Proteins specific for calcitonin gene-related peptide

Patent metadata

Field	Value
Publication number	US-11034737-B2
Application number	US-201916504761-A
Country	US
Kind code	B2
Filing date	Jul 8, 2019
Priority date	Dec 10, 2015
Publication date	Jun 15, 2021
Grant date	Jun 15, 2021

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 method of treating, preventing, or ameliorating a disease or disorder associated with deregulated protein plasma extravasation in a subject, comprising administering to the subject a human neutrophil gelatinase associated lipocalin (hNGAL) mutein that is capable of binding CGRP with detectable affinity.

2. The method of claim 1 , wherein the disease or disorder is selected from the group consisting of migraine, temporomandibular joint disorder, cardiac failure, hypertension and sepsis.

3. The method of claim 1 , wherein the disease or disorder is migraine.

4. The method of claim 1 , wherein the hNGAL mutein is capable of binding CGRP with a KD of about 5 nM or lower.

5. The method of claim 1 , wherein the hNGAL mutein is crossreactive with both human CGRP and rat CGRP.

6. The method of claim 1 , wherein the hNGAL mutein is capable of inhibiting or reducing CGRP-induced cAMP production with an IC50 value of about 5 nM or lower.

7. The method of claim 1 , wherein the hNGAL 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 hNGAL mutein has at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-40 and 87-93.

8. The method 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; Gln 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; 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→Arg; Trp 79→His; Arg 81→Glu; Cys 87→Ser; Asn 96→Thr; Tyr 100→His; 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;
(k) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gln 49→Ile; Tyr 52→Gln; Thr 54→Met; 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→Val; Ser 146→Asn;
(l) 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; Val 111→Met; Lys 125→Gly; Ser 127→Asn; Tyr 132→Ile; Lys 134→Glu; Thr 136→Ile; Ser 146→Asn;
(m) Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gln 49→Ile; Tyr 52→Gln; Thr 54→Met; Ser 68→Trp; Leu 70→Tyr; Arg 72→Ala; Lys 73→Asp; Cys 76→Arg; 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→Val; Ser 146→Asn; Cys 175→Phe;
(n) Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gln 49→Ile; Tyr 52→Gln; Thr 54→Met; Ser 68→Trp; Leu 70→Tyr; Arg 72→Ala; Lys 73→Asp; Cys 76→Met; 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→Val; Ser 146→Asn; Cys 175→Tyr;
(o) Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gln 49→Ile; Tyr 52→Gln; Thr 54→Met; Ser 68→Trp; Leu 70→Tyr; Arg 72→Ala; Lys 73→Asp; Cys 76→Leu; 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→Val; Ser 146→Asn; Cys 175→Trp;
(p) Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gln 49→Ile; Tyr 52→Gln; Thr 54→Met; Ser 68→Trp; Leu 70→Tyr; Arg 72→Ala; Lys 73→Asp; Cys 76→Ile; 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→Val; Ser 146→Asn; Cys 175→Glu;
(q) Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gln 49→Ile; Tyr 52→Gln; Thr 54→Met; Ser 68→Trp; Leu 70→Tyr; Arg 72→Ala; Lys 73→Asp; Cys 76→Val; 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→Val; Ser 146→Asn; Cys 175→Tyr;
(r) Leu 36→Trp; Ala 40→Thr; Ile 41→Leu; Gln 49→Ile; Tyr 52→Gln; Thr 54→Met; Ser 68→Trp; Leu 70→Tyr; Arg 72→Ala; Lys 73→Asp; Cys 76→Arg;

Assignees

• Pieris Pharmaceuticals Gmbh

Inventors

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

Classifications

• A61P25/06

  Antimigraine agents · CPC title

• A61P19/02

  for joint disorders, e.g. arthritis, arthrosis · CPC title

• A61P37/02

  Immunomodulators · CPC title

• A61P31/00

  Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics · CPC title

• 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