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WO2011161127A1 - Variants de protéase - Google Patents

Variants de protéase Download PDF

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Publication number
WO2011161127A1
WO2011161127A1 PCT/EP2011/060385 EP2011060385W WO2011161127A1 WO 2011161127 A1 WO2011161127 A1 WO 2011161127A1 EP 2011060385 W EP2011060385 W EP 2011060385W WO 2011161127 A1 WO2011161127 A1 WO 2011161127A1
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WIPO (PCT)
Prior art keywords
neprilysin
polypeptide
hsa
peptide
variant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/EP2011/060385
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English (en)
Inventor
Carl Innes Webster
Lutz Ulrich Jochen Wilhelm Jermutus
Jorg Birkenfeld
Andrea Eicker
Per-Ola Freskgard
Claudia Gotzberger-Schad
Joanna Grudzinska
Ulrich Haupts
Josi Innig
Christoph Mahlert
Andreas Scheidig
Michael Strerath
Jan Tebbe
Johan Per Wallin
Nina Wobst
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MedImmune LLC
Original Assignee
MedImmune LLC
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Publication date
Priority claimed from PCT/US2010/039392 external-priority patent/WO2010148413A2/fr
Priority claimed from PCT/EP2010/070615 external-priority patent/WO2011160732A1/fr
Application filed by MedImmune LLC filed Critical MedImmune LLC
Publication of WO2011161127A1 publication Critical patent/WO2011161127A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C12N9/6494—Neprilysin (3.4.24.11), i.e. enkephalinase or neutral-endopeptidase 24.11
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24011—Neprilysin (3.4.24.11), i.e. enkephalinase or neutral endopeptidase 24.11
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to polypeptides comprising a half-life extension moiety and a variant of human neprilysin with increased specificity for cleavage of amyloid beta ( ⁇ ) peptides compared to wild-type human neprilysin and to the use of such polypeptides in pharmaceutical compositions.
  • Polypeptides and compositions of the invention may be used in the treatment of diseases associated with accumulation of amyloid beta, in particular
  • Engineered proteases are desirable as therapeutics because the cleavage of a substrate peptide or protein associated with a disease will often lead to its irreversible inactivation or activation.
  • a protease must have a sufficient activity on the target, but must not cleave other substrates to an extent that leads to unacceptable toxic side effects under treatment conditions.
  • proteases i.e., their ability to recognize and hydro lyze preferentially certain peptide substrates, can be expressed qualitatively and quantitatively. Qualitatively, proteases that act on one or a small number of peptides have a high specificity, whereas proteases that act on many different peptides are deemed to have low specificity. In quantitative terms, the specificity profile of a protease is given by the respective kcat/Km ratios for all substrates, including potentially kcat/Km ratios for several cleavage sites in a given substrate. Modern methods of protein engineering permit modulation of the specificity of a given protease, potentially enabling the generation of proteases with desired specificities for use as prophylactic or therapeutic protein drugs.
  • An accumulation or increase in the activity of a polypeptide compared to the "normal" level may contribute to the cause or symptoms of a disease; in such cases the inactivation of the polypeptide by proteolytic cleavage may be beneficial for the patient.
  • AD Alzheimer's disease
  • the neuropathological hallmarks that occur in the brains of individuals suffering from AD are senile plaques and profound cytoskeletal changes coinciding with the appearance of abnormal filamentous structures.
  • the neuronal loss is accompanied by extracellular deposition of amyloid beta ( ⁇ ) peptides in the form of senile plaques and intracellular accumulation of neurofibrillary tangles made of a hyperphosphorylated form of the microtubule-associated protein tau.
  • ⁇ amyloid beta
  • Both familial and sporadic cases share the deposition in brain of extracellular, fibrillary ⁇ -amyloid as a common pathological hallmark that is believed to be associated with impairment of neuronal functions and neuronal loss (Younkin S. G., Ann. Neurol.
  • ⁇ -amyloid deposits are composed of several species of amyloid- ⁇ peptides ( ⁇ ); especially ⁇ _ 42 , which is deposited progressively in amyloid plaques. Genetic evidence suggests that increased amounts of ⁇ _ 42 are produced in many, if not all, genetic conditions that cause familial AD (Borchelt D. R. et al, Neuron 17, 1005- 1013, 1996; Duff K. et al, Nature 383, 710-713, 1996; Scheuner D. et al, Nat. Med.
  • DeMattos PNAS 98: 8850-8855, 2001 have described the sink hypothesis, which states that ⁇ peptides can be removed from CNS indirectly by lowering the concentration of the peptides in the plasma.
  • De Mattos used an antibody that binds ⁇ in the plasma. By preventing influx of ⁇ from the plasma to CNS and/or changing the equilibrium between the plasma and CNS (due to a lowering of the free ⁇ concentration in plasma) ⁇ is sequestered from the CNS.
  • Two other ⁇ binding agents, gelsolin and GM1, unrelated to antibodies, have also been shown through binding in plasma to be effective in removing ⁇ from CNS and reducing or preventing brain amyloidosis (Matsuoka et al. (J. Neuroscience 23: 29-33, 2003).
  • An alternative approach to remove ⁇ is to use an enzyme that degrades ⁇ into smaller fragments that have lower toxicological effects and are more readily cleared. It is postulated that this enzymatic digestion of the ⁇ will also work through the sink hypothesis mechanism by lowering the free concentration of ⁇ in plasma. However, this approach also provides a possibility of direct clearance of ⁇ in the CNS and/or CSF. This approach will not only lower the free concentration of ⁇ but also directly clear the full-length peptide from the environment. This approach is advantageous because it will not increase the total (free and bound) concentration of ⁇ in the plasma as has been seen in cases when using ⁇ peptide binding agents such as antibodies.
  • Neprilysin is an enzyme described in the literature that degrades the ⁇ peptide at multiple cleavage sites, generating small fragments that are cleared from the blood stream easily (Leissring et al., JBC. 278: 37314-37320, 2003). Neprilysin has also been reported to play a key role in regulating the level of ⁇ peptide in the brain.
  • proteases have been described that degrade ⁇ peptide including insulin degrading enzyme, plasmin, ACE and others.
  • Anti- ⁇ peptide antibodies have been applied to effectively reduce free ⁇ levels in the blood leading to a decreased plaque deposition in the brain.
  • systemic application of proteases that degrade and inactivate ⁇ peptide may be an alternative; but such protease would need to be sufficiently specific for ⁇ peptide to be effective and to avoid induction of toxic side effects due to off-target activity.
  • Human neprilysin (also termed NEP, neutral endopeptidase, CD 10, common acute lymphoblastic leukemia antigen (CALLA), enkephalinase; SwissProt accession P08473) is a 94 kD, type two membrane-bound Zn-metallopeptidase composed of 750 residues, or 749 residues due to the removal of the initial methionine (SEQ ID NO: 1).
  • the 749 amino acid nomenclature (pdb numbering) will be used throughout this text. It is present in peptidergic neurons in the CNS, and its expression in brain is regulated in a cell-specific manner (Roques B. P. et al, Pharmacol. Rev.
  • the proteolytic domain (extracellular catalytic domain, ECD) comprises amino acids 51 to 749 and contains an active site containing a zinc-binding motif (HEXXH).
  • HEXXH zinc-binding motif
  • Neprilysin is capable of degrading a number of peptidic substrates, including monomeric and (possibly) oligomeric forms of ⁇ peptides and can act as an endopeptidase as well as a carboxypeptidase, although the relevance of these different activities under physiological conditions has not been determined in detail.
  • Peptides that are degraded include, but are not limited to, (Table 1):
  • Neprilysin belongs to the M13 class of metalloproteases and is characterized by a mostly a-helical, two-domain structure. These two domains enclose an integral cavity that includes the active site. The size of the cavity limits the majority of natural substrates to ⁇ 5kDa. However, it is largely unknown which residues of neprilysin interact with the substrate and thus influence protease specificity. A few amino acids in contact with the inhibitors that might be considered as part of the active site of the protease include (Table 2):
  • Neprilysin also degrades many vasoactive peptides, including bradykinin, angiotensin II, endothelin I, and the natriuretic peptides (atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)) (Reid Ian A., Vasoactive Peptides, in "Basic and Clinical
  • Angiotensin, bradykinin, endothelins and natriuretic peptides are involved in the regulation of arterial pressure.
  • Angiotensin II is a vasoconstrictive
  • Bradykinin is a vasodilator nonapeptide.
  • Endothelins are vasoconstrictive polypeptides of about 20 amino acids with two disulfide bridges connecting cysteine residues.
  • ANP 28-amino acid
  • BNP 32-amino acid
  • neprilysin is a 36 amino acid polypeptide neurotransmitter distributed in the mammalian central nervous system.
  • NT Neurotensin
  • DA dopaminergic
  • NT is primarily produced throughout the mucosa and regulates a number of digestive processes.
  • Other organs that produce NT include the heart and adrenals (Sarret and Kitabgi, Encyclopedia of Neuroscience, 1021-1034, 2009; Pons, J., et al., Current Opinion in Investigational Drugs 5, 957-962, 2004).
  • neprilysin cleaves a multitude of peptide substrates, many if not all of which play important physiological roles, it would be desirable to identify neprilysin variants that have enhanced specificity for cleavage of one of the substrate peptides, such as ⁇ , relative to cleavage of the other (off-target) peptide substrates.
  • Mutants of neprilysin with a changed specificity profile have been described. Namely, mutating arginine 102 to glutamine (R102Q) leads to a differential catalytic efficiency with respect to the carboxypeptidase activity of neprilysin (Beaumont et al. (1992) J. Biol. Chem.
  • WO 2007/040437 describes fusion proteins of the form A-L-M, in which "A” is a protease capable of cleaving amyloid beta peptide, “L” is a linker and “M” is a component that modulates in-vivo half-life, such as the Fc part of an antibody; “A” may be human neprilysin.
  • WO 2008/118093 describes a fusion protein that cleaves amyloid beta peptide wherein a half- life modulating moiety is attached to the N-terminal end of human neprilysin, and a method to reduce ⁇ peptide concentrations by administration of such a fusion protein as a medical therapy.
  • WO 2005/123119 provides a method of making a recombinant truncated mammalian neprilysin and the method of treating inflammatory bowel disease in mammals with a pharmaceutical composition comprising such truncated protein.
  • US2003/0083277 and US2003/0165481 describe a method of preventing formation of growth of amyloid fibrils by administration of effective amounts of an inactivating enzyme, e.g. neprilysin. Treatment can be either by administration of purified protein or viral or plasmid vector. Administration is made to the brain. US2003/0083277 describes insulin degrading enzyme for the same application.
  • fusion proteins comprising a half-life modulator moiety and a neprilysin variant with increased specificity for amyloid beta ( ⁇ ) peptides, which is suitably well-expressed to enable efficient and economic manufacture and which will provide an acceptable half-life in circulation, to permit an acceptable dosing regimen.
  • the present invention provides a polypeptide comprising a half-life modulator moiety (M) provided N-terminal to a neprilysin protease variant (A), wherein said half-life modulator moiety is human serum albumin HSA or variant or fragment thereof, and wherein said neprilysin protease variant comprises a variant of wild type human neprilysin extracellular catalytic domain (SEQ ID NO: 2) wherein G399 is replaced by another naturally-occurring amino acid and / or G714 is replaced by another naturally-occurring amino acid.
  • polypeptides of the invention have an enhanced specificity for cleavage of ⁇ than other substrates of wild type neprilysin.
  • a polypeptide according to the invention suitably is capable of digesting one or more peptide selected from Angiotensin- 1 and -2, ANP, BNP, bradykinin, Endothelin-1 and -2, Neuropeptide Y,
  • Such molecules when administered as a therapeutic may have a similar or an enhanced effect at degrading ⁇ than wild type neprilysin, but a reduced effect at degrading the other neprilysin ligand substrates when compared to wild type neprilysin, thus minimising or reducing any unwanted or
  • the present invention provides a polypeptide comprising a variant human neprilysin extracellular domain or a fragment thereof, said variant or fragment thereof having an amino acid sequence that differs from the wild-type human neprilysin extracellular domain shown in SEQ ID NO: 2 by at least one amino acid, wherein the polypeptide is capable of digesting an amyloid beta polypeptide with a higher specificity than wild-type neprilysin.
  • the amyloid beta polypeptide can be human Amyloid ⁇ 1-40 , and/or human Amyloid Bi _ 42 .
  • the amino acid G399 and / or G714 is replaced by another naturally occurring amino acid, said naturally occurring amino acid may be an amino acid other than Ala; G399 may be replaced by Valine (V) and/or G714 may be replaced by Lysine (K); the amino acid residue numbering is based on the wild type human neprilysin sequence shown in SEQ ID NO: 1.
  • the polypeptide comprises a human neprilysin protease variant wherein G399 is replaced by Valine (V) and/or G714 is replaced by Lysine (K), most preferably wherein G399 is replaced by Valine (V) and G714 is replaced by Lysine (K).
  • the polypeptide of the invention may further comprise replacement of an amino acid at one or more of the following positions in the neprilysin sequence: S227, R228, F247, E419, D590, G593, F596, G600, G645, D709 or 1718.
  • the replacement of an amino acid at one or more positions in the neprilysin sequence may be selected from the group consisting of: S227R, S227L, R228G, F247L, F247C, E419M, E419L, D590W, D590M, D590F, G593V, F596P, G600W, G600V, G600D, G600L, G645Q, D709K, D709V and I718L.
  • a polypeptide in accordance the invention comprises a moiety capable of modulation, i.e., extending, half-life of the polypeptide in plasma, which moiety is a human serum albumin, or variant or fragment thereof, provided N-terminally to the variant human neprilysin
  • the human serum albumin can be a variant HSA, such as the variant HSA C34S in which a cysteine residue has been replaced by a serine.
  • HSA human serum albumin
  • embodiments of the invention may be of the form: N HSA or N HSA variant (such as HSA C34S) or fragment thereof -neprilysin variant 0 .
  • the half-life modulator moiety and neprilysin protease variant may, optionally, be joined by a linker peptide.
  • Particularly suitable linker is particularly suitable
  • polypeptides comprise (Gly) 5 Ser (SEQ ID NO: 32) or multimers thereof, or (Gly) 4 Ser (SEQ ID NO: 31) linkers,or multimers thereof, with linkers comprising (Gly) 4 S (SEQ ID NO: 31) or multimers thereof being particularly preferred.
  • embodiments of the invention may be of the form: N HSA or HSA variant (such as HSA C34S) or fragment thereof - linker, e . g . , (Gly) 4 S or multimer thereo f - neprilysin variant 0 .
  • a polypeptide according to the invention may comprise a pro-HSA amino acid sequence "pro" positioned N-terminally to the protease variant.
  • pro amino acid sequence "pro”
  • embodiments of the invention may be of the form: N pro sequence of HSA - HSA or HSA variant (such as HSA C34S) or fragment thereof - (optional linker, e.g., (Gly) 4 S or multimer thereof) - neprilysin variant 0 .
  • a polypeptide of the invention may comprise a leader amino acid sequence positioned N- terminally to the protease variant.
  • embodiments of the invention may be of the form: N Leader - (optional pro sequence of HSA) - HSA or HSA variant (such as HSA C34S) or fragment thereof - (optional linker, e.g., (Gly) 4 S or multimer thereof) - neprilysin variant 0 .
  • a polypeptide according to any the invention may comprise a leader sequence amino acid positioned N-terminally to a pro-HSA sequence.
  • embodiments of the invention may be of the form: N Leader - pro sequence of HSA - HSA or HSA variant (such as HSA C34S) or fragment thereof - (optional linker, e.g., (Gly) 4 S or multimer thereof) - neprilysin variant 0 .
  • Suitable leader amino acid sequences include a HSA leader (or signal) amino acid sequence or an IgG leader amino acid sequence.
  • the present invention provides a polypeptide comprising N HSA C34S, a GGGGS (SEQ ID NO: 31) linker and a G399V / G714K variant human neprilysin extracellular domain 0 , such as is shown in SEQ ID NO: 28.
  • the present invention provides a polypeptide comprising: N pro sequence of HSA, HSA C34S, a GGGGS (SEQ ID NO: 31) linker and a G399V / G714K variant human neprilysin extracellular domain 0 , such as is shown herein.
  • a polypeptide of the invention suitably has a greater specificity for an ⁇ peptide compared to wild type human neprilysin.
  • the invention further provides an isolated nucleic acid sequence (polynucleotide) encoding a polypeptide of the invention described herein.
  • Nucleic acid may include DNA and/or RNA.
  • the present invention also provides constructs in the form of vectors, transcription or expression cassettes which comprise a polynucleotide of the invention and thereby encode a polypeptide of the invention described herein.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids e.g. phagemid, or viral e.g. 'phage, as appropriate [Sambrook and Russell, Molecular Cloning: a Laboratory Manual: 3rd edition, 2001 , Cold Spring Harbor Laboratory Press].
  • a host cell comprising a nucleic acid of the invention, preferably a host cell comprising a vector of the invention.
  • a further aspect of the present invention provides a host cell containing nucleic acid, e.g., a vector, as disclosed herein. Such a host cell may be in vitro and may be in culture.
  • Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems and transgenic plants and animals.
  • the expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. For a review, see for example Pluckthun [Pluckthun, A. Bio/Technology 9: 545-551 (1991)].
  • a common bacterial host is E. coli.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells and many others.
  • Another aspect provides a method comprising introducing nucleic acid of the invention into a host cell.
  • the introduction may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus.
  • Introducing nucleic acid in the host cell in particular a eukaryotic cell may use a viral or a plasmid based system.
  • the plasmid system may be maintained episomally or may be incorporated into the host cell or into an artificial chromosome. Incorporation may be either by random or targeted integration of one or more copies at single or multiple loci.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
  • the introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.
  • the purification of the expressed product may be achieved by methods known to one of skill in the art.
  • Nucleic acid encoding a polypeptide of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences that promote recombination with the genome, in accordance with standard techniques.
  • the present invention also provides a method that comprises using a vector construct as described above in an expression system in order to express a polypeptide of the invention.
  • the invention thus provides a method for producing a polypeptide according to the invention, wherein the method comprises the following steps:
  • composition comprising a polypeptide of the invention and a pharmaceutically acceptable excipient.
  • Polypeptides of the invention with enhanced specificity for ⁇ peptide may be useful in the treatment of Alzheimer's disease and other diseases mediated by ⁇ accumulation, due to excessive ⁇ formation or decreased ⁇ degradation.
  • the invention provides a method for treating a human neprilysin substrate- related disease, such as an ⁇ -related pathology, such as Alzheimer's disease, comprising administering to a patient in need thereof a therapeutically effective dose of a polypeptide of the invention, whereby a symptom of the human neprilysin substrate-related disease is ameliorated.
  • the invention provides a polypeptide of the invention for use as a medicament for a human neprilysin substrate-related disease, such as an ⁇ -related pathology, such as Alzheimer's disease.
  • the invention provides a polypeptide for use to prevent and/or treat an ⁇ -related pathology, such as Alzheimer's disease.
  • SEQ ID NO: 1 shows the amino acid sequence of wild type human neprilysin without the initial methionine (Wt-full length neprilysin).
  • the first amino acid (Y) of the human soluble Neprilysin sequence occurs at position 51.
  • SEQ ID NO:2 shows the amino acid sequence of wild type soluble human neprilysin
  • SEQ ID NO: 3 shows the amino acid sequence of soluble human neprilysin with amino terminal 3xHA-tag and dipeptide-linker.
  • the first amino acid (Y) of the human soluble Neprilysin sequence occurs at position 30.
  • SEQ ID NO:4 shows the nucleotide-sequence of wild type soluble human neprilysin
  • SEQ ID NO: 5 shows the nucleotide-sequence of soluble human neprilysin with amino terminal 3xHA-tag and dipeptide linker. The first codon triplet of the human soluble
  • Neprilysin sequence occurs at positions 88-90.
  • SEQ ID NO: 6 shows the nucleotide-sequence of full-length wild type human
  • SEQ ID NO: 7 shows the nucleotide sequence of human soluble neprilysin sequence N-terminal fused to sequences encoding a secretion leader, secretion site, triple HA-tag and a dipeptide linker in expression vector pYES2.
  • the alpha secretion leader sequence including the secretion site is at position 507-773, the 3xHA tag sequence is at position 774-854; the
  • Gly/Ser linker (Dipeptide-linker) is at position 855 -860; the sNeprilysin sequence is at position 861-2960; and the CYY1 terminator sequence is at position 3090-3338.
  • SEQ ID NO: 28 shows a human variant neprilysin extracellular domain that has two amino acid changes from wild-type human neprilysin: Glycine 399 to Valine and Glycine 714 to Lysine; this variant has enhanced stability and specificity:
  • SEQ ID NO: 29 shows (N terminus to C-terminus) HSA (C34S variant) - GGGGS linker (SEQ ID NO: 31) - human neprilysin variant with two amino acid changes from wild type neprilysin: G399V and G714K.
  • HSA leader 55-72 HSA propep
  • 73-1827 HSA
  • 1828-1842 G4S
  • 1843-3945 NEPG399V-G714K
  • SEQ ID NO: 52 shows the sequence for HSA leader-HSA propeptide - HSA (C34S variant) - G4S-NEPG399V/G714K Protein
  • HSA leader 18-23 HSA propep
  • 24-609 HSA
  • 610-614 G4S
  • 615-1313 NEPG399V-G714K
  • SEQ ID NO: 54 shows the sequence for IgG leader- HSA propeptide - HSA (C34S variant) - G4S-NEPG399V/G714K Protein
  • NFRIIGTLQNSAEFSEAFHCRK SYMNPEK CRVW SEQ ID NO: 55 shows the sequence for HSA propeptide - HSA (C34S variant) -G4S- NEPG399V/G714K DNA
  • SEQ ID NO: 56 shows the sequence for HSA propeptide - HSA (C34S variant) - G4S- NEPG399V/G714K Protein
  • AACCCCGAGAAGAAATGCCGCGTGTGGTGATAA SEQ ID NO: 58 shows the sequence for HSA (C34S variant) - G4S-NEPG399V/G714K Protein
  • SEQ ID NO: 59 shows the sequence for IgG leader- 10HisNEPG399V/G714K DNA
  • PEK CRVW SEQ ID NO: 61 shows the sequence for lOHisNEP (ECD) G399V/G714K DNA
  • SEQ ID NO: 62 shows the sequence for lOHisNEP (ECD) G399V/G714K Protein
  • SEQ ID NO: 63 shows the sequence for:
  • SEQ ID NO: 64 shows the sequence for: IgG leader-HSAC34S-G4S-NEP G399V-G714K Protein
  • SEQ ID NO: 65 shows a HSA signal (leader) amino acid sequence
  • SEQ ID NO: 66 shows the nucleic acid sequence for vector pEU23.3 HSA propeptide (pro)- HSA (C34S variant) - G4S-neprilysin variant G399V/ G714K (yeast codon optimised).
  • Figure 1 shows the nucleotide sequence of yeast expression vector pYES2
  • the pYES2 vector is designed for native expression of your protein of interest in S. cerevisiae. It contains the URA3 gene for selection in yeast and 2 ⁇ origin for high-copy maintenance.
  • Figure 2 shows nucleotide sequences of yeast expression vector pESC-URA
  • Figure 3 shows nucleotide sequence of expression vector p427-TEF (Dualsystems Biotech), 6702 bp (SEQ ID NO:24).
  • Figure 4 shows a Western blot analysis of a culture supernatant of cells expressing human sNeprilysin (detection antibody: goat-polyclonal anti-h neprilysin (R&D)).
  • Figure 8 Abeta degradation of human Abeta 1-42 in plasma from TG2576 mice after
  • Figure 10 Abeta degradation of rat Abeta 1-40 in plasma from Sprague Dawley rats after 1 hour incubation at RT °C using 1 uM to 0.1 nM of enzyme.
  • Figure 11 Abeta degradation of Abeta 1-42 in human plasma after 1 hour incubation at RT °C using 3 uM to 0.1 nM of enzyme.
  • Figure 12 Abeta degradation of Abeta 1-40 in human plasma after 1 hour incubation at RT °C using 1 uM to 0.1 nM of enzyme.
  • Figure 13 Abeta degradation of Abeta 1-40 in buffer after 1 hour incubation at RT °C using 1 uM to 1 nM of enzyme.
  • Figure 14 Vector map for pEU23.3 (SEQ ID NO: 65) with a signal sequence of MGDNDIHFAFLSTGAHS, as shown for expression of HSA propeptide - HSA C34S- (Gly) 4 S-neprilysin (ECD) G399V / G714K.
  • Figure 15 Vector map for pEE12.4 (Lonza Biologies) encoding HSA C34S - (Gly) 4 S - neprilysin (ECD) G399V / G714K.
  • amyloid beta peptide means any form of the peptide that correlates to amino acid sequence (one letter code) DAEFRHDSG YEVHHQKLVF FAEDVGSNKG AIIGLMVGGV VIAT (SEQ ID NO: 33) in the human ⁇ A4 protein [Precursor], corresponding to amino acid 672 to 714 in the sequence (amino acid 1-43; ⁇ 1-43).
  • this peptide also includes any shorter forms of this peptide, such as ⁇ 1-40, ⁇ 1-41, ⁇ 1-42, ⁇ 1-39, ⁇ 1-38, ⁇ 1-43, and modified peptides such as N-terminal truncated forms as ⁇ 3-42, ⁇ 1-40 and ⁇ 1-42, ⁇ peptides with pyroglutamyl formation as ⁇ ( ⁇ 3-42) and ⁇ 1-42) and ⁇ peptides which are modified by oxidation, isomerisation, racemization, and/or covalently linkage (ID 17274, ID 17231, ID 17850).
  • the term comprises also ⁇ with substitutions of residues such Glu22 for Gin (references in Soto, C. and
  • polynucleotide corresponds to any genetic material of any length (greater than two nucleotides) and any sequence, comprising single-stranded and double-stranded DNA and RNA molecules, including regulatory elements, structural genes, groups of genes, plasmids, whole genomes, and fragments thereof.
  • site in a polynucleotide or polypeptide refers to a certain position or region in the sequence of the polynucleotide or polypeptide, respectively.
  • position in a polynucleotide or polypeptide refers to specific single bases or amino acids in the sequence of the polynucleotide or polypeptide, respectively.
  • region in a polynucleotide or polypeptide refers to stretches of several bases or amino acids in the sequence of the polynucleotide or polypeptide, respectively.
  • polypeptide comprises proteins such as enzymes, antibodies and the like, medium-length polypeptides such as peptide inhibitors, cytokines and the like, as well as short peptides down to an amino acid sequence length below ten (provided it is two or more amino acids), such as peptidic receptor ligands, peptide hormones, and the like.
  • protease means any protein molecule catalyzing the hydrolysis of peptide bonds. It includes naturally-occurring proteolytic enzymes, as well as protease variants and derivatives thereof. It also comprises any fragment of a proteolytic enzyme, and variants engineered by insertion, deletion, recombination and/or any other method, that leads to proteases that differ in their amino acid sequence from the naturally-occurring protease or the protease variants. It also comprises protein molecules with posttranslational and/or chemical modifications, e.g. glycosylation, PEGylation, HESylation, gamma carboxylation and acetylation, any molecular complex or fusion protein comprising one of the aforementioned proteins.
  • protease molecule with an amino acid sequence that differs from the wild type amino acid sequence and which can be obtained by mutagenesis, preferably by site-directed or random mutagenesis with an altered amino acid sequence compared to the respective wild type sequence, which retains protease activity and may have a different substrate specificity profile when compared to the wild-type sequence.
  • proteases means the ability of an enzyme to recognize and convert preferentially certain substrates.
  • the specificity of proteases i.e. their ability to recognize and hydro lyze preferentially certain peptide substrates, can be expressed qualitatively and quantitatively. Qualitatively, proteases that digest one or a small number of peptides have a high specificity, whereas proteases that digest numerous polypeptides have a low specificity. In quantitative terms, the specificity profile of a protease is given by the respective kcat/Km ratios for all substrates, including potentially kcat/Km ratios for several cleavage sites in a given substrate.
  • a variant enzyme is able to cleave amyloid beta ( ⁇ ) peptides to a greater degree and/or other peptides (including ANP, BNP, angiotensin- 1, bradykinin, endothelin 1 , neuropeptide Y, neurotensin, adrenomedullin and insulin ⁇ -chain) to a lesser degree as compared to the wild-type enzyme.
  • ⁇ amyloid beta
  • the variant neprilysin cleaves ⁇ _ 40 and/or ⁇ _ 42 peptide to a greater degree than any one of the following peptide substrates: ANP, BNP, angiotensin- 1, bradykinin, endothelin 1 , neuropeptide Y, neurotensin, adrenomedullin and insulin ⁇ -chain.
  • catalytic activity describes quantitatively the conversion of a given substrate under defined reaction conditions and is proportional to kcat/Km.
  • substrate or "peptide substrate” comprises any peptide, oligopeptide, or protein molecule of any amino acid composition, sequence or length, and post-translational or chemically-modified forms of these molecules that contain a peptide bond that can be hydro lyzed catalytically by a protease.
  • the peptide bond that is hydro lyzed is referred to as the "cleavage site”.
  • modulator refers to a molecule that prevents degradation and/or increases plasma half-life, reduces toxicity, reduces immunogenicity, or increases biological activity of a therapeutic protein.
  • exemplary modulators are a human serum albumin (HSA) binding component, such as wild type human HSA or a variant human HAS, such as HSA C34S which thereby prolong the plasma half-life of the polypeptide.
  • HSA human serum albumin
  • fusion refers to a molecule that is composed of a modulator molecule and a protein molecule.
  • the modulator may be covalently linked to the protein part to create the fusion protein.
  • a non-covalent approach can also be used to connect the protein to the modulator part.
  • degrade refers to a process where one starting molecule is divided in two or more molecule(s). More specifically, the amyloid ⁇ peptide (in any size from amino acid 1-43 and smaller) is cleaved to generate smaller fragments compared to the starting molecule. The cleavage can be accomplished through hydrolysis of peptide bonds or other type of reaction, which split the molecule in smaller parts.
  • pharmacologically active means that a substance so described is determined to have activity that affects a medical parameter (e.g., blood pressure, blood cell count, cholesterol level) or disease state (e.g., cancer, autoimmune disorders, dementia).
  • half- life is defined as the time taken for the removal of half the initial concentration of the protein or polypeptide from the plasma.
  • This invention describes ways of modulating the half-life of neprilysin variant polypeptides in plasma. Such modification can produce fusion proteins with improved pharmacokinetic properties (e.g., increased in vivo serum half- life). Prolonging the half- life means that it takes a longer time for clearance of half of the initial concentration of the protein from the plasma.
  • the half-life of a pharmaceutical or chemical compound is a well defined and well known term of the art.
  • connection means a covalent or a reversible linkage between two or more parts.
  • a covalent linkage can for example be a peptide bond, disulfide bond, carbon-carbon coupling or any type of linkage that is based of a covalent linkage between to atoms.
  • a reversible linkage can for example be biotin-streptavidin, antibody-antigen or a linkage which is classified as a reversible linkage known in the art.
  • a covalent linkage is directly obtained when the half-life modulator part and protease part of the fusion protein is produced in a recombinant form from the same plasmid, thus the connection is designed on DNA level.
  • covalently connected means a chemical link between two atoms in which electrons are shared between them.
  • bonds covalently connected are a peptide bond, disulfide bond, carbon-carbon coupling.
  • a fusion protein can be linked together by a polypeptide bond where the linkage can be accomplished during the translational process on the ribosome when the fusion protein is produced.
  • Other type of covalently connected component could be modification with a pegylation reagent that is covalently linked to an amino residue (for example lysine) on the protein.
  • the chemical coupling reaction can, for example, be acylation or other suitable coupling reaction which link the two components together into a fusion protein.
  • Covalently connected can also mean a linkage of a linker at two sites in which the modulator is linked together with the protein part.
  • cleavage sites means a specific location/site in a peptide sequence that can be cleaved by a protein or an enzyme. Cleavage is normally produced by hydrolysis of the peptide bond connecting two amino acids. Cleavage can also take place at multiple sites in the same peptide using a single or a combination of proteins or enzymes. A cleavage site can also be other site than the peptide bond. This invention describes the cleavage of the amyloid ⁇ peptide in detail.
  • the polypeptide comprising the protease variant e.g. a fusion polypeptide, or a derivative of any of the aforesaid, or a nucleic acid encoding same is isolated.
  • An isolated biological component such as a nucleic acid molecule or protein such as a protease
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • Amino acids are referred to herein using the name of the amino acid, the three-letter abbreviation or the single letter abbreviation.
  • the table below provides a list of the standard amino acids together with their abbreviations.
  • conservative amino acid substitutions of the variations are provided herein. Such substitutions are those, which are conservative, for example, wherein the variant amino acid is replaced by another amino acid of the same class.
  • Amino acids can be classified as acidic, basic, neutral and polar, or neutral and nonpolar and/or aromatic, depending on their side chain.
  • Preferred substitutions of a variant amino acid position include those that have one or more classifications that are the same as the variant amino acid at that position.
  • amino acids Lys, Arg, and His are basic; amino acids aspartic and glutamic are acidic; amino acids Ser, Thr, Cys, Gin, and Asn are neutral polar; amino acids Gly, Ala, Val, He, and Leu are non-polar aliphatic, and amino acids Phe, Trp, and Tyr are aromatic.
  • Gly and Ala are small amino acids and Val, He and Leu are aliphatic amino acids.
  • nucleic acids provided herein also include alternate sequences that use different codons to encode the same amino acid sequence. Furthermore, the nucleic acids provided herein also include both the coding sequence and the complementary sequence of nucleic acids encoding a variant neprilysin polypeptides provided herein.
  • a polypeptide provided herein can be prepared by recombinant expression of nucleic acid sequences encoding the same in a host cell.
  • a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide such that the polypeptide is expressed in the host cell.
  • Standard recombinant DNA methodologies are used prepare and/or obtain nucleic acids encoding the polypeptide, incorporate these nucleic acids into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989).
  • protease-encoding nucleic acids can be operatively linked to another fragment encoding a flexible linker such that the protease and other polypeptide sequences can be expressed as a contiguous single-chain protein, with the protease and other polypeptide regions joined by the flexible linker.
  • DNA encoding the desired polypeptide can be inserted into an expression vector which is then transfected into a suitable host cell. It is understood that the design of the expression vector, including the selection of regulatory sequences is affected by factors such as the choice of the host cell, the level of expression of protein desired and whether expression is constitutive or inducible.
  • Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • the recombinant expression vectors can also include origins of replication and selectable markers (see e.g., U.S. 4,399,216, 4,634,665 and U.S.
  • Suitable selectable markers include genes that confer resistance to drugs such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • drugs such as G418, hygromycin or methotrexate
  • DHFR dihydrofolate reductase
  • neo gene confers resistance to G418.
  • Transfection of the expression vector into a host cell can be carried out using standard techniques such as electroporation, calcium-phosphate precipitation, and DEAE-dextran transfection.
  • Suitable mammalian host cells for expressing the polypeptides provided herein include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells.
  • the expression vector is designed such that the expressed protein is secreted into the culture medium in which the host cells are grown.
  • the polypeptide thereof can be recovered from the culture medium using standard protein purification methods.
  • polypeptide can also be produced in prokaryotic cells using suitable vectors as described, for example, in U.S. 6,204,023 to Robinson, et al. and in (Carter et al,
  • the expression vector can be designed to allow the expressed polypeptide to be secreted into the periplasmic space, or the polypeptide can be retained within the cell, for example, in inclusion bodies.
  • the expressed polypeptide can be isolated from the periplasmic space or the inclusion bodies can be isolated from the host cell, respectively.
  • Suitable host cells for cloning or expressing the DNA in the vectors described herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • prokaryotes such as filamentous fungi or yeast are suitable cloning or expression hosts for the antibodies, antigen binding portions, or derivatives thereof provided herein.
  • Saccharomyces cerevisiae is a suitable eukaryotic host microorganism.
  • Another suitable yeast host is Schizosaccharomyces pombe.
  • Suitable host cells for the expression of a glycosylated protease or derivative thereof provided herein include mammalian, plant, and insect cells.
  • Host cells are transformed with the above-described expression or cloning vectors for the polypeptide and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Commercially available media such as Ham's F10, Minimal Essential Medium ((MEM), RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM), are suitable for culturing the host cells.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • polypeptide composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography.
  • polypeptides described herein have pharmacological activity resulting from their ability to process/degrade pharmacological active substrates.
  • An altered activity and/or specificity by a factor of two is sufficient to change the pharmacological activity of polypeptide comprising a human neprilysin variant compared to wild type.
  • activity/specificity of a polypeptide comprising a protease variant can be determined by assays known in the art. In vivo assays are known in the art and further described in the examples section.
  • compositions according to the invention may be for administration by injection, or for oral, pulmonary, nasal, transdermal, sub-cutaneous or other forms of administration.
  • the invention encompasses pharmaceutical compositions comprising effective amounts of a polypeptide of the invention together with
  • compositions include diluents of various buffer content, pH and ionic strength; additives such as detergents and solubilising agents, anti-oxidants, preservatives and bulking substances; incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes.
  • Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the polypeptide of the invention. See, e.g. Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa.
  • polypeptide may be prepared in liquid form, or may be in dried powder, such as lyophilized form.
  • Implantable sustained release formulations are also contemplated, as are transdermal formulations. These administration alternatives are well known in the art.
  • polypeptides provided herein can be administered to a patient in need thereof.
  • routes can be used to administer the polypeptide.
  • Any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects can be used to administer the protease or derivative thereof.
  • modes of administration include oral, sublingual, topical, nasal, transdermal or parenteral routes.
  • parenteral includes subcutaneous, intravenous, intramuscular, or infusion.
  • the polypeptides can be administered once, continuously, such as by continuous pump, or at periodic intervals.
  • the periodic interval may be weekly, bi-weekly, or monthly.
  • the dosing can occur over the period of one month, two months, three months or more to elicit an appropriate response. Desired time intervals of multiple doses of a particular composition can be determined without undue experimentation by one skilled in the art.
  • Other protocols for the administration of a polypeptide will be known to one of ordinary skill in the art, in which the dose amount, schedule of administration, sites of administration, mode of administration and the like vary from the foregoing.
  • the present invention relates to polypeptides, which comprise variants of human neprilysin extracellular catalytic domain having an altered activity and/or specificity.
  • the polypeptides have an improved specificity and / or activity against ⁇ peptide.
  • Protease variant G399V shows a 1.43-fold increased activity on Peptide- 1 ( ⁇ peptide derivative), a 1.21-fold increased activity on Peptide-2 ( ⁇ peptide derivative), a 1.32-fold increased activity on Peptide-7 (NPY derivative), a 50-fold decreased activity on Peptide-8 (neurotensin derivative) and Peptide-13 (Bradykinin derivative), and a 12.5-fold decrease on Peptide-5 (angiotensin derivative).
  • this variant G399V shows an approximate 70-fold increased specificity for Peptide- 1 vs. Peptide-13.
  • Protease variant G714K shows a 6.91 -fold increased activity on Peptide- 1 ( ⁇ peptide derivative), a 3.99-fold increased activity on Peptide-2 ( ⁇ peptide derivative), a 1.31 -fold increased activity on Peptide-6 (endothelin derivative), and a 5-fold decreased activity on Peptide-13 (Bradykinin derivative) and Peptide-4 (BNP derivative).
  • this variant G714K shows an approximate 35-fold increased specificity for Peptide- 1 vs. Peptide-13.
  • amino acid positions identified herein relate to those in full-length wild-type neprilysin (minus the initiating methionine), as disclosed in SEQ ID NO: 1.
  • G399 refers to the Glycine at position 399 in full length wild-type neprilysin.
  • mutant neprilysin polypeptides that comprise two substitutions being at positions 399 and 714 were especially specific for ⁇ relative to any of the off-peptide substrates, when compared to wild-type neprilysin, thus a particularly preferred variant polypeptide is one that comprises the G399V and G714K substitutions in the human neprilysin extracellular catalytic domain.
  • polypeptide comprising a variant human neprilysin extracellular catalytic domain which, compared to wild type neprilysin having the sequence according to the position in SEQ ID NO: 1, possesses an amino acid other than Glycine (G) at position 399 and/or an amino acid other than Glycine (G) at position 714, and optionally one or more substitutions relative to wild type neprilysin.
  • the one or more optional substitutions are at any of the following positions: 227, 228, 247, 419, 590, 593, 596, 600, 645, 709 or 718, with particular substitutions being any of: S227R, S227L, R228G, F247L, F247C, E419M, E419L, D590W, D590M, D590F, G593V, F596P, G600W, G600V, G600D, G600L, G645Q, D709K, D709V or I718L.
  • an isolated polypeptide which comprises a variant human neprilysin extracellular catalytic domain (compared to wild type neprilysin) having the sequence according to the position in SEQ ID NO: 1, with a valine (V) at position 399 and/or a lysine (K) at position 714, and optionally one or more substitutions relative to wild type neprilysin.
  • the one or more optional substitutions are selected from the group consisting of: S227R, S227L, R228G, F247L, F247C, E419M,
  • the one or more optional substitutions are selected from the group consisting of: S227R, R228G, F247L, E419M, D590M, D590F, G593V, F596P, G600V, G600D, G600L, G645Q and D709V.
  • the polypeptide comprises a neprilysin variant polypeptide which compared to wild type neprilysin having the sequence according to the position in SEQ ID NO: 1, possesses a valine (V) at position 399 and a lysine (K) at position 714, and one or more optional substitutions at one or more of the following positions: 227, 228, 247, 419, 590, 593, 596, 600, 645, 709, and 718, particular substitutions being any of: S227R, S227L, R228G, F247L, F247C, E419M, E419L, D590W, D590M, D590F, G593V, F596P, G600W, G600V, G600D, G600L, G645Q, D709K, D709V and I718L.
  • V valine
  • K lysine
  • Another embodiment encompasses a nucleic acid encoding an aforementioned polypeptide.
  • a further embodiment is a vector comprising the aforementioned nucleic acid.
  • another embodiment is a host cell comprising the aforementioned vector, such as one into which the vector has been transformed or transfected.
  • One embodiment is a method for producing a polypeptide of the invention, wherein the method comprises the following steps: culturing the aforementioned host cell comprising the vector housing the nucleic acid encoding the polypeptide, under conditions suitable for the expression of the polypeptide; and recovering the polypeptide from the host cell culture.
  • the polypeptide or nucleic acid encoding same is isolated.
  • An isolated biological component such as a nucleic acid molecule or protein such as a protease
  • An isolated biological component is one that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, e.g., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • Polypeptides of the invention may be described in the form M-A, wherein A is the neprilysin variant polypeptide as described herein and M is the moiety that prolongs the half- life of the neprilysin polypeptide.
  • the M polypeptide is attached to the N-terminus of the neprilysin variant.
  • the invention provides a polypeptide, wherein M is human serum albumin (HSA) or a HSA binding domain or peptide or a variant HSA with one or more mutations, preferably the variant HSA is C34S.
  • HSA human serum albumin
  • polypeptide wherein M and A are linked together with a linker, L.
  • polypeptide wherein L is selected from a peptide and a chemical linker.
  • Therapeutic methods of the invention encompass a method for reducing ⁇ peptide concentration, wherein said reduction of ⁇ peptide is accomplished in plasma, or in cerebrospinal fluid (CSF), or in the CNS.
  • CSF cerebrospinal fluid
  • neprilysin variants of the present invention may be derived or based on the full length neprilysin protein, or on the extra-cellular part of the protein which houses the regions capable of peptide cleavage.
  • the extra-cellular part is defined as the part of neprilysin that is defined as outside the membrane region.
  • the invention also comprises smaller fragments of neprilysin as long as the catalytic activity is preserved against the ⁇ peptide.
  • a neprilysin variant polypeptide or derivative thereof provided herein can be prepared by recombinant expression of nucleic acid sequences encoding the same in a host cell.
  • a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the neprilysin or derivative thereof such that the neprilysin or derivative is expressed in the host cell.
  • Standard recombinant DNA methodologies are used to prepare and/or obtain nucleic acids encoding the neprilysin or derivative thereof; to incorporate these nucleic acids into recombinant expression vectors; and, to introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989).
  • the neprilysin variants described herein have pharmacological activity resulting from their ability to process/degrade pharmacological active substrates. An altered activity and/or specificity by a factor of two is sufficient to change the pharmacological activity of the variant compared to wild type.
  • the activity/specificity of the neprilysin variants can be determined by assays known in the art. In vivo assays are known in the art and further described in the examples section.
  • DNA sequence of any portion of a polypeptide of the invention may be changed to codons more compatible with the chosen host cell.
  • optimized codons are known in the art. Codons may be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell.
  • the vehicle, linker and peptide DNA sequences may be modified to include any of the foregoing sequence changes.
  • Linkers Any "linker” group is optional. When present, its chemical structure is not critical, since it serves primarily as a spacer.
  • the linker is preferably made up of amino acids linked together by peptide bonds.
  • the linker is made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art.
  • the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine.
  • a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine.
  • preferred linkers are polyglycines (particularly (Gly) 4
  • a particularly useful linker is (Gly) 5 Ser (SEQ ID NO: 32) or (Gly) 4 Ser (SEQ ID NO: 31).
  • a polypeptide of the invention may be administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of the polypeptide of the invention in the plasma of the patient. In some methods, dosage is adjusted to achieve a plasma fusion protein concentration of 1-1000 ug/ml and in some methods 25-300 ug/ml. Also in some methods, dosage is adjusted to achieve a plasma polypeptide concentration of 1- 1000 ng/ml and in some methods 25-300 ng/ml. Alternatively, polypeptide can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.
  • the dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic.
  • a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.
  • a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a
  • a catalytically-active amyloid-P-peptide-degrading polypeptide can be administrated at a lower dose compare to a binding agent, such as for example an antibody.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a neprilysin variant with enhanced specificity for ⁇ relative to an off-target ( ⁇ - ⁇ ) peptide substrate, and / or relative to wild type human neprilysin, which variant possess one or more amino acid substitutions located at positions: 227, 228, 247, 399, 419, 590, 593, 596, 600, 709, 714 and 718, relative to the position in SEQ ID NO: 1.
  • Particular variants have one or both of residues at positions 399 and 714 substituted for a non-wild type codon.
  • the wild type codons are those present in SEQ ID NO: 1.
  • the introduction of a mutation into the polynucleotide sequence to exchange one nucleotide for another nucleotide optionally resulting in a mutation in the corresponding polypeptide sequence may be accomplished by site-directed mutagenesis using any of the methods known in the art. Such techniques are explained in the literature, for example:
  • Particularly useful is the procedure that utilizes a supercoiled, double-stranded DNA vector with the polynucleotide sequence of interest and two polynucleotide primers harboring the mutation of interest.
  • the primers are complementary to opposite strands of the vector and are extended during a thermocycling reaction using, for example, Pfu DNA polymerase.
  • a mutated plasmid containing nicks is generated.
  • this plasmid is digested with Dpnl, which is specific for methylated and hemimethylated DNA to digest the start plasmid without destroying the mutated plasmid (see Example 2.1).
  • polynucleotides including genomic DNA, genomic RNA, cDNA and mRNA; double stranded as well as +ve and -ve strands, which encode the polypeptides of the invention.
  • polynucleotides can be synthesised chemically, or isolated by one of several approaches known to the person skilled in the art such as polymerase chain reaction (PCR) or ligase chain reaction (LCR) or by cloning from a genomic or cDNA library.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • expression vector/host systems may be used to express polypeptides of the invention. These include, but are not limited to
  • microorganisms such as bacteria expressed with plasmids, cosmids or bacteriophage; yeasts transformed with expression vectors; insect cell systems transfected with baculovirus expression systems; plant cell systems transfected with plant virus expression systems, such as cauliflower mosaic virus; or mammalian cell systems (for example those transfected with adenoviral vectors); selection of the most appropriate system is a matter of choice.
  • Expression vectors usually include an origin of replication, a promoter, a translation initiation site, optionally a signal peptide, a polyadenylation site, and a transcription termination site. These vectors also usually contain one or more antibiotic resistance marker gene(s) for selection.
  • suitable expression vectors may be plasmids, cosmids or viruses such as phage or retroviruses. Examples of suitable retroviral vectors that could be used include: pLNCX2 (Clontech, BD Biosciences, Cat# 631503), pVPac-Eco (Stratagene, Cat# 217569) or pFB-neo (Statagene, Cat# 217561).
  • packaging cell lines examples include: BD EcoPack2-293 (Clontech, BD Biosciences, Cat# 631507), BOSC 23 (ATCC, CRL-11270), or Phoenix-Eco (Nolan lab, Stanford University).
  • the coding sequence of the polypeptide is placed under the control of an appropriate promoter (i.e. HSV, CMV, TK, RSV, SV40 etc), control elements and transcription terminator so that the nucleic acid sequence encoding the polypeptide is transcribed into RNA in the host cell transformed or transfected by the expression vector construct.
  • the coding sequence may or may not contain a signal peptide or leader sequence for secretion of the polypeptide out of the host cell.
  • Preferred vectors will usually comprise at least one multiple cloning site.
  • Such cloning sites can be used to create N-terminal fusion proteins by cloning a second nucleic acid sequence into the cloning site so that it is contiguous and in- frame with the gene of interest.
  • there may be a cloning site or multiple cloning site situated immediately downstream of the gene of interest to facilitate the creation of C-terminal fusions in a similar fashion to that for N- terminal fusions described above, may be expressed in a variety of hosts such as bacteria, plant cells, insect cells, fungal cells and human and animal cells. Eukaryotic recombinant host cells are particularly suitable. Examples include yeast, mammalian cells including cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including
  • Drosophila, army fallworm and silkworm derived cell lines Drosophila, army fallworm and silkworm derived cell lines.
  • a variety of mammalian expression vector/host systems may be used to express the neprilysin variant polypeptides of the present invention. Particular examples include those adapted for expression using a recombinant adenoviral, adeno-associated viral (AAV) or retroviral system. Vaccinia virus, cytomegalovirus, herpes simplex virus, and defective hepatitis B virus systems, amongst others may also be used.
  • L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), HEK 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), CI 271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
  • mammalian expression systems are used for expression of the neprilysin variant polynucleotide sequence
  • vector and host cell systems such as, bacterial, yeast, plant, fungal, insect are also possible.
  • the vectors containing the DNA coding for the polypeptides of the invention can be introduced into host cells to express a polypeptide of the present invention via any one of a number of techniques, including calcium phosphate transformation, DEAE-dextran transformation, cationic lipid mediated lipofection, electroporation or infection. Performance of the invention is neither dependent on nor limited to any particular strain of host cell or vector; those suitable for use in the invention will be apparent to, and a matter of choice for, the person skilled in the art.
  • Host cells genetically modified to include a nucleotide sequence encoding a polypeptide of the invention may be cultured under conditions suitable for the expression and recovery of the encoded polypeptides from the cell culture. Such expressed polypeptides may be secreted into the culture medium, or they may be contained intracellularly, depending on the sequences used, i.e. whether or not suitable secretion signal / leader sequences were present.
  • polypeptides of the invention can be easily performed using methods well known in the art (for example as described in Sambrook et al., ibid).
  • the invention provides for cells and cell lines transformed or transfected with the nucleic acids encoding polypeptides or vectors of the present invention.
  • the transformed cells may, for example, be mammalian, bacterial, yeast or insect cells.
  • a host cell adapted to express a polypeptide of the invention.
  • a plasmid comprising a nucleotide sequence encoding a polypeptide of the invention represents a further aspect of the invention.
  • a host cell adapted to express a polypeptide of the invention from the nucleic acid sequence of the invention.
  • Preferred host cells are mammalian such as CHO-K1 or Phoenix cells. Human cells are most preferred for expression purposes.
  • the polypeptides of this invention may be made in transformed host cells using recombinant DNA techniques. To do so, a recombinant DNA molecule coding for the polypeptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences coding for the modulator and protein could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidate method. Also, a
  • the invention also includes a vector capable of expressing the polypeptide in an appropriate host.
  • the vector comprises the DNA molecule that codes for the polypeptide operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known.
  • Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation.
  • the resulting vector having the DNA molecule therein is used to transform an appropriate host. This transformation may be performed using methods well-known in the art.
  • Any of a large number of available and well-known host cells may be used in the practice of this invention.
  • the selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the fusion encoded by the DNA molecule, rate of
  • useful microbial hosts include bacteria (such as E. coli sp.), yeast (such as
  • Saccharomyces sp. Saccharomyces sp.
  • Host cells may be cultured under conventional fermentation conditions so that the desired polypeptides are expressed. Such fermentation conditions are well known in the art.
  • the polypeptide is purified from the culture by methods well known in the art.
  • the polypeptide may also be made by synthetic methods. For example, solid phase synthesis techniques may be used. Suitable techniques are well known in the art, and include those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl.
  • compositions comprising effective amounts of a polypeptide of the invention together with pharmaceutically-acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • Such compositions may include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc.
  • buffer content e.g., Tris-HCl, acetate, phosphate
  • additives such as detergents and so
  • Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation.
  • Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present polypeptide. See, e.g. Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack
  • compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form.
  • Implantable sustained release formulations are also contemplated, as are transdermal formulations. These administration alternatives are well known in the art.
  • the dosage regimen involved in a method for treating the above-described conditions may be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body weight, sex and diet of the patient, the severity of disease, time of administration and other clinical factors.
  • the daily regimen should be in the range of 0.1-1000 micrograms of the polypeptide per kilogram of body weight, preferably 0.1-150 micrograms per kilogram.
  • the present invention provides a method for the treatment of ⁇ -related pathologies such as Downs syndrome and ⁇ -amyloid angiopathy, such as but not limited to cerebral amyloid angiopathy, systemic amyloidosis, inclusion body myositis, hereditary cerebral hemorrhage, disorders associated with cognitive impairment, such as but not limited to MCI ("mild cognitive impairment"), Alzheimer Disease, memory loss, attention deficit symptoms associated with Alzheimer disease, neurodegeneration associated with diseases such as Alzheimer disease or dementia including dementia of mixed vascular and degenerative origin, pre-senile dementia, senile dementia and dementia associated with Parkinson's disease, progressive supranuclear palsy or cortical basal degeneration, comprising administering to a mammal (including human) a therapeutically effective amount of a fusion protein according to the present invention.
  • ⁇ -related pathologies such as Downs syndrome and ⁇ -amyloid angiopathy, such as but not limited to cerebral amyloid angiopathy, systemic amyloido
  • a human wt-s neprilysin sequence comprising the codons for aa5 l-aa749 (PDB numbering) was cloned into a yeast expression vector (pYES2 Invitrogen, SKU# V825-20; see SEQ ID NO:22).
  • yeast expression vectors beside pYES2 like pESC- URA (Stratagen; see SEQ ID NO:23) or p427-TEF(Dualsystems Biotech; see SEQ ID NO:24) can be used.
  • the neprilysin sequence in the resulting construct is N-terminally fused to sequences encoding a secretion leader, secretion site, triple HA-tag and a linker (see SEQ ID NO:5).
  • the triple HA-tag serves for purification of expressed s neprilysin.
  • a His-tag can be used. Nucleotide and amino acid sequences of the wt-s neprilysin construct with tag and linker are shown in SEQ ID NO: 5 and 3 respectively.
  • Variants were generated by oligo based site-specific mutagenesis.
  • 3xHA-tag was introduced via 2-step PCR.
  • a first PCR was performed using primer NEP-85A and NEP-24
  • the PCR amplification product and the vector were digested with Xhol and Notl with a subsequent ligation reaction using standard molecular biology protocols, resulting in a construct with the nucleotide sequence shown in SEQ ID NO: 7, wherein the alpha secretion leader sequence including the secretion site is at position 507-773, the 3xHA tag sequence is at position 774-854; the Gly/Ser linker (linker) is at position 855 - 860; the s neprilysin sequence is at position 861-2960 (wt sequence shown); and the CYY1 terminator sequence is at position 3090-3338.
  • HA-tag monoclonal Antibody HA. l 1, # MMS-101P
  • His-tagged protease by metal-chelate affinity chromatography.
  • HA-tag monoclonal Antibody HA. l 1, # MMS-101P
  • metal-chelate affinity chromatography Coligan, J. E., Dunn, B. M. , Ploegh, H. L. , Speicher, D. W., Wingfield, P. T. (Eds.), Current Protocols in Protein Science, John Wiley & Sons, New York (1996) 9.4 and 9.5, respectivily).
  • pre loading the protease in the yeast supernatant was re-buffered using a cross- filtration device (VIVAFLOW 200, 10k MWCO, Satorius, #512-4069).
  • Un-tagged protease can be purified by ion exchange chromatography on resource Q (Amersham Pharmacia Biotech) followed by gel filtration chromatography on Superdex 200 (Amersham Pharmacia Biotech) (Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., Wingfield, P. T. (Eds.), Current Protocols in Protein Science, John Wiley & Sons, New York (1999) 8.2 and (1998) 8.3, respectively).
  • Example 3 Determination of catalytic activity and specificity
  • This k app was measured as kinetic changes in fluorescence anisotropy for every single substrate. All substrates were customized (Thermo Fisher Scientific GmbH) and were labelled with a fluorophore and a biotin at the N- and C- termini, respectively. The biotin serves to increase the molecular size of uncleaved molecules after addition of streptavidin, thereby increasing the assay window and the measurable signals.
  • the assay was performed by incubating the protease sample in a microtitre plate with an assay solution composed of 60nM peptide substrate in 50mM Hepes (sigma, #H4034), 150mM NaCl (Merck, #1.06404.5000) and 0.05% PluronicF68 (Sigma, #P7061-500), pH7.0.
  • Table 5 the G714K substitution (the single mutation in B9) is included in all clones, Bl to B12.
  • Table 6 lists relative activities of the protease variants vs. mutant G714K on different substrates determined as ratio of the two corresponding k app - values.
  • Bl to B8 (most of them have the mutation G399V), exhibiting a particularly desirable profile of cleavage against the various peptides (in terms of an improved specificity for ⁇ vs. the off- peptides, such as peptide-5, -8, -13, -3, -6 and -10; see Table 6).
  • the G399V/G714K double mutant shows an improved specificity for ⁇ vs. peptide-5, -8, -13 and -3 by a factor of >100; vs. peptide-4 by a factor of -50; and, vs. peptide-6, -10 and -7 by a factor of >10.
  • Table 7 lists relative activities of the certain protease variants vs. Bl on different substrates determined as ratio of the two corresponding k app -values.
  • CI to C23 exhibit an increased activity on Peptide -1 and - 2, apart from C2 and C3, and a reduced activity on peptide-6, -5 and -3.
  • peptide 5 angiotensin
  • peptide 3 A P
  • peptide 6a one of the endothelin peptides
  • peptide 1 ABi_ 4o
  • peptide 2 ABi_ 42
  • the extra-cellular domain of a variant neprilysin containing one or more mutations that impact the specificity of the protease for one or more of its substrates, is fused to the human IgGl Fc domain (including the hinge region).
  • a signal sequence - MGWSCIILFLVATATGAHS (SEQ ID NO: 25) is introduced to enable secretion of the protein into the culture media during expression.
  • the sequence of the hinge region is
  • THTCPPCP SEQ ID NO: 26
  • IgGl Fc domain is shown in SEQ ID NO: 27.
  • the complete fusion protein (excluding the signal sequence) with a human neprilysin variant has predicted molecular weights of 211 kDa (Fc-Nep as a dimer).
  • the complete gene (encoding the Fc-Neprilysin variant) including the signal sequence is inserted into a suitable mammalian expression vector, such as pCEP4, pEAKlO, pEF5/FRT7V5-DEST and pcDNA5/FRT/TO (Gateway adapted). All these are standard mammalian expression vectors based on a CMV promoter (pCEP4, pEAKlO and
  • the protein NEP (extra-cellular domain only) and Fc-NEP (Fc-Nep) are transiently expressed in suspension-adapted mammalian cells.
  • the cell lines used in the production experiments may be cell lines derived from HEK293, including HEK293S, HEK293S-T and HEK293S-EBNA cells. Transfection is performed at cell density of approximately 0.5-lxlO 6 and with plasmid DNA at concentrations ranging from 0.3-0.8 ⁇ g/ml cell suspension (final concentration). Expression is performed in cell culture volumes of 30 ml to 1000 ml (shaker flasks), and 5L to 10L Wave Bioreactor. Cell cultures are harvested after 4 to 14 days by centrifugation.
  • Purification of the fusion protein can be performed using cell media from expression in mammalian cells.
  • the purification can be performed by Affinity chromatography (Protein A) followed by low pH elution, on AKTA Chromatography systems (Explorer or Purifier, GE Healthcare).
  • rProtein A Sepharose FF (GE Healthcare) in an XK26 column (GE Healthcare) is equilibrated with 10 column volumes (CV) of PBS (2.7 mM KC1, 138 mM NaCl, 1.5 mM KH 2 PO 4 , 8 mM Na 2 HP0 4 -7H 2 0, pH 6.7-7.0, Invitrogen).
  • Cell culture media with expressed fusion protein (Fc-Neprilysin) is applied onto the column.
  • the column is washed with 20 CV PBS before bound protein is eluted with Elution buffer (0.1 M Glycine, pH 3.0). Purified fractions are immediately neutralized by adding 50 ⁇ of 1M Tris Base to 1 ml of eluted protein. Purified fractions are pooled and buffer is exchanged to 50 mM Tris-HCl, pH 7.5, 150 mM NaCl using PD10 Columns (GE Healthcare).
  • Elution buffer 0.1 M Glycine, pH 3.0
  • Purified fractions are immediately neutralized by adding 50 ⁇ of 1M Tris Base to 1 ml of eluted protein. Purified fractions are pooled and buffer is exchanged to 50 mM Tris-HCl, pH 7.5, 150 mM NaCl using PD10 Columns (GE Healthcare).
  • Example 6 Degradation of amyloid ⁇ peptidel-40 in human plasma by neprilysin or neprilysin variants.
  • Degradation of human amyloid ⁇ peptidel-40 ( ⁇ 40) and human amyloid ⁇ peptide 1- 42 ( ⁇ 42) by Neprilysin is investigated using heparinised plasma from healthy volunteer humans.
  • Human heparin plasma is prepared by centrifugation for 20 min at 4°C at 2500 x g within 30 minutes of sampling. Plasma samples are transferred to pre-chilled polypropylene tubes and immediately frozen and stored at -70°C prior to use.
  • Neprilysin or Neprilysin variants (0.1-300 ⁇ g/ml) or 5 ⁇ g/ml recombinant human Neprilysin (R&D systems) with corresponding vehicles (50 mM Tris-HCl, 150 mM NaCl pH 7.5 or 25 mM Tris-HCl, 0.1 M NaCl pH 8.0 or 50 mM HEPES, 100 mM NaCl, 0.05% BSA pH 7.4) are incubated with a pool of plasma in presence or absence of 10 ⁇ phosphoramidon (BIOMOL) or 2 mM 1,10- phenantroline (Sigma- Aldrich) at room temperature for 0, 1 h and 4h.
  • corresponding vehicles 50 mM Tris-HCl, 150 mM NaCl pH 7.5 or 25 mM Tris-HCl, 0.1 M NaCl pH 8.0 or 50 mM HEPES, 100 mM NaCl, 0.05% BSA pH 7.4
  • Example 7 Degradation of amyloid ⁇ peptidel-40 in C57BL/6 mice by Neprilysin or neprilysin variants (in vivo studies).
  • Example 8 Degradation of mouse amyloid ⁇ peptidel-40 in mouse C57BL/6 plasma by neprilysin or neprilysin variants.
  • Degradation of mouse amyloid ⁇ peptidel-40 ( ⁇ 40) by neprilysin is investigated using heparinized plasma from male and female C57BL/6 mice (20-30 g). Blood is withdrawn from anaesthetized mice by heart puncture. The blood is collected into prechilled microtainer tubes containing heparin and centrifuged for 10 min at 4°C at 3000 x g within 20 minutes of sampling. Plasma samples are transferred to pre-chilled polypropylene tubes and immediately frozen on dry ice and stored at -70°C prior to use.
  • neprilysin or neprilysin variants (0.1-300 ⁇ g/ml) or 5 ⁇ g/ml recombinant human neprilysin (R&D systems) with corresponding vehicles (50 mM Tris-HCl, 150 mM NaCl pH 7.5 or 25 mM Tris-HCl, 0.1 M NaCl pH 8.0 or 50 mM HEPES, 100 mM NaCl, 0.05% BSA pH 7.4) are incubated with a pool of plasma in presence or absence of 10 ⁇
  • Example 9 Treatment of APPswF-transgenic mice with neprilysin or neprilysin variants and subsequent analysis on ⁇ levels in plasma and CNS.
  • Tg2576 mice In vivo studies in APPsw E -transgenic (Tg2576) mice are performed in order to test the in vivo efficacy of neprilysin or neprilysin variants.
  • the primary read-outs are amyloid beta ( ⁇ ) levels in plasma and CNS as well as plasma drug concentration.
  • the Tg2576 mice, 20- 25g, are weighed and administrated intravenously (i.v.) or intraperitoneally (i.p.) with a single or repeated administration.
  • transgenic mice 25-27 weeks of age
  • 5-6 animals for each group.
  • Each time point has its own vehicle group.
  • Blood is withdrawn from anaesthetized mice by heart puncture into pre-chilled microtainer tubes containing EDTA. Blood samples are immediately put on ice prior to centrifugation. Plasma is prepared by centrifugation for 10 minutes at approximately 3000 x g at +4°C. After blood sampling, mice are sacrificed by decapitation and brain samples are collected. One brain hemisphere is homogenized with 0.2% diethylamine (DEA) and 50 mM NaCl (18 ⁇ /mg tissue). Brain homogenates are centrifuged at 133,000 x g for 1 hour at +4°C.
  • DEA diethylamine
  • Recovered supernatants are neutralised to pH 8.0 with 2 M Tris-HCl.
  • ⁇ 40 and ⁇ 42 levels in plasma and brain are analyzed by commercial ELISA kit obtained from Biosource or Innogenetics, respectively. All plasma samples are analysed to determine drug exposure with mesoscale technology.
  • transgenic mice 25-27 weeks of age at study start
  • 30 animals for each group Each time point has its own vehicle group.
  • blood is withdrawn from mice every second week into pre-chilled microtainer tubes containing EDTA. Blood samples are immediately put on ice prior to centrifugation. Plasma is prepared by centrifugation for 10 minutes at
  • mice are sacrificed by decapitation and brain samples are collected.
  • One brain hemisphere is homogenized with 0.2% diethylamine (DEA) and 50 mM NaCl (18 ⁇ /mg tissue).
  • Brain homogenates are centrifuged at 133,000 x g for 1 hour at +4°C. Recovered supernatants are neutralised to pH 8.0 with 2 M Tris-HCl. The insoluble pellet is further sonicated with 70% formic acid (FA) (18 ⁇ /mg tissue).
  • Brain homogenates are centrifuged at 133,000 x g for 1 hour at +4°C. Recovered supernatants are neutralised to pH 8.0 with 1 M Tris.
  • ⁇ 40 and ⁇ 42 levels in plasma, brain and CSF are analyzed by commercial ELISA kit obtained from Biosource or Innogenetics, respectively. All plasma samples are analysed to determine drug exposure.
  • Example 10 Degradation of amyloid ⁇ mouse peptidel-40., amyloid ⁇ human peptidel- 40 and amyloid ⁇ human peptidel-42 in Tg2576 mouse plasma by neprilysin or neprilysin variants.
  • mice by heart puncture.
  • the blood is collected into prechilled microtainer tubes containing heparin and centrifuged for 10 min at 4°C at 3000 x g within 20 minutes of sampling.
  • Plasma samples are transferred to pre-chilled polypropylene tubes and immediately frozen on dry ice and stored at -70°C prior to use.
  • neprilysin or neprilysin variants (0.1-300 ⁇ g/ml) or 5 ⁇ g/ml recombinant human Neprilysin (R&D systems) with corresponding vehicles (50 mM Tris-HCl, 150 mM NaCl pH 7.5 or 25 mM Tris-HCl, 0.1 M NaCl pH 8.0 or 50 mM HEPES, 100 mM NaCl, 0.05% BSA pH 7.4) are incubated with a pool of plasma in presence or absence of 10 ⁇
  • SD rats In vivo studies in male Sprague Dawley (SD) rats are performed in order to test the in vivo efficacy of neprilysin or neprilysin variants.
  • the read-outs are soluble amyloid beta ( ⁇ ) levels in plasma, csf and brain as well as plasma drug concentration.
  • the male SD rats 250- 350 g are weighed and given single or repeated intravenous administration of appropriate doses. 8-10 animals are included in each time point and each time point has its own vehicle group. Blood is withdrawn from anaesthetized rats by heart puncture into pre-chilled microtainer tubes containing EDTA. Blood samples are immediately put on ice prior to centriiugation.
  • Plasma is prepared by centriiugation for 10 minutes at approximately 3000 x g at +4°C.
  • CSF is aspirated from the cisterna magna and transferred to pre-chilled eppendorf tubes prior to centrifugation.
  • CSF is centrifuged for 1 minute at approximately 3000g at +4 °C.
  • the supernatant is collected and put in new pre-chilled eppendorf tubes.
  • the tubes are immediately frozen on dry ice and stored frozen at -70 °C.
  • rats are sacrificed by decapitation and brain samples are collected.
  • One brain hemisphere is homogenized with 0.2% diethylamine (DEA) and 50 mM NaCl (18 ⁇ /mg tissue).
  • DEA diethylamine
  • Brain homogenates are centrifuged at 133,000 x g for 1 hour at +4°C. Recovered supernatants are neutralised to pH 8.0 with 2 M Tris-HCl. Soluble ⁇ 40 in plasma as well as soluble ⁇ 40 and ⁇ 42 levels in brain and CSF are analyzed by commercial ELISA kit obtained from Biosource. All plasma samples are analysed to determine drug exposure with Mesoscale technology.
  • Example 12 Degradation of amyloid ⁇ rat peptidel-40 in rat plasma by neprilysin or neprilysin variants.
  • rat amyloid ⁇ peptidel-40 ( ⁇ 40) by Neprilysin is investigated using heparinised plasma from male Sprague Dawley rats (250-350 g). Blood is withdrawn from anaesthetized rats by heart puncture. The blood is collected into prechilled microtainer tubes containing heparin and centrifuged for 10 min at 4°C at 3000 x g within 20 minutes of sampling. Plasma samples are transferred to pre-chilled polypropylene tubes and immediately frozen on dry ice and stored at -70°C prior to use. The experiments are performed on a pool of plasma.
  • Neprilysin or Neprilysin variants (0.1-300 ⁇ g/ml) or 5 ⁇ g/ml recombinant human Neprilysin (R&D systems) with corresponding vehicles (50 mM Tris-HCl, 150 mM NaCl pH 7.5 or 25 mM Tris-HCl, 0.1 M NaCl pH 8.0 or 50 mM HEPES, 100 mM NaCl, 0.05% BSA H 7.4) are incubated with a pool of plasma in presence or absence of 10 ⁇ phosphoramidon (BIOMOL) or 2 mM 1,10-phenantroline (Sigma- Aldrich) at room temperature for 0, 1 h and 4h. A final concentration of 5 mM EDTA is added to the tubes before the amount of ⁇ 40 is analysed using a commercial ELISA kit obtained from
  • Example 13 Degradation of amyloid ⁇ peptides in Guinea pigs by neprilysin or neprilysin variants (in vivo studies).
  • CSF is centrifuged for 1 minute at approximately 3000g at +4 °C.
  • the supernatant is collected and put in new pre- chilled eppendorf tubes.
  • the tubes are immediately frozen on dry ice and stored frozen at - 70 °C.
  • blood is collected by heart puncture into pre- labeled and pre-chilled microtainer tubes containing EDTA. Blood samples are immediately put on ice prior to centrifugation. It is important that the exact sampling times are recorded.
  • Plasma is prepared by centrifugation for 10 minutes at approximately 3000g at 4°C within 20 minutes from sampling. After sampling, the animals are sacrificed by decapitation and brain samples are collected.
  • One brain hemisphere is homogenized with 0.2% diethylamine (DEA) and 50 mM NaCl (20 ⁇ /mg wet weight tissue). Brain homogenates are centrifuged at 133,000 x g for 1 hour at +4°C. Recovered supernatants are neutralised to pH 8.0 with 2 M Tris-HCl. Soluble ⁇ 40 and ⁇ 42 levels in plasma, brain and CSF are analyzed by commercial ELISA kit obtained from Biosource and Innogenetics, respectively. All plasma samples are analysed to determine drug exposure with Mesoscale technology.
  • Example 14 Treatment of APPswF-transgenic mice with Neprilysin or neprilysin variants and subsequent analysis on soluble ⁇ levels in plasma.
  • the objective with this study is to evaluate the time and dose-response effect of neprilysin variants in plasma of female APPsw E -tg mice after acute intravenous treatment.
  • the specific purpose is to find an effect on plasma ⁇ 40 and ⁇ 42 .
  • mice 25-31 weeks old female APPsw E -transgenic mice (10 mice/group) receive vehicle or the neprilysin variants at 1 or 5 mg/kg as a single intravenous injections. The animals are treated in 3 hours (4 mice). A blank group is also included in the study. Blood is sampled from vehicle- and compound-treated animals at 1.5 and 3 hours after dose. Blood is withdrawn from anaesthetized mice by heart puncture into pre-chilled microtainer tubes containing EDTA. Blood samples are immediately put on ice prior to centrifugation. Plasma is prepared by centrifugation for 10 minutes at approximately 3000 x g at +4 °C within 20 minutes from sampling. After blood sampling, mice are terminated.
  • ⁇ 40 and ⁇ 42 levels in plasma are analyzed by commercial ELISA kit obtained from Biosource and Innogenetics, respectively.
  • Example 15 EEG study in APPs -transgenic mice with Neprilysin or neprilysin variants (in vivo studies).
  • mice can be complemented with a read-out with EEG.
  • Mice are implanted with an indwelling electrode consisting of three polyimide-coated wires with bare tips that are implanted at depths 3 mm, 1 mm, and 1 mm from the dorsal surface of the brain to target the CA3 region of the hippocampus (2.5 mm posterior and 2.0 mm lateral from Bregma) and cortical surfaces (1 and 2 mm rostral from hippocampal wire), respectively. Electrode location is verified in a subset of animals to show proper targeting of the hippocampal area. Data is recorded continuously during the dark (night; active) cycle (6pm- 6am). Normally data is analysed from the first two hours of the dark cycle separately and presented as representative.
  • PSDs Power spectral densities
  • FFT Fast Fourier Transform
  • Spike2 Spike Electronic Design
  • PSDs are calculated from the entire recording. Spectrograms are generated and power spectra are calculated for each one second using an FFT of 128Hz and color-mapped as terms of Log of PSD calculated as 10*logl0(raw PSD), where raw PSD is normalized so that the sum of all the spectrum values equals to the mean squared value of the signal.
  • Power scales are globalised and a boxcar filter was used to smooth the resulting spectrogram for visualization.
  • PSDs are generated as above for every 30 seconds for each individual recording.
  • the DF for each 30 second epoch is the frequency that has the greatest power in that epoch.
  • the average DF represents the average of the DFs from all the mice in each group.
  • the object recognition task has been designed to assess the effects of experimental manipulations on the cognitive performance of rodents.
  • a rat is placed in an open field, in which two identical objects are present.
  • the rats inspects both objects during the first trial of the object recognition task.
  • a second trial after a retention interval of for example 24 hours, one of the two objects used in the first trial, the 'familiar' object, and a novel object are placed in the open field.
  • the inspection time at each of the objects is registered.
  • the basic measures in the OR task is the time spent by a rat exploring the two object the second trial. Good retention is reflected by higher exploration times towards the novel than the 'familiar' object.
  • Administration of the putative cognition enhancer prior to the first trial predominantly allows assessment of the effects on acquisition, and eventually on consolidation processes.
  • Administration of the testing compound after the first trial allows to assess the effects on consolidation processes, whereas administration before the second trial allows to measure effects on retrieval processes.
  • the passive avoidance task assesses memory performance in rats and mice.
  • the inhibitory avoidance apparatus consists of a two compartment box with a light compartment and a dark compartment. The two compartments are separated by a guillotine door that can be operated by the experimenter. When the door is open, the illumination in the dark
  • the compartment is about 2 lux.
  • the light intensity is usually about 500 lux at the centre of the floor of the light compartment.
  • Two habituation sessions, one shock session, and a retention session are given, separated by inter session intervals of 24 hours.
  • the rat is allowed to explore the apparatus for 300 sec.
  • the rat is placed in the light compartment, facing the wall opposite to the guillotine door. After an accommodation period of 15 sec. the guillotine door is opened so that all parts of the apparatus can be visited freely. Rats normally avoid brightly lit areas and will enter the dark compartment within a few seconds.
  • the guillotine door between the compartments is lowered as soon as the rat has entered the dark compartment with its four paws, and a scrambled 0.3 -1 mA foot shock is administered for 2 sec.
  • the rat is removed from the apparatus and put back into its home cage.
  • the procedure during the retention session is identical to that of the habituation sessions.
  • the step through latency which is the first latency of entering the dark compartment (in sec.) during the retention session, is an index of the memory performance of the animal; the longer the latency to enter the dark compartment, the better the retention is.
  • a testing compound is given half an hour before the shock session, together with scopolamine.
  • Scopolamine impairs the memory performance during the retention session 24 hours later. If the test compound increases the enter latency compared with the scopolamine treated controls, is likely to possess cognition enhancing potential.
  • Contextual fear conditioning measures aversive memory in rats and mice.
  • An observation box with distinctive contextual features is used (light, texture etc)
  • the box is equipped with a gridded floor and stimulus lights located in each compartment.
  • the chamber is made of transparent Plexiglas and illuminated by a 60-W bulb (including dimmers).
  • the animals On the day of training and testing the animals are first allowed to habituate to the experimental room for 60 minutes. On the first day of experiment (training trial), the animal is placed in the illuminated chamber where it is left to explore the compartment. After a defined time (180s) a foot shock (usually 0.7 mA, 2s duration, constant current) is delivered to the animal's feet. The animal is left in the light chamber for an additional 30s before being returned to its home cage immediately after the training trial. Behavior is recorded again 24 h later (test trial), in the same manner as described above with the exception that no chock is delivered on the test day and the cut off time is 180s. The readout used is freezing response (i.e.
  • the boxes are controlled by software from the manufacturer.
  • the animals are videotaped and the freezing response is scored manually afterwards. Animals are evenly distributed over doses and time of day.
  • the testing compound is given together with scopolamine. Scopolamine impairs the memory performance during the retention session 24 hours later. If the test compound increases the enter latency compared with the scopolamine treated controls, is likely to possess cognition enhancing potential.
  • the Morris water escape task
  • the Morris water escape task measures spatial orientation learning in rodents. It is a test system that has extensively been used to investigate the effects of putative therapeutic on the cognitive functions of rats and mice. The performance of an animal is assessed in a circular water tank with an escape platform that is submerged about 1 cm below the surface of the water. The escape platform is not visible for an animal swimming in the water tank.
  • Abundant extra maze cues are provided by the furniture in the room, e.g. desks, computer equipment.
  • the animals receive four trials during five daily acquisition sessions.
  • a trial is started by placing an animal into the pool, facing the wall of the tank. Each of four starting positions in the quadrants north, east, south, and west is used once in a series of four trials; their order is randomized.
  • the escape platform is always in the same position.
  • a trial is terminated as soon as the animal had climbs onto the escape platform or when 90 seconds have elapsed, whichever event occurs first. The animal is allowed to stay on the platform for 30 seconds. Then it is taken from the platform and the next trial is started. If an animal did not find the platform within 90 seconds it is put on the platform by the experimenter and is allowed to stay there for 30 seconds.
  • an additional trial is given as a probe trial: the platform is removed, and the time the animal spends in the four quadrants is measured for 30 or 60 seconds.
  • the probe trial all animals start from the same start position, opposite to the quadrant where the escape platform had been positioned during acquisition.
  • rats or mice with specific brain lesions which impair cognitive functions, or animals treated with compounds such as scopolamine or MK 801, which interfere with normal learning, or aged animals which suffer from cognitive deficits, are used.
  • the T maze spontaneous alternation task assesses the spatial memory performance in mice.
  • the start arm and the two goal arms of the T maze are provided with guillotine doors which can be operated manually by the experimenter.
  • a mouse is put into the start arm at the beginning of training.
  • the guillotine door is closed.
  • the 'forced trial' either the left or right goal arm is blocked by lowering the guillotine door.
  • the mouse After the mouse has been released from the start arm, it will negotiate the maze, eventually enter the open goal arm, and return to the start position, where it will be confined for 5 seconds, by lowering the guillotine door.
  • the animal can choose freely between the left and right goal arm (all guillotine doors opened) during 14 'free choice' trials. As soon as the mouse has entered one goal arm, the other one is closed. The mouse eventually returns to the start arm and is free to visit whichever go alarm it wants after having been confined to the start arm for 5 seconds. After completion of 14 free choice trials in one session, the animal is removed from the maze. During training, the animal is never handled.
  • the percent alternations out of 14 trials is calculated. This percentage and the total time needed to complete the first forced trial and the subsequent 14 free choice trials (in s) is analyzed.
  • Cognitive deficits are usually induced by an injection of scopolamine, 30 min before the start of the training session. Scopolamine reduced the per cent alternations to chance level, or below.
  • a cognition enhancer which is always administered before the training session, will at least partially, antagonize the scopolamine induced reduction in the spontaneous alternation rate. 2.
  • Neuropathic pain is induced by different variants of unilateral sciatic nerve injury mainly in rats.
  • the operation is performed under anaesthesia.
  • the first variant of sciatic nerve injury is produced by placing loosely constrictive ligatures around the common sciatic nerve.
  • the second variant is the tight ligation of about the half of the diameter of the common sciatic nerve.
  • a group of models is used in which tight ligations or transections are made of either the L5 and L6 spinal nerves, or the L% spinal nerve only.
  • the fourth variant involves an axotomy of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact whereas the last variant comprises the axotomy of only the tibial branch leaving the sural and common nerves uninjured. Control animals are treated with a sham operation.
  • the nerve injured animals develop a chronic mechanical allodynia, cold allodynioa, as well as a thermal hyperalgesia.
  • Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc. Life Science Instruments, Woodland Hills, SA, USA; Electronic von Frey System, Somedic Sales AB, Horby, Sweden).
  • Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy), or by means of a cold plate of 5 to 10°C where the nocifensive reactions of the affected hind paw are counted as a measure of pain intensity.
  • a further test for cold induced pain is the counting of nocifensive reactions, or duration of nocifensive responses after plantar administration of acetone to the affected hind limb.
  • Chronic pain in general is assessed by registering the circadanian rhythms in activity (Surjo and Arndt, Universitat zu Koln, Cologne, Germany), and by scoring differences in gait (foot print patterns; FOOTPRINTS program, Klapdor et al., 1997. A low cost method to analyze footprint patterns. J. Neurosci. Methods 75, 49 54).
  • Protease variants are tested against sham operated and vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to pain testing. 3. In vivo testing of cardiovascular effects of protease variants
  • a tracheotomy is performed, and catheters are inserted into the femoral artery for blood pressure and heart rate measurements (Gould pressure transducer and recorder, model RS 3400) and into the femoral vein for substance administration.
  • the animals are ventilated with room air and their body temperature is controlled.
  • Test protease variants are
  • Female conscious SHR (Moellegaard/Denmark, 220 - 290 g) are equipped with implantable radiotelemetry, and a data acquisition system (Data Sciences, St. Paul, MN, USA), comprising a chronically implantable transducer/transmitter unit equipped with a fluid- filled catheter is used.
  • the transmitter is implanted into the peritoneal cavity, and the sensing catheter is inserted into the descending aorta.
  • test protease variant Single administration of test protease variant is performed intravenously.
  • the animals of control groups only receive the vehicle.
  • mean blood pressure and heart rate of treated and untreated control groups are measured.
  • Example 17 Construction of the gene encoding the lOHistidine tag (SEP ID NO: 36) fused to a Neprilysin variant, its expression and purification
  • the extra-cellular domain of a variant Neprilysin containing one or more mutations that impact the specificity of the protease for one or more of its substrates, is fused to an N- terminal lOHis Tag (SEQ ID NO: 36).
  • a signal sequence -MGWSCIILFLVATATGAHS (SEQ ID NO 25) is introduced to enable secretion of the protein into the culture media during expression.
  • the complete fusion protein (excluding the signal sequence) with a human Neprilysin variant has a predicted molecular weight of approximately 81 kDa.
  • the complete gene (encoding the lOHis-Neprilysin variant ('lOHis' disclosed as SEQ ID NO: 36)) including the signal sequence is inserted into a suitable mammalian expression vector, such as pDEST12.2, pCEP4, pEAKlO, pEF5/FRT/V5-DEST and pcDNA5/FRT/TO (Gateway adapted). All these are standard mammalian expression vectors based on a CMV promoter (pDEST12.2, pCEP4, pEAKlO and pcDNA5/FRT/TO) or EF-la promoter
  • the lOHis-Neprilysin variant ECD ('lOHis' disclosed as SEQ ID NO: 36) is transiently expressed in suspension-adapted CHO cells.
  • the cell lines used in the production experiments may be cell lines derived from CHO-K1. Transfection is performed at cell density of approximately 0.5-lxlO 6 and with plasmid DNA at a concentration of 1 ⁇ g/ml cell suspension (final concentration). Expression is performed in cell culture volumes of 30 ml to 500 ml (shaker flasks), and 5L to 25L Wave Bioreactor. Cell cultures are harvested after 4 to 14 days by centrifugation.
  • Purification of the fusion protein can be performed using cell media from expression in mammalian cells.
  • the purification can be performed by immobilized metal ion adsorption chromatography (IMAC) using for example, a HisTrap HP or Ni-Sepharaose on an AKTA Chromatography system (Explorer or Purifier, GE Healthcare).
  • IMAC immobilized metal ion adsorption chromatography
  • the column is equilibrated with 10 column volumes (CV) of 2 x PBS (5.4 mM KC1, 276 mM NaCl, 3 mM KH 2 P0 4 , 16 mM Na 2 HP0 4 -7H 2 0, pH 7.4, Invitrogen).
  • Cell culture media with expressed fusion protein (lOHis-Neprilysin ECD ( * 10His * disclosed as SEQ ID NO: 36)) is applied onto the column.
  • the column is then washed with 20 CV 2xPBS and 10 CV 2xPBS with 40 mM imidazole before being eluted using an imidazole gradient from 40 to 400 mM imidazole over 10 CV.
  • Fractions containing the lOHis-Neprilysin ECD ('lOHis' disclosed as SEQ ID NO: 36) protein are pooled and concentrated and further purified using size exclusion chromatography. This can be performed using a Superdex 200 16/60 column (GE Healthcare) on an AKTA
  • the protein is eluted in lx PBS 2.7 mM KC1, 138 mM NaCl, 1.5 mM KH 2 P0 4 , 8 mM Na 2 HP0 4 -7H 2 0, pH 7.4, Invitrogen) and the fractions containing lOHisNeprilysin ('lOHis' disclosed as SEQ ID NO: 36) pooled, frozen and stored at -80C.
  • Example 18 Construction of the gene encoding the fusion protein HSA-Neprilysin variant, its expression and purification
  • the extra-cellular domain of a variant neprilysin containing one or more mutations that impact the specificity of the protease for one or more of its substrates is fused to the human serum albumin (HSA) with or without its propeptide (+ / - pro-HAS).
  • HSA human serum albumin
  • a signal (leader) sequence such as the endogenous HSA leader, MGWSCIILFLVATATGAHS (SEQ ID NO 25) or MGWSCIILFLVATATGVHS (SEQ ID NO: 65) .
  • the complete fusion protein (excluding the signal sequence) with a human neprilysin variant has predicted molecular weight of approximately 147 kDa.
  • the complete gene (encoding the HSA-neprilysin variant ECD) including the signal sequence is inserted into a suitable mammalian expression vector, such as pDEST12.2, pCEP4, pEAKlO, pEE12.4, pEF5/FRT7V5-DEST and pcDNA5/FRT/TO (Gateway adapted). All these are standard mammalian expression vectors based on a CMV promoter
  • pEU23.3 (figure 14; SEQ ID NO: 66) with a signal sequence of MGDNDIHFAFLSTGAHS (alternative HSA leader as shown in SEQ ID NO: 65) was used to express proHSA-G4S-Neprilysin G399VG714K in suspension-adapted CHO cells derived from CHO-K1.
  • the protein NEP extra-cellular domain only
  • HSA-NEP extracellular domain
  • pro-HSA-NEP extracellular domain
  • the cell lines used in the production experiments may be cell lines derived from CHO- Kl . Transfection is performed at cell density of approximately 0.5-lxlO 6 and with plasmid DNA at a concentration of 1 ⁇ / ⁇ 1 cell suspension (final concentration). Expression is performed in cell culture volumes of 30 ml to 500 ml (shaker flasks), and 5L to 25L Wave Bioreactor. Cell cultures are harvested after 4 to 14 days by centrifugation.
  • Stable CHO cell lines expressing HSA-NEP can be created.
  • Cells are transfected with vector DNA encoding HSA-NEP along with a selectable marker gene such as the glutamine synthetase gene (Bebbington, 1991 : Methods Enzymol 2: 136-145) as shown for pEE12.4 (Lonza Biologies) in Figurel5.
  • Transfectants expressing the marker gene are identified using selective cell culture conditions, such as glutamine-free medium containing methionine sulphoximine for the glutamine synthetase marker gene.
  • Transfectant cell lines are then screened for expression of HSA-NEP and can be scaled up for production of HSA-NEP.
  • Purification of the fusion protein can be performed using cell media from expression in mammalian cells.
  • the purification can be performed by affinity chromatography using an anti-HSA Affibody column.
  • the Affibody is coupled to Sulfolink resin (Pierce) via its free cysteine and is equilibrated with 10 column volumes (CV) of Buffer A (50 mM Tris, 250 mM NaCl, pH 8).
  • Buffer A 50 mM Tris, 250 mM NaCl, pH 8.
  • Cell culture media with expressed fusion protein (HSA-neprilysin variant / pro- HSA - neprilysin variant) is applied onto the resin.
  • the column is washed with Buffer A before bound protein is eluted with Buffer B (100 mM Glycine, pH 2.7).
  • Purified fractions are immediately neutralized by adding 1 ml of 1 M Tris, pH 8.5 to 10 ml of eluted protein. Purified fractions are pooled, concentrated and further purified using size exclusion chromatography. This can be performed using a Superdex 200 16/60 column (GE Healthcare) on an AKTA Chromatography system (Explorer or Purifier, GE Healthcare). The protein is eluted in lx PBS 2.7 mM KC1, 138 mM NaCl, 1.5 mM KH 2 P0 4 , 8 mM Na 2 HP0 4 -7H 2 0, pH 7.4, Invitrogen) and the fractions containing HSA-Neprilysin pooled, frozen and stored at - 80C.
  • the purification can be performed using the affinity adsorbent, mimetic blue SA (Prometic).
  • the adsorbent is equilibrated with 5 column volumes (CV) of Buffer A (50mM Na 2 HP0 4 .12H 2 0, 17mM NaH 2 P0 4 .2H 2 0, 76mM NaCl pH7.2).
  • Cell culture media with expressed fusion protein (HSA-Neprilysin) is loaded onto the adsorbent and then washed with 5CV of Buffer A and bound product subsequently eluted in 5CV of Buffer B (50mM Na 2 HP0 4 .12H 2 0, 17mM NaH 2 P0 4 .2H 2 0, 76mM NaCl, C 8 Hi 5 Na0 2 pH7.2).
  • Buffer B 50mM Na 2 HP0 4 .12H 2 0, 17mM NaH 2 P0 4 .2H 2 0, 76mM NaCl, C 8 Hi 5 Na0 2 pH7.2.
  • Buffer B 50mM Na 2 HP0 4 .12H 2 0, 17mM NaH 2 P0 4 .2H 2 0, 76mM NaCl, C 8 Hi 5 Na0 2 pH7.2.
  • Buffer B 50mM Na 2 HP0 4 .12H 2 0, 17mM NaH 2 P0 4 .2H 2 0, 76mM NaCl, C 8 Hi 5
  • Partially purified fusion protein (HSA-Neprilysin) is first diluted by a ratio of 1 :6 with Buffer C, loaded onto the adsorbent and then washed with 5CV of Buffer C. Bound product, to the Poros 50HS adsorbent, is subsequently eluted in 5CV of Buffer D (50mM Tris-HCl, lOOmM NaCl pH7.5). Post chromatography, the purified product is formulated into Buffer A using tangential flow filtration (30KDa MWCO, Millipore Biomax or equivalent) and stored at -80°C.
  • Buffer D 50mM Tris-HCl, lOOmM NaCl pH7.5
  • the assay was performed in a 96-well micro titre plate and contained 50 mM HEPES (pH 7.4, Sigma, # H3375), 150 mM NaCl, 0.05% (w/v) BSA (Sigma, # A9576), 1-200 ⁇ peptide substrate and 1-500 nM protease variant.
  • Assays with endothelin la, endothelin lb and ANP contained 2 mM tris(2-carboxyethyl)phosphine (Sigma, # C4706) in addition.
  • Table 12 shows k cat /K M values for cleavage of a panel of peptides by wild type Nep, Nep-G399V/G714K, HSA-NepG399V/G714K with the propeptide (pro-HSA-Nep- G399V/G714K) and HSA-Nep-G399V/G714K without the propeptide (Mature HSA-Nep- G399V/G714K).
  • G399V/G714K mutant compared to wild type Nep.
  • a similar increase in k cat /K M on ⁇ 1-40 was observed with pro-HSA-Nep-G399V/G714K.
  • the k cat /K M for ⁇ 1-40 cleavage observed with the mature HSA-Nep-G399V/G714K was 6.2-fold higher than the value for wild type Nep.
  • k cat /K M values for cleavage of bradykinin, neurotensin, somatostatin 1-28, angiotensin and ANP were reduced by factors of 3200, 330, 140, 71 and 11, respectively in the G399V/G714K mutant.
  • k cat /K M values for cleavage of bradykinin, neurotensin, angiotensin, somatostatin 1-28 and glucagon by mature HSA-Nep-G399V/G714K were reduced by factors of 2900, 190, 77, 39 and 1.9. Overall, the presence or absence of the propeptide in HSA-Nep-G399V/G714K had very little effect on the peptide cleavage activity of the protein.
  • the k cat /K M values are averages of data from at least two independent experiments.
  • the k cat /K M represents the average of values determined in duplicate for the la and lb iso forms.

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Abstract

La présente invention concerne des polypeptides comprenant des variants de protéase de la néprilysine humaine de type sauvage ayant une spécificité et/ou une activité altérée(s). En particulier, cette invention concerne des polypeptides comprenant un fragment d'allongement de la demi-vie et un variant de protéase dérivé de la néprilysine humaine ayant une spécificité et/ou une activité accrue(s) vis-à-vis de certains substrats, en particulier, vis-à-vis de l'amyloïde bêta.
PCT/EP2011/060385 2010-06-21 2011-06-21 Variants de protéase Ceased WO2011161127A1 (fr)

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EPPCT/EP2010/070615 2010-12-22
PCT/EP2010/070615 WO2011160732A1 (fr) 2010-06-21 2010-12-22 Variantes de la néprilysine humaine de type protéase

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US8748380B2 (en) 2009-10-30 2014-06-10 Novozymes Biopharma Dk A/S Albumin variants
WO2014096803A1 (fr) * 2012-12-21 2014-06-26 Immunocore Limited Méthode permettant de prédire la liaison non ciblée d'un peptide qui se lie à un peptide cible présenté par un complexe majeur d'histocompatibilité
US8822417B2 (en) 2011-05-05 2014-09-02 Novozymes Biopharma DIC A/S Albumin variants
US9944691B2 (en) 2012-03-16 2018-04-17 Albumedix A/S Albumin variants
US10233228B2 (en) 2010-04-09 2019-03-19 Albumedix Ltd Albumin derivatives and variants
US10501524B2 (en) 2012-11-08 2019-12-10 Albumedix Ltd Albumin variants
US10633428B2 (en) 2015-08-20 2020-04-28 Albumedix Ltd Albumin variants and conjugates
US10711050B2 (en) 2011-11-18 2020-07-14 Albumedix Ltd Variant serum albumin with improved half-life and other properties
US11555061B2 (en) 2009-02-11 2023-01-17 Albumedix, Ltd Albumin variants and conjugates

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