Fusion molecules

Patent metadata

Field	Value
Publication number	US-10947284-B2
Application number	US-201916393410-A
Country	US
Kind code	B2
Filing date	Apr 24, 2019
Priority date	Jul 7, 2015
Publication date	Mar 16, 2021
Grant date	Mar 16, 2021

Abstract

The present invention relates to fusion molecules that have binding specificity for pyoverdine type I, II and III and pyochelin and can be used in various applications, including diagnostic and/or therapeutic applications, for example, to inhibit or reduce growth of P. aeruginosa and/or to prevent or treat P. aeruginosa biofilm infection as well as diseases or disorders associated with P. aeruginosa biofilm infection. The present invention also concerns methods of producing the fusion molecules described herein as well as compositions and kits comprising such fusion molecules. The present invention further relates to nucleic acid molecules encoding the fusion molecules described herein.

First claim

What is claimed is:

1. A fusion molecule having binding specificity for pyoverdine type I, II and III and pyochelin, comprising
(a) a first polypeptide comprising a first human neutrophil gelatinase-associated lipocalin (hNGAL) mutein that binds pyoverdine type I, wherein the first hNGAL mutein comprises at least ten of the following mutated amino acid residues in comparison with the linear polypeptide sequence of hNGAL (SEQ ID NO: 1): Leu 36-Asn, Thr, Val, Trp or Phe; Ala 40-Gly, Asn, Thr or Phe; Ile 41-Arg, Ala, Thr, Phe or Trp; Gln 49-Ile, Leu, Val, Ala or Pro; Tyr 52-Met, Trp or Pro; Ser 68-Asp, Val or Glu; Leu 70-Gln, Trp, Asp or Thr; Arg 72-Trp, Ala, Ser, Leu, Pro or Glu; Lys 73-Asp, Leu, Ala, Glu or Asn; Asp 77-Arg, Leu, Tyr, Ser, Gln, Thr, Ile or Asn; Trp 79-Gln, Asp, Ser, Arg, Met or Glu; Arg 81-Gln, Gly, Ile, Glu, His or Asp; Asn 96-His, Ile, Gly, Tyr or Asp; Tyr 100-Lys, Glu, Asn, Ser, Phe or Tyr; Leu 103-Lys, Pro, Gln, His, Asp, Tyr, Glu, Trp or Asn; Tyr 106-His, Gln or Phe; Lys 125-Arg, Ser, Trp, Tyr, Val or Gly; Ser 127-Trp, Asn, Ala, Thr, Tyr, His, Ile, Val or Asp; Tyr 132-Trp, Asn, Gly or Lys; and Lys 134-Asn, His, Trp, Gly, Gln or Asp, and wherein the first hNGAL mutein has at least 82% sequence identity to the sequence shown in SEQ ID NO: 16;
(b) a second polypeptide comprising a second hNGAL mutein that binds pyoverdine type II, wherein the second hNGAL mutein comprises at least ten of the following mutated amino acid residues in comparison with the linear polypeptide sequence of hNGAL (SEQ ID NO: 1): Leu 36-Asn, Ile or Val; Ala 40-Glu, Gly, Asn, Thr or His; Ile 41-Arg, Val or Thr; Gln 49-Gly, Ala or Pro; Tyr 52-Asn, Gly, Trp or Pro; Ser 68-Asp, Arg or Glu; Leu 70-Arg or Trp; Arg 72-His, Ile, Ala, Ser or Gly; Lys 73-Asn, Met, Pro, Phe, Gln or Arg; Asp 77-His, Ile, Met, Lys, Gly or Asn; Trp 79-Ser, Tyr, Ala, Asp, Phe or Trp; Arg 81-Glu, Ser, Tyr or Asp; Asn 96-Met, Ile, Arg, Asp, Lys, Asn or Ala; Tyr 100-Lys, Glu, Asn, Ser, Phe or Tyr; Leu 103-Thr, Ile, Gln, Gly, Met, His, Trp or Val; Tyr 106-Met, Gln, Ala, Ile, Asn, Gly, Met or Phe; Lys 125-Ala, Ile or Asn; Ser 127-Lys, Arg, Ser, Met, Asp or Asn; Tyr 132-Met, Phe, Asn, Ala, Ile, Gly or Val; and Lys 134-Trp or Tyr, and wherein the second hNGAL mutein has at least 84% sequence identity to the sequence shown in SEQ ID NO: 36;
(c) a third polypeptide comprising a third hNGAL mutein that binds pyoverdine type III, wherein the third hNGAL mutein comprises at least ten of the following mutated amino acid residues in comparison with the linear polypeptide sequence of hNGAL (SEQ ID NO: 1): Leu 36-Phe or Glu; Ala 40-Trp, Leu or Arg; Ile 41-Met, Arg, Ala, Leu or Trp; Gln 49-His, Ile, Arg, Lys, Met or Pro; Tyr 52-Asn, Tyr, Arg, Ser or Met; Ser 68-Asp, Asn, Glu or Gln; Leu 70-Lys, Asn or Arg; Arg 72-Leu, Arg, Gln or Tyr; Lys 73-His, Leu, Ala, Pro, Gln or Tyr; Asp 77-Ala, Ile, Lys, Gln or Arg; Trp 79-Ser or Asp; Arg 81-His, Ala, Ser or Val; Asn 96-Met, Ile, Arg, Gly, Leu or Val; Tyr 100-Ala, Ile, Asn, Pro or Asp; Leu 103-Gln, Gly, Phe or Pro; Tyr 106-Glu; Lys 125-Trp or Thr; Ser 127-Val, His, Ile, Phe or Ala; Tyr 132-Phe; and Lys 134-Trp, Gln or Glu, and wherein the third hNGAL mutein has at least 84% sequence identity to the sequence shown in SEQ ID NO: 53; and
(d) a fourth polypeptide comprising a fourth hNGAL mutein that binds pyochelin, pyochelin wherein the fourth hNGAL mutein comprises at least ten of the following mutated amino acid residues in comparison with the linear polypeptide sequence of hNGAL (SEQ ID NO: 1): Leu 36-His, Met or Val; Ala 40-Ile, Gln, Tyr or Phe; Ile 41-Leu, His or Trp; Gln 49-His, Arg, Ser or Ala; Tyr 52-Leu, Trp or Pro; Ser 68-Asp or His; Leu 70-Arg or Trp; Arg 72-His, Ile, Ala, Ser or Gly; Lys 73-Asn, Met, Pro, Phe, Gln or Arg; Asp 77-Arg, Thr, Pro or Asp; Trp 79-Ala, Arg, Lys or Asp; Arg 81-Thr, Ile or Trp; Asn 96-Met, Asn, Pro or Ala; Tyr 100-Gly, His or Glu; Leu 103-Gly, Met, His or Gln; Tyr 106-Met, Gly, Arg or Trp; Lys 125-Trp, Phe, Gly or Leu; Ser 127-Arg, Trp, Asp or Ile; Tyr 132-Ala, Glu or Thr; and Lys 134-Leu, Val, Asn or Phe, and wherein the fourth hNGAL mutein has at least 84% sequence identity to the sequence shown in SEQ ID NO: 62; and wherein the first, second, third and fourth polypeptides are covalently linked.

2. The fusion molecule of claim 1 , wherein the first hNGAL mutein comprises one of the following sets of mutated amino acid residues in comparison with the linear polypeptide sequence of hNGAL (SEQ ID NO: 1):
(a) Gln 28→His; Leu 36→Asn; Ala 40→Gly; Ile 41→Trp; Gln 49→Ile; Tyr 52→Met; Ser 68→Val; Leu 70→Gln; Arg 72→Trp; Lys 73→Asp; Asp 77→Leu; Trp 79→Gln; Arg 81→Gln; Cys 87→Ser; Asn 96→His; Tyr 100→Lys; Leu 103→His; Tyr 106→His; Lys 125→Arg; Ser 127→Trp; Tyr 132→Trp; Lys 134→Asp;
(b) Gln 28→His; Leu 36→Thr; Ala 40→Gly; Ile 41→Phe; Gln 49→Leu; Tyr 52→Trp; Leu 70→Trp; Arg 72→Ala; Lys 73→Leu; Asp 77→Tyr; Trp 79→Asp; Arg 81→Gly; Cys 87→Ser; Asn 96→Ile; Tyr 100→Glu; Leu 103→His; Tyr 106→Gln; Lys 125→Trp; Ser 127→Asn; Tyr 132→Asn; Lys 134→Gln;
(c) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro; Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu; Asp 77→Ser; Trp 79→Ser; Arg 81→Ile; Cys 87→Ser; Asn 96→Gly; Tyr 100→Asn; Leu 103→Lys; Tyr 106→His; Lys 125→Tyr; Ser 127→Ala; Tyr 132→Gly; Lys 134→Asn;
(d) Gln 28→His; Leu 36→Phe; Ala 40→Asn; Ile 41→Arg; Gln 49→Pro; Tyr 52→Met; Ser 68→Asp; Leu 70→Thr; Arg 72→Glu; Lys 73→Ala; Asp 77→Arg; Trp 79→Arg; Arg 81→Ile; Cys 87→Ser; Asn 96→Tyr; Tyr 100→Lys; Leu 103→Pro; Tyr 106→Phe; Lys 125→Ser; Ser 127→Thr; Tyr 132→Trp; Lys 134→Gly;
(e) Gln 28→His; Ala 40→Gly; Ile 41→Trp; Gln 49→Val; Tyr 52→Met; Ser 68→Val; Leu 70→Asp; Arg 72→Glu; Lys 73→Leu; Asp 77→Arg; Trp 79→Met; Arg 81→Glu; Cys 87→Ser; Asn 96→Asp; Tyr 100→Phe; Leu 103→Trp; Tyr 106→Gln; Lys 125→Gly; Ser 127→Tyr; Tyr 132→Trp; Lys 134→His;
(f) Gln 28→His; Leu 36→Val; Ala 40→Phe; Ile 41→Phe; Gln 49→Ala; Tyr 52→Pro; Ser 68→Glu; Leu 70→Trp; Arg 72→Leu; Lys 73→Asn; Asp 77→Gln; Trp 79→Glu; Arg 81→His; Cys 87→Ser; Asn 96→Tyr; Leu 103→Tyr; Tyr 106→His; Lys 125→Val; Ser 127→His; Tyr 132→Lys; Lys 134→Trp;
(g) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro; Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu; Asp 77→Ser; Trp 79→Ser; Ile 80→Thr; Arg 81→Ile; Cys 87→Ser; Asn 96→Gly; Tyr 100→Ser; Leu 103→Gln; Tyr 106→His; Lys 125→Tyr; Ser 127→Ile; Tyr 132→Gly; Lys 134→Asn;
(h) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro; Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Asp; Asp 77→Ser; Trp 79→Ser; Arg 81→Ile; Cys 87→Ser; Asn 96→Gly; Tyr 100→Asn; Leu 103→Asp; Tyr 106→His; Lys 125→Tyr; Ser 127→Val; Tyr 132→Gly; Lys 134→Asn;
(i) Gln 28→His; Leu 36→Trp; Ala→40 Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro; Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu; Asp 77→Thr; Trp 79→Ser; Arg 81→Ile; Cys 87→Ser; Asn 96→Asp; Tyr 100→Asn; Leu 103→Glu; Tyr 106→His; Lys 125→Tyr; Ser 127→Asp; Tyr 132→Gly; Lys 134→Asn;
(j) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro; Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Asp; Asp 77→Val; Trp 79→Ser; Arg 81→Ile; Cys 87→Ser; Asn 96→Gly; Tyr 100→Asn; Leu 103→Asn; Tyr 106→His; Lys→125 Tyr; Ser 127→Val; Tyr 132→Gly; Lys 134→Asn;
(k) Gln 28→His; Ala 40→Gly; Ile 41→Trp; Gln 49→Leu; Tyr 52→Met; Ser 68→Val; Leu 70→Asp; Arg 72→Glu; Lys 73→Leu; Asp 77→Arg; Trp 79→Met; Arg 81→Glu; Cys 87→Ser; Asn 96→Asp; Tyr 100→Ser; Leu 103→Trp; Tyr 106→Gln; Lys 125→Gly; Ser 127→Tyr; Tyr 132→Trp; Lys 134→His;
(l) Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro; Thr 54→Val; Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu; Lys 75→Glu; Asp 77→Ser; Trp 79→Ser; Ile 80→Thr; Arg 81→Ile; Cys 87→Ser; Asn 96→Gly; Tyr 100→Ser; Leu 103→Gln; Tyr 106→His; Lys 125→Tyr; Ser 127→Thr; Tyr 132→Gly; Lys 134→Asn;
(m) Gln 28→His; Ala 40→Gly; Ile 41→Trp; Lys 46→Glu; Gln 49→Leu; Tyr 52→Met; Thr 54→Ala; Ile 55→Val; Lys 59→Arg; Ser 68→Val; Leu

Assignees

• Pieris Pharmaceuticals Gmbh

Inventors

• Corvey Carsten
• Stump Heike
• Kruip Jochen
• Lange Christian
• Focken Ingo
• Rat Dorothea
• Stuedemann Thomas
• Rasser Hans-Falk
• Schaefer Juergen
• Calandra Bernard
• Rey Astrid
• Mourez Michael
• Fraisse Laurent
• Rothe Christine
• Allersdorfer Andrea
• Wiedenmann Alexander
• Hinner Marlon
• Lunde Bradley
• Jensen Kristian
• Hülsmeyer Martin

Classifications

• C07K2319/22

  containing a Strep-tag · CPC title

• C07K14/47 Primary

  from mammals · CPC title

• C07K2319/50

  containing protease site · 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

• C07K2319/70

  containing domain for protein-protein interaction · CPC title