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WO2007118889A1 - Protéase spécifique pour l'inactivation du facteur de nécrose tumorale alpha humain - Google Patents

Protéase spécifique pour l'inactivation du facteur de nécrose tumorale alpha humain Download PDF

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Publication number
WO2007118889A1
WO2007118889A1 PCT/EP2007/053788 EP2007053788W WO2007118889A1 WO 2007118889 A1 WO2007118889 A1 WO 2007118889A1 EP 2007053788 W EP2007053788 W EP 2007053788W WO 2007118889 A1 WO2007118889 A1 WO 2007118889A1
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Prior art keywords
substituted
seq
protease
sdr
amino acid
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PCT/EP2007/053788
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English (en)
Inventor
Ulrich Kettling
Oliver Hesse
Peter Scholz
Uwe Gritzan
Georg Zeidler
Rolf Kalmbach
André Koltermann
Ulrich Haupts
Markus Rarbach
Jan Tebbe
Christian Votsmeier
Andreas Scheidig
Wayne M. Coco
Antonio Da Silva
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Direvo Biotech Ag
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Priority claimed from EP06112706A external-priority patent/EP1847600A1/fr
Application filed by Direvo Biotech Ag filed Critical Direvo Biotech Ag
Publication of WO2007118889A1 publication Critical patent/WO2007118889A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • 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
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is directed to specific proteases, fragments and derivatives thereof that specifically inactivate tumour necrosis factor-alpha, methods for their preparation, pharmaceutical and diagnostic compositions comprising such a protease.
  • the proteases of the invention are useful for the treatment of clinical conditions resulting from the detrimental activity of tumour necrosis factor.
  • the invention further relates to nucleotide sequences thereof encoding the protease, as well as vectors and hosts containing such sequences.
  • tumour necrosis factor-alpha TNF-alpha, TNF ⁇
  • TNF-alpha The tumour necrosis factor-alpha (TNF-alpha, TNF ⁇ ) is a homotrimeric protein (Eck et al., Journal of Biological Chemistry, 264(29) : 17595- 17605 ( 1989)) expressed primarily by cells of the im m une system , namely macrophages and monocytes. Exposure of host cells to antigen, cytokines, infectious pathogens, or stress signals initiate signalling pathways that lead to the activation of transcription factors in the nucleus that orchestrate the tightly regulated induction of TNF-alpha expression.
  • TNF-alpha is a pleiotropic, m ultifunctional cytokine that mediates key roles in acute and chronic inflam mation, cell growth, antiviral activity, vasculature and extracellular matrix remodelling, anti-tumour responses, pathogenesis of many infections and bone resorption, among others.
  • the primary biological function of TNF- alpha is that of a pro-inflam matory cytokine (Tracey et al., Annual Review in Medicine, 45:491 -503 ( 1994)) .
  • TNF-alpha exerts its activity by binding to two structurally distinct TNF receptors, TNF- R1 and TNF- R2, on effector cells, triggering signalling cascades that include TRAF-2 and the more downstream kinases I KK, p38, ERK, and JNK. These signals in turn mediate the activation of the target cell effector functions, namely cytokine expression such as l nterleukin ( I L) - 6 , granulocyte-macrophage colony-stim ulating factor (GM- CSF) and I L- 8, cellular proliferation and apoptosis among others.
  • cytokine expression such as l nterleukin ( I L) - 6 , granulocyte-macrophage colony-stim ulating factor (GM- CSF) and I L- 8, cellular proliferation and apoptosis among others.
  • TNF-alpha IL-6 IL-6
  • TNF-alpha human TNF-alpha
  • hTNF ⁇ a key role for hTNF ⁇ in the initiation and/or perpetuation of the inflam matory processes in RA has been confirmed by several studies in pharmacologically relevant experimental animal models and validated clinically in RA, CD and psoriatic patients treated with biologicals that neutralize its activity. These studies elucidated the role of the cytokine in the induction of other pro- inflam matory cytokine cascades by macrophages and fibroblasts, which consequently lead to chronic inflam mation. Therefore, an anti- hTNF ⁇ treatment is expected to affect a broad range of physiological networks. Significant efforts have been applied to the development of medicinal com pounds that specifically modulate the activity of hTNF ⁇ in physiologically relevant disease settings.
  • Etanercept represents one such approach to targeting hTNF ⁇ for neutralization of its disease inducing activity.
  • This hTNF ⁇ antagonist consists of two recombinant copies of the human soluble TNF- receptor I l (TNF- R2, p75) fused to the Fc portion of human IgGL
  • the Fc component contains the CH2 domain, the C H 3 domain and hinge region, but not the C N I domain of IgGL Etanercept is produced by recombinant methods in a Chinese hamster ovary (CHO) mammalian cell expression system .
  • Etanercept binds both hTNF ⁇ and human lym photoxin-alpha but it does not fix com plement or lyse cells.
  • the terminal half- life is approximately three days, which is half that of infliximab.
  • I nfliximab (Rem icade ® ) is a chimeric monoclonal antibody in which the murine constant region has been replaced by human equivalent sequences. It neutralizes the biological activity of hTNF ⁇ by binding to both the soluble and transmembrane forms of hTNF ⁇ and inhibits binding of hTNF ⁇ to its receptors (Reimold A.M., Current Drug Targets- I nflam mation & Allergy 1 : 377-392 (2002)) .
  • I n addition to receptor binding antagonism infliximab is also capable of directly inducing apoptosis of membrane hTNF ⁇ expressing cells.
  • Adalimumab (Hum ira ® ) is a fully human antibody generated by phage-display technology, resulting in an antibody with human heavy- and light-chain variable regions, and human imm unoglobulin IgGI - K constant regions.
  • Adalim umab also binds to hTNF ⁇ and blocks its interaction with the TNF receptor and lyse hTNF ⁇ -expressing cells in the presence of complement.
  • the half- life of Adalim umab is 1 0-20 days, which is longer than non- human antibodies (Bain B. and Brazil M., Nature Reviews (2003) 2: 693-694) .
  • Adalim umab has been shown to directly induce apoptosis of membrane bound hTNF ⁇ .
  • im munoglobulin derived therapeutics The disadvantage of im munoglobulin derived therapeutics is that the directly interfere with the function of imm une effector cells, namely those expressing Fc- receptors such as macrophages which play a crucial role in innate im munity. This can cause im m une related adverse events seen with antibody based therapies, namely induction of cytokine release syndrome. There is also a potential risk of infectious diseases such as tuberculosis due to its interference with the innate imm une system . Additionally, hTNF ⁇ antibodies have been reported to cause the formation of anti- dsDNA antibodies (ADA), and after repeated treatment the cum ulative ADA incidence can be as high as 50% .
  • ADA anti- dsDNA antibodies
  • Demyelinising disease and aplastic anaem ia have been reported in a small number of I nfliximab and Etanercept treated patients (van Deventer, S.J.H., Gut 51 : 362-363 (2002); Kuruvilla J., Eur J Haematol. 71 (5) : 396 -8 (2003)) .
  • a likely cause is induction of apoptotic cell death via engagement of the membrane form of hTNF ⁇ or Fc- receptors.
  • acute exposure to genomic DNA induces an im mune response.
  • NBE ® provides a platform for the provision of proteases with novel functions that do not exist in the com ponents used as source material of such proteins.
  • the NBE ® platform is characterised by the re-engineering of a protease scaffold retaining its basic catalytic function and combining it with one or more specificity determ ining regions (SDFf) thus rendering it specific for a particular substrate molecule.
  • the one or more SDRs are located at sites within the protein scaffold that enables the resulting engineered protein to discriminate between at least one target substrate and one or more different substrates, which would not have been discriminated by the unmodified protease.
  • a clear benefit of target-specific proteases is the irreversible inactivation of the target substrate by hydrolysis whereas target neutralization by binding of therapeutic antibodies is reversible.
  • target-specific proteases have a higher efficiency due to the catalytic substrate turnover. Therefore, one protease molecule can inactivate a very high number of hTNF ⁇ molecules while antibodies exhibit only a stoichiometric neutralization by antigen- antibody binding.
  • Antibody- based therapeutics are produced in mam malian cell culture that is a costly process.
  • the objective of the present invention to provide a hTNF ⁇ targeting protease, preferentially expressed in a m icrobial expression system .
  • the invention provides proteases with a scaffold capable of hydrolyzing the protein hTNF ⁇ and one or more specificity determ ining regions (SDR ® ) to discrim inate hTNF ⁇ from other potential protein substrates.
  • SDR ® specificity determ ining regions
  • Another objective of the present invention is to provide nucleotide sequences or fragments thereof encoding the protease, as well as vectors and hosts containing such sequences.
  • the present invention is thus directed to (1) a protease capable of inactivating human tumour necrosis factor-alpha (hTNF ⁇ ) of SEQ ID NO: 11
  • a method for the preparation of the protease of (1) above which comprises culturing host cells of (4) above and isolating the protease from the culture;
  • a pharmaceutical or diagnostic composition comprising the protease of (1) above; (7) the use of the protease of (1) above for preparing a medicament for the treatment of clinical conditions resulting from the detrimental activity of tumour necrosis factor; and
  • Figure 1 shows a mass spectrum of the cleavage product of hTNF ⁇ verifying the targeted cleavage site.
  • Figure 2 deconvoluted mass spectrum of the cleavage product of hTNF ⁇ verifying the targeted cleavage site.
  • Figure 3 demonstrates the progression in specific activity of a panel of variants.
  • Figure 4 demonstrates that pegylation does not abrogate the activity of variant E.
  • Figure 5 demonstrates the specificity of different variants in a biochemical assay based on substrate competition
  • Figure 6 demonstrates the specificity of unpegylated variant E on different human serum proteins in comparison to the unspecific original trypsin scaffold.
  • Figure 7 demonstrates the activity of a pegylated variant of the protease in a human
  • TNF ⁇ transgenic mouse model of polyarthritis In this particular study, treatment was initiated after the onset of clinical expression (joint inflammation).
  • TRY1 HUMAN is human cationic trypsin (SEQ ID NO:1)
  • TRY2 HUMAN is human anionic trypsin (trypsin -2 precursor; SEQ ID NO:25)
  • TRY3_HUMAN is human mesotrypsin (trypsin -3 precursor; SEQ ID NO: 26)
  • protease means any protein molecule catalyzing the hydrolysis of peptide bonds. It includes naturally-occurring proteolytic enzymes, as well as protease variants obtained by site-directed or random m utagenesis, insertion, deletion, recombination and/or any other protein engineering method, that leads to novel proteases that differ in their amino acid sequence, physical characteristics and functional properties from the parent protease. It also com prises any fragment of a proteolytic enzyme, any molecular com plex, conjugated protease or fusion protein com prising one of the aforementioned proteases.
  • TNF-alpha specific protease describes a protein molecule catalyzing specifically the hydrolysis of human TNF-alpha. It is a non- naturally occurring proteolytic enzyme obtained by site-directed or random m utagenesis, insertion, deletion, recombination and/or any other protein engineering method, that leads to a TNF-alpha specific protease that differ in the am ino acid sequence by insertion, deletion or substitution of one or more am ino acids, physical characteristics and functional properties from the parent protease. It also com prises any fragment of a proteolytic enzyme, any molecular com plex, conjugated protease or fusion protein com prising one of the aforementioned protease.
  • human TNF-alpha refers to the cytokine with the structure and biological function as described further in for exam ple (Tracey et al. ( 1994) Tumor necrosis factor: a pleiotropic cytokine and therapeutic target; Annual Review in Medicine, 45:491 -503) . Its definition includes the endogenously expressed secreted soluble cytokine or its cell surface expression in a transmembrane form . Additionally, the term also includes recombinantly expressed and purified protein following standard procedures or purchased com suddenly from suppliers.
  • pharmacokinetics means the study of the way the hTNF ⁇ specific protease behaves in the body after adm inistration. It assesses the ADME ( absorption, distribution, metabolism and excretion) processes by exam ining the time course of drug concentration profiles in readily accessible body fluids such as blood, plasma, serum or urine.
  • ADME absorption, distribution, metabolism and excretion
  • pharmacodynam ics refers to the relationship between protease concentrations in e.g. plasma or at the effect site with the type and magnitude of pharmacological effects of the protease.
  • terminal half- life or more sim ply "half- life” is a parameter of pharmacokinetics and indicates the time necessary for the concentrations of detectable drug in any given body fluid, for exam ple blood serum , to decrease by 50 percent.
  • effcacy relates to the concentration of a compound needed to induce a certain degree of a physiological response. A compound X is said to possess higher efficiency than a comparator compound Y, if the concentration of X needed to induce the same response as Y is lower.
  • substrate or "peptide substrate” means any peptide, oligopeptide, or protein molecule of any am ino acid com position, sequence or length, that contains a peptide bond that can be hydrolyzed catalytically by a protease.
  • the peptide bond that is hydrolyzed is referred to as the "cleavage site”.
  • proteases that act selectively on a single peptide or protein among all possible peptide or protein substrates have a "high specificity”. Proteases that accept almost any peptide or protein substrate have a “low specificity”. Proteases with very low specificity are also referred to as “unspecific proteases”.
  • defined specificity refers to a certain type of specificity, i.e. to a certain target substrate or a set of certain target substrates that are preferentially converted versus other substrates.
  • protein scaffold or "scaffold protein” refers to a variety of primary, secondary and tertiary polypeptide structure s.
  • the scaffold is a protease structure capable of hydrolyzing a specific site in a protein target that leads to activation or inactivation of the protein target.
  • m utation refers to the substitution or replacement of single or m ultiple nucleotide triplets, insertions or deletions of one or more codons, homologous or heterologous recombination between different genes, fusion of additional coding sequences at either end of the encoding sequence, or insertion of additional encoding sequences or any combination of these methods, which result in a polynucleic acid sequence encoding the desired protein.
  • m utations also refers to all of the changes in the polypeptide sequence encoded by the polynucleic acid sequence modified by one or more of the above described changes.
  • Amino acid residues are abbreviated according to the following Table 1 either in one- or in three-letter code.
  • Mutations are described by use of the following nomenclature: amino acid residue in the protein scaffold; position; substituted amino acid residue(s). According to this nomenclature, the substitution of, for instance, an alanine residue for a glycine residue at position 20 is indicated as Ala20Gly or A20G. The deletion of alanine in the same position is shown as Ala20 * or A20 * . The insertion of an additional amino acid residue (e.g. a glycine) is indicated as Ala20AlaGly or A20AG. The deletion of a consecutive stretch of am ino acid residues (e.g.
  • a com ma or a slash separates these residues.
  • substitution of alanine at position 30 with either glycine or glutamic acid is indicated as A20G,E or A20G/E, or A20G, A20E.
  • a position suitable for modification is identified herein without any specific modification being suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position.
  • the alanine may be deleted or substituted for any other am ino acid residue (i.e.
  • the term "conservative mutation” refers to an amino acid m utation that a person skilled in the art would consider to be conservative to a first m utation. "Conservative” in this context means a sim ilar am ino acid in terms of the am ino acid characteristics. If, for exam ple, a m utation leads at a specific position to a substitution of a non- aliphatic am ino acid residue (e.g. Ser) with an aliphatic am ino acid residue (e.g.
  • a substitution at the same position with a different aliphatic am ino acid is referred to as a conservative m utation.
  • Further amino acid characteristics include size of the residue, hydrophobicity, polarity, charge, pK-value, and other am ino acid characteristics known in the art. Accordingly, a conservative m utation may include substitution such as basic for basic, acidic for acidic, polar for polar etc.
  • the sets of am ino acids thus derived are likely to be conserved for structural reasons. These sets can be described in the form of a Venn diagram (Livingstone CD. and Barton G. J., Comput.Appl Biosci.
  • peptide sequence refers to any sequence of one or more amino acids that is part of the scaffold protein, deleted form the scaffold protein, inserted in the protein scaffold, substituted with the protein scaffold or combined with the protein scaffold. Insertion, substitution or combination of peptide sequences with the protein scaffold are generated by insertion, substitution or combination of single or multiple nucleotide triplets into or with a polynucleotide encoding the protein scaffold.
  • synthetic in combination with the term “peptide sequence” refers to stretches of peptide sequence(s) that are not present in the protein scaffold at the position at which the peptide sequences are inserted or substituted or with which they are combined.
  • fragment when relating to proteins refers to a portion of the original protein, a homologue or a derivative thereof.
  • fragments include for example binding domains, regulatory domains or pro-sequences. Fragments may also be deletions of the N-terminus or the C-terminus or both.
  • Fragments of proteins comprise at least 20 consecutive amino acids from the original protein.
  • a fragment can be 20 amino acids or more, such as 25 amino acids or more, for example 30 amino acids or more, such as 50 amino acids or more, for example 100 amino acids or more, such as 150 amino acids or more, for example 200 amino acids or more, such as 220 amino acids or more, for example 250 amino acids in length or any integer in between these amounts.
  • derived from or “derivative thereof” when relating to proteins refer to derivatives of proteins that are m utated at one or more am ino acid positions and/or have a homology of at least 70% , such as at least 80% homology, preferably at least 90% homology, such as at least 91 % homology, for exam ple at least 92% homology, such as at least 93% homology, for exam ple at least 94% homology, such as at least 95% homology, for example at least 96% homology, such as at least 97% homology, for example at least 98% homology, such as at least 99% homology, for example at least 99.5% homology to the original protein, and/or that are proteolytically processed, and/or that have an altered glycosylation pattern, and/or that are covalently linked to non- protein substances, and/or that are fused with further protein domain s, and/or that have C-terminal and/or N-term inal truncations, and/or that have
  • the tern "region" refers to a structural domain of a protein like for example a beta barrel or a imm unoglobulin fold.
  • the term "functional equivalent” refers to a protease having the same proteolytic function as the parent protease. For exam ple will a functional equivalent of Trypsin preferentially cleave on the C -term inal side of arginine or lysine at the Pi position.
  • the term “structural equivalent” refers to a protease having the same structural features as the parent protease. For exam ple will a structural equivalent of Trypsin have a tertiary structure or fold equal or sim ilar to the tertiary structure or fold of the S1 structural subclass of serine proteases.
  • SDR ® or "Specificity Determ ining Region” refers to a synthetic peptide sequence that enhances specificity for the target when combined with the protein scaffold at sites that enable the resulting proteases to discrim inate between the target substrate and one or more other substrates. Such sites are termed “SDR ® sites”.
  • SDR ® sites Preferably the SDR sequences are combined with the protein scaffold by inserting them into the protein scaffold at sites that enable the result ing proteases to discrim inate between the target substrate and one or more other substrates.
  • the SDR sequences are inserted between residues 21 and 22, 42 and 43, and 123 and 124 in the scaffold, respectively, wherein the numbering refers to the wild- human cationic trypsin as shown in SEQ I D NO: 1 .
  • conjugate or “conjugated protease” is meant a com posite or chimeric molecule formed by the covalent attachment of a protease variant to one or more pharmaceutically acceptable non-polypeptide or polypeptide moieties to improve the pharmacokinetic and pharmacodynamic properties of the protease, e.g. increase terminal half- life in physiological fluids such as blood.
  • Covalent attachment means that the protease variant and the non- polypeptide or polypeptide moiety are either directly joined covalently to one another, or are alternatively indirectly joined covalently to one another through an intervening moiety or moieties, such as a bridge, spacer, or linkage moiety or moieties.
  • the non-polypeptide or polypeptide moiety may be covalently linked in a specific way to a defined attachment group or in a statistical way to a number of attachment groups.
  • the conjugation does not significantly alter the specific activity or specificity of the protease and the conjugated protease variant is soluble at relevant concentrations and conditions, i.e. soluble in physiological fluids such as blood.
  • Non- polypeptide moieties Polymer molecules, lipophilic com pounds, sugar moieties or organic derivatising agents are only a few exam ples for such non- polypeptide moieties. They may or may not have there own biological activity.
  • the non- polypeptide moiety is linked to the protease variant through an attachment group of the variant.
  • catalytic activity or "activity” describes quantitatively the conversion of a given substrate under defined reaction conditions.
  • inhibitor describes all substances that, when present in the reaction mixture, physically interact with the protease and decrease its catalytic activity com pared to the activity in the absence of the substance when all other parameters and concentrations are kept constant.
  • a protease variant is termed " inhibitor insensitive” when the relative activity in the presence of a given amount of inhibitor is higher than for a com parative protease variant.
  • library of protease variants describes a m ixture of proteases, whereby every single artificial protease is encoded by a different polynucleotide sequence.
  • gene library indicates a library of polynucleotides that encodes the library of protease variants.
  • composition or “pharmaceutical composition” refers to com positions comprising the TNF-alpha specific protease.
  • the com positions are prepared by m ixing the TNF-alpha specific protease with any pharmaceutically acceptable com ponent, such as, for exam ple, a carrier, a medicinal agent, an adjuvant, a diluent, and the like, as well as combinations thereof.
  • com ponent such as, for exam ple, a carrier, a medicinal agent, an adjuvant, a diluent, and the like, as well as combinations thereof.
  • vehicle refers to a diluent, adjuvant, excipient, or carrier with which a com position of the invention is administered.
  • DNA coding sequences are said to be “joined” or “fused” when, as a result of in-frame fusions between the DNA coding sequences, the DNA coding sequences are translated into a fusion polypeptide.
  • a "fusion protein” comprises a protease operatively linked to a second peptide or protein.
  • the present invention is directed to protease variants capable of inactivating human tumour necrosis factor-alpha (hTNF- ⁇ ) by hydrolysis. These variants have been generated by procedures described in WO 2004/ 1 13521 and WO 2004/ 1 13522.
  • the NBE ® platform is characterised by taking a protease scaffold with the basic enzymatic functionality and capable of catalyzing a chemical reaction on a substrate and combining it with one or more specificity determ ining regions (SDR ® ) .
  • the one or more SDRs are located at sites in the protein scaffold that enables the resulting engineered protein to discriminate between at least one target substrate and one or more different substrates, which would not have been discrim inated by the unmodified protease.
  • any protease can be used that has at least 70% homology to wild-type human cationic trypsin having the amino acid sequence shown in SEQ I D NO: 1 . however, human proteases are preferred. It is particularly preferred that the primary structure is based on a scaffold having at least 80% , preferably 90% homology to wild-type human cationic trypsin having the am ino acid sequence shown in SEQ I D NO: 1 . Also it is preferred that the scaffold is based on wild-type human cationic trypsin, human anionic trypsin or human mesotrypsin shown in SEQ I D NOs: 1 , 25 and 26, respectively.
  • the primary structure of the protease of the invention is based the wild-type human cationic trypsin having the am ino acid sequence shown in SEQ I D NO: 1 . and m ost preferred hum an cationic Trypsin is chosen as a scaffold.
  • the primary structure of the proteases variants of the invention is form ed by a hum an cationic trypsin scaffold having the am ino acid sequence shown in SEQ I D N0: 1 or from a functional and/or structural equivalent thereof (such as human anionic trypsin und human mesotrypsin shown in SEQ I D NOs: 25 and 26, respectively) and com prising at least three SDRs located in the scaffold, namely the first, second and third SDR mentioned above.
  • Said scaffold m ay contain further SDRs and/or one or m ore additional am ino acid substitutions.
  • the scaffold has a fourth SDR having a length of up to 8, preferably 2 to 5 am ino acid residues located between residues 1 28 and 1 29 of the scaffold, wherein the num bering refers to the wild-type human cationic trypsin as shown in SEQ I D NO: 1 .
  • said fourth SDR com prises at least one P and/or one T residue.
  • I n a preferred embodim ent, protease variants having one or m ore of the following SDRs or a com bination of the following SDRs are provided:
  • the first SDR ® has a length of up to 5 amino acid residues and/or com prises at least two serine residues.
  • X 1 and X 3 within the structure -X 1 -X 2 -X 3 - are serine residues, m ost preferably the sequence of said first SDR is SNS or SDS.
  • the general structure -X 4 - Xs -Xe-Xy Xs-Xg- of the second SDR X 4 is a tiny am ino acid residue
  • X 5 is selected from F, A, I , L, P and N
  • X 6 is selected from F, L, V, W, A, I and G
  • X 7 is selected from P, M, V, G, D and N
  • X 8 is selected from A, L, V, F, P, T, R, Y and G
  • X 9 is selected from D, S, N, T, A, L and E.
  • X 4 is A or S
  • X 3 is F or L
  • X 6 is F
  • X 7 is P or N
  • X 8 is A, L, V or G
  • X 9 is D or E.
  • I n the general structure of the third SDR X 1 0 is selected from A, G, S, T, V, R and L
  • X 1 1 is selected from K
  • X 1 2 is selected from D, G, L and N
  • X 13 is selected from P, F and I
  • X 14 is an aromatic am ino acid residue, L, G, E, S, T or K, or represents a peptide having four am ino acid residues, in which peptide the first and second residues are independently selected from charged and sm all am ino acid residues, the third residue is a small or hydrophobic am ino acid residue and the fourth residue is a hydrophobic am ino acid residue.
  • X 10 is A, G, S, or L
  • X 1 1 is K or R
  • X 1 2 is D
  • G or N
  • X 13 is P
  • X 4 is Y, W, RDPY (SEQ I D NO: 1 8) or GALY (SEQ I D NO: 1 9)
  • the fourth SDR has the general structure -X 15 -X 16 -X 17 -, wherein X 5 is P, X 6 is an arbitrary amino acid residue and X 17 is T.
  • the sequence of said fourth SDR is
  • protease variants having one or more of the following SDRs or a combination of the following SDRs are provided:
  • the sequence of said first SDR is SNS or SDS.
  • X 1 is A or S
  • X 5 is F
  • X 6 is F or L
  • X 7 is P
  • X 8 is A or L
  • X 9 is D
  • the second SDR has the sequence
  • X 3 is P
  • X 14 is Y, RDPY (SEQ ID NO: 18) or GALY (SEQ ID NO: 19).
  • the third SDR has the sequence SKDPY (SEQ ID NO:22), SKGPRDPY(SEQ ID NO:22), SKGPRDPY(SEQ ID NO:22), SKGPRDPY(SEQ ID NO:22), SKGPRDPY(SEQ ID NO:22), SKGPRDPY(SEQ ID NO:22), SKGPRDPY(SEQ ID NO:22), SKGPRDPY(SEQ ID NO:22), SKGPRDPY(SEQ ID NO:22), SKGPRDPY(SEQ ID NO:22), SKGPRDPY(SEQ
  • the sequence of said fourth SDR is PST or PPT.
  • the proteases of the invention may optionally comprise one or more of the following amino acid substitutions:
  • G at position 21 is preferably substituted by A, D, S or V, more preferably D or V
  • Y at position 22 is preferably substituted by T, H, Q, S, W, G or A, more preferably by T or H;
  • H at position 23 is preferably substituted by T, N, G, D, R or Y, more preferably by T or
  • F at position 24 is preferably substituted by I, V, Q, T, L or A, more preferably by I or
  • S at position 28 is preferably substituted by A;
  • S at position 37 is preferably substituted by T;
  • I at position 46 is preferably substituted by V, N, L or T, more preferably by V;
  • E at position 52 is preferably substituted by V or M, more preferably by V;
  • N at position 54 is preferably substituted by S;
  • I at position 55 is preferably substituted by T, N or R, more preferably by T or N;
  • Fat position 64 is preferably substituted by I or T, more preferably by I;
  • R at position 78 is preferably substituted by W;
  • S at position 92 is preferably substituted by T;
  • R at position 93 is preferably substituted by P;
  • a at position 98 is preferably substituted by D;
  • R at position 99 is preferably substituted by H
  • T at position 1 12 is preferably substituted by A or P, more preferably by A;
  • K at position 1 15 is preferably substituted by M
  • a at position 125 is preferably substituted by P or S, more preferably by P;
  • G at position 128 is preferably substituted by R, K or T, more preferably by R;
  • Y at position 131 is preferably substituted by F, N or H, more preferably by F;
  • D at position 133 is preferably substituted by G;
  • V at position 163 is preferably substituted by A;
  • S at position 172 is preferably substituted by T;
  • Q at position 174 is preferably substituted by R;
  • C at position 183 is preferably substituted by H, Q or R, preferably by H;
  • D at position 195 is preferably substituted by E and /or and
  • K at position 214 is preferably substituted by E, D, R, T or V, most preferably by E;
  • Particularly preferred embodiments of the invention provide TNF-alpha specific proteases based on the human cationic trypsin scaffold with one or more of the above detailed insertions in combination with one or more of the listed substitutions.
  • Specific variants are given in SEQ I D N0:2, SEQ I D N0:3, SEQ I D N0:4, SEQ I D N0:5, SEQ I D
  • said proteases are capable of specifically inactivating human TNF-alpha of the SEQ I D NO: 1 1 , that is, proteolytic cleavage of hTNF-alpha by the protease variant renders hTNF-alpha incapable of exerting its biologic effects.
  • said protease variants, a fusion construct of said protease variants or conjugated protease variants are capable of hydrolysing the peptide bonds between positions 31 /32 or 32/33 in hTNF-alpha.
  • variants are selective for either soluble or transmembrane human TNF-alpha and in a more preferred embodiment they are selective for soluble TNF-alpha and do not inactivate transmembrane TNF-alpha. Since the action of transmembrane TNF-alpha has been associated with beneficial effects with respect to resistance to bacterial infection and anti- inflam matory effects. The selectivity of variants of the invention provides them with an advantage over currently marketed antibodies that neutralize both soluble and transmembrane TNF-alpha.
  • the variants of the present invention have a reduced inhibitor sensitivity com pared to the parent protease scaffold.
  • the protease variant is sufficiently insensitive against inhibitors naturally present in the body com partments where its activity is desired, leading to a residual activity that results in efficacy in the intended application.
  • Body compartments where activity may be desired comprise, but are not lim ited to blood, synovial fluid, interstitial fluid, m ucosal fluid, peritoneal fluid, extracellular matrix, the eye, cerebrospinal fluid, the brain, epidermal tissue, different organs as well as epithelial and m ucosal surfaces of the body and the intracellular space including cytoplasm or cellular organelles such as lysosomes, endosomes, endoplasmic reticulum , Golgi apparatus, nucleus and mitochondria.
  • the protease variant is insensitive to protease inhibitors present in human blood or synovial fluid.
  • protease variants with the desired properties in terms of specificity, activity, inhibitor insensitivity or any other property are identified in a screening process as described in WO 2004/ 1 13521 A1 and WO 2004/ 1 13522 A1 .
  • the result of the screening process is a culture of a clone of the organism expressing the protease variant of interest. From th is culture deoxyribonucleic acid (DNA) sequence coding for said protease can be extracted by standard molecular cloning techniques known to anyone skilled in the art (e.g. Sambrook, J. F; Fritsch, E.F. ; Maniatis, T.
  • the DNA encoding such TNF-alpha specific protease is ligated into a suitable expression vector by standard molecular cloning techniques (e.g. Sambrook, J.F; Fritsch, E.F. ; Maniatis, T. ; Cold Spring Harbor Laboratory Press, Second Edition, 1989, New York) .
  • the vector is introduced in a suitable expression host cell that expresses the corresponding TNF-alpha specific protease.
  • Particularly suitable expression hosts are bacterial expression hosts such as Escherichia coli, Pseudomonas fluorescence or Bacillus subtilis, or yeast expression hosts such as Saccharomyces cerevisiae, Kluveromyces lactis, Hansenula polymorpha or Pichia pastoris, other fungal expression hosts such as Aspergillus niger or Trichoderma reesei or mammalian expression hosts such as mouse (e.g., NSO) , Chinese Hamster Ovary (CHO) or Baby Hamster Kidney (BHK) cell lines, transgenic mam malian systems such as rabbit, goat or cattle, other eukaryotic hosts such as insect cells or viral expression systems such as bacteriophages like M13, T7 phage or Lambda, or viruses such as vaccinia and baculovirus expression systems.
  • yeast expression hosts such as Saccharomyces cerevisiae, Kluveromyces lactis, Hansenula polymorpha or Pichia pastoris
  • the DNA is ligated into an expression vector behind a suitable signal sequence that leads to secretion of the TNF-alpha specific protease into the extracellular space, thereby allowing direct detection of enzyme activity in the cell supernatant.
  • suitable signal sequences for Escherichia coli, other Gram negative bacteria and other organisms known in the art include those that drive expression of the HIyA, DsbA, PhoA, PeIB, Om pA, OmpT or M13 phage GI I I genes.
  • particularly suitable signal sequences include those that drive expression of the AprE, NprB, Mpr, AmyA, AmyE, Blac, SacB, and for S. cerevisiae or other yeast, include the killer toxin, BaM , Suc2, Mat ⁇ , I nu1 A or Ggplp signal sequence.
  • the enzyme variants are expressed intracellular ⁇ .
  • a permeabilisation or lysis step is used to release the TNF-alpha specific protease into the supernatant.
  • the disruption of the membrane barrier is effected by the use of mechanical means such as ultrasonic waves, French press, cavitation or the use of membrane-digesting enzymes such as lysozyme.
  • the genes encoding the TNF-alpha specific protease are expressed cell-free by the use of a suitable cell-free expression system .
  • a suitable cell-free expression system For exam ple, the S30 extract from Escherichia coli cells is used for this purpose as described by Lesly et al. (Methods in Molecular Biology 37 ( 1995) 265 -278) .
  • the gene of interest is typically transcribed with the assistance of a promoter, but ligation to form a circular expression vector is optional. Regardless of the presence of a circular vector or the final host organism , the DNA sequence of the protease expression construct is determ ined us ing techniques that are standard in the art.
  • the pharmaceutical proteins are expressed in a variety of expression systems and the appropriate down- stream processing and purification procedures are selected accordingly.
  • the protease variant is expressed in a m icrobial host and the protein is secreted into the periplasmic or extracellular space.
  • Cells carrying an appropriate expressing construct for the protease variants may be preserved as cryo stocks, well known to anyone skilled in the art.
  • Cultures for protein expression are inoculated from a cryo stock and the volume of the culture increased successively in the appropriate container.
  • the cells are grown in a fermenter under controlled conditions of pH, tem perature, oxygen and nutrient supply.
  • a first step com prises the separation of cells from supernatant using one or more of several techniques, such as sedimentation, m icrofiltration, centrifugation, flocculation or other. I n a preferred embodiment the method applied is microfiltration.
  • I n a preferred embodiment of the invention the protein is secreted into the supernatant and a further step of purification comprises the concentration of the supernatant by ultrafiltration.
  • Protein purification from the supernatant or concentrated supernatant is performed with one or more of several preferred chromatographic methods including but not lim ited to ion-exchange, hydrophobic interaction, hydroxyapatite, size fractionation by gel-filtration and affinity chromatography or any combination thereof.
  • I n a more preferred method the protein is purified by combining several ion-exchange chromatographic steps to obtain a high purity protein.
  • An even more preferred method com prises the combination of a cation- exchange and an anion-exchange chromatography, optionally combined with further cation or anion-exchange chromatographies.
  • An appropriate purification method yields a purity of the protein of > 50% , in a more preferred method the purity is > 80% , in an even more preferred method the purity is > 90% , in a yet more preferred method the purity is > 95% and in a most preferred method the purity is > 98% .
  • pharmacokinetic and pharmacodynam ic properties of the protease of the present invention are further im proved by one or more of the following strategies or any combination thereof: a) fusion to a peptidic component, preferably being selected from , but not limited to the group consisting of binding domains, receptors, antibodies, regulatory domains, pro -sequences, serum album in, or fragments or derivatives thereof, and/or b) covalent conj ugation to a natural or synthetic polypeptide or non-polypeptide moiety, preferably being selected from the group consisting of polyethylenglycols, carbohydrates, lipids, fatty acids, nucleic acids, metals, metal chelates, nano - particles, liposomes, dendrimers or fragments or derivatives thereof, and/or c) introduction of consensus glycosylation sites into the protease variant that are glycosylated during the post-translational processing of the protease variant during biosynthesis and/or d) introduction of
  • the protease is fused to a proteinacious com ponent.
  • the proteinacious component is an album in, in particular human serum albumin, fragments or derivatives or variants thereof without significantly im pairing the biological properties of the protease. It causes increased terminal half- life and thus enables the protease of the present invention to maintain a given biological activity in vivo for a prolonged period.
  • Useful album ins and fusion methods are exem plary described in US 5,876,969.
  • Other acceptable fusion partners include the Fc portion of human immunoglobulin and human transferrin.
  • the protease variant is fused to human serum album in or the Fc portion of a human IgG im munoglobulin.
  • said proteinacious com ponent is not imm unogenic.
  • fusion proteins are also generated that promote localisation of the protease to desired locations in the body such as tissues, organs or cell subtypes.
  • the fusion partner of the protease comprises, but is not limited to, antibody fragments, for exam ple scFv fragments with specificity for certain cellular surface antigens or other markers for a particular cell type, organ or tissue.
  • fusion partners may include sequences corresponding to the binding domain within certain ligands or receptors that facilitate recognition by the protease of certain cellular surface expressed target proteins or moieties, or to circulating cytokines, growth factors, hormones, enzymes and other serum proteins.
  • the protease is fused to certain cytokines, growth factors, hormones, or fragments thereof, that will promote attachment to specific receptors on certain cell types to promote intake of the fusion protein to the intracellular compartment of the target cells by for example receptor mediated endocytosis.
  • the protease is fused to the whole or a fragment of the granulocyte-colony stim ulating factor (G-CSF) for targeting of the G-CSF receptor on granulocytes to facilitate intracellular penetration.
  • G-CSF granulocyte-colony stim ulating factor
  • the protease is covalently conjugated to one or more polypeptide or non-polypeptide moieties such as polymer molecules, selected from the group described in EP- B- 1062230 consisting of, but not lim ited to, polyethylene glycol (PEG) , polyvinyl pyrrolidone, polyvinyl alcohol, polyam ino acids, divinylether maleic anhydride, N- (2-Hydroxypropyl) - methacrylam ide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polyoxyethylated polyol, heparin, heparin fragments, sugar moieties such as polysaccharides, cellulose and cellulose derivatives, including methylcellulose and carboxymethyl cellulose, starch and starch derivatives, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethy
  • protease is covalently conjugated to polypeptides or proteins, such as but not lim ited to binding domains, receptors, antibodies, regulatory domains, pro-sequences, serum album in, transferrin, the Fc portion of imm unoglobulins or fragments or derivatives thereof.
  • the non- polypeptide polymer is PEG.
  • Conjugation of PEG to proteins is a widely used technique to stabilize proteins.
  • the PEG molecules require a chem ical activation before the conjugation reaction by the introduction of a chem ical activating group such as but not lim ited to dichlorotriazine, chlorotriazine, tresylate, succinim idyl carbonate, benzotriazole carbonate, p- nitrophenyl carbonate, trichlorophenyl carbonate, carbonylimidazole, succinim idyl succinate, propionaldehyde the generation of active esters of PEG carboxylic acids such as N-hydroxysuccinim ide or carbodiim ide.
  • a chem ical activating group such as but not lim ited to dichlorotriazine, chlorotriazine, tresylate, succinim idyl carbonate, benzotriazole carbon
  • a specific labelling of cysteines is obtained by reaction with PEG-derivatives such as maleim ide, iodoacetam ide, vinylsulfone or orthopyridyl disulfide.
  • PEG-derivatives such as maleim ide, iodoacetam ide, vinylsulfone or orthopyridyl disulfide.
  • Am ine-derivatives of PEG can be covalently linked to glutam ines by enzymatic reactions using enzymes such as transglutam inase (H. Sato Enzymatic procedure for site-specific pegylation of proteins. Advanced Drug Delivery Reviews (2002) 54: 487-504) .
  • electrophilic groups are introduced to the polymer which can than be coupled to the nucleophilic amino -groups of lysine residues or the N-terminal am ino group on the peptide or protein.
  • the coupling moiety is either incorporated as a part of the PEG- protein conjugate or lost fully or partially during the coupling step.
  • Different pegylation methods and chem istries and their effect on biopharmaceuticals are well known by a person skilled in the art (Roberts MJ. et al. Chemistry for peptide and protein PEGylation. Advanced Drug Delivery Reviews (2002) 54:459-476; Harris J.M. and Chess R.B.
  • the PEG moiety can be of different structure.
  • I n a preferred embodiment of the invention the term inal hydroxyl group is methylated (m PEG) .
  • the structure of the PEG molecules is either linear, bifurcated or branched.
  • I n a branched PEG two or more PEG molecules are connected to a moiety providing the branching point and the connectivity to a reactive chemical group for attachment to the target protein.
  • the average molecular weight of the reactant PEG is preferably between about 5,000 and about 60,000 Daltons, more preferably between about 10,000 and about 50,000 Daltons, and most preferably between about 20,000 and about 40,000 Daltons.
  • PEGs having nom inal average sizes of about 20,000 and about 40,000 Daltons.
  • the method of attachment is not critical, but preferably does not alter, or only m.lly alters, the activity of the biologically active molecule. Preferably the increase in terminal half- life outweighs any decrease in biological activity.
  • the PEG- moiety is covalently linked to lysine residues using a N- hydroxysuccinim ide-activated, methylated PEG-derivative of a nominal size between 5kD and 6OkD.
  • non-polypeptide moiety is a glycoside.
  • Covalent in- vitro coupling of glycosides to am ino acid residues of the protease may be used to modify or increase the number or profile of carbohydrate substituents.
  • the one or more sugar moieties may be attached to a) lysine, arginine and histidine, b) free carboxyl groups, c) free sulfhydryl groups such as those of cysteine, d) free hydroxyl groups such as those of serine, threonine, tyrosine or hydroxyproline, e) aromatic residues such as those of phenylalanine or tryptophan or f) the amide group of glutam ine.
  • These amino acid residues constitute exam ples of attachment groups for a sugar moiety, which may be introduced and/or removed in the protease of the present invention.
  • Suitable methods of in vitro coupling are described, for exam ple, in WO8705330 A1 .
  • the in vitro coupling of sugar moieties or PEG to protein-and peptide-bound Gin- residues can also be carried out by transglutam inases, e. g. as described in EP-A-725145.
  • the non-polypeptide moiety is a carbohydrate molecule attached by in- vivo or in-vitro post-translational glycosylation, such as N- or O-glycosylation.
  • the in- vivo glycosylation consensus sequence is asparagine-x-serine or asparagine-x-threonine throughout the eukaryotic kingdom , where x stands for any am ino acid except proline.
  • the im provement of the pharmacokinetic properties of several proteins through the introduction of consensus glycosylation sequences has been reported (Elliot et al. Enhancement of therapeutic protein in vivo activities through glycoengineering. Nature Biotech (2003) 21 : 414- 421 ) .
  • I n order to obtain a glycosylation structure sim ilar or identical to human glycosylation a yeast host has been engineered that expresses human enzymes involved in the generation of glycosyl structures. This strain produces glycosylated protein that is virtually identical to human protein (Ham ilton et al. Production of complex human glycoproteins in yeast, Science (2003) 301 : 1244- 1246) .
  • I n a preferred aspect of this embodiment of the invention the 9 base pairs coding for the consensus glycosylation sites are introduced in frame into the coding region of the gene for the protein variant where the 9 base pairs can replace either none, three, six or nine base pairs at the position of insertion.
  • the insertion site can be random or based on rational design.
  • the engineered gene of the protease variant containing additional glycosylation sites is introduced in an appropriate expression vector in an expression host, selected from filamentous fungi or yeast, insect or animal cells, from transgenic plant cells or any other suitable eukaryotic expression host.
  • the glycosylation may be achieved in the human body when using a nucleotide sequence encoding the polypeptide of the invention in gene therapy.
  • the host cell is a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a BHK cell or a HEK cell, e. g. a HEK293 cell, or an insect cell, such as an SF9 cell, or a yeast cell, e. g. Saccharomyces cerevisiae, Pichia pastoris, Kluveromyces lactis or any other suitable glycosylating host.
  • CHO Chinese Hamster Ovary
  • a BHK cell or a HEK cell e. g. a HEK293 cell
  • insect cell such as an SF9 cell
  • yeast cell e. g. Saccharomyces cerevisiae, Pichia pastoris, Kluveromyces lactis or any other suitable glycosylating host.
  • sugar moieties attached to the protease by in vivo glycosylation are further modified by use of glycosyltransferases or other processing enzymes.
  • m utations, insertions or deletions are introduced into the coding gene of the protease variant leading to one or more amino acid changes in the protein.
  • said mutant protein has an increased terminal half - life compared to the non-m utated variant.
  • I n a preferred aspect of this embodiment said mutation renders the variant less sensitive to proteolytic degradation by proteases that the protease variant of the invention comes into contact with in the human body.
  • the introduction of the mutation is either based on rational design after experimentally identifying cleavage sites or by screening a library of protease variants under suitable conditions allowing the selection of protease variants less sensitive to degradation.
  • am ino acid exchange can be introduced based on rational design or m utated protease variants can be selected in a screening process comprising a library of protease variants and an appropriate screening process, allowing the identification of protease variants characterized by a reduction in an undesired interaction with certain components of the surroundings.
  • the protease may be form ulated in such a way that the protease is protected from premature clearance and/or degradation by external factors and is released into circulation in a controlled manner, in order to optim ize its pharmacokinetic profile such as the systemic exposure of the drug, the area under the curve or term inal half- life of the protease.
  • These form ulations allow for increased dosing levels of the drug that can be administered, reduce concentration differences between peak and trough levels and allow a more efficient use of the therapeutic window.
  • Therapeutic formulations are typically sterile and exhibit long-term stability under the conditions of manufacture and storage.
  • Examples such as the entrapment in a matrix or the encapsulation in a sem ipermeable membrane represent some of the most widely used stabilisation techniques ( I ngemann M. et al. Peptide and Protein Drug Delivery Systems for Non- parenteral Routes of Administration. Pharmaceutical Form ulation Development of Peptides and Proteins by Frokjaer S. and Hovgaard L., 1 st edition (2000) chapter 10 (pages: 189-205)) .
  • the entrapment or encapsulation may be done by a polymeric drug delivery system , such as hydrogel or nanocapsule / m icrosphere, or lipid drug delivery system such as liposomes and m icroem ulsions.
  • m icropumps for the controlled release of the drug in a suitable form ulation.
  • hTNF-alpha has been im plicated in the ethyology and/or progression of m ultiple diseases.
  • the protease variants of the invention can be used for preparing medicinal drugs for the treatment of diseases and various disorders associated with TNF-alpha.
  • the invention provides methods to reduce the hTNF-alpha activity in individuals suffering from said diseases, com prising the adm inistration of protease variants to the subject in a suitable form and dosage which results in a reduction of hTNF-alpha activity to a degree that ameliorates or reduces signs and sym ptoms of the disease or disorder.
  • the subject is an animal or more preferably a mam mal, which expresses TNF-alpha, that serves as a target for the protease variant of the invention. Most preferable the subject is a human.
  • Diseases that can be treated with the protease variants include, but are not lim ited to, the following: Autoimmune diseases: rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing sponndylitis, sclerotic arthritis, Behcet's disease, adult- onset Still's disease, autoim mune uveitis, multiple sclerosis, insulin resistance in diabetes, Sjogren's Syndrome, system ic lupus erythematosus, Systemic inflam matory response syndrome (SI RS) which leads to distant organ damage and m ultiple organ dysfunction syndrome (MODS) ,
  • SI RS Systemic inflam matory response syndrome
  • Intestinal diseases inflammatory bowel diseases, Crohn's disease, ulcerative colitis;
  • Infectious diseases sepsis, viral diseases and infections, encephalitis, bacterial meningitis, cerebral malaria, AI DS and AI DS- related com plex, cytomegalovirus infection secondary to transplantation;
  • Pulmonary disorders chronic obstructive pulmonary disease, asthma, pulmonary sarcoidosis, pulmonary fibrosis, silicosis, shock lung;
  • Malignancy neoplastic diseases associated with abnormal im m une function, classical Hodgkin's Lym phoma (cHL) , cachexia secondary to malignancy;
  • cHL Hodgkin's Lym phoma
  • Cardio- vascular disorders arterial sclerosis, hypertension, vasculatitis, disorders of haematopoietic cells, heart failure, stroke, vasodilation, intravascular coagulation
  • osteoporosis scleroderma
  • polymyositis polymyositis
  • dermatomyositis Grave's disease
  • Hashimoto's thyroiditis eosinophilia
  • neurodegenerative disease closed head injury and m ultiple organ failure.
  • the invention is indicated for the treatm ent of rheumatoid arthritis, j uvenile rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, ankylosing sponndylitis, system ic lupus erythematosus and ulcerative colitis, where blocking anti- TNF-alpha im munoglobulin derived biotherapeutics have validated the target in a clinical setting.
  • Second most favoured indications are chronic obstructive pulmonary disease, sepsis, Behcet's disease, adult-onset Still's disease, polymyositis, dermatomyositis, osteoporosis, uveitis and vasculatitis, as supported by preclinical evidence for a role for TNF-alpha in the modulation of the underlying molecular mechanisms of these inflam matory diseases.
  • the preferred dosing regimen for the protease is adm inistration as a monotherapy.
  • the second most favoured regimen com prises the co- adm inistration of non-biological therapies that are standard of care in the treatment of the above diseases and disorders. These include but are not restricted to disease modifying anti-rheumatic drugs, corticosteroids, non-steroidal anti- inflam matory drugs and analgesics. Exam ples of these com pounds include methotrexate, cyclosporine A, azathioprine, and mesalazine. The most preferred com pound is methotrexate. The second most preferred com pound is cyclosporine A.
  • the protease of the invention is co -adm inistered with protein-based, anti- TNF-alpha specific or otherwise, therapeutics to provide additive or synergistic efficacy.
  • protein-based, anti- TNF-alpha specific or otherwise, therapeutics to provide additive or synergistic efficacy.
  • examples include among others, Fc-fusion proteins such as Etanercept, monoclonal antibodies such as infliximab and adalim umab in the treatment of for example RA, or efalizumab and alefacept in the treatment of for exam ple psoriasis.
  • the invention is com patible with co-adm inistration with other non- biological compounds or agents that are utilized in the clinic to ameliorate the symptoms of the disease or reduce the scope of drug induced adverse events.
  • the diseases are treated by adm inistering to a patient a therapeutically effective amount of a com position com prising the TNF-alpha specific protease.
  • the specific amount and frequency of the active com pound adm inistered will depend on the indication, supporting data from clinical trial studies in the given indication, the subject being treated, the subject's weight, the form ulation and manner of adm inistration, and the judgment of the prescribing physician. I nformation concerning dosages of various pharmacological agents is found in standard pharmaceutical reference books, e.g. Remington: The Science And Practice Of Pharmacy, 21 st edition (2005).
  • compositions of the invention that will be effective in the treatment of a particular disorder or condition disclo sed herein will depend on the nature of the disorder or condition, and is determined by standard clinical techniques. I n addition, in vitro or in vivo assays are optionally em ployed to help identify optimal dosage ranges. The precise dose of the compositio ns to be em ployed will also depend on the route of adm inistration, and the seriousness of the disease or disorder, and should be decided according to the j udgment of clinical trial results, the practitioner and each patient's circumstances.
  • the effective dose of the TNF-alpha specific protease may be determined in a number of ways, including dosages calculated to alleviate symptoms associated with a specific disease state in a patient. Alternatively, dosages are calculated to comprise an effective amount of the TNF-alpha specific protease to induce a detectable change in TNF-alpha levels in the blood, synovial fluid or any other body fluid or compartment.
  • Such detectable changes in blood cytokine concentrations include a decrease in hTNF- alpha levels of between about 1 % and 99% , or between about 5% and about 80% , such as between about 10% and about 70% , for example between about 20% and about 60% , such as between about 30% and about 50% or such as between about 1 % and about 40% , for exam ple between about 1 % and about 30% , like between about 1 % and about 20% , such as between about 1 % and about 10% .
  • the protease variant is given at a dose sufficient to detect activity of the hTNF-alpha specific protease in the patient through the measurement of defined secondary pharmacodynamic endpoints.
  • Formulations are also calculated using a unit measurement of activity of hTNF-alpha specific protease. The measurements by weight or activity can be calculated using known standards.
  • suitable dosage ranges for intravenous, epidural or intracerebral adm inistration are preferably between about 0.1 to 50 mg per kg body weight, such as between 0.5 to 40 mg per kg body weight, like between 1 .0 to 30 mg per kg body weight, such as between 1 .5 to 20 mg per kg body weight, like between 2.0 to 15 mg per kg body weight, such as between 2.5 to 10 mg per kg body weight, and especially in the range of from about 0.1 to 10 mg per kg body weight such as in the range of from about 0.1 to 5 mg per kg body weight.
  • Suitable dosage ranges for intranasal and pulmonary administration are generally about 0.5 to 100 mg per kg body weight, such as about 0.5 to 90 mg per kg body weight, for example about 0.5 to 80 mg per kg body weight, such as about 0.5 to 70 mg per kg body weight, for example 0.5 to 60 mg per kg weight, such as about 0.5 to 50 mg per kg body weight, for exam ple 0.5 to 40 mg per kg body weight, such as 0.5 to 30 mg per kg body weight, for exam ple 0.5 to 20 mg per kg body weight, such as 0.5 to 10 mg per kg body weight.
  • Suppositories and orally adm inistered tablets and pills generally contain 0.5 to 100 mg per kg of the protease of the invention per kg body weight, such as about 0.5 to 90 mg per kg of the protease of the invention per kg body weight, for example about 0.5 to 80 mg per kg of the protease of the invention per kg body weight, such as about 0.5 to 70 mg per kg of the protease of the invention per kg body weight, for example 0.5 to 60 mg per kg of the protease of the invention per kg body weight, such as about 0.5 to 50 mg per kg of the protease of the invention per kg body weight, for example 0.5 to 40 mg per kg of the protease of the invention per kg body weight, such as 0.5 to 30 mg per kg of the protease of the invention per kg body weight, for exam ple 0.5 to 20 mg per kg of the protease of the invention per kg body weight, such as 0.5 to 10 mg per kg of the protease of the invention per kg body weight.
  • Recom mended dosages for intradermal, intram uscular, intraperitoneal, subcutaneous, sublingual, intravaginal or transdermal adm inistration are in the range of 1 to 100 mg per kg of body weight, such as in the range of 1 to 90 mg per kg of body weight, for exam ple 1 to 80 m g per kg of body weight, such as in the range of 1 to 70 mg per kg of body weight, for exam ple 1 to 60 mg per kg of body weight, such as in the range of 1 to 50 mg per kg of body weight, for exam ple 1 to 40 mg per kg of body weight, such as in the range of 1 to 30 mg per kg of body weight, for exam ple 1 to 20 mg per kg of body weight, such as in the range of 1 to 10 mg per kg of body weight.
  • Adm inistrations by inhalation are in the range of 1 to 100 mg per kg of body weight such as in the range of 1 to 90 mg per kg of body weight, for exam ple 1 to 80 mg per kg of body weight, such as in the range of 1 to 70 mg per kg of body weight, for exam ple 1 to 60 mg per kg of body weight, such as in the range of 1 to 50 mg per kg of body weight, for exam ple 1 to 40 mg per kg of body weight, such as in the range of 1 to 30 mg per kg of body weight, for exam ple 1 to 20 mg per kg of body weight, such as in the range of 1 to 10 m g per kg of body weight.
  • Suitable doses of the com pounds of the invention for topical adm inistration are in the range 0.25 to 25 mg per kg body weight, such as 0.25 to 20 mg per kg body weight, for exam ple in the range of 0.25 to 15 mg per kg body weight, such as 0.25 to 10 mg per kg body weight, for exam ple in the range of 0.25 to 5 mg per kg body weight, such as 1 to 5 mg per kg body weight or like in the range of 1 to 20 m g per kg body weight, such as in the range of 5 to 15 mg per kg body weight, depending on the surface area to which the compound is adm inistered.
  • composition com prising the protease of the invention may, though not necessarily, be administered daily, in an effective amount to ameliorate a disease sym ptom .
  • it is adm inistered once every other day, or more preferably once a week, or even more preferably every other week, or even more preferably at intervals longer than two weeks.
  • the hTNF-alpha specific protease is adm inistered to achieve efficacious concentration levels in target tissues that maxim ize its disease modulating effects. It may be adm inistered by any convenient route, for example, intradermal, intramuscular, intraperitoneal, intravenous infusion or bolus injection, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, by absorption through epithelial or mucocutaneous linings (e.g., oral m ucosa, rectal and intestinal m ucosa, etc.) .
  • epithelial or mucocutaneous linings e.g., oral m ucosa, rectal and intestinal m ucosa, etc.
  • the form ulation has the broadest applicability.
  • protease in a given formulation can interchangeably be adm inistered subcutaneously, intram uscularly or intravenously.
  • I n a further embodiment of this aspect, it may be desirable to adm inister one or more com positions of the invention locally to the area in need of treatment. This is achieved, for exam ple, and not by way of lim itation, by topical application (e. g., as a cream) , by local infusion during surgery, by injection, by means of a catheter, by means of a suppository, by means of inhalation or by means of an im plant.
  • administration can be by direct injection at the site (or former site) of the tissue.
  • the administration and com positions of the invention are preferably assayed in vitro and in vivo in relevant animal disease models, for the desired therapeutic or prophylactic activity, prior to use in humans.
  • in vitro assays are used to determ ine whether adm inistration of a specific com position of the invention or a combination of compositions of the invention is preferred for treating or ameliorating a disease or disorder as described herein.
  • the com positions of the invention are also demonstrated to be effective and safe using animal model.
  • Therapeutic com positions typically must be sterile and stable under the conditions of manufacture, storage and application.
  • Adm inistration is system ic or local.
  • Various delivery systems are known, for exam ple, encapsulation in liposomes, m icroparticles, microcapsules, capsules, etc. as described above, and used to adm inister a composition of the invention.
  • more than one com position of the invention is adm inistered to a patient.
  • the preferred mode of adm inistration is chosen according to the results of clinical trial data and left to the discretion of the practitioner, and will depend in- part upon the site and severity of the medical condition. I n most instances, adm inistration will result in the release of the composition of the inventio n for maximum efficacy.
  • the com positions may be encapsulated in a liposome envelope that is coupled to a (poly)peptide (e.g. an antibody) or chemical moiety directed against specific proteins and other cell surface structures so as to provide target tissue selectivity.
  • a (poly)peptide e.g. an antibody
  • the specific nature of the form ulation is also determined by the desired route of adm inistration, e.g., topical, parenteral, oral, rectal, surgical implantation or by other means of local (intraprostatic) delivery.
  • the dosage is determined for each route of administration to maxim ize pharmacokinetic profile and efficacy.
  • the amount of TNF-alpha specific protease in the com position ranges from about 0.01 to 99% by weight of the com position.
  • a further aspect of the present invention is directed to pharmaceutical com positions comprising the TNF-alpha specific protease.
  • suitable forms of the compositions are solutions, suspensions, em ulsion, tablets, pills, pellets, capsules, capsules containing liqu ids, powders, sustained- release form ulations, suppositories, em ulsions, aerosols, sprays, suspensions, or any other form suitable for use.
  • Other exam ples of suitable pharmaceutical vehicles are described in Rem ington: The Science And Practice Of Pharmacy, 21 st edition (2005) .
  • compositions are prepared by mixing the TNF-alpha specific protease with any pharmaceutically acceptable component, such as, for exam ple, a carrier, a medicinal agent, an adjuvant, a diluent, and the like, as well as combinations thereof.
  • Suitable pharmaceutical carriers, medicinal agents, adjuvants, and diluents are also described in Remington: The Science And Practice Of Pharmacy, 21 st edition (2005) .
  • the com position is preferably sterile. Therefore, water is a preferred vehicle when the com pound of the invention is adm inistered intravenously.
  • Other applicable liquid vehicles are preferably derived from natural sources, e.g. of petroleum , animal or vegetable origin.
  • Chem ically derived liquids such as m ineral oils, talc, colloidal silica, aqueous dextrose and glycerol solutions among others are also suitable.
  • Suitable pharmaceutical vehicles also include excipients such as, but not lim ited to, starch, glucose, lactose, sucrose, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim m ilk, glycerol, propylene, glycol, ethanol, mannitol, polysorbate 20, polysorbate 80, trehalose and the like.
  • excipients such as, but not lim ited to, starch, glucose, lactose, sucrose, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim m ilk, glycerol, propylene, glycol, ethanol, mannitol, polysorbate 20, polysorbate 80, trehalose and the like.
  • the com position may comprise stabilisers such as but not lim ited to sucrose, glucose, polyols, trehalose, mannitol, polysorbate 20, polysorbate 80, sodium chloride, buffer salts, ion exchange polymers and others. Further com ponents of the com position are wetting or em ulsifying agents, surfactants or pH buffering agents.
  • the com positions of the invention are form ulated in accordance with routine procedures as a pharmaceutical com position adapted for intravenous administration to human beings.
  • com positions of the invention for intravenous adm inistration are solutions in sterile isotonic aqueous buffer.
  • the compositions also include a solubilising agent.
  • Com positions for intravenous administration optionally include a local anaesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette, each containing the exact quantity of active agent for a single application.
  • the ingredients are reconstituted to the final injectable concentrations by the addition of sterile water or saline provided separately and m ixed just prior to adm inistration.
  • the com position of the invention is to be adm inistered by intravenous infusion, it is dispensed by a bolus injection or by continuous injection, for exam ple, with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • Slow release form ulations for parenteral administration I n order to increase systemic exposure of the drug, the area under the curve or term inal half- life of the protease, a particular preferred embodiment comprises slow- release formulations. They reduce concentration differences between peak and trough levels and allow a more efficient use of the therapeutic window. E ⁇ xamples for slow - release form ulations are the entrapment in a matrix or the encapsulation in a sem ipermeable membrane, the most widely used stabilization techniques ( I ngemann M. et al. Peptide and Protein Drug Delivery Systems for Non- parenteral Routes of Administration. Pharmaceutical Formulation Development of Peptides and Proteins by Frokjaer S.
  • the entrapment or encapsulation is done by a polymeric drug delivery system , such as hydrogel or nanocapsule / m icrosphere, or lipid drug delivery system such as liposomes and m icroem ulsions.
  • a polymeric drug delivery system such as hydrogel or nanocapsule / m icrosphere, or lipid drug delivery system such as liposomes and m icroem ulsions.
  • lipid drug delivery system such as liposomes and m icroem ulsions.
  • m icropumps for the controlled release of the drug in a suitable form ulation.
  • hydrogels are crosslinked via hydrophilic polymers of natural or synthetic origin to entrap the protease drug.
  • Hydrogels have the ability to swell in an aqueous environment without dissolving.
  • hydrogel entrapment monomers form a crosslinked polymeric network around the material to be entrapped.
  • the surrounding polymer matrix perm its entrapment of large quantities of protein drugs.
  • the polymerization reaction is carried out in bulk, solution or suspension by free radical polymerization initiated by the generation of free radicals by thermal, ionization or redox means. This is performed by mixing of monomers and cross-linking agent in an organic solvent or buffered solution followed by the addition of a catalyst system , which initiates the polymerization process.
  • the protease to be entrapped can be introduced into the hydrogel matrix during or after synthesis. Entrapment after synthesis is done by soaking of pre-washed hydrogels in a concentrated drug solution. I n a preferred variant the protease is introduced after cross-linking.
  • the polymerized protease complex may be produced in a wide variety of geometric shapes including films, membranes, rods and particles. Exam ples of synthetic and natural polymers which are used for hydrogels are acrylics, vinyl alcohols, ethylene oxides, cellulose ethers and starch, album in or dextran. I n addition hydrogels with enzyme digestible crosslinkers or polymer backbones are used as biodegradable drug delivery systems.
  • a further exam ple for an encapsulating material could com prise peptidic com ponents that are slowly degraded by the proteolytic action of the protease variants of the invention.
  • These peptidic com ponents could for exam ple contain the target sequence or variants thereof of the specific protease.
  • the peptidic com ponents are connected in such a way as to effectively entrap the proteases and cleavage of the peptidic component leads to a release of the protease.
  • the protease of the present invention is encapsulated by nanocapsules, nanospheres, m icrocapsules or m icrospheres which mainly differ by their size of below and above 1 ⁇ m and the process used for preparation.
  • Nanocapsules and m icrocapsules are vesicular systems in which the drug is confined to a cavity surrounded by a polar membrane, whereas nanospheres or microspheres are matrix systems in which the protease is dispersed throughout the particle.
  • nanocapsules and m icrocapsules There are two common preparation methods in preparation of nanocapsules and m icrocapsules according to whether the formation requires an in situ polymerization reaction or whether it is achieved directly from a preformed polymer or a natural macromolecule via precipitation of synthetic polymers or by denaturation of natural macromolecules. I n contrast, common processes to prepare nanospheres and microspheres include spray-drying, solvent-evaporation and phase-separation techniques.
  • Microspheres comprising the protease of the invention are com monly made out of polystyrene, polymethylmethacrylate, poly- hydroxybutyrate, poly- D,L- lactic acid, poly- L-lactic acid, poly- D,L- lactide-coglycolide, ethyl cellulose, cellulose acetate, hydrogen phthalate or cellulose triacetate I n a third variant of this embodiment the protease is included into liposomes which are considered for parenteral adm inistration (Torchilin, "Recent advances with liposomes as pharmaceutical carriers" Nat. Rev. Drug Discov. (2005) 4 : 145-160) .
  • Liposomes are vesicles in which an aqueous core is enclosed by phospholipid bilayers. They are broadly classified by size and lamellarity as small unilamellar vesicles (SUVs: size 25- 50 nm) , large unilamellar vesicles (LUVs: 100 nm) and multilamellar vesicles (MLVs: 50- 10000 nm) . Liposomes are spontaneously formed when phospholipbs are dispersed in excess water, arranging themselves in bilayers with the enclosure of an aqueous core. For SUVs and MLVs is a lipid hydration step involved, whereas LUVs are made by solvent dispersion methods.
  • the surface of the liposomes is modified to increase circulating half- life in the blood. Suitable surface modifications com prise but are not lim ited to covalent coupling of poly-ethylene glycol (PEG) , poly[ N-(2- hydroxypropyl)methacrylamide] , poly- N-vinylpyrrolidones, L-am ino- acid-based biodegradable polymer- lipid conjugates, polyvinyl alcohols, mucins or sugar residues such as mannosyl.
  • the liposome surface is modified to include a targeting moiety or moieties. Examples are antibodies, receptors, receptor ligands, binding domains or fragments thereof.
  • the liposomes are pH-sensitive, i.e. they are stable at the blood pH but destabilized at either lower or higher pH compared to the pH in the blood.
  • pH- sensitive liposomes can be generated by methods known in the art, for exam ple by use of anionic phospahtidylethanolamine or terminally alkylated co- polymer of N- isopropylacrylam ide and methyacrylic acid.
  • the protease is included in liposomes with any combination of surface modification, targeting moiety and pH-sensitivity.
  • the liposomes are pegylated and/or carry a targeting moiety on the surface that allows direction to the desired locus and/or are destabilised at low pH. Liposomes are also a preferred form ulation for oral or pulmonary delivery.
  • Emulsions are two imm iscible phases dispersed h one another.
  • Microem ulsions are more complex quarternary or ternary systems, containing a surfactant/co-surfactant blend added to a two-phase hydrophilic / lipophilic m ixture.
  • Microem ulsions are spontaneously formed at room temperature, appear transparent, and consist of m icro-droplets of a size less than 100 nm .
  • the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be form ulated according to the known art using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparations may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvate, for exam ple as a solution in 1 ,3-butanediol.
  • acceptable vehicles and solvents that may be em ployed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium .
  • the com positions of the invention are delivered in a controlled release system , such as a pum p or m icropump.
  • a controlled release system such as a pum p or m icropump.
  • these include devices consisting of an osmotic core with the drug surrounded by a sem ipermeable membrane drilled with a delivery orifice in which the drug is delivered passively or by the use of different channeling agents in the coating to control release.
  • the protease is delivered for extended periods using diffusion-controlled im planted tubes. These devices are designed to release drugs at various dosages and for both interm ittent and continuous delivery. I nfusion times are designed to operate for varying periods of time from days to several months.
  • microscale pum ps are used in which the protease is delivered by ingestion, injected into tissue, inhaled or even released into circulation.
  • any of the controlled- release systems described above is placed in proxim ity of the target area to be treated, thus requiring only a fraction of the system ic dose.
  • All compositions used for controlled- release formulations will contain a therapeutically effective amount of the hTNF-alpha specific protease, optionally with an additional therapeutic, preferably in purified form , together with a suitable amount of a pharmaceutically acceptable vehicle so as to provide the form for proper adm inistration to the patient.
  • Topical/ transdermal adm inistration I n another embodiment, the com position is prepared in a form suitable for adm inistration directly or indirectly to surface areas of the body for direct application to affected areas.
  • This formulation includes, but is not limited to, anti-drying agents (e. g. pantethine) , penetration enhancers (e. g dimethyl isosorbide) , accelerants (e.g., isopropylmyristate) or other com mon known additives used for topical applications (e. g. glycerin, propylene glycol, polyethylene glycols, ethyl alcohol, liposomes, lipids, oils, creams, or emollients) .
  • anti-drying agents e. g. pantethine
  • penetration enhancers e. g dimethyl isosorbide
  • accelerants e.g., isopropylmyristate
  • penetration enhancers Alcohols, sulphoxides, fatty acids, esters, azone, pyrrolidones, urea and polyols are j ust som e of the members of this class of com pounds (Kalbitz et al. , Modulation of drug penetration in the skin, Pharmazie 1996; 51 (9) : 619-637) .
  • the objectives of these penetration enhancers are to change the solubility and diffusivity of the drug in the stratum corneum , th us some modulate their effects through the lipid pathway while others modify diffusion via the polar pathway.
  • DMI dimethyl isosorbide
  • the present invention also provides formulations of TNF-alpha specific protease for transdermal delivery.
  • Transdermal systems deliver therapeutic formulations through the skin into the bloodstream , making them easy to adm inister.
  • Passive and active transdermal delivery systems are used to deliver medicines in even concentrations in a way that is painless and results in few adverse side effects.
  • the fusion proteins could be delivered transdermal ⁇ using a skin patch and other means such as m icroneedles, i.e. mechanically puncturing the skin in order to increase the permeability of the skin to a drug.
  • Pulmonary administration includes pulmonary delivery of the TNF-alpha specific protease form ulations. Pulmonary delivery is particularly prom ising for the delivery of macromolecules, which are difficult to deliver by other routes of administration. Such pulmonary delivery can be effective both for system ic delivery and for localized delivery to treat diseases of the lungs, since drugs delivered to the lung are readily absorbed through the alveolar region directly into the blood circulation.
  • the invention further provides com positions suitable for forming a drug dispersion for oral inhalation (pulmonary delivery) to treat various conditions or diseases.
  • TNF- alpha specific protease form ulation could be delivered by different approaches such as liquid nebulizers, aerosol based metered dose inhalers (MDI 's) , and dry powder dispersion devices.
  • MDI 's aerosol based metered dose inhalers
  • I n form ulating com positions for pulmonary delivery pharmaceutically acceptable carriers including surface active agents or surfactants and bulk carriers are com monly added to provide stability, dispersibility, consistency, and/or bulking characteristics to enhance uniform pulmonary delivery of the com position to the subject.
  • Surface active agents or surfactants promote absorptio n of polypeptide through mucosal membrane or lining.
  • Useful surface active agents or surfactants include fatty acids and salts thereof, bile salts, phospholipids, or an alkyl saccharide. Exam ples of fatty acids and salts thereof include sodium , potassium and lysine salts of caprylate (C8) , caprate (CIO) , laurate (C12) and myristate (CI4) .
  • bile salts include cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, lithocholic acid, and ursodeoxycholic acid.
  • phospholipids include single -chain phospholipids, such as lysophosphatidylcholine, lysophosphatidylglycerol, lysophosphatidylethanolamine, lysophosphatidylinositol and lysophosphatidylserine; or double-chain phospho- lipids, such as diacylphosphatidylcholines, diacylphosphatidylglycerols, diacyl- phosphatidyl- ethanolam ines, diacylphosphatidylinositols and diacylphosphatidyl-serines.
  • alkyl saccharides include alkyl glucosides or alkyl maltosides, such as decyl glucoside and dodecyl maltoside.
  • compositions that are useful as carriers include stabilizers such as human serum album in (HSA) or recombinant human album in; bulking agents such as carbohydrates, am ino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a m ixture of the two.
  • HSA human serum album in
  • recombinant human album in such as recombinant human album in
  • bulking agents such as carbohydrates, am ino acids and polypeptides
  • pH adjusters or buffers such as sodium chloride
  • salts such as sodium chloride
  • carbohydrates for use as bulking agents include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-beta-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextran, and the like; alditols, such as mannitol, xylitol, and the like.
  • Exam ples of polypeptides for use as bulking agents include aspartame.
  • Am ino acids include alanine and glycine, with glycine being preferred.
  • Additives which are m inor components of the com position, are included for conformational stability during spray drying and for im proving dispersibility of the powder.
  • additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine, and the like.
  • Suitable pH adj usters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.
  • the TNF-alpha specific protease com positions for pulmonary delivery are packaged as unit doses where a therapeutically effective amount of the com position is present in a unit dose receptacle, such as a blister pack, gelatin capsule, or the like.
  • a unit dose receptacle such as a blister pack, gelatin capsule, or the like.
  • the manufacture of blister packs or gelatin capsules is typically carried out by methods that are generally well known in the packaging art.
  • One approach for the pulmonary delivery of dry powder drugs utilizes a hand- held device with a hand pum p for providing a source of pressurized gas. The pressurized gas is abruptly released through a powder dispersion device, such as a venturi nozzle, and the dispersed powder made available for patient inhalation.
  • the present invention provides formulating TNF-alpha specific protease for oral inhalation.
  • the form ulation comprises TNF-alpha specific protease and suitable pharmaceutical excipients for pulmonary delivery.
  • the present invention also provides adm inistering the fusion protein com position via oral inhalation to subjects in need thereof.
  • Rectal administration Com positions for rectal adm inistration are prepared with any of the usual pharmaceutical excipients, including for exam ple, binders, lubricants and disintegrating agents.
  • the com position may also include cell penetration enhancers, such as aliphatic sulphoxides. I n a preferred embodiment, the composition is in the form of a suppository.
  • Proteins and peptides are prone to chem ical and conformational instability and are often degraded by the acidic conditions in the stomach, as well as by enzymes in the stomach and gastrointestinal tract.
  • certain technologies for oral delivery have been developed, such as encapsulation in nanoparticles composed of polymers with a hydrophobic backbone and hydrophilic branches as drug carriers, encapsulation in m icroparticles, insertion into liposomes in emulsions, and conj ugation to other molecules.
  • the protease variant is formulated for oral delivery exploiting one or several of the technologies developed for oral delivery of proteins.
  • Com positions of the invention for oral delivery are in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, em ulsions, capsules, syrups, or elixirs. Com pounds and compositions of the invention for oral delivery can also be form ulated in foods and food mixes. Orally administered com positions contain one or more optionally agents, for exam ple, sweetening agents such as fructose, aspartame or saccharin ; flavouring agents such as peppermint, oil of wintergreen, or cherry; colouring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
  • sweetening agents such as fructose, aspartame or saccharin
  • flavouring agents such as peppermint, oil of wintergreen, or cherry
  • colouring agents such as peppermint, oil of wintergreen, or cherry
  • preserving agents to provide a pharmaceutically palatable preparation.
  • the com positions are coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time.
  • Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally adm inistered com positions of the invention. I n these later platforms, fluid from the environment surrounding the capsule is imbedded by the driving com pound, which swells to displace the agent or agent com position through an aperture.
  • These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of im mediate release form ulations.
  • a time delay material such as glycerol monostearate or glycerol stearate may also be used.
  • Oral com positions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such vehicles are preferably of pharmaceutical grade.
  • the TNF-alpha specific protease that is used to treat certain classes of a diseases or medical conditions are particularly amenable for oral form ulation and delivery, e.g. inflam matory diseases of the gastro -intestinal tract such as Crohn's disease and ulcerative colitis. I n many chronic inflam matory diseases as described above, oral formulations of the TNF-alpha specific protease and methods of administration are particularly useful because they allow long-term patient care and therapy via home oral adm inistration without reliance on injectable treatment or drug protocols.
  • Oral form ulations and delivery methods com prising the TNF-alpha specific protease take advantage of, in part, various receptor mediated transcytosis across the gastrointestinal (Gl ) epithelium .
  • Gl gastrointestinal
  • the transferrin receptor is found at a very high density in the human Gl epithelium , transferrin is highly resistant to tryptic and chymotryptic digestion and transferrin chemical conjugates have been used to successfully deliver proteins and peptides across the Gl epithelium (Xia et al., (2000) J. Pharmacol. Experiment. Therap. , 295: 594-600; Xia et al.
  • the protease is fused to the constant region of an imm unoglobulin (Fc) which can interact with the Fc- receptor in the gut epithelium and be transported to the blood stream .
  • Oral formulations of TNF-alpha specific proteases are prepared so that they are suitable for transport to the Gl epithelium and protection of the fusion protein component and other active components in the stomach.
  • Such form ulations include carrier and dispersant com ponents and be in any suitable form , including syrups, elixirs, tablets, including chewable tablets, hard or soft capsules, troches, lozenges, aqueous or oily suspensions, em ulsions, cachets or pellet granulates and dispersible powders.
  • formulations com prising the TNF-alpha specific protease are em ployed in solid dosage forms suitable for simple, and preferably oral, adm inistration of precise dosages.
  • Solid dosage forms for oral adm inistration are preferably tablets, capsules, or the like.
  • TNF-alpha specific protease For oral administration in the form of a tablet or capsule, care is taken to ensure that the com position enables sufficient active ingredient to be absorbed by the host to produce an effective response. Thus, for exam ple, the amount of the TNF-alpha specific protease is increased over that theoretically required or other known measures such as coating or encapsulation taken to protect the polypeptides from enzymatic action in the stomach.
  • oral pharmaceutical com positions comprising the TNF-alpha specific protease are formulated in buffered liquid form which is then encapsulated into soft or hard-coated gelatin capsules which are then coated with an appropriate enteric coating.
  • the location of release is anywhere in the Gl system , including the small intestine (the duodenum , the jejunum , or the ileum) , or the large intestine.
  • nanoparticles include m ucoadhesive nanoparticles coated with chitosan and Carbopol (Takeuchi et al., Adv. Drug DeNv. Rev. 47 ( 1 ) : 39-54, 2001 ) and nanoparticles containing charged combination polyesters, poly (2-sulfobutyl-vinyl alcohol) and poly (D, L- lactic-co-glycolic acid) (Jung et al., Eur. J. Pharm . Biopharm . 50 ( 1 ) : 147-160,2000) .
  • Nanoparticles containing surface polymers with poly- N- isopropylacrylamide regions and cationic poly-vinylam ine groups showed improved absorption of salmon calcitonin when administered orally to rats.
  • Drug delivery particles com posed of alginate and pectin, strengthened with polylysine, are relatively acid and base resistant and can be used as a carrier for drugs. These particles combine the advantages of bioadhesion, enhanced absorption and sustained release (Liu et al., J. Pharm . Pharmacol. 51 (2) : 141 -149, 1999) .
  • Another form ulation exam ple is in poly (vinyl alcohol) gel spheres, such as aprotinin or bacitracin.
  • Com positions comprising TNF-alpha specific proteases intended for oral use are prepared according to any method known for the manufacture of pharmaceutical com positions and such com positions contain one or more agents including, but not limited to, sweetening agents in order to provide a pharmaceutically elegant and palatable preparation.
  • a TNF-alpha specific protease is m ixed with at least one pharmaceutical excipient, and the solid form ulation is compressed to form a tablet according to known methods, for delivery to the gastrointestinal tract.
  • the tablet composition is typically form ulated with additives, (e.g.
  • a saccharide or cellulose carrier a binder such as starch paste or methyl cellulose, a filler, a disintegrator, or other additives typically usually used in the manufacture of medical preparations.
  • protease is m ixed with at least one pharmaceutical excipient, and the solid form ulation is placed in a capsular container suitable for delivery to the gastrointestinal tract.
  • Compositions com prising a fusion protein are prepared as described generally in Rem ington: The Science And Practice Of Pharmacy, 21 st edition (2005) .
  • many of the oral formulations of the invention contain inert ingredients, which allow for protection against the stomach environment, and release of the biologically active material in the intestine.
  • Such form ulations, or enteric coatings are well known in the art.
  • tablets containing the TNF-alpha specific protease in adm ixture with non-toxic pharmaceutically acceptable excipients which are suitable for manufacture of tablets are used.
  • excipients are inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for exam ple, maize starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid, or talc.
  • the tablets aer uncoated or coated with known techniques to delay disintegration and absorption in the gastrointestinal track and thereby provide a sustained action over a longer period of time.
  • a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be em ployed.
  • Formulations for oral use are also presented as hard gelatin capsules, wherein the active ingredient is m ixed with an inert solid diluent, for exam ple, calcium carbonate, calcium phosphate, or kaolin or as soft gelatin capsules wherein the active ingredient is mixed with an aqueous or an oil medium , for example, arachis oil, peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions contain a TNF-alpha specific protease in the adm ixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for exam ple, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally occurring phosphatide, for example, lecithin, or condensation products of an alkylen oxide with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for exam ple, heptadecylethyloxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids
  • the aqueous suspensions also contain one or more preservatives for exam ple, ethyl or n- propel p- hydroxybenzoate, one or more colouring agents, one or more flavouring agents and one or more sweetening agents such as sucrose or saccharin.
  • Oily suspensions are form ulated by suspending the active ingredient in a vegetable oil, for example, arachis ol, olive oil, sesame oil or coconut oil, or in a m ineral oil such as liquid paraffin.
  • the oil suspensions may contain a thickening agent, for exam ple, beeswax, hard paraffin or acetyl alcohol.
  • Sweetening agents, such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation.
  • These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
  • the pharmaceutical com positions containing the TNF-alpha specific protease are also in the form of oil- in-water em ulsions.
  • the oil phase may be a vegetable oil, for exam ple, olive oil or arachis oil, or a m ineral oil for exam ple, gum acacia or gum tragacanth, naturally-occurring phosphatides, for exam ple soybean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for exam ple, sorbitol monooleate, and condensation products of the same partial esters with ethylene oxide, for exam ple, polyoxyethylene sorbitol monooleate.
  • the em ulsions may also contain sweetening and flavo uring agents.
  • the proportion of pharmaceutically active TNF-alpha specific protease to carrier and/or other substances varies from about 0.5 to about 100 wt. % (weight percent) .
  • the pharmaceutical form ulation will generally contain from about 5 to about 100% by weight of the active material.
  • the form ulation will generally have from about 0.5 to about 50 wt. % of the active material.
  • oral com positions of the invention are form ulated to slowly release the active ingredients, including the fusion proteins of the invention, in the Gl system using known delayed release form ulations.
  • the fusion protein is engineered to contain a cleavage site between the TNF-alpha specific protease and the second peptide moiety.
  • cleavable sites or linkers are known in the art.
  • Pharmaceutical compositions and methods may include the addition of a transcytosis enhancer to facilitate transfer of the TNF-alpha specific protease across the Gl epithelium .
  • enhancers are known in the art. See Xia etal., (2000) J. Pharmacol. Experiment. Therap., 295: 594-600 ; and Xia et al. (2001 ) Pharmaceutical Res., 18 (2) : 191 -195.
  • Figure 1 Mass spectrometric verification of the proteolytic cleavage site of TNF-alpha.
  • TNF-alpha was digested at a concentration of 1 mg/m l with protease variant E for 1 h at 37 °C in PBS buffer.
  • the short fragment of the cleavage reaction was isolated on a
  • Figure 2 Deconvoluted mass spectrometric analysis. The small cleavage product has been isolated and the determ ined mass represents the first 32 amino acids of SEQ I D NO: 1 1 , confirm ing the targeted cleavage site.
  • FIG. 3 Inactivation of recombinant human TNF-alpha activity by a panel of variants in a cell-based caspase assay.
  • the cytokine was pre- incubated at 1 ng/m l for four hours at 37 °C in the presence of the different proteases.
  • proteolytic cleavage of hTNF ⁇ by the protease digestions were added to the WEHI 13VAR cells in wells of a m icrotiter plate. Detection of hTNF ⁇ -induced caspase activation was measured by a lum inescence based read-out. Data are depicted as remaining TNF- alpha activity as a percentage of activity of undigested TNF-alpha.
  • A depicts maximal detected inactivation of hTNF ⁇
  • B depicts dose dependency of the effects of the hTNF ⁇ specific protease.
  • Figure 4 Effect of pegylation on activity.
  • Variant E was subjected to protein modification by chem ical attachment of 20 kDa poly(ethyleneglycol) .
  • the activity of the resulting pegylated protease was assessed against a recombinant hTNF ⁇ (A) or by a caspase assay that measures levels of remaining TNF-alpha activity following digestion of the cytokine by the protease (B) .
  • Pegylation does not abrogate activity, although a slight reduction in specific activity can be observed.
  • Figure 5 Specificity measurements of different Variants. Specificity of the protease variants was compared to unspecific trypsin in a biochem ical assay.
  • the substrate is a fluorescently labelled peptide representing the cleavage site in TNF-alpha. Cleavage is assessed by following the changes in the f luorescent signal.
  • the assay was performed in the presence and absence of com petitor substrate. Specific variants have a higher residual activity in the presence of com petitor (protein hydrolysate) compared to activity without competitor. Variant G shows a slightly reduced specificity com pared to the other variants, however, wild- type trypsin has a m uch lower relative activity in the presence of competitor.
  • FIG. 6 Proteolytic activity on different serum proteins. Potential activity towards serum proteins is tested for variant E in comparison with trypsin. The proteins were incubated with protease at a concentration of 50 ⁇ g/m l for two hours in PBS buffer, followed by SDS- page analysis of potential cleavage. A close inspection of the gel demonstrates no detectable cleavage of the serum proteins by variant E while substantial degradation is observed in the case of trypsin.
  • Figure 7 Efficacy of protease variant in a mouse model. Efficacy of pegylated TNF- alpha specific protease variant E is demonstrated in a human TNF-alpha transgenic mouse model of arthritis.
  • mice Six week old transgenic mice (Tg197) were random ly distributed into three dose groups: vehicle alone which served as negative control (PBS) , pegylated variant E at 50 mg/kg, and infliximab ( Rem icade ® ) at 8 mg/kg which served as positive control. At six weeks of age the m ice already expressed mild signs of the disease and hence the study demonstrates efficacy in an established disease setting. Mice were injected three times per week for five weeks. Study was term inated when vehicle control animals showed severe sym ptoms. Efficacy was determ ined by using a standard clinical scoring system for ankle joint inflam mation.
  • the sem iquantitative scoring system uses a scale of 0 to 3, in which 0 indicates no disease sym ptoms and 3 extremely debilitating disease sym ptoms. Asterixes ( * ) indicate statistically significant differences of specific drug data points when compared to the PBS control (p ⁇ 0.01 ) .
  • Figure 8 Membrane-bound TNF-alpha is not cleaved by variant G.
  • a cell line expressing a variant of human TNF-alpha that is not proteolytically processed and released from the membrane is incubated with an anti-TNF antibody (panel B) or with different amounts of protease variant G or pegylated variant G for 4 h.
  • a reporter cell line WEHI 13VAR is added and apoptosis is induced due to direct contact between membrane-bound TNF and reporter cells.
  • Figure 9 CLUSTAL W ( 1 .7) m ultiple sequence alignment between human trypsin variants, namely human cationic trypsin (SEQ I D NO: 1 ; top) , human Anionic trypsin (Trypsin-2 precursor; SEQ I D NO: 25; middle) and human Mesotrypsin (Trypsin -3 precursor, SEQ I D NO: 26; bottom) . * matching position.
  • Products were purified by standard DNA cleanup colum ns and the yield and purity assessed by agarose gel eletrophoresis as well as by UV absorbance at 260 and 280 nm .
  • the fragment amplified with BF and BR was used as tem plate (diluted 1 : 100) in a further PCR containing primers DF and DR (SEQ I D NOs: 33 and 34, respectively) in order to introduce the desired variant loops.
  • the PCR and downstream steps were as above.
  • the isolated products from the AF/ AR, DF/DR and CF/CR PCRs were then spliced by overlap extension using the above cycling parameters with 40 ng of each fragment and 0.3 ⁇ M of the outside primers, AF and CR (SEQ I D NOs: 27 and 32, respectively) .
  • the product of this reaction was gel isolated and cloned into standard expression vectors, introduced into respective expression hosts and characterized for the protease properties. I n addition, oligonucleotide primers AF and CR (SEQ ID NOs:27 and 32, respectively) were used to amplify 20 ng of wild-type human trypsin as well as proteases containing alternative SDRs using KOD polymerase and the manufacturers recommended buffer.
  • the oligo pair (300 nM each) was used with the cycling conditions 95 0 C, 2 min, followed by 25 cycles of 95 0 C, 60 s; 58 0 C, 45 s and 68 0 C, 45 s. Products were purified by standard DNA cleanup columns and the yield and purity assessed by agarose gel electrophoresis as well as by UV absorbance at 260 and 280 nm. The resulting fragment was then amplified with OF and OR using the GeneMorphll kit (Stratagene) as recommended by the manufacturer. The resulting products were gel isolated and cloned into standard expression vectors, introduced into respective expression hosts and characterized for the protease properties.
  • I ndividual candidate proteases were isolated according to their enzymatic examined characteristics (e.g., specificity, activity, resistance to inhibitors or inactivation, etc.) .
  • I ndividual genes were cloned into E col 7 (see Sambrook, J.F; Fritsch, E.F. ; Maniatis, T. ; Cold Spring Harbor Laboratory Press, Second Edition, 1989, New York) , plated to result in single colonies on solid LB (Luria- Bertani Medium : 1 % Trypton, 0.5% yeast extract, 1 .0% NaCI with a final pH of 7.0) with 1 .5% agar and containing selective marker ("selective solid medium”) .
  • I ndividual protease variants were purified by a combination of ion-exchange chromatographic steps to 99% homogeniety.
  • I ndidvidual variants were pegylated at a concentration of 1 mg/m l in 150 m M bicarbonate buffer (pH 8.3) with an excess of methoxy- poly(ethyleneglycol) - ⁇ - methyl butanoic acid (20 kD, NEKTAR Therapeutics, Huntsville, AL) .
  • the activated PEG was added successively over a period of 1 h as solid material.
  • the reaction mixture was stirred for additional 30 min at room temperature.
  • the reaction was stopped by dialysis (3 x 45 m in., MWCO 12000) against 20 m M NaOAc, 150 mM NaCI, pH 5.0.
  • the products were separated from non- pegylated protein and unreacted PEG by gel filtration chromatography.
  • the material was dialysed against PBS buffer (MWCO 12000) and lyophilised. This type of chem istry allows covalent coupling of PEG to one or several lysine residues on the surface of the protein.
  • the calculated mass for the fragment is 3606.0 Dalton which is identical with the mass of the third isotope peak. This confirms the cleavage of TNF-alpha after the twin arginine motive by the protease variants. Sim ilar result were obtained with various variants.
  • hTNF ⁇ induced caspase activation was measured by a luminescence based read-out (Figs. 3, 4B) .
  • the two forms were com pared in parallel in an activity assay against a synthetic peptide (Fig. 4A) or in a caspase assay which measures the functional activity of the cytokine upon binding to its receptor expressed on WEHI 13VAR cells (Fig. 4B) .
  • Two concentrations of the protease variants were used in the caspase assay (50 and 100 ⁇ g/m l) .
  • the pegylation has only a marginal effect on the activity of the protease variant.
  • Fig. 5 shows the relative specificities of different protease variants.
  • the activity on a peptidic substrate which represents the TNF-alpha cleavage site is measured in absence and presence of a peptide mixture as com petitor substrate.
  • the peptide (20 nM) was incubated with protease in PBS buffer at 37 °C for 10 to 30 m in. Cleavage is followed by changes in fluorescence properties of the labelled target peptide. Specificity is expressed as the ratio of cleavage rates in the presence and absence of com petitor (slope) .
  • Example 8 Efficacy in an animal model.
  • TNF-alpha specific protease was tested in a human TNF-alpha transgenic mouse model of arthritis.
  • the Tg197 strain has been extensively used to test TNF-alpha biopharmaceuticals (etanercept, infliximab, adalim umab) thus validating the model.
  • Six week old m ice were random ly distributed among four groups of 5 to 6 an imals each.
  • the mice were treated as follows: phosphate buffered saline ( PBS) vehicle alone which served as negative control, pegylated variant E at 50 mg/m l and infliximab at 8 mg/m l which served as positive control.
  • PBS phosphate buffered saline
  • mice were injected intraperitoneal ⁇ three times per week for 5 weeks when the animals were sacrificed. Arthritis was evaluated in ankle joints in a blinded manner using a sem i quantitative arthritis score ranging from 0 to 3, where 0 means no arthritis (normal appearance and grip strength) , 1 indicates m ild arthritis (joint swelling) , 2 indicates moderate arthritis (severe joint swelling and digit deformation, no grip strength) and 3 indicates severe arthritis (ankylosis detected on flexion and severely im paired movement) .
  • the mice had expressed clinical sym ptoms of the disease and as such this exam ple can be used to illustrate the efficacy of the protease in an established disease setting (Fig. 7) .
  • Example 9 Membrane-bound TNF-alpha is not cleaved.
  • a cell line expressing a m utant version of TNF with a deletion of am ino acids 1 to 12 of the mature soluble protein is incapable of shedding TNF-alpha into solution via cleavage with TNF-alpha converting enzyme (TACE) (Grell et al. ( 1995)
  • TACE TNF-alpha converting enzyme
  • the transmembrane form of tum or necrosis factor is the prime activating ligand of the 8OkDa tumor necrosis factor receptor.
  • Cell, 83: 793-802 was either incubated with infliximab or with protease variant G of the invention or pegylated variant G at different concentrations for 4 h.
  • transmembrane TNF induces apoptosis in these TNF- susceptible cells unless it is neutralised by either an antibody or by proteolytic cleavage.
  • Figure 8B shows the neutralization of TNF by a commercial antibody while variant G or pegylated variant G do not inhibit apoptosis demonstrating that transmembrane TNF-alpha is not inactivated by the protease.
  • SEQ I D NO: 1 human TNF-alpha, soluble form
  • SEQ I D NO: 26 human Mesotrypsin (Trypsin-3 precursor)

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Abstract

La présente invention concerne des protéases spécifiques, des fragments et des dérivés associés qui inactivent spécifiquement le facteur de nécrose tumorale alpha, des méthodes liées à leur préparation, des compositions pharmaceutiques et diagnostiques renfermant une telle protéase. Les protéases de cette invention sont utilisées pour traiter des troubles cliniques résultant de l'activité déficitaire du facteur de nécrose tumorale. Ladite invention a aussi pour objet des séquences de nucléotides codant la protéase, ainsi que des vecteurs et des hôtes comprenant de telles séquences.
PCT/EP2007/053788 2006-04-18 2007-04-18 Protéase spécifique pour l'inactivation du facteur de nécrose tumorale alpha humain WO2007118889A1 (fr)

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WO2004113522A1 (fr) * 2003-06-18 2004-12-29 Direvo Biotech Ag Nouvelles entites biologiques et leur utilisation a des fins pharmaceutiques ou diagnostiques
WO2004113521A1 (fr) * 2003-06-18 2004-12-29 Direvo Biotech Ag Nouvelles entites biologiques et leur utilisation

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004113522A1 (fr) * 2003-06-18 2004-12-29 Direvo Biotech Ag Nouvelles entites biologiques et leur utilisation a des fins pharmaceutiques ou diagnostiques
WO2004113521A1 (fr) * 2003-06-18 2004-12-29 Direvo Biotech Ag Nouvelles entites biologiques et leur utilisation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KIM Y J ET AL: "Determination of the limited trypsinolysis pathways of tumor necrosis factor-alpha and its mutant by electrospray ionization mass spectrometry.", ANALYTICAL BIOCHEMISTRY. 15 FEB 1999, vol. 267, no. 2, 15 February 1999 (1999-02-15), pages 279 - 286, XP002412016, ISSN: 0003-2697 *
VAN KESSEL K P ET AL: "Inactivation of recombinant human tumor necrosis factor-alpha by proteolytic enzymes released from stimulated human neutrophils", JOURNAL OF IMMUNOLOGY, THE WILLIAMS AND WILKINS CO. BALTIMORE, US, vol. 147, no. 11, 1 December 1991 (1991-12-01), pages 3862 - 3868, XP002304822, ISSN: 0022-1767 *

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