Nothing Special   »   [go: up one dir, main page]

WO2011160732A1 - Protease variants of human neprilysin - Google Patents

Protease variants of human neprilysin Download PDF

Info

Publication number
WO2011160732A1
WO2011160732A1 PCT/EP2010/070615 EP2010070615W WO2011160732A1 WO 2011160732 A1 WO2011160732 A1 WO 2011160732A1 EP 2010070615 W EP2010070615 W EP 2010070615W WO 2011160732 A1 WO2011160732 A1 WO 2011160732A1
Authority
WO
WIPO (PCT)
Prior art keywords
neprilysin
polypeptide
peptide
seq
hsa
Prior art date
Application number
PCT/EP2010/070615
Other languages
French (fr)
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
Original Assignee
Medimmune, Llc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from PCT/US2010/039392 external-priority patent/WO2010148413A2/en
Application filed by Medimmune, Llc. filed Critical Medimmune, Llc.
Priority to PCT/EP2011/060385 priority Critical patent/WO2011161127A1/en
Publication of WO2011160732A1 publication Critical patent/WO2011160732A1/en

Links

Classifications

    • 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

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 toxico logical 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.
  • Evidence suggests that down-regulation of neprilysin at the early stages of AD development, accompanied with aging, genetic deficiency (knockout), or treatment with neprilysin inhibitors, results in increasing accumulation of ⁇ peptide in the brain leading to memory impairment.
  • overexpression of neprilysin leads to a reduction of plaque accumulation in the brain of transgenic AD mice.
  • 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 ⁇ 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 or 749 residues due to the removal of the initial methionine (SEQ ID NO:l).
  • the 749 aa 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. 45, 87-146, 1993; Lu B.
  • the proteolytic domain (extracellular catalytic domain, ECD) comprises aa 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 metallo proteinases 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 might be considered as part of the active site of the protease and 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 is a human serum albumin, or variant or fragment thereof, provided N-terminal to the variant human neprilysin extracellular domain or fragment thereof.
  • 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.
  • the half-life modulator moiety and neprilysin protease variant may, optionally, be joined by a linker peptide.
  • linker polypeptides are (Gly) 5 Ser (SEQ ID NO: 32) or (Gly) 4 Ser (SEQ ID NO: 31) linkers, with (Gly) 4 S (SEQ ID NO: 31) being particularly preferred.
  • a polypeptide according to the invention may comprise a pro-HSA amino acid sequence positioned N-terminally to the protease variant.
  • a polypeptide to the invention may comprise a leader amino acid sequence positioned N- terminally to the protease variant.
  • a polypeptide according to any the invention may comprise a leader sequence amino acid positioned N-terminally to a pro-HSA sequence.
  • Suitable leader amino acid sequences include a HSA leader 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-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 a nucleic acid encoding a polypeptide of the invention. Additionally, the invention provides a vector comprising a nucleic acid of the invention. Further provided by the invention is a host cell comprising a vector of the invention.
  • the invention also provides a method for producing a polypeptide according to the invention, wherein the method comprises the following steps:
  • compositions 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. Detailed description of key sequences
  • SEQ ID NO: l 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-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-G4S- NEPG399V/G714K Protein
  • IgG leader 20-25 HSA propep
  • 26-611 HSA
  • 612-616 G4S
  • 617-1315 NEPG399V-G714K
  • NFRIIGTLQNSAEFSEAFHCRK SYMNPEK CRVW SEQ ID NO: 55 shows the sequence for HSA-propeptide - HSA-G4S-NEPG399V/G714K DNA
  • SEQ ID NO: 56 shows the sequence for HSA-propeptide - HSA-G4S-NEPG399V/G714K Protein
  • SEQ ID NO: 57 shows the sequence for HSA-G4S-NEPG399V/G714K DNA
  • SEQ ID NO: 59 shows the sequence for IgG leader- 10HisNEPG399V/G714K DNA
  • SEQ ID NO: 61 shows the sequence for 10HisNEPG399V/G714K DNA
  • SEQ ID NO: 62 shows the sequence for 10HisNEPG399V/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
  • 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
  • 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)).
  • mice C57BL/6 mice after 1 hour incubation at RT°C using 1 uM to 0.1 nM of enzyme.
  • Figure 8 Abeta degradation of human Abeta 1-42 in plasma from TG2576 mice after 1 hour incubation at RT°C using 1 uM to 1 nM of enzyme.
  • 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.
  • 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). It 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 (ID17274, ID17231, ID17850).
  • the term comprises also ⁇ with substitutions of residues such Glu22 for Gin (references in Soto, C. and Castano, M., (1996) Biochem. J. 314:701-707) and oligomeric forms and aggregates.
  • polynucleotide corresponds to any genetic material of any length and any sequence, comprising single-stranded and double-stranded DNA and R A 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, 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 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 hydrolyze 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-trans lational or chemically-modified forms of these molecules that contain a peptide bond that can be hydrolyzed catalytically by a protease.
  • the peptide bond that is hydrolyzed 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.
  • pharmaceutically 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).
  • a medical parameter e.g., blood pressure, blood cell count, cholesterol level
  • 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 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 R A, 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.
  • a 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.
  • 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.
  • the 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. In a more preferred embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Even more preferably, 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 (SEQ ID NO: 34), (Gly) 5 (SEQ ID NO: 35)), poly(Gly-Ala), and polyalanines.
  • 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).
  • isolated 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 baculo virus 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 combination of these techniques could be used.
  • 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, polygly colic acid, etc.
  • buffer content e.g., Tris-HCl, acetate, phosphate
  • pH and ionic strength e
  • 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 aa51-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
  • 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.
  • SEQ ID NO: 7 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)
  • 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).
  • 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.
  • Peptide-6 Dy505 a) GCS S S SLMDKES VYFCHLDII WK(SEQ ID Endothelin
  • 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 ANP
  • peptide 6a one of the endothelin peptides
  • peptide 1 ABi_ 40
  • 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 PD 10 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 PD 10 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 ⁇ peptidel- 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 ⁇
  • Tg2576 mice In vivo studies in APP S wE-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 After 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. 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.
  • 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 pH 7.4) are incubated with a pool of plasma in presence or absence of 10 ⁇
  • Example 11 Degradation of amyloid ⁇ peptides in Sprague Dawley rats by neprilysin or neprilysin variants (in vivo studies).
  • 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.
  • Plasma samples are withdrawn from anaesthetized rats 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. 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. After sampling, 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). 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.
  • DEA diethylamine
  • 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 pH 7.4) are incubated with a pool of plasma in presence or absence of 10 ⁇
  • Biosource/Invitrogen ( ⁇ 1-40).
  • 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.
  • DEA diethylamine
  • 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 .
  • 25-31 weeks old female APP S wE-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.
  • Plasma 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 APPswF-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 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 that 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.
  • 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 are 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 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.
  • 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 Anesthesio meter, 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.
  • application routes i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal
  • 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 (SEQ 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 ('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 ('lOHis' 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 ('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,
  • Example 18 Construction of the gene encoding the fusion protein HSA-Neprilysin variant, its expression and purification A. Construction of HSA-neprilysin variant expression system
  • 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.
  • HSA human serum albumin
  • 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 predicted molecular weight of approximately 147 kDa.
  • the complete gene (encoding the HSA-neprilysin variant) including the signal sequence is inserted into a suitable mammalian expression vector, such as pDEST12.2, pCEP4, pEAKlO, pEF5/FRT7V5-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-l promotor (pEF5/FRT/V5-DEST). After all cloning steps, it is advisable to sequence the genes to verify that the correct sequence exists in the vector.
  • a suitable mammalian expression vector such as pDEST12.2, pCEP4, pEAKlO, pEF5/FRT7V5-DEST and pcDNA5/FRT/TO (Gateway adapted). All these are standard mammalian
  • the protein NEP (extra-cellular domain only) and HSA-NEP are 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 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).
  • Cell culture media with expressed fusion protein (HSA-Neprilysin) 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,
  • 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,
  • the assay was performed in a 96-well microtitre 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 wild type neprilysin, the G399V/G714K mutant and the fusion of the G399V/G714K mutant with HSA.
  • the ratios of the G399V/G714K variant and HSA fusion of the mutant k cat /K M values to those of wild type neprilysin are shown in Table 13.
  • Catalytic efficiency on ⁇ 1-40 was increased by a factor of 4.5 in the
  • G399V/G714K mutant compared to wild type neprilysin.
  • 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. Similar reductions in catalytic efficiency on these substrates were observed with the HSA fusion of the mutant.
  • k cat /K M values for cleavage of endothelin- 1, GIP, and glucagon were reduced by 2-4-fold in the G399V/G714K mutant compared to wild type neprilysin. Similar reductions in catalytic efficiency on these substrates were observed with the HSA fusion of the mutant. Table 12 - kcat M values for peptide cleavage by neprilysin variants.
  • 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 1 and lb iso forms.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Psychiatry (AREA)
  • Hospice & Palliative Care (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

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

Description

PROTEASE VARIANTS OF HUMAN NEPRILYSIN
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 (Αβ) 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
Alzheimer's disease. Background of the Invention
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. However, for use as a drug, 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.
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 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.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by a loss of neurons in discrete regions of the brain, particularly in the cortex and
hippocampus. 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 (Αβ) peptides in the form of senile plaques and intracellular accumulation of neurofibrillary tangles made of a hyperphosphorylated form of the microtubule-associated protein tau. Both familial and sporadic cases share the deposition in brain of extracellular, fibrillary β-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. 37, 287- 288, 1995; Selkoe, D. J., Nature 399, A23-A31, 1999; Borchelt D. R. et al, Neuron 17, 1005-1013, 1996). β-amyloid deposits are composed of several species of amyloid-β peptides (Αβ); especially Αβι_42, which is deposited progressively in amyloid plaques. Genetic evidence suggests that increased amounts of Αβι_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. 2, 864-870, 1996; Citron M. et al, Neurobiol. Dis. 5, 107-116, 1998), suggesting that amyloid formation may be caused either by increased generation of Αβι_42, or decreased degradation, or both (Glabe, C, Nat. Med. 6, 133-134, 2000).
Currently, there is no cure for AD. However, Αβ has become a major target for the development of drugs, both with the aim to reduce formation (Vassar, R. et al, Science 286, 735-41, 1999), and to activate mechanisms that accelerate its clearance from brain. Although considerable effort has focused on reducing the generation of Αβ, considerably less emphasis has been placed on the clearance of these peptides.
Bard et al (Nature Medicine, Vol. 6, Number 8, 916-919, 2000) report that peripheral administration of antibodies against Αβ is sufficient to reduce amyloid burden. The passively administered antibodies were able to cross the blood-brain barrier and enter the central nervous system, bind to ("decorate") plaques and induce clearance of pre-existing amyloid. However, even a passive immunisation against Αβ may cause undesirable side effects in human patients.
DeMattos (PNAS 98: 8850-8855, 2001) have described the sink hypothesis, which states that Αβ peptides can be removed from CNS indirectly by lowering the concentration of the peptides in the plasma. De Mattos used an antibody that binds Αβ in the plasma. By preventing influx of Αβ from the plasma to CNS and/or changing the equilibrium between the plasma and CNS (due to a lowering of the free Αβ concentration in plasma) Αβ is sequestered from the CNS. Two other Αβ binding agents, gelsolin and GM1, unrelated to antibodies, have also been shown through binding in plasma to be effective in removing Αβ from CNS and reducing or preventing brain amyloidosis (Matsuoka et al. (J. Neuroscience 23: 29-33, 2003).
An alternative approach to remove Αβ is to use an enzyme that degrades Αβ into smaller fragments that have lower toxico logical effects and are more readily cleared. It is postulated that this enzymatic digestion of the Αβ will also work through the sink hypothesis mechanism by lowering the free concentration of Αβ in plasma. However, this approach also provides a possibility of direct clearance of Αβ in the CNS and/or CSF. This approach will not only lower the free concentration of Αβ 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 Αβ in the plasma as has been seen in cases when using Αβ peptide binding agents such as antibodies. Neprilysin is an enzyme described in the literature that degrades the Αβ -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 Αβ peptide in the brain. Evidence suggests that down-regulation of neprilysin at the early stages of AD development, accompanied with aging, genetic deficiency (knockout), or treatment with neprilysin inhibitors, results in increasing accumulation of Αβ peptide in the brain leading to memory impairment. Conversely, overexpression of neprilysin, leads to a reduction of plaque accumulation in the brain of transgenic AD mice.
Several other proteases have been described that degrade Αβ peptide including insulin degrading enzyme, plasmin, ACE and others.
Anti-Αβ peptide antibodies have been applied to effectively reduce free Αβ levels in the blood leading to a decreased plaque deposition in the brain. However, systemic application of proteases that degrade and inactivate Αβ peptide may be an alternative; but such protease would need to be sufficiently specific Αβ 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 or 749 residues due to the removal of the initial methionine (SEQ ID NO:l). The 749 aa 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. 45, 87-146, 1993; Lu B. et al, J. Exp. Med. 181, 2271-2275, 1995; Lu B. et al., Ann. N.Y. Acad. Sci. 780, 156-163, 1996). The proteolytic domain (extracellular catalytic domain, ECD) comprises aa 51 to 749 and contains an active site containing a zinc- binding motif (HEXXH). A soluble form lacking the transmembrane and intracellular domains is known to be present in the circulation. Neprilysin is capable of degrading a number of peptidic substrates, including monomeric and (possibly) oligomeric forms of Αβ 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):
Table 1 :
Figure imgf000005_0001
Rice et al. (2004) Biochem. J. 383:45 [4] Dion et al. (1995) Biochem J. 311(2):623-7; Marie- Claire et al. (2000) Proteins, 39:365-71 [5] Vanneste et al. (1988) Biochem. J. 254:531-7 [6] Brenda database
The structure of neprilysin in complex with inhibitors has been solved (Oefner et al. (2000) J. Mol. Biol. 296:341-9; Sahli et al. (2005) Helv.Chim.Acta. 88:731; PDB entries 1Y8J, 1DMT, 1R1H). Neprilysin belongs to the M13 class of metallo proteinases 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 might be considered as part of the active site of the protease and include (Table 2):
Figure imgf000006_0001
[9] Marie-Claire et al. (2000) Proteins, 39:365-71; [10] Voisin et al. (2004) JBC 279:46172-
81; [11] Vijayaraghavan et al, (1990) Biochemistry 29: 8052-8056
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
Pharmacology", (1998), The McGraw-Hill Companies).
Angiotensin, bradykinin, endothelins and natriuretic peptides (ANP and BNP) are involved in the regulation of arterial pressure. Angiotensin II is a vasoconstrictive
octapeptide. 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) and BNP (32-amino acid) are vasodilator peptides synthesized in the heart and are primarily destroyed by neprilysin in kidney brush-border cells, liver, and lungs
(Rademaker M.T. and Richards A.M. Clinical Science. 108, 23-36, 2005). ANP and BNP produce vasodilation and decrease blood pressure. Thus, therapeutic administration of a recombinant neprilysin molecule may shorten the half-life of natriuretic peptides and thereby aggravate hypertension or chronic heart failure. Neprilysin also degrades some signalling peptides, including neuropeptide Y and neurotensin. Neuropeptide Y is a 36 amino acid polypeptide neurotransmitter distributed in the mammalian central nervous system. Known physiological functions within the CNS include the regulation of social and feeding behaviour, circadian rhythm and central cardiovascular function (Gray, W., Molecular and Cellular Endocrinology 288, 52-62, 2008). Neurotensin (NT), is a 30 amino acid peptide. In the brain, NT is expressed in neurons where it acts as a neuromodulator. The effects of centrally administered NT include the interaction of the peptide with dopaminergic (DA) systems, the ability to induce opioid-independent analgesia, inhibition of food intake, and modulation of pituitary hormone release. In the periphery, 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).
In the development of a potential therapeutic agent, because 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 Αβ, 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.
267:2138-41; Kim et al. (1992) J. Biol. Chem. 267: 12330-35; Barros et al. (2007) Biol. Chem.
388:447-455). R747 has been found to influence selectivity as well (Beaumont et al. (1991)
J. Biol. Chem. 266:214-220). Positions F563, F564, M579, F716 and 1718 have been described to influence kcat/Km for the hydrolysis of an enkephalin derivative (Marie-Claire et al (2000) Proteins 39:365-371). Positions R102 and N542 were also found to influence inhibition by small compounds (Dion et al. (1997) FEBS Lett. 411 : 140:144).
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 Αβ 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.
It is desirable to produce fusion proteins comprising a half life modulator moiety and a neprilysin variant with increased specificity for amyloid beta (Αβ) 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.
Summary of the Invention 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.
Compared to wild type neprilysin, the polypeptides of the invention have an enhanced specificity for cleavage of Αβ 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,
Neurotensin, Adrenomedullin, Bombesin, BLP, CGRP, Enkephalins, FGF-2, fMLP, GRP, Neurokinin A, Neuromedin C, Oxytocin, PAMP, Substance P and VIP with a lower specificity than wild-type human neprilysin. Such molecules, when administered as a therapeutic may have a similar or an enhanced effect at degrading Αβ 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
disadvantageous or toxic effects that might arise through degradation of these other substrates.
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 β1-40, and/or human Amyloid Bi _ 42. In the variant human neprilysin extracellular domain 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.
Preferably 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 is a human serum albumin, or variant or fragment thereof, provided N-terminal to the variant human neprilysin extracellular domain or fragment thereof. 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.
In a polypeptide of the invention, the half-life modulator moiety and neprilysin protease variant may, optionally, be joined by a linker peptide. Particularly suitable linker polypeptides are (Gly)5Ser (SEQ ID NO: 32) or (Gly)4Ser (SEQ ID NO: 31) linkers, with (Gly)4S (SEQ ID NO: 31) being particularly preferred.
A polypeptide according to the invention may comprise a pro-HSA amino acid sequence positioned N-terminally to the protease variant.
A polypeptide to the invention may comprise a leader amino acid sequence positioned N- terminally to the protease variant. A polypeptide according to any the invention may comprise a leader sequence amino acid positioned N-terminally to a pro-HSA sequence.
Suitable leader amino acid sequences include a HSA leader amino acid sequence or an IgG leader amino acid sequence.
The present invention provides a polypeptide comprising NHSA C34S, a GGGGS (SEQ ID NO: 31) linker and a G399V / G714K variant human neprilysin extracellular domain0 , such as is shown in SEQ ID NO: 28. The present invention provides a polypeptide comprising Npro-HSA, HSA C34S, a GGGGS (SEQ ID NO: 31) linker and a G399V / G714K variant human neprilysin extracellular domain0 , such as is shown herein.
A polypeptide of the invention suitably has a greater specificity for an Αβ peptide compared to wild type human neprilysin.
The invention further provides a nucleic acid encoding a polypeptide of the invention. Additionally, the invention provides a vector comprising a nucleic acid of the invention. Further provided by the invention is a host cell comprising a vector of the invention.
The invention also provides a method for producing a polypeptide according to the invention, wherein the method comprises the following steps:
a. culturing a host cell of the invention under conditions suitable for the expression of the polypeptide; and
b. recovering the polypeptide from the host cell culture.
Also encompassed within the invention is a pharmaceutical composition comprising a polypeptide of the invention and a pharmaceutically acceptable excipient. Polypeptides of the invention with enhanced specificity for Αβ peptide may be useful in the treatment of Alzheimer's disease and other diseases mediated by Αβ accumulation, due to excessive Αβ formation or decreased Αβ degradation.
Accordingly, the invention provides a method for treating a human neprilysin substrate- related disease, such as an Αβ-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 Αβ-related pathology, such as Alzheimer's disease.
The invention provides a polypeptide for use to prevent and/or treat an Αβ-related pathology such as Alzheimer's disease. Detailed description of key sequences
SEQ ID NO: l 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
(Wt-sNeprilysin;), i.e., the extracellular catalytic domain.
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
(Wt-sNeprilysin) .
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 (TAC) occurs at positions 88-90.
SEQ ID NO: 6 shows the nucleotide-sequence of full-length wild type human
Neprilysin without the codon triplet for initial methionine.
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:
YDDGICKSSDCIKSAARLIQNMDATTEPCTDFFKYACGGWLKRNVIPETSSRYGNFDI
LRDELE V VLKD VLQEPKTEDI V AVQKAKAL YRS CINE S AID SRGGEPLLKLLPDI YG W
PVATENWEQKYGASWTAEKAIAQLNSKYGK VLINLFVGTDDKNSVNHVIHIDQPR
LGLPSRDYYECTGIYKEACTAYVDFMISVARLIRQEERLPIDENQLALEMNKVMELEK EIANATAKPEDRNDPMLLYNKMTLAQIQNNFSLEINGKPFSWLNFTNEIMSTVNISITN
EEDVVVYAPEYLTKLKPILTKYSARDLQNLMSWRFIMDLVSSLSRTYKESRNAFRKA
LYVTTSETATWRRCANYVNGNMMNAVGRLYVEAAFAGESKHVVEDLIAQIREVFIQ
TLDDLTWMDAETKKRAEEKALAIKERIGYPDDIVSNDNKLNNEYLELNYKEDEYFEN IIQNLKF S Q SKQLKKLREKVDKDE WIS G AA V VN AF YS S GRNQI VFP AGILQPPFF S AQQ SNSLNYGGIGMVIGHEITHGFFDNGRNPNKDDDLVDWWTQQSASNFKEQSQCMVYQ YGNFSWDLAGGQHLNGINTLGENIADNGGLGQAYRAYQNYIKK GEEKLLPGLDLN HKQLFFLNFAQVWCGTYRPEYAVNSIKTDVHSPK FRIIGTLQNSAEFSEAFHCRK S YMNPEK CRVW
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.
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADE SAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLP RLVRPEVDVMCTAFHDNEETFLK YLYEIARRHPYFYAPELLFFAKRYKAAFTECCQ AADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKA EFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLL EKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPD YSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQ LGEYKFQNALLVRYTK VPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYL SVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHA DICTLSEKERQIK QTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETC FAEEGKKLVAASQAALGLGGGGSYDDGICKSSDCIKSAARLIQNMDATTEPCTDFFK YACGGWLKRNVIPETSSRYGNFDILRDELEVVLKDVLQEPKTEDIVAVQKAKALYRS CINESAIDSRGGEPLLKLLPDIYGWPVATENWEQKYGASWTAEKAIAQLNSKYGK V LINLFVGTDDKNSVNHVIHIDQPRLGLPSRDYYECTGIYKEACTAYVDFMISVARLIR QEERLPIDENQLALEMNKVMELEKEIANATAKPEDRNDPMLLYNKMTLAQIQNNFSL EINGKPFSWLNFTNEIMSTVNISITNEEDVVVYAPEYLTKLKPILTKYSARDLQNLMS WRFIMDLVSSLSRTYKESRNAFRKALYVTTSETATWRRCANYVNGNMMNAVGRLY VEAAFAGESKHVVEDLIAQIREVFIQTLDDLTWMDAETK RAEEKALAIKERIGYPD DI VSNDNKLNNE YLELN YKEDE YFENIIQNLKF S Q SKQLKKLREKVDKDE WI SGAAV VNAFYSSGRNQIVFPAGILQPPFFSAQQSNSLNYGGIGMVIGHEITHGFFDNGRNPNKD DDLVDWWTQQSASNFKEQSQCMVYQYGNFSWDLAGGQHLNGINTLGENIADNGGL GQAYRAYQNYIKKNGEEKLLPGLDLNHKQLFFLNFAQVWCGTYRPEYAVNSIKTDV HSPKNFRIIGTLQNSAEFSEAFHCRKNSYMNPEKKCRVW SEQ ID NO: 30 shows the sequence for the human serum albumin variant HSA C34S:
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADE SAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLP RLVRPEVDVMCTAFHDNEETFLK YLYEIARRHPYFYAPELLFFAKRYKAAFTECCQ AADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKA EFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLL EKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPD YSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQ LGEYKFQNALLVRYTK VPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYL SVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHA DICTLSEKERQIK QTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETC FAEEGKKLVAASQAALGL SEQ ID NO: 51 shows the DNA sequence for HSA leader-HSA-propeptide - HSA-G4S- NEPG399V/G714K DNA
1-54 = HSA leader 55-72 =HSA propep, 73-1827 = HSA , 1828-1842=G4S, 1843-3945 = NEPG399V-G714K
>HSA leader HSA propeptide HSA-G4S-NEP G399V-G714K DNA
ATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTATTCCAGG GGTGTGTTTCGTCGAGATGCCCA
CAAGTCCGAGGTGGCCCACCGGTTCAAGGACCTGGGCGAGGAAAACTTCAAGGC CCTGGTGCTGATCGCCTTCGCCCAGT
ACCTGCAGCAGAGCCCCTTCGAGGACCATGTGAAGCTGGTGAACGAAGTGACCG AGTTCGCCAAGACCTGCGTGGCCGAC
GAGTCCGCCGAGAACTGCGACAAGTCCCTGCACACCCTGTTCGGCGACAAGCTGT GTACCGTGGCCACCCTGCGGGAAAC
CTACGGCGAGATGGCCGACTGCTGCGCCAAGCAGGAACCCGAGCGGAACGAGTG CTTCCTGCAGCACAAGGACGACAACC CCAACCTGCCCCGGCTGGTGCGACCTGAGGTGGACGTGATGTGTACCGCCTTCCA CGACAACGAGGAAACCTTCCTGAAG
AAGTACCTGTACGAGATCGCCAGACGGCACCCCTACTTCTACGCCCCCGAGCTGC TGTTCTTCGCCAAGCGGTACAAGGC CGCCTTCACCGAGTGCTGCCAGGCCGCCGATAAGGCCGCCTGCCTGCTGCCTAAG CTGGACGAGCTGCGGGACGAGGGCA
AGGCCTCCTCCGCCAAGCAGAGACTGAAGTGCGCCTCCCTGCAGAAGTTCGGCGA GCGGGCCTTTAAGGCCTGGGCCGTG GCCCGGCTGTCCCAGAGATTCCCCAAGGCCGAGTTTGCCGAGGTGTCCAAGCTGG TGACAGACCTGACCAAAGTGCATAC
AGAGTGTTGCCACGGCGACCTGCTGGAATGCGCCGACGACAGAGCCGACCTGGC CAAGTACATCTGCGAGAACCAGGACT
CCATCTCCTCCAAGCTGAAAGAGTGCTGCGAGAAGCCCCTGCTGGAAAAGTCCCA CTGTATCGCCGAGGTGGAAAACGAC
GAGATGCCCGCCGACCTGCCTTCTCTGGCCGCCGACTTCGTGGAATCCAAGGACG TGTGCAAGAACTACGCCGAGGCCAA
GGATGTGTTCCTGGGCATGTTCCTGTACGAGTACGCCCGCAGACACCCCGACTAC TCCGTGGTGCTGCTGCTGCGGCTGG CC AAGACCTACGAGACAACCCTGGAAAAGTGCTGCGCCGCTGCCGACCCCC ACG AGTGTTACGCCAAGGTGTTCGACGAG
TTCAAGCCTCTGGTGGAAGAACCCCAGAACCTGATCAAGCAGAACTGCGAGCTGT TCGAGCAGCTGGGCGAGTACAAGTT
CCAGAACGCCCTGCTGGTGCGATACACCAAGAAAGTGCCCCAGGTGTCCACCCCC ACCCTGGTGGAAGTGTCCCGGAACC
TGGGCAAAGTGGGCTCCAAGTGCTGCAAGCACCCTGAGGCCAAGCGGATGCCCT GCGCCGAGGACTACCTGAGCGTGGTG
CTGAACCAGCTGTGCGTGCTGCACGAAAAGACCCCCGTGTCCGACAGAGTGACC AAGTGCTGTACCGAGTCCCTGGTGAA CAGACGGCCCTGCTTCTCCGCCCTGGAAGTGGACGAGACATACGTGCCCAAAGA GTTCAACGCCGAGACATTCACCTTCC
ACGCCGACATCTGCACCCTGTCCGAGAAAGAGCGGCAGATCAAGAAACAGACCG CACTGGTGGAACTGGTGAAACACAAG
CCCAAGGCCACCAAAGAACAGCTGAAGGCCGTGATGGACGACTTCGCCGCCTTT GTGGAAAAGTGTTGCAAGGCCGACGA
CAAAGAGACATGCTTCGCCGAAGAGGGCAAGAAACTGGTGGCCGCTTCCCAGGC TGCTCTGGGACTGGGAGGCGGCGGAT CCTACGACGACGGCATCTGCAAGTCCTCCGACTGCATCAAGTCCGCCGCCAGACT GATCCAGAACATGGACGCCACCACC
GAGCCCTGCACCGATTTCTTTAAGTACGCCTGCGGCGGCTGGCTGAAGCGGAACG TGATCCCCGAGACATCCTCCAGATA CGGCAACTTCGACATCCTGAGGGACGAGCTGGAAGTGGTGCTGAAGGACGTGCT GCAGGAACCCAAGACCGAGGACATCG
TGGCCGTGCAGAAGGCCAAGGCCCTGTACCGGTCCTGCATCAACGAGAGCGCCA TCGACTCCAGAGGCGGCGAGCCTCTG
CTGAAGCTGCTGCCCGACATCTACGGCTGGCCTGTGGCCACCGAGAACTGGGAGC AGAAGTACGGCGCCTCCTGGACCGC
CGAGAAGGCTATCGCCCAGCTGAACTCTAAGTACGGCAAGAAGGTGCTGATCAA CCTGTTCGTGGGCACCGACGACAAGA
ACTCCGTGAACCATGTGATCCACATCGACCAGCCTCGGCTGGGCCTGCCTTCCCG GGACTACTACGAGTGTACCGGCATC TACAAAGAGGCCTGCACCGCCT ACGTGGACTTCATGATCTCCGTGGCCAGGCTGA TCCGGCAGGAAGAGAGACTGCCCAT
CGACGAGAACCAGCTGGCCCTGGAAATGAACAAAGTGATGGAACTGGAAAAAGA GATCGCCAACGCTACCGCCAAGCCCG
AGGACCGGAACGACCCCATGCTGCTGTACAACAAGATGACCCTGGCCCAGATTC AGAACAACTTCTCCCTGGAAATCAAC
GGCAAGCCCTTCTCCTGGCTGAACTTCACCAACGAGATCATGTCCACCGTGAACA TCTCCATCACCAACGAAGAGGACGT
GGTGGTGTACGCCCCTGAGTACCTGACCAAGCTGAAGCCCATCCTGACCAAGTAC TCCGCCAGGGACCTGCAGAATCTGA TGTCCTGGCGGTTCATCATGGACCTGGTGTCCTCCCTGTCCCGGACCTATAAAGA GTCCCGGAACGCCTTTCGGAAGGCT
CTGTACGTGACCACCTCCGAGACAGCCACCTGGCGGAGATGCGCCAACTACGTGA ACGGCAACATGGAAAACGCCGTGGG
CAGACTGTACGTGGAAGCCGCCTTCGCCGGCGAGTCCAAACATGTGGTGGAAGA TCTGATCGCCCAGATCAGAGAGGTGT
TCATCCAGACCCTGGACGACCTGACCTGGATGGACGCCGAGACTAAGAAGCGGG CCGAGGAAAAGGCCCTGGCCATCAAA GAGCGGATCGGCTACCCCGACGACATCGTGTCCAACGACAACAAGCTGAACAAC GAGTACCTGGAACTGAATTACAAAGA
GGACGAGTACTTCGAGAACATCATCCAGAATCTGAAGTTCTCCCAGTCCAAGCAG CTGAAGAAACTGCGCGAGAAGGTGG ACAAGGACGAGTGGATCTCCGGCGCTGCCGTGGTGAACGCCTTCTACTCCTCCGG CCGGAACCAGATCGTGTTCCCTGCC
GGAATCCTGCAGCCCCCATTCTTCAGCGCCCAGCAGTCCAACTCCCTGAACTACG GCGGCATCGGCATGGTGATCGGCCA
CGAGATCACCCACGGCTTCGACGACAACGGCCGGAACTTCAACAAGGACGGCGA TCTGGTGGATTGGTGGACCCAGCAGA
GCGCCTCCAACTTCAAAGAACAGTCCCAGTGCATGGTGTACCAGTACGGCAATTT CTCCTGGGACCTGGCTGGCGGACAG
CACCTGAACGGCATCAACACCCTGGGCGAGAATATCGCCGACAACGGCGGACTG GGCCAGGCTTACAGAGCCTACCAGAA CTACATCAAGAAGAACGGCGAAGAGAAACTGCTGCCCGGCCTGGACCTGAACCA CAAGCAGCTGTTCTTTCTGAACTTCG
CCCAGGTCTGGTGCGGCACCTACCGGCCTGAGTACGCCGTGAACTCCATCAAGAC CGATGTGCATTCCCCCAAGAACTTC
CGGATCATCGGCACCCTGCAGAACTCCGCCGAGTTCTCCGAGGCCTTCCACTGCC GGAAGAACTCCTACATGAACCCCGA GAAGAAATGCCGCGTGTGGTGATAA
SEQ ID NO: 52 shows the sequence for HSA leader-HSA-propeptide - HSA-G4S- NEPG399V/G714K Protein
1-17 = HSA leader 18-23 =HSA propep, 24-609 = HSA , 610-614=G4S, 615-1313 = NEPG399V-G714K
>HSA leader HSA propeptide HSA-G4S-NEP G399V-G714K Protein
MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQ SPFEDHVKLVNEVTEFAKTCVAD
ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNL PRLVRPEVDVMCTAFHDNEETFLK
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS AKQPvLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS KLKECCEKPLLEKSHCIAEVEND
EMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKT YETTLEKCCAAADPHECYAKVFDE FKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTK VPQVSTPTLVEVSRNLGKV GSKCCKHPEAKRMPCAEDYLSVV
LNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADIC TLSEKERQIK QTALVELVKHK
PKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSY DDGICKS SDCIKS AARLIQNMD ATT
EPCTDFFKYACGGWLKRNVIPETSSRYGNFDILRDELEVVLKDVLQEPKTEDIVAVQK AKAL YRS CINE S AID SRGGEPL
LKLLPDIYGWPVATENWEQKYGASWTAEKAIAQLNSKYGK VLINLFVGTDDKNSV NHVIHIDQPRLGLPSRDYYECTGI YKE ACT AYVDFMIS VARLIRQEERLPIDENQLALEMNKVMELEKEIANATAKPEDRN DPMLLYNKMTLAQIQNNFSLEIN
GKPFSWLNFTNEIMSTVNISITNEEDVVVYAPEYLTKLKPILTKYSARDLQNLMSWRF IMDLVSSLSRTYKESRNAFRKA
LYVTTSETATWRRCANYVNGNMENAVGRLYVEAAFAGESKHVVEDLIAQIREVFIQ TLDDLTWMDAETKKRAEEKALAIK
EMGYPDDIVSNDNKLNNEYLELNYKEDEYFENIIQNLKFSQSKQLKKLPvEKVDKDE WI S G AAV VN AF YS S GRNQI VFP A
GILQPPFFSAQQSNSLNYGGIGMVIGHEITHGFDDNGRNFNKDGDLVDWWTQQSASN FKEQSQCMVYQYGNFSWDLAGGQ HLNGINTLGENIADNGGLGQAYRAYQNYIK NGEEKLLPGLDLNHKQLFFLNFAQV WCGTYRPEYAVNSIKTDVHSPKNF RIIGTLQNSAEFSEAFHCRKNSYMNPEK CRVW SEQ ID NO: 53 shows the sequence for IgG leader-HSA-propeptide - HSA-G4S-
NEPG399V/G714K DNA 1-48, 128-139 = IgG leader 140-158 =HSA propep, 159-1912 =
HSA , 1913-1927=G4S, 1928-4030 = NEPG399V-G714K
>IgG leader HSA propeptide HSA-G4S-NEP G399V-G714K DNA ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGT TAACAGTAGCAGGCTTGAGGTCTGG
ACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGCGTGCAC TCCAGGGGTGTGTTTCGTCGAGAT GCCCACAAGTCCGAGGTGGCCCACCGGTTCAAGGACCTGGGCGAGGAAAACTTC AAGGCCCTGGTGCTGATCGCCTTCGC
CCAGTACCTGCAGCAGAGCCCCTTCGAGGACCATGTGAAGCTGGTGAACGAAGT GACCGAGTTCGCCAAGACCTGCGTGG
CCGACGAGTCCGCCGAGAACTGCGACAAGTCCCTGCACACCCTGTTCGGCGACAA GCTGTGTACCGTGGCCACCCTGCGG
GAAACCTACGGCGAGATGGCCGACTGCTGCGCCAAGCAGGAACCCGAGCGGAAC GAGTGCTTCCTGCAGCACAAGGACGA
CAACCCCAACCTGCCCCGGCTGGTGCGACCTGAGGTGGACGTGATGTGTACCGCC TTCCACGACAACGAGGAAACCTTCC TGAAGAAGTACCTGTACGAGATCGCCAGACGGC ACCCCTACTTCTACGCCCCCGA GCTGCTGTTCTTCGCCAAGCGGTAC
AAGGCCGCCTTCACCGAGTGCTGCCAGGCCGCCGATAAGGCCGCCTGCCTGCTGC CTAAGCTGGACGAGCTGCGGGACGA
GGGCAAGGCCTCCTCCGCCAAGCAGAGACTGAAGTGCGCCTCCCTGCAGAAGTTC GGCGAGCGGGCCTTTAAGGCCTGGG
CCGTGGCCCGGCTGTCCCAGAGATTCCCCAAGGCCGAGTTTGCCGAGGTGTCCAA GCTGGTGACAGACCTGACCAAAGTG
CATACAGAGTGTTGCCACGGCGACCTGCTGGAATGCGCCGACGACAGAGCCGAC CTGGCCAAGTACATCTGCGAGAACCA GGACTCCATCTCCTCCAAGCTGAAAGAGTGCTGCGAGAAGCCCCTGCTGGAAAA GTCCCACTGTATCGCCGAGGTGGAAA
ACGACGAGATGCCCGCCGACCTGCCTTCTCTGGCCGCCGACTTCGTGGAATCCAA GGACGTGTGCAAGAACTACGCCGAG
GCCAAGGATGTGTTCCTGGGCATGTTCCTGTACGAGTACGCCCGCAGACACCCCG ACTACTCCGTGGTGCTGCTGCTGCG
GCTGGCCAAGACCTACGAGACAACCCTGGAAAAGTGCTGCGCCGCTGCCGACCC CCACGAGTGTTACGCCAAGGTGTTCG ACGAGTTCAAGCCTCTGGTGGAAGAACCCCAGAACCTGATCAAGCAGAACTGCG AGCTGTTCGAGCAGCTGGGCGAGTAC
AAGTTCCAGAACGCCCTGCTGGTGCGATACACCAAGAAAGTGCCCCAGGTGTCCA CCCCCACCCTGGTGGAAGTGTCCCG GAACCTGGGCAAAGTGGGCTCCAAGTGCTGCAAGCACCCTGAGGCCAAGCGGAT GCCCTGCGCCGAGGACTACCTGAGCG
TGGTGCTGAACCAGCTGTGCGTGCTGCACGAAAAGACCCCCGTGTCCGACAGAGT GACCAAGTGCTGTACCGAGTCCCTG
GTGAACAGACGGCCCTGCTTCTCCGCCCTGGAAGTGGACGAGACATACGTGCCCA AAGAGTTCAACGCCGAGACATTCAC
CTTCCACGCCGACATCTGCACCCTGTCCGAGAAAGAGCGGCAGATCAAGAAACA GACCGCACTGGTGGAACTGGTGAAAC
ACAAGCCCAAGGCCACCAAAGAACAGCTGAAGGCCGTGATGGACGACTTCGCCG CCTTTGTGGAAAAGTGTTGCAAGGCC GACGACAAAGAGACATGCTTCGCCGAAGAGGGCAAGAAACTGGTGGCCGCTTCC CAGGCTGCTCTGGGACTGGGAGGCGG
CGGATCCTACGACGACGGCATCTGCAAGTCCTCCGACTGCATCAAGTCCGCCGCC AGACTGATCCAGAACATGGACGCCA
CCACCGAGCCCTGCACCGATTTCTTTAAGTACGCCTGCGGCGGCTGGCTGAAGCG GAACGTGATCCCCGAGACATCCTCC
AGATACGGCAACTTCGACATCCTGAGGGACGAGCTGGAAGTGGTGCTGAAGGAC GTGCTGCAGGAACCCAAGACCGAGGA
CATCGTGGCCGTGCAGAAGGCCAAGGCCCTGTACCGGTCCTGCATCAACGAGAG CGCCATCGACTCCAGAGGCGGCGAGC CTCTGCTGAAGCTGCTGCCCGACATCTACGGCTGGCCTGTGGCCACCGAGAACTG GGAGCAGAAGTACGGCGCCTCCTGG
ACCGCCGAGAAGGCTATCGCCCAGCTGAACTCTAAGTACGGCAAGAAGGTGCTG ATCAACCTGTTCGTGGGCACCGACGA
CAAGAACTCCGTGAACCATGTGATCCACATCGACCAGCCTCGGCTGGGCCTGCCT TCCCGGGACTACTACGAGTGTACCG
GCATCTACAAAGAGGCCTGCACCGCCTACGTGGACTTCATGATCTCCGTGGCCAG GCTGATCCGGCAGGAAGAGAGACTG CCCATCGACGAGAACCAGCTGGCCCTGGAAATGAACAAAGTGATGGAACTGGAA AAAGAGATCGCCAACGCTACCGCCAA
GCCCGAGGACCGGAACGACCCCATGCTGCTGTACAACAAGATGACCCTGGCCCA GATTCAGAACAACTTCTCCCTGGAAA TCAACGGCAAGCCCTTCTCCTGGCTGAACTTCACCAACGAGATCATGTCCACCGT GAACATCTCCATCACCAACGAAGAG
GACGTGGTGGTGTACGCCCCTGAGTACCTGACCAAGCTGAAGCCCATCCTGACCA AGTACTCCGCCAGGGACCTGCAGAA
TCTGATGTCCTGGCGGTTCATCATGGACCTGGTGTCCTCCCTGTCCCGGACCTATA AAGAGTCCCGGAACGCCTTTCGGA
AGGCTCTGTACGTGACCACCTCCGAGACAGCCACCTGGCGGAGATGCGCCAACTA CGTGAACGGCAACATGGAAAACGCC
GTGGGCAGACTGTACGTGGAAGCCGCCTTCGCCGGCGAGTCCAAACATGTGGTG GAAGATCTGATCGCCCAGATCAGAGA GGTGTTCATCC AGACCCTGGACGACCTGACCTGGATGGACGCCGAGACTAAGAA GCGGGCCGAGGAAAAGGCCCTGGCCA
TCAAAGAGCGGATCGGCTACCCCGACGACATCGTGTCCAACGACAACAAGCTGA ACAACGAGTACCTGGAACTGAATTAC
AAAGAGGACGAGTACTTCGAGAACATCATCCAGAATCTGAAGTTCTCCCAGTCCA AGCAGCTGAAGAAACTGCGCGAGAA
GGTGGACAAGGACGAGTGGATCTCCGGCGCTGCCGTGGTGAACGCCTTCTACTCC TCCGGCCGGAACCAGATCGTGTTCC
CTGCCGGAATCCTGCAGCCCCCATTCTTCAGCGCCCAGCAGTCCAACTCCCTGAA CTACGGCGGCATCGGCATGGTGATC GGCCACGAGATCACCCACGGCTTCGACGACAACGGCCGGAACTTCAACAAGGAC GGCGATCTGGTGGATTGGTGGACCCA
GCAGAGCGCCTCCAACTTCAAAGAACAGTCCCAGTGCATGGTGTACCAGTACGGC AATTTCTCCTGGGACCTGGCTGGCG
GACAGCACCTGAACGGCATCAACACCCTGGGCGAGAATATCGCCGACAACGGCG GACTGGGCCAGGCTTACAGAGCCTAC
CAGAACTACATCAAGAAGAACGGCGAAGAGAAACTGCTGCCCGGCCTGGACCTG AACCACAAGCAGCTGTTCTTTCTGAA CTTCGCCCAGGTCTGGTGCGGCACCTACCGGCCTGAGTACGCCGTGAACTCCATC AAGACCGATGTGCATTCCCCCAAGA
ACTTCCGGATCATCGGCACCCTGCAGAACTCCGCCGAGTTCTCCGAGGCCTTCCA CTGCCGGAAGAACTCCTACATGAAC CCCGAGAAGAAATGCCGCGTGTGGTGATAA
SEQ ID NO: 54 shows the sequence for IgG leader-HSA-propeptide - HSA-G4S- NEPG399V/G714K Protein
1-19 = IgG leader 20-25 =HSA propep, 26-611 = HSA , 612-616=G4S, 617-1315 = NEPG399V-G714K
>IgG leader HSA propeptide HSA-G4S-NEP G399V-G714K Protein
MGWSCIILFLVATATGVHSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQ QSPFEDHVKLVNEVTEFAKTCV
ADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNP NLPRLVRPEVDVMCTAFHDNEETF
LK YLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKA SSAKQRLKCASLQKFGERAFKAW
AVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSI SSKLKECCEKPLLEKSHCIAEVE NDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA KTYETTLEKCCAAADPHECYAKVF
DEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTK VPQVSTPTLVEVSRNLG KVGSKCCKHPEAKRMPCAEDYLS
VVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHA DICTLSEKERQIK QTALVELVK
HKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGG S YDDGICKS SDCIKS AARLIQNMD A
TTEPCTDFFKYACGGWLKRNVIPETSSRYGNFDILRDELEVVLKDVLQEPKTEDIVAV QKAKALYRSCINESAIDSRGGE PLLKLLPDIYGWPVATENWEQKYGASWTAEKAIAQLNSKYGK VLINLFVGTDDKN SVNHVIHIDQPRLGLPSRDYYECT GIYKEACTAYVDFMISVARLIRQEERLPIDENQLALEMNKVMELEKEIANATAKPEDR NDPMLLYNKMTLAQIQNNFSLE
INGKPFSWLNFTNEIMSTVNISITNEEDVVVYAPEYLTKLKPILTKYSARDLQNLMSW RFIMDLVSSLSRTYKESRNAFR KALYVTTSETATWRRCANYVNGNMENAVGRLYVEAAFAGESKHVVEDLIAQIREVF IQTLDDLTWMDAETKKRAEEKALA
IKERIGYPDDIVSNDNKL NEYLELNYKEDEYFENIIQNLKFSQSKQLKKLREKVDKD EWISGAAVVNAFYSSGRNQIVF
PAGILQPPFFSAQQSNSLNYGGIGMVIGHEITHGFDDNGRNFNKDGDLVDWWTQQSA SNFKEQ S QCM V YQ YGNF S WDL AG
GQHLNGINTLGENIADNGGLGQAYRAYQNYIKK GEEKLLPGLDLNHKQLFFLNFA
QVWCGTYRPEYAVNSIKTDVHSPK
NFRIIGTLQNSAEFSEAFHCRK SYMNPEK CRVW SEQ ID NO: 55 shows the sequence for HSA-propeptide - HSA-G4S-NEPG399V/G714K DNA
1-18 =HSA propep, 19-1773 = HSA , 1774-1788 = G4S, 1789-3891 = NEPG399V-G714K HSA propeptide HSA-G4S-NEP G399V-G714K DNA
AGGGGTGTGTTTCGTCGAGATGCCCACAAGTCCGAGGTGGCCCACCGGTTCAAGG ACCTGGGCGAGGAAAACTTCAAGGC
CCTGGTGCTGATCGCCTTCGCCCAGTACCTGCAGCAGAGCCCCTTCGAGGACCAT GTGAAGCTGGTGAACGAAGTGACCG
AGTTCGCCAAGACCTGCGTGGCCGACGAGTCCGCCGAGAACTGCGACAAGTCCCT GCACACCCTGTTCGGCGACAAGCTG TGTACCGTGGCCACCCTGCGGGAAACCTACGGCGAGATGGCCGACTGCTGCGCCA AGCAGGAACCCGAGCGGAACGAGTG
CTTCCTGCAGCACAAGGACGACAACCCCAACCTGCCCCGGCTGGTGCGACCTGAG GTGGACGTGATGTGTACCGCCTTCC
ACGACAACGAGGAAACCTTCCTGAAGAAGTACCTGTACGAGATCGCCAGACGGC ACCCCTACTTCTACGCCCCCGAGCTG
CTGTTCTTCGCCAAGCGGTACAAGGCCGCCTTCACCGAGTGCTGCCAGGCCGCCG ATAAGGCCGCCTGCCTGCTGCCTAA GCTGGACGAGCTGCGGGACGAGGGCAAGGCCTCCTCCGCCAAGCAGAGACTGAA GTGCGCCTCCCTGCAGAAGTTCGGCG
AGCGGGCCTTTAAGGCCTGGGCCGTGGCCCGGCTGTCCCAGAGATTCCCCAAGGC CGAGTTTGCCGAGGTGTCCAAGCTG GTGACAGACCTGACCAAAGTGCATACAGAGTGTTGCCACGGCGACCTGCTGGAA TGCGCCGACGACAGAGCCGACCTGGC
CAAGTACATCTGCGAGAACCAGGACTCCATCTCCTCCAAGCTGAAAGAGTGCTGC GAGAAGCCCCTGCTGGAAAAGTCCC
ACTGTATCGCCGAGGTGGAAAACGACGAGATGCCCGCCGACCTGCCTTCTCTGGC CGCCGACTTCGTGGAATCCAAGGAC
GTGTGCAAGAACTACGCCGAGGCCAAGGATGTGTTCCTGGGCATGTTCCTGTACG AGTACGCCCGCAGACACCCCGACTA
CTCCGTGGTGCTGCTGCTGCGGCTGGCCAAGACCTACGAGACAACCCTGGAAAAG TGCTGCGCCGCTGCCGACCCCCACG AGTGTTACGCCAAGGTGTTCGACGAGTTCAAGCCTCTGGTGGAAGAACCCC AGAA CCTGATCAAGCAGAACTGCGAGCTG
TTCGAGCAGCTGGGCGAGTACAAGTTCCAGAACGCCCTGCTGGTGCGATACACCA AGAAAGTGCCCCAGGTGTCCACCCC
CACCCTGGTGGAAGTGTCCCGGAACCTGGGCAAAGTGGGCTCCAAGTGCTGCAA GCACCCTGAGGCCAAGCGGATGCCCT
GCGCCGAGGACTACCTGAGCGTGGTGCTGAACCAGCTGTGCGTGCTGCACGAAA AGACCCCCGTGTCCGACAGAGTGACC
AAGTGCTGTACCGAGTCCCTGGTGAACAGACGGCCCTGCTTCTCCGCCCTGGAAG TGGACGAGACATACGTGCCCAAAGA GTTCAACGCCGAGACATTCACCTTCCACGCCGACATCTGCACCCTGTCCGAGAAA GAGCGGCAGATCAAGAAACAGACCG
CACTGGTGGAACTGGTGAAACACAAGCCCAAGGCCACCAAAGAACAGCTGAAGG CCGTGATGGACGACTTCGCCGCCTTT
GTGGAAAAGTGTTGCAAGGCCGACGACAAAGAGACATGCTTCGCCGAAGAGGGC AAGAAACTGGTGGCCGCTTCCCAGGC
TGCTCTGGGACTGGGAGGCGGCGGATCCTACGACGACGGCATCTGCAAGTCCTCC GACTGCATCAAGTCCGCCGCCAGAC TGATCCAGAACATGGACGCCACCACCGAGCCCTGCACCGATTTCTTTAAGTACGC CTGCGGCGGCTGGCTGAAGCGGAAC
GTGATCCCCGAGACATCCTCCAGATACGGCAACTTCGACATCCTGAGGGACGAGC TGGAAGTGGTGCTGAAGGACGTGCT GCAGGAACCCAAGACCGAGGACATCGTGGCCGTGCAGAAGGCCAAGGCCCTGTA CCGGTCCTGCATCAACGAGAGCGCCA
TCGACTCCAGAGGCGGCGAGCCTCTGCTGAAGCTGCTGCCCGACATCTACGGCTG GCCTGTGGCCACCGAGAACTGGGAG
CAGAAGTACGGCGCCTCCTGGACCGCCGAGAAGGCTATCGCCCAGCTGAACTCTA AGTACGGCAAGAAGGTGCTGATCAA
CCTGTTCGTGGGCACCGACGACAAGAACTCCGTGAACCATGTGATCCACATCGAC CAGCCTCGGCTGGGCCTGCCTTCCC
GGGACTACTACGAGTGTACCGGCATCTACAAAGAGGCCTGCACCGCCTACGTGG ACTTCATGATCTCCGTGGCCAGGCTG ATCCGGC AGGAAGAGAGACTGCCCATCGACGAGAACCAGCTGGCCCTGGAAATG AACAAAGTGATGGAACTGGAAAAAGA
GATCGCCAACGCTACCGCCAAGCCCGAGGACCGGAACGACCCCATGCTGCTGTA CAACAAGATGACCCTGGCCCAGATTC
AGAACAACTTCTCCCTGGAAATCAACGGCAAGCCCTTCTCCTGGCTGAACTTCAC CAACGAGATCATGTCCACCGTGAAC
ATCTCCATCACCAACGAAGAGGACGTGGTGGTGTACGCCCCTGAGTACCTGACCA AGCTGAAGCCCATCCTGACCAAGTA
CTCCGCCAGGGACCTGCAGAATCTGATGTCCTGGCGGTTCATCATGGACCTGGTG TCCTCCCTGTCCCGGACCTATAAAG AGTCCCGGAACGCCTTTCGGAAGGCTCTGTACGTGACCACCTCCGAGACAGCCAC CTGGCGGAGATGCGCCAACTACGTG
AACGGCAACATGGAAAACGCCGTGGGCAGACTGTACGTGGAAGCCGCCTTCGCC GGCGAGTCCAAACATGTGGTGGAAGA
TCTGATCGCCCAGATCAGAGAGGTGTTCATCCAGACCCTGGACGACCTGACCTGG ATGGACGCCGAGACTAAGAAGCGGG
CCGAGGAAAAGGCCCTGGCCATCAAAGAGCGGATCGGCTACCCCGACGACATCG TGTCCAACGACAACAAGCTGAACAAC GAGTACCTGGAACTGAATTACAAAGAGGACGAGTACTTCGAGAACATCATCCAG AATCTGAAGTTCTCCCAGTCCAAGCA
GCTGAAGAAACTGCGCGAGAAGGTGGACAAGGACGAGTGGATCTCCGGCGCTGC CGTGGTGAACGCCTTCTACTCCTCCG GCCGGAACCAGATCGTGTTCCCTGCCGGAATCCTGCAGCCCCCATTCTTCAGCGC CCAGCAGTCCAACTCCCTGAACTAC
GGCGGCATCGGCATGGTGATCGGCCACGAGATCACCCACGGCTTCGACGACAAC GGCCGGAACTTCAACAAGGACGGCGA
TCTGGTGGATTGGTGGACCCAGCAGAGCGCCTCCAACTTCAAAGAACAGTCCCAG TGCATGGTGTACCAGTACGGCAATT
TCTCCTGGGACCTGGCTGGCGGACAGCACCTGAACGGCATCAACACCCTGGGCGA GAATATCGCCGACAACGGCGGACTG
GGCCAGGCTTACAGAGCCTACCAGAACTACATCAAGAAGAACGGCGAAGAGAAA CTGCTGCCCGGCCTGGACCTGAACCA CAAGCAGCTGTTCTTTCTGAACTTCGCCCAGGTCTGGTGCGGCACCTACCGGCCT GAGTACGCCGTGAACTCCATCAAGA
CCGATGTGCATTCCCCCAAGAACTTCCGGATCATCGGCACCCTGCAGAACTCCGC CGAGTTCTCCGAGGCCTTCCACTGC
CGGAAGAACTCCTACATGAACCCCGAGAAGAAATGCCGCGTGTGGTGATAA
SEQ ID NO: 56 shows the sequence for HSA-propeptide - HSA-G4S-NEPG399V/G714K Protein
1-6 =HSA propep, 7-592 = HSA , 593-597 =G4S, 598-1296 = NEPG399V-G714K
>HSA propeptide HSA-G4S-NEP G399V-G714K Protein
SRGVFR DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFA KTCVADESAENCDKSLHTLFGDK
LCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHD NEETFLK YLYEIARRHPYFYAPE LLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGER AFKAWAVARLSQRFPKAEFAEVSK
LVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIA EVENDEMPADLPSLAADFVESK DVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHEC YAKVFDEFKPLVEEPQNLIKQNCE
LFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCA EDYLSVVLNQLCVLHEKTPVSDRV TKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIK QTALVEL VKHKPKATKEQLKAVMDDFAA
FVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSYDDGICKSSDCIKSAARLIQ NMDATTEPCTDFFKYACGGWLKR
NVIPETSSRYGNFDILRDELEVVLKDVLQEPKTEDIVAVQKAKALYRSCINESAIDSRG GEPLLKLLPDIYGWPVATENW
EQKYGASWTAEKAIAQLNSKYGK VLINLFVGTDDK SVNHVIHIDQPRLGLPSRDY YECTGIYKEACTAYVDFMISVAR
LIRQEERLPIDENQLALEMNKVMELEKEIANATAKPEDRNDPMLLYNKMTLAQIQNN FSLEINGKPFSWLNFTNEIMSTV NISITNEEDVVVYAPEYLTKLKPILTKYSARDLQNLMSWRFIMDLVSSLSRTYKESRN AFRKAL Y VTT SET AT WRRC AN Y
VNGNMENAVGRLYVEAAFAGESKHVVEDLIAQIREVFIQTLDDLTWMDAETKKRAE EKALAIKERIGYPDDIVSNDNKLN
NEYLELNYKEDEYFENIIQNLKFSQSKQLKKLREKVDKDEWISGAAVVNAFYSSGRN QIVFPAGILQPPFFSAQQSNSLN
YGGIGMVIGHEITHGFDDNGRNFNKDGDLVDWWTQQSASNFKEQSQCMVYQYGNF SWDLAGGQHLNGINTLGENIADNGG
LGQAYRAYQNYIK NGEEKLLPGLDLNHKQLFFLNFAQVWCGTYRPEYAVNSIKTD VHSPKNFRIIGTLQNSAEFSEAFH CRKNSYMNPEK CRVW
SEQ ID NO: 57 shows the sequence for HSA-G4S-NEPG399V/G714K DNA
1-1755 = HSA , 1774-1788 = G4S, 1789-3873 = NEPG399V-G714K
>HSA-G4S-NEP G399V-G714K Protein DNA
GATGCCCACAAGTCCGAGGTGGCCCACCGGTTCAAGGACCTGGGCGAGGAAAAC TTCAAGGCCCTGGTGCTGATCGCCTT CGCCCAGTACCTGCAGCAGAGCCCCTTCGAGGACCATGTGAAGCTGGTGAACGA AGTGACCGAGTTCGCCAAGACCTGCG
TGGCCGACGAGTCCGCCGAGAACTGCGACAAGTCCCTGCACACCCTGTTCGGCGA CAAGCTGTGTACCGTGGCCACCCTG CGGGAAACCTACGGCGAGATGGCCGACTGCTGCGCCAAGCAGGAACCCGAGCGG AACGAGTGCTTCCTGCAGCACAAGGA
CGACAACCCCAACCTGCCCCGGCTGGTGCGACCTGAGGTGGACGTGATGTGTACC GCCTTCCACGACAACGAGGAAACCT
TCCTGAAGAAGTACCTGTACGAGATCGCCAGACGGCACCCCTACTTCTACGCCCC CGAGCTGCTGTTCTTCGCCAAGCGG
TACAAGGCCGCCTTCACCGAGTGCTGCCAGGCCGCCGATAAGGCCGCCTGCCTGC TGCCTAAGCTGGACGAGCTGCGGGA
CGAGGGCAAGGCCTCCTCCGCCAAGCAGAGACTGAAGTGCGCCTCCCTGCAGAA GTTCGGCGAGCGGGCCTTTAAGGCCT GGGCCGTGGCCCGGCTGTCCC AGAGATTCCCCAAGGCCGAGTTTGCCGAGGTGTC CAAGCTGGTGACAGACCTGACCAAA
GTGCATACAGAGTGTTGCCACGGCGACCTGCTGGAATGCGCCGACGACAGAGCC GACCTGGCCAAGTACATCTGCGAGAA
CCAGGACTCCATCTCCTCCAAGCTGAAAGAGTGCTGCGAGAAGCCCCTGCTGGAA AAGTCCCACTGTATCGCCGAGGTGG
AAAACGACGAGATGCCCGCCGACCTGCCTTCTCTGGCCGCCGACTTCGTGGAATC CAAGGACGTGTGCAAGAACTACGCC
GAGGCCAAGGATGTGTTCCTGGGCATGTTCCTGTACGAGTACGCCCGCAGACACC CCGACTACTCCGTGGTGCTGCTGCT GCGGCTGGCCAAGACCTACGAGACAACCCTGGAAAAGTGCTGCGCCGCTGCCGA CCCCCACGAGTGTTACGCCAAGGTGT
TCGACGAGTTCAAGCCTCTGGTGGAAGAACCCCAGAACCTGATCAAGCAGAACT GCGAGCTGTTCGAGCAGCTGGGCGAG
TACAAGTTCCAGAACGCCCTGCTGGTGCGATACACCAAGAAAGTGCCCCAGGTGT CCACCCCCACCCTGGTGGAAGTGTC
CCGGAACCTGGGCAAAGTGGGCTCCAAGTGCTGCAAGCACCCTGAGGCCAAGCG GATGCCCTGCGCCGAGGACTACCTGA GCGTGGTGCTGAACCAGCTGTGCGTGCTGCACGAAAAGACCCCCGTGTCCGACAG AGTGACCAAGTGCTGTACCGAGTCC
CTGGTGAACAGACGGCCCTGCTTCTCCGCCCTGGAAGTGGACGAGACATACGTGC CCAAAGAGTTCAACGCCGAGACATT CACCTTCCACGCCGACATCTGCACCCTGTCCGAGAAAGAGCGGCAGATCAAGAA ACAGACCGCACTGGTGGAACTGGTGA
AAC AC AAGCC C AAGGC C ACC AAAG AAC AGCTG AAGGCC GTG ATGG AC G ACTTC G CCGCCTTTGTGGAAAAGTGTTGCAAG
GCCGACGACAAAGAGACATGCTTCGCCGAAGAGGGCAAGAAACTGGTGGCCGCT TCCCAGGCTGCTCTGGGACTGGGAGG
CGGCGGATCCTACGACGACGGCATCTGCAAGTCCTCCGACTGCATCAAGTCCGCC GCCAGACTGATCCAGAACATGGACG
CCACCACCGAGCCCTGCACCGATTTCTTTAAGTACGCCTGCGGCGGCTGGCTGAA GCGGAACGTGATCCCCGAGACATCC TCCAGATACGGCAACTTCGACATCCTGAGGGACGAGCTGGAAGTGGTGCTGAAG GACGTGCTGCAGGAACCCAAGACCGA
GGACATCGTGGCCGTGCAGAAGGCCAAGGCCCTGTACCGGTCCTGCATCAACGA GAGCGCCATCGACTCCAGAGGCGGCG
AGCCTCTGCTGAAGCTGCTGCCCGACATCTACGGCTGGCCTGTGGCCACCGAGAA CTGGGAGCAGAAGTACGGCGCCTCC
TGGACCGCCGAGAAGGCTATCGCCCAGCTGAACTCTAAGTACGGCAAGAAGGTG CTGATCAACCTGTTCGTGGGCACCGA
CGACAAGAACTCCGTGAACCATGTGATCCACATCGACCAGCCTCGGCTGGGCCTG CCTTCCCGGGACTACTACGAGTGTA CCGGCATCTACAAAGAGGCCTGCACCGCCTACGTGGACTTCATGATCTCCGTGGC CAGGCTGATCCGGCAGGAAGAGAGA
CTGCCCATCGACGAGAACCAGCTGGCCCTGGAAATGAACAAAGTGATGGAACTG GAAAAAGAGATCGCCAACGCTACCGC
CAAGCCCGAGGACCGGAACGACCCCATGCTGCTGTACAACAAGATGACCCTGGC CCAGATTCAGAACAACTTCTCCCTGG
AAATCAACGGCAAGCCCTTCTCCTGGCTGAACTTCACCAACGAGATCATGTCCAC CGTGAACATCTCCATCACCAACGAA GAGGACGTGGTGGTGTACGCCCCTGAGTACCTGACCAAGCTGAAGCCCATCCTGA CCAAGTACTCCGCCAGGGACCTGCA
GAATCTGATGTCCTGGCGGTTCATCATGGACCTGGTGTCCTCCCTGTCCCGGACCT ATAAAGAGTCCCGGAACGCCTTTC GGAAGGCTCTGTACGTGACCACCTCCGAGACAGCCACCTGGCGGAGATGCGCCA ACTACGTGAACGGCAACATGGAAAAC
GCCGTGGGCAGACTGTACGTGGAAGCCGCCTTCGCCGGCGAGTCCAAACATGTG GTGGAAGATCTGATCGCCCAGATCAG
AGAGGTGTTCATCCAGACCCTGGACGACCTGACCTGGATGGACGCCGAGACTAA GAAGCGGGCCGAGGAAAAGGCCCTGG
CCATCAAAGAGCGGATCGGCTACCCCGACGACATCGTGTCCAACGACAACAAGC TGAACAACGAGTACCTGGAACTGAAT
TACAAAGAGGACGAGTACTTCGAGAACATCATCCAGAATCTGAAGTTCTCCCAGT CCAAGCAGCTGAAGAAACTGCGCGA GAAGGTGGAC AAGGACGAGTGGATCTCCGGCGCTGCCGTGGTGAACGCCTTCTA CTCCTCCGGCCGGAACCAGATCGTGT
TCCCTGCCGGAATCCTGCAGCCCCCATTCTTCAGCGCCCAGCAGTCCAACTCCCTG AACTACGGCGGCATCGGCATGGTG
ATCGGCCACGAGATCACCCACGGCTTCGACGACAACGGCCGGAACTTCAACAAG GACGGCGATCTGGTGGATTGGTGGAC
CCAGCAGAGCGCCTCCAACTTCAAAGAACAGTCCCAGTGCATGGTGTACCAGTAC GGCAATTTCTCCTGGGACCTGGCTG
GCGGACAGCACCTGAACGGCATCAACACCCTGGGCGAGAATATCGCCGACAACG GCGGACTGGGCCAGGCTTACAGAGCC TACCAGAACTACATCAAGAAGAACGGCGAAGAGAAACTGCTGCCCGGCCTGGAC CTGAACCACAAGCAGCTGTTCTTTCT
GAACTTCGCCCAGGTCTGGTGCGGCACCTACCGGCCTGAGTACGCCGTGAACTCC ATCAAGACCGATGTGCATTCCCCCA
AGAACTTCCGGATCATCGGCACCCTGCAGAACTCCGCCGAGTTCTCCGAGGCCTT CCACTGCCGGAAGAACTCCTACATG
AACCCCGAGAAGAAATGCCGCGTGTGGTGATAA SEQ ID NO: 58 shows the sequence for HSA-G4S-NEPG399V/G714K Protein 1-585 = HSA , 586-590=G4S, 591-1289 = NEPG399V-G714K
>HSA-G4S-NEP G399V-G714K Protein
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADE SAENCDKSLHTLFGDKLCTVATL
RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK KYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWA VARLSQRFPKAEFAEVSKLVTDLTK
VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDE MPADLPSLAADFVESKDVCKNYA
EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDE FKPLVEEPQNLIKQNCELFEQLGE
YKFQNALLVRYTK VPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVV LNQLCVLHEKTPVSDRVTKCCTES
LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIK QTALVELVKHKPK ATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGKKLVAASQAALGLGGGGSYDDGICKSSDCIKSAARLIQNMDATTE PCTDFFKYACGGWLKRNVIPETS
SRYGNFDILRDELEVVLKDVLQEPKTEDIVAVQKAKALYRSCINESAIDSRGGEPLLK LLPDIYGWPVATENWEQKYGAS
WTAEKAIAQLNSKYGK VLINLFVGTDDKNSVNHVIHIDQPRLGLPSRDYYECTGIY KE ACT AY VDFMI S V ARLIRQEER
LPIDENQLALEMNKVMELEKEIANATAKPEDRNDPMLLYNKMTLAQIQNNFSLEING KPFSWLNFTNEIMSTVNISITNE
EDVVVYAPEYLTKLKPILTKYSARDLQNLMSWRFIMDLVSSLSRTYKESRNAFRKAL YVTTSETATWRRCANYVNGNMEN AVGRLYVEAAFAGESKHVVEDLIAQIREVFIQTLDDLTWMDAETKKRAEEKALAIKE RIGYPDDIVSNDNKLNNEYLELN YKEDEYFENIIQNLKFSQSKQLKKLREKVDKDEWISGAAVVNAFYSSGRNQIVFPAGI LQPPFFSAQQSNSLNYGGIGMV
IGHEITHGFDDNGRNFNKDGDLVDWWTQQSASNFKEQSQCMVYQYGNFSWDLAGG QHLNGINTLGENIADNGGLGQAYRA YQNYIKK GEEKLLPGLDLNHKQLFFLNFAQVWCGTYRPEYAVNSIKTDVHSPK FR IIGTLQNSAEFSEAFHCRK SYM NPEK CRVW
SEQ ID NO: 59 shows the sequence for IgG leader- 10HisNEPG399V/G714K DNA
1-48, 128-139 = IgG leader, 140-169 = lOHis , 170-2272 = NEPG399V-G714K
>IgG leader lOHisNEP G399V-G714K DNA
ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGT TAACAGTAGCAGGCTTGAGGTCTGG
ACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGCGTGCAC TCCCATCATCATCATCATCATCAT CATCATCATTACGACGACGGCATCTGCAAGTCCTCCGACTGC ATC AAGTCCGCCG CCAGACTGATCCAGAACATGGACGC
CACCACCGAGCCCTGCACCGATTTCTTTAAGTACGCCTGCGGCGGCTGGCTGAAG CGGAACGTGATCCCCGAGACATCCT
CCAGATACGGCAACTTCGACATCCTGAGGGACGAGCTGGAAGTGGTGCTGAAGG ACGTGCTGCAGGAACCCAAGACCGAG
GACATCGTGGCCGTGCAGAAGGCCAAGGCCCTGTACCGGTCCTGCATCAACGAG AGCGCCATCGACTCCAGAGGCGGCGA
GCCTCTGCTGAAGCTGCTGCCCGACATCTACGGCTGGCCTGTGGCCACCGAGAAC TGGGAGCAGAAGTACGGCGCCTCCT GGACCGCCGAGAAGGCTATCGCCCAGCTGAACTCTAAGTACGGCAAGAAGGTGC TGATCAACCTGTTCGTGGGCACCGAC
GACAAGAACTCCGTGAACCATGTGATCCACATCGACCAGCCTCGGCTGGGCCTGC CTTCCCGGGACTACTACGAGTGTAC
CGGCATCTACAAAGAGGCCTGCACCGCCTACGTGGACTTCATGATCTCCGTGGCC AGGCTGATCCGGCAGGAAGAGAGAC
TGCCCATCGACGAGAACCAGCTGGCCCTGGAAATGAACAAAGTGATGGAACTGG AAAAAGAGATCGCCAACGCTACCGCC AAGCCCGAGGACCGGAACGACCCCATGCTGCTGTACAACAAGATGACCCTGGCC CAGATTCAGAACAACTTCTCCCTGGA
AATCAACGGCAAGCCCTTCTCCTGGCTGAACTTCACCAACGAGATCATGTCCACC GTGAACATCTCCATCACCAACGAAG AGGACGTGGTGGTGTACGCCCCTGAGTACCTGACCAAGCTGAAGCCCATCCTGAC CAAGTACTCCGCCAGGGACCTGCAG
AATCTGATGTCCTGGCGGTTCATCATGGACCTGGTGTCCTCCCTGTCCCGGACCTA TAAAGAGTCCCGGAACGCCTTTCG
GAAGGCTCTGTACGTGACCACCTCCGAGACAGCCACCTGGCGGAGATGCGCCAA CTACGTGAACGGCAACATGGAAAACG
CCGTGGGCAGACTGTACGTGGAAGCCGCCTTCGCCGGCGAGTCCAAACATGTGGT GGAAGATCTGATCGCCCAGATCAGA
GAGGTGTTCATCCAGACCCTGGACGACCTGACCTGGATGGACGCCGAGACTAAG AAGCGGGCCGAGGAAAAGGCCCTGGC CATCAAAGAGCGGATCGGCTACCCCGACGAC ATCGTGTCC AACGAC AACAAGCT GAACAACGAGTACCTGGAACTGAATT
ACAAAGAGGACGAGTACTTCGAGAACATCATCCAGAATCTGAAGTTCTCCCAGTC CAAGCAGCTGAAGAAACTGCGCGAG
AAGGTGGACAAGGACGAGTGGATCTCCGGCGCTGCCGTGGTGAACGCCTTCTACT CCTCCGGCCGGAACCAGATCGTGTT
CCCTGCCGGAATCCTGCAGCCCCCATTCTTCAGCGCCCAGCAGTCCAACTCCCTG AACTACGGCGGCATCGGCATGGTGA
TCGGCCACGAGATCACCCACGGCTTCGACGACAACGGCCGGAACTTCAACAAGG ACGGCGATCTGGTGGATTGGTGGACC CAGCAGAGCGCCTCCAACTTCAAAGAACAGTCCCAGTGCATGGTGTACCAGTACG GCAATTTCTCCTGGGACCTGGCTGG
CGGACAGCACCTGAACGGCATCAACACCCTGGGCGAGAATATCGCCGACAACGG CGGACTGGGCCAGGCTTACAGAGCCT
ACCAGAACTACATCAAGAAGAACGGCGAAGAGAAACTGCTGCCCGGCCTGGACC TGAACCACAAGCAGCTGTTCTTTCTG
AACTTCGCCCAGGTCTGGTGCGGCACCTACCGGCCTGAGTACGCCGTGAACTCCA TCAAGACCGATGTGCATTCCCCCAA GAACTTCCGGATCATCGGCACCCTGCAGAACTCCGCCGAGTTCTCCGAGGCCTTC
CACTGCCGGAAGAACTCCTACATGA
ACCCCGAGAAGAAATGCCGCGTGTGGTGATAA SEQ ID NO: 60 shows the sequence for IgG leader- 10HisNEPG399V/G714K Protein 1-19 = IgG leader, 20-29 = lOHis, 29-728 = NEPG399V-G714K
>IgG leader lOHisNEP G399 V-G714K Protein
MGWSCIILFLVATATGVHSHHHHHHHHHHYDDGICKSSDCIKSAARLIQNMDATTEP CTDFFKYACGGWLKRNVIPETSS RYGNFDILRDELEVVLKD VLQEPKTEDIVAVQKAKALYRSCINES AIDSRGGEPLLKL LPDIYGWPVATENWEQKYGASW
TAEKAIAQLNSKYGK VLINLFVGTDDKNSVNHVIHIDQPRLGLPSRDYYECTGIYKE ACT AY VDFMI S V ARLIRQEERL
PIDENQLALEMNKVMELEKEIANATAKPEDRNDPMLLYNKMTLAQIQNNFSLEINGK PFSWLNFTNEIMSTVNISITNEE
DVVVYAPEYLTKLKPILTKYSARDLQNLMSWRFIMDLVSSLSRTYKESRNAFRKALY VTTSETATWRRCANYVNGNMENA
VGRLYVEAAFAGESKHVVEDLIAQIREVFIQTLDDLTWMDAETKKRAEEKALAIKER IGYPDDIVSNDNKLNNEYLELNY KEDEYFENIIQNLKFSQSKQLKKLREKVDKDEWISGAAVVNAFYSSGRNQIVFPAGIL QPPFFSAQQSNSLNYGGIGMVI
GHEITHGFDDNGRNFNKDGDLVDWWTQQSASNFKEQSQCMVYQYGNFSWDLAGG QHLNGINTLGENIADNGGLGQAYRAY
QNYIKKNGEEKLLPGLDLNHKQLFFLNFAQVWCGTYRPEYAVNSIKTDVHSPKNFRII GTLQNSAEFSEAFHCRKNSYMN PEK CRVW
SEQ ID NO: 61 shows the sequence for 10HisNEPG399V/G714K DNA
1-30 = lOHis , 31-2133 = NEPG399V-G714K
>10HisNEP G399V-G714K DNA
CATCATCATCATCATCATCATCATCATCATTACGACGACGGCATCTGCAAGTCCTC CGACTGCATCAAGTCCGCCGCCAG ACTGATCCAGAACATGGACGCCACCACCGAGCCCTGCACCGATTTCTTTAAGTAC GCCTGCGGCGGCTGGCTGAAGCGGA
ACGTGATCCCCGAGACATCCTCCAGATACGGCAACTTCGACATCCTGAGGGACGA GCTGGAAGTGGTGCTGAAGGACGTG CTGCAGGAACCCAAGACCGAGGACATCGTGGCCGTGCAGAAGGCCAAGGCCCTG TACCGGTCCTGCATCAACGAGAGCGC
CATCGACTCCAGAGGCGGCGAGCCTCTGCTGAAGCTGCTGCCCGACATCTACGGC TGGCCTGTGGCCACCGAGAACTGGG
AGCAGAAGTACGGCGCCTCCTGGACCGCCGAGAAGGCTATCGCCCAGCTGAACT CTAAGTACGGCAAGAAGGTGCTGATC
AACCTGTTCGTGGGCACCGACGACAAGAACTCCGTGAACCATGTGATCCACATCG ACCAGCCTCGGCTGGGCCTGCCTTC
CCGGGACTACTACGAGTGTACCGGCATCTACAAAGAGGCCTGCACCGCCTACGTG GACTTCATGATCTCCGTGGCCAGGC TGATCCGGC AGGAAGAGAGACTGCCC ATCGACGAGAACCAGCTGGCCCTGGAAA TGAACAAAGTGATGGAACTGGAAAAA
GAGATCGCCAACGCTACCGCCAAGCCCGAGGACCGGAACGACCCCATGCTGCTG TACAACAAGATGACCCTGGCCCAGAT
TCAGAACAACTTCTCCCTGGAAATCAACGGCAAGCCCTTCTCCTGGCTGAACTTC ACCAACGAGATCATGTCCACCGTGA
ACATCTCCATCACCAACGAAGAGGACGTGGTGGTGTACGCCCCTGAGTACCTGAC CAAGCTGAAGCCCATCCTGACCAAG
TACTCCGCCAGGGACCTGCAGAATCTGATGTCCTGGCGGTTCATCATGGACCTGG TGTCCTCCCTGTCCCGGACCTATAA AGAGTCCCGGAACGCCTTTCGGAAGGCTCTGTACGTGACCACCTCCGAGACAGCC ACCTGGCGGAGATGCGCCAACTACG
TGAACGGCAACATGGAAAACGCCGTGGGCAGACTGTACGTGGAAGCCGCCTTCG CCGGCGAGTCCAAACATGTGGTGGAA
GATCTGATCGCCCAGATCAGAGAGGTGTTCATCCAGACCCTGGACGACCTGACCT GGATGGACGCCGAGACTAAGAAGCG
GGCCGAGGAAAAGGCCCTGGCCATCAAAGAGCGGATCGGCTACCCCGACGACAT CGTGTCCAACGACAACAAGCTGAACA ACGAGTACCTGGAACTGAATTACAAAGAGGACGAGTACTTCGAGAACATCATCC AGAATCTGAAGTTCTCCCAGTCCAAG
CAGCTGAAGAAACTGCGCGAGAAGGTGGACAAGGACGAGTGGATCTCCGGCGCT GCCGTGGTGAACGCCTTCTACTCCTC CGGCCGGAACCAGATCGTGTTCCCTGCCGGAATCCTGCAGCCCCCATTCTTCAGC GCCCAGCAGTCCAACTCCCTGAACT
ACGGCGGCATCGGCATGGTGATCGGCCACGAGATCACCCACGGCTTCGACGACA ACGGCCGGAACTTCAACAAGGACGGC
GATCTGGTGGATTGGTGGACCCAGCAGAGCGCCTCCAACTTCAAAGAACAGTCCC AGTGCATGGTGTACCAGTACGGCAA
TTTCTCCTGGGACCTGGCTGGCGGACAGCACCTGAACGGCATCAACACCCTGGGC GAGAATATCGCCGACAACGGCGGAC
TGGGCCAGGCTTACAGAGCCTACCAGAACTACATCAAGAAGAACGGCGAAGAGA AACTGCTGCCCGGCCTGGACCTGAAC CAC AAGCAGCTGTTCTTTCTGAACTTCGCCCAGGTCTGGTGCGGCACCTACCGGC CTGAGTACGCCGTGAACTCCATCAA
GACCGATGTGCATTCCCCCAAGAACTTCCGGATCATCGGCACCCTGCAGAACTCC GCCGAGTTCTCCGAGGCCTTCCACT
GCCGGAAGAACTCCTACATGAACCCCGAGAAGAAATGCCGCGTGTGGTGATAA
SEQ ID NO: 62 shows the sequence for 10HisNEPG399V/G714K Protein
1-10 = lOHis, 11-709 = NEPG399V-G714K
>10HisNEP G399V-G714K DNA
HHHHHHHHHHYDDGICKSSDCIKSAARLIQNMDATTEPCTDFFKYACGGWLKRNVI PET S SRYGNFDILRDELE V VLKD V
LQEPKTEDIVAVQKAKALYRSCINESAIDSRGGEPLLKLLPDIYGWPVATENWEQKY GASWTAEKAIAQLNSKYGKKVLI
NLFVGTDDKNSVNHVIHIDQPRLGLPSRDYYECTGIYKEACTAYVDFMISVARLIRQE ERLPIDENQLALEMNKVMELEK EIANATAKPEDRNDPMLLYNKMTLAQIQNNFSLEINGKPFSWLNFTNEIMSTVNISITN EEDVVVYAPEYLTKLKPILTK YSARDLQNLMSWRFIMDLVSSLSRTYKESRNAFRKALYVTTSETATWRRCANYVNG NMENAVGRLYVEAAFAGESKHVVE
DLIAQIREVFIQTLDDLTWMDAETKKRAEEKALAIKERIGYPDDIVSNDNKL NEYLE LNYKEDEYFENIIQNLKFSQSK QLKKLREKVDKDEWISGAAVVNAFYSSGRNQIVFPAGILQPPFFSAQQSNSLNYGGIG MVIGHEITHGFDDNGRNFNKDG
DLVDWWTQQSASNFKEQSQCMVYQYGNFSWDLAGGQHLNGINTLGENIADNGGLG QAYRAYQNYIKK GEEKLLPGLDLN
HKQLFFLNFAQVWCGTYRPEYAVNSIKTDVHSPK FRIIGTLQNSAEFSEAFHCRK S YMNPEK CRVW
SEQ ID NO: 63 shows the sequence for:
IgG leader -HSAC34S-G4S-NEP G399V-G714K DNA
ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTGCACT CCGATGCCC ACAAGTCCGAGGTGGCCC ACCGGTTCAAGGACCTGGGCGAGGAAA ACTTCAAGGCCCTGGTGCTGATCGCCTTCGCCCAGTACCTGCAGCAGAGCCCCTT CGAGGACCATGTGAAGCTGGTGAACGAAGTGACCGAGTTCGCCAAGACCTGCGT GGCCGACGAGTCCGCCGAGAACTGCGACAAGTCCCTGCACACCCTGTTCGGCGAC AAGCTGTGTACCGTGGCCACCCTGCGGGAAACCTACGGCGAGATGGCCGACTGCT GCGCCAAGCAGGAACCCGAGCGGAACGAGTGCTTCCTGCAGCACAAGGACGACA ACCCCAACCTGCCCCGGCTGGTGCGACCTGAGGTGGACGTGATGTGTACCGCCTT CCACGACAACGAGGAAACCTTCCTGAAGAAGTACCTGTACGAGATCGCCAGACG GCACCCCTACTTCTACGCCCCCGAGCTGCTGTTCTTCGCCAAGCGGTACAAGGCC GCCTTCACCGAGTGCTGCCAGGCCGCCGATAAGGCCGCCTGCCTGCTGCCTAAGC TGGACGAGCTGCGGGACGAGGGCAAGGCCTCCTCCGCCAAGCAGAGACTGAAGT GCGCCTCCCTGCAGAAGTTCGGCGAGCGGGCCTTTAAGGCCTGGGCCGTGGCCCG GCTGTCCCAGAGATTCCCCAAGGCCGAGTTTGCCGAGGTGTCCAAGCTGGTGACA GACCTGACCAAAGTGCATACAGAGTGTTGCCACGGCGACCTGCTGGAATGCGCC GACGACAGAGCCGACCTGGCCAAGTACATCTGCGAGAACCAGGACTCCATCTCCT CCAAGCTGAAAGAGTGCTGCGAGAAGCCCCTGCTGGAAAAGTCCCACTGTATCG CCGAGGTGGAAAACGACGAGATGCCCGCCGACCTGCCTTCTCTGGCCGCCGACTT CGTGGAATCCAAGGACGTGTGCAAGAACTACGCCGAGGCCAAGGATGTGTTCCT GGGCATGTTCCTGTACGAGTACGCCCGCAGACACCCCGACTACTCCGTGGTGCTG CTGCTGCGGCTGGCCAAGACCTACGAGACAACCCTGGAAAAGTGCTGCGCCGCT GCCGACCCCCACGAGTGTTACGCCAAGGTGTTCGACGAGTTCAAGCCTCTGGTGG AAGAACCCCAGAACCTGATCAAGCAGAACTGCGAGCTGTTCGAGCAGCTGGGCG AGTACAAGTTCCAGAACGCCCTGCTGGTGCGATACACCAAGAAAGTGCCCCAGG TGTCCACCCCCACCCTGGTGGAAGTGTCCCGGAACCTGGGCAAAGTGGGCTCCAA GTGCTGCAAGCACCCTGAGGCCAAGCGGATGCCCTGCGCCGAGGACTACCTGAG CGTGGTGCTGAACCAGCTGTGCGTGCTGCACGAAAAGACCCCCGTGTCCGACAGA GTGACCAAGTGCTGTACCGAGTCCCTGGTGAACAGACGGCCCTGCTTCTCCGCCC TGGAAGTGGACGAGACATACGTGCCC AAAGAGTTCAACGCCGAGACATTCACCT TCCACGCCGACATCTGCACCCTGTCCGAGAAAGAGCGGCAGATCAAGAAACAGA CCGCACTGGTGGAACTGGTGAAACACAAGCCCAAGGCCACCAAAGAACAGCTGA AGGCCGTGATGGACGACTTCGCCGCCTTTGTGGAAAAGTGTTGCAAGGCCGACGA CAAAGAGACATGCTTCGCCGAAGAGGGCAAGAAACTGGTGGCCGCTTCCCAGGC TGCTCTGGGACTGGGAGGCGGCGGATCCTACGACGACGGC ATCTGC AAGTCCTCC GACTGCATCAAGTCCGCCGCCAGACTGATCCAGAACATGGACGCCACCACCGAG CCCTGCACCGATTTCTTTAAGTACGCCTGCGGCGGCTGGCTGAAGCGGAACGTGA TCCCCGAGACATCCTCCAGATACGGCAACTTCGACATCCTGAGGGACGAGCTGGA AGTGGTGCTGAAGGACGTGCTGCAGGAACCCAAGACCGAGGACATCGTGGCCGT GCAGAAGGCCAAGGCCCTGTACCGGTCCTGCATCAACGAGAGCGCCATCGACTC CAGAGGCGGCGAGCCTCTGCTGAAGCTGCTGCCCGACATCTACGGCTGGCCTGTG GCCACCGAGAACTGGGAGCAGAAGTACGGCGCCTCCTGGACCGCCGAGAAGGCT ATCGCCCAGCTGAACTCTAAGTACGGCAAGAAGGTGCTGATCAACCTGTTCGTGG GCACCGACGACAAGAACTCCGTGAACCATGTGATCCACATCGACCAGCCTCGGCT GGGCCTGCCTTCCCGGGACTACTACGAGTGTACCGGCATCTACAAAGAGGCCTGC ACCGCCTACGTGGACTTCATGATCTCCGTGGCCAGGCTGATCCGGCAGGAAGAGA GACTGCCCATCGACGAGAACCAGCTGGCCCTGGAAATGAACAAAGTGATGGAAC TGGAAAAAGAGATCGCCAACGCTACCGCCAAGCCCGAGGACCGGAACGACCCCA TGCTGCTGTACAACAAGATGACCCTGGCCCAGATTCAGAACAACTTCTCCCTGGA AATCAACGGCAAGCCCTTCTCCTGGCTGAACTTCACCAACGAGATCATGTCCACC GTGAACATCTCCATCACCAACGAAGAGGACGTGGTGGTGTACGCCCCTGAGTACC TGACCAAGCTGAAGCCCATCCTGACCAAGTACTCCGCCAGGGACCTGCAGAATCT GATGTCCTGGCGGTTCATCATGGACCTGGTGTCCTCCCTGTCCCGGACCTATAAA GAGTCCCGGAACGCCTTTCGGAAGGCTCTGTACGTGACCACCTCCGAGACAGCCA CCTGGCGGAGATGCGCCAACTACGTGAACGGCAACATGGAAAACGCCGTGGGCA GACTGTACGTGGAAGCCGCCTTCGCCGGCGAGTCCAAACATGTGGTGGAAGATCT GATCGCCCAGATCAGAGAGGTGTTCATCCAGACCCTGGACGACCTGACCTGGATG GACGCCGAGACTAAGAAGCGGGCCGAGGAAAAGGCCCTGGCCATCAAAGAGCG GATCGGCTACCCCGACGACATCGTGTCCAACGACAACAAGCTGAACAACGAGTA CCTGGAACTGAATTACAAAGAGGACGAGTACTTCGAGAACATCATCCAGAATCT GAAGTTCTCCCAGTCCAAGCAGCTGAAGAAACTGCGCGAGAAGGTGGACAAGGA CGAGTGGATCTCCGGCGCTGCCGTGGTGAACGCCTTCTACTCCTCCGGCCGGAAC CAGATCGTGTTCCCTGCCGGAATCCTGCAGCCCCCATTCTTCAGCGCCCAGCAGT CCAACTCCCTGAACTACGGCGGCATCGGCATGGTGATCGGCCACGAGATCACCCA CGGCTTCGACGACAACGGCCGGAACTTCAACAAGGACGGCGATCTGGTGGATTG GTGGACCCAGCAGAGCGCCTCCAACTTCAAAGAACAGTCCCAGTGCATGGTGTAC CAGTACGGCAATTTCTCCTGGGACCTGGCTGGCGGAC AGCACCTGAACGGCATCA ACACCCTGGGCGAGAATATCGCCGACAACGGCGGACTGGGCCAGGCTTACAGAG CCTACCAGAACTACATCAAGAAGAACGGCGAAGAGAAACTGCTGCCCGGCCTGG ACCTGAACCACAAGCAGCTGTTCTTTCTGAACTTCGCCCAGGTCTGGTGCGGCAC CTACCGGCCTGAGTACGCCGTGAACTCCATCAAGACCGATGTGCATTCCCCCAAG AACTTCCGGATCATCGGCACCCTGCAGAACTCCGCCGAGTTCTCCGAGGCCTTCC ACTGCCGGAAGAACTCCTACATGAACCCCGAGAAGAAATGCCGCGTGTGGTGAT AA
SEQ ID NO: 64 shows the sequence for: IgG leader-HSAC34S-G4S-NEP G399V-G714K Protein
MGWSCIILFLVATATGVHSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFED HVKLVNEVTEFAKTCV
ADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNP NLPRLVRPEVDVMCTAFHDNEETF LK YLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKA SSAKQRLKCASLQKFGERAFKAW AVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSI SSKLKECCEKPLLEKSHCIAEVE
NDEMPADLPSLAADFVESKDVCK YAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA KTYETTLEKCCAAADPHECYAKVF DEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTK VPQVSTPTLVEVSRNLG KVGSKCCKHPEAKRMPCAEDYLS
VVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHA DICTLSEKERQIK QTALVELVK
HKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGG S YDDGICKS SDCIKS AARLIQNMD A
TTEPCTDFFKYACGGWLKRNVIPETSSRYGNFDILRDELEVVLKDVLQEPKTEDIVAV QKAKALYRSCINESAIDSRGGE
PLLKLLPDIYGWPVATENWEQKYGASWTAEKAIAQLNSKYGK VLINLFVGTDDK SVNHVIHIDQPRLGLPSRDYYECT GIYKEACTAYVDFMIS VARLIRQEERLPIDENQLALEMNKVMELEKEIANAT AKPEDR NDPMLLYNKMTLAQIQNNFSLE
INGKPFSWLNFTNEIMSTVNISITNEEDVVVYAPEYLTKLKPILTKYSARDLQNLMSW RFIMDLVSSLSRTYKESRNAFR
KALYVTTSETATWRRCANYVNGNMENAVGRLYVEAAFAGESKHVVEDLIAQIREVF IQTLDDLTWMDAETKKRAEEKALA
IKERIGYPDDIVSNDNKL NEYLELNYKEDEYFENIIQNLKFSQSKQLKKLREKVDKD EWISGAAVVNAFYSSGRNQIVF
PAGILQPPFFSAQQSNSLNYGGIGMVIGHEITHGFDDNGRNFNKDGDLVDWWTQQSA SNFKEQ S QCM V YQ YGNF S WDL AG GQHLNGINTLGENIADNGGLGQAYRAYQNYIKK GEEKLLPGLDLNHKQLFFLNFA QVWCGTYRPEYAVNSIKTDVHSPK NFRIIGTLQNSAEFSEAFHCRK SYMNPEK CRVW
Detailed description of the Figures
Figure 1 shows the nucleotide sequence of yeast expression vector pYES2
(Invitrogen, SKU# V825-20), 5856bp (SEQ ID NO: 22). 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 2μ origin for high-copy maintenance.
Figure 2 shows nucleotide sequences of yeast expression vector pESC-URA
(Stratagen), 6631 bp (SEQ ID NO:23).
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 5 shows the cleavage of five of the peptide substrates (peptide 5 = angiotensin; peptide 3 = ANP; peptide 6a = one of the endothelin peptides; peptide 1 = ABi_40 ; and, peptide 2 = ABi_42) by various mutants relative to the G399V/G714K parent mutant (see Table 8), illustrating the increased cleavage of the amyloid beta peptides (ABi_4o and ABi_42) and reduced cleavage of the three off-peptides (ANP, endothelin and angiotensin).
Figure 6 shows the cleavage of six of the peptide substrates (peptide 5 = angiotensin; peptide 4 = BNP; peptide 7 = neuropeptide Y; peptide 6a = one of the endothelin peptides; peptide 1 = ABi_40 ; and, peptide 2 = ABi_42) by various mutants from Table 10 relative to the G399V/G714K parent mutant.
Figure 7: Abeta degradation of endogenous mouse Abeta 1-40 in plasma from
C57BL/6 mice after 1 hour incubation at RT°C using 1 uM to 0.1 nM of enzyme. A) wildtype neprilysin. B) neprilysin variant G399V/G714K fused to HAS (N-HSA - hNepG399V/G714K-C in this and the following examples).
Figure 8: Abeta degradation of human Abeta 1-42 in plasma from TG2576 mice after 1 hour incubation at RT°C using 1 uM to 1 nM of enzyme. A) wildtype neprilysin. B) neprilysin variant G399V/G714K fused to HSA.
Figure 9: Abeta degradation of human Abeta 1-40 in plasma from TG2576 mice after
1 hour incubation at RT °C using 3 uM to 10 nM of enzyme. A) wildtype neprilysin. B) neprilysin variant G399V/G714K fused to HSA.
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. A) wildtype neprilysin. B) neprilysin variant G399V/G714K fused to HSA. 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. A) wildtype neprilysin. B) neprilysin variant G399V/G714K fused to HSA.
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. A) wildtype neprilysin. B) neprilysin variant G399V/G714K fused to HSA.
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. A) wildtype neprilysin. B) neprilysin variant G399V/G714K fused to HSA.
Detailed description of the Invention
In the framework of this invention the following abbreviations, terms and definitions are used:
aa amino acid
HA-tag Haemagglutinin epitope tag
3xHA tag 3 -times the HA epitope
Nt nucleotide
PCR Polymerase Chain Reaction
sNeprilysin soluble Neprilysin
wt wild type
The term "amyloid beta peptide", "Αβ peptide" or "amyloid β 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 Αβ A4 protein [Precursor], corresponding to amino acid 672 to 714 in the sequence (amino acid 1-43; Αβ1-43). It also includes any shorter forms of this peptide, such as Αβ1-40, Αβ1-41,
Αβ1-42, Αβ1-39, Αβ1-38, Αβ1-43, and modified peptides such as N-terminal truncated forms as Αβ3-42, Αβΐ 1-40 and Αβΐ 1-42, Αβ peptides with pyroglutamyl formation as Αβ(ρν3-42) and Αβ^Ι 1-42) and Αβ peptides which are modified by oxidation, isomerisation, racemization, and/or covalently linkage (ID17274, ID17231, ID17850). The term comprises also Αββ with substitutions of residues such Glu22 for Gin (references in Soto, C. and Castano, M., (1996) Biochem. J. 314:701-707) and oligomeric forms and aggregates. The term "polynucleotide" corresponds to any genetic material of any length and any sequence, comprising single-stranded and double-stranded DNA and R A molecules, including regulatory elements, structural genes, groups of genes, plasmids, whole genomes, and fragments thereof.
The term "site" in a polynucleotide or polypeptide refers to a certain position or region in the sequence of the polynucleotide or polypeptide, respectively.
The term "position" in a polynucleotide or polypeptide refers to specific single bases or amino acids in the sequence of the polynucleotide or polypeptide, respectively.
The term "region" in a polynucleotide or polypeptide refers to stretches of several bases or amino acids in the sequence of the polynucleotide or polypeptide, respectively.
The term "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, such as peptidic receptor ligands, peptide hormones, and the like.
The term "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.
The term "protease variant" means any protease molecule 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.
The term "specificity" means the ability of an enzyme to recognize and convert preferentially certain substrates. The specificity of proteases, i.e. their ability to recognize and hydrolyze 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.
M JSubstrate_
M J Substrate _k JVai
M J Substrate _i
M JSubstrate k WT
This equation with "Var" = protease (e.g. Neprilysin) variant and "WT" = wild type (e.g. Neprilysin) protease describes the relative activities of a protease variant on
"Substrate i" and "Substrate k" in comparison to the wild-type protease. An increased specificity is expressed by ratios of 1.5, 2, 3, 4, 5, 7, 10, 20, 30, 40, 50, 100, 200 or higher. In practice, the reaction velocity kapp=(kcat/Km)*[E] ([E] = enzyme concentration) is measured. But since all measurements are done at the same enzyme concentration, the specificity as defined is independent of [E].
By enhanced specificity we mean that a variant enzyme is able to cleave amyloid beta (Αβ) peptides to a greater degree and/or other peptides (including ANP, BNP, angiotensin- 1, bradykinin, endothelin 1 , neuropeptide Y, neurotensin, adrenomedullin and insulin β-chain) to a lesser degree as compared to the wild-type enzyme.
By enhanced specificity for amyloid beta (Αβ), we mean that compared to wild type neprilysin, the variant neprilysin cleaves Αβι_40 and/or Αβι_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 β-chain.
The term "catalytic activity" describes quantitatively the conversion of a given substrate under defined reaction conditions and is proportional to kcat/Km.
The term "substrate" or "peptide substrate" comprises any peptide, oligopeptide, or protein molecule of any amino acid composition, sequence or length, and post-trans lational or chemically-modified forms of these molecules that contain a peptide bond that can be hydrolyzed catalytically by a protease. The peptide bond that is hydrolyzed is referred to as the "cleavage site".
The term "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.
The term "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.
The term "degrade", "degrading" or "degradation" refers to a process where one starting molecule is divided in two or more molecule(s). More specifically, the amyloid β 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.
The term "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).
The term "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 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.
The term "connect" 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. For example, 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.
The term "covalently connected" means a chemical link between two atoms in which electrons are shared between them. Examples of 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.
The term "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 β peptide in detail.
In some embodiments, 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) 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.
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. Alanine A Ala
Cysteine C Cys
Aspartic acid D Asp
Glutamic acid E Glu
Phenylalanine F Phe
Glycine G Gly
Histidine H His
Isoleucine I He
Lysine K Lys
Leucine L Leu
Methionine M Met
Asparagine N Asn
Proline P Pro
Glutamine Q Gin
Arginine R Arg
Serine S Ser
Threonine T Thr
Valine V Val
Tryptophan w Trp
Tyrosine Y Tyr
Cysteine C Cys
In addition to the specific amino acid variations and nucleic acids encoding the variations, 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. Thus, in general, 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.
It is well known to one of ordinary skill in the art that the genetic code is degenerate, that a particular amino acid can be encoded by more than one codon triplet. Therefore, the 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. To express a polypeptide recombinantly, 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).
To create a polynucleotide sequence that encodes a protease or derivative thereof fused to another polypeptide, 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.
To express the polypeptide standard recombinant DNA expression methods can be used (see, for example, Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). For example, 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
promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. 5,168,062 by Stinski, U.S. 4,510,245 by Bell et al. and U.S. 4,968,615 by Schaffner et al. 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. 5,179,017, by Axel et al). 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. For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate and the 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. In some embodiments, 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.
The 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,
Bio/Technology 10: 163-167 (1992). 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. In addition to
prokaryotes, eukaryotic microbes 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. Where the polypeptide is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. The polypeptide composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography.
In general, the 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. The
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.
Pharmaceutical compositions according to the invention may be for administration by injection, or for oral, pulmonary, nasal, transdermal, sub-cutaneous or other forms of administration. In general, the invention encompasses pharmaceutical compositions comprising effective amounts of a polypeptide of the invention together with
pharmaceutically acceptable diluents, preservatives, solubilisers, emulsifiers, adjuvants and/or carriers. Such 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. 18042) pages 1435-1712. The 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.
The polypeptides provided herein can be administered to a patient in need thereof. A variety of 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. Such modes of administration include oral, sublingual, topical, nasal, transdermal or parenteral routes. The term "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. In a preferred embodiment the polypeptides have an improved specificity and / or activity against Αβ peptide.
The following table lists relative activities of human neprilysin protease variants vs. wild type neprilysin on different substrates determined from the ratio of the two
corresponding kapp-values (see example 3).
For the purpose of exemplary illustration:
Protease variant G399V shows a 1.43-fold increased activity on Peptide- 1 (Αβ peptide derivative), a 1.21-fold increased activity on Peptide-2 (Αβ 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). With the specificity definition above comparing a protease variant with the wild type protease 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 (Αβ peptide derivative), a 3.99-fold increased activity on Peptide-2 (Αβ 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). With the specificity definition above comparing a protease variant with the wild type protease this variant G714K shows an approximate 35 -fold increased specificity for Peptide-1 vs. Peptide-13.
Table 3
Figure imgf000052_0001
Unless indicated otherwise, the 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. Thus, for example, G399 refers to the Glycine at position 399 in full length wild-type neprilysin.
The inventors have found that mutant neprilysin polypeptides that comprise two substitutions being at positions 399 and 714 were especially specific for Αβ 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.
According to the invention there is provided a 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. In a particular embodiment 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.
According to the invention there is provided 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. In particular embodiments the one or more optional substitutions are 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. In one particular embodiment 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. Preferably , 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.
Another embodiment encompasses a nucleic acid encoding an aforementioned polypeptide. A further embodiment is a vector comprising the aforementioned nucleic acid. Yet, 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.
In some embodiments, 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) 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 R A, 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.
As used herein, 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.
In another aspect of the present invention, there is provided a polypeptide, wherein M and A are linked together with a linker, L.
In another aspect of the present invention, there is provided a polypeptide, wherein L is selected from a peptide and a chemical linker. Therapeutic methods of the invention encompass a method for reducing Αβ peptide concentration, wherein said reduction of Αβ peptide is accomplished in plasma, or in cerebrospinal fluid (CSF), or in the CNS.
The 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 Αβ 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. To express a neprilysin variant polypeptide or derivative thereof recombinantly, 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).
In general, 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.
The DNA sequence of any portion of a polypeptide of the invention may be changed to codons more compatible with the chosen host cell. For E. coli, 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. Thus, in preferred embodiments, 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. In a more preferred embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Even more preferably, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Thus, preferred linkers are polyglycines (particularly (Gly) 4 (SEQ ID NO: 34), (Gly)5 (SEQ ID NO: 35)), poly(Gly-Ala), and polyalanines. A particularly useful linker is (Gly)5Ser (SEQ ID NO: 32) or (Gly)4Ser (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. In prophylactic applications, 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. In therapeutic applications, 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
prophylactic regime. It is predicted that 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.
According to a further aspect of the present invention there is provided an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a neprilysin variant with enhanced specificity for Αβ relative to an off-target (ηοη-Αβ) 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:
Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY (2002).
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. On incorporation of the primers, a mutated plasmid containing nicks is generated. Subsequently, 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).
Other procedures know in the art for creating, identifying and isolating mutants may also be used, such as, for example, gene shuffling or phage display techniques. According to another aspect of the invention there are provided isolated 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.
The 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.
Once isolated or synthesised, a variety of 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 baculo virus 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. As noted above, 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). Examples of packaging cell lines that might be used with these vectors 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. In certain embodiments there will be a cloning site or multiple cloning site situated between the promoter and the gene of interest. 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. In other embodiments 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. 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. Particular cell lines derived from mammalian species which may be used and which are commercially available include, 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).
Although it is preferred that mammalian expression systems are used for expression of the neprilysin variant polynucleotide sequence, it will be understood that other 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.
Expression and purification of the polypeptides of the invention can be easily performed using methods well known in the art (for example as described in Sambrook et al., ibid).
Thus, in another aspect, 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.
According to a further aspect of the invention there is provided 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.
According to a further aspect of the invention there is provided 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 combination of these techniques could be used.
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
transformation, ease of recovery of the fusion, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (such as E. coli sp.), yeast (such as
Saccharomyces sp.) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.
Next, the transformed host is cultured and purified. 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. Finally, 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. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105- 253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527.
The invention encompasses pharmaceutical 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, polygly colic acid, etc. or into liposomes. 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
Publishing Co., Easton, Pa. 18042) pages 1435-1712. The 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. Generally, 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.
In some embodiments, the present invention provides a method for the treatment of Αβ-related pathologies such as Downs syndrome and β-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.
Examples:
Example 1: Cloning
A human wt-s neprilysin sequence comprising the codons for aa51-aa749 (PDB numbering) was cloned into a yeast expression vector (pYES2 Invitrogen, SKU# V825-20; see SEQ ID NO:22). Alternative other 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. Alternatively 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
NEP-85A
5*GAC GTC CCA GAC TAT GCT TAc CCt TAc GAt GTa CCt GAt TAc GCa GGA TCC TAC GAT GAT GGT ATT TGC AAG (SEQ ID NO : 19)
NEP-24
5 ATA GTT TAG CGG CCG CTC ACC AAA CCC GGC ACT T (SEQ ID NO: 20) A second PCR was performed on the foregoing PCR amplification product using primers NEP-85B and NEP-24, introducing additionally Xhol and Notl restriction
endonuclease sites.
NEP-85B
5* GTA TCT CTC GAG AAA AGA GAG GCT GAA GCT TAT CCA TAT GAC GTC CCA GAC TAT GCT TAT CCA TAT GAC GTC CCA GAC TAT GCT TAC (SEQ ID NO: 21) Underlined sequence is Xhol site.
NEP-24
5 ATA GTT TAG CGG CCG CTC ACC AAA CCC GGC ACT T (SEQ ID NO: 20)
Underlined sequence is Notl site.
For ligation of PCR amplification product into the expression vector pYES2 containing a secretion leader, 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. Example 2: Expression and purification
Expression of mammalian neprilysin in yeast is described in the literature for
Schizosaccharomyces pombe and Pichia pasoris (Beaulieu et al. (1999), Oefner et al. (1999)). Using the construct described in Example 1 s neprilysin and variants with mutations were expressed in Saccharomyces cerevisiae YMR307w (EUROSCARF) cultured in SC-Media (YB-Yeast, Nitrogen Base (Becton, Dickinson, #291920), CSM-Ura (MPBio, #4511 -222), 0.5% casein hydrolysate, 0.2M HEPES (Merck, #1.010110.1000); pH7.0) with 2% galactose (Merck, #1.04061.1000) for induction of expression for 55-70h at 30°C (Fig. 4).
Purification of HA-tagged protease can be achieved by immunoaffinity
chromatography specific for the HA-tag (monoclonal Antibody HA. l 1, # MMS-101P) or alternatively for His-tagged protease by 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). In the latter case pre loading the protease in the yeast supernatant was re-buffered using a cross- filtration device (VIVAFLOW 200, 10k MWCO, Satorius, #512-4069).
Eluted chromatography samples were re-buffered into 50mM Hepes (sigma, #H4034),
300mM NaCl (Merck, #1.06404.5000), pH7, by dialysis or the use of desalting columns (Sephadex G-25, Amersham Pharmacia Biotech).
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
The kcat/kM ratio of a proteolytic activity is proportional to the apparent kinetic constant kapp of the determined substrate degradation and is proportional to kcat/Km*[E] ([E]=enzyme concentration). As all measurements are performed at the same enzyme concentration [E], thus the specificity as defines is independent of [E] eliminates from the calculation of relative kcat/Km ratios. This kapp 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.
Table 4
Substrate Label Amino acid sequence (SEQ ID NO:) Derivative of
Peptide- 1 Dy647 D AEFRHD S G YE VHHQKL VFF AED VG SNKG AI Αβ1-40
IGLMVGGVVK (SEQ ID NO: 8)
Peptide-2 Dy647 D AEFRHD S G YE VHHQKL VFF AED VG SNKG AI Αβ1-42
IGLMVGGVVIAK (SEQ ID NO:9)
Peptide-3 Dy505 SLRRSSCFGGRMDRIGAQSGLGCNSFRYK ANP
(SEQ ID NO: 10)
Peptide-4 Dy505 SPKMVQGSGCFGRKMDRISSSSGLGCKVLRR BNP
HK (SEQ ID NO: 11)
Peptide-5 Dy505 CDRVYIHPFHLK (SEQ ID NO: 12) Angiotensin
Peptide-6 Dy505 a) GCS S S SLMDKES VYFCHLDII WK(SEQ ID Endothelin
NO: 13) or
b) GSSCSSLMDKECVYFSHLDIIWK (SEQ ID
NO: 14)
Peptide-7 Dy505 CYPSKPDNPGEDAPAEDMARYYSALRHYINL Neuropeptide Y
Figure imgf000066_0001
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. After incubation of this assay at 37°C suitable for dynamic measurements (turnover of 5 to 90% of the substrate molecules) the assay was stopped by diluting the sample with an equal volume of 1.2μΜ Streptavidin (Calbiochem, #D36271), in the case of assays with peptide-3 or peptide-6 this solution contained lOmM DTT (Sigma, #117K0663) in addition. A typical incubation time for peptide- 1 and -2 was 21h, for peptide-4 and -7 24 h, for peptide- 10 6h, for peptide-3 and -6 2.5h and digests of peptide-5, -8, -13 were incubated for 40min. The anisotropy in the sample was measured in a MTP -reader with an appropriate setup of polarisation filters (Tecan infinite F500; filters: 485/20, 535/25, 625/35, 670/25). Peptides 1- 6a, 6b, 7, 8, 10 and 13 correspond to SEQ ID NO: 8 - 18, respectively. Table 3 depicts the specific activities of a variety of mutants against each of the peptides substrates shown in Table 4.
Example 4: Multiple substitution mutants
The specific activities against the various peptides that each of the mutants exhibited (Table 3) identified certain locations and particular substitutions as conferring enhanced activity on amyloid beta and reduced activity on the off-peptides. One of the most effective individual substitutions (in terms in increased activity on Αβ from the first set of experiments was found to be G714K, however other mutants exhibit a stronger decrease in activity on certain of the off-target peptides. It was postulated that combining the best individual substitutions might generate mutants with even greater activity on Αβ and less activity on the off-target-peptides. Accordingly, variants with a combination of mutations were generated (Table 5).
In 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 kapp- 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 Αβ vs. the off- peptides, such as peptide-5, -8, -13, -3, -6 and -10; see Table 6).
A particular embodiment, the G399V/G714K double mutant, shows an improved specificity for Αβ 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 5 - Mutants:
Figure imgf000067_0001
Table 6 - activity data
Peptid Peptid Peptid Peptide- Peptid Peptid Peptide- Peptid Peptid
e-1 e-5 e -8 13 e -3 e- 6 10 e- 7 e- 4
fold fold fold fold fold fold fold fold fold
CLONE G714 G714 G714 G714K G714 G714 G714K G714 G714
Figure imgf000068_0001
On the basis of Bl (G399V/G714K double mutant) further variants with a
combination of additional substitutions were generated (Table 7). Table 8 lists relative activities of the certain protease variants vs. Bl on different substrates determined as ratio of the two corresponding kapp-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. Hence all show an improved specificity for peptide- 1 and -2 vs. peptide-6, -5 and -3 compared to Bl. The differences in activities on peptide-7, -4, -13 and -8 between the variants are not significant in many cases, but they all are lying in the range of the respective activities of Bl, hence the specificity of these variants for peptide- 1 and -2 vs. peptide-7, -4, -13 and -8 is improved compared to Bl .
Table 7. Sequences of variants CLONE 227 228 247 399 419 590 593 596 600 709 714 718 No. of
Mutations wt S R F G E D G F G D G I 0
Bl V K 2
CI V M V K 4
C2 R G L V M F P D K 9
C3 R G L V F P D K 8
C4 R G V M F V V K 8
C5 R G V M F P D K 8
C6 R G V M M V V K 8
C7 R G V F V L K 7
C8 R G V F W K 6
C9 R G V M V L K 7
CIO R L V M M V P D K 9
Cl l R L V M M P W K 8
C12 R L V M V K 6
C13 G L V M M V K 7
C14 G L V M M D K 7
C15 G V M M V D K 7
C16 G V M M V P W K 8
C17 G V M M V P K 7
C18 G V M M V W K 7
C19 G V M M P L K 7
C20 G V F V L K 6
C21 L V M M V K 6
C22 V M F P D K 6
C23 V F P D K 5
Variants with particularly interesting profiles are shown in Table 8.
Peptide- Peptide- Peptide- Peptid Peptid Peptid Peptid Peptid Peptide- Peptid
1 2 6a e-6b e-5 e-3 e-7 e-4 13 e-8 fold fold fold fold fold fold
G399 G399 G399 G399 G399 G399 fold fold V / V / V / V / V / V /
G399V / G399V / fold G714 G714 G714 G714 G714 fold G714
G714K G714K G399V / K K K K K G399V / K
CLON (= B1) activity G714K activit activit activit activit activit G714K activit
E activity activity y y y y y activity y
Bl 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
CI 1.28 1.23 0.97 0.76 1.02 0.36 0.90 0.51 0.52 0.62
C2 1.06 0.88 0.14 0.10 0.19 0.24 0.47 0.72 0.68 0.34
C3 0.86 0.81 0.18 0.09 0.15 0.24 0.64 0.79 0.85 0.37
C4 2.41 2.43 0.37 0.29 0.27 0.24 0.44 0.44 0.62 0.40
C5 1.95 1.92 0.23 0.18 0.22 0.24 0.72 0.74 0.63 0.41
C6 2.35 2.36 0.28 0.19 0.29 0.24 0.61 1.26 0.85 0.37
C7 2.77 2.83 0.42 0.28 0.34 0.24 0.47 0.96 0.43 0.46
C8 1.86 1.64 0.32 0.24 0.27 0.24 0.69 1.17 0.43 0.36
C9 2.33 2.44 0.31 0.21 0.22 0.24 0.52 0.67 0.52 0.46
CIO 2.35 2.60 0.27 0.19 0.17 0.24 0.49 0.80 0.68 0.34
Cll 2.19 2.14 0.28 0.11 0.19 0.24 0.66 0.49 0.61 0.35
C12 2.11 2.04 0.32 0.18 0.26 0.24 0.84 0.79 0.83 0.43
C13 1.78 1.56 0.30 0.18 0.18 0.24 0.36 0.91 0.39 0.35
C14 1.98 2.01 0.30 0.24 0.20 0.24 0.59 0.72 0.71 0.43
C15 2.52 2.72 0.46 0.24 0.25 0.24 0.75 0.70 0.44 0.52
C16 2.24 2.19 0.35 0.23 0.21 0.24 0.70 0.70 0.44 0.40
C17 3.28 3.13 0.47 0.33 0.27 0.24 0.57 0.70 0.49 0.33
C18 2.56 2.29 0.36 0.26 0.16 0.24 0.67 0.59 0.50 0.73
C19 2.33 2.34 0.29 0.19 0.15 0.24 0.34 0.67 0.33 0.29
C20 2.18 2.25 0.37 0.26 0.43 0.24 0.72 1.21 0.32 0.41
C21 2.30 2.76 0.41 0.24 0.22 0.24 0.50 0.65 0.74 0.49
C22 2.85 2.72 0.40 0.28 0.18 0.24 0.76 0.98 0.70 0.43
C23 2.39 2.42 0.41 0.23 0.32 0.34 0.75 1.15 0.45 0.55 mean
error 19% 24% 18% 18% 54% 180% 35% 90% 79% 61%
Table 8.
Figure 5 also illustrates the cleavage of five of the peptide substrates (peptide 5 = angiotensin; peptide 3 = ANP; peptide 6a = one of the endothelin peptides; peptide 1 = ABi_40 ; and, peptide 2 = ABi_42) by various mutants relative to the G399V/G714K parent mutant, illustrating the increased cleavage of the amyloid beta peptides (ABi_4o and ABi_42) and reduced cleavage of the three off-peptides (ANP, endothelin and angiotensin).
Two of these mutants (C22 and CIO) were selected as parent molecules and further mutants with one or more of D377G, A287S and G645Q were introduced therein.
Table 9.
Figure imgf000071_0001
Specificity data are shown in Tab e 10.
Peptide- 1 Peptide-2 Peptide-6a Peptide-5 Peptide-7 Peptide-4
fold
G399V / fold fold fold fold fold
G714K (= G399V / G399V / G399V / G399V / G399V /
Bl) G714K G714K G714K G714K G714K clone activity activity activity activity activity activity
Bl 1.00 1.00 1.00 1.00 1.00 1.00
CI 1.14 1.12 0.76 0.86 0.96 1.10
C22 2.29 2.27 0.30 0.08 0.57 1.07
Dl 1.11 1.24 0.12 0.08 0.61 0.95
D2 0.83 1.06 0.08 0.08 0.40 0.90
D3 3.11 3.42 0.47 0.10 0.95 1.04
D4 2.43 2.71 0.28 0.08 0.51 0.91
D5 2.94 3.37 0.35 0.08 0.90 0.90
CIO 2.05 2.39 0.28 0.08 0.68 0.83
D6 2.24 2.37 0.35 0.08 0.68 0.89
D7 0.88 1.18 0.13 0.09 0.66 0.91 D8 2.58 2.66 0.44 0.09 0.82 0.95
D9 1.07 1.11 0.06 0.08 0.43 0.86
D10 1.33 1.26 0.08 0.08 0.33 0.84
The data for representative clones in Table 10 is illustrated in Figure 6.
Example 5. Construction of the gene encoding the fusion protein Fc-Neprilysin variant, its expression and purification
A. Construction of Fc-neprilysin variant expression system
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) and the 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
pcDNA5/FRT/TO) or EF-l promoter (pEF5/FRT7V5-DEST). After all cloning steps, it is advisable to sequence the genes to verify that the correct sequence exists in the vector.
B. Expression of extra-cellular domain of NEP and fusion protein Fc-NEP in HEK293 cells
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-lxlO6 and with plasmid DNA at concentrations ranging from 0.3-0.8 μg/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.
C. Purification of expressed Fc-Neprilysin protein by ajfinity chromatography
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 KH2PO4, 8 mM Na2HP04-7H20, 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 μΐ 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 PD 10 Columns (GE Healthcare) .
Example 6. Degradation of amyloid β peptidel-40 in human plasma by neprilysin or neprilysin variants.
Degradation of human amyloid β peptidel-40 (Αβ40) and human amyloid β peptidel- 42 (Αβ42) 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 μg/ml) or 5 μg/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 μΜ 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 Αβ40 and Αβ42 is analysed using a commercial ELISA kit obtained from Biosource/Invitrogen (Αβ1-40) or Innogenetics (Αβΐ- 42). Example 7. Degradation of amyloid β peptidel-40 in C57BL/6 mice by Neprilysin or neprilysin variants (in vivo studies).
In vivo studies in C57BL/6 mice are performed in order to test the in vivo efficacy of neprilysin or neprilysin variants. The read-outs are soluble amyloid beta (Αβ) levels in plasma as well as plasma drug concentration. The C57BL/6 mice, 17-21g, are weighed and given single intravenous administration of appropriate doses. 5 animals are included in each time point and 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. Αβ40 levels in plasma are analyzed by commercial ELISA kit obtained from Biosource. All plasma samples are analysed to determine drug exposure with Mesoscale technology.
Example 8. Degradation of mouse amyloid β peptidel-40 in mouse C57BL/6 plasma by neprilysin or neprilysin variants.
Degradation of mouse amyloid β peptidel-40 (Αβ40) 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. The experiments are performed on a pool of plasma, neprilysin or neprilysin variants (0.1-300 μg/ml) or 5 μg/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 μΜ
phosphoramidon (BIOMOL) or 2 mM 1, 10-phenantro line (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 mouse Αβ40 is analysed using a commercial ELISA kit obtained from Biosource (Αβ1-40). Example 9. Treatment of APPswF-transgenic mice with neprilysin or neprilysin variants and subsequent analysis on Αβ levels in plasma and CNS.
In vivo studies in APPSwE-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 (Αβ) 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.
Single administration of appropriate doses are given to transgenic mice (25-27 weeks of age), including 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 μΐ/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. Αβ40 and Αβ42 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.
Repeated administration of appropriate doses are given to transgenic mice (25-27 weeks of age at study start), including 30 animals for each group. Each time point has its own vehicle group. During the time of the study, 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
approximately 3000 x g at +4°C. Drug concentration and immunogenicity are measured in the plasma during the study period with mesoscale technology. At termination, blood is withdrawn from anaesthetized mice by heart puncture into pre-chilled microtainer tubes containing EDTA and plasma is prepared as described above. 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. After 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 μΐ/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 μΐ/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. Αβ40 and Αβ42 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 β mouse peptidel-40., amyloid β human peptidel- 40 and amyloid β human peptidel-42 in Tg2576 mouse plasma by neprilysin or neprilysin variants.
Degradation of mouse amyloid β peptidel-40 (Αβ40), human amyloid β peptidel-40 (Αβ40) and human amyloid β peptidel-42 (Αβ42) by neprilysin is investigated using heparinised plasma from female Tg2576 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. The experiments are performed on a pool of plasma, neprilysin or neprilysin variants (0.1-300 μg/ml) or 5 μg/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 μΜ
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 Αβ40 and Αβ42 is analysed using a commercial ELISA kit obtained from Biosource/Invitrogen (Αβ1-40) or Innogenetics (Αβ1-42).
Example 11. Degradation of amyloid β peptides in Sprague Dawley rats by neprilysin or neprilysin variants (in vivo studies). 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 (Αβ) 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 centrifugation. Plasma is prepared by centrifugation 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. After sampling, 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 μΐ/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. Soluble Αβ40 in plasma as well as soluble Αβ40 and Αβ42 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 β rat peptidel-40 in rat plasma by neprilysin or neprilysin variants.
Degradation of rat amyloid β peptidel-40 (Αβ40) 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 μg/ml) or 5 μg/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 μΜ
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 Αβ40 is analysed using a commercial ELISA kit obtained from
Biosource/Invitrogen (Αβ1-40). Example 13. Degradation of amyloid β peptides in Guinea pigs by neprilysin or neprilysin variants (in vivo studies).
In vivo studies in male Dunkin Hartley (DH) Guinea pigs are performed in order to test the in vivo efficacy of neprilysin or neprilysin variants. The read-outs are soluble amyloid beta (Αβ) levels in plasma, csf and brain as well as plasma drug concentration. The male DH guinea pigs (200-4000 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. CSF is aspirated from the cisterna magna from anaesthetized animals 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. Immediately after the CSF sampling, 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 μΕ/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 Αβ40 and Αβ42 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 Αβ levels in plasma,
The objective with this study is to evaluate the time and dose-response effect of neprilysin variants in plasma of female APPswE-tg mice after acute intravenous treatment. The specific purpose is to find an effect on plasma Αβ40 and Αβ42. 25-31 weeks old female APPSwE-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. Αβ40 and Αβ42 levels in plasma are analyzed by commercial ELISA kit obtained from Biosource and Innogenetics, respectively.
Example 15. EEG study in APPswF-transgenic mice with Neprilysin or neprilysin variants (in vivo studies).
The studies in 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.
Signals are interpolated to 128Hz and band-passed filtered 1-64 Hz (second order Butterworth). Power spectral densities (PSDs) are calculated with Fast Fourier Transform (FFT) to convert the waveform data into a power spectrum with 0.5Hz resolution (FFT size of 256) using Spike2 (Cambridge 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. To calculate the dominant frequency (DF) at a specific Hz interval, 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. An average DF is calculated for each mouse from each DF in each 30 second epoch (3600/30 s=120 epochs) in its recording. The average DF represents the average of the DFs from all the mice in each group.
Example 16. In vivo testing of protease variants
1. Dementia
The object recognition task
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. In 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
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
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. In the habituation sessions and the retention session 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.
In the shock session 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, that 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 in 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.
The Contextual Fear Conditioning task
Contextual fear conditioning measures aversive memory in rats and mice. An observation box with distinctive contextual features are 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).
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. no movement of the animal) and is used as a measure of memory of the previously aversive event in this context. 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. Sometimes, 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. After the fourth trial of the fifth daily session, 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. In the probe trial, all animals start from the same start position, opposite to the quadrant where the escape platform had been positioned during acquisition.
Four different measures are taken to evaluate the performance of an animal during acquisition training: escape latency, travelled distance, distance to platform, and swimming speed. The following measures are evaluated for the probe trial: time (s) in quadrants and travelled distance (cm) in the four quadrants. The probe trial provides additional information about how well an animal learned the position of the escape platform. If an animal spends more time and swims a longer distance in the quadrant where the platform had been positioned during the acquisition sessions than in any other quadrant, one concludes that the platform position has been learned well.
In order to assess the effects of putative cognition enhancing protease variants, 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
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. In the first trial, the 'forced trial', either the left or right goal arm is blocked by lowering the guillotine door. 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. Then, 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 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. In the next variant, 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.
Postoperatively, 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 Anesthesio meter, 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
Hemodynamics in anesthetized rats
Male Wistar rats weighing 300-350 g (Harlan Winkelmann, Borchen, Germany) are anesthetized with thiopental "Nycomed" (Nycomed, Munich, Germany) 100 mg kg-1 i.p.
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
administered intravenously.
Hemodynamics in conscious SHR
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.
Single administration of test protease variant is performed intravenously. The animals of control groups only receive the vehicle. Before treatment, mean blood pressure and heart rate of treated and untreated control groups are measured.
Example 17. Construction of the gene encoding the lOHistidine tag (SEQ ID NO: 36) fused to a Neprilysin variant, its expression and purification
A. Construction of lOHis-neprilysin variant ('lOHis' disclosed as SEQ ID NO: 36) expression system
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
(pEF5/FRT/V5-DEST). After all cloning steps, it is advisable to sequence the genes to verify that the correct sequence exists in the vector. B. Expression of extra-cellular domain of NEP and fusion protein 10His-NEP ('lOHis' disclosed as SEQ ID NO: 36) in CHO cells
The lOHis-Neprilysin variant ('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-lxlO6 and with plasmid DNA at a concentration of 1 μg/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.
C. Purification of expressed lOHis-Neprilysin ('lOHis ' disclosed as SEQ ID NO: 36) protein by affinity chromatography
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). The column is equilibrated with 10 column volumes (CV) of 2 x PBS (5.4 mM KC1, 276 mM NaCl, 3 mM KH2P04, 16 mM Na2HP04-7H20, pH 7.4, Invitrogen). Cell culture media with expressed fusion protein (lOHis-Neprilysin ('lOHis' 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 ('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
Chromatography system (Explorer or Purifier, GE Healthcare). The protein is eluted in lx PBS 2.7 mM KC1, 138 mM NaCl, 1.5 mM KH2P04, 8 mM Na2HP04-7H20, 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 A. Construction of HSA-neprilysin variant expression system
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. 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 predicted molecular weight of approximately 147 kDa.
The complete gene (encoding the HSA-neprilysin variant) including the signal sequence is inserted into a suitable mammalian expression vector, such as pDEST12.2, pCEP4, pEAKlO, pEF5/FRT7V5-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-l promotor (pEF5/FRT/V5-DEST). After all cloning steps, it is advisable to sequence the genes to verify that the correct sequence exists in the vector.
B. Expression of extra-cellular domain of NEP and fusion protein HSA-NEP in CHO cells
The protein NEP (extra-cellular domain only) and HSA-NEP are 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-lxlO6 and with plasmid DNA at a concentration of 1 μg/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.
C. Purification of expressed HSA-Neprilysin protein by affinity chromatography
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). Cell culture media with expressed fusion protein (HSA-Neprilysin) 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 KH2P04, 8 mM Na2HP04-7H20, pH 7.4,
Invitrogen) and the fractions containing HSA-Neprilysin pooled, frozen and stored at -80C.
Example 19: Kinetic analysis of peptide cleavage by protease variants
Kinetic parameters Vmax, KM, kcat and kcat/KM for cleavage of peptides by protease variants were determined using a fluorescence polarisation assay that measured cleavage of synthetic peptide substrates labelled at the N- and C-termini with fluorescein and biotin, respectively. The biotin serves for increasing the molecular size of uncleaved molecules after addition of avidin, thereby increasing the assay window and the measurable signals. The peptides substrate are shown in Table 11.
Table 11. Synthetic peptide substrates. Peptides were labelled at their N-termini with fluorescein and their C-termini with Lys-biotin.
Peptide Sequence SEQ ID NO: Supplier
A-beta 1-40 D AEFRHD S G YE VHHQKL VFF AED V 37 Bachem
GSNKGAIIGLMVGGVV
Neurotensin QLYENKPR PYIL 38 Alta Bioscience
ANP SLRRSSCFGGRMDRIGAQSGLGCNS 39 Bachem
FRY
Endothelin- CSSSSLMDKESVYFCHLDIIW 40 Alta Bioscience la
Endothelin- SSCSSLMDKECVYFSHLDIIW 41 Alta Bioscience lb
GLP-1 H AEGTFT SD V S S YLEGQ AAKEFI A W 42 Alta Bioscience
LVKGRG
Angiotensin DRVYIHPFHL 43 Alta Bioscience
Bradykinin RPPGFSPFR 44 Alta Bioscience
GIP YAEGTFISDYSIAMDKIHQQDFVNW 45 Alta Bioscience
LLAQKGKKNDWKHNITQ
Somatostatin SANSNPAMAPRERKAGCKNFFWKT 46 Alta Bioscience 1-28 FTSC
Glucagon HSQGTFTSDYSKYLDSRRAQDFVQ 47 Alta Bioscience
WLMNT The assay was performed in a 96-well microtitre plate and contained 50 mM HEPES (pH 7.4, Sigma, # H3375), 150 mM NaCl, 0.05% (w/v) BSA (Sigma, # A9576), 1-200 μΜ 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.
Reactions were incubated at 37°C before being stopped at various time points between 2 and 360 min by transferring 5 aliquots to 245 50 mM HEPES buffer containing 2 mM 1,10-phenanethroline monohydrate (Sigma, # P9375) and 2 μΜ avidin (Invitrogen, # A2667). The fluorescence polarisation of the resulting solution was measured on a Victor plate reader and the amount of substrate cleaved was determined with reference to substrate-only controls with and without avidin. Initial rates were obtained by linear regression of the linear regions of time courses. Enzyme velocity was plotted as a function of substrate concentration and the Michaelis-Menten equation was used to fit the data, giving the parameters Vmax and KM. kcat was calculated by dividing Vmax by the enzyme concentration. Catalytic efficiency on a particular substrate was assessed by the second order rate constant kcat/KM, which was expressed in units of M"1 s"1.
Table 12 shows kcat/KM values for wild type neprilysin, the G399V/G714K mutant and the fusion of the G399V/G714K mutant with HSA. The ratios of the G399V/G714K variant and HSA fusion of the mutant kcat/KM values to those of wild type neprilysin are shown in Table 13. Catalytic efficiency on Αβ 1-40 was increased by a factor of 4.5 in the
G399V/G714K mutant, compared to wild type neprilysin. A similar increase in kcat/KM on Αβ 1-40 was observed with the HSA fusion of the G399V/G714K mutant. kcat/KM 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. Similar reductions in catalytic efficiency on these substrates were observed with the HSA fusion of the mutant. kcat/KM values for cleavage of endothelin- 1, GIP, and glucagon were reduced by 2-4-fold in the G399V/G714K mutant compared to wild type neprilysin. Similar reductions in catalytic efficiency on these substrates were observed with the HSA fusion of the mutant. Table 12 - kcat M values for peptide cleavage by neprilysin variants.
Figure imgf000090_0001
The kcat/KM values are averages of data from at least two independent experiments. For endothelin-1, the kcat/KM represents the average of values determined in duplicate for the 1 and lb iso forms.
Table 13 - Ratios oi f mutant and wild type neprilysin kcat/KM (
Peptide Ratio mutant vs wild type
derivative
Nep- HSA-Nep-
G399V/G714K G399V/G714K
A-beta 1-40 4.5 4.2
Neurotensin 0.003 0.004
ANP 0.088 0.15
Endothelin-1 0.29 0.21
GLP-1 0.27 0.25
Angiotensin 0.014 0.012
Bradykinin 0.00031 0.00017
GIP 0.34 0.28
Somatostatin 1-28 0.0073 0.0054
Glucagon 0.41 0.35

Claims

Claims
1. A polypeptide comprising a half-life modulator moiety (M) provided N-terminally to a neprilysin protease variant (A), wherein said half-life modulator moiety is human serum albumin HSA or a variant or fragment thereof, such as HSAC34S, and wherein said neprilysin protease variant comprises a variant of wild type human neprilysin extracellular catal ytic domain (SEQ ED NO: 2) wherein G399 is replaced by another naturally-occurring amino acid and / or G714 is replaced by another naturally-occurring amino acid.
2. A polypeptide according to claim 1 , comprising a neprilysin protease variant (SEQ ED NO: 50) wherein G399 is replaced by Valine (V) and/or G714 is replaced by Lysine ( ).
3. A polypeptide according to claim 1 , comprising a neprilysin protease variant (SEQ ED NO: 50) wherein G399 is replaced by Valine (V) and G714 is replaced by Lysine ( ).
4. A polypeptide according to any preceding claim, comprising a neprilysin protease variant (SEQ ID NO: 50) further comprising replacement of an amino acid at one or more of the foUowing positions: S227, R228, F247, E419, D590, G593, F596, G600, G645, D709 or I7l£.
5. A polypeptide according to claim 4, wherein said replacement of an amino acid at one or more positions is 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.
6. A polypeptide according to any preceding claim, wherein said half-life modulator moiety and neprilysin protease variant are joined by a linker peptide.
7. A polypeptide according to any preceding claim, wherein said linker peptide is a (Gly)sSer (SEQ ID NO: 32) or (Gly)4Ser (SEQ ID NO: 31) linker.
8. A polypeptide according to any preceding claim, wherein said linker peptide is (Gly)4S (SEQ ID NO: 31).
RECTIFIED SHEET (RULE 91)
ISA/EP
9. A polypeptide according to any preceding claim, comprising a pro-HSA amino acid sequence positioned N-terminally to the neprilysin protease variant.
10. A polypeptide according to any preceding claim, comprising a leader amino acid sequence positioned N-terminally to the neprilysin protease variant.
11. A polypeptide according to any preceding claim, comprising a leader amino sequence acid positioned N-terminally to a pro-HSA sequence.
12. A polypeptide according to any preceding claim, wherein the leader amino acid sequence is a HSA leader amino acid sequence or an IgG leader amino acid sequence.
13. A polypeptide according to any preceding claim having a greater specificity for an Αβ peptide compared to wild type human neprilysin.
14. A nucleic acid encoding a polypeptide of any one of claims 1 to 13.
15. A vector comprising the nucleic acid of claim 14.
16. A host cell comprising the vector of claim 15.
17. A method for producing a polypeptide according to any one of claims 1 to 13, wherein the method comprises the following steps:
a. cultaring the host cell of claim 16 under conditions suitable for the expression of the polypeptide; and
b. recovering the polypeptide from the host cell culture.
18. A pharmaceutical composition comprising a polypeptide of any one of claims 1 to 13.
19. A method for treating a human neprilysin substrate-related disease, such as an Αβ-related pathology, such as Alzheimer's disease, comprising administering to a patient in need thereof
RECTIFIED SHEET (RULE 91)
ISA/EP a therapeutically effective dose of a polypeptide according to any one of claims 1 to 13, whereby a symptom of the human nepnlysin substrate-related disease is ameliorated.
20. A polypeptide according to any one of claims 1 to 13 for use as a medicament for a human nepnlysin substrate-related disease, such as an Αβ-related pathology, such as Alzheimer's disease.
21. A polypeptide according to any one of claims 1 to 13 for use to prevent and/or treat an Αβ-related pathology such as Alzheimer's disease.
RECTIFIED SHEET (RULE 91)
ISA/EP
PCT/EP2010/070615 2010-06-21 2010-12-22 Protease variants of human neprilysin WO2011160732A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/EP2011/060385 WO2011161127A1 (en) 2010-06-21 2011-06-21 Protease variants of human neprilysin

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
USPCT/US2010/039392 2010-06-21
PCT/US2010/039392 WO2010148413A2 (en) 2009-06-19 2010-06-21 Protease variants

Publications (1)

Publication Number Publication Date
WO2011160732A1 true WO2011160732A1 (en) 2011-12-29

Family

ID=43880972

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2010/070615 WO2011160732A1 (en) 2010-06-21 2010-12-22 Protease variants of human neprilysin

Country Status (1)

Country Link
WO (1) WO2011160732A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2832854A1 (en) * 2013-08-02 2015-02-04 F. Hoffmann-La Roche AG Method for improving the recombinant expression of a polypeptide by C-terminal fusion to human neprilysin
WO2015014884A1 (en) * 2013-08-02 2015-02-05 F. Hoffmann-La Roche Ag Therapeutic fusion protein

Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3941763A (en) 1975-03-28 1976-03-02 American Home Products Corporation PGlu-D-Met-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH2 and intermediates
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4510245A (en) 1982-11-18 1985-04-09 Chiron Corporation Adenovirus promoter system
US4634665A (en) 1980-02-25 1987-01-06 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4968615A (en) 1985-12-18 1990-11-06 Ciba-Geigy Corporation Deoxyribonucleic acid segment from a virus
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
US5179017A (en) 1980-02-25 1993-01-12 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US6204023B1 (en) 1985-11-01 2001-03-20 Xoma Ltd. Modular assembly of antibody genes, antibodies prepared thereby and use
US20030083277A1 (en) 2000-02-24 2003-05-01 Hersh Louis B. Use of insulin degrading enzyme (IDE) for the treatment of alzheimer's disease in patients
US20030165481A1 (en) 2000-02-24 2003-09-04 Hersh Louis B. Amyloid peptide inactivating enzyme to treat Alzheimer's disease
WO2005123119A2 (en) 2004-06-10 2005-12-29 Catalyst Biosciences, Inc. Administration of neutral endopeptidase to treat inflammatory bowel disease
WO2007040437A1 (en) 2005-10-03 2007-04-12 Astrazeneca Ab Fusion proteins having a modulated half-life in plasma
WO2008118093A1 (en) 2007-03-28 2008-10-02 Astrazeneca Ab Fusion protein capable of degrading amyloid beta peptide
WO2010148413A2 (en) * 2009-06-19 2010-12-23 Medimmune, Llc Protease variants

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3941763A (en) 1975-03-28 1976-03-02 American Home Products Corporation PGlu-D-Met-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH2 and intermediates
US5179017A (en) 1980-02-25 1993-01-12 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4634665A (en) 1980-02-25 1987-01-06 The Trustees Of Columbia University In The City Of New York Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4510245A (en) 1982-11-18 1985-04-09 Chiron Corporation Adenovirus promoter system
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
US6204023B1 (en) 1985-11-01 2001-03-20 Xoma Ltd. Modular assembly of antibody genes, antibodies prepared thereby and use
US4968615A (en) 1985-12-18 1990-11-06 Ciba-Geigy Corporation Deoxyribonucleic acid segment from a virus
US20030083277A1 (en) 2000-02-24 2003-05-01 Hersh Louis B. Use of insulin degrading enzyme (IDE) for the treatment of alzheimer's disease in patients
US20030165481A1 (en) 2000-02-24 2003-09-04 Hersh Louis B. Amyloid peptide inactivating enzyme to treat Alzheimer's disease
WO2005123119A2 (en) 2004-06-10 2005-12-29 Catalyst Biosciences, Inc. Administration of neutral endopeptidase to treat inflammatory bowel disease
WO2007040437A1 (en) 2005-10-03 2007-04-12 Astrazeneca Ab Fusion proteins having a modulated half-life in plasma
WO2008118093A1 (en) 2007-03-28 2008-10-02 Astrazeneca Ab Fusion protein capable of degrading amyloid beta peptide
WO2010148413A2 (en) * 2009-06-19 2010-12-23 Medimmune, Llc Protease variants

Non-Patent Citations (49)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", 1989, GREENE PUBLISHING ASSOCIATES
"Current Protocols in Molecular Biology", 2002, JOHN WILEY & SONS
"Current Protocols in Protein Science", 1996, JOHN WILEY & SONS, pages: 9.4,9.5
"Current Protocols in Protein Science", 1999, JOHN WILEY & SONS
"Molecular Cloning; A Laboratory Manual", 1989, COLD SPRING HARBOR
"Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING CO., pages: 1435 - 1712
BARD ET AL., NATURE MEDICINE, vol. 6, no. 8, 2000, pages 916 - 919
BARROS ET AL., BIOL. CHEM., vol. 388, 2007, pages 447 - 455
BEAUMONT ET AL., J. BIOL. CHEM., vol. 266, 1991, pages 214 - 220
BEAUMONT ET AL., J. BIOL. CHEM., vol. 267, 1992, pages 2138 - 41
BORCHELT D. R. ET AL., NEURON, vol. 17, 1996, pages 1005 - 1013
CARTER ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 163 - 167
CITRON M. ET AL., NEUROBIOL. DIS., vol. 5, 1998, pages 107 - 116
DAVIS ET AL., BIOCHEM. INTL., vol. 10, 1985, pages 394 - 414
DEMATTOS, PNAS, vol. 98, 2001, pages 8850 - 8855
DION ET AL., FEBS LETT., vol. 411, 1997, pages 140 - 144
DUFF K. ET AL., NATURE, vol. 383, 1996, pages 710 - 713
ERICKSON ET AL.: "The Proteins", vol. 2, 1976, pages: 257 - 527
FINN ET AL.: "The Proteins", vol. 2, 1976, pages: 105 - 253
GLABE, C., NAT. MED., vol. 6, 2000, pages 133 - 134
GOEDDEL: "Methods in Enzymology", vol. 185, 1990, ACADEMIC PRESS, article "Gene Expression Technology"
GRAY, W., MOLECULAR AND CELLULAR ENDOCRINOLOGY, vol. 288, 2008, pages 52 - 62
GUAN HANJUN ET AL: "Peripherally Expressed Neprilysin Reduces Brain Amyloid Burden: A Novel Approach for Treating Alzheimer's Disease", JOURNAL OF NEUROSCIENCE RESEARCH, WILEY-LISS, US, vol. 87, no. 6, 1 May 2009 (2009-05-01), pages 1462 - 1473, XP002589911, ISSN: 0360-4012 *
HAMA EMI ET AL: "Effects of neprilysin chimeric proteins targeted to subcellular compartments on amyloid beta peptide clearance in primary neurons", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, INC, US, vol. 279, no. 29, 16 July 2004 (2004-07-16), pages 30259 - 30264, XP002523061, ISSN: 0021-9258, [retrieved on 20040420], DOI: DOI:10.1074/JBC.M401891200 *
KIM ET AL., J. BIOL. CHEM., vol. 267, 1992, pages 12330 - 35
KLAPDOR ET AL.: "A low cost method to analyze footprint patterns", J. NEUROSCI. METHODS, vol. 75, 1997, pages 49 54
LEISSRING ET AL., JBC., vol. 278, 2003, pages 37314 - 37320
LU B. ET AL., ANN. N.Y. ACAD. SCI., vol. 780, 1996, pages 156 - 163
LU B. ET AL., J. EXP. MED., vol. 181, 1995, pages 2271 - 2275
MARIE-CLAIRE, PROTEINS, vol. 39, 2000, pages 365 - 371
MATSUOKA ET AL., J. NEUROSCIENCE, vol. 23, 2003, pages 29 - 33
MERRIFIELD, J. AM. CHEM. SOC., vol. 85, 1963, pages 2149
MERRIFIELD: "Chem. Polypeptides", 1973, pages: 335 - 61
OEFNER ET AL., J. MOL. BIOL., vol. 296, 2000, pages 341 - 9
PONS, J. ET AL., CURRENT OPINION IN INVESTIGATIONAL DRUGS, vol. 5, 2004, pages 957 - 962
R. J. KAUFMAN; P. A. SHARP, MOL. BIOL., vol. 159, 1982, pages 601 - 621
RADEMAKER M.T.; RICHARDS A.M., CLINICAL SCIENCE, vol. 108, 2005, pages 23 - 36
REID IAN A.: "Basic and Clinical Pharmacology", 1998, THE MCGRAW-HILL COMPANIES, article "Vasoactive Peptides"
ROQUES B. P. ET AL., PHARMACOL. REV., vol. 45, 1993, pages 87 - 146
SAHLI ET AL., HELV.CHIM.ACTA., vol. 88, 2005, pages 731
SARRET; KITABGI: "Encyclopedia of Neuroscience", 2009, pages: 1021 - 1034
SCHEUNER D. ET AL., NAT. MED., vol. 2, 1996, pages 864 - 870
SELKOE, D. J., NATURE, vol. 399, 1999, pages A23 - A31
SHIROTANI K ET AL: "Neprilysin degrades both amyloid beta peptides 1-40 and 1-42 most rapidly and efficiently among thiorphan- and phosphoramidon-sensitive endopeptidases.", THE JOURNAL OF BIOLOGICAL CHEMISTRY 15 JUN 2001 LNKD- PUBMED:11278416, vol. 276, no. 24, 15 June 2001 (2001-06-15), pages 21895 - 21901, XP002634357, ISSN: 0021-9258 *
SOTO, C.; CASTANO, M., BIOCHEM. J., vol. 314, 1996, pages 701 - 707
STEWART; YOUNG, SOLID PHASE PEPTIDE SYNTHESIS, 1969
URLAUB; CHASIN, PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4216 - 4220
VASSAR, R. ET AL., SCIENCE, vol. 286, 1999, pages 735 - 41
YOUNKIN S. G., ANN. NEUROL., vol. 37, 1995, pages 287 - 288

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2832854A1 (en) * 2013-08-02 2015-02-04 F. Hoffmann-La Roche AG Method for improving the recombinant expression of a polypeptide by C-terminal fusion to human neprilysin
WO2015014884A1 (en) * 2013-08-02 2015-02-05 F. Hoffmann-La Roche Ag Therapeutic fusion protein
CN105431203A (en) * 2013-08-02 2016-03-23 豪夫迈·罗氏有限公司 Therapeutic fusion protein

Similar Documents

Publication Publication Date Title
WO2011161127A1 (en) Protease variants of human neprilysin
US20210277378A1 (en) Coagulation factor ix compositions and methods of making and using same
US20080274096A1 (en) Fusion Proteins Having a Modulated Half-Life in Plasma
AU2008230177B2 (en) Fusion protein capable of degrading amyloid beta peptide
AU2005235635B2 (en) Bone delivery conjugates and method of using same to target proteins to bone
US20120237496A1 (en) Protease variants
WO2011160732A1 (en) Protease variants of human neprilysin

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10809151

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10809151

Country of ref document: EP

Kind code of ref document: A1