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 a 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 pharmaceutical composition comprising a lipocalin mutein having binding affinity to hepcidin, 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), which are Asn 96→Gly, Tyr 100→Gly, 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. 
 
     
     
         80 . The pharmaceutical composition of  claim 79 , wherein the lipocalin mutein further comprises the following amino acid substitutions with respect to the linear polypeptide sequence of mature hNGAL: Tyr 52→Trp, Ser 68→Ile, Arg 81→Gln and Ser 127→Trp. 
     
     
         81 . The pharmaceutical composition of  claim 79 , wherein the lipocalin mutein further comprises the following amino acid substitutions with respect to the linear polypeptide sequence of mature hNGAL: Ala 40→Glu; Ile 41→Val; Gln 49→Met; Leu 70→Met; Arg 72→Leu; Lys 73→Ala; Asp 77→Glu; Trp 79→Leu; Leu 103→Arg; Lys 125→Val; Tyr 132→Val; and Lys 134→Trp. 
     
     
         82 . The pharmaceutical composition of  claim 80 , wherein the lipocalin mutein further comprises the following amino acid substitutions with respect to the linear polypeptide sequence of mature hNGAL: Ala 40→Glu; Ile 41→Val; Gln 49→Met; Leu 70→Met; Arg 72→Leu; Lys 73→Ala; Asp 77→Glu; Trp 79→Leu; Leu 103→Arg; Lys 125→Val; Tyr 132→Val; and Lys 134→Trp. 
     
     
         83 . The pharmaceutical composition of  claim 79 , wherein the lipocalin mutein further comprises the following amino acid substitutions with respect to the linear polypeptide sequence of mature hNGAL: Lys 59→Glu; Phe 71→Pro; Lys 74→Glu; Ile 80→Phe; Cys 87→Ser; and Ile 135→Val. 
     
     
         84 . The pharmaceutical composition of  claim 80 , wherein the lipocalin mutein further comprises the following amino acid substitutions with respect to the linear polypeptide sequence of mature hNGAL: Lys 59→Glu; Phe 71→Pro; Lys 74→Glu; Ile 80→Phe; Cys 87→Ser; and Ile 135→Val. 
     
     
         85 . The pharmaceutical composition of  claim 81 , wherein the lipocalin mutein further comprises the following amino acid substitutions with respect to the linear polypeptide sequence of mature hNGAL: Lys 59→Glu; Phe 71→Pro; Lys 74→Glu; Ile 80→Phe; Cys 87→Ser; and Ile 135→Val. 
     
     
         86 . The pharmaceutical composition of  claim 82 , wherein the lipocalin mutein further comprises the following amino acid substitutions with respect to the linear polypeptide sequence of mature hNGAL: Lys 59→Glu; Phe 71→Pro; Lys 74→Glu; Ile 80→Phe; Cys 87→Ser; and Ile 135→Val. 
     
     
         87 . The pharmaceutical composition of  claim 79 , wherein the lipocalin mutein comprises, at sequence positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 106, 125, 127, 132 and 134, with respect to the linear polypeptide sequence of mature hNGAL, the same set of amino acid residues that the mutein set forth in sequence 13 of  FIG. 3  (SEQ ID NO: 49) has at the respective positions. 
     
     
         88 . The pharmaceutical composition of  claim 79 , wherein the lipocalin mutein comprises, at sequence positions 33 to 54, 66 to 83, 94 to 106, 123 to 136, with respect to the linear polypeptide sequence of mature hNGAL, the same set of amino acid residues that the mutein set forth in sequence 13 of  FIG. 3  (SEQ ID NO: 49) has at the respective positions. 
     
     
         89 . The pharmaceutical composition of  claim 79 , wherein the lipocalin mutein comprises, at 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, with respect to the linear polypeptide sequence of mature hNGAL, the same set of amino acid residues that the mutein set forth in sequence 13 of  FIG. 3  (SEQ ID NO: 49) has at the respective positions. 
     
     
         90 . The pharmaceutical composition of  claim 79 , wherein the lipocalin mutein has at least 90% sequence identity to sequence 13 of  FIG. 3  (SEQ ID NO: 49). 
     
     
         91 . The pharmaceutical composition of  claim 79 , wherein the lipocalin mutein has at least 95% sequence identity to sequence 13 of  FIG. 3  (SEQ ID NO: 49). 
     
     
         92 . The pharmaceutical composition of  claim 79 , wherein the lipocalin mutein comprises the polypeptide sequence of sequence 13 of  FIG. 3  (SEQ ID NO: 49). 
     
     
         93 . The pharmaceutical composition of  claim 79 , 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. 
     
     
         94 . The pharmaceutical composition of  claim 79 , wherein the lipocalin mutein is conjugated to a compound that extends the serum half-life of the mutein.