Human neutrophil gelatinase-associated lipocalin (hngal) muteins that bind hepcidin and nucleic acid encoding such

Abstract

The present invention relates to novel, specific-binding therapeutic and/or diagnostic proteins directed against Hepcidin, which proteins preferably are muteins of lipocalin protein. The invention also relates to nucleic acid molecules encoding such proteins and to methods for generation and use of such proteins and nucleic acid molecules. Accordingly, the invention also is directed to pharmaceutical and/or diagnostic compositions comprising such a lipocalin proteins, including uses of these proteins.

1 - 78 . (canceled) 
     
     
         79 . A method of producing a lipocalin mutein that is capable of binding hepcidin with an affinity by a KD of about 10 nM or lower, wherein the lipocalin mutein comprises:
 (i) a set of mutated amino acid residues at the sequence positions 96, 100, and/or 106 of the linear polypeptide sequence of mature human neutrophil gelatinase-associated lipocalin (hNGAL), selected from the group consisting of (a) Asn 96→Val, Tyr 100→Gln, and Tyr 106→unchanged (b) Asn 96→Arg, Tyr 100→Glu, and Tyr 106→Phe, (c) Asn 96→Asp, Tyr 100→Ser, and Tyr 106→Gly, (d) Asn 96→Gly, Tyr 100→Gly, and Tyr 106→Gly, (e) Asn 96→Lys, Tyr 100→Ala, and Tyr 106→Ile, (f) Asn 96→Ser, Tyr 100→Arg, and Tyr 106→Val, (g) Asn 96→Ser, Tyr 100→Val, and Tyr 106→Arg, and (h) Asn 96→Thr, Tyr 100→Val, and Tyr 106→Gly; and   (ii) 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 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 103, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL,   
       wherein the lipocalin mutein is produced starting from the nucleic acid coding for the lipocalin mutein by means of genetic engineering methods. 
     
     
         80 . A pharmaceutical or diagnostic composition comprising a lipocalin mutein that is capable of binding hepcidin with an affinity by a KD of about 10 nM or lower, wherein the lipocalin mutein comprises:
 (i) a set of mutated amino acid residues at the sequence positions 96, 100, and/or 106 of the linear polypeptide sequence of mature human neutrophil gelatinase-associated lipocalin (hNGAL), selected from the group consisting of (a) Asn 96→Val, Tyr 100→Gln, and Tyr 106→unchanged (b) Asn 96→Arg, Tyr 100→Glu, and Tyr 106→Phe, (c) Asn 96→Asp, Tyr 100→Ser, and Tyr 106→Gly, (d) Asn 96→Gly, Tyr 100→Gly, and Tyr 106→Gly, (e) Asn 96→Lys, Tyr 100→Ala, and Tyr 106→Ile, (f) Asn 96→Ser, Tyr 100→Arg, and Tyr 106→Val, (g) Asn 96→Ser, Tyr 100→Val, and Tyr 106→Arg, and (h) Asn 96→Thr, Tyr 100→Val, and Tyr 106→Gly; and   (ii) 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 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 103, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL,   
       and a pharmaceutically acceptable excipient. 
     
     
         81 . A lipocalin mutein that comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-14. 
     
     
         82 . A lipocalin mutein that is capable of binding hepcidin with an affinity by a KD of about 10 nM or lower, wherein the mutein has at least 90% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 1-14. 
     
     
         83 . The lipocalin mutein according to  claim 81 , wherein the lipocalin mutein is fused at its N-terminus and/or its C-terminus to a fusion partner which is a protein, or a protein domain or a peptide. 
     
     
         84 . The lipocalin mutein according to  claim 81 , wherein the mutein is conjugated to a compound that extends the serum half-life of the mutein. 
     
     
         85 . A nucleic acid molecule comprising a nucleotide sequence encoding the lipocalin mutein according to  claim 81 . 
     
     
         86 . An isolated host cell containing a nucleic acid molecule of  claim 85 . 
     
     
         87 . The lipocalin mutein according to  claim 82 , wherein the lipocalin mutein is fused at its N-terminus and/or its C-terminus to a fusion partner which is a protein, or a protein domain or a peptide. 
     
     
         88 . The lipocalin mutein according to  claim 82 , wherein the mutein is conjugated to a compound that extends the serum half-life of the mutein. 
     
     
         89 . A nucleic acid molecule comprising a nucleotide sequence encoding the lipocalin mutein according to  claim 82 . 
     
     
         90 . An isolated host cell containing a nucleic acid molecule of  claim 89 . 
     
     
         91 . A method of forming a complex with hepcidin and inhibiting the ability of hepcidin to bind to or interact with ferroportin in a subject, comprising the step of administrating a lipocalin mutein that is capable of binding hepcidin with an affinity by a KD of about 10 nM or lower to the subject, wherein the lipocalin mutein comprises:
 (i) a set of mutated amino acid residues at the sequence positions 96, 100, and/or 106 of the linear polypeptide sequence of mature human neutrophil gelatinase-associated lipocalin (hNGAL), selected from the group consisting of (a) Asn 96→Val, Tyr 100→Gln, and Tyr 106→unchanged (b) Asn 96→Arg, Tyr 100→Glu, and Tyr 106→Phe, (c) Asn 96→Asp, Tyr 100→Ser, and Tyr 106→Gly, (d) Asn 96→Gly, Tyr 100→Gly, and Tyr 106→Gly, (e) Asn 96→Lys, Tyr 100→Ala, and Tyr 106→Ile, (f) Asn 96→Ser, Tyr 100→Arg, and Tyr 106→Val, (g) Asn 96→Ser, Tyr 100→Val, and Tyr 106→Arg, and (h) Asn 96→Thr, Tyr 100→Val, and Tyr 106→Gly; and   (ii) 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 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 103, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL.   
     
     
         92 . A method of treatment or diagnosis of a disease or disorder involving a disorder of iron homeostasis or an inflammatory condition associated with an elevated level of hepcidin, comprising the step of administering to a subject in need thereof a lipocalin mutein that is capable of binding hepcidin with an affinity by a KD of about 10 nM or lower to the subject, wherein the lipocalin mutein comprises:
 (i) a set of mutated amino acid residues at the sequence positions 96, 100, and/or 106 of the linear polypeptide sequence of mature human neutrophil gelatinase-associated lipocalin (hNGAL), selected from the group consisting of (a) Asn 96→Val, Tyr 100→Gln, and Tyr 106→unchanged (b) Asn 96→Arg, Tyr 100→Glu, and Tyr 106→Phe, (c) Asn 96→Asp, Tyr 100→Ser, and Tyr 106→Gly, (d) Asn 96→Gly, Tyr 100→Gly, and Tyr 106→Gly, (e) Asn 96→Lys, Tyr 100→Ala, and Tyr 106→Ile, (f) Asn 96→Ser, Tyr 100→Arg, and Tyr 106→Val, (g) Asn 96→Ser, Tyr 100→Val, and Tyr 106→Arg, and (h) Asn 96→Thr, Tyr 100→Val, and Tyr 106→Gly; and   (ii) 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 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 103, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL.   
     
     
         93 . A diagnostic or analytical kit comprising a lipocalin mutein that is capable of binding hepcidin with an affinity by a KD of about 10 nM or lower, wherein the lipocalin mutein comprises:
 (i) a set of mutated amino acid residues at the sequence positions 96, 100, and/or 106 of the linear polypeptide sequence of mature human neutrophil gelatinase-associated lipocalin (hNGAL), selected from the group consisting of (a) Asn 96→Val, Tyr 100→Gln, and Tyr 106→unchanged (b) Asn 96→Arg, Tyr 100→Glu, and Tyr 106→Phe, (c) Asn 96→Asp, Tyr 100→Ser, and Tyr 106→Gly, (d) Asn 96→Gly, Tyr 100→Gly, and Tyr 106→Gly, (e) Asn 96→Lys, Tyr 100→Ala, and Tyr 106→Ile, (f) Asn 96→Ser, Tyr 100→Arg, and Tyr 106→Val, (g) Asn 96→Ser, Tyr 100→Val, and Tyr 106→Arg, and (h) Asn 96→Thr, Tyr 100→Val, and Tyr 106→Gly; and   (ii) 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 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 103, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL.