmic reticulum of eukaryotic cells. A large number of signal sequences are known in the art. An exemplary signal sequence for secreting a polypeptide into the periplasm of E. coli is the OmpA signal sequence.

The present invention also relates to nucleic acid molecules (DNA and RNA) comprising nucleotide sequences encoding mutant proteins as described herein. Because the degeneracy of the genetic code allows other codons specifying the same amino acid to replace certain codons, the present invention is not limited to the specific nucleic acid molecules encoding the mutant proteins of the present invention, but rather encompasses all nucleic acid molecules comprising nucleotide sequences encoding functional mutant proteins.

Therefore, the present invention also includes a nucleic acid sequence encoding a mutant protein according to the present invention, which has a mutation at at least one codon at any one of amino acid sequence positions 26-34, 56-58, 80, 83, 104-106 and 108 of the native mature human tear lipocalin linear polypeptide sequence, wherein the codons encoding at least one cysteine residue at sequence positions 61 and 153 of the mature human tear lipocalin linear polypeptide sequence have been mutated to encode any other amino acid residue.

The invention described herein also includes nucleic acid molecules encoding tear lipocalin muteins that include additional mutations beyond the indicated sequence positions that were experimentally mutagenized. Such mutations are often tolerated or may even prove beneficial, for example, if they contribute to improving the folding efficiency, serum stability, thermal stability, or ligand binding affinity of the mutein.

The nucleic acid molecules disclosed herein may be "operably linked" to regulatory sequences to allow for expression of the nucleic acid molecule.

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" or "capable of permitting expression of a nucleotide sequence" if it includes sequence elements that contain information for transcriptional and/or translational regulation, and such sequences are "operably linked" to a nucleotide sequence encoding a polypeptide. An operable linkage is one in which the regulatory sequence elements and the sequence to be expressed are linked in such a way that the gene is expressed. The exact nature of the regulatory regions required for gene expression may vary between species, but generally these regions include promoters, which in prokaryotes contain both the promoter itself (i.e., the DNA element that directs the initiation of transcription) and a DNA element that signals the initiation of translation when transcribed into RNA. Such promoter regions typically include 5' non-coding sequences involved in the initiation of transcription and translation, such as the -35/-10 box, the Shine-Dalgarno element or the TATA box in prokaryotes, CAAT sequences, and 5'-capping elements in eukaryotes. These regions may also include enhancer or repressor elements, as well as translation signals and leader sequences for targeting the native polypeptide to a specific compartment of the host cell.

Furthermore, the 3' non-coding sequences may contain regulatory elements involved in transcription termination, polyadenylation, etc. However, if these termination sequences do not function satisfactorily in a particular host cell, they may be replaced by signals that are functional in that cell.

Therefore, nucleic acid molecules of the present invention can include regulatory sequences, such as promoter sequences. In some embodiments, nucleic acid molecules of the present invention include promoter sequences and transcription termination sequences. Suitable prokaryotic promoters, for example, tet promoters, lacUV5 promoters or T7 promoters. Examples of promoters that can be used for expression in eukaryotic cells include SV40 promoters or CMV promoters.

The nucleic acid molecule of the invention may also be part of a vector or any other type of cloning vehicle, such as a plasmid, phagemid, phage, baculovirus, cosmid or artificial chromosome.

In one embodiment, the nucleic acid molecule is contained in a phagemid. A phagemid vector represents a vector encoding an intergenic region of a temperate phage such as M13 or f1, or a functional portion thereof fused to a target cDNA. After superinfection of a bacterial host cell with such a phagemid vector and an appropriate helper phage (e.g., M13K07, VCS-M13, or R408), a complete phage particle is generated, thereby physically coupling the encoded heterologous cDNA to its corresponding polypeptide displayed on the phage surface (e.g., see Lowman, H.B. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 401-424, or Rodi, D.J. and Makowski, L. (1999) Curr. Opin. Biotechnol. 10, 87-93).

In addition to the above-mentioned regulatory sequences and the nucleic acid sequence encoding the lipocalin mutein of the present invention, such cloning vectors may include replication and control sequences derived from species compatible with the host cell used for expression, as well as a selectable marker that confers a selectable phenotype on the transformed or transfected cells. A large number of suitable cloning vectors are known in the art and are commercially available.

DNA molecules encoding the lipocalin muteins of the present invention, in particular cloning vectors comprising the coding sequence for such lipocalin muteins, can be transformed into host cells capable of expressing the gene. Transformation can be performed using standard techniques. Thus, the present invention also relates to host cells comprising the nucleic acid molecules disclosed herein.

The transformed host cell is cultured under conditions suitable for the nucleotide sequence encoding the fusion protein of the present invention. Suitable host cells can be prokaryotic, such as Escherichia coli or Bacillus subtilis, or eukaryotic, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells, immortalized mammalian cell lines (such as HeLa cells or CHO cells) or primary mammalian cells.

The present invention also relates to a method for producing a mutein according to the present invention, wherein the mutein, a fragment of the mutein, or a fusion protein of the mutein with another polypeptide is produced by genetic engineering methods starting from a nucleic acid encoding the mutein. The method can be performed in vivo, for example, the mutein can be produced in a bacterial or eukaryotic host organism and then isolated from the host organism or a culture thereof. Proteins can also be produced in vitro, for example, by using an in vitro translation system.

When producing mutans in vivo, the nucleic acid encoding mutans of the present invention is introduced into suitable bacteria and eukaryotic host organisms (as listed above) by recombinant DNA technology. For this purpose, first, a cloning vector comprising the nucleic acid molecule encoding mutans of the present invention is used to transform the host cell with the standard method of having established. The host cell is then cultivated under the conditions of allowing exogenous DNA to express and then synthesize the corresponding polypeptide. Subsequently, the polypeptide is reclaimed from the cell or from the culture medium.

In some tear lipocalin muteins of the present invention, the naturally occurring disulfide bond between Cys 61 and Cys 153 is removed. Thus, such muteins (or any other tear lipocalin muteins that do not include an intramolecular disulfide bond) can be produced in cellular compartments with a reduced redox environment, such as the cytoplasm of Gram-negative bacteria. Where the lipocalin muteins of the present invention include an intramolecular disulfide bond, it may be desirable to direct the native polypeptide to a cellular compartment with a redox environment using an appropriate signal sequence. This oxidizing environment can be provided by the periplasm of Gram-negative bacteria such as Escherichia coli, in the extracellular environment of Gram-positive bacteria, or within the lumen of the endoplasmic reticulum of eukaryotic cells, and generally favors the formation of structural disulfide bonds. However, the muteins of the present invention can also be produced in the cytosol of host cells such as E. coli. In such cases, the polypeptide can be obtained directly in a soluble and folded state or recovered as inclusion bodies and then renatured in vitro. Another option is to use specific host strains with an oxidative intracellular environment, which may thus allow the formation of disulfide bonds in the cytosol (Venturi M et al. (2002) J. Mol. Biol. 315, 1-8).

However, the muteins of the present invention need not necessarily be generated or produced solely by using genetic engineering. Instead, lipocalin muteins can also be obtained by chemical synthesis, such as Merrifield solid-phase peptide synthesis, or by in vivo transcription and translation. For example, it is feasible that promising mutations are identified using molecular modeling, and then the desired (designed) polypeptide is synthesized in vitro and tested for binding affinity to a given target. Methods for solid-phase and/or liquid-phase protein synthesis are well known in the art (e.g., see Bruckdorfer, T. et al. (2004) Curr. Pharm. Biotechnol. 5, 29-43).

In another embodiment, the muteins of the present invention can be produced by in vitro transcription/translation using established methods known to those skilled in the art.

The present invention also relates to a pharmaceutical composition comprising at least one human tear lipocalin mutant protein or its fusion protein or conjugate according to the present invention and a pharmaceutically acceptable excipient.

The lipocalin mutants according to the present invention can be administered via parenteral or non-parenteral (enteral) routes that are therapeutically effective for proteinaceous drugs. The mutants of the present invention are administered systemically or locally in formulations containing conventional non-toxic pharmaceutically acceptable excipients or carriers, such as desired additives and solvents.

In one embodiment of the present invention, the drug is administered parenterally to a mammal, particularly a human. The pharmaceutical composition may be an aqueous solution, an oil-in-water emulsion, or a water-in-oil emulsion.

In this regard, it should be noted that transdermal delivery techniques such as iontophoresis, ultrasound permeation, or microneedle-facilitated delivery, as described in Meidan VM and Michniak BB (2004) Am. J. Ther. 11, 4, 312-316, can also be used for transdermal delivery of the mutant proteins described herein. The mutant proteins of the present invention can be delivered systemically or locally in formulations containing a variety of conventional non-toxic pharmaceutically acceptable excipients or carriers, additives, and solvents.

The dose of the administered mutant protein can be varied within wide limits to achieve the desired preventive effect or therapeutic response. This will depend, for example, on the affinity of the compound for the selected ligand and the in vivo half-life of the complex between the mutant protein and the ligand. In addition, the ideal dose will depend on the biodistribution of the mutant protein or its fusion protein or its conjugate, the mode of administration, the severity of the disease/disorder to be treated, and the patient's medical condition. For example, when used in a topical ointment, a high concentration of the tear lipocalin mutant protein can be used. However, if desired, the mutant protein can also be provided in a sustained release formulation, such as a liposomal dispersion or a hydrogel-based polymer microsphere, such as PolyActive ^™ or OctoDEX ^™ (see Bos et al., Business Briefing: Pharmatech 2003: 1-6). Other sustained release formulations available are, for example, PLGA-based polymers (PR pharmaceuticals), PLA-PEG-based hydrogels (Medincell), and PEA-based polymers (Medivas).

Therefore, the mutant proteins of the present invention can be formulated into compositions using pharmaceutically acceptable ingredients and established preparation methods. The pharmaceutical compositions may also contain additives such as fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers, and other solvents or cosolvents or formulations for achieving sustained efficacy. In the latter case, the fusion protein can be incorporated into slow or sustained-release or targeted delivery systems such as liposomes and microcapsules.

The formulations can be sterilized by various means, including filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile medium immediately before use.

The lipocalin muteins described herein can be administered to an organism, including a human patient, per se, or in a pharmaceutical composition, which can include or be admixed with a pharmaceutically active ingredient or a suitable carrier or excipient. The formulation and administration techniques for each lipocalin mutein composition are similar to or identical to those established in the art for low molecular weight compounds. Exemplary routes include, but are not limited to, oral, transdermal, and parenteral delivery.

Compositions comprising the lipocalin muteins of the invention can, for example, be applied to the skin or applied to a wound. In some embodiments, the lipocalin muteins or respective compositions can be administered in a local rather than systemic manner, for example, by injection.

Pharmaceutical compositions comprising the lipocalin muteins of the invention can be prepared in a manner known per se, for example, by conventional mixing, dissolving, granulating, sugar-coating, grinding, emulsifying, encapsulating, fixing or lyophilizing processes. Pharmaceutical compositions for use according to the invention can therefore be prepared by conventional means using one or more physiologically acceptable carriers (including excipients and adjuvants that facilitate processing of the hydrogel and/or peptide/peptoid into a pharmaceutically acceptable formulation). The appropriate formulation depends on the chosen route of administration.

For injection, the lipocalin muteins or respective compositions can be formulated in aqueous solutions, for example, in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used for the formulation. Such penetrants are conventionally known in the art.

For oral administration, the lipocalin muteins or respective compositions can be readily prepared by combining them with pharmaceutically acceptable carriers well known in the art. Such carriers enable the lipocalin muteins or respective compositions and the pharmaceutically active compound (if present) to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient to be treated. Pharmaceutical preparations for oral use can be obtained by adding solid excipients, optionally grinding the resulting mixture, and treating the granulated mixture (after adding suitable adjuvants, if desired) to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.

The dragee cores are provided with a suitable coating. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carboxyvinyl polymer gel, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablet or dragee coating to identify or indicate different combinations of active ingredients.

Pharmaceutical formulations that can be used orally include push-fit capsules made of gelatin and soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredient in admixture with a filler, such as lactose, a binder, such as starch, and/or a lubricant, such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the peptide/peptoid can be suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers can be added. All formulations for oral administration should be available in a dosage suitable for such administration.

Lipocalin muteins can be formulated for parenteral administration by injection, for example, by intramuscular injection or bolus injection or continuous infusion. Injectable formulations can be presented in unit dosage form, for example, in ampoules or multi-dose containers, with added preservatives. The respective compositions can take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can include formulation agents such as suspended, stabilized, and/or dispersed formulations.

A subject in need of such treatment can be a mammal, such as a human, dog, mouse, rat, pig, or ape, for example, cymologous, to name a few illustrative examples. The muteins of the present invention can be used to treat any disease or disorder involving the 11-4 receptor alpha, in which the expression products of the nucleic acid library of the present invention or a separately obtained tear lipocalin mutein are expressed in the progression of the disease or disorder.

It is shown herein that a variety of tumor cells express much more high-affinity IL-4 receptors than normal cells. Such cells include human solid tumors such as melanoma, breast cancer, ovarian cancer, mesothelioma, glioblastoma, astrocytoma, renal cell carcinoma, head and neck cancer, AIDS-related Kaposi's sarcoma = AIDS KS, hormone-dependent and -independent prostate cancer cells, and primary cultures of prostate tumors (see, for example, Garland, L et al. (2005) Journal of Immunotherapy 28, 4, 376-381, Rand, RW et al. Clinical Cancer Research. (2000) 6, 2157-2165; Husain SR et al. (1999) Nature Medicine 5, 817-822; Puri RK et al. Cancer Research (1996) 56, 5631-5637; Debinski W et al. or Husain SR et al. Cancer Research (1998) 58, 3649-3653, Kawakami K et al. (2000) Cancer Research, 60, 2981-2987; or Strome SE et al. Clinical Cancer Research (2002) 8, 281-286. Specific examples of cells in which IL-4 receptor overexpression is documented include, but are not limited to, Burkitt lymphoma cell line Jijoye (B cell lymphoma), prostate cancer (LNCaP, DU145), head and neck cancer (SCC, KCCT873), pancreatic cancer (PANC-1 cell line), SCC-25: 13000 (+/- 500) h head and neck cancer cell line (ATCC). IL4 receptor α chain It plays an important role in IL4 internalization. Therefore, when fused or conjugated to a toxin, the tear lipocalin mutant protein that binds to the IL-4 receptor α chain can also be used to treat tumors (cancer). Examples of suitable toxins include Pseudomonas exotoxin, pertussis toxin, diphtheria toxin, ricin, saporin, Pseudomonas exotoxin, calicheamicin or its derivatives, taxanes, maytansine, tubulysin and auristatin analogs. Examples of auristatin analogs include, but are not limited to, auristatin E, monomethylauristatin E, auristatin PYE and auristatin PHE.

For the treatment of cancer, mutant proteins that bind to the IL-4 receptor alpha chain can also be conjugated to cytostatics. Examples of such cytostatics include cisplatin, carboplatin, oxaliplatin, pentafluorouracil, taxotere (docetaxel), paclitaxel, anthracyclines (adriamycin), methotrexate, vinblastine, vincristine, vindesine, vinorelbine, dacarbazine, cyclophosphamide, etoposide, adriamycin, Camptotecine, Combretatastin A-4 related compounds, sulfonamides, oxadiazolins, benzo[b]thiophene synthetic spiroketal pyrans, monotetrahydrofuran compounds, curacin and curacin derivatives, methoxyestradiol derivatives, and folinic acid.

It is obvious from the above disclosure that the mutant protein or fusion protein or its conjugate of the present invention can be used in many applications. Generally speaking, such mutant protein can be used in all applications using antibodies, except those that specifically rely on glycosylation of the Fc portion.

Therefore, in another aspect of the present invention, the human tear lipocalin mutant protein is used to detect a given non-natural ligand of human tear lipocalin. This use may include the steps of contacting the mutant protein with a sample suspected of containing the given ligand under suitable conditions, and detecting the complexed mutant protein via a suitable signal.

The detectable signal can be caused by labeling, as explained above, or by a change in a physical property due to the binding itself (i.e. complex formation). An example is surface plasmon resonance, the value of which changes during binding of binding partners, one of which is immobilized on a surface such as gold foil.

The human tear lipocalin muteins disclosed herein can also be used to isolate a given non-natural ligand of human tear lipocalin. Such use may include the steps of contacting the mutein with a sample presumed to contain the ligand under suitable conditions, thereby allowing a complex to form between the mutein and the given ligand, and isolating the mutein/ligand complex from the sample.

In both the use of the mutein to detect a given non-natural ligand and in the isolation of a given ligand, the mutein and/or the target may be immobilized on a suitable solid phase.

The human tear lipocalin mutants of the present invention can also be used to target compounds to preselected sites. For such purposes, the mutant protein and the target compound are contacted to allow complex formation. The complex comprising the mutant protein and the target compound is then delivered to the preselected site. This use is particularly suitable for, but not limited to, (selectively) delivering drugs to preselected sites in an organism, such as infected body parts, tissues or organs that are believed to be treated with the drug. In addition to forming a complex between the mutant protein and the target compound, the mutant protein can also react with a given compound to produce a conjugate of the mutant protein and the compound. Similar to the complex above, this conjugate can be suitable for delivering the compound to a preselected target site. This conjugate of the mutant protein and the compound can also include a linker that covalently links the mutant protein and the compound to each other. Optionally, this linker is stable in the bloodstream but cleavable in the cellular environment.

Muten disclosed herein and derivatives thereof can therefore be similar to antibodies or their fragments and be used in many fields.Except that they are attached to the purposes of support (allowing the target of given muten or conjugate or the fusion protein of this target to fix or separate), muten can be used for labeling together with enzyme, antibody, radioactive substance or any other group with biochemical activity or definite binding characteristic.By doing so, their respective targets or their conjugates or fusion proteins can be detected or contacted with them.For example, muten of the present invention can be used to detect chemical structure by established analytical method (such as ELISA or Western blotting) or by microscope or immune perception (immunosensorics).Here, detection signal can be directly generated by using suitable muten conjugate or fusion protein or indirectly generated by immunochemical detection via the combination muten of antibody.

Numerous possible applications of the muteins of the present invention also exist in medicine. In addition to their use in diagnosis and drug delivery, mutant polypeptides of the present invention can be generated that bind, for example, tissue- or tumor-specific surface molecules. Such muteins can, for example, be used in conjugated form or as fusion proteins for "tumor imaging" or directly for cancer treatment. Therefore, the present invention provides a diagnostic composition comprising the muteins of the invention and diagnostic means, such as excipients, buffers, and labels (which can be used to label the muteins).

Therefore, the present invention also relates to the use of the human tear lipocalin mutein of the present invention to form a complex with a given non-natural ligand.

Another related use of the mutant proteins described herein is target confirmation, that is, analyzing whether the polypeptides assumed to be involved in the development or process of a disease or disorder have actually induced this disease or disorder to some extent. This is used to verify that the purposes of the protein as a pharmacological drug target utilizes the ability of the mutant proteins of the present invention to specifically recognize the surface area of the native conformation protein, for example, to be bound to the native epitope. In this respect, it should be noted that this ability has only been reported for a limited number of recombinant antibodies. However, the purposes of the mutant proteins of the invention for the confirmation of drug targets are not limited to the detection of proteins as targets, but also include the detection of protein domains, peptides, nucleic acid molecules, organic molecules or metal complexes.

Exemplary embodiments of the present invention

Figure 1 shows the primary structure of a previously described human tear lipocalin mutant (S191.4-B24) that exhibits binding affinity for IL-4 receptor alpha. The first 21 residues (underlined) constitute the signal sequence, which is cleaved after periplasmic expression. The N-terminal T7 tag (italicized) and C-terminal Streptag-II (bold) are part of the characterized protein. Figure 1 also shows that the four N-terminal amino acid residues (His1, His2, Leu3, Ala4) and the last two C-terminal amino acid residues (Ser157 and Asp158) of wild-type tear lipocalin are missing from this mutant.

Figure 2 shows the polypeptide sequences of exemplary mutant proteins with high affinity for IL-4 receptor α (SEQ ID Nos. 2-11). The numbers indicated by 'SwissProt P31025' indicate the corresponding amino acid position numbers of the unprocessed precursor sequence from the SwissProt database entry. Wild-type TLc26 corresponds substantially to the wild-type tear lipocalin sequence in the vector pTLc2. However, wild-type TLc26 does not include disulfide bonds because the cysteine residues at positions 61 and 153 of the mature protein are replaced by serine residues. Similarly, the serine residue at position 101 of the mature protein is replaced by a serine residue. In addition, the arginine residue at position 111 of the mature protein is replaced by a proline residue, and the lysine residue at position 114 of the mature protein is replaced by a tryptophan residue. Furthermore, the two C-terminal amino acids included in the sequence of SwissProt entry P31025 are not included in the sequence. AB4004 is a random library disclosed in International Patent Application WO 2008/015239, where the mutation positions are indicated in bold. The randomized sequence was identical to J14 except for positions 53 and 55. M3-B24 (PSM) indicates the hotspot from PSM B24.

Figure 3 shows the results of a TF-1 cell proliferation assay. TF-1 cells were incubated with serial dilutions of the indicated mutant proteins of the invention (S276.2K04, S308.5F08, S308.5N01, S308.5L20, S308.5L04, S308.5N20, S308.3O10, S191.4B24 [SEQ ID Nos: 2-11]) at 37°C for 1 hour, followed by the addition of 0.8 ng/ml IL-4 (A) or 12 ng/ml IL-13 (B) for 72 hours. Proliferation was measured using ^3H -thymidine incorporation.

Figure 4 depicts the _IC50 values from Figure 3 and Biacore measurements of human tear lipocalin muteins with affinity for IL-4 receptor α. Approximately 400 RU of IL-4 receptor α-Fc was captured onto a CM-5 chip pre-coated with anti-human Fc monoclonal antibody. Subsequently, a single concentration of 25 nM of the mutein was passed through the flow cell, and the change in resonance units was recorded. The baseline signal from an identically treated flow cell, except for the absence of any IL-4 receptor α-Fc, was subtracted, and the resulting data were fitted to a 1:1 Langmuir model using BIAevaluation software. Due to slow dissociation, double-referenced kinetics were used by subtracting the signal from an identically treated flow cell, except for the absence of any IL-4 receptor α-Fc, as well as the signal from an experiment in which only sample buffer was injected. The resulting data were fitted to a 1:1 Langmuir model with mass transport restrictions using BIAevaluation software.

Unless otherwise stated, established methods in the field of recombinant gene technology were used.

Example

Example 1: Affinity maturation of mutant protein S191.4-B24 using a site-directed randomization approach

A variant library based on the mutant protein S191.4-B24 (SEQ ID NO: 2) described in PCT application WO 2008/015239 was designed by randomizing positions 26, 32, 34, 55, 56, 58 and 63 to allow for all 20 amino acids at these positions. The library was constructed essentially as described in Example 1 of WO 2008/015239.

Phagemid screening was performed as described in Example 2 of WO2008/015239, using a competing monoclonal antibody against IL-4 receptor α (MAB230, R&D Systems; 1 hour wash), using limited target concentrations (0.5 nM and 0.1 nM IL-4 receptor α, Peprotech) in combination with extended wash times or short incubation times (10 minutes), respectively. Three or four rounds of screening were performed.

For the production of IL-4 receptor α-specific mutant proteins, Escherichia coli K12 strain JM83 was used, which carries the corresponding mutant protein encoded on the expression vector pTLPC10 (SEQ ID No: 1), or when larger amounts of protein are required, Escherichia coli strain W3110 is used, which carries the corresponding expression vector as described in WO2008/015239.

Example 2: Affinity determination using Biacore

Affinity measurements were performed essentially as described in Example 9 of WO2006/56464, with the modifications that approximately 400 RU of IL-4 receptor α-Fc (R&D Systems) were immobilized (instead of 2000 RU of human CTLA-4 or murine CTLA-4-Fc used as target in WO2006/56464) and 100 μl of the mutein at a concentration of 25 nM was injected (instead of 40 μl of the sample-purified lipocalin mutein at a concentration of 5-0.3 μM used in WO2006/56464).

Example 3: TF-1 cell proliferation assay

IL-4 and IL-13 stimulated TF-1 cell proliferation assays were performed essentially as described in Lefort et al. (Lefort S. et al. (1995) FEBS Lett. 366, 2-3, 122-126) and in Example 10 of PCT application WO 2008/015239. TF-1 cells were incubated with serial dilutions of the indicated mutant proteins of the invention (S276.2K04, S308.5F08, S308.5N01, S308.5L20, S308.5L04, S308.5N20, S308.3O10, S191.4B24 [SEQ ID Nos: 3-11]) at 37°C for 1 hour, followed by the addition of 0.8 ng/ml IL-4 (a) or 12 ng/ml IL-13 (b) for 72 hours. Proliferation was measured using ³ H-thymidine nucleotide incorporation. Results from the TF-1 proliferation assay are depicted in the figure and show that the high affinity variants S276.2K04, S308.5F08, S308.5N01, S308.5L20, S308.5L04, S308.5N20, and S308.3O10 are potent antagonists of IL-4 and IL-13 induced signaling and proliferation.

Those skilled in the art will readily appreciate that the present invention is well suited to carry out the subject matter and obtain the results and advantages mentioned, as well as those inherent therein. In addition, various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention, as will be apparent to those skilled in the art. The compositions, methods, processes, treatments, molecules and specific compounds described herein are representative of certain embodiments currently exemplified and are not intended to be limiting of the scope of the invention. Variations and other uses that occur to those skilled in the art that are within the spirit of the invention are defined by the scope of the claims. The listing or discussion of previously published documents in this specification should not necessarily be construed as an admission that the documents are part of the state of the art or are common general knowledge.

The invention exemplarily described herein may be appropriately implemented in the absence of any elements or limitations not specifically disclosed herein. Thus, for example, the terms "comprise," "include," "contain," etc. should be understood broadly and without limitation. Furthermore, the terms and expressions employed herein are used as descriptive rather than restrictive terms, and are not intended to exclude any equivalents of the features shown and described, or portions thereof, but it is understood that various modifications are possible within the scope of the invention claimed. Therefore, it should be understood that although the invention has been specifically disclosed by way of exemplary embodiments and optional features, those skilled in the art may seek modifications and changes to the embodiments disclosed herein, and it is believed that such modifications and changes are within the scope of the invention.

The invention has been described broadly and generically herein. Each narrower class and subclass grouping falling within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a prerequisite or negative limitation removing any subject matter from that generic class, regardless of whether the excluded material is specifically described herein.

Other embodiments are within the scope of the following claims. Also, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subset of members of the Markush group.

Claims (8)

1. A mutant human tear lipid transporter protein, wherein the amino acid sequence of the mutant protein is: 2. A fusion protein, wherein the fusion protein is the mutant protein of claim 1, wherein the mutant protein is fused to a signal sequence and/or an affinity tag at its N-terminus or its C-terminus. 3. A method for generating the mutant human tear lipid transporter protein of claim 1, wherein the mutant protein binds to IL-4 receptor α, the method comprising: (a) Mutagenesis is performed on the nucleic acid molecule encoding human tear lipid transport protein at amino acid positions 27, 28, 30, 31, 33, 53, 57, 61, 64, 66, 80, 83, 104-106, and 108 of the linear polypeptide sequence of mature human tear lipid transport protein, and further, the nucleic acid molecule encoding human tear lipid transport protein is mutagenesis is performed at amino acid positions 26, 32, 34, 55, 56, 58, and 63 of the linear polypeptide sequence of mature human tear lipid transport protein. (b) Express one or more nucleic acid molecules obtained in step (a) in an expression system to obtain one or more mutant proteins, and (c) Enrich one or more mutant proteins obtained in step (b) and bound to human IL-4 receptor α by means of screening and/or isolation. 4. The method of claim 3, wherein step (c) comprises: (ci) provides IL-4 receptor α or its immunogenic fragment, (cii) Contact the one or more mutant proteins with the IL-4 receptor α or its immunogenic fragment, thereby allowing the formation of a complex between the IL-4 receptor α or its immunogenic fragment and the mutant protein having a binding affinity for it, and (ciii) Remove mutant proteins that have no binding affinity or no substantial binding affinity. 5. The method according to claim 3 or 4, wherein the screening in step (c) is performed under competitive conditions. 6. A nucleic acid molecule comprising a nucleotide sequence encoding a mutant protein according to claim 1 or a fusion protein according to claim 2. 7. A host cell comprising the nucleic acid molecule according to claim 6. 8. A pharmaceutical composition comprising the human tear lipid transporter mutant protein as defined in claim 1 or the fusion protein as described in claim 2 and a pharmaceutically acceptable excipient.