Muteins of human lipocalin 2 (LcnC,hNGAL) with affinity for a given target

摘要

The present invention relates to a novel library for the generation of muteins and to novel muteins derived from human lipocalin 2 (Lcn2, hNGAL) and related proteins that bind a given target with detectable affinity. The invention also relates to corresponding nucleic acid molecules encoding such a mutein and to a method for their generation. The invention further relates to a method for producing such a mutein. For example, such muteins may serve to bind and deplete pathological forms of natural biomolecules such as the amyloid beta peptide in Alzheimer's disease or may target the fibronectin extra-domain B, which is associated with tumor neovasculature.

主权项

1. A mutein derived from human neutrophil gelatinase-associated lipocalin (hNGAL), comprising a mutated amino acid residue at one or more amino acid sequence positions selected from the group consisting of amino acid sequence positions 96, 100, and 106 of mature hNGAL (SEQ ID NO:44), wherein the mutein binds a given non-natural target with detectable affinity, and wherein the mutein comprises with respect to mature hNGAL (SEQ ID NO:44) at least three amino acid replacements that are:
(A) selected from the group consisting of Leu36→Val or Cys; Ala40→Tyr or Lys or Val; Ile41→Thr or Ser or Leu; Gln49→Leu or Trp; Leu70→Gly; Arg72→Gly or Asp; Lys73→Leu or Thr or Asp; Asp77→Asn or His or Leu; Trp79→Lys; Asn96→Ile or Arg; Tyr100→Gln or Arg or Glu; Leu103→Met or Arg or Gly; Tyr106→Tyr or Ala or Trp; Lys125→Thr or Val or Glu; Ser127→Gly or Gln or Ala; Tyr132→Met or Ser or Thr; and Lys134→Asn, or (B) selected from the group consisting of Leu36→Lys or Glu or Arg or Ala; Ala40→His or Met or Thr or Ser; Ile41→Asp or Arg or Trp or Leu; Gln49→Arg or Ala or Tyr; Leu70→Leu or Arg or Met; Arg72→Val or Arg or Gln or Met; Lys73→His or Arg or Ser; Asp77→Asn or His or Lys or Arg; Trp79→Arg or Met or Leu; Asn96→Lys or Ala or Ser; Tyr100→Trp or Pro or Lys; Leu103→His or Pro; Tyr106→Phe or Trp or Thr; Lys125→Arg or His or Thr; Ser127→Tyr or Phe or Ala; Tyr132→Leu or Phe; Lys134→Glu or His or Gly or Phe; and Ser146→Asn.