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

Patent metadata

Field	Value
Publication number	US-9549968-B2
Application number	US-201013514133-A
Country	US
Kind code	B2
Filing date	Dec 7, 2010
Priority date	Dec 7, 2009
Publication date	Jan 24, 2017
Grant date	Jan 24, 2017

Abstract

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.

First claim

The invention claimed is:

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.

2. The mutein of claim 1 , wherein the mutein comprises a mutated amino acid residue at two or three 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).

3. The mutein of claim 1 , wherein the mutein comprises a mutated amino acid residue at 18, 19, or 20 amino acid sequence positions selected from the group consisting of amino acid sequence positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 106, 125, 127, 132, and 134 of mature hNGAL (SEQ ID NO:44).

4. The mutein of claim 1 , wherein said non-natural target is selected from the group consisting of a peptide, a protein, a fragment or a domain of a protein, and a small organic molecule.

5. The mutein of claim 4 , wherein the small organic molecule is a compound showing the features of an immunological hapten.

6. The mutein of claim 4 , wherein the peptide has a length of 2-45 amino acids.

7. The mutein of claim 6 , wherein the peptide is an amyloid beta peptide.

8. The mutein of claim 7 , wherein the amyloid beta peptide is an Aβ40 peptide or an Aβ42 peptide.

9. 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 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.

10. The mutein of claim 9 , wherein the mutein has the sequence of SEQ ID NO:39 (S1-A4), SEQ ID NO:41 (US7), or SEQ ID NO:43 (H1-G1).

11. The mutein of claim 4 , wherein the protein is fibronectin or a domain thereof.

12. The mutein of claim 11 , wherein the fibronectin domain is extra-domain B or a fragment thereof.

13. 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 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.

14. The mutein of claim 13 , wherein the mutein has the sequence of SEQ ID NO:20 (N7A), SEQ ID NO:22 (N7E), SEQ ID NO:24 (N9B), or SEQ ID NO:26 (N10D).

15. The mutein of claim 1 , wherein the mutein further comprises with respect to mature hNGAL (SEQ ID NO:44) amino acid replacements selected from the group consisting of Gln28→His and Cys87→Ser.

16. The mutein of claim 1 , wherein the mutein comprises with respect to mature hNGAL (SEQ ID NO:44) one or more amino acid replacements selected from the group consisting of Tyr52→Gln or Val; Ser68→Lys or Asn; Arg81→Trp or Asn or His among the group of amino acid replacements according to claim 1 .

17. The mutein of claim 1 , wherein the mutein is conjugated to a compound selected from the group consisting of an organic molecule, enzyme label, radioactive label, colored label, fluorescent label, chromogenic label, luminescent label, hapten, digoxigenin, biotin, cytostatic agent, toxin, metal complex, metal, and colloidal gold.

18. The mutein of claim 1 , wherein the 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.

19. The mutein of claim 1 , wherein the mutein is conjugated to a compound that extends the serum half-life of the mutein.

20. The mutein of claim 19 , wherein the compound that extends the serum half-life is selected from the group consisting of a polyalkylene glycol molecule, hydroethylstarch, a Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, an albumin binding peptide, and an albumin binding protein.

21. The mutein of claim 20 , wherein the polyalkylene glycol is polyethylene (PEG) or an activated derivative thereof.

22. The mutein of claim 18 , wherein the fusion partner of the mutein is a protein domain that extends the serum half-life of the mutein.

23. The mutein of claim 22 , wherein the protein domain is a Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, an albumin binding peptide, or an albumin binding protein.

24. The mutein of claim 20 , wherein the albumin binding protein is a bacterial albumin domain or a lipocalin mutein.

25. The mutein of claim 24 , wherein the bacterial albumin binding domain is the albumin binding domain of streptococcal protein G.

26. The mutein of claim 20 , wherein the albumin binding peptide has the formula Cys-Xaa 1 -Xaa 2 -Xaa 3 -Xaa 4 -Cys, wherein Xaa 1 is Asp, Asn, Ser, Thr, or Trp; Xaa 2 is Asn, Gln, His, Ile, Leu, or Lys; Xaa 3 is Ala, Asp, Phe, Trp, or Tyr; and Xaa 4 is Asp, Gly, Leu, Phe, Ser, or Thr.

27. The mutein of claim 1

Assignees

• Skerra Arne
• Gebauer Michaela
• Hinz Dominik
• Rauth Sabine
• Matschiner Gabriele
• Huelsmeyer Martin
• Pieris Pharmaceuticals Gmbh

Inventors

• Skerra Arne
• Gebauer Michaela
• Hinz Dominik
• Rauth Sabine
• Matschiner Gabriele
• Huelsmeyer Martin

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• A61P25/28

  for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia · CPC title

• A61P29/00

  Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] · CPC title

• A61K47/60

  the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol · CPC title

• C07K14/47 Primary

  from mammals · CPC title