WO2011069992A2 - Muteins of human lipocalin 2 (lcn2, hngal) with affinity for a given target - Google Patents

Muteins of human lipocalin 2 (lcn2, hngal) with affinity for a given target Download PDF

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WO2011069992A2
WO2011069992A2 PCT/EP2010/069028 EP2010069028W WO2011069992A2 WO 2011069992 A2 WO2011069992 A2 WO 2011069992A2 EP 2010069028 W EP2010069028 W EP 2010069028W WO 2011069992 A2 WO2011069992 A2 WO 2011069992A2
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mutein
sequence
lcn2
protein
muteins
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PCT/EP2010/069028
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English (en)
French (fr)
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WO2011069992A3 (en
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Arne Skerra
Michaela Gebauer
Dominik Hinz
Sabine Rauth
Gabriele Matschiner
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Pieris Ag
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Priority to CN201080055370.5A priority Critical patent/CN102770764B/zh
Priority to JP2012542505A priority patent/JP5913120B2/ja
Priority to EP10784824.4A priority patent/EP2510357B1/en
Priority to KR1020187007781A priority patent/KR101974044B1/ko
Priority to EP15186647.2A priority patent/EP2990798B1/en
Priority to US13/514,133 priority patent/US9549968B2/en
Priority to DK10784824.4T priority patent/DK2510357T3/en
Priority to KR1020127017730A priority patent/KR101842076B1/ko
Application filed by Pieris Ag filed Critical Pieris Ag
Priority to EP19198835.1A priority patent/EP3660510A3/en
Priority to RU2012128586/10A priority patent/RU2564125C2/ru
Priority to BR112012013662-3A priority patent/BR112012013662B1/pt
Priority to CA2779562A priority patent/CA2779562C/en
Priority to AU2010329995A priority patent/AU2010329995B2/en
Publication of WO2011069992A2 publication Critical patent/WO2011069992A2/en
Publication of WO2011069992A3 publication Critical patent/WO2011069992A3/en
Priority to US15/380,168 priority patent/US10618941B2/en
Priority to US16/812,069 priority patent/US11827681B2/en

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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00041—Use of virus, viral particle or viral elements as a vector

Definitions

  • the present invention relates to a novel library for the generation of muteins and to novel muteins derived from human lipocalin 2 (Lcn2, hNGAL) and related proteins that bind a given target with detectable affinity.
  • the invention also relates to corresponding nucleic acid molecules encoding such a mutein and to a method for their generation.
  • the invention further relates to a method for producing such a mutein.
  • the invention is directed to a pharmaceutical composition comprising such a lipocalin mutein as well as to various uses of the mutein.
  • Lcn2 muteins according to this invention show potential as therapeutic and/or diagnostic reagents in several disease areas. For example, they may serve to bind and deplete pathological forms of natural biomolecules such as the amyloid beta peptide in Alzière's disease. In another example they may be employed for the specific targeting of various labels or toxins to disease-related cell surface markers such as the fibronectin extra-domain B, which is associated with tumor neovasculature. Due to the particular benefits of the Lcn2 muteins that can be generated according to this invention various other applications or examples are possible.
  • AD Alzheimer's disease
  • ⁇ amyloid beta peptide
  • non-Ig binding reagents such as Anticalins
  • the identification of an Affibody molecule with specificity for amyloid beta is one example to illustrate the potential of engineered non-Ig binding proteins (Gronwall et al (2007) J. Biotechnol. 128, 162-183; Hoyer et al. (2008) Proc. Natl. Acad. Sci. USA 105, 5099-5104).
  • Fibronectin plays an essential role in cell adhesion, migration, proliferation, and differentiation.
  • FN is a large, modular, dimeric glycoprotein comprising multiple domains of type I, II, and III.
  • Alternative splice variants of FN such as its extra-domain B (ED-B), which is incorporated between the FN in 7 and FN in 8 domains, are expressed in a tissue- specific and developmental stage-dependent manner (Zardi et al. (1987) EMBO J 6, 2337- 2342).
  • ED-B is absent from normal adult tissue except during wound healing and neoplastic vascularization. Consequently, ED-B containing fibronectin is abundantly expressed in many different tumor types that attract neovascularization and undergo aberrant angiogenesis. While the actual biological function of ED-B in angiogenesis remains elusive, its incorporation into FN serves as excellent marker for tumorgenesis. Generally, discrimination between malignant tissues and healthy organs is an advantageous therapeutic strategy as the selective targeting of drugs directly to the tumor tissue leads to an increased local drug concentration.
  • Such Lcn2 muteins specifically recognize ED-B containing fibronectin on human cells with high sensitivity and thus show pospects as therapeutic reagents for the diagnosis and treatment of tumor diseases.
  • the inventors were also able to identify specific Lcn2 muteins with high affinity and specificity for the ⁇ peptide. Such Lcn2 muteins can even inhibit ⁇ aggregation and thus show pospects, possibly after further improvement and modification, as therapeutic reagents to treat AD.
  • the present invention also describes new random libraries based on the human Lcn2 scaffold which permit the efficient generation of muteins, such as the muteins of the present invention, with high affinity and specificity for a given target in general. Examples for such libraries or sections thereof are shown in Figures 1 and 2.
  • nucleotide triplet(s) encoding for any of the sequence positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of hLcn2 a random mutagenesis was carried out by allowing substitution at this positions by a subset of nucleotide triplets.
  • nucleotide triplets coding for cystein are not used for substitution during the mutagenesis. That means that the mutagenesis does not lead to muteins carrying new cysteins which were not already included in the original not mutagenized sequenze.
  • the present invention is directed to a method of generating a mutein derived from human Lipocalin 2 (Lcn2; also known as human neutrophil gelatinase- associated lipocalin, hNGAL, or as siderocalin).
  • Lcn2 also known as human neutrophil gelatinase- associated lipocalin, hNGAL, or as siderocalin.
  • the muteins obtained with this method can bind to a non-natural target with detectable affinity.
  • the method comprises subjecting a nucleic acid molecule encoding for human Lipocalin 2 (Lcn2, hNGAL) to mutagenesis at a nucleotide triplet coding for at least one of any one of the sequence positions corresponding to the sequence positions 96, 100, and 106, of the linear polypeptide sequence of human Lipocalin 2, resulting in one or more mutein nucleic acid molecule(s).
  • a nucleic acid molecule encoding for human Lipocalin 2 (Lcn2, hNGAL)
  • human Lipocalin 2 or “human neutrophil gelatinase-associated lipocalin (hNGAL)” includes structural homologues, already identified or yet to be isolated, from other species which have an amino acid sequence homology or sequence identity of more than about 60%.
  • these human lipocalins described above comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 mutated amino acid residues at any of the sequence positions corresponding to the sequence positions 33, 36, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 55, 59, 65, 68, 70, 72, 73, 75, 77, 78, 79, 80, 81, 86, 87, 98, 96, 99, 100, 103, 106, 107, 110, 111, 125, 127, 132, 134, 136 and 138 of the linear polypeptide sequence of hNGAL.
  • sequence identity means the percentage of pair-wise identical residues - following homology alignment of a sequence of a polypeptide of the present invention with a sequence in question - with respect to the number of residues in the longer of these two sequences.
  • the percentage of sequence homology or sequence identity is determined herein using the program BLASTP, version blastp 2.2.5 (November 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res. 25, 3389-3402).
  • the percentage of homology is based on the alignment of the entire polypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1; cutoff value set to 10 " ) including the propeptide sequences, using the human Lipocalin 2 as reference in a pairwise comparison. It is calculated as the percentage of numbers of "positives" (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment. It is noted in this connection that this total number of selected amino acids can differ from the length of the human Lipocalin 2.
  • a protein other tha human Lipocalin 2 is used in the present invention
  • the definition of the mutated sequence positions given for human Lipocalin 2 can be assigned to the other lipocalin with the help of published sequence alignments or alignments methods which are available to the skilled artisan.
  • a sequence alignment can, for example, be carried out as explained in WO 99/16873 (cf. Fig. 3 therein), using an published alignment such as the one in Fig. 1 of Redl, B. (2000) Biochim. Biophys. Acta 1482, 241-248.
  • the three- dimensional structure of the lipocalins is available structural superpositions can also be used for the determination of those sequence positions that are to be subjected to mutagenesis in the present invention.
  • Other methods of structural analysis such as multidimensional nuclear magnetic resonance spectroscopy can also be employed for this purpose.
  • the homologue of human Lipocalin 2 can also be a mutein protein of human Lipocalin 2 itself, in which amino acid substitutions are introduced at positions other than the positions selected in the present invention.
  • a mutein can be a protein in which positions at the solvent exposed surface of the ⁇ -barrel are mutated compared to the wild type sequence of the human Lipocalin 2 in order to increase the solubility or the stability of the protein.
  • the term "human Lipocalin 2" includes all proteins that have a sequence homology or sequence identity of more than 60%, 70% 80%, 85%, 90%, or 95% in relation to the human Lipocalin 2.
  • these human lipocalins described above comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 mutated amino acid residues at any of the sequence positions corresponding to the sequence positions 33, 36, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 55, 59, 65, 68, 70, 72, 73, 75, 77, 78, 79, 80, 81, 86, 87, 98, 96, 99, 100, 103, 106, 107, 110, 111, 125, 127, 132, 134, 136 and 138 of the linear polypeptide sequence of hNGAL.
  • the present invention provides a mutein derived from human Lipocalin 2 (i.e., human lipocalin mutein or a mutated human lipocalin, preferably a mutated mature hNGAL, wherein said mature hNGAL has the SWISS-PROT Data Bank Accession Number P80188, more preferably said mature hNGAL has the amino acid sequence shown in SEQ ID NO:44).
  • This mutein includes at least one or two mutated amino acid residue at any of the sequence positions corresponding to the sequence positions 96, 100 and 106 of the linear polypeptide sequence of human Lcn2, and wherein the mutein binds a given non-natural target with detectable affinity.
  • a "mutein,” a “mutated” entity (whether protein or nucleic acid) or “mutant” refers to the exchange, deletion, or insertion of one or more nucleotides or amino acids, respectively, compared to the naturally occurring (wild- type) nucleic acid or protein "reference" scaffold.
  • the number of nucleotides or amino acids, respectively, that is exchanged, deleted or inserted is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more such as 25, 30, 35, 40, 45 or 50.
  • human neutrophil gelatinase-associated lipocalin or "hNGAL” or “lipocalin 2" or “Lcn2” as used herein to refer to the mature hNGAL with the SWISS-PROT Data Bank Accession Number P80188 (exemplified herein as SEQ ID NO:44).
  • the invention is based on the surprising finding that subjecting human Lipocalin 2, rat 2-micro globulin-related protein (A2m) and mouse 24p3/uterocalin (24p3) to mutagenesis at one or more of these above-mentioned 3 sequence position provides for muteins that have a sufficiently affine binding to pre-defined target which can include, but is not limited to a peptide, a protein, a fragment or a domain of a protein, and a small organic molecule.
  • the given target may be any desired non-natural target/ligand.
  • non-natural ligand refers to a compound, which does not bind to native mature hNGAL under physiological conditions.
  • organic molecule or "small organic molecule” as used herein for the non-natural target denotes an organic molecule comprising at least two carbon atoms, but preferably not more than 7 or 12 rotatable carbon bonds, having a molecular weight in the range between 100 and 2000 Dalton, preferably between 100 and 1000 Dalton, and optionally including one or two metal atoms.
  • peptide refers to a dipeptide or an oligopeptide with 2 to 45 amino acids.
  • the peptide has 2-40, 2-35, 2- 30, 2-25, 2-20, 2-15 or 2-10 amino acid residues.
  • the peptide may be a naturally occurring or synthetic peptide and may comprise - besides the 20 naturally occurring L-amino acids - D- amino acids, non-naturally occurring amino acids and amino acid analogs.
  • the peptide is an amyloid beta peptide (Abeta or ⁇ ).
  • the amyloid beta peptide is an A ?40 peptide or an A ?42 peptide.
  • the small organic molecule is a compound showing the features of an immunological hapten.
  • the non-natural target which is a protein is fibronection or a domain thereof, such as the EB -domain or a fragment of the EB -domain.
  • a human Lipocalin 2 mutein of the invention may comprise the wild type (natural) amino acid sequence outside the mutated amino acid sequence positions.
  • the lipocalin muteins disclosed herein may also contain amino acid mutations outside the sequence positions subjected to mutagenesis as long as those mutations do not interfere with the binding activity and the folding of the mutein.
  • Such mutations can be accomplished very easily on DNA level using established standard methods (Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • Possible alterations of the amino acid sequence are insertions or deletions as well as amino acid substitutions. Such substitutions may be conservative, i.e.
  • an amino acid residue is replaced with a chemically similar amino acid residue.
  • conservative substitutions are the replacements among the members of the following groups: 1) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan.
  • modifications of the amino acid sequence include directed mutagenesis of single amino acid positions in order to simplify sub-cloning of the mutated lipocalin gene or its parts by incorporating cleavage sites for certain restriction enzymes.
  • these mutations can also be incorporated to further improve the affinity of a lipocalin mutein for a given target.
  • mutations can be introduced in order to modulate certain characteristics of the mutein such as to improve folding stability, serum stability, protein resistance or water solubility or to reduce aggregation tendency, if necessary.
  • cysteine residues may be mutated to other amino acids to prevent disulphide bridge formation.
  • cysteine residues are introduced into the amino acid sequence of a human Lipocalin 2 mutein.
  • cysteine residues are introduced at least at one of the sequence positions that correspond to sequence positions 14, 21, 60, 84, 88, 116, 141, 145, 143, 146 or 158 of the wild type sequence of hNGAL.
  • the generated thiol moiety at the side of any of the amino acid positions 14, 21, 60, 84, 88, 116, 141, 145, 143, 146 and/or 158 may be used to PEGylate or HESylate the mutein, for example, in order to increase the serum half-life of a respective human Lipocalin 2 mutein.
  • the mutein of the present invention includes mutated amino acid residues at least at any 2 or all 3 of the sequence positions corresponding to the sequence positions 96, 100 and 106 of the linear polypeptide sequence of human Lipocalin 2.
  • the mutein includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 mutated amino acid residues at any of the sequence positions corresponding to the sequence positions 33, 36, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 55, 59, 65, 68, 70, 72, 73, 75, 77, 78, 79, 80, 81, 86, 87, 98, 96, 99, 100, 103, 106, 107, 110, 111, 125, 127, 132, 134, 136 and 138 of the linear polypeptide sequence of hNGAL.
  • the mutein includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 mutated amino acid residues at any one of the sequence positions 33, 36, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 55, 59, 65, 68, 70, 72, 73, 75, 77, 78, 79, 80, 81, 86, 87, 98, 96, 99, 100, 103, 106, 107, 110, 111, 125, 127, 132, 134, 136 and 138 of the linear polypeptide sequence of hNGAL.
  • the mutein includes 18, 19 or 20 mutated amino acid residues at any one of the sequence positions 33, 36, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 55, 59, 65, 68, 70, 72, 73, 75, 77, 78, 79, 80, 81, 86, 87, 98, 96, 99, 100, 103, 106, 107, 110, 111, 125, 127, 132, 134, 136 and 138 of the linear polypeptide sequence of human Lipocalin 2.
  • the mutein includes mutated amino acid residues at at least any 10, 14, 15, 20, 22, 24, 26, 28, 29, 30, 31, 32, 33, 35 or all 45 of the above-listed sequence positions.
  • a mutein of the invention which binds to an amyloid beta peptide can comprise with respect to the mature human Lipocalin 2 wild type amino acid sequence illustrated in Figure 17 (Lcn2) at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid replacements which include, but are not limited to Leu36 ⁇ Val or Cys; Ala40 ⁇ Tyr or Lys or Val; Ile41 ⁇ Thr or Ser or Leu; Gln49 ⁇ Leu or Trp; Leu70 ⁇ Gly; Arg72 ⁇ Gly or Asp; Lys73 ⁇ Leu or Thr or Asp; Asp77 ⁇ Asn or His or Leu; Trp79 ⁇ Lys; Asn96 ⁇ He or Arg; TyrlOO ⁇ Gin or Arg or Glu; Leul03 ⁇ Met or Arg or Gly; Tyrl06 ⁇ Tyr or Ala or Trp; Lys 125 ⁇ Thr or Val or Glu; Serl27 ⁇ Gly or Gin
  • muteins can diminish ⁇ aggregation in vitro or in vivo. It is preferred that the mutein described herein binds to and inhibits aggregation of ⁇ , preferably ⁇ 40, more preferably under assay conditions as specified in Example 11 (preferably including the ratio 1: 10 mutein : ⁇ 40).
  • the present invention also relates to muteins as described above having a comparable biological function when compared with mutein US7.
  • “Comparable biological function” means that these muteins are able to bind to and inhibit ⁇ , preferably ⁇ 40, with a deviation of the aggregation inhibiting activity in respect to US7 of not more than about 40%, 30%, 20%, 15%, 10%, 5%, 2,5%, 2% or 1%, for example under conditions which equate to or are identical with those set out in Example 11 (preferably including the ratio 1 : 10 mutein : ⁇ 40).
  • the present invention also relates to the muteins described herein for use in the treatment or prevention of neurodegenerative diseases such as Alzheimer's disease.
  • a mutein of the invention which binds to an amyloid beta peptide, such as A ?40 peptide or an A ?42 peptide comprises the following amino acid replacements Leu36 ⁇ Val; Ala40 ⁇ Tyr; Ile41 ⁇ Thr; Gln49 ⁇ Leu; Leu70 ⁇ Gly; Lys73 ⁇ Leu; Asp77 ⁇ Asn; Trp79 ⁇ Lys; Asn96 ⁇ He; TyrlOO ⁇ Gin; Leul03 ⁇ Met; Lysl25 ⁇ Thr; Serl27 ⁇ Gly; Tyrl32 ⁇ Met; and Lys 134 ⁇ Asn.
  • An example of a mutein including such amino acid replacements is S1-A4 illustrated in Figure 17 (SEQ ID NO: 39).
  • a mutein of the invention which binds to an amyloid beta peptide, such as A ?40 peptide or an A ?42 peptide comprises the following amino acid replacements Leu36 ⁇ Val; Ala40 ⁇ Lys; Ile41 ⁇ Ser; Gln49 ⁇ Trp; Leu70 ⁇ Gly; Arg72 ⁇ Gly; Lys73 ⁇ Thr; Asp77 ⁇ His; Trp79 ⁇ Lys; Asn96 ⁇ Arg; TyrlOO ⁇ Arg; Leul03 ⁇ Arg; Tyrl06 ⁇ Ala; Lysl25 ⁇ Val; Serl27 ⁇ Gin; Tyrl32 ⁇ Ser; and Lysl34 ⁇ Asn.
  • An example of a mutein including such amino acid replacements is US-7 illustrated in Figure 17 (SEQ ID NO: 41).
  • the invention also envisages a mutated mature hNGAL lipocalin (as deposited in SWISS-PROT Data Bank under Accession Number P80188, preferably having the amino acid sequence shown in SEQ ID NO:40) comprising at a position corresponding to a position 36, 40, 41, 49, 70, 72, 73, 77, 79, 96, 100, 103, 106, 125, 127, 132, 134 of the linear polypeptide sequence of wild type hNGAL one or more mutated amino acids as described herein.
  • a mutein of the invention which binds to an amyloid beta peptide comprises the following amino acid replacements Leu36 ⁇ Cys; Ala40 ⁇ Val; Ile41 ⁇ Leu; Gln49 ⁇ Leu; Leu70 ⁇ Gly; Arg72 ⁇ Asp; Lys73 ⁇ Asp; Asp77 ⁇ Leu; Trp79 ⁇ Lys; Asn96 ⁇ Arg; TyrlOO ⁇ Glu; Leul03 ⁇ Gly; Tyrl06 ⁇ Trp; Lys 125 ⁇ Glu; Serl27 ⁇ Ala; Tyrl32 ⁇ Thr; and Lys 134 ⁇ Asn.
  • An example of a mutein including such amino acid replacements is Hl-Gl illustrated in Figure 17 (SEQ ID NO: 43).
  • a mutein of the invention which binds to an amyloid beta peptide, such as A ?40 peptide or an A ?42 peptide comprises the following amino acid replacements Leu36 ⁇ Ala; Ala40 ⁇ Val; Ile41 ⁇ Leu; Gln49 ⁇ Leu; Leu70 ⁇ Gly; Arg72 ⁇ Asp; Lys73 ⁇ Asp; Asp77 ⁇ Leu; Trp79 ⁇ Lys; Asn96 ⁇ Arg; TyrlOO ⁇ Glu; Leul03 ⁇ Gly; Tyrl06 ⁇ Trp; Lys 125 ⁇ Glu; Serl27 ⁇ Ala; Tyrl32 ⁇ Thr; and Lys 134 ⁇ Asn.
  • An example of a mutein including such amino acid replacements is H1GA illustrated SEQ ID NO: 50.
  • the mutein described herein above binds to and inhibits aggregation of ⁇ , preferably ⁇ 40, more preferably under assay conditions as specified in Example 23 (preferably in a ratio of 10:2 ⁇ 40 : H1GA).
  • the present invention also relates to muteins as described above having a comparable biological function when compared with mutein H1GA.
  • “Comparable biological function” means that these muteins are able to bind to and inhibit ⁇ , preferably ⁇ 40 with a deviation of the aggregation inhibiting activity in respect to H1GA of not more than about 40%, 30%, 20%, 15%, 10%, 5%, 2,5%, 2% or 1%, for example under conditions which equate to or are identical with those set out in Example 23 (preferably in a ratio of 10:2 ⁇ 40 : H1GA).
  • the present invention also relates to the muteins described herein for use in the treatment or prevention of neurodegenerative diseases such as Alzheimer's disease.
  • a mutein of the invention which binds to an amyloid beta peptide, such as A ?40 peptide or an A ?42 peptide comprises the following amino acid replacements Leu36 ⁇ Val; Ala40 ⁇ Val; Ile41 ⁇ Leu; Gln49 ⁇ Leu; Leu70 ⁇ Gly; Arg72 ⁇ Asp; Lys73 ⁇ Asp; Asp77 ⁇ Leu; Trp79 ⁇ Lys; Asn96 ⁇ Arg; TyrlOO ⁇ Glu; Leul03 ⁇ Gly; Tyrl06 ⁇ Trp; Lys 125 ⁇ Glu; Serl27 ⁇ Ala; Tyrl32 ⁇ Thr; and Lys 134 ⁇ Asn.
  • An example of a mutein including such amino acid replacements is HIGV illustrated SEQ ID NO: 52.
  • the mutein described herein above binds to ⁇ 40 under assay conditions as specified in Example 21.
  • the present invention also relates to muteins as described above having a comparable biological function when compared with mutein HIGV.
  • “Comparable biological function” means that these muteins are able to bind to ⁇ , preferably ⁇ 40 with a deviation of the aggregation inhibiting activity in respect to HIGV of not more than about 40%, 30%, 20%, 15%, 10%, 5%, 2,5%, 2% or 1%, for example under conditions which equate to or are identical with those set out in Example 21.
  • the present invention also relates to the muteins described herein for use in the treatment or prevention of neurodegenerative diseases such as Alzheimer' s disease.
  • a mutein of the invention which bind to extra-domain B or a fragment thereof comprises the following amino acid replacements Leu36 ⁇ Lys; Ala40 ⁇ His; Ile41 ⁇ Asp; Gln49 ⁇ Arg; Tyr52 ⁇ Gin; Ser68 ⁇ Asn; Leu70 ⁇ Arg; Arg72 ⁇ Val; Lys73 ⁇ His; Asp77 ⁇ Asn; Trp79 ⁇ Arg; Arg81 ⁇ Trp; TyrlOO ⁇ Trp; Tyrl06 ⁇ Trp; Lys 125 ⁇ Arg; Serl27 ⁇ Tyr; Tyrl32 ⁇ Leu; Lys 134 ⁇ Glu and Serl46 ⁇ Asn.
  • An example of a mutein including such amino acid replacements is N7A illustrated in Figure 17 (SEQ ID NO: 20).
  • a mutein of the invention which binds to extra-domain B or a fragment thereof comprises the following amino acid replacements Leu36 ⁇ Arg; Ala40 ⁇ Met; Ile41 ⁇ Arg; Gln49 ⁇ Ala; Tyr52 ⁇ Val; Ser68 ⁇ Lys; Leu70 ⁇ Met; Arg72 ⁇ Gin; Lys73 ⁇ Arg; Asp77 ⁇ Lys; Trp79 ⁇ Met; Arg81 ⁇ Asn; Asn96 ⁇ Ala; TyrlOO ⁇ Pro; Leul03 ⁇ Pro; Tyrl06 ⁇ Thr; Lys l25 ⁇ His; Serl27 ⁇ Phe; and Lysl34 ⁇ His.
  • An example of a mutein including such amino acid replacements is N9B illustrated in Figure 17 (SEQ ID NO: 24).
  • a mutein of the invention which binds to extra-domain B or a fragment thereof comprises the following amino acid replacements Leu36 ⁇ Ala; Ala40 ⁇ Thr; Ee41 ⁇ Trp; Gln49 ⁇ Tyr; Tyr52 ⁇ Gin; Ser68 ⁇ Asn; Arg72 ⁇ Met; Lys73 ⁇ Ser; Asp77 ⁇ Arg; Trp79 ⁇ Met; Arg81 ⁇ His; Asn96 ⁇ Ser; TyrlOO ⁇ Trp; Tyrl06 ⁇ Trp; Lysl25 ⁇ Arg; Serl27 ⁇ Tyr; Tyrl32 ⁇ Phe; and Lys l34 ⁇ Gly.
  • An example of a mutein including such amino acid replacements is N10D illustrated in Figure 17 (SEQ ID NO: 26).
  • a mutein of the invention which bind to extra-domain B or a fragment thereof comprises the following amino acid replacements Leu36 ⁇ Glu; Ala40 ⁇ Ser; Ile41 ⁇ Leu; Gln49 ⁇ Arg; Leu70 ⁇ Arg; Lys73 ⁇ Ser; Asp77 ⁇ His; Trp79 ⁇ Leu; Asn96 ⁇ Leu; TyrlOO ⁇ Lys; Leul03 ⁇ His; Tyrl06 ⁇ Phe; Lys 125 ⁇ Thr; Serl27 ⁇ Ala; and Lys 134 ⁇ Phe.
  • An example of a mutein including such amino acid replacements is N7E illustrated in Figure 17 (SEQ ID NO: 22).
  • the above muteins described with reference to Figure 17 can include further amino acid replacements.
  • the muteins can further comprise amino acid replacements which can include, but are not limited to Gln28 ⁇ His or Cys87 ⁇ Ser.
  • Other possible amino acid replacements include, but are not limited to Tyr52 ⁇ Gin or Val; Ser68 ⁇ Lys or Asn; or Arg81 ⁇ Trp or Asn or His.
  • the lipocalin muteins of the invention are able to bind the desired non-natural target with detectable affinity, i.e. with a dissociation constant (K D ) of at least 200 nM.
  • the mutein binds the given non-natural target with a Kj) of 1 ⁇ or less, or 100 ⁇ or less, or 1 ⁇ or less, or 500 nM, or 200 nM or less, or 100, nM or less, or 50 nM or less, or 10 nM or less, or 1 nM or less.
  • lipocalin muteins bind the desired target with a dissociation constant for a given target of at least 100, 20, 1 nM or even less.
  • the binding affinity of a mutein to the desired target can be measured by a multitude of methods such as fluorescence titration, competition ELISA or surface plasmon resonance (Biacore).
  • a mutein of the invention typically exists as monomeric protein. However, it is also possible that an inventive lipocalin mutein is able to spontaneously dimerise or oligomerise. Although the use of lipocalin muteins that form stable monomers may be preferred for some applications, e.g. because of faster diffusion and better tissue penetration, the use of lipocalin muteins that form stable homodimers or multimers may be advantageous in other instances, since such multimers can provide for a (further) increased affinity and/or avidity to a given target. Furthermore, oligomeric forms of the lipocalin mutein may have slower dissociation rates or prolonged serum half-life.
  • the complex formation between the respective mutein and its ligand is influenced by many different factors such as the concentrations of the respective binding partners, the presence of competitors, pH and the ionic strength of the buffer system used, and the experimental method used for determination of the dissociation constant K D (for example fluorescence titration, competition ELISA or surface plasmon resonance, just to name a few) or even the mathematical algorithm which is used for evaluation of the experimental data.
  • the K D values (dissociation constant of the complex formed between the respective mutein and its target/ligand) may vary within a certain experimental range, depending on the method and experimental setup that is used for determining the affinity of a particular lipocalin mutein for a given ligand. This means, there may be a slight deviation in the measured K D values or a tolerance range depending, for example, on whether the K D value was determined by surface plasmon resonance (Biacore) or by competition ELISA.
  • the muteins disclosed herein can be linked, either N- or C- terminal to an affinity tag such as pentahistidine tag, a hexahistidine tag or a Strep-tag ® .
  • an affinity tag such as pentahistidine tag, a hexahistidine tag or a Strep-tag ® .
  • fragment as used in the present invention in connection with the feature lipocalin mutein fragment relates to proteins or peptides derived from full-length mature Lcn2 that are N-terminally and/or C-terminally shortened, i.e. lacking at least one of the N-terminal and/or C-terminal amino acids.
  • Such fragments comprise preferably at least 10, more preferably 20, most preferably 30 or more consecutive amino acids of the primary sequence of mature Lcn2 and are usually detectable in an immunoassay of mature Lcn2.
  • Cytotoxic T-cells recognize peptide antigens on the cell surface of an antigen- presenting cell in association with a class I major histocompatibility complex (MHC) molecule.
  • MHC major histocompatibility complex
  • the ability of the peptides to bind to MHC molecules is allele specific and correlates with their immunogenicity. In order to reduce immunogenicity of a given protein, the ability to predict which peptides in a protein have the potential to bind to a given MHC molecule is of great value.
  • Approaches that employ a computational threading approach to identify potential T-cell epitopes have been previously described to predict the binding of a given peptide sequence to MHC class I molecules (Altuvia et al. (1995) J. Mol. Biol. 249: 244-250).
  • Such an approach may also be utilized to identify potential T-cell epitopes in the muteins of the invention and to make depending on its intended use a selection of a specific mutein on the basis of its predicted immunogenicity. It may be furthermore possible to subject peptide regions which have been predicted to contain T-cell epitopes to additional mutagenesis to reduce or eliminate these T-cell epitopes and thus minimize immunogenicity.
  • the removal of amphipathic epitopes from genetically engineered antibodies has been described (Mateo et al. (2000) Hybridoma 19(6):463-471) and may be adapted to the muteins of the present invention.
  • the muteins thus obtained may possess a minimized immunogenicity, which is desirable for their use in therapeutic and diagnostic applications, such as those described below.
  • the invention is also directed to lipocalin muteins which are conjugated to a compound which can include, but is not limited to organic molecules, an enzyme label, a colored label, a cytostatic agent, a toxin, a label that can be photoactivated and which is suitable for use in photodynamic therapy, a fluorescent label, a radioactive label, a chromogenic label, a luminescent label, metal complexes, metal, such as colloidal gold, haptens, digoxigenin, biotin, a chemo therapeutic metal, or a chemo therapeutic metal, to name only a few evocative examples.
  • the mutein may also be conjugated to an organic drug molecule. The conjugation can be carried out using any conventional coupling method known in the art.
  • a Lcn2 mutein described herein with any appropriate chemical substance or enzyme, which directly or indirectly generates a detectable compound or signal in a chemical, physical, optical, or enzymatic reaction.
  • An example for a physical reaction and at the same time optical reaction/marker is the emission of fluorescence upon irradiation.
  • Alkaline phosphatase, horseradish peroxidase or ⁇ -galactosidase are examples of enzyme labels (and at the same time optical labels) which catalyze the formation of chromogenic reaction products.
  • all labels commonly used for antibodies can also be used for conjugation to the muteins of the present invention.
  • the muteins of the invention may also be conjugated with any suitable therapeutically active agent, e.g., for the targeted delivery of such agents to a given cell, tissue or organ or for the selective targeting of cells, e.g., of tumor cells without affecting the surrounding normal cells.
  • suitable therapeutically active agents include radionuclides, toxins, small organic molecules, and therapeutic peptides (such as peptides acting as agonists/antagonists of a cell surface receptor or peptides competing for a protein binding site on a given cellular target).
  • Suitable toxins include, but are not limited to pertussis-toxin, diphtheria toxin, ricin, saporin, pseudomonas exotoxin, calicheamicin or a derivative thereof, a taxoid, a maytansinoid, a tubulysin or a dolastatin analogue.
  • the dolastatin analogue may be auristatin E, monomethylauristatin E, auristatin PYE and auristatin PHE.
  • cytostatic agent examples include, but are not limited to Cisplatin, Carboplatin, Oxaliplatin, 5-Fluorouracil, Taxotere (Docetaxel), Paclitaxel, Anthracycline (Doxorubicin), Methotrexate, Vinblastin, Vincristine, Vindesine, Vinorelbine, dacarbazine, Cyclophosphamide, Etoposide, Adriamycine, Camptotecine, Combretatastin A-4 related compounds, sulfonamides, oxadiazolines, benzo[b]thiophenes synthetic spiroketal pyrans, monotetrahydrofuran compounds, curacin and curacin derivatives, methoxyestradiol derivatives and Leucovorin.
  • the lipocalin muteins of the invention may also be conjugated with therapeutically active nucleic acids such as antisense nucleic acid molecules, small interfering RNAs, micro RNAs or ribozymes. Such conjugates can be produced by methods well known in the art.
  • the muteins of the invention may also be coupled to a targeting moiety that targets a specific body region in order to deliver the inventive muteins to a desired region or area within the body.
  • a targeting moiety that targets a specific body region in order to deliver the inventive muteins to a desired region or area within the body.
  • One example wherein such modification may be desirable is the crossing of the blood-brain-barrier.
  • the muteins of the invention may be coupled to moieties that facilitate the active transport across this barrier (see Gaillard PJ, et al. (2005) International Congress Series. 1277,185-198 or Gaillard PJ, et al. (2005) Expert Opin Drug Deliv. 2(2), 299-309).
  • targeting molecules to which the muteins of the present invention may be coupled include antibodies, antibody fragments or lipocalin muteins with affinity for a desired target molecule.
  • the target molecule of the targeting moieties may, for example, be a cell-surface antigen.
  • Cell-surface antigens may be specific for a cell or tissue type, such as, for example, cancer cells.
  • Illustrative examples of such cell surface proteins are HER-2 or proteoglycans such as NEU-2.
  • a mutein of the invention may in some embodiments be conjugated to a compound that extends the serum half-life of the mutein (in this regard see also PCT publication WO 2006/56464 where such conjugation strategies are described with references to muteins of human neutrophil gelatinase-associated lipocalin with binding affinity for CTLA-4).
  • the compound that extends the serum half-life may be a polyalkylene glycol molecule, such as polyethylene (PEG) or an activated derivative thereof; hydroxyethyl starch, fatty acid molecules, such as palmitic acid (Vajo & Duckworth (2000) Pharmacol. Rev.
  • an Fc part 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 an albumin binding protein, transferrin to name only a few.
  • the albumin binding protein may be a bacterial albumin binding protein, an antibody, an antibody fragment including domain antibodies (see US patent 6,696,245, for example), or a lipocalin mutein with binding activity for albumin.
  • suitable conjugation compounds for extending the half-life of a lipocalin mutein of the invention include albumin (Osborn et al. (2002) J. Pharmacol. Exp. Ther.
  • an albumin binding protein for example, a bacterial albumin binding domain, such as the one of streptococcal protein G (Konig, T. and Skerra, A. (1998) J. Immunol. Methods 218, 73-83).
  • albumin binding peptides that can be used as conjugation partner are, for instance, those having a Cys-Xaa 1 -Xaa 2 -Xaa 3 -Xaa 4 -Cys consensus sequence, wherein Xaai is Asp, Asn, Ser, Thr, or Trp; Xaa 2 is Asn, Gin, His, He, Leu, or Lys; Xaa is Ala, Asp, Phe, Trp, or Tyr; and Xaa 4 is Asp, Gly, Leu, Phe, Ser, or Thr as described in US patent application 2003/0069395 or Dennis et al. (Dennis et al. (2002) J. Biol. Chem. 277, 35035-35043).
  • albumin itself or a biological active fragment of albumin can be used as compound of a lipocalin mutein of the invention that extends the serum half-life of the mutein.
  • the term "albumin” comprises all mammal albumins such as human serum albumin or bovine serum albumin or rat albumin.
  • the albumin or fragment thereof can be recombinantly produced as described in US patent 5,728,553 or European patent applications EP 0 330 451 and EP 0 361 991.
  • Recombinant human albumin (Recombumin®) for use as a protein stabilizer is for example available from Novozymes Delta Ltd. (Nottingham, UK).
  • albumin-binding protein is an antibody fragment it may be a domain antibody.
  • Domain Antibodies are engineered to allow precise control over biophysical properties and in vivo half-life to create the optimal safety and efficacy product profile. Domain Antibodies are for example commercially available from Domantis Ltd. (Cambridge, UK and MA, USA).
  • the muteins can be genetically fused to the N or C terminus, or both, of non- glycosylated transferrin.
  • Non-glycosylated transferrin has a half-life of 14-17 days, and a transferrin fusion protein will similarly have an extended half-life.
  • the transferrin carrier also provides high bioavailability, biodistribution and circulating stability. This technology is commercially available from BioRexis (BioRexis Pharmaceutical Corporation, PA, USA). Recombinant human transferrin (DeltaFerrinTM) for use as a protein stabilizer is also commercially available from Novozymes Delta Ltd. (Nottingham, UK).
  • an Fc part of an immunoglobulin is used for the purpose to prolong the serum half-life of the muteins of the invention
  • the SynFusionTM technology commercially available from Syntonix Pharmaceuticals, Inc (MA, USA)
  • the use of this Fc-fusion technology allows the creation of longer-acting biopharmaceuticals and may for example consist of two copies of the mutein linked to the Fc region of an antibody to improve pharmacokinetics, solubility, and production efficiency.
  • Yet another alternative to prolong the half-life of a mutein of the invention is to fuse the N- or C-terminus of a mutein of the invention to long, unstructured, flexible glycine- rich sequences (for example poly-glycine with about 20 to 80 consecutive glycine residues).
  • This approach disclosed in WO2007/038619, for example, has also been term "rPEG" (recombinant PEG).
  • polyalkylene glycol is used as compound that extends the half-life of the mutein, the polyalkylene glycol can be substituted or unsubstituted. It can also be an activated polyalkylene derivative.
  • suitable compounds are polyethylene glycol (PEG) molecules as described in WO 99/64016, in US Patent 6,177,074 or in US Patent 6,403,564 in relation to interferon, or as described for other proteins such as PEG-modified asparaginase, PEG-adenosine deaminase (PEG-ADA) or PEG- superoxide dismutase (see for example, Fuertges et al.
  • the molecular weight of such a polymer may range from about 300 to about 70.000 Dalton, including, for example, polyethylene glycol with a molecular weight of about 10.000, of about 20.000, of about 30.000 or of about 40.000 Dalton.
  • carbohydrate oligo- and polymers such as starch or hydroxyethyl starch (HES) can be conjugated to a mutein of the invention for the purpose of serum half-life extension.
  • artificial amino acids may be introduced by mutagenesis.
  • such artificial amino acids are designed to be more reactive and thus to facilitate the conjugation to the desired moiety.
  • an artificial amino acid that may be introduced via an artificial tRNA is para-acetyl- phenylalanine.
  • the inventive mutein is fused at its N-terminus and/or it's C-terminus to a protein, a protein domain or a peptide such as a signal sequence and/or an affinity tag.
  • a mutein of the invention may be fused to a fusion partner that extends the in vivo serum half-life of the mutein (see again PCT publication WO 2006/56464 where suitable fusion partner are described with references to muteins of human neutrophile gelatinase- associated lipocalin with binding affinity for CTLA-4).
  • the fusion partner may be an Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunogloubulin, albumin, an albumin binding peptide or an albumin binding protein, to name only a few.
  • the albumin binding protein may be a bacterial albumin binding protein or a lipocalin mutein with binding activity for albumin.
  • suitable fusion partners for extending the half-life of a lipocalin mutein of the invention include albumin (Osborn, B.L. et al. (2002) supra J. Pharmacol. Exp. Ther. 303, 540-548), or an albumin binding protein, for example, a bacterial albumin binding domain, such as the one of streptococcal protein G (Konig, T. and Skerra, A. (1998) supra J. Immunol. Methods 218, 73- 83).
  • albumin itself or a biological active fragment of albumin as fusion partner of a lipocalin mutein of the invention.
  • the term "albumin” comprises all mammal albumins such as human serum albumin or bovine serum albumin or rat serum albumin.
  • the recombinant production of albumin or fragments thereof is well known in the art and for example described in US patent 5,728,553, European patent application EP 0 330 451 or EP 0 361 991.
  • the fusion partner may confer new characteristics to the inventive lipocalin mutein such as enzymatic activity or binding affinity for other molecules.
  • fusion proteins examples include alkaline phosphatase, horseradish peroxidase, gluthation-S-transferase, the albumin-binding domain of protein G, protein A, antibody fragments, oligomerization domains, lipocalin muteins of same or different binding specificity (which results in the formation of "duocalins", cf. 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.
  • a lipocalin mutein of the invention may be fused with a separate enzyme active site such that both "components" of the resulting fusion protein together act on a given therapeutic target.
  • the binding domain of the lipocalin mutein attaches to the disease-causing target, allowing the enzyme domain to abolish the biological function of the target.
  • Affinity tags such as the Strep-tag® or Strep-tag® II (Schmidt, T.G.M. et al. (1996) J. Mol. Biol. 255, 753-766), the myc-tag, the FLAG-tag, the His6-tag or the HA-tag or proteins such as glutathione-S-transferase also allow easy detection and/or purification of recombinant proteins are further examples of preferred fusion partners. Finally, proteins with chromogenic or fluorescent properties such as the green fluorescent protein (GFP) or the yellow fluorescent protein (YFP) are suitable fusion partners for a lipocalin mutein of the invention as well.
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • fusion protein also comprises lipocalin muteins according to the invention containing a signal sequence.
  • Signal sequences at the N-terminus of a polypeptide direct this polypeptide to a specific cellular compartment, for example the periplasm of E. coli or the endoplasmatic reticulum of eukaryotic cells. A large number of signal sequences is known in the art.
  • a preferred signal sequence for secretion 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 coding for muteins as described herein. Since the degeneracy of the genetic code permits substitutions of certain codons by other codons specifying the same amino acid, the invention is not limited to a specific nucleic acid molecule encoding a mutein of the invention but includes all nucleic acid molecules comprising nucleotide sequences encoding a functional mutein.
  • the present invention also includes a nucleic acid sequence encoding a mutein according to the invention including a mutation at least at one codon of any of the amino acid sequence positions 96, 100 and 106 of the linear polypeptide sequence of Lcn2.
  • the invention as disclosed herein also includes nucleic acid molecules encoding Lcn2 muteins, which comprise additional mutations outside the indicated sequence positions of experimental mutagenesis. Such mutations are often tolerated or can even prove to be advantageous, for example if they contribute to an improved folding efficiency, serum stability, thermal stability or ligand binding affinity of the mutein.
  • a nucleic acid molecule disclosed in this application may be "operably linked" to a regulatory sequence (or regulatory sequences) to allow expression of this nucleic acid molecule.
  • a nucleic acid molecule such as DNA
  • An operable linkage is a linkage in which the regulatory sequence elements and the sequence to be expressed are connected in a way that enables gene expression. The precise nature of the regulatory regions necessary for gene expression may vary among species, but in general these regions comprise a promoter which, in prokaryotes, contains both the promoter per se, i.e.
  • promoter regions normally include 5' non-coding sequences involved in initiation of transcription and translation, such as the -35/- 10 boxes and the Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequences, and 5'-capping elements in eukaryotes. These regions can also include enhancer or repressor elements as well as translated signal and leader sequences for targeting the native polypeptide to a specific compartment of a host cell.
  • the 3' non-coding sequences may contain regulatory elements involved in transcriptional termination, polyadenylation or the like. If, however, these termination sequences are not satisfactory functional in a particular host cell, then they may be substituted with signals functional in that cell.
  • a nucleic acid molecule of the invention can include a regulatory sequence, preferably a promoter sequence.
  • a nucleic acid molecule of the invention comprises a promoter sequence and a transcriptional termination sequence.
  • Suitable prokaryotic promoters are, for example, the tet promoter, the ZacUV5 promoter or the T7 promoter. Examples of promoters useful for expression in eukaryotic cells are the SV40 promoter or the CMV promoter.
  • the nucleic acid molecules of the invention can also be part of a vector or any other kind of cloning vehicle, such as a plasmid, a phagemid, a phage, a baculovirus, a cosmid or an artificial chromosome.
  • the nucleic acid molecule is comprised in a phasmid.
  • a phasmid vector denotes a vector encoding the intergenic region of a temperent phage, such as Ml 3 or fl, or a functional part thereof fused to the cDNA of interest.
  • an appropriate helper phage e.g. M13K07, VCS-M13 or R408
  • intact phage particles are produced, thereby enabling physical coupling of the encoded heterologous cDNA to its corresponding polypeptide displayed on the phage surface (reviewed, e.g., in Kay, B.K. et al.
  • Such cloning vehicles can include, aside from the regulatory sequences described above and a nucleic acid sequence encoding a lipocalin mutein of the invention, replication and control sequences derived from a species compatible with the host cell that is used for expression as well as selection markers conferring a selectable phenotype on transformed or transfected cells. Large numbers of suitable cloning vectors are known in the art, and are commercially available.
  • the DNA molecule encoding lipocalin muteins of the invention, and in particular a cloning vector containing the coding sequence of such a lipocalin mutein can be transformed into a host cell capable of expressing the gene. Transformation can be performed using standard techniques (Sambrook, J. et al. (1989), supra).
  • the invention is also directed to a host cell containing a nucleic acid molecule as disclosed herein.
  • the transformed host cells are cultured under conditions suitable for expression of the nucleotide sequence encoding a fusion protein of the invention.
  • Suitable host cells can be prokaryotic, such as Escherichia coli (E. coli) or Bacillus subtilis, or eukaryotic, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells, immortalized mammalian cell lines (e.g. HeLa cells or CHO cells) or primary mammalian cells.
  • the invention also relates to a method for the production of a mutein of the invention, wherein the mutein, a fragment of the mutein or a fusion protein of the mutein and another polypeptide is produced starting from the nucleic acid coding for the mutein by means of genetic engineering methods.
  • the method can be carried out in vivo, the mutein can for example be produced in a bacterial or eucaryotic host organism and then isolated from this host organism or its culture. It is also possible to produce a protein in vitro, for example by use of an in vitro translation system.
  • a nucleic acid encoding a mutein of the invention is introduced into a suitable bacterial or eukaryotic host organism by means of recombinant DNA technology (as already outlined above).
  • the host cell is first transformed with a cloning vector comprising a nucleic acid molecule encoding a mutein of the invention using established standard methods (Sambrook, J. et al. (1989), supra).
  • the host cell is then cultured under conditions, which allow expression of the heterologous DNA and thus the synthesis of the corresponding polypeptide.
  • the polypeptide is recovered either from the cell or from the cultivation medium.
  • the present invention relates to a method for the generation of a mutein of the invention, comprising:
  • the method can further comprise:
  • mutagenesis means that the experimental conditions are chosen such that the amino acid naturally occurring at a given sequence position of Lcn2 (hNGAL; Swiss-Prot data bank entry P80188) can be substituted by at least one amino acid that is not present at this specific position in the respective natural polypeptide sequence.
  • mutagenesis also includes the (additional) modification of the length of sequence segments by deletion or insertion of one or more amino acids.
  • one amino acid at a chosen sequence position is replaced by a stretch of three random mutations, leading to an insertion of two amino acid residues compared to the length of the respective segment of the wild type protein.
  • Such an insertion of deletion may be introduced independently from each other in any of the peptide segments that can be subjected to mutagenesis in the invention.
  • an insertion of several mutations may be introduced into the loop AB of the chosen lipocalin scaffold (cf. International Patent Application WO 2005/019256 which is incorporated by reference its entirety herein).
  • random mutagenesis means that no predetermined single amino acid (mutation) is present at a certain sequence position but that at least two amino acids can be incorporated with a certain probability at a predefined sequence position during mutagenesis.
  • the coding sequence of human Lipocalin 2 is used as a starting point for the mutagenesis of the peptide segments selected in the present invention.
  • the person skilled in the art has at his disposal the various established standard methods for site-directed mutagenesis (Sambrook, J. et al. (1989), supra).
  • a commonly used technique is the introduction of mutations by means of PCR (polymerase chain reaction) using mixtures of synthetic oligonucleotides, which bear a degenerate base composition at the desired sequence positions.
  • codons for other amino acids such as selenocystein or pyrrolysine can also be incorporated into a nucleic acid of a mutein. It is also possible, 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, to use "artificial" codons such as UAG which are usually recognized as stop codons in order to insert other unusual amino acids, for example o-methyl-L-tyrosine or p-aminophenylalanine.
  • nucleotide building blocks with reduced base pair specificity as for example inosine, 8-oxo-2'deoxyguanosine or 6(2-deoxy-B-D-ribofuranosyl)-3,4-dihydro-8H- pyrimindo-l,2-oxazine-7-one (Zaccolo et al. (1996) J. Mol. Biol. 255, 589-603), is another option for the introduction of mutations into a chosen sequence segment.
  • a further possibility is the so-called triplet-mutagenesis.
  • This method uses mixtures of different nucleotide triplets, each of which codes for one amino acid, for incorporation into the coding sequence (Virnekas B, Ge L, Pliickthun A, Schneider KC, Wellnhofer G, Moroney SE. 1994 Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Res 22, 5600-5607).
  • One possible strategy for introducing mutations in the selected regions of the respective polypeptides is based on the use of four oligonucleotides, each of which is partially derived from one of the corresponding sequence segments to be mutated.
  • oligonucleotides each of which is partially derived from one of the corresponding sequence segments to be mutated.
  • a person skilled in the art can employ mixtures of nucleic acid building blocks for the synthesis of those nucleotide triplets which correspond to the amino acid positions to be mutated so that codons encoding all natural amino acids randomly arise, which at last results in the generation of a lipocalin peptide library.
  • the first oligonucleotide corresponds in its sequence - apart from the mutated positions - to the coding strand for the peptide segment to be mutated at the most N-terminal position of the lipocalin polypeptide.
  • the second oligonucleotide corresponds to the non-coding strand for the second sequence segment following in the polypeptide sequence.
  • the third oligonucleotide corresponds in turn to the coding strand for the corresponding third sequence segment.
  • the fourth oligonucleotide corresponds to the non-coding strand for the fourth sequence segment.
  • a polymerase chain reaction can be performed with the respective first and second oligonucleotide and separately, if necessary, with the respective third and fourth oligonucleotide.
  • both of these reactions can be combined by various known methods into a single nucleic acid comprising the sequence from the first to the fourth sequence segments, in which mutations have been introduced at the selected positions.
  • both of the products can for example be subjected to a new polymerase chain reaction using flanking oligonucleotides as well as one or more mediator nucleic acid molecules, which contribute the sequence between the second and the third sequence segment.
  • flanking oligonucleotides as well as one or more mediator nucleic acid molecules, which contribute the sequence between the second and the third sequence segment.
  • nucleic acid molecules defined above can be connected by ligation with the missing 5'- and 3'-sequences of a nucleic acid encoding a lipocalin polypeptide and/or the vector, and can be cloned in a known host organism.
  • a multitude of established procedures are available for ligation and cloning (Sambrook, J. et al. (1989), supra).
  • recognition sequences for restriction endonucleases also present in the sequence of the cloning vector can be engineered into the sequence of the synthetic oligonucleotides.
  • Longer sequence segments within the gene coding for the protein selected for mutagenesis can also be subjected to random mutagenesis via known methods, for example by use of the polymerase chain reaction under conditions of increased error rate, by chemical mutagenesis or by using bacterial mutator strains. Such methods can also be used for further optimization of the target affinity or specificity of a lipocalin mutein. Mutations possibly occurring outside the segments of experimental mutagenesis are often tolerated or can even prove to be advantageous, for example if they contribute to an improved folding efficiency or folding stability of the lipocalin mutein.
  • the above method includes subjecting the nucleic acid molecule encoding a human Lipocalin 2 protein to mutagenesis at least at 2 or 3 nucleotide triplets coding for any of the above indicated sequence positions of human Lipocalin 2.
  • the method further includes subjecting the nucleic acid molecule to mutagenesis at at least one nucleotide triplet coding for any of the sequence positions corresponding to the sequence positions 33, 36, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 55, 59, 65, 68, 70, 72, 73, 75, 77, 78, 79, 80, 81, 86, 87, 98, 96, 99, 100, 103, 106, 107, 110, 111, 125, 127, 132, 134, 136 and 138 of the linear polypeptide sequence of hNGAL.
  • the method includes subjecting the nucleic acid molecule to mutagenesis at nucleotide triplets coding for at least any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the sequence positions corresponding to the sequence positions 33, 36, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 55, 59, 65, 68, 70, 72, 73, 75, 77, 78, 79, 80, 81, 86, 87, 98, 96, 99, 100, 103, 106, 107, 110, 111, 125, 127, 132, 134, 136 and 138 of the linear polypeptide sequence of human Lipocalin 2.
  • a mutein is obtained starting from a nucleic acid encoding hNGAL.
  • a nucleic acid is subjected to mutagenesis and introduced into a suitable bacterial or eukaryotic host organism by means of recombinant DNA technology.
  • Obtaining a nucleic acid library of human Lipocalin 2 can be carried out using any suitable technique that is known in the art for generating lipocalin muteins with antibody-like properties, i.e. muteins that have affinity towards a given target.
  • 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.
  • step (c) further comprises in another embodiment of the above methods:
  • the target/ligand is a non-natural target which includes, but is not limited to a peptide, a protein, a fragment or a domain of a protein, or a small organic molecule.
  • the small organic molecule is a compound showing the features of an immunological hapten.
  • the peptide is an amyloid beta peptide, such as A ?40 peptide or an A ?42 peptide.
  • the non-natural targe is fibronectin or a domain thereof, such as the EB-domain or a fragment of the EB-domain.
  • the selection in step (c) is carried out under competitive conditions.
  • Competitive conditions as used herein means that selection of muteins encompasses at least one step in which the muteins and the given non- natural ligand of human Lipocalin 2 (target) are brought in contact in the presence of an additional ligand, which competes with binding of the muteins to the target.
  • This additional ligand may be a physiological ligand of the target, an excess of the target itself or any other non-physiological ligand of the target that binds at least an overlapping epitope to the epitope recognized by the muteins of the invention and thus interferes with target binding of the muteins.
  • the additional ligand competes with binding of the muteins by complexing an epitope distinct from the binding site of the muteins to the target by allosteric effects.
  • Ml 3 phagemids are produced which allow the expression of the mutated lipocalin nucleic acid sequence as a fusion protein with a signal sequence at the N-terminus, preferably the OmpA-signal sequence, and with the capsid protein pill of the phage M13 or fragments thereof capable of being incorporated into the phage capsid at the C-terminus.
  • the C-terminal fragment ⁇ of the phage capsid protein comprising amino acids 217 to 406 of the wild type sequence is preferably used to produce the fusion proteins.
  • a C-terminal fragment of pill in which the cysteine residue at position 201 is missing or is replaced by another amino acid.
  • a further embodiment of the methods of the invention involves operably fusing a nucleic acid coding for the plurality of muteins of human Lipocalin 2 and resulting from mutagenesis at the 3' end with a gene coding for the coat protein pill of a filamentous bacteriophage of the M13-family or for a fragment of this coat protein, in order to select at least one mutein for the binding of a given ligand.
  • the fusion protein may comprise additional components such as an affinity tag, which allows the immobilization, detection and/or purification of the fusion protein or its parts.
  • a stop codon can be located between the sequence regions encoding the lipocalin or its muteins and the phage capsid gene or fragments thereof, wherein the stop codon, preferably an amber stop codon, is at least partially translated into an amino acid during translation in a suitable suppressor strain.
  • the phasmid vector pTLPC27 now also called pTlc27 that is described here can be used for the preparation of a phagemid library encoding human Lipocalin 2 muteins.
  • the inventive nucleic acid molecules coding for the hNGAL muteins are inserted into the vector using the two BstXI restriction sites. After ligation a suitable host strain such as E. coli XL 1 -Blue is transformed with the resulting nucleic acid mixture to yield a large number of independent clones.
  • a respective vector can be generated for the preparation of a hyperphagemid library, if desired.
  • the resulting library is subsequently superinfected in liquid culture with an appropriate M13-helper phage or hyperphage in order to produce functional phagemids.
  • the recombinant phagemid displays the lipocalin mutein on its surface as a fusion with the coat protein pill or a fragment thereof, while the N-terminal signal sequence of the fusion protein is normally cleaved off.
  • it also bears one or more copies of the native capsid protein pill supplied by the helper phage and is thus capable of infecting a recipient, in general a bacterial strain carrying an F- or F'-plasmid.
  • the hyperphagemids display the lipocalin muteins on their surface as a fusion with the infective coat protein pill but no native capsid protein.
  • gene expression of the fusion protein between the lipocalin mutein and the capsid protein pill can be induced, for example by addition of anhydrotetracycline.
  • the induction conditions are chosen such that a substantial fraction of the phagemids obtained displays at least one lipocalin mutein on their surface.
  • hyperphage display induction conditions result in a population of hyperphagemids carrying between three and five fusion proteins consisting of the lipocalin mutein and the capsid protein pill.
  • Various methods are known for isolating the phagemids, such as precipitation with polyethylene glycol. Isolation typically occurs after an incubation period of 6-8 hours.
  • the isolated phasmids can then be subjected to selection by incubation with the desired target, wherein the target is presented in a form allowing at least temporary immobilization of those phagemids which carry muteins with the desired binding activity as fusion proteins in their coat.
  • the target can, for example, be conjugated with a carrier protein such as serum albumin and be bound via this carrier protein to a protein binding surface, for example polystyrene.
  • a carrier protein such as serum albumin
  • Microtiter plates suitable for ELISA techniques or so-called "immuno-sticks" can preferrably be used for such an immobilization of the target.
  • conjugates of the target with other binding groups can be used.
  • the target can then be immobilized on a surface which selectively binds this group, for example microtiter plates or paramagnetic particles coated with streptavidin, neutravidin or avidin. If the target is fused to an Fc portion of an immunoglobulin, immobilization can also be achieved with surfaces, for example microtiter plates or paramagnetic particles, which are coated with protein A or protein G.
  • Non-specific phagemid-binding sites present on the surfaces can be saturated with blocking solutions as they are known for ELISA methods.
  • the phagemids are then typically brought into contact with the target immobilized on the surface in the presence of a physiological buffer. Unbound phagemids are removed by multiple washings. The phagemid particles remaining on the surface are then eluted.
  • the phagemids can be eluted by addition of proteases or in the presence of acids, bases, detergents or chaotropic salts or under moderately denaturing conditions.
  • a preferred method is the elution using buffers of pH 2.2, wherein the eluate is subsequently neutralized.
  • a solution of the free target can be added in order to compete with the immobilized target for binding to the phagemids or target- specific phagemids can be eluted by competition with immunoglobulins or natural liganding proteins which specifically bind to the target of interest.
  • E. coli cells are infected with the eluted phagemids.
  • the nucleic acids can be extracted from the eluted phagemids and used for sequence analysis, amplification or transformation of cells in another manner.
  • fresh phagemids or hyperphagemids are again produced by superinfection with Ml 3 helper phages or hyperphage according to the method described above and the phagemids amplified in this way are once again subjected to a selection on the immobilized target. Multiple selection cycles are often necessary in order to obtain the phagemids with the muteins of the invention in sufficiently enriched form.
  • the number of selection cycles is preferably chosen such that in the subsequent functional analysis at least 0.1 % of the clones studied produce muteins with detectable affinity for the given target. Depending on the size, i.e. the complexity of the library employed, 2 to 8 cycles are typically required to this end.
  • an E. coli strain is infected with the phagemids obtained from the selection cycles and the corresponding double stranded phasmid DNA is isolated.
  • the nucleic acid sequences of the selected muteins of the invention can be determined by the methods known in the art and the amino acid sequence can be deduced therefrom.
  • the mutated region or the sequence of the entire hNGAL mutein can be subcloned on another expression vector and expressed in a suitable host organism.
  • the vector pTLPC26 now also called pTlc26 can be used for expression in E.
  • the muteins of human Lipocalin 2 thus produced can be purified by various biochemical methods.
  • the hNGAL muteins produced, for example with pTlc26, carry the affinity peptide Strep-tag II (Schmidt et al., supra) at their C-termini and can therefore preferably be purified by streptavidin affinity chromatography.
  • the selection can also be carried out by means of other methods. Many corresponding embodiments are known to the person skilled in the art or are described in the literature. Moreover, a combination of methods can be applied. For example, clones selected or at least enriched by "phage display” can additionally be subjected to "colony screening". This procedure has the advantage that individual clones can directly be isolated with respect to the production of a human Lipocalin 2 mutein with detectable binding affinity for a target.
  • error-prone PCR results in point mutations over a selected range of sequence positions of the lipocalin mutein.
  • the error-prone PCR can be carried out in accordance with any known protocol such as the one described by Zaccolo et al. (1996) J. Mol. Biol. 255, 589-603.
  • Other methods of random mutagenesis thatare suitable for such purposes include random insertion/deletion (RID) mutagenesis as described by Murakami et al. (2002) Nat. Biotechnol. 20, 76-81 or nonhomologous random recombination (NRR) as described by Bittker et al. (2002) Nat. Biotechnol.
  • affinity maturation can also be carried out according to the procedure described in WO 00/75308 or Schlehuber et al. (2000) J. Mol. Biol. 297, 1105- 1120, where muteins of the bilin-binding protein having high affinity to digoxigenin were obtained.
  • a further approach for improving the affinity is to carry out positional saturation mutagenesis.
  • "small" nucleic acid libraries can be created in which amino acid exchanges/mutations are only introduced at single positions within any of the four loop segments. These libraries are then directly subjected to a selection step (affinity screening) without further rounds of panning. This approach allows the identification of residues that contribute to improved binding of the desired target and allows identification of "hot spots" that are important for the binding.
  • the above method for modifying a mutein further includes introducing a Cys residue at at least one of any of the sequence positions that correspond to sequence positions 14, 21, 60, 84, 88, 116, 141, 145, 143, 146 or 158 of the wild type sequence of human Lipocalin 2 and coupling a moiety that is able to modify the serum half time of said mutein via the thiol group of a Cys residue introduced at at least one of any of the sequence positions that correspond to sequence positions 14, 21, 60, 84, 88, 116, 141, 145, 143, 146 or 158 of the wild type sequence of hNGAL.
  • the moiety that is able to modify the serum half time of said mutein may be selected from the group consisting of a polyalkylene glycol molecule and hydroxyethylstarch.
  • the present invention is directed to a mutein of human Lipocalin 2 having detectable binding affinity to a given non-natural ligand of human Lipocalin 2, which is obtainable by or obtained by the above-detailed methods of the invention.
  • muteins of the invention the naturally occurring disulfide bond between Cys 76 and Cys 175 is removed. Accordingly, such muteins (or any other human Lipocalin 2 mutein that does not comprise an intramolecular disulfide bond) can be produced in a cell compartment having a reducing redox milieu, for example, in the cytoplasma of Gram-negative bacteria.
  • a lipocalin mutein of the invention comprises intramolecular disulfide bonds
  • an oxidizing environment may be provided by the periplasm of Gram-negative bacteria such as E. coli, in the extracellular milieu of Gram-positive bacteria or in the lumen of the endoplasmatic reticulum of eukaryotic cells and usually favors the formation of structural disulfide bonds.
  • a mutein of the invention in the cytosol of a host cell, preferably E. coli.
  • the polypeptide can either be directly obtained in a soluble and folded state or recovered in form of inclusion bodies, followed by renaturation in vitro.
  • a further option is the use of specific host strains having an oxidizing intracellular milieu, which may thus allow the formation of disulfide bonds in the cytosol (Venturi et al. (2002) J. Mol. Biol. 315, 1-8.).
  • a mutein of the invention may not necessarily be generated or produced only by use of genetic engineering.
  • a lipocalin mutein can also be obtained by chemical synthesis such as Merrifield solid phase polypeptide synthesis or by in vitro transcription and translation. It is for example possible that promising mutations are identified using molecular modeling and then to synthesize the wanted (designed) polypeptide in vitro and investigate the binding activity for a given target.
  • Methods for the solid phase and/or solution phase synthesis of proteins are well known in the art (reviewed, e.g., in Lloyd- Williams et al. (1997) Chemical Approaches to the Synthesis of Peptides and Proteins. CRC Press, Boca Raton, Fields, GB, and Colowick (1997) Solid-Phase Peptide Synthesis. Academic Press, San Diego, or Bruckdorfer et al. (2004) Curr. Pharm. Biotechnol. 5, 29-43).
  • the muteins of the invention may be produced by in vitro transcription/translation employing well-established methods known to those skilled in the art.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one inventive mutein referred to in the claims or a fusion protein or conjugates thereof and, optionally, a pharmaceutically acceptable excipient.
  • the lipocalin muteins according to the invention can be administered via any parenteral or non-parenteral (enteral) route that is therapeutically effective for proteinaceous drugs.
  • Parenteral application methods comprise, for example, intracutaneous, subcutaneous, intramuscular or intravenous injection and infusion techniques, e.g. in the form of injection solutions, infusion solutions or tinctures, as well as aerosol installation and inhalation, e.g. in the form of aerosol mixtures, sprays or powders.
  • Non-parenteral delivery modes are, for instance, orally, e.g. in the form of pills, tablets, capsules, solutions or suspensions, or rectally, e.g. in the form of suppositories.
  • the muteins of the invention can be administered systemically or topically in formulations containing conventional non-toxic pharmaceutically acceptable excipients or carriers, additives and vehicles as desired.
  • the pharmaceutical is administered parenterally to a mammal, and in particular to humans.
  • Corresponding administration methods include, but are not limited to, for example, intracutaneous, subcutaneous, intramuscular or intravenous injection and infusion techniques, e.g. in the form of injection solutions, infusion solutions or tinctures as well as aerosol installation and inhalation, e.g. in the form of aerosol mixtures, sprays or powders.
  • a combination of intravenous and subcutaneous infusion and /or injection might be most convenient in case of compounds with a relatively short serum half life.
  • the pharmaceutical composition may be an aqueous solution, an oil-in water emulsion or a water-in-oil emulsion.
  • transdermal delivery technologies e.g. iontophoresis, sonophoresis or microneedle-enhanced delivery, as described in Meidan and Michniak (2004) Am. J. Ther. 11(4), 312-316, can also be used for transdermal delivery of the muteins described herein.
  • Non-parenteral delivery modes are, for instance, oral, e.g. in the form of pills, tablets, capsules, solutions or suspensions, or rectal administration, e.g. in the form of suppositories.
  • the muteins of the invention can be administered systemically or topically in formulations containing a variety of conventional non-toxic pharmaceutically acceptable excipients or carriers, additives, and vehicles.
  • the dosage of the mutein applied may vary within wide limits to achieve the desired preventive effect or therapeutic response. It will, for instance, depend on the affinity of the compound for a chosen ligand as well as on the half-life of the complex between the mutein and the ligand in vivo. Further, the optimal dosage will depend on the biodistribution of the mutein or its fusion protein or its conjugate, the mode of administration, the severity of the disease/disorder being treated as well as the medical condition of the patient. For example, when used in an ointment for topical applications, a high concentration of the human Lipocalin 2 mutein can be used.
  • the mutein may also be given in a sustained release formulation, for example liposomal dispersions or hydrogel-based polymer microspheres, like PolyActiveTM or OctoDEXTM (cf. Bos et al., Business Briefing: Pharmatech 2003: 1-6).
  • a sustained release formulation for example liposomal dispersions or hydrogel-based polymer microspheres, like PolyActiveTM or OctoDEXTM (cf. Bos et al., Business Briefing: Pharmatech 2003: 1-6).
  • the muteins of the present invention can be formulated into compositions using pharmaceutically acceptable ingredients as well as established methods of preparation (Gennaro and Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA).
  • pharmaceutically inert inorganic or organic excipients can be used.
  • pills, powders, gelatine capsules or suppositories for example, lactose, talc, stearic acid and its salts, fats, waxes, solid or liquid polyols, natural and hardened oils can be used.
  • Suitable excipients for the production of solutions, suspensions, emulsions, aerosol mixtures or powders for reconstitution into solutions or aerosol mixtures prior to use include water, alcohols, glycerol, polyols, and suitable mixtures thereof as well as vegetable oils.
  • the pharmaceutical composition may also contain additives, such as, for example, fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers, and furthermore solvents or solubilizers or agents for achieving a depot effect.
  • additives such as, for example, fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers, and furthermore solvents or solubilizers or agents for achieving a depot effect.
  • fusion proteins may be incorporated into slow or sustained release or targeted delivery systems, such as liposomes and microcapsules.
  • the formulations can be sterilized by numerous means, including filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile medium just prior to use.
  • a mutein of the present invention or a fusion protein or a conjugate thereof can be employed in many applications. In general, such a mutein can be used in all applications antibodies are used, except those with specifically rely on the glycosylation of the Fc part.
  • the invented muteins of human Lipocalin 2 are used for the binding and/or detection of a given non-natural ligand of human Lipocalin 2.
  • Such use may comprise the steps of contacting the mutein with a sample suspected of containing the given ligand under suitable conditions, thereby allowing formation of a complex between the mutein and the given ligand, and detecting the complexed mutein by a suitable signal.
  • the detectable signal can be caused by a label, as explained above, or by a change of physical properties due to the binding, i.e. the complex formation, itself.
  • a label as explained above
  • a change of physical properties due to the binding i.e. the complex formation, itself.
  • plasmon surface resonance the value of which is changed during binding of binding partners from which one is immobilized on a surface such as a gold foil.
  • the muteins of human Lipocalin 2 disclosed herein may also be used for the separation of a given non-natural ligand of human Lipocalin 2. Such use may comprise the steps of contacting the mutein with a sample supposed to contain said ligand under suitable conditions, thereby allowing formation of a complex between the mutein and the given ligand, and separating the mutein/ligand complex from the sample.
  • the mutein and/or the target may be immobilized on a suitable solid phase.
  • the human Lipocalin 2 muteins of the invention may also be used to target a compound to a pre-selected site.
  • a mutein of human Lipocalin 2 is used for the targeting of a pharmaceutically active compound to a pre-selected site in an organism or tissue, comprising of:
  • the mutein is contacted with the compound of interest in order to allow complex formation. Then the complex comprising the mutein and the compound of interest are delivered to the pre-selected site. This may, for example, be achieved by coupling the mutein to a targeting moiety, such as an antibody, antibody fragment or lipocalin mutein or lipocalin mutein fragment wit binding affinity for the selected target.
  • a targeting moiety such as an antibody, antibody fragment or lipocalin mutein or lipocalin mutein fragment wit binding affinity for the selected target.
  • This use is in particular suitable, but not restricted to, for delivering a drug (selectively) to a pre-selected site in an organism, such as an infected body part, tissue or organ which is supposed to be treated with the drug.
  • the mutein can also be reacted with the given compound to yield a conjugate of mutein and compound. Similar to the above complex, such a conjugate may be suitable to deliver the compound to the pre-selected target site.
  • Such a conjugate of mutein and compound may also include a linker that covalently links mutein and compound to each other.
  • a linker is stable in the bloodstream but is cleavable in a cellular environment.
  • muteins disclosed herein and its derivatives can thus be used in many fields similar to antibodies or fragments thereof.
  • the muteins can be used for labeling with an enzyme, an antibody, a radioactive substance or any other group having biochemical activity or defined binding characteristics.
  • their respective targets or conjugates or fusion proteins thereof can be detected or brought in contact with them.
  • muteins of the invention can serve to detect chemical structures by means of established analytical methods (e.g. ELISA or Western Blot) or by microscopy or immunosensorics.
  • the detection signal can either be generated directly by use of a suitable mutein conjugate or fusion protein or indirectly by immunochemical detection of the bound mutein via an antibody.
  • a mutant polypeptide of the invention which binds, for example, tissue- or tumor-specific cellular surface molecules can be generated.
  • Such a mutein may, for example, be employed in conjugated form or as a fusion protein for "tumor imaging" or directly for cancer therapy.
  • the present invention also involves the use of the human Lipocalin 2 muteins of the invention for complex formation with a given non-natural ligand or target.
  • the present invention also encompasses the use of a mutein according to the invention for the manufacture of a pharmaceutical composition.
  • the pharmaceutical composition thus obtained may be suited for the treatment of neugrodegenerative disorders, such as Alzheimer disease.
  • the pharmaceutical composition can also be used for the treatment of cancer, the treatment of fibrosis ro the treatment of an inflammation.
  • the pharmaceutical composition may be used as monotherapy or as combination therapy.
  • the present invention features a diagnostic or analytical kit comprising a mutein according to the present invention.
  • Another aspect of the present invention relates to a method of treating a subject suffering from a neurodegenerative disease, or cancer, or fibrosis, or an inflammation, including administering a respective mutein of the invention or a pharmaceutical composition comprising a mutein of the invention to a subject in need thereof.
  • the subject in need of such a treatment may be a mammal, such as a human, a dog, a mouse, a rat, a pig, an ape such as cymologous monkeys to name only a few illustrative examples.
  • the present invention features a method for in vivo imaging in a subject, including administering to said subject a mutein of the invention or a pharmaceutical composition comprising a mutein of the invention.
  • the subject may be defined as above.
  • Figure 1 illustrates the PCR assembly strategy for the simultaneous random mutagenesis of the 20 amino acid positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79 81, 96, 100, 103, 106, 125, 127, 132, and 134 (underlined and numbered) in the amino acid sequence of the mature Lcn2. These 20 positions were divided into four sequence subsets. For randomization of the amino acids in each subset an oligodeoxynucleotide was synthesized (SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4) wherein NNK mixtures of the nucleotides were employed at the mutated codons.
  • N means a mixture of all four bases A, C, G, and T while K means a mixture of only the two bases G and T; hence such a triplet encodes all 20 natural amino acids as well as the amber stop codon TAG, which is translated as glutamine in the E. coli supE-strains XLl-blue (Bullock et al., BioTechniques 5 (1987), 376-378) or TGI (Sambrook et al., Molecular Cloning. A Laboratory Manual (1989), Cold Spring Harbor Press) that were used for phagemid production and gene expression.
  • the two flanking primers each encompassed a BstXI restriction site (CCANNNNNNTGG), giving rise to mutually non-compatible overhangs upon enzyme digest.
  • This special arrangement of restriction sites enabled a particularly efficient ligation and cloning of the synthetic gene.
  • Substitution of the amino acid Gln28 to His with respect to the original Lcn2 sequence was necessary to introduce the first BstXI site, while the second one naturally occurs in the cDNA of Lcn2.
  • the unpaired residue Cys87 was replaced by Ser during the gene assembly.
  • the resulting gene fragment was inserted into a vector providing the missing parts of the Lcn2 structural gene.
  • This illustration also depicts two short primers (SEQ ID NO: 45 and SEQ ID NO: 46) upstream and downstream, respectively, of the cassette flanked by the two BstXI restriction sites, which served for double stranded DNA sequencing.
  • Figure 2 illustrates the nucleotide sequence of a library of synthetic Lcn2 genes (only the central cassette flanked by the two BstXI restriction sites, as in Figure 1, is shown). This gene fragment was prepared by Sloning BioTechnology GmbH. Compared with the DNA library described in Figure 1 there are two differences. First, whenever possible codons optimized for E. coli expression were used throughout for the non-mutated amino acid positions.
  • Figure 3 shows the SEC elution profiles of the recombinant ⁇ fusion proteins ⁇ - ⁇ 28 (A) and ⁇ - ⁇ 40 (B) after expression in E. coli.
  • the purity of both the synthetic ⁇ 40 and the recombinant ⁇ fusion proteins was assessed by SDS-PAGE analysis (C). Both fusion proteins were expressed in the cytoplasm of E. coli JM83. After disruption of the cells with a French Pressure Cell the proteins were purified via His 6 -tag affinity chromatography and then further applied to a Superdex 75 HR 10/30 column in the case of ⁇ - ⁇ 28 or to a Superdex 200 HR 10/30 column in the case of ⁇ - ⁇ 40.
  • Figure 4 represents an overlay of the SEC elution profiles of the Lcn2 muteins Hl- Gl, S1-A4, and US7 (A) and the analysis of the purified proteins via 15% SDS-PAGE (B).
  • the three Lcn2 muteins Hl-Gl, S1-A4, and US7 were expressed in the periplasm of E. coli JM83 or TGl-F " (as shown here) and purified via Strep-tag II affinity chromatography. All three muteins eluted predominantly as monomeric proteins from the Superdex 75 HR 10/30 column with a retention volume of 10 to 11 ml. Peak intensities differed depending on the expression yield of the particular mutein.
  • (B) shows a 15% SDS-PAGE analysis of recombinant wild-type Lcn2 (lanes 1, 5) and the muteins US7 (lanes 2, 6), Hl-Gl (lanes 3, 7), and S1-A4 (lanes 4, 8) after Strep-tag II affinity purification and SEC.
  • Lanes 1-4 show the Lcn2 muteins reduced with 2-mercaptoethanol, lanes 5-8 show the proteins under non- reducing conditions.
  • M represents the molecular weight marker with the corresponding band sizes in kDa displayed on the left of the gel.
  • Figure 5 depicts the binding activity of the Lcn2 muteins Hl-Gl, S1-A4, and US7 in capture ELISAs with different biotinylated ⁇ targets.
  • StrepMAB-Immo at 10 g/ml was immobilized on microtitre plates and employed for capturing of 1 ⁇ Lcn2 muteins via the Strep-tag II.
  • a to C the binding of the Lcn2 muteins S1-A4 and US7 to (A) biotinylated ⁇ 40, (B) biotinylated ⁇ - ⁇ 28, and (C) biotinylated ⁇ - ⁇ 40 are shown.
  • Binding was detected by incubation with ExtrAvidin/AP and a subsequent chromogenic reaction with pNPP.
  • Ovalbumin (Ova), thioredoxin (Trx), and maltose binding protein (MBP) were used as negative controls. Additionally, binding of wild- type (wt) lipocalin was tested. The data were fitted to a monovalent binding model.
  • Figure D shows the same ELISA setup performed with Lcn2 Hl-Gl and biotinylated ⁇ 40 and ⁇ - ⁇ 28 as targets.
  • K D values determined for ⁇ 40 were 2.7 nM for S1-A4, 6.8 nM for US7, and 16.2 nM for Hl-Gl, corresponding values for ⁇ - ⁇ 28 were 1.9 nM, 2.4 nM, and 24.3 nM. Binding to MBP- ⁇ 40 showed a K D value of 4.7 nM for S1-A4 and 11.4 nM for US7. Accordingly K D values for ⁇ 16-27 were 2.6 nM and 2.1 nM.
  • Figure 6 shows the binding activity of the Lcn2 muteins S1-A4 and US7 in a direct ELISA.
  • the ⁇ targets ⁇ - ⁇ 28 and ⁇ - ⁇ 40 as well as the control protein maltose binding protein (MBP) were immobilized at 2 ⁇ in PBS over night.
  • Bound Lcn2 muteins were detected via their Strep-tag II using Streptavidin/AP followed by a chromogenic reaction with pNPP.
  • wt wild-type lipocalin was tested.
  • the data were fitted to a monovalent binding model.
  • K D values determined for ⁇ - ⁇ 28 were 16.2 nM for S1-A4 and 9.6 nM for US7, corresponding values for ⁇ - ⁇ 40 were 149 nM and 49.7 nM.
  • Figure 7 shows the binding activity of the Lcn2 muteins S1-A4 (A) and US7 (B) in a competitive ELISA.
  • StrepMAB-Immo was immobilized at 10 g/ml on microtitre plates and employed for capturing of 1 ⁇ Lcn2 mutein via the Strep-tag II.
  • a constant concentration of biotinylated ⁇ - ⁇ 28 as tracer was mixed with varying concentrations of unlabeled ⁇ - ⁇ 28.
  • Bound biotinylated ⁇ - ⁇ 28 was subsequently detected via incubation with ExtrAvidin/AP followed by a chromogenic reaction with pNPP.
  • the data were fitted using a sigmoidal equation.
  • the K D values were 21.7 nM for S1-A4 and 76.9 nM for US7.
  • Figure 8 depicts the kinetic real time analysis of Lcn2 muteins S1-A4 (A) and US7 (B) measured on a Biacore instrument at a flow rate of 10 ⁇ /min.
  • the measured signal is shown as a grey line whereas the curve fit is depicted as a black line in each case.
  • Figure 9 shows the functional activity of the Lcn2 muteins S1-A4 and US7 tested in a thioflavin T aggregation assay.
  • 100 ⁇ ⁇ 40 was incubated with 10 ⁇ of US7, S1-A4, wild-type (wt) lipocalin or with PBS at 37 °C without shaking.
  • 20 ⁇ of each sample were mixed with 180 ⁇ of 5 ⁇ thioflavin T and fluorescence was measured with an excitation wavelength of 450 nm and an emission wavelength of 482 nm.
  • the Lcn2 mutein US7 showed potent inhibition of aggregation at a ratio of 1: 10 (US7:A 40).
  • S1-A4 was not able to significantly inhibit aggregation at this ratio but exerted clear inhibition at a ratio of 1:2 (81- ⁇ 4: ⁇ 40) (not shown).
  • the wild type Lcn2 did not reveal an inhibitory effect on ⁇ aggregation.
  • Figure 10 shows SDS-PAGE analysis, after staining with Coomassie brilliant blue, of recombinant ED-B (lane 1), FN7B8 (lane 2), and FN789 (lane 3) after ion exchange chromatography. All samples were reduced with 2-mercaptoethanol.
  • M molecular weight marker (Fermentas, St. Leon-Rot, Germany).
  • Figure 11 shows SDS-PAGE analysis, after staining with Coomassie brilliant blue, of Lcn2 muteins N7A (lane 1), N7E (lane 2), N9B (lane 3), and N10D (lane 4) after Strep-tag II affinity purification. All samples were reduced with 2-mercaptoethanol.
  • M molecular weight marker (Fermentas, St. Leon-Rot, Germany).
  • Figure 12 depicts binding activity in the ELISA.
  • a microtiter plate was coated with the purified proteins FN7B8 and FN789 and incubated with a dilution series of the selected Lcn2 muteins, followed by detection with Streptavidin-alkaline-phosphatase conjugate and pNPP substrate.
  • Recombinant Lcn2 muteins N7A, N9B, and N10D revealed negligible signals in this assay for FN789, which lacked the ED-B and served as a negative control.
  • Figure 13 depicts the kinetic real time analysis of Lcn2 mutein N9B measured on a Biacore instrument.
  • the measured signal is shown as a grey line whereas the curve fit is depicted as a black line in each case.
  • the kinetic constants determined from this set of curves are listed in Table 2 (Example 17).
  • Figure 14 depicts the kinetic real time analysis of Lcn2 mutein N7E measured on a Biacore instrument.
  • Figure 15 depicts the kinetic real time analysis of Lcn2 mutein N7A measured on a Biacore instrument.
  • the measured signal is shown as a grey line whereas the curve fit is depicted as a black line in each case.
  • the kinetic constants determined from this set of curves are listed in Table 2 (Example 17).
  • Figure 16 depicts the kinetic real time analysis of Lcn2 mutein N10D measured on a Biacore instrument.
  • the measured signal is shown as a grey line whereas the curve fit is depicted as a black line in each case.
  • the kinetic constants determined from this set of curves are listed in Table 2 (Example 17).
  • Figure 17 illustrates a sequence alignment of human Lcn2 muteins designated Sl- A4 (SEQ ID NO: 39), US-7 (SEQ ID NO: 41), Hl-Gl (SEQ ID NO: 43), N7A (SEQ ID NO: 20), N7E (SEQ ID NO: 22), N9B (SEQ ID NO: 24) and N10D (SEQ ID NO: 26) with Lcn2.
  • Sl- A4 SEQ ID NO: 39
  • US-7 SEQ ID NO: 41
  • Hl-Gl SEQ ID NO: 43
  • N7A SEQ ID NO: 20
  • N7E SEQ ID NO: 22
  • N9B SEQ ID NO: 24
  • N10D SEQ ID NO: 26
  • Figure 18 represents an overlay of the SEC elution profiles of the Lcn2 muteins HIGA and HIGV (A), and the analysis of the purified proteins via 15% SDS-PAGE (B).
  • the two Lcn2 muteins HIGA and HIGV were expressed in the periplasm of E. coli JM83 and purified via Strep-tag II affinity chromatography. Both muteins eluted predominantly as monomeric proteins from the Superdex 75 HR 10/30 column with a retention volume of 10 to 11 ml. Peak intensities differed depending on the expression yields of the muteins.
  • (B) shows a 15% SDS-PAGE analysis of recombinant Lcn2 muteins Hl-Gl, HIGA, and HIGV after Strep-tag II affinity purification and SEC. All three Lcn2 muteins are shown under reduced conditions using 2-mercaptoethanol. M represents the molecular weight marker.
  • Figure 19 depicts the binding activity of the Lcn2 muteins HIGA, HIGV, and Hl- Gl in capture ELISAs with the biotinylated ⁇ targets ⁇ 40 (A) and ⁇ - ⁇ 28 (B).
  • Sire/?MAB-Immo was immobilized at 10 g/ml on microtitre plates and employed for capturing of 1 ⁇ Lcn2 muteins via the Strep-tag II. Binding of the biotinylated targets, applied in a dilution series, was then detected by incubation with ExtrAvidin/AP and a subsequent chromogenic reaction with pNPP.
  • Biotinylated ovalbumin (Ova) and thioredoxin (Trx) served as negative controls and exhibited no binding to the Lcn2 muteins (not shown).
  • the data were fitted to a monovalent binding model.
  • K D values determined for ⁇ 40 were 4.2 nM for HIGA, 4.9 nM for H1GV, and 21.5 nM for Hl-Gl ; corresponding values for Trx- ⁇ 28 were 3.8 nM, 4.2 nM, and 24.4 nM, respectively.
  • Figure 20 depicts the kinetic real time analysis of Lcn2 muteins HIGA (A) and H1GV (B) measured on a Biacore X instrument at a flow rate of 20 ⁇ /min.
  • the measured signal is shown as a grey line while the curve fit is depicted as a black line in each case.
  • the overall KD values were 0.476 nM for HIGA and 1.06 nM for H1GV.
  • Figure 21 depicts the kinetic real time analysis of the Lcn2 mutein HIGA measured on a Biacore T100 instrument with immobilized ⁇ 40 using an extended dissociation time for a trace measured at high concentration.
  • ARU 325 chip
  • kon rates were determined using the dilution series of the Lcn2 mutein HIGA with both association and dissociation times of 300 s.
  • Figure 22 (A) shows the functional activity of the Lcn2 mutein HIGA tested in a thioflavin T aggregation assay.
  • 500 ⁇ of 1 mg/ml monomeric ⁇ was incubated in the absence or presence of different molar ratios of Lcn2 mutein HIGA in 0.5 x PBS at 37 °C with stirring. Aggregation reactions were prepared in triplicates.
  • 20 ⁇ of a sample taken at periodic intervals was each mixed with 180 ⁇ ThT at a final concentration of 50 ⁇ in 0.5 x PBS and analysed at an excitation wavelength of 450 nm and an emission wavelength of 482 nm.
  • the Lcn2 mutein HIGA showed potent inhibition of aggregation at an equimolar ratio. At a subequimolar ratio of 10:2 ( ⁇ 40: ⁇ 1 ⁇ ) aggregation was still significantly reduced. (B) In contrast, equimolar amounts of neither BSA nor Lcn2 as negative controls had an inhibitory effect on the aggregation of ⁇ 40.
  • Figure 23 summarizes the KD values determined in the different ELISA setups as well as in surface plasmon resonance measurements for the Lcn2 muteins HIGA and H1GV.
  • Figure 24 illustrates a sequence alignment of the Lcn2 muteins S1-A4 (SEQ ID NO: 39), US7 (SEQ ID NO: 41), Hl-Gl (SEQ ID NO: 43), H1GA (SEQ ID NO: 50), H1GV (SEQ ID NO: 52), N7A (SEQ ID NO: 20), N7E (SEQ ID NO: 22), N9B (SEQ ID NO: 24), and N10D (SEQ ID NO: 26) with Lcn2 (SEQ ID NO: 44).
  • Figure 25 illustrates a mutual sequence alignment of the muteins H1GA (SEQ ID NO: 50) and H1GV (SEQ ID NO: 52) with their precursor Hl-Gl (SEQ ID NO: 43) and the wt Lcn2 (SEQ ID NO: 44).
  • Figure 26 illustrates specific binding activity of the Lcn2 Variant N7E for FN7B8 in an ELISA.
  • a microtiter plate was coated with FN7B8, FN789, BSA or ovalbumin and incubated with a dilution series of N7E, followed by detection with streptavidin-alkaline- phosphatase conjugate and pNPP substrate.
  • the Lcn2 variant N7E revealed negligible signals in this assay for FN789, BSA and ovalbumin.
  • Figure 27 depicts the kinetic real time analysis of Lcn2 variants N7A (A), N7E (B), N9B (C) and N10D (D) measured on a Biacore X instrument.
  • the measured signals are shown as traces together with the curve fits.
  • the kinetic constants determined from these sensorgrams are listed in Table 3 (Example 24).
  • Figure 28 depicts the analytical size exclusion chromatography of Lcn2 variants N7A (A), N10D (B), N9B (C) and N7E (D).
  • Affinity-purified protein was applied to a Superdex S75 10/30 column equilibrated with TBS. The arrows indicate the exclusion volume of the column (6.7 ml).
  • Analytical gel filtration resulted in a prominent peak for each of the four Lcn2 variants.
  • the apparent molecular weight of the proteins was 21.0 kDa for N7A, 21.3 kDa for N10D, 21.7 kDa for N9B and 21.7 kDa for N7E, indicating the presence of only monomeric species.
  • Example 1 Construction of a mutant Lcn2 phage display library
  • Oligodeoxynucleotides were designed such that the primers with SEQ ID NO: 1- 4 corresponded to the coding strand and carried degenerate codons at the amino acid positions 36, 40, 41, 49, 52, or 68, 70, 72, 73, 77, 79, 81, or 96, 100, 103, 106, or 125, 127, 132, 134 respectively, while primers with SEQ ID NO: 5-8 corresponded to the non-coding strand and did not carry degenerate codons or anticodons.
  • the two flanking primers with SEQ ID NO: 9 and SEQ ID NO: 10 were used in excess and served for the amplification of the assembled randomized gene fragment. All PCR steps were performed using Go-Taq Hot Start DNA polymerase (Promega, Mannheim, Germany) as described (Schlehuber et al. (2000) J. Mol. Biol. 297, 1105-1120).
  • Oligodeoxynucleotides that did not carry degenerate codons were purchased in HPLC grade from Metabion (Munich, Germany). NNK-containing oligodeoxynucleotides were purchased desalted from the same vendor and further purified by urea PAGE. The resulting DNA library was cut with BstXI (Promega, Mannheim, Germany) and cloned on the phagemid vector phNGAL102 (SEQ ID NO: 11), which is based on the generic expression vector pASKl l l (Vogt and Skerra (2001) J. Mol. Recognit.
  • phNGAL108 which is based on the generic expression vector pASK75 (Skerra (1994) Gene 151, 131-135), codes for a fusion protein composed of the OmpA signal peptide, the modified mature Lcn2, the Strep-tag followed by an amber codon, and the full length gene III coat protein of the filamentous bacteriophage M13 (Vogt and Skerra (2004) ChemBioChem 5, 191-199).
  • VCS-M13 helper phage (Agilent Technologies, La Jolla, USA) at a multiplicity of infection of approximately 10
  • the culture was shaken for additional 30 min at 37 °C, 100 rpm.
  • the incubator temperature was lowered to 26 °C and the shaker speed was increased again to 160 rpm, after 10 min kanamycin (70 ⁇ g/ml) was added, followed by induction of gene expression via addition of anhydrotetracycline (ACROS Organics, Geel, Belgium) at 25 ⁇ g/l (125 ⁇ of a 200 ⁇ g/ml stock solution in dimethylformamide, DMF per liter of culture).
  • ACROS Organics anhydrotetracycline
  • Example 2 Preparation of ⁇ targets in different formats
  • ⁇ 40 peptide (SEQ ID NO: 29), corresponding to amino acids 1 to 40 of the mature beta- amyloid sequence (Dodel et al. (2003) Lancet Neurology 2, 215-220), was purchased as synthetic, lyophilized peptide either with a C-terminal biotin group attached via a lysine spacer (Peptide Speciality Laboratory, Heidelberg, Germany) or in a non-labeled form (W. M. Keck Laboratory, New Haven, USA).
  • Homogeneously monomeric ⁇ 40 was obtained by dissolving up to 5 mg of the peptide in 0.5 mL of 1,1, 1,3,3, 3-hexafluoro-2- propanol (HFIP; Sigma-Aldrich, Steinheim, Germany) at room temperature for at least 0.5 h. Subsequently, HFIP was evaporated in a SpeedVac concentrator, and ⁇ 40 was dissolved in a suitable volume of distilled H 2 0 followed by sonication (Bandelin, Sonorex, RK100, Germany) in cold water for 15 min.
  • HFIP 1,1, 1,3,3, 3-hexafluoro-2- propanol
  • beta- amyloid peptide such as the N-terminal peptide ⁇ - 11 (SEQ ID NO: 30) and the central peptide ⁇ 16-27 (SEQ ID NO: 31), were also purchased from the Peptide Speciality Laboratory. These shorter versions were dissolved straight in the buffer of choice without previous HFIP treatment.
  • a fusion protein with the maltose binding protein (MBP, pMBP-His, SEQ ID NO: 32) was constructed according to Hortschansky et al. ((2005) Protein Sci. 14, 1753-1759) resulting in pASK75- MBP-Abeta40 (SEQ ID NO: 33).
  • pASK75-TrxAbeta28H6 (SEQ ID NO: 34), in which amino acids 1 to 28 of the mature amyloid peptide were inserted into the active site loop of thioredoxin (Trx, pASK75-TrxH6, SEQ ID NO: 35), was constructed according to Moretto et al. ((2007) J. Biol. Chem. 282, 11436-11445).
  • the cell pellets from a 2 L culture were resuspended in 20 ml lysis buffer (100 mM Tris/HCl pH 8.0, 50 mM NaCl, 1 mM EDTA) and the resulting suspension was homogenized by three passages through a French Pressure Cell. Insoluble material was removed by centrifugation (34500 g, 4 °C, 20 min), the supernatant was filtered with a 0.45 ⁇ filter (Filtropur S 0.45; Sarstedt, Nuembrecht, Germany) and used for affinity purification via the His 6 -tag of each protein. After immobilized metal affinity chromatography (IMAC) the fusion proteins were further purified via size exclusion chromatography (SEC).
  • IMAC immobilized metal affinity chromatography
  • Protein concentrations were determined by absorption measurement at 280 nm using calculated extinction coefficients of 66350 M ' 1 for MBP (SEQ ID NO: 32) 69330 M-'cm "1 for ⁇ - ⁇ 40 (SEQ ID NO: 33), 15470 M ' 1 for ⁇ - ⁇ 28 (SEQ ID NO: 34), and 13980 M ' 1 for Trx (SEQ ID NO: 35) (Gasteiger et al., supra).
  • D-biotinoyl-e-aminocaproic acid-N-hydroxysuccinimide ester or digoxigenin-3-0-methylcarbonyl-e-aminocaproic acid- N-hydroxysuccinimide ester (Roche Diagnostics, Mannheim, Germany) dissolved in water- free dimethylformamide (DMF; Sigma-Aldrich, Steinheim, Germany) or dimethylsulfoxide (DMSO; Sigma-Aldrich, Steinheim, Germany) was added in a 2-fold molar ratio to the protein in PBS (4 mM KH 2 P0 4 , 16 mM Na 2 HP0 4 , 115 mM NaCl, pH 7.4).
  • DMF water- free dimethylformamide
  • DMSO dimethylsulfoxide
  • Example 3 Selection of Lcn2 muteins with affinity to the ⁇ 40 peptide by phage display
  • Streptavidin-coated magnetic bead suspension Portions of 50 and of 25 ⁇ L of Streptavidin-coated magnetic bead suspension (Dynabeads M-280 Streptavidin; Dynal Biotech, Invitrogen, Düsseldorf, Germany and Streptavidin Magnetic Particles; Roche Diagnostics, Mannheim, Germany) were separately washed with PBS/T and blocked with 2 % (w/v) BSA in PBS/T for 1 h. The 25 of blocked beads were used for preadsorption of the blocked phagemids to remove phagemids specific for the beads, the 50 were used later for the selection cycle.
  • the blocked phagemids were incubated for 30 min with the 25 washed and blocked Streptavidin-coated magnetic beads. The beads were then pulled down with a single tube magnetic stand (Promega, Mannheim, Germany) for 2 min, and the supernatant containing phagemids not bound to the beads was incubated for 1-2 h with 100 nM biotinylated ⁇ 40 from Example 2 in a total volume of 400 ⁇ ⁇ . The mixture of phagemids and peptide target was then incubated for 0.5 h with the 50 blocked beads and subsequently pulled down with a single tube magnetic stand for 2 min. The supernatant containing unbound phagemids was discarded.
  • the target/phagemid complexes bound to magnetic beads were washed 10 times with 400 ⁇ PBS/T, and then bound phagemids were eluted under rotation for 10 min with 350 ⁇ 0.1 M glycine/HCl, pH 2.2, followed by immediate neutralization with 55 0.5 M Tris base. Alternatively, elution was performed under denaturing conditions with 400 4 M urea in PBS for 30 min, followed by dilution with 1 mL PBS.
  • the bacterial pellet was resuspended in a suitable volume of 2x YT medium (16 g/L Bacto Tryptone, 10 g/L Bacto Yeast Extract, 5 g/L NaCl, pH 7.5), plated onto LB-Cam plates (10 g/L Bacto Tryptone, 5 g/L Bacto Yeast Extract, 5 g/L NaCl, 15 g/L Bacto Agar, 35 mg/L chloramphenicol, pH 7.5), and incubated for 14-16 h at 32 °C. Cells were then scraped off the plates and employed for rescue and reamplification of the recombinant phagemids.
  • 2x YT medium 16 g/L Bacto Tryptone, 10 g/L Bacto Yeast Extract, 5 g/L NaCl, pH 7.5
  • LB-Cam plates 10 g/L Bacto Tryptone, 5 g/L Bacto Yeast Extract, 5 g/L NaCl, 15
  • Screening of the enriched phagemid pools was performed by a screening ELISA (Example 5) after the fourth panning step.
  • Example 4 Selection of Lcn2 muteins with affinity to the ⁇ - ⁇ 28 fusion protein by phage display
  • anti-DIG-IgG coated magnetic bead suspension (Europa Bioproducts Ltd, Cambridge, UK) were washed with PBS/T, and separately blocked for 1 h with 2 % (w/v) BSA in PBS/T or skim milk.
  • the blocked phagemids were incubated for 30 min with the washed and blocked 25 ⁇ L ⁇ anti-DIG-IgG coated magnetic beads.
  • the beads were then pulled down with a single tube magnetic stand (Promega, Mannheim, Germany) for 2 min, and the supernatant containing phagemids not bound to the beads was incubated for 30 min with 15 ⁇ non-labeled Trx fromExample 2 to remove phagemids specific for Trx.
  • Digoxigenated and carboxymethylated ⁇ - ⁇ 28 from Example 2 was then added to a final concentration of 100 nM, and the mixture was incubated for 1-2 h in a total volume of 400 ⁇ .
  • the mixture of phagemids, Trx, and ⁇ - ⁇ 28 was then incubated with the drained blocked beads from the 50 ⁇ ⁇ portion for 0.5 h.
  • the beads were then pulled down with a single tube magnetic stand for 2 min.
  • the supernatant containing unbound phagemids was discarded.
  • the magnetic beads with the bound phagemids were washed 10 times with 500 PBS/T.
  • bound phagemids were eluted under rotation for 30 min in 400 4 M urea in PBS, followed by dilution with 1 mL PBS. In total, 6 selection cycles were performed.
  • the bacterial pellet was resuspended in a suitable volume of 2x YT medium (16 g/L Bacto Tryptone, 10 g/L Bacto Yeast Extract, 5 g/L NaCl, pH 7.5), plated onto LB-Cam plates (10 g/L Bacto Tryptone, 5 g/L Bacto Yeast Extract, 5 g/L NaCl, 15 g/L Bacto Agar, 35 mg/L chloramphenicol, pH 7.5), and incubated for 14-16 h at 32 °C. Cells were then scraped off the plates and employed for rescue and reamplification of the recombinant phagemids.
  • 2x YT medium 16 g/L Bacto Tryptone, 10 g/L Bacto Yeast Extract, 5 g/L NaCl, pH 7.5
  • LB-Cam plates 10 g/L Bacto Tryptone, 5 g/L Bacto Yeast Extract, 5 g/L NaCl, 15
  • Screening of the enriched phagemid pools was performed by a filter- sandwich colony screening assay (Example 6) after the sixth panning step.
  • Example 5 Identification of Lcn2 muteins specific for ⁇ via screening ELISA
  • single colonies from the enriched pool were grown in 96- well plates (Multiple Well Plate 96 round bottom with lid; Sarstedt, Nuembrecht, Germany) in 100 ⁇ ⁇ TB-Amp medium (12 g/L Bacto Tryptone, 24 g/L Bacto Yeast Extract, 55 mM glycerol, 17 mM KH 2 P0 4 , 72 mM K 2 HP0 4 , 100 mg/L ampicillin) at 37 °C over night.
  • 100 ⁇ TB-Amp medium was inoculated with the overnight cultures, and grown to exponential phase at 22 °C or 37 °C.
  • Periplasmic expression of Lcn2 muteins was induced with 20 ⁇ ⁇ 0.2 ⁇ g/mL anhydrotetracycline (aTc; Acros, Geel, Belgium) dissolved in water- free dimethylformamide (DMF; Sigma- Aldrich, Steinheim, Germany) for 13-17 h at 20 °C. Periplasmic proteins were released with 40 ⁇ .
  • BBS 800 mM Na-borate, 640 mM NaCl, 8 mM EDTA, pH 8.0
  • BBS 800 mM Na-borate, 640 mM NaCl, 8 mM EDTA, pH 8.0
  • 0.5 ⁇ non-biotinylated ⁇ 40 from Example 2 was directly immobilized onto a 96-well MaxiSorp polystyrene microtitre plate (Nunc, Langenselbold, Germany), followed by blocking. After incubation with the cell extract from above and washing, bound Lcn2 muteins were detected via the Strep-tag II using a 1: 1500 dilution of Streptactin/AP conjugate (IB A, Gottingen, Germany). As a control, 0.5 ⁇ Ova was immobilized onto the ELISA plate.
  • Example 6 Identification of Lcn2 muteins specific for ⁇ - ⁇ 28 via filter- sandwich colony screening assay
  • the mutagenized Lcn2 gene cassette was subcloned via BstXI (Fermentas, St. Leon-Rot, Germany) on the plasmid phNGAL124 (SEQ ID NO: 42), which encodes a fusion of the OmpA signal peptide, the Lcn2-coding region with the C-terminal Strep-tag II (Schmidt and Skerra (2007) Nat. Protoc. 2, 1528-1535) followed by an amber stop codon as well as a gene for the albumin-binding domain (ABD) from streptococcal protein G (Schlehuber et al. (2000) J. Mol.
  • BstXI Fermentas, St. Leon-Rot, Germany
  • This membrane was probed with 100 nM of DIG-labeled ⁇ - ⁇ 28 from Example 2 in PBS/T for 1 h.
  • the bound target conjugate was detected with an anti-DIG Fab/alkaline phosphatase (AP) conjugate (Roche Diagnostics, Mannheim, Germany) followed by a chromogenic reaction with 5-bromo-4-chloro-3-indolyl phosphate, 4-toluidine salt (BCIP; AppliChem, Darmstadt, Germany) and nitro blue tetrazolium (NBT; AppliChem, Darmstadt, Germany). Having identified spots with intense colour signals on this membrane, the corresponding colonies were picked from the first filter and propagated for side by side comparison in a secondary colony screen.
  • AP anti-DIG Fab/alkaline phosphatase conjugate
  • Example 7 Soluble production and purification of Lcn2 muteins specific for ⁇ and ED-B
  • the plasmid phNGAL98 (SEQ ID NO: 27) was used, encoding a fusion of the OmpA signal peptide with the mature Lcn2 protein (SEQ ID NO: 28) and the C-terminal Strep-tag II, whereby the plasmid carries the two non- compatible BstXI restriction sites for unidirectional subcloning of the mutated gene cassette.
  • the soluble protein was affinity-purified by means of the Strep-tag II (Schmidt and Skerra (2007) Nat. Protoc. 2, 1528-1535), followed by size exclusion chromatography (SEC) on a Superdex 75 HR 10/30 column (Amersham- Pharmacia, Freiburg, Germany) using PBS buffer. Protein purity was checked by SDS-PAGE (Fling and Gregerson (1986) Anal. Biochem. 155, 83-88).
  • Protein concentrations were determined by absorption measurement at 280 nm using calculated extinction coefficients of 31400 M _1 cm _1 for wt Lcn2 (SEQ ID NO: 44) and of 26930 M ' 1 for the ⁇ specific muteins Hl-Gl (SEQ ID NO: 43), 24410 M " 1 for S1-A4 (SEQ ID NO: 38), and 26930 M ⁇ crn 1 for US7 (SEQ ID NO: 41).
  • the calculated extinction coefficients of the ED-B specific Lcn2 muteins were 37930 M _1 cm _1 for mutein N7A (SEQ ID NO: 20), 22920 M ' 1 for N7E (SEQ ID NO: 22), 21430 M ' 1 for N9B (SEQ ID NO: 24), and 39420 M ' 1 for N10D (SEQ ID NO: 26) (Gasteiger et al. , supra).
  • Example 8 Measurement of binding activity for different ⁇ targets in ELISA experiments
  • [L] tot represents the concentration of the applied ligand conjugate and K D is the dissociation constant (Voss and Skerra (1997) Protein Eng. 10, 975-982).
  • a "competitive" ELISA was performed in a similar manner as the "capture” ELISA described above. Again, after immobilizing StrepMAB-Immo and washing, 50 ⁇ ⁇ of a 1 ⁇ solution of the purified Lcn2 mutein from Example 7 was applied to all wells for 1 h. After washing, the biotinylated target ⁇ - ⁇ 28 from Example 2 was applied at a fixed concentration of 5 nM (for S1-A4) or 40 nM (for US7) in the presence of varying concentrations of the free non-labeled ⁇ - ⁇ 28 target as competitor. Starting with a 100-fold excess, the competitor concentration was decreased by the factor 3 in each step. In this case, the data were fitted to the sigmoidal equation
  • Example 9 Measurement of binding activity for ⁇ via surface plasmon resonance (SPR)
  • the SPOT membrane (Amino-PEG500-UC540 sheet, 100 x 150 mm, Mavis, Koln, Germany) for mapping the ⁇ epitope of the Lcn2 muteins S1-A4 and US7 was prepared in an automated procedure as described by Frank ((2002) J. Immunol. Methods 262, 13-26) using a MultiPep RS instrument (Intavis, Koln, Germany) and activated amino acids from the same vendor.
  • the ⁇ 40 amino acid sequence (SEQ ID NO: 29) was synthesized on the membrane in successive hexamers, decamers, and pentadecamers, each with a dislocation of 1 amino acid, thus covering the entire sequence.
  • the C-termini of these peptides became covalently attached to the membrane whereas their N-termini were acetylated.
  • Immobilized Strep-tag II served as a positive control for the detection method.
  • the membrane was washed once with ethanol, 3 times with PBS (4 mM KH 2 P0 4 , 16 mM Na 2 HP0 4 , 115 mM NaCl, pH 7.4), and blocked with 3 % (w/v) BSA in PBS/T (PBS containing 0.1 % (v/v) Tween 20) for 1 h. After washing 3 times with PBS/T, the membrane was incubated with the Lcn2 mutein S1-A4 (50 nM), US7 (100 nM) or wild- type Lcn2 (100 nM) in PBS/T for 1 h and washed 3 times with PBS/T.
  • the membrane was incubated with a 1: 1500 dilution of Streptavidin/AP conjugate (GE Healthcare, Buckinghamshire, UK) in PBS/T for 1 h for the protein detection via the Strep-tag II. Subsequently, the membrane was washed 3 times with PBS/T and once in AP buffer (100 mM Tris/HCl, pH 8.8, 100 mM NaCl, 5 mM MgCl 2 ), followed by a chromogenic reaction with 5-bromo-4-chloro-3-indolyl phosphate, 4-toluidine salt (BCIP; AppliChem, Darmstadt, Germany) and nitro blue tetrazolium (NBT; AppliChem, Darmstadt, Germany) in AP buffer as described by Schlehuber et al. ((2000) J. Mol. Biol. 297, 1105-1120).
  • AP buffer 100 mM Tris/HCl, pH 8.8, 100 mM NaCl, 5 m
  • Example 11 Functional analysis of Lcn2 muteins with affinity to ⁇ in a ThT aggregation assay
  • Solubilized and homogenously monomeric, non-biotinylated ⁇ 40 from Example 2 was subjected to a thioflavin T aggregation assay in the presence or absence of various Lcn2 muteins.
  • Thioflavin T (ThT; Sigma- Aldrich, Steinheim, Germany) specifically binds to ⁇ - sheet-rich amyloid fibrils and oligomeric precursors but not to the monomeric ⁇ peptide, which is accompanied by an increase in ThT fluorescence (Khurana et al. (2005) J. Struct. Biol. 151, 229-238).
  • ThT was dissolved in distilled H 2 0 to a concentration of 1 mM.
  • the working solution was prepared by diluting the 1 mM stock solution to a final concentration of 5 ⁇ with 5 mM glycine/NaOH, pH 8.5.
  • samples were vortexed shortly 10 times and 20 ⁇ ⁇ of the respective sample were mixed with 180 ⁇ ⁇ of the ThT working solution. Fluorescence intensity was measured with an excitation wavelength of 450 nm and an emission wavelength of 482 nm in a luminescence spectrometer (LS 50 B, Perkin Elmer, Waltham, USA).
  • Example 12 Preparation of recombinant fibronectin fragments containing the extra domain B (ED-B)
  • Coding sequences of FN7B8, FN789, and ED-B were cloned on the vector pASK75 (Skerra (1994) Gene 151, 131-135) or its derivative pASG-IBA-33 (IBA, Gottingen, Germany), yielding pASK75-FN7B8 (SEQ ID NO: 13), pASK75-FN789 (SEQ ID NO: 17), and pASG-IBA-33-EDB (SEQ ID NO: 15), respectively. All constructs provide a His 6 -tag at the carboxy terminus for the purification via immobilized metal affinity chromatography (EVIAC) and were produced as soluble proteins in the cytoplasm of E.
  • EVIAC immobilized metal affinity chromatography
  • E. coli TG1/F [a derivative of E. coli K12 TGI (Gibson (1984) Studies on the Epstein-Barr virus genome, Cambridge University, England)] or BL21 (Studier and Moffatt (1986), 189, 113-130). Therefore, a 2-L culture of E. coli harboring the expression plasmid was grown to mid-log phase at 37 °C in LB-medium containing 100 ⁇ g/ml ampicillin. After addition of 200 ⁇ g/L anhydrotetracycline (Acros, Geel, Belgium) growth was continued for 5 to 7 h at 37°C.
  • Cells were concentrated by centrifugation, resuspended in 35 ml ice-cold chromatography buffer (40 niM Hepes/NaOH, 1 M NaCl, pH 7.5) and lysed by sonification (S250D, Branson, Danbury CT, USA).
  • Fractions containing recombinant protein were identified by SDS-PAGE, mixed with EDTA to a final concentration of 1 mM, and dialysed against 20 mM Hepes/NaOH, pH 7.4 overnight. Subsequently, the protein was loaded on an ion-exchange chromatography column (Resource Q, GE Healthcare, Kunststoff, Germany) equilibrated with 20 or 40 mM Hepes/NaOH, pH 7.4. To elute bound fibronectin fragments a gradient between 0 and 300 mM NaCl was used.
  • Example 13 Selection of Lcn2 variants with affinity to ED-B by Phage Display
  • Example 14 Selection of Lcn2 variants with affinity to ED-B by screening ELISA
  • Soluble expression of individual Lcn2 muteins in 96-well plates was performed as follows: 100 ⁇ TB medium (Tartof and Hobbs (1987) Bethesda Research Laboratory Focus 9, 12) containing 100 ⁇ g/ml ampicillin was inoculated with a single, randomly picked colony and incubated for 5 h at 37°C with shaking (500 to 800 rpm; Thermomixer comfort; Eppendorf, Hamburg, Germany). For each clone 100 ⁇ of fresh medium was inoculated with 10 ⁇ of this culture and incubated for 1 to 2 h at 37 °C with shaking followed by lowering the temperature to 22°C.
  • Lcn2 muteins were induced in exponentially growing cells for 12-14 h with 0.2 ⁇ g/ml anhydrotetracycline (Acros, Geel, Belgium).
  • Periplasmic release of proteins was effected by the addition of 40 ⁇ 2xBBS (0.2 M borate/NaOH, 160 mM NaCl, 1 mM EDTA, pH 8.0) containing lmg/ml lysozyme (Roche Diagnostics, Mannheim, Germany) for 1 h at room temperature with shaking. Lysates were blocked with 40 ⁇ 10 % w/v BSA (Applichem, Darmstadt, Germany) in TBS and 0,5 % Tween-20 for 1 h and cell debris was removed from the crude extract by centrifugation for 10 min.
  • Bound Lcn2 muteins were detected with Streptavidin-alkaline phosphatase conjugate (1: 1500 in TBS/T; GE Healthcare, Munich, Germany) using the substrate 4-nitrophenyl phosphate (pNpp, 0.5 mg/ml; AppliChem GmbH, Darmstadt, Germany) in 0.1 M Tris/HCl, 0.1 M NaCl, 5 mM MgCl 2 , pH 8.8 (see Example 16).
  • Lcn2 muteins were found to also show specific binding activity for FN7B8 in ELISA, SPR-analysis, and in immunofluorescence microscopy of ED-B positive human colon cancer cells. These Lcn2 muteins were designated as N7A (SEQ ID NO: 20), N7E (SEQ ID NO: 22), N9B (SEQ ID NO: 24), and N10D (SEQ ID NO: 26).
  • Example 15 Soluble protein production and purification of Lcn2 and its variants
  • Example 16 Measurement of binding activity for ED-B in an ELISA
  • N7A, N9B, and N10D were found to specifically bind to FN7B8 with K D values of 14.8 nM (N7A), 40.1 nM (N9B), 30.0 nM (N7E), and 51.2 (N10D) but not to FN789 or BSA.
  • Example 17 Measurement of binding activity for recombinant Fibronectin FN7B8 via surface plasmon resonance (SPR)
  • Example 18 Immunostaining of ED-B positive CaCo2 cells
  • Human cancer colon cells (CaCo2 cells kindly provided by H. Daniel, Technische Universitat Miinchen, Germany; Pujuguet et al., Am. J. Pathol. 148, 579-592) were cultured on the Nunc Lab-TekTM II chamber slideTM system (4 chambers per slide; Thermo Fisher Scientific, Langenselbold, Germany) in MEM with Earle's Salts and L-Glutamine, supplemented with 10 % Fetal Bovine Serum, lx MEM non-essential amino acids, and 50 ⁇ g/ml gentamycin (PAA Laboratories, Pasching, Austria) at 37°C in a humidified atmosphere until cell confluence was about 50 to 70 %.
  • CaCo2 cells kindly provided by H. Daniel, Technische Universitat Miinchen, Germany; Pujuguet et al., Am. J. Pathol. 148, 579-592
  • Nunc Lab-TekTM II chamber slideTM system 4 chambers per slide;
  • Example 19 Generation of the ⁇ -specific Lcn2 muteins H1GA and H1GV via exchange of a free cysteine residue in Hl-Gl at position 36
  • Cys36 was replaced with Ala or Val by site-directed mutagenesis.
  • a PCR was conducted with the degenerate oligodeoxynucleotide DH-4 (SEQ ID NO: 45) and the second oligodeoxynucleotide J08rev (SEQ ID NO: 48) as well as plasmid DNA encoding the Lcn2 mutein Hl-Gl as template (SEQ ID NO: 37).
  • the amplified fragments were subcloned via BstXI on the expression plasmid phNGAL98 and sequenced to identify the individual substitution variants.
  • the resulting Lcn2 variants were named H1GA (SEQ ID NO: 49, 50) and H1GV (SEQ ID NO: 51, 52).
  • Example 20 Soluble production and purification of the new ⁇ -specific Lcn2 muteins H1GA and H1GV using E. coli cultures with a high optical density
  • the soluble protein was affinity-purified by means of the Strep-tag II, followed by size exclusion chromatography (SEC) on a Superdex 75 HR 10/30 column using PBS buffer (4 mM KH 2 P0 4 , 16 mM Na 2 HP0 4 , 115 mM NaCl, pH 7.4). Protein purity was checked by SDS-PAGE . Protein concentrations were determined by absorption measurement at 280 nm using a calculated extinction coefficient of 27055 M-lcm-1 for the ⁇ specific muteins H1GA (SEQ ID NO: 50) and H1GV (SEQ ID NO: 52).
  • Example 21 Measurement of binding activity for ⁇ - ⁇ 40 via surface plasmon resonance on a BiacoreX instrument
  • the purified Lcn2 muteins H1GA and H1GV from Example 20 were applied in concentrations ranging from 4 nM to 128 nM at a flow rate of 20 ⁇ / ⁇ .
  • the data were double-referenced by subtraction of the corresponding signals measured for the control channel, which had been activated and blocked with ethanolamine, as well as subtraction of the measured signals for an average of buffer injections.
  • Kinetic data were fitted globally using the BIAevaluation software V 3.0 (Karlsson et al. (1991) J. Immunol. Methods 145, 229-240).
  • Example 22 Measurement of binding activity for ⁇ 40 via surface plasmon resonance on a Biacore T100 instrument
  • the purified Lcn2 mutein H1GA from Example 20 was applied in concentrations ranging from 1 nM to 32 nM at a flow rate of 30 ⁇ 7 ⁇ .
  • the dilution series of H1GA was injected with both association and dissociation times of 300 s to obtain k on information. For exact determination of the low k 0 ff rate the highest concentration was analysed using a dissociation time of 7200 s.
  • Example 21 The data were double-referenced as in Example 21. k on and k Q ff for the binding reaction were determined from the entire data set using Biacore T100 Evaluation Software V2.0.3 for data processing and kinetic fitting. The data was globally fitted using a 1: 1 binding model.
  • Example 23 Functional analysis of the Lcn2 mutein H1GA in a ThT aggregation assay
  • Thioflavin T (ThT) aggregation assay synthetic ⁇ peptide (SEQ ID NO: 29) was dissolved in l,l,l,3,3,3-hexafluoro-2-propanol (HFIP; Sigma- Aldrich, Steinheim, Germany) for 12 h. HFIP was evaporated under vacuum, and ⁇ was dissolved in a suitable volume of H 2 0 dd, sonicated for 15 min at 4 °C, and filtrated (Costar Spin-X centrifuge tube filter cellulose acetate membrane, 0.45 ⁇ ; Corning Inc., Corning, NY). The solubilized monomeric ⁇ was then immediately used for the aggregation assays.
  • HFIP Thioflavin T
  • Example 24 Measurement of binding activity for the recombinant fibronectin single domain ED-B via surface plasmon resonance (SPR)
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RU2012128586/10A RU2564125C2 (ru) 2009-12-07 2010-12-07 Мутеины липокалина 2 человека (lcn2, hngal) с аффинностью для определенной мишени
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BR112012013662-3A BR112012013662B1 (pt) 2009-12-07 2010-12-07 Muteínas de lipocalina associada à gelatinase de neutrófilo humano (lcn2, hngal), seu uso e seus métodos de geração e produção, molécula de ácido nucleico, célula hospedeira, composição farmacêutica e kit de diagnóstico ou analítico
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013174783A1 (en) * 2012-05-23 2013-11-28 Pieris Ag Lipocalin muteins with binding-affinity for glypican-3 (gpc-3) and use of lipocalin muteins for target-specific delivery to cells expressing gpc-3
WO2016142314A1 (en) 2015-03-06 2016-09-15 Deutsches Krebsforschungszentrum FUSION PROTEINS COMPRISING A BINDING PROTEIN AND AN INTERLEUKIN-15 POLYPEPTIDE HAVING A REDUCED AFFINITY FOR IL15Ra AND THERAPEUTIC USES THEREOF
CN107403074A (zh) * 2017-06-09 2017-11-28 天津市湖滨盘古基因科学发展有限公司 一种突变蛋白的检测方法及装置
WO2018015380A1 (en) 2016-07-18 2018-01-25 Philippe Ulsemer Natural microorganisms which are naturally capable of binding toxins and/or toxin receptors
US10273275B2 (en) 2015-05-18 2019-04-30 Pieris Pharmaceuticals Gmbh Muteins of human lipocalin 2 with affinity for glypican-3 (GPC3) and methods of use thereof
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WO2020201038A1 (en) 2019-03-29 2020-10-08 Pieris Pharmaceuticals Gmbh Inhaled administration of lipocalin muteins
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US10947284B2 (en) 2015-07-07 2021-03-16 Pieris Pharmaceuticals Gmbh Fusion molecules

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3660510A3 (en) * 2009-12-07 2020-07-08 Pieris Pharmaceuticals GmbH Muteins of human lipocalin 2 (lcn2, hngal) with affinity for a given target
WO2012022742A1 (en) * 2010-08-16 2012-02-23 Pieris Ag Binding proteins for hepcidin
US9758554B2 (en) 2012-01-31 2017-09-12 Technische Universitaet Muenchen Muteins of α1m lipocalin and method of production therefor
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AU2016258977C1 (en) 2015-05-04 2022-07-14 Pieris Pharmaceuticals Gmbh Anti-cancer fusion polypeptide
US11261221B2 (en) 2015-05-04 2022-03-01 Pieris Pharmaceuticals Gmbh Proteins specific for CD137
BR112018010887A2 (pt) * 2015-11-30 2018-11-21 Pieris Australia Pty Ltd polipeptídeo de fusão
TW201725212A (zh) * 2015-12-10 2017-07-16 第一三共股份有限公司 特異性於降鈣素基因相關胜肽的新穎蛋白
GB201615254D0 (en) 2016-09-08 2016-11-23 Ge Healthcare Bio Sciences Ab Novel Ligand and use thereof
EP3715370A1 (en) * 2019-03-28 2020-09-30 Technische Universität München High affinity anticalins directed against human cd98hc

Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0330451A2 (en) 1988-02-22 1989-08-30 Toa Nenryo Kogyo Kabushiki Kaisha A cDNA coding for human normal serum albumin a, and a process for production of the albumin
EP0361991A2 (en) 1988-08-05 1990-04-04 Rhone-Poulenc Sante Method for the microbiological preparation of human serum albumin and other heterologous proteins from a yeast
US5728553A (en) 1992-09-23 1998-03-17 Delta Biotechnology Limited High purity albumin and method of producing
WO1999016873A1 (de) 1997-09-26 1999-04-08 Arne Skerra Anticaline
WO1999064016A1 (en) 1998-06-08 1999-12-16 F. Hoffmann-La Roche Ag Use of peg-ifn-alpha and ribavirin for the treatment of chronic hepatitis c
WO2000075308A1 (de) 1999-06-08 2000-12-14 Pieris Proteolab Ag Muteine des bilin-bindungsproteins
US6177074B1 (en) 1995-11-02 2001-01-23 Schering Corporation Polyethylene glycol modified interferon therapy
US6403564B1 (en) 1998-10-16 2002-06-11 Schering Corporation Ribavirin-interferon alfa combination therapy for eradicating detectable HCV-RNA in patients having chronic hepatitis C infection
US6500930B2 (en) 1998-03-31 2002-12-31 Hemosol Inc. Hemoglobin-polysaccharide conjugates
WO2003029471A1 (en) 2001-09-27 2003-04-10 Pieris Proteolab Ag Muteins of apolipoprotein d
WO2003029462A1 (en) 2001-09-27 2003-04-10 Pieris Proteolab Ag Muteins of human neutrophil gelatinase-associated lipocalin and related proteins
US20030069395A1 (en) 2001-03-09 2003-04-10 Sato Aaron K. Serum albumin binding moieties
US6620413B1 (en) 1995-12-27 2003-09-16 Genentech, Inc. OB protein-polymer chimeras
US6696245B2 (en) 1997-10-20 2004-02-24 Domantis Limited Methods for selecting functional polypeptides
WO2005019256A2 (en) 2003-08-25 2005-03-03 Pieris Proteolab Ag Muteins of tear lipocalin
WO2005019254A1 (en) 2003-08-25 2005-03-03 Pieris Proteolab Ag Muteins of a bilin-binding protein with affinity for a given target
WO2006056464A2 (en) 2004-11-26 2006-06-01 Pieris Ag Compound with affinity for the cytotoxic t lymphocyte-associated antigen (ctla-4)
WO2007038619A2 (en) 2005-09-27 2007-04-05 Amunix, Inc. Proteinaceous pharmaceuticals and uses thereof

Family Cites Families (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4376110A (en) 1980-08-04 1983-03-08 Hybritech, Incorporated Immunometric assays using monoclonal antibodies
EP0257956B2 (en) 1986-08-19 2000-11-22 Genentech, Inc. Use of polypeptide growth factors and cytokines for the manufacture of a device and of a dispersion
AU696387B2 (en) 1994-05-18 1998-09-10 Inhale Therapeutic Systems, Inc. Methods and compositions for the dry powder formulation of interferons
DE4417598A1 (de) 1994-05-19 1995-12-14 Max Planck Gesellschaft Verwendung des Tetracyclinpromotors zur stringent regulierten Produktion von rekombinanten Proteinen in prokaryontischen Zellen
AU5132096A (en) 1995-01-30 1996-08-21 Terrapin Technologies, Inc. Glubodies - multiplicities of proteins capable of binding a variety of small molecules
DE19641876B4 (de) 1996-10-10 2011-09-29 Iba Gmbh Streptavidinmuteine
WO1998016873A1 (fr) 1996-10-14 1998-04-23 Firm Forsat Ltd. Procede de preparation de dispersions a base de composants chromogenes
US6020163A (en) 1997-08-06 2000-02-01 Zymogenetics, Inc. Lipocalin homolog
US20140080177A1 (en) 1997-09-26 2014-03-20 Pieris Ag Anticalins
CA2343917A1 (en) 1998-10-19 2000-04-27 Ariad Gene Therapeutics, Inc. Materials and methods involving conditional retention domains
ES2290023T3 (es) * 1999-03-04 2008-02-16 Praecis Pharmaceuticals Incorporated Moduladores de la agregacion del peptido beta-amiloide que comprenden d-aminoacidos.
JP2007284351A (ja) * 2004-07-27 2007-11-01 Osaka Bioscience Institute アミロイド蛋白質の凝集を抑制する物質とその作用
CA2659413C (en) * 2006-08-01 2016-06-14 Pieris Ag Muteins of tear lipocalin and methods for obtaining the same
CN101516907B (zh) * 2006-08-01 2015-08-26 皮里斯股份公司 泪液脂质运载蛋白的突变蛋白及其获得方法
JP2011501673A (ja) * 2007-10-19 2011-01-13 アボット・ラボラトリーズ グリコシル化された哺乳動物ｎｇａｌ及びその使用
AU2009264214B2 (en) 2008-06-24 2013-06-06 Technische Universitaet Muenchen Muteins of hNGAL and related proteins with affinity for a given target
EP3660510A3 (en) * 2009-12-07 2020-07-08 Pieris Pharmaceuticals GmbH Muteins of human lipocalin 2 (lcn2, hngal) with affinity for a given target
WO2012022742A1 (en) * 2010-08-16 2012-02-23 Pieris Ag Binding proteins for hepcidin
JP6100694B2 (ja) 2010-11-15 2017-03-22 ピエリス ファーマシューティカルズ ゲーエムベーハー グリピカン−３（ｇｐｃ３）に対して親和性を有するヒトリポカリン２の突然変異タンパク質
EP3441400B1 (en) * 2012-11-19 2022-07-20 Pieris Pharmaceuticals GmbH Novel specific-binding polypeptides and uses thereof
US10526382B2 (en) * 2015-01-28 2020-01-07 Pieris Pharmaceuticals Gmbh Human neutrophil gelatinase-associated lipocalin (hNGAL) muteins capable of binding angiopoietin-2 (Ang-2) and methods of use thereof
AU2016221816B2 (en) 2015-02-18 2020-07-02 Pieris Pharmaceuticals Gmbh Novel proteins specific for pyoverdine and pyochelin
US11261221B2 (en) 2015-05-04 2022-03-01 Pieris Pharmaceuticals Gmbh Proteins specific for CD137
CN107787327B (zh) 2015-05-18 2022-02-08 皮里斯制药有限公司 对磷脂酰肌醇聚糖-3（gpc3）具有亲和力的人脂质运载蛋白2的突变蛋白
TW201725212A (zh) 2015-12-10 2017-07-16 第一三共股份有限公司 特異性於降鈣素基因相關胜肽的新穎蛋白

Patent Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0330451A2 (en) 1988-02-22 1989-08-30 Toa Nenryo Kogyo Kabushiki Kaisha A cDNA coding for human normal serum albumin a, and a process for production of the albumin
EP0361991A2 (en) 1988-08-05 1990-04-04 Rhone-Poulenc Sante Method for the microbiological preparation of human serum albumin and other heterologous proteins from a yeast
US5728553A (en) 1992-09-23 1998-03-17 Delta Biotechnology Limited High purity albumin and method of producing
US6177074B1 (en) 1995-11-02 2001-01-23 Schering Corporation Polyethylene glycol modified interferon therapy
US6620413B1 (en) 1995-12-27 2003-09-16 Genentech, Inc. OB protein-polymer chimeras
WO1999016873A1 (de) 1997-09-26 1999-04-08 Arne Skerra Anticaline
US6696245B2 (en) 1997-10-20 2004-02-24 Domantis Limited Methods for selecting functional polypeptides
US6500930B2 (en) 1998-03-31 2002-12-31 Hemosol Inc. Hemoglobin-polysaccharide conjugates
WO1999064016A1 (en) 1998-06-08 1999-12-16 F. Hoffmann-La Roche Ag Use of peg-ifn-alpha and ribavirin for the treatment of chronic hepatitis c
US6403564B1 (en) 1998-10-16 2002-06-11 Schering Corporation Ribavirin-interferon alfa combination therapy for eradicating detectable HCV-RNA in patients having chronic hepatitis C infection
WO2000075308A1 (de) 1999-06-08 2000-12-14 Pieris Proteolab Ag Muteine des bilin-bindungsproteins
US20030069395A1 (en) 2001-03-09 2003-04-10 Sato Aaron K. Serum albumin binding moieties
WO2003029462A1 (en) 2001-09-27 2003-04-10 Pieris Proteolab Ag Muteins of human neutrophil gelatinase-associated lipocalin and related proteins
WO2003029463A2 (en) 2001-09-27 2003-04-10 Pieris Proteolab Ag Muteins of human neutrophil gelatinase-associated lipocalin and related proteins
WO2003029471A1 (en) 2001-09-27 2003-04-10 Pieris Proteolab Ag Muteins of apolipoprotein d
WO2005019256A2 (en) 2003-08-25 2005-03-03 Pieris Proteolab Ag Muteins of tear lipocalin
WO2005019254A1 (en) 2003-08-25 2005-03-03 Pieris Proteolab Ag Muteins of a bilin-binding protein with affinity for a given target
WO2005019255A1 (en) 2003-08-25 2005-03-03 Pieris Proteolab Ag Muteins of tear lipocalin
WO2006056464A2 (en) 2004-11-26 2006-06-01 Pieris Ag Compound with affinity for the cytotoxic t lymphocyte-associated antigen (ctla-4)
WO2007038619A2 (en) 2005-09-27 2007-04-05 Amunix, Inc. Proteinaceous pharmaceuticals and uses thereof

Non-Patent Citations (78)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL, S. F. ET AL., NUCL. ACIDS RES., vol. 25, 1997, pages 3389 - 3402
ALTUVIA ET AL., J. MOL. BIOL., vol. 249, 1995, pages 244 - 250
AMSTUTZ, P. ET AL., CURR. OPIN. BIOTECHNOL., vol. 12, 2001, pages 400 - 405
BACHMANN, MICROBIOL. REV., vol. 54, 1990, pages 130 - 197
BESTE ET AL., PNAS, vol. 96, 1999, pages 1898 - 903
BESTE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 96, 1999, pages 1898 - 1903
BITTKER ET AL., NAT. BIOTECHNOL., vol. 20, 2002, pages 1024 - 1029
BOS ET AL., BUSINESS BRIEFING: PHARMATECH, 2003, pages 1 - 6
BREUSTEDT ET AL., BIOCHIM. BIOPHYS. ACTA, vol. 1764, 2006, pages 161 - 173
BRODERS ET AL.: "Hyperphage. Improving antibody presentation in phage display", METHODS MOL. BIOL., vol. 205, 2003, pages 295 - 302
BRODY; HOLTZMANN, ANNU. REV. NEUROSCI., vol. 31, 2008, pages 175 - 193
BRUCKDORFER ET AL., CURR. PHARM. BIOTECHNOL., vol. 5, 2004, pages 29 - 43
BULLOCK ET AL., BIOTECHNIQUES, vol. 5, 1987, pages 376 - 378
CARNEMOLLA ET AL., INT. J. CANCER, vol. 68, 1996, pages 397 - 405
COLOWICK: "Solid-Phase Peptide Synthesis", 1997, ACADEMIC PRESS
DENNIS ET AL., J. BIOL. CHEM., vol. 277, 2002, pages 35035 - 35043
DODEL ET AL., LANCET NEUROLOGY, vol. 2, 2003, pages 215 - 220
EBBINGHAUS ET AL., CURR. PHARM. DES., vol. 10, 2004, pages 1537 - 1549
FLING; GREGERSON, ANAL. BIOCHEM., vol. 155, 1986, pages 83 - 88
FRANK, J. IMMUNOL. METHODS, vol. 262, 2002, pages 13 - 26
FUERTGES ET AL.: "The Clinical Efficacy of Poly(Ethylene Glycol)-Modified", PROTEINS J. CONTROL. RELEASE, vol. 11, 1990, pages 139 - 148
GAILLARD PJ ET AL., EXPERT OPIN DRUG DELIV., vol. 2, no. 2, 2005, pages 299 - 309
GAILLARD PJ ET AL., INTERNATIONAL CONGRESS SERIES, vol. 1277, 2005, pages 185 - 198
GASTEIGER ET AL., NUCLEIC ACIDS RES., vol. 31, 2003, pages 3784 - 3788
GASTEIGER ET AL.: "ExPASy: the proteomics server for in-depth protein knowledge and analysis", NUCLEIC ACIDS RES., vol. 31, 2003, pages 3784 - 3788
GENNARO; GENNARO: "Remington: The Science and Practice of Pharmacy", 2000, LIPPINCOTT WILLIAMS & WILKINS
GOETZ ET AL., BIOCHEMISTRY, vol. 39, 2000, pages 1935 - 1941
GRONWALL ET AL., J. BIOTECHNOL., vol. 128, 2007, pages 162 - 183
HAASS; SELKOE, NAT. REV. MOL. CELL. BIOL., vol. 8, 2007, pages 101 - 112
HORTSCHANSKY ET AL., PROTEIN SCI., vol. 14, 2005, pages 1753 - 1759
HOYER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 105, 2008, pages 5099 - 5104
KARLSSON ET AL., J. IMMUNOL. METHODS, vol. 145, 1991, pages 229 - 240
KASPAR ET AL., INT. J. CANCER, vol. 118, 2006, pages 1331 - 1339
KAY, B.K. ET AL.: "Phage Display of Peptides and Proteins - A Laboratory Manual", 1996, ACADEMIC PRESS
KHURANA ET AL., J. STRUCT. BIOL., vol. 151, 2005, pages 229 - 238
KIM ET AL., J. AM. CHEM. SOC., vol. 131, 2009, pages 3565 - 3576
KONIG, T.; SKERRA, A., J. IMMUNOL. METHODS, vol. 218, 1998, pages 73 - 83
LEAHY ET AL., PROTEINS, vol. 19, 1994, pages 48 - 54
LICHTLEN; MOHAJERI, J. NEUROCHEM., vol. 104, 2007, pages 859 - 874
LLOYD-WILLIAMS ET AL.: "Chemical Approaches to the Synthesis of Peptides and Proteins", 1997, CRC PRESS
LOWMAN, H.B., ANNU. REV. BIOPHYS. BIOMOL. STRUCT., vol. 26, 1997, pages 401 - 424
MATEO ET AL., HYBRIDOMA, vol. 19, no. 6, 2000, pages 463 - 471
MEIDAN; MICHNIAK, AM. J. THER., vol. 11, no. 4, 2004, pages 312 - 316
MORETTO ET AL., J. BIOL. CHEM., vol. 282, 2007, pages 11436 - 11445
MURAKAMI ET AL., NAT. BIOTECHNOL., vol. 20, 2002, pages 76 - 81
OSBORN ET AL., J. PHARMACOL. EXP. THER., vol. 303, 2002, pages 540 - 548
OSBORN, B.L. ET AL., J. PHARMACOL. EXP. THER., vol. 303, 2002, pages 540 - 548
PINI ET AL., J. BIOL. CHEM., vol. 273, 1998, pages 21769 - 21776
PINI, A. ET AL., COMB. CHEM. HIGH THROUGHPUT SCREEN., vol. 5, 2002, pages 503 - 510
PUJUGUET ET AL., AM. J. PATHOL., vol. 148, pages 579 - 592
REDL, B., BIOCHIM. BIOPHYS. ACTA, vol. 1482, 2000, pages 241 - 248
RODI, D.J.; MAKOWSKI, L., CURR. OPIN. BIOTECHNOL., vol. 10, 1999, pages 87 - 93
SAMBROOK ET AL.: "Molecular Cloning. A Laboratory Manual", 1989, COLD SPRING HARBOR PRESS
SAMBROOK, J. ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SCHLEHUBER ET AL., J MOL BIOL, vol. 297, 2000, pages 1109 - 20
SCHLEHUBER ET AL., J. MOL. BIOL., vol. 297, 2000, pages 1105 - 1120
SCHLEHUBER, S.; SKERRA, A.: "Duocalins, engineered ligand-binding proteins with dual specificity derived from the lipocalin fold", BIOL. CHEM., vol. 382, 2001, pages 1335 - 1342
SCHLIEMANN; NERI, BIOCHIM. BIOPHYS. ACTA, vol. 1776, 2007, pages 175 - 192
SCHMIDT, T.G.M. ET AL., J. MOL. BIOL., vol. 255, 1996, pages 753 - 766
SCHMIDT; SKERRA, NAT. PROTOC., vol. 2, 2007, pages 1528 - 1535
SCHONFELD ET AL., PROC. NATL. ACAD. SCI. USA, vol. 106, 2009, pages 8198 - 8203
SKERRA, GENE, vol. 151, 1994, pages 131 - 135
SKERRA, J. BIOTECHNOL., vol. 74, 2001, pages 257 - 275
STUDIER; MOFFAT, J. MOL. BIOL., vol. 189, 1986, pages 113 - 130
TARTOF; HOBBS, BETHESDA RESEARCH LABORATORY FOCUS, vol. 9, 1987, pages 12
VAJO; DUCKWORTH, PHARMACOL. REV., vol. 52, 2000, pages 1 - 9
VENTURI ET AL., J. MOL. BIOL., vol. 315, 2002, pages 1 - 8
VIRNEKÄS B; GE L; PLIICKTHUN A; SCHNEIDER KC; WELLNHOFER G; MORONEY SE: "Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis", NUCLEIC ACIDS RES, vol. 22, 1994, pages 5600 - 5607
VOGT; SKERRA, CHEMBIOCHEM, vol. 5, 2004, pages 191 - 199
VOGT; SKERRA, J. MOL. RECOGNIT., vol. 14, no. 1, 2001, pages 79 - 86
VOSS; SKERRA, PROTEIN ENG., vol. 10, 1997, pages 975 - 982
WANG, L. ET AL., SCIENCE, vol. 292, 2001, pages 498 - 500
WANG, L.; SCHULTZ, P.G., CHEM. COMM., vol. 1, 2002, pages 1 - 11
WILSON, D.S. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 98, 2001, pages 3750 - 3755
YANISCH-PERRON ET AL., GENE, vol. 33, 1985, pages 103 - 119
ZACCOLO ET AL., J. MOL. BIOL., vol. 255, 1996, pages 589 - 603
ZARDI ET AL., EMBO J, vol. 6, 1987, pages 2337 - 2342
ZARDI ET AL., EMBO JOURNAL, vol. 6, 1987, pages 2337 - 2342

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