Muteins of tear lipocalin and methods for obtaining the same

Abstract

The present invention relates to novel muteins derived from human tear lipocalin. The invention also refers to a corresponding nucleic acid molecule encoding such mutein and to a method for its generation. The invention further refers to a method for producing such a mutein. Finally, the invention is directed to a pharmaceutical composition comprising such a lipocalin mutein as well as to various uses of the mutein.

1 - 147 . (canceled) 
     
     
         148 . A mutein of human tear lipocalin capable of binding a non-natural ligand of human tear lipocalin with a K D  of about 200 nM or lower, wherein the mutein comprises a mutated amino acid residue at any one or both of the sequence positions 80 and 104 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 71), and wherein the mutein comprises at least 10 mutated amino acid residues at any of the sequence positions 26-34, 56-58, 83, 105-106, and 108 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 71). 
     
     
         149 . A mutein of human tear lipocalin capable of binding a non-natural ligand of human tear lipocalin with a K D  of about 200 nM or lower, wherein the mutein comprises 2 or 3 mutated amino acid residues at any of the sequence positions 61, 101, and 153 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 71), and wherein the mutein comprises at least 12 mutated amino acid residues at any of the sequence positions 26-34, 56-58, 80, 83, 104-106, and 108 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 71). 
     
     
         150 . The mutein of  claim 148 , wherein the mutein comprises 1, 2, or 3 mutated amino acid residues at any of the sequence positions 61, 101, and 153 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 71). 
     
     
         151 . The mutein of  claim 148 , wherein the mutein comprises at least one of the following mutated amino acid residues with respect to the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 71): Cys 61→Ala, Phe, Lys, Arg, Thr, Asn, Tyr, Met, Ser, Pro or Trp; Cys 153→Ser or Ala; and Cys 101→Ser. 
     
     
         152 . The mutein of  claim 148 , wherein the mutein comprises at least 15 mutated amino acid residues at any of the sequence positions 26-34, 56-58, 80, 83, 104-106, and 108 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 71). 
     
     
         153 . The mutein of  claim 149 , wherein the mutein comprises at least 15 mutated amino acid residues at any of the sequence positions 26-34, 56-58, 80, 83, 104-106, and 108 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 71). 
     
     
         154 . The mutein  claim 148 , wherein the non-natural ligand is selected from the group consisting of a small molecule, a peptide, and a protein or protein domain. 
     
     
         155 . The mutein of  claim 148 , wherein the non-natural ligand is selected from the group consisting of interleukin 4 receptor alpha chain (IL-4 receptor alpha), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor 2 (VEGF-R2). 
     
     
         156 . The mutein of  claim 148 , wherein the mutein comprises one of the following sets of mutated amino acid residues at sequence positions corresponding to the linear wild type amino acid sequence of mature human tear lipocalin (SEQ ID NO: 71):
 (a) Arg 26→Ser, Glu 27→Arg, Phe 28→Cys, Glu 30→Arg; Met 31→Ala, Leu 33→Tyr, Leu 56→Gln, Ile 57→Arg, Asp 80→Ser, Lys 83→Arg, Glu 104→Leu, Leu 105→Cys, His 106→Pro, and Lys 108→Gln.   (b) Glu 27→Gly, Phe 28→Ala, Pro 29→Leu, Glu 30→Arg, Met 31→Cys, Asn 32→Leu, Leu 33→Ala, Glu 34→Gly, Asp 80→Ile, Lys 83→Ile, Glu 104→Cys, and Lys 108→Val.   (c) Arg 26→Ser, Glu 27→Ile, Glu 30→Ser, Met 31→Gly, Asn 32→Arg, Leu 33→Ile, Glu→34 Tyr, Ile 57→Phe, Ser 58→Arg, Lys 83→Glu, Glu 104→Leu, Leu 105→Ala, His 106→Val, and Lys 108→Thr.   
     
     
         157 . The mutein of  claim 148 , wherein the mutein has at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8, 26-33 and 44-47. 
     
     
         158 . The mutein of  claim 148 , wherein the mutein is in a conjugated form or as a fusion protein. 
     
     
         159 . The mutein of  claim 148 , wherein the mutein is, at its N-terminus or its C-terminus, conjugated with or fused to a molecule selected from the group consisting of a toxin, a signal sequence, an affinity tag, a peptide, a protein or a protein domain, an antibody, and a mutein of human lipocalin. 
     
     
         160 . The mutein of  claim 148 , wherein the mutein is fused to a moiety that extends the serum half-life of the mutein. 
     
     
         161 . The mutein of  claim 160 , wherein the moiety that extends the serum half-life is selected from the group consisting of an Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, albumin or an albumin fragment, an albumin binding peptide, an albumin binding protein, and transferrin. 
     
     
         162 . A nucleic acid molecule comprising a nucleotide sequence encoding a mutein of  claim 148 . 
     
     
         163 . A method for the production of the mutein of  claim 148 , wherein the mutein is produced starting from the nucleic acid coding for the mutein. 
     
     
         164 . A pharmaceutical composition comprising one or more mutein(s) of  claim 148 . 
     
     
         165 . A nucleic acid molecule comprising a nucleotide sequence encoding a mutein of  claim 149 . 
     
     
         166 . A method for the production of the mutein of  claim 149 , wherein the mutein is produced starting from the nucleic acid coding for the mutein. 
     
     
         167 . A pharmaceutical composition comprising one or more mutein(s) of  claim 149 .