Patent ID: 8313924
Filing Date: 2012-11-20
Classification: A61K,A61P,C07K,C12N,G01N

Abstract:
1. A method for the generation of one or more mutein(s) of human tear lipocalin, wherein the one or more mutein(s) bind a given non-natural ligand of human tear lipocalin with detectable binding affinity, comprising: (a) subjecting a nucleic acid molecule encoding a human tear lipocalin to mutagenesis at least 12, 14, or 16 of the codons of any of the amino acid sequence positions 26-34, 56-58, 80, 83, 104-106 and 108 of the linear wild type amino acid sequence of mature human tear lipocalin (SEQ ID NO: 71), wherein at least one of the codons encoding cysteine residues at sequence positions 61 and 153 of the linear wild type amino acid sequence of the mature human tear lipocalin (SEQ ID NO: 71) has been mutated to encode any other amino acid residue, thereby obtaining a plurality of nucleic acids encoding muteins of human tear lipocalin, (b) expressing the nucleic acid molecule(s) obtained in (a) in an expression system, thereby obtaining one or more mutein(s); wherein the expression system comprises a bacterial or isolated eukaryotic host cell, or an in vitro translation system, (c) contacting the one or more muteins of step (b) with a non-natural ligand of human tear lipocalin to allow formation of complexes between said ligand and one or more mutein(s) having binding affinity for said ligand, wherein the non-natural ligand is a compound selected from the group consisting of a chemical compound in free or conjugated form that exhibits features of an immunological hapten, a peptide, a protein or another macromolecule, and (d) selecting the one or more mutein(s) that have detectable binding affinity to said non-natural ligand.