Muteins of human lipocalin 2 (Lcn2,hNGAL) with affinity for a given target

Patent metadata

Field	Value
Publication number	US-10618941-B2
Application number	US-201615380168-A
Country	US
Kind code	B2
Filing date	Dec 15, 2016
Priority date	Dec 7, 2009
Publication date	Apr 14, 2020
Grant date	Apr 14, 2020

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 human neutrophil gelatinase-associated lipocalin (hNGAL) mutein, 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) and at nine or more 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, 103, 125, 127, 132, and 134 of mature hNGAL (SEQ ID NO: 44), wherein the mutein has at least 80% sequence identity to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 44), and wherein the mutein demonstrates binding with detectable affinity to a target which is a non-natural ligand in that it does not bind to the mature hNGAL (SEQ ID NO: 44) with detectable affinity under physiological conditions.

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

3. The mutein of claim 1 , wherein the mutated amino acid residue at one or more amino acid sequence positions selected from the group consisting of 96, 100, and 106 of mature hNGAL (SEQ ID NO:44) comprises a mutated amino acid residue at each of the amino acid sequence positions.

4. The mutein of claim 1 , wherein the mutated amino acid residue at nine or more 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) comprises a mutated amino acid residue at eighteen or more of the amino acid sequence positions.

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

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

7. The mutein of claim 1 , wherein the mutein comprises with respect to the 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; and Arg81→Trp or Asn or His.

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

9. 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.

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

11. The mutein of claim 10 , wherein the compound that extends the serum half-life is selected from the group consisting of a polyalkylene glycol molecule, hydroxyethyl starch, 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.

12. The mutein of claim 11 , wherein the polyalkylene glycol is polyethylene glycol (PEG) or an activated derivative thereof.

13. The mutein of claim 9 , wherein the fusion partner of the mutein is a protein domain that extends the serum half-life of the mutein.

14. The mutein of claim 13 , 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.

15. The mutein of claim 1 , wherein the mutein binds the target with a K D of 200 nM or less.

16. A nucleic acid molecule comprising a nucleotide sequence encoding a mutein of claim 1 .

17. The nucleic acid molecule of claim 16 , wherein the nucleic acid molecule is operably linked to a regulatory sequence to allow expression of said nucleic acid molecule.

18. A host cell containing a nucleic acid molecule of claim 17 .

19. A method for the production of a product that is the mutein of claim 1 , a fragment of the mutein or a fusion protein of the mutein and another polypeptide, wherein the method comprises:
subjecting a nucleic acid molecule encoding mature hNGAL to mutagenesis at a nucleotide triplet coding for the at least one amino acid sequence positions 96, 100, and 106 of the mature hNGAL (SEQ ID NO:44), and for the at least nine 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, 103, 125, 127, 132, and 134 of the mature hNGAL (SEQ ID NO:44), resulting in one or more nucleic acid molecule(s) encoding the mutein; and
expressing the product from the one or more nucleic acid molecule(s) encoding the mutein.

20. The method of claim 19 , wherein the product is produced in a bacterial or eukaryotic host organism and is isolated from said host organism or its culture.

Assignees

• Pieris Pharmaceuticals Gmbh

Inventors

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

Classifications

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• 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

• 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