Proteins specific for calcitonin gene-related peptide

Patent metadata

Field	Value
Publication number	US-10400016-B2
Application number	US-201615371698-A
Country	US
Kind code	B2
Filing date	Dec 7, 2016
Priority date	Dec 10, 2015
Publication date	Sep 3, 2019
Grant date	Sep 3, 2019

Abstract

The present disclosure provides hNGAL muteins that bind CGRP and can be used in various application including pharmaceutical applications, for example, migraine. The present disclosure also concerns methods of making one or more muteins described herein as well as compositions and combinations comprising one or more of such muteins. The present disclosure further relates to nucleic acid molecules encoding such muteins and to methods for generation of such muteins and nucleic acid molecules. In addition, the application discloses therapeutic and/or diagnostic uses of these muteins as well as compositions and combinations comprising one or more of such muteins.

First claim

The invention claimed is:

1. A human neutrophil gelatinase associated lipocalin (hNGAL) mutein that is capable of binding CGRP with detectable affinity, wherein the mutein comprises eleven or more of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 1): Leu 36→Ile, Phe, Trp, Arg or Glu; Ala 40→Asp, Glu, Met, Trp or Thr; Ile 41→Ala, Arg, Val, Thr, Leu, Trp, Gly or Glu; Gln 49→Ile, Pro, Leu, Phe, Lys, Glu or Thr; Tyr 52→Ala, Gly, Glu or Gln; Ser 68→Trp, His or Asp; Leu 70→Met, Trp, Tyr, Gly or Gln; Arg 72→Val, Ala, Met, Ile, Trp, Glu or Ser; Lys 73→Arg, Asp, Ala, Glu, Thr or Gln; Asp 77→Met, Arg, Ile or Asn; Trp 79→Val, Gly, His or Thr; Arg 81→Gly, His, Glu or Asn; Asn 96→Leu, Ala, Gly or Thr; Tyr 100→His, Ile, Pro or Glu; Leu 103→Val, Met, Glu, Thr or Gln; Tyr 106→Gly, Leu, Ile, Ala, His or Asn; Lys 125→Leu, Val, Phe, Gly or Glu; Ser 127→Asn, Gly, Lys, Phe, Trp or Arg; Tyr 132→Ser, Leu, Ile or Trp; and Lys 134→Trp, His or Glu, and wherein the mutein has at least 80% sequence identity to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 1).

2. The hNGAL mutein of claim 1 , wherein said mutein is capable of
(a) binding CGRP with a K D of about 5 nM or lower, or
(b) inhibiting or reducing CGRP induced cAMP production with an IC50 value of about 5 nM or lower.

3. The hNGAL mutein of claim 1 , wherein said mutein is crossreactive with both human CGRP and rat CGRP.

4. The hNGAL mutein of claim 3 , wherein said mutein is capable of
(a) binding rat CGRP with detectable affinity,
(b) binding rat CGRP with a K D of about 5 nM or lower, or
(c) inhibiting or reducing rat CGRP induced cAMP production with an IC50 value of about 5 nM or lower.

5. The hNGAL mutein of claim 1 , wherein said mutein comprises a mutated amino acid residue at one or more positions corresponding to a sequence positions 8, 9, 28, 38, 42, 44, 46, 47, 54, 62, 65, 66, 71, 75, 76, 80, 83, 87, 97, 98, 105, 108, 111, 112, 114, 123, 126, 129, 135, 136, 145, 146, 175, 176, 177 and 178 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 1).

6. The hNGAL mutein of claim 1 , wherein the amino acid sequence of the hNGAL mutein comprises at least one of the following mutated amino acid residue in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 1): Gln 28→His; and Cys 87→Ser.

7. The hNGAL mutein of claim 1 , wherein the hNGAL mutein comprises one of the following sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 1):
(a) Gln 28→His; Leu 36→Glu; Ala 40→Trp; Ile 41→Gly; Gln 49→Lys; Tyr 52→Ala; Ser 68→Asp; Leu 70→Gln; Arg 72→Ile; Lys 73→Glu; Arg 81→Gly; Cys 87→Ser; Asn 96→Ala; Tyr 100→Glu; Leu 103→Gln; Tyr 106→Asn; Lys 125→Glu; Ser 127→Trp; Tyr 132→Leu; Lys 134→Trp;
(b) Gln 28→His; Leu 36→Phe; Ala 40→Met; Ile 41→Trp; Gln 49→Phe; Tyr 52→Gly; Ser 68→Trp; Leu 70→Trp; Arg 72→Glu; Lys 73→Ala; Trp 79→Gly; Arg 81→Asn; Cys 87→Ser; Asn 96→Gly; Tyr 100→Pro; Leu 103→Met; Tyr 106→His; Lys 125→Glu; Ser 127→Phe; Tyr 132→Trp; Lys 134→Trp;
(c) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gln 49→Thr; Tyr 52→Gln; Ser 68→Trp; Leu 70→Tyr; Arg 72→Ser; Lys 73→Glu; Asp 77→Asn; Trp 79→His; Arg 81→Glu; Cys 87→Ser; Asn 96→Thr; Leu 103→Glu; Tyr 106→Ile; Lys 125→Gly; Tyr 132→Ile; Lys 134→Glu;
(d) Gln 28→His; Leu 36→Arg; Ile 41→Glu; Gln 49→Glu; Tyr 52→Glu; Ser 68→Asp; Leu 70→Gly; Arg 72→Trp; Lys 73→Gln; Asp 77→Ile; Trp 79→Val; Arg 81→His; Cys 87→Ser; Leu 103→Thr; Tyr 106→Ala; Lys 125→Val; Ser 127→Arg; Tyr 132→Trp; Lys 134→Glu; or
(e) Gln 28→His; Leu 36→Ile; Ala 40→Trp; Ile 41→Trp; Gln 49→Leu; Ser 68→His; Leu 70→Met; Arg 72→Met; Lys 73→Thr; Trp 79→Thr; Cys 87→Ser; Tyr 100→Ile; Leu 103→Met; Tyr 106→Leu; Lys 125→Phe; Ser 127→Trp; Tyr 132→Trp; Lys 134→His.

8. The hNGAL mutein of claim 1 , wherein the amino acid sequence of the hNGAL mutein comprises at least one of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 1): Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Leu 42→Arg; Asp 47→Asn; Gln 49→Ile, Pro or Thr; Tyr 52→Gln; Thr 54→Met, Ile or Lys; Lys 62→Arg; Asn 65→Asp; Val 66→Ala; Ser 68→Trp; Leu 70→Tyr; Phe 71→Leu; Arg 72→Ala or Ser; Lys 73→Asp or Glu; Lys 75→Arg; Asp 77→Arg or Asn; Trp 79→His; Arg 81→Glu; Phe 83→Ser; Cys 87→Ser; Asn 96→Leu or Thr; Ile 97→Thr; Lys 98→Gln; Tyr 100→His; Leu 103→Glu; Ser 105→Pro; Tyr 106→Ile; Val 111→Met; Lys 125→Gly; Val 126→Met; Ser 127→Gly or Asn; Tyr 132→Ile; Lys 134→Glu; Thr 136→Ile or Val; Thr 145→Ala; and Ser 146→Asn.

9. The hNGAL mutein of claim 1 , wherein the hNGAL mutein comprises one of the following sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 1):
(a) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gln 49→Ile; Tyr 52→Gln; Ser 68→Trp; Leu 70→Tyr; Arg 72→Ser; Lys 73→Glu; Lys 75→Arg; Asp 77→Asn; Trp 79→His; Arg 81→Glu; Phe 83→Ser; Cys 87→Ser; Asn 96→Thr; Leu 103→Glu; Tyr 106→Ile; Lys 125→Gly; Tyr 132→Ile; Lys 134→Glu;
(b) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Leu 42→Arg; Asp 47→Asn; Gln 49→Thr; Tyr 52→Gln; Ser 68→Trp; Leu 70→Tyr; Arg 72→Ser; Lys 73→Glu; Asp 77→Asn; Trp 79→His; Arg 81→Glu; Phe 83→Ser; Cys 87→Ser; Asn 96→Thr; Leu 103→Glu; Tyr 106→Ile; Lys 125→Gly; Tyr 132→Ile; Lys 134→Glu;
(c) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gln 49→Ile; Tyr 52→Gln; Asn 65→Asp; Ser 68→Trp; Leu 70→Tyr; Phe 71→Leu; Arg 72→Ser; Lys 73→Glu; Asp 77→Asn; Trp 79→His; Arg 81→Glu; Phe 83→Ser; Cys 87→Ser; Asn 96→Thr; Leu 103→Glu; Tyr 106→Ile; Lys 125→Gly; Val 126→Met; Tyr 132→Ile; Lys 134→Glu; Thr 145→Ala;
(d) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Asp 47→Asn; Gln 49→Thr; Tyr 52→Gln; Val 66→Ala; Ser 68→Trp; Leu 70→Tyr; Phe 71→Leu; Arg 72→Ser; Lys 73→Glu; Asp 77→Asn; Trp 79→His; Arg 81→Glu; Phe 83→Ser; Cys 87→Ser; Asn 96→Thr; Ile 97→Thr; Leu 103→Glu; Ser 105→Pro; Tyr 106→Ile; Lys 125→Gly; Tyr 132→Ile; Lys 134→Glu;
(e) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gln 49→Ile; Tyr 52→Gln; Thr 54→Ile; Lys 62→Arg; Ser 68→Trp; Leu 70→Tyr; Arg 72→Ser; Lys 73→Glu; Asp 77→Asn; Trp 79→His; Arg 81→Glu; Cys 87→Ser; Asn 96→Thr; Leu 103→Glu; Ser 105→Pro; Tyr 106→Ile; Lys 125→Gly; Ser 127→Asn; Tyr 132→Ile; Lys 134→Glu; Thr 136→Ile; Ser 146→Asn;
(f) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gln 49→Pro; Tyr 52→Gln; Lys 62→Arg; Ser 68→Trp; Leu 70→Tyr; Arg 72→Ser; Lys 73→Glu; Asp 77→Asn; Trp 79→His; Arg 81→Glu; Phe 83→Ser; Cys 87→Ser; Asn 96→Thr; Lys 98→Gln; Tyr 100→His; Leu 103→Glu; Ser 105→Pro; Tyr 106→Ile; Lys 125→Gly; Val 126→Met; Ser 127→Gly; Tyr 132→Ile; Lys 134→Glu;
(g) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gln 49→Ile; Tyr 52→Gln; Thr 54→Lys; Lys 62→Arg; Ser 68→Trp; Leu 70→Tyr; Arg 72→Ala; Lys 73→Asp; Asp 77→Asn; Trp 79→His; Arg 81→Glu; Cys 87→Ser; Asn 96→Leu; Leu 103→Glu; Ser 105→Pro; Tyr 106→Ile; Lys 125→Gly; Ser 127→Asn; Tyr 132→Ile; Lys 134→Glu; Thr 136→Ile; Ser 146→Asn;
(h) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gin 49→Ile; Tyr 52→Gln; Thr 54→Lys; Lys 62→Arg; Ser 68→Trp; Leu 70→Tyr; Arg 72→Ala; Lys 73→Glu; Asp 77→Asn; Trp 79→His; Arg 81→Glu; Cys 87→Ser; Asn 96→Thr; Leu 103→Glu; Ser 105→Pro; Tyr 106→Ile; Lys 125→Gly; Ser 127→Asn; Tyr 132→Ile; Lys 134→Glu; Thr 136→Ile; Ser 146→Asn;
(i) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gln 49→Ile; Tyr 52→Gln; Thr 54→Lys; Lys 62→Arg; Ser 68→Trp; Leu 70→Tyr; Arg 72→Ala; Lys 73→Asp; Asp 77→Asn; Trp 79→His; Arg 81→Glu; Cys 87→Ser; Asn 96→Thr; Leu 103→Glu; Ser 105→Pro; Tyr 106→Ile; Lys 125→Gly; Ser 127→Asn; Tyr 132→Ile; Lys 134→Glu; Thr 136→Ile; Ser 146→Asn;
(j) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gin 49→Ile; Tyr 52→Gln; Thr 54→Lys; Lys 62→Arg; Ser 68→Trp; Leu 70→Tyr; Arg 7

Assignees

• Pieris Pharmaceuticals Gmbh

Inventors

• Matschiner Gabriele
• Rothe Christine
• Wiedenmann Alexander
• Aiba Rachida Siham Bel
• Hinner Marlon
• Allersdorfer Andrea
• Lunde Bradley
• Kubota Kazufumi
• Makino Mitsuhiro
• Takahashi Sakiko
• Hashimoto Ryuji
• Takahashi Tohru
• Otoyo Mamoru

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P37/02

  Immunomodulators · CPC title

• A61P9/12

  Antihypertensives · CPC title

• A61P9/00

  Drugs for disorders of the cardiovascular system · CPC title

• A61P31/04

  Antibacterial agents · CPC title