# US 9,040,020 B2 · Muteins of hNGAL and related proteins with affinity for a given target

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- Number: US 9,040,020 B2
- Document ID: US9040020B2
- Status: Granted
- Kind: B2
- Application number: 13/859,119
- Filed: 2013-04-09
- Published: 2013-11-28
- Granted: 2015-05-26
- Earliest claimed: 2008-06-24
- Assignees: Technische Universitaet Muenchen
- Applicants: Technische UniversitäT MüNchen
- Inventors: Arne Skerra; Andreas Eichinger; Hyun-Jin Kim
- Web page: https://patentgrep.com/patent/US9040020B2

## CPC

- C07K 14/47: from mammals
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61K 51/081: {the protein being an albumin, e.g. human serum albumin [HSA], bovine serum albumin [BSA], ovalbumin}
- A61K 51/088: {conjugates with carriers being peptides, polyamino acids or proteins (antibodies A61K51/10)}
- A61P 35/00: Antineoplastic agents
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- C07K 2319/01: containing a localisation/targetting motif
- C07K 2319/20: containing a tag with affinity for a non-protein ligand
- C07K 2319/40: containing a tag for immunodetection, or an epitope for immunisation
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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 G1u28→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; Ile55→Thr; of Lys59→Arg; Asn65→Asp; Ser68→Ala; Leu70→Arg; Lys75→Met; Asp77Glu; Tyr78→His; Trp79→Ala; Trp79→Leu; Ile 80→Thr; Leu; Arg81→Met; Gly86→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.

### Claim 6 (depends on 1)

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; 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; 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; Lys75→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.

### Claim 7 (depends on 1)

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.

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 1)

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.

### Claim 10 (depends on 1)

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.

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 11)

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.

### Claim 13 (depends on 1)

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.

### Claim 14 (depends on 1)

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

### Claim 15 (depends on 14)

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

### Claim 16 (depends on 14)

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

### Claim 17 (depends on 1)

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 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), resulting in one or more mutein nucleic acid molecule(s),
(b) expressing the one more mutein nucleic acid molecule(s) obtained in (a) in a suitable expression system, and
(c) identifying one or mutein nucleic acid molecules encoding a mutein having a detectable binding affinity for a given target by means of selection and/or isolation.

### Claim 18 (depends on 1)

The mutein of claim 1, which is recombinantly expressed.

### Claim 19 (depends on 1)

A pharmaceutical composition comprising the mutein of claim 1 and a pharmaceutically acceptable excipient.

### Claim 20 (depends on 1)

A diagnostic or analytical kit comprising the mutein of claim 1 in a container, optionally containing instructions for its use.