Muteins of tear lipocalin and methods for obtaining the same
AssigneePieris AG
InventorsKristian Jensen, Martin Huelsmeyer, Steffen Schlehuber, Andreas Hohlbaum, Arne Skerra, Eric Boudreau, Richard Jones, Ian Kimber, Rebecca Dearman
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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 a 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.
Claims44
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.
A mutein of human tear lipocalin having detectable binding affinity to a non-natural ligand of human tear lipocalin, wherein at least one of the cysteine residues occurring at sequence positions 61 and 153 of the linear wild type amino acid sequence of mature human tear lipocalin (SEQ ID NO:
- 71) is replaced by other amino acids and wherein at least 12, 14, or 16 mutated amino acid residues are present at any of the 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 set forth in SEQ ID NO:
- 71.
A method for producing a mutein of human tear lipocalin having detectable binding affinity to a non-natural ligand of human tear lipocalin, comprising:
- (i) expressing a nucleic acid encoding the mutein in an expression system which comprises a bacterial or isolated eukaryotic host cell, or an in vitro translation system, and
- (ii) isolating the mutein from the host cell or its culture, or the in vitro translation system;
- wherein the mutein of human tear lipocalin having detectable binding affinity to a non-natural ligand of human tear lipocalin is mutated in at least one of the cysteine residues occurring at sequence positions 61 and 153 of the linear wild type amino acid sequence of mature human tear lipocalin (SEQ ID NO:
- 71) by replacement with other amino acids;
- and at least 12, 14, or 16 mutated amino acid residues are present at any of the 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 set forth in SEQ ID NO:
- 71.
Dependencies are inferred from the claim text.