PROTEASE VARIANTS

20120237496

13/378764

June 21, 2010

The present invention relates to polypeptides comprising protease variants of wild type human neprilysin having an altered specificity and/or activity. In particular the present invention relates to polypeptides comprising protease variants derived from human neprilysin having an increased specificity and/or activity against certain substrates, in particular against amyloid beta.

Birkenfeld, Joerg (Duesseldorf, DE); Eicker, Andrea (Moenchengladbach, DE); Freskgard, Per-Ola (Sodertalje, SE); Gotzberger-Schad, Claudia (Leichlingen, DE); Grudzinska, Joanna (Bonn, DE); Haupts, Ulrich (Odenthal, DE); Innig, Josi (Bergisch Gladbach, DE); Mahlert, Christoph (Koln, DE); Scheidig, Andreas (Koln, DE); Strerath, Michael (Koln, DE); Tebbe, Jan (Koln, DE); Per-Wallin, Johan (Sodertalje, SE); Wobst, Nina (Wuppertal, DE); Webster, Carl Innes (Cambridge, GB); Jermutus, Lutz (Cambridge, GB)

1. A polypeptide comprising a protease variant of wild type human neprilysin extracellular catalytic domain (SEQ ID NO: 2), said polypeptide having a greater specificity for an Aβ peptide compared to wild type human neprilysin (SEQ ID NO: 1), wherein G399 is replaced by another naturally occurring amino acid and/or G714 is replaced by another naturally occurring amino acid, optionally said naturally occurring amino acid is other than Ala (A).

2. A polypeptide comprising a protease variant according to claim 1, wherein G399 is replaced by Valine (V) and/or G714 is replaced by Lysine (K).

3. A polypeptide comprising a protease variant according to claim 1, wherein G399 is replaced by Valine (V) and G714 is replaced by Lysine (K).

4. A polypeptide according to claim 1 comprising a protease variant of wild-type human neprilysin extracellular catalytic domain as shown in SEQ ID NO: 2, said polypeptide having an altered specificity against Amyloid β40, Amyloid β42, Angiotensin-1 and -2, ANP, BNP, bradykinin, Endothelin-1 and -2, Neuropeptide Y, Neurotensin, Adrenomedullin, Bombesin, BLP, CGRP, Enkephalins, FGF-2, fMLP, GRP, Neurokinin A, Neuromedin C, Oxytocin, PAMP, Substance P or VIP.

5. A polypeptide according to claim 1 comprising a protease variant of wild type human neprilysin extracellular domain of SEQ ID NO: 2 having an altered specificity against Amyloid β40, Amyloid β42, Angiotensin-1 and -2, ANP, BNP, bradykinin, Endothelin-1 and -2, Neuropeptide Y or Neurotensin.

6. A polypeptide according to claim 1 comprising a half-life modulator moiety provided N-terminal to the protease variant, preferably said half-life modulator moiety is selected from an Fc domain and a human serum albumin (HSA) or variant thereof, optionally said half-life modulator moiety and protease variant are joined by a linker.

7. A nucleic acid encoding a polypeptide of claim 1.

8. A vector comprising the nucleic acid of claim 7.

9. A host cell comprising the vector of claim 8.

10. A method for producing a polypeptide according to claim 1, wherein the method comprises the following steps: a. culturing the host cell of claim 9 under conditions suitable for the expression of the protease variant; and b. recovering the protease variant from the host cell culture.

11. A pharmaceutical composition comprising a polypeptide of claim 1.

12. A method for treating a human neprilysin substrate related disease, such as an Aβ-related pathology, such as Alzheimer's disease, comprising administering to a patient in need thereof a therapeutically effective dose of a polypeptide comprising a protease variant according to claim 1, whereby a symptom of the human neprilysin substrate related disease is ameliorated.

13. (canceled)

14. A polypeptide with increased specificity for Aβ according to claim 4 or 5 for use to prevent and/or treat an Aβ-related pathology such as Alzheimer's disease.

15. (canceled)

424/9/467

61; 12; 12; 61; 12; 12; 12; 12; 12

edspap.20120237496