Muteins of hNGAL and related proteins with affinity for a given target

Patent metadata

Field	Value
Publication number	US-9040020-B2
Application number	US-201313859119-A
Country	US
Kind code	B2
Filing date	Apr 9, 2013
Priority date	Jun 24, 2008
Publication date	May 26, 2015
Grant date	May 26, 2015

Abstract

The present invention relates to novel muteins derived from human lipocalin 2 (hNGAL) and related proteins that bind a given non-natural ligand with detectable affinity. The invention also related 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. Furthermore, the invention is directed to a pharmaceutical composition comprising such a lipocalin mutein as well as to various uses of the mutein.

First claim

The invention claimed is:

1. A mutein derived human neutrophil gelatinase-associated lipocalin (hNGAL) comprising at least 3 mutated amino acid residues at any of the sequence positions corresponding to the sequence positions 33, 36, 41, 52, 54, 68, 70, 79, 81, 134, 136 and 138 of the linear polypeptide sequence of hNGAL (SEQ ID NO: 1), wherein the mutein binds a given target with detectable affinity.

2. The mutein of claim 1 , which has an amino acid sequence further differing from the linear polypeptide sequence of hNGAL (SEQ ID NO: 1) by at least one mutated amino acid residue at at least one of the sequence positions corresponding to the sequence positions 42, 43, 44, 46, 47, 48, 49, 50, 51, 55, 59, 65, 71, 73, 74, 75, 77, 78, 80, 86, 87, 98, 99, 103, 107, 110,111, 116, 125, 127 and 135 of the linear polypeptide sequence of hNGAL.

3. The mutein of claim 1 , wherein the mutein comprises at least 4mutated amino acid residues at at least one of the sequence positions corresponding to the sequence positions 33, 36, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 55, 59, 65, 68, 70, 75, 77, 78, 79, 80, 81, 86, 87, 98, 99, 103, 107, 110, 111, 127, 134, 136 and 138 of the linear polypeptide sequence of hNGAL.

4. The mutein of claim 1 , wherein a Cys residue is introduced at at least one of the sequence positions that correspond to sequence positions 14, 21, 60, 84, 88, 116, 141, 145, 143, 146 or 158 of the wild type sequence of hNGAL.

5. The mutein of claim 1 , wherein the mutein comprises with respect to the linear polypeptide sequence of hNGAL at least one amino acid replacement selected from the group consisting of G 1 u28→His; Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro; Glu44→Val; Glu44→Met; Lys46→Pro; Asp47→Glu; Pro48Leu; Gln49→Leu; Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile 55 →Thr; of Lys59→Arg; Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77Glu; Tyr78→His; Trp79→Ala; Trp79→Leu; Ile 80→Thr; Leu; Arg 81 →Met; Gly 86 →Ser; Ser87→Pro; Ser87→Phe; Cys87→Ser; Lys98→Glu; Ser99→Asn; Leu103→Ile; Leu107→Phe; Val110→Met; Val111→Ala; Ser127→Gln; Lys134→Ser; Thr136→Ser; Tyr138→Leu and Thr145→Ala.

6. The mutein of claim 1 , wherein the mutein comprises with respect to the linear polypeptide sequence of hNGAL any one of the following amino acid replacements:
(a). Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln; Ser68→Ala; Leu70→Arg; Trp79→Ala; Ile 80→Thr; Arg81→Met; Lys134→Ser; and Tyr138→Leu;
(b). Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln; Ser68→Ala; Leu70→Arg; Trp79→Leu; Ile 80Thr; Arg81→Met; Lys134→Ser; and Tyr138→Leu;
(c). Val33→Gln; Leu36Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln; Ser68→Ala; Leu70→Arg; Trp79→Leu; Ile 80→Thr; Arg81→Met; Ser127→Gln; Lys134→Ser; and Tyr138→Leu;
(d). Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln; Ser68→Ala; Leu70→Arg; Trp79→Leu; Ile 80→Thr; Arg81→Met; Lys134→Ser; Thr136→Ser; and Tyr138→Leu;
(e). Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln; Ser68→Ala; Leu70→Arg; Trp79→Leu; Ile 80→Thr; Arg81→Met; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu;
(f). Val33→Gln; Leu36→Arg; Ile41→Ala; Tyr52→Thr; Thr54→Gln; Ser68→Ala; Leu70→Arg; Asp77→Glu; Trp79→Leu; Ile 80→Thr; Arg81→Met; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu;
(g). Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Pro48→Leu; Gln49→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu; Trp79→Leu; Ile 80→Thr; Arg81→Met; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu;
(h). Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro; Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu; Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu; Trp79→Leu; Ile 80→Thr; Arg81→Met; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu;
(i). Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg 43 →Pro; Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu; Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr;Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu; Trp79→Leu; Ile 80→Thr; Arg81→Met; Lys98→Glu; Va110→Met; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu;
(j). Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro; Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu; Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr; Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu; Trp79→Leu; Ile 80→Thr; Arg81→Met; Gly86→Ser; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu;
(k). Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro; Glu44→Met; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu; Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr; Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys 75 →Met; Asp77→Glu; Trp79→Leu; Ile 80→Thr; Arg81→Met; Gly86→Ser; Ser87→Pro; Ser99→Asn; Leu107→Phe; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu;
(l). Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro; Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu; Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr; Lys59→Arg; Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu; Trp79→Leu; Ile 80→Thr; Arg81→Met; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu;
(m). Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro; Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu; Lys50→Leu; Met51→Leu; Tyr52Thr; Thr54→Gln; Ile55→Thr; Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu; Trp79→Leu; Ile 80→Thr; Arg81→Met; Ser87→Phe; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu; or
(n). Val33→Gln; Leu36→Arg; Ile41→Ala; Leu42→Pro; Arg43→Pro; Glu44→Val; Lys46→Pro; Asp47→Glu; Pro48→Leu; Gln49→Leu; Lys50→Leu; Met51→Leu; Tyr52→Thr; Thr54→Gln; Ile55→Thr; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77→Glu; Tyr78→His; Trp79→Leu; Ile 80→Thr; Arg81→Met; Leu103→Ile; Leu107→Phe; Val111→Ala; Ser127→Gln; Lys134→Ser; Thr136→Ser; and Tyr138→Leu.

7. The mutein of claim 1 , wherein the mutein has an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 2-10 and 28-34.

8. The mutein of claim 1 , wherein the mutein binds a small organic molecule or a peptide.

9. The mutein of claim 1 , wherein the mutein is conjugated to targeting moiety with binding affinity for a chosen target molecule, wherein said targeting moiety targets a specific body region in a mammal.

10. The mutein of claim 1 , wherein the mutein is conjugated to a label selected from the group consisting of organic molecules, enzyme labels, radioactive labels, colored labels, fluorescent labels, chromogenic labels, luminescent labels, haptens, digoxigenin, biotin, metal complexes, metals, and colloidal gold.

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

12. The mutein of claim 11 , wherein the moiety that extends the serum half-life is selected from the group consisting of a polyalkylene glycol molecule, hydroxyethylstarch, a Fc part of an immunoglobulin, a CH3domain of an immunoglobulin, a CH4domain of an immunoglobulin, an albumin binding peptide, and an albumin binding protein.

13. The mutein of claim 1 , wherein the mutein is fused at its N-terminus and/or its C-terminus to a protein, a protein domain or a peptide.

14. An isolated nucleic acid molecule encoding a mutein of claim 1 .

15. The isolated nucleic acid molecule of claim 14 comprised in a phagemid vector.

16. An isolated cell containing the nucleic acid molecule of claim 14 .

17. A method for the generation of a mutein of claim 1 , comprising:
(a) subjecting a nucleic acid molecule encoding an hNGAL protein to mutagenesis at a nucleotide triplet coding for at least 3 of the sequence posit

Assignees

• Univ Muenchen Tech
• Univ Muenchen Tech

Inventors

• Skerra Arne
• Eichinger Andreas
• Kim Hyun-Jin

Classifications

• C07K2319/01

  containing a localisation/targetting motif · CPC title

• C12N15/1037

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C07K14/47 Primary

  from mammals · CPC title

• A61K51/088

  conjugates with carriers being peptides, polyamino acids or proteins (antibodies A61K51/10) · CPC title