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WO2009143493A2 - Modulateurs des voies de signalisation dépendantes de tnfa et leurs utilisations - Google Patents

Modulateurs des voies de signalisation dépendantes de tnfa et leurs utilisations Download PDF

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Publication number
WO2009143493A2
WO2009143493A2 PCT/US2009/045084 US2009045084W WO2009143493A2 WO 2009143493 A2 WO2009143493 A2 WO 2009143493A2 US 2009045084 W US2009045084 W US 2009045084W WO 2009143493 A2 WO2009143493 A2 WO 2009143493A2
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Prior art keywords
tnfα
spintl
activity
tmprs
polypeptide
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PCT/US2009/045084
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WO2009143493A3 (fr
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Marcin Bugno
Jeremy Rouse
Lioubov Medvedeva
Sergie Makarov
Sergie Romanov
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Attagene Inc.
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Publication of WO2009143493A2 publication Critical patent/WO2009143493A2/fr
Publication of WO2009143493A3 publication Critical patent/WO2009143493A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8114—Kunitz type inhibitors
    • 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)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/525—Tumor necrosis factor [TNF]

Definitions

  • Embodiments of the present disclosure are directed to inhibitors of TNF ⁇ and methods of use thereof.
  • TNF ⁇ is a pleiotropic pro-inflammatory cytokine. In concert to other members of the cytokine family, TNF ⁇ plays a critical role in the host immune system which provides defense from variety of environmental pathogens, including viruses and bacteria. As pro-inflammatory cytokine, TNF ⁇ is primarily involved in the acute phase of immune responses. TNF ⁇ can be produced in response to injury or infection by a broad variety of cell types, but mainly by macrophages. Newly synthesized TNF ⁇ molecules form stable homotrimers which may exist in both membrane-integrated and soluble forms (Pennica et al., 1984; Tang et al, 1996; reviewed in Locksley et al, 2001).
  • TNF ⁇ The membrane bound form of TNF ⁇ exerts its signaling function via direct cell-cell contacts. Soluble TNF ⁇ is released from the membrane by proteolytic cleavage that is mediated by the TNF ⁇ converting enzyme (Black et al, 1997). Once released into a bloodstream, along with other cytokines, TNF ⁇ may cause numerous physiological responses. Most significantly, it promotes chemotaxis of neutrophiles attracting them to the sites of primary infection and stimulating phagocytosis. Additionally, released TNF ⁇ initiates various systemic effects by influencing function of hypothalamus. Such effects include fewer and loss of appetite and are largely mediated by the ability of TNF ⁇ to stimulate secretion of corticotropin releasing hormone and production inflammatory prostaglandin PGE2.
  • TNF ⁇ participates in a variety of additional physiological processes, most importantly in a day-to-day tissue remodeling and wound healing. These functions of TNF ⁇ are mediated by its ability to induce apoptosis in certain cell types, to promote proliferation in others, and to inhibit cell differentiation. These processes, however, require proper and timely regulation of TNF ⁇ expression. Loss of proper regulation of TNF ⁇ may lead to development of serious pathological conditions including septic shock, chronic inflammation, autoimmunity, osteoporosis, cancer and diabetes.
  • TNF ⁇ -mediated cell signaling (reviewed in Chen and Goeddel, 2002).
  • TNF ⁇ signaling is mediated by two distinct receptors, TNFaRl and TNF ⁇ R2.
  • TNFaRl is constitutively expressed in most cell types, and can be fully activated by both the membrane-bound and soluble forms of TNF ⁇ .
  • TNF ⁇ R2 is only found in cells of the immune system and responds to the membrane-bound form of the TNF ⁇ . It is commonly thought that the binding of TNF ⁇ to TNFaRl initiates most biological effects of TNF ⁇ , while the role of TNF ⁇ R2 in TNF ⁇ signaling remains largely under-investigated.
  • TNF ⁇ receptors Upon contact with their ligand, TNF ⁇ receptors undergo a conformational change triggering dissociation of the inhibitory protein silencer, SODD, from the intracellular domain of the receptor and recruitment of the adaptor protein TRADD.
  • TRADD recruits additional adapter proteins to the receptor complex, TRAF2, RIP and FADD, which serve as signaling branching points for activation of three major pathways: NF- ⁇ B pathway, MAPK pathway and apoptotic pathway.
  • NF- ⁇ B transcription factor NF- ⁇ B
  • the NF- ⁇ B family of transcription factors comprises five members, NF- ⁇ Bl, NF- ⁇ B2, c-Rel, ReIA and ReIB (reviewed in Luo et al., 2005). These proteins demonstrate constrained DNA binding specificities provided by ReI homology domain which their share, and can form homo- and heterodimers, with the most frequent being the RelA/NF- ⁇ Bl (p65/p50) heterodimer.
  • NF- ⁇ B proteins complex with inhibitory factors, IkBa, IkB ⁇ and IkB ⁇ , and reside in the cytoplasm in an inactive form.
  • IKK IkB kinase
  • the multi-component protein kinase IKK becomes activated, enabling initial phosphorylation and then dissociation and degradation of the IKBS.
  • This allows NF- ⁇ B to translocate to the nucleus and activate transcription of target genes. While translocation to the nucleus is essential step in NF- ⁇ B activation, several reports have suggested that some additional, posttranslational modifications of the NF -KB are required to achieve its maximum transcriptional activity. To this end, at least three distinct serine residues within p65/RelA protein may become phosphorylated in response to TNF ⁇ .
  • Ser-276 located in the ReI homology domain, can be phosphorylated by the catalytic subunit of protein kinase A (PKAc) as well as by mitogen- and stress-activated protein kinase- 1 (MSKl).
  • Ser-529 or Ser-536 located in the transactivation domain, can be phosphorylated by casein kinase II (CK II) or by the IKK ⁇ in response to TNF ⁇ .
  • NF- ⁇ B-dependent genes encode inhibitors of the TNF ⁇ /NF- ⁇ B pathway, including IkBa and A20.
  • IkBa and A20 IkBa and A20.
  • TNF ⁇ Activation of the MAPK pathway by TNF ⁇ .
  • TNF ⁇ strongly induces the stress-related JNK pathway which leads to activation of transcription factor c-Jun (Brenner et ah, 1989).
  • TRAF2 activates the JNK-inducing upstream kinases, such as extracellular signal regulated kinase kinase MEKKl and apoptosis stimulated kinase ASKl.
  • This activates a cascade of additional kinases that ultimately leads to activation of C-JUn-NH 2 terminal kinase, JNK.
  • Activated JNK translocates to the nucleus and phosphorylates c-Jun as well some additional transcription factors whereby increasing their trans-activation potential.
  • the JNK pathway is involved in cell differentiation, proliferation, and is generally pro-apoptotic.
  • TNFaRl is involved in death signaling.
  • TNF ⁇ -induced pathway of cell death is mediated by death domains (DDs) of TNFaRl and TRADD.
  • DDs death domains
  • TRADD Fas-associated death domain
  • apoptotic signaling transduced by TNF ⁇ is, however, often counteracted by the transcriptional activity of NF- ⁇ B, which induces expression of anti- apoptotic proteins, such as inhibitor of caspese-8 FLIP L (Micheau and Tschopp, 2003).
  • TNF ⁇ /NF-KB pathway as a pharmaceutical target. Because the TNF ⁇ /NF- ⁇ B pathway is implicated in so many pathological conditions, the ability to specifically modulate its activity has been a long standing goal of pharmacology. To this end two major classes of therapeutic agents are being developed: TNF ⁇ blockers and NF- ⁇ B inhibitors.
  • NF- ⁇ B inhibitors Since many adverse effects of deregulated TNF ⁇ signaling are mediated thru transcription factor NF -KB, several intracellular components of the TNF ⁇ /NF- KB pathway, including IKKs and NF- ⁇ B itself have become attractive potential targets for pharmacological intervention. It has been found that many traditionally used medications such as sulfasalazine, aspirin and other NSAIDs, glucocorticoids as well as some naturally- derived products, such as trans-resveratrol, curcumin and 1-acetoxychavicol acetate, exhibit inhibitory effects on NF -KB activity (reviewed in Luo et al., 2005).
  • NF- ⁇ B inhibitors however have never been systematically selected as NF- ⁇ B inhibitors, and therefore are not specific to TNF ⁇ /NF- ⁇ B signaling. They generally would reduce, to some extent, NF- ⁇ B activation in response to a variety of stimuli including IL- l ⁇ , bacterial inducers of toll-like receptors (TLRs), viruses and growth factors, while also affecting many other cellular functions. Frequently, their inhibitory effects are seen only at very high concentrations that may approach or even exceed cytotoxic levels.
  • TLRs toll-like receptors
  • BAY 11-7082 and BAY 11-7085 block NF- ⁇ B signaling by inhibiting TNF ⁇ -induced phosphorylation and degradation of IkBa.
  • SN50 peptide comprising the nuclear localization signal of NF- ⁇ Bl/p50 inhibits NF- KB activity by interfering with its translocation through the nuclear pore (Lin et ah, 1995).
  • Another peptide, NBD comprises NEMO-biding domain that is essential for interaction of IKK ⁇ with IKK ⁇ and - ⁇ .
  • NBD peptide interferes with proper assembly of IKK complex thereby inhibiting NF -KB activation pathway (May et ah, 2000).
  • Pl and P6 comprising amino acids surrounding Ser-276 and Ser-529 and -536 phosphorylation sites of RelA/p65, have been also shown to inhibit TNF ⁇ -induced NF- ⁇ B activation in vivo by blocking TNF ⁇ -induced phosphorylation and nuclear translocation of RelA/p65 while not affecting upstream components of the pathway, such as IkBa degradation and TNF ⁇ -induced activation of c-Jun (Takada et ah, 2004).
  • TNF ⁇ blockers comprise either monoclonal anti-TNF ⁇ antibodies (found in drugs marketed as Infliximab and Adalimumab) that bind specifically to human TNF ⁇ and neutralize its function, or fusion proteins (such as Etanercept) that act as a soluble decoy receptor for TNF ⁇ and inhibit the binding of TNF ⁇ to its endogenous receptor (reviewed in Chang and Girgis, 2007).
  • monoclonal anti-TNF ⁇ antibodies found in drugs marketed as Infliximab and Adalimumab
  • fusion proteins such as Etanercept
  • Approved clinical indications for the use of TNF ⁇ blockers include various severe forms of rheumato logic indications (such as active rheumatoid arthritis, ankylosing spondylitis, polyarticular juvenile idiopathic arthritis and psoriatic arthritis), and gastrointestinal indications (Crohn's disease), which are refractory to conventional therapies.
  • rheumato logic indications such as active rheumatoid arthritis, ankylosing spondylitis, polyarticular juvenile idiopathic arthritis and psoriatic arthritis
  • gastrointestinal indications Crohn's disease
  • TNF ⁇ tumor necrosis factor alpha
  • the present invention provides for inhibitors of a TNF ⁇ pathway identified by a genetic screening technology. Considering a critical role of TNF ⁇ /NF- ⁇ B pathway in inflammation, as well as acute and chronic diseases related to abnormal regulation of inflammatory responses, this discovery will be useful for creating new anti-inflammatory reagents, drugs and(or) therapies. This inhibitors will be useful for the modulation of other TNF ⁇ and NF -KB regulated processes under both normal and pathological conditions.
  • Inhibitors of TNF ⁇ /NF- ⁇ B pathway disclosed in this application represent plasma membrane bound proteins that likely target the TNF ⁇ receptor complex.
  • SPINTl and (or) TMPRS S4-specific drug candidates may represent valuable alternatives for currently used TNF ⁇ blockers.
  • simultaneous targeting of TNF ⁇ (with described anti-TNF ⁇ drugs) and TNF ⁇ receptor (with SPINTl and/or TMPRS S4 -derived recombinant proteins) may provide a complementary strategy for efficient inhibition of TNF ⁇ signaling.
  • the compounds of the present invention are useful in vitro as unique tools for understanding the biological role of molecules involved in the TNF ⁇ /NF- ⁇ B pathway.
  • the antagonists of the present invention are also useful in the development of other compounds that bind and/or inhibit TNF ⁇ because the peptide antagonists of the present invention provide important information on the relationship between structure and activity that will facilitate such development.
  • the antagonists of the present invention can also be used in assays as probes for determining the expression of molecules involved in the TNF ⁇ /NF- ⁇ B pathway. Such assays may be useful, for example, for determining the degree of cellular inflammatory response to tissue infection or injury.
  • assays may be useful, for example, for determining the degree of cellular inflammatory response to tissue infection or injury.
  • the cells under study are exposed to the peptides or peptidomimetics of the present invention and reacted cells are visualized (e.g., after wash, cell sorting, affinity chromatography, immunohistochemistry, autoradiography etc.).
  • the compounds of the present invention can be administered to a subject to completely or partially inhibit the effects of TNF ⁇ in vivo.
  • the methods of the present invention are useful in the therapeutic treatment of TNF ⁇ related disorders.
  • the compositions of the present invention can be administered in a therapeutically effective amount to treat symptoms related to inappropriate production of TNF ⁇ or inappropriate response to TNF ⁇ .
  • a pharmaceutical composition comprising a therapeutically effective amount of a SPINTl polypeptide or derivative, variants, or fragments thereof, and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising a therapeutically effective amount of a TMPRS S4 polypeptide or derivative, variants, or fragments thereof, and a pharmaceutical acceptable excipient.
  • polypeptide having 95% sequence identity to the human spintl deletion variants and having TNF ⁇ inhibitory activity there is provided a polypeptide having 95% sequence identity to the human spintl deletion variants and having TNF ⁇ inhibitory activity.
  • polypeptide having 95% sequence identity to the mouse spintl deletion variants and having TNF ⁇ inhibitory activity there is provided a polypeptide having 95% sequence identity to the mouse spintl deletion variants and having TNF ⁇ inhibitory activity.
  • polypeptide having 98% sequence identity to the human spintl deletion variant and having TNF ⁇ inhibitory activity there is provided a polypeptide having 98% sequence identity to the human spintl deletion variant and having TNF ⁇ inhibitory activity.
  • a polypeptide having 98% sequence identity to the mouse spintl deletion variant and having TNF ⁇ inhibitory activity comprising administrating a subject in need thereof with the compositions or compounds described herein.
  • a method for screening for compounds that have TNF ⁇ modulating activity comprising contacting a cell expressing the SPINTl and TMPRSS4 polypeptides of the present invention with a test agent and detecting whether TNF ⁇ activity is affected.
  • a method for screening for compounds that have SPINTl and TMPRS S4 polypeptide binding activity comprising contacting a SPINTl and TMPRS S4 polypeptide of the present invention with a test agent and detecting whether the SPINTl and TMPRS S4 polypeptide binds to the test agent.
  • FIG. 1 Illustration of TNF ⁇ -mediated cellular signaling.
  • Figure 2A DNA sequence and open reading frame (ORF) of cDNA inserts coding for a SPINTl deletion variant isolated by pLCX-MGC-3'del library screening.
  • Sail and Pad are vector-derived flanking restriction sites. See SEQ ID NO: 1 and SEQ ID NO: 2.
  • Figure 2B DNA sequence and open reading frame (ORF) of cDNA inserts coding for a TMPRSS4 isolated by pLCX-MGC-3'del library screening. EcoRI and Pad are vector- derived flanking restriction sites. See SEQ ID NO: 3 and SEQ ID NO: 4.
  • FIG. 3 TMPRSS4 and SPINTl cDNA variants identified by pLCX-MGC-3'del library screening inhibit TNF ⁇ induced NF -KB activation.
  • Activity of NF- ⁇ B was assayed in HEK293 cells co-transfected with NF- ⁇ B-dependent luciferase reporter gene construct 4xkB- Luc and pSV40-SEAP reporter plasmids along with: - pLCX-MGC-3'del (a plasmid pool containing original/non-selected 3 ' deletion cDNA library), - pLCX-Ctrl (vector control plasmid), - pLCX-IkBaSR (pLCX-based IkBa Super Repressor construct), - pLCX-IkBa-del (pLCX-based 3' cDNA deletion coding for a C-termial truncation of IkBa that produces a strong dominant negative phen
  • Transfected cells were stimulated with 3 ng/ml of TNF ⁇ for 0 (no stimulation control), 4 or 16 hrs. Activity of luciferase reporter in control and TNF ⁇ -stimulated samples was normalized to SEAP activity measured at 0 hrs. Data were expressed as average of three experiments +/- standard deviation.
  • FIG. 4 Stable expression of SPINT 1 -del and TMPRSS4 inhibits TNF ⁇ induction of NF -KB.
  • Pools of HEK293kB-EGFP reporter cells stably overexpressing identified cDNA deletion variant of SPINTl (SPINTl -del), a frame-shift mutant version of SPINTl -del ((fs)- SPINTl -del) and full length TMPRSS4 were treated with TNF ⁇ (3ng/ml) for 16 hrs. in parallel with a vector control cell poll (Ctrl).
  • NF- ⁇ B activity was monitored through FACS analysis of EGFP expression.
  • Basal EGFP profiles for analyzed cDNA clones were similar to the basal profile of control cells and, for clarity, they have been omitted.
  • FIG. 5 Stable expression of SPINT 1 and TMPRSS4 cDNA clones inhibits TNF ⁇ dependent NF -KB activity in the presence of high TNF ⁇ concentrations.
  • Pools of HEK293kB-EGFP reporter cells stably overexpressing cDNA deletion variants of IkBa (IkBa-del), IKKg (IKKg-del), SPINTl (SPINTl -del) and full length TMPRSS4 were treated with increasing TNF ⁇ concentrations (0, 0.3, 3, and 30 ng/ml) for 16 hrs. in parallel with a vector control cell poll (Ctrl). NF- ⁇ B activity was monitored through FACS analysis of EGFP expression.
  • Figure 5 shows representative data for the highest TNF ⁇ concentration tested.
  • FIG. 6 Stable expression of SPINTl and TMPRSS4 clones inhibits TNF ⁇ /NF- KB pathway but does not inhibit IL-Ib or flagellin induced NF -KB activity.
  • Pools of HEK293kB-EGFP reporter cells stably overexpressing cDNA deletion variants of IkBa (IkBa-del), IKKg (IKKg-del), SPINTl (SPINTl -del) and full length TMPRSS4 were treated with TNF ⁇ (3 ng/ml) (B), IL-Ib (10 ng/ml) (C) or flagellin (100 ng/ml) (D) for 16 hrs. in parallel with vector control cell poll (Ctrl).
  • NF -KB activity was monitored through FACS analysis of EGFP expression. Basal EGFP profiles for tested cell pools are shown in (A).
  • FIG. 7 Serine proteinase inhibitors: Pefabloc and aprotinin reverse inhibition of TNF ⁇ /NF- ⁇ B pathway in cells expressing TMPRSS4 but not SPINTl. Pools of HEK293kB- EGFP reporter cells stably overexpressing cDNA deletion variants of SPINTl (SPINTl -del) and full length TMPRSS4 were treated with TNF ⁇ (3 ng/ml) for 16 hrs. in parallel with vector control cell poll (Vect.CtrL). NF- ⁇ B activity was monitored through FACS analysis of EGFP expression.
  • Basal EGFP profiles for tested cell pools are shown in (A, C, E) for VectCtrL, TMPRSS4 and SPINTl -del, respectively.
  • EGFP profiles of TNF ⁇ treated reporter cell pools are shown in (B, D, F) for Vect.CtrL, TMPRSS4 and SPINTl -del, respectively.
  • Figure 8 Engineered human spintl deletion variant that lacks spintl intracellular domain (h-spintl-dID) inhibits TNF ⁇ induced NF -KB activation.
  • Activity of NF- ⁇ B was assayed in HEK293 cells co-transfected with NF- ⁇ B-dependent luciferase reporter gene construct 4xkB-Luc and pSV40-SEAP reporter plasmids along with constructs expressing: h- spintl FL: wild type human spintl, h-spintl_dID: human spintl without intracellular domain; a.a.
  • h-spintl ORF h-spintl dTM: human spintl without intracellular domain and transmembrane domain
  • a.a. 1-443 of h-spintl ORF m-spintl-dl604: mouse spintl deletion variant identified in the screening without intracellular domain
  • Transfected cells were stimulated with 3 ng/ml of TNF ⁇ for 24 hrs.
  • Activity of luciferase reporter in control and TNF ⁇ -stimulated samples was normalized to SEAP activity measured at 0 hrs. Data were expressed as average of three experiments +/- standard deviation.
  • Figure 10 Human spintl domains (SEQ ID NO: 6).
  • Figure 1 IA-B. Amino acid sequence of a mouse (SEQ ID NO: 7) and human (SEQ ID NO: 8) spintl deletion variant exhibiting anti-TNF ⁇ activity.
  • Figure 12. Alignment of mouse (SEQ ID NO: 7) and human (SEQ ID NO: 8) spintl deletion variants exhibiting anti-TNF ⁇ activity (including predicted signal peptide sequences .
  • Figure 13 Alignment of mouse (SEQ ID NO: 9) and human (SEQ ID NO: 10) spintl deletion variants exhibiting anti-TNF ⁇ activity (excluding predicted signal peptide sequences).
  • FIGS 14A and 14B TMPRS S4 dependent shedding of TNF ⁇ receptor I (TNFRI).
  • TNFRI TNF ⁇ receptor I
  • Control PE413 cells and the cells ectopically expressing TMPRS S4 were cultured in serum- free DMEM media in the presence or absence of TNF ⁇ (3ng/ml) for 24 hours. Cell lysates and conditioned media collected from the respective cell samples were analyzed by Western blot with antibodies specific to the extracellular part of hTNFRI.
  • Control PE413 cells and the cells ectopically expressing TMPRSS4 were cultured for 24 hours in serum- free DMEM media with the indicated concentrations of serine protease inhibitors. Conditioned media collected from the respective cells samples were analyzed by Western blot with antibodies specific to the extracellular part of hTNFRI.
  • Figure 15 Alignment of mouse (SEQ ID NO: 5) spintl ORF (top sequence) and human (SEQ ID NO: 6) spintl ORF (bottom sequence).
  • Figures 16 A - F Functional validation of SPINT 1 -dl 604 and TMPRSS4 as inhibitors of TNF ⁇ /NF- ⁇ B pathway
  • A Inhibitory effects of wild type (wt) SPINTl, SPINT 1-dl 604 and a frame-shifted (fs) variant of SPINT 1-d 1604 on TNF ⁇ /NF- ⁇ B pathway were compared in a transient transfection assay performed as described above.
  • NF- ⁇ B/GFP reporter cells stably transduced with a vector control construct SPINT 1-dl 604 - or TMPRSS4 expression construct were used for RT Q-PCR analysis of endogenous NF- ⁇ B regulated targets (TNF ⁇ and I ⁇ B ⁇ ); quantitation of TGF ⁇ l mRNA was used as a negative control.
  • Total RNA was isolated for each experimental group (prepared in quadruplicates) at 0, 2 and 6 hrs. after treatment with TNF ⁇ (10 ng/ml). RT-Q-PCR measurements of TGF ⁇ l, I ⁇ B ⁇ and TNF ⁇ mRNA levels were normalized to that of GAPDH mRNA and expressed as mean values (+/- standard deviation).
  • NF- ⁇ B dependent GFP reporter in response to TNF ⁇ [3 ng/ml] and IL- l ⁇ [100 u/ml] was analyzed in vector control NF- ⁇ B/GFP cells (D), SPINT 1-d 1604 (E) - and TMPRS S4-expressing cells (F). The cells were stimulated with cytokines for 18 hrs and the NF-kB dependent induction GFP reporter was analyzed by FACS.
  • This invention pertains to novel methods for modulating TNF ⁇ function.
  • the invention also pertains to new means of modulating TNF ⁇ /NF- ⁇ B, ILl-R/NF- ⁇ B and to TLR5/NF- ⁇ B signaling pathways.
  • the invention is based on a discovery that ectopic expression of variants of mouse SPINTl cDNA results in inhibition of TNF ⁇ -inducible activation of transcription factor NF -KB.
  • the invention is also based on a discovery that ectopic expression of full length cDNA encoding human TMPRS S4 protein results in inhibition of TNF ⁇ -inducible activation of transcription factor NF -KB.
  • AS SPINTl and TMPRS S4 proteins are evolutionary conserved and share significant degree of similarity across mammalian species, the methods of current invention equally apply to any of mammalian SPINTl and TMPRSS4 protein homologs and orthologs.
  • the invention provides new method of modulating function of TNF ⁇ , by using the SPINTl and TMPRS S4 cDNA variants of the present invention.
  • the modulation of TNF ⁇ activity is achieved by ectopic expression of the cDNA variants of the present invention and their derivatives in a biological system of interest, including a cell, a tissue, an organ, or an organism of interest.
  • the ectopic expression can be achieved by using any available methods for delivery and expression of genes in a biological system.
  • transient and stable trans fection-reagent mediated techniques include, but not limited to: transient and stable trans fection-reagent mediated techniques, retrovirus-mediated gene transfer, adenovirus-mediated gene transfer, hydrodynamic gene delivery, electroporation and other methods known to the knowledgeable in the art of molecular cell biology and medicine.
  • the invention provides new methods for modulating TNF ⁇ function, by using SPINTl and TMPRSS4 proteins and variants, derivatives, fragments, mimetics, and homologues thereof.
  • the modulation of TNF ⁇ activity is achieved by providing the biological system of interest with purified or synthesized peptides or polypeptides that encompass full length or partial regions of SPINTl or TMPRSS4 proteins.
  • the purified or synthesized proteins can be delivered into the biological system of interest by variety of methods, including simple addition to the tissue medium, systemic and local injection (intravenous (iv), intraperitoneal (ip) and subcutaneous (sc) injections), micro-injection, electrophoresis and by any other method known to the knowledgeable in the art of molecular cell biology and medicine.
  • the invention provides new methods for modulating TNF ⁇ function, by using reagents that modulate expression of function of endogenous SPINTl or TMPRS S4 proteins.
  • reagents exist or can be produced for this purpose, including small molecular weight inhibitors of SPINTl or TMPRSS4, antibodies against SPINTl or TMPRSS4, siRNA and anti-sense oligonucleotides designed to diminish expression of SPINTl or TMPRSS4 genes and other reagents known to the knowledgeable in the art of molecular cell biology and medicine.
  • the reagents can be delivered into the biological system of interest by variety of methods, including simple addition to the tissue medium, systemic and local injection (iv, ip and sc injections), microinjection, electrophoresis and by any other method known to the knowledgeable in the art of molecular cell biology and medicine.
  • the invention provides new methods for treatment of disease condition by using the described new methods of modulating TNF ⁇ function.
  • the disease condition is any pathological condition associated with TNF ⁇ function, including, but not limited to inflammation, autoimmunity, cancer and any other condition that is or will be recognized as being associated with TNF ⁇ function.
  • the treatment comprises administration of pharmacological reagents that modulate function of SPINTl or TMPRSS4 proteins.
  • the pharmacological reagents are not limited to, but may include any reagents described above.
  • Various vehicles and methods of delivery of the pharmacological reagents exist, including oral tablets, injectable solutions, liposomes, porous beads, nanoparticles and others known to the knowledgeable in the art of molecular cell biology and medicine.
  • the present invention provides for amino acid sequences derived from SPINTl and TMPRS S4 that have the ability to modulate TNF ⁇ activity.
  • the protein or peptides possess the ability to modulate TNF ⁇ /NF- ⁇ B, ILl-R/NF- ⁇ B and TLR5/NF- ⁇ B signaling pathways.
  • the polypeptides of the present invention modulate the TNF ⁇ /NF- ⁇ B signaling pathway.
  • the polypeptides of the present invention are inhibitors of TNF ⁇ activity.
  • the present invention provides SPINTl and TMPRS S4 polypeptides that have the ability to modulate TNF ⁇ activity.
  • the protein or peptides possess the ability to modulate TNF ⁇ /NF- ⁇ B, ILl-R/NF- ⁇ B and to TLR5/NF- ⁇ B signaling pathways.
  • the present application is directed to SPINTl and TMPRSS4 polypeptides at least 80%, 85%, 88%, 95%, 96%, 97%, 98% or 99% identical to the SPINTl and TMPRSS4 amino acid sequences disclosed herein.
  • the present application is directed to fragments, derivative, variants, homologues, and mimetics of the SPINTl and TMPRS S4 amino acid sequences disclosed herein having TNF ⁇ modulating activity, preferably TNF ⁇ inhibitory activity.
  • fragment refers to a protein or polypeptide that consists of a continuous subsequence of the amino acid sequence of a protein or peptide and includes naturally occurring fragments such as splice variants and fragments resulting from naturally occurring in vivo protease activity. Such a fragment may be truncated at the amino terminus, the carboxy terminus, and/or internally (such as by natural splicing). Such fragments may be prepared with or without an amino terminal methionine.
  • fragment includes fragments, whether identical or different, from the same protein or peptide, with a contiguous amino acid sequence in common or not, joined together, either directly or through a linker.
  • Such fragments may comprise at least 5 to about 50 contiguous amino acids that are identical to the amino acid sequence of the present invention.
  • the SPINTl and TMPRSS4 peptides or fragments comprise at least 7 to about 50 contiguous amino acids that are identical to the amino acid sequence of the present invention.
  • the SPINTl and TMPRSS4 peptides or fragments comprise at least 5, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 50 contiguous amino acids that are identical to the SPINTl and TMPRSS4 amino acid sequences disclosed herein.
  • the SPINTl and TMPRSS4 peptides or fragments are 95%, 96%, 97%, 98% or 99% identical to the contiguous amino acids of SPINTl and TMPRSS4 amino acid sequences.
  • the 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, etc. amino acids may substituted or deleted while maintaining the desired activity of the derived SPINTl and TMPRS S4 peptides.
  • the amino acid substitutions are conservative amino acid substitutions.
  • variant refers to a protein or polypeptide in which one or more amino acid substitutions, deletions, and/or insertions are present as compared to the amino acid sequence of an protein or peptide and includes naturally occurring allelic variants or alternative splice variants of an protein or peptide.
  • variant includes the replacement of one or more amino acids in a peptide sequence with a similar or homologous amino acid(s) or a dissimilar amino acid(s). There are many scales on which amino acids can be ranked as similar or homologous. (Gunnar von Heijne, Sequence Analysis in Molecular Biology, p. 123-39 (Academic Press, New York, N.Y.
  • Preferred variants include alanine substitutions at one or more amino acid positions.
  • Other preferred substitutions include conservative substitutions that have little or no effect on the overall net charge, polarity, or hydrophobicity of the protein.
  • Conservative substitutions are set forth in the table below.
  • the SPINTl and TMPRSS4 polypeptides have at least 80%, 85%, 88%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences of the preferred embodiments.
  • variants can consist of less conservative amino acid substitutions, such as selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • substitutions that in general are expected to have a more significant effect on function are those in which (a) glycine and/or proline is substituted by another amino acid or is deleted or inserted; (b) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (c) a cysteine residue is substituted for (or by) any other residue; (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) a residue having an electronegative charge, e.g., glutamyl or aspartyl; or (e) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not
  • variants include those designed to either generate a novel glycosylation and/or phosphorylation site(s), or those designed to delete an existing glycosylation and/or phosphorylation site(s).
  • Variants include at least one amino acid substitution at a glycosylation site, a proteolytic cleavage site and/or a cysteine residue.
  • Variants also include proteins and peptides with additional amino acid residues before or after the protein or peptide amino acid sequence on linker peptides.
  • variant also encompasses polypeptides that have the amino acid sequence of the proteins/peptides of the present invention with at least one and up to 25 (e.g., 5, 10, 15, 20) or more (e.g., 30, 40, 50, 100) additional amino acids flanking either the N-terminal or C-terminal end of the amino acid sequence.
  • the SPINTl peptides or fragments at least comprise a sequence derived from the transmembrane domain (e.g., SEQ ID NO: 11)
  • the SPINTl peptides or fragments at least comprise a sequence derived from the transmembrane domain and can also further comprise a sequence derived from the extracellular domain of SPINTl .
  • the SPINTl peptides or fragments at least comprise a sequence derived from the transmembrane domain and optionally at least 5, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 50, at least 100, at least 200, at least 300, etc. up to 444 amino acids of the N-terminus region.
  • the SPINTl peptides or fragments at least comprise a sequence derived from the transmembrane domain and optionally at least 5, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 50, at least 100, at least 200, at least 300, etc. amino acids of the extracellular domain.
  • the SPINTl peptides or fragments at least comprise of at least 5, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 50, at least 100, at least 200, at least 300, etc amino acids of the extracellular domain.
  • the peptides, peptide derivatives, or peptidomimetics of the present invention may be represented by the following formula: Xl- X2-X3, wherein Xl represents an amino acid sequence of 0 to 50 amino acids in length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40 or 50, etc.), X2 is a Spintl or TMPRSS4 polypeptide sequence according to the present invention, and X3 represents an amino acid sequence of 0 to 50 amino acids in length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40 or 50, etc.)
  • homologue refers to a protein that is at least 80 percent identical in its amino acid sequence of the proteins of the present invention as determined by standard methods that are commonly used to compare the similarity in position of the amino acids of two polypeptides.
  • the degree of similarity or identity between two proteins can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • Preferred computer program methods useful in determining the identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al, Nucleic Acids Research, 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA, Atschul, S. F. et al, J. Molec. Biol, 215: 403-410 (1990).
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al, J. MoL Biol, 215: 403-410 (1990).
  • the two proteins or polypeptides for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the "matched span", as determined by the algorithm).
  • a gap opening penalty (which is calculated as 3x (times) the average diagonal; the "average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually ⁇ fraction (1/10) ⁇ times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.
  • a standard comparison matrix see Dayhoff et al. in: Atlas of Protein Sequence and Structure, vol. 5, supp.3 for the PAM250 comparison matrix; see Henikoff et al, Proc. Natl. Acad.
  • fusion protein refers to a protein where one or more peptides are recombinantly fused or chemically conjugated (including covalently and non-covalently) to a protein such as (but not limited to) an antibody or antibody fragment like an Fab fragment or short chain Fv.
  • fusion protein also refers to multimers ⁇ i.e. dimers, trimers, tetramers and higher multimers) of peptides. Such multimers comprise homomeric multimers comprising one peptide, heteromeric multimers comprising more than one peptide, and heteromeric multimers comprising at least one peptide and at least one other protein.
  • Such multimers may be the result of hydrophobic, hyrdrophilic, ionic and/or covalent associations, bonds or links, may be formed by cross-links using linker molecules or may be linked indirectly by, for example, liposome formation.
  • Peptide Derivatives and Peptidomimetics may be the result of hydrophobic, hyrdrophilic, ionic and/or covalent associations, bonds or links, may be formed by cross-links using linker molecules or may be linked indirectly by, for example, liposome formation.
  • peptide mimetic refers to biologically active compounds that mimic the biological activity of a peptide or a protein but are no longer peptidic in chemical nature, that is, they no longer contain any peptide bonds (that is, amide bonds between amino acids).
  • peptide mimetic is used in a broader sense to include molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi- peptides and peptoids. Examples of peptide mimetics in this broader sense (where part of a peptide is replaced by a structure lacking peptide bonds) are described below.
  • peptide mimetics provide a spatial arrangement of reactive chemical moieties that closely resemble the three- dimensional arrangement of active groups in the peptide on which the peptide mimetic is based. As a result of this similar active-site geometry, the peptide mimetic has effects on biological systems that are similar to the biological activity of the peptide.
  • the peptide mimetics of the embodiments are preferably substantially similar in both three-dimensional shape and biological activity to the peptides described herein.
  • Examples of methods of structurally modifying a peptide known in the art to create a peptide mimetic include the inversion of backbone chiral centers leading to D-amino acid residue structures that may, particularly at the N-terminus, lead to enhanced stability for proteolytic degradation without adversely affecting activity.
  • An example is given in the paper "Tritriated D-ala 1 -Peptide T Binding", Smith C. S. et al, Drug Development Res., 15, pp. 371-379 (1988).
  • a second method is altering cyclic structure for stability, such as N to C interchain imides and lactames (Ede et al in Smith and Rivier (Eds.) "Peptides: Chemistry and Biology", Escom, Leiden (1991), pp. 268-270).
  • An example of this is given in conformationally restricted thymopentin-like compounds, such as those disclosed in U.S. Pat. No. 4,457,489 (1985), Goldstein, G. et al, the disclosure of which is incorporated by reference herein in its entirety.
  • a third method is to substitute peptide bonds in the peptide by pseudopeptide bonds that, confer resistance to proteolysis.
  • derivative refers to a chemically modified protein or polypeptide that has been chemically modified either by natural processes, such as processing and other post- translational modifications, but also by chemical modification techniques, as for example, by addition of one or more polyethylene glycol molecules, sugars, phosphates, and/or other such molecules, where the molecule or molecules are not naturally attached to wild-type proteins.
  • Derivatives include salts.
  • Such chemical modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given protein or polypeptide. Also, a given protein or polypeptide may contain many types of modifications.
  • Modifications can occur anywhere in a protein or polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycos
  • peptide derivatives containing additional chemical moieties not normally part of the peptide, provided that the derivative retains the desired functional activity of the peptide.
  • examples of such derivatives include (i) N-acyl derivatives of the amino terminal or of another free amino group, wherein the acyl group may be an alkanoyl group (e.g.
  • acetyl, hexanoyl, octanoyl an aroyl group (e.g., benzoyl) or a blocking group such as F-moc (fluorenylmethyl-O ⁇ CO ⁇ ); (ii) esters of the carboxy terminal or of another free carboxy or hydroxyl group; (iii) amide of the carboxy terminus or of another free carboxyl group produced by reaction with ammonia or with a suitable amine; (iv) phosphorylated derivatives; (v) derivatives conjugated to an antibody or other biological ligand and other types of derivatives.
  • an aroyl group e.g., benzoyl
  • a blocking group such as F-moc (fluorenylmethyl-O ⁇ CO ⁇
  • esters of the carboxy terminal or of another free carboxy or hydroxyl group amide of the carboxy terminus or of another free carboxyl group produced by reaction with ammonia or with a suitable amine; (iv
  • derivatives include chemical modifications resulting in the protein or polypeptide becoming branched or cyclic, with or without branching. Cyclic, branched and branched circular proteins or polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well. A cyclic derivative containing an intramolecular disulfide bond may be prepared by conventional solid phase synthesis while incorporating suitable S-protected cysteine or homocysteine residues at the positions selected for cyclization such as the amino and carboxy termini.
  • cyclization can be performed either (1) by selective removal of the S-protecting group with a consequent on-support oxidation of the corresponding two free SH-functions, to form a S--S bonds, followed by conventional removal of the product from the support and appropriate purification procedure; or (2) by removal of the peptide from the support along with complete side chain deprotection, followed by oxidation of the free SH-functions in highly dilute aqueous solution.
  • the cyclic derivative containing an intramolecular amide bond may be prepared by conventional solid phase synthesis while incorporating suitable amino and carboxyl side chain protected amino acid derivatives, at the position selected for cyclization.
  • the cyclic derivatives containing intramolecular -S-- alkyl bonds can be prepared by conventional solid phases while incorporating an amino acid residue with a suitable amino-protected side chain, and a suitable S-protected cysteine or homocysteine residue at the position selected for cyclization.
  • a peptide derivative or peptidomimetic of the present invention may be all L, all D or mixed D, L peptide.
  • the peptides consist of all D-amino acids. The presence of an N-terminal or C-terminal D-amino acid increases the in vivo stability of a peptide since peptidases cannot utilize a D-amino acid as a substrate.
  • Substitution of unnatural amino acids for natural amino acids in a subsequence of the peptides can also confer resistance to proteolysis. Such a substitution can, for instance, confer resistance to proteolysis by exopeptidases acting on the N-terminus. Such substitutions have been described and these substitutions do not affect biological activity. Examples of non-naturally occurring amino acids include ⁇ , ⁇ -disubstituted amino acids, N-alkyl amino acids, lactic acids, C- ⁇ -methyl amino acids, and ⁇ -methyl amino acids.
  • Amino acid analogs useful in the present invention may include but are not limited to ⁇ -alanine, norvaline, norleucine, 4-aminobutyric acid, orithine, hydroxyproline, sarcosine, citrulline, cysteic acid, cyclohexylalanine, 2-aminoisobutyric acid, 6-aminohexanoic acid, t-butylglycine, phenylglycine, o-phosphoserine, N-acetyl serine, N-formylmethionine, 3-methylhistidine and other unconventional amino acids. Furthermore, the synthesis of peptides with unnatural amino acids is routine and known in the art.
  • One other effective approach to confer resistance to peptidases acting on the N- terminal or C-terminal residues of a peptide is to add chemical groups at the peptide termini, such that the modified peptide is no longer a substrate for the peptidase.
  • One such chemical modification is glycosylation of the peptides at either or both termini.
  • Certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of peptides in human serum.
  • N-terminal alkyl group consisting of a lower alkyl of from 1 to 20 carbons, such as an acetyl group, and/or the addition of a C- terminal amide or substituted amide group.
  • the present invention includes modified peptides consisting of peptides bearing an N-terminal acetyl group and/or a C- terminal amide group.
  • the present invention relates to a method for inhibiting TNF ⁇ .
  • the peptides, peptide derivatives and peptidomimetics of the present invention are useful in the treatment of conditions or diseases associated with TNF ⁇ .
  • methods of the present invention comprise administering to a subject in need thereof or at risk of being in need thereof an effective amount of a peptide, peptide derivative or peptidomimetic, or a composition comprising a peptide, peptide derivative or peptidomimetic to a subject, to inhibit TNF ⁇ biological activity.
  • an effective amount of a therapeutic composition comprising a peptide or peptide derivative thereof and a suitable pharmaceutical carrier is administered to a subject to inhibit TNF ⁇ biological activity to prevent, ameliorate symptoms or treat a disorder, disease or condition related to abnormal signaling through TNF ⁇ a pathway.
  • the subject is an animal.
  • the subject is a mammal, and preferably a human.
  • the peptides, peptide derivatives and peptidomimetics of the present invention are used in the treatment, prophylaxis or amelioration of symptoms in any disease condition or disorder where the inhibition of TNF ⁇ biological activity might be beneficial.
  • compositions within the scope of the present invention should contain the active agent (e.g. peptide, peptide derivative or peptidomimetic) in an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects.
  • the active agent e.g. peptide, peptide derivative or peptidomimetic
  • Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art.
  • the amount administered should be chosen so as to avoid adverse side effects.
  • the amount of the therapeutic or pharmaceutical composition which is effective in the treatment of a particular disease, disorder or condition will depend on the nature and severity of the disease, the target site of action, the patient's weight, special diets being followed by the patient, concurrent medications being used, the administration route and other factors that will be recognized by those skilled in the art.
  • the dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 100 mg/kg/day will be administered to the subject. Effective doses may be extrapolated from dose response curves derived from in vitro or animal model test systems. For example, in order to obtain an effective mg/kg dose for humans based on data generated from rat studies, the effective mg/kg dosage in rat is divided by six.
  • compositions of the present invention can be administered by any suitable route including, intravenous or intramuscular injection, intraventricular or intrathecal injection (for central nervous system administration), orally, topically, subcutaneously, subconjunctivally, or via intranasal, intradermal, sublingual, vaginal, rectal or epidural routes.
  • compositions of the present invention can be delivered in a controlled release system.
  • polymeric materials can be used, in another embodiment, a pump may be used.
  • Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
  • the compounds of the present invention may also be coupled to a class of biodegradable polymers useful in achieving controlled release of the drug, non- limiting examples, include: polylactic acid, polyorthoesters, cross-linked amphipathic block copolymers and hydrogels, polyhydroxy butyric acid and polydihydropyrans.
  • compositions of the present invention comprise a peptide, peptide derivative or peptidomimetic combined with a pharmaceutically acceptable carrier.
  • carrier refers to diluents, adjuvants, excipients such as a filler or a binder, a disintegrating agent, a lubricant a silica flow conditioner a stabilizing agent or vehicles with which the peptide, peptide derivative or peptidomimetic is administered.
  • Such pharmaceutical carriers include sterile liquids such as water and oils including mineral oil, vegetable oil (e.g. , peanut oil, soybean oil, sesame oil, canola oil), animal oil or oil of synthetic origin.
  • Aqueous glycerol and dextrose solutions as well as saline solutions may also be employed as liquid carriers of the pharmaceutical compositions of the present invention.
  • the choice of the carrier depends on the nature of the peptide, peptide derivative or peptidomimetic, its solubility and other physiological properties as well as the target site of delivery and application.
  • carriers that can penetrate the blood brain barrier are used for treatment, prophylaxis or amelioration of symptoms of diseases or conditions (e.g. inflammation) in the central nervous system.
  • suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy by Alfonso R. Gennaro, 2003, 21st edition, Mack Publishing Company.
  • compositions of the present invention include absorption enhancers, pH regulators and buffers, osmolarity adjusters, preservatives, stabilizers, antioxidants, surfactants, thickeners, emollient, dispersing agents, flavoring agents, coloring agents and wetting agents.
  • suitable pharmaceutical excipients include, water, glucose, sucrose, lactose, glycol, ethanol, glycerol monostearate, gelatin, rice, starch, flour, chalk, sodium stearate, malt, sodium chloride and the like.
  • the pharmaceutical compositions of the present invention can take the form of solutions, capsules, tablets, creams, gels, powders, sustained release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides (see Remington: The Science and Practice of Pharmacy by Alfonso R. Gennaro, 2003, 21.sup.th edition, Mack Publishing Company).
  • compositions contain a therapeutically effective amount of the therapeutic composition, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • the formulations are designed so as to suit the mode of administration and the target site of action (e.g., a particular organ or cell type).
  • compositions of the present invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those that form with free amino groups and those that react with free carboxyl groups.
  • Non-toxic alkali metal, alkaline earth metal and ammonium salts commonly used in the pharmaceutical industry include sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine zinc salts, which are prepared by methods well known in the art.
  • the term also includes non-toxic acid addition salts, which are generally prepared by reacting the compounds of the present invention with suitable organic or inorganic acid.
  • Representative salts include the hydrobromide, hydrochloride, valerate, oxalate, oleate, laureate, borate, benzoate, sulfate, bisulfate, acetate, phosphate, tysolate, citrate, maleate, fumarate, tartrate, succinate, napsylate salts and the like.
  • fillers or binders examples include acacia, alginic acid, calcium phosphate (dibasic), carboxymethylcellulose, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulf ⁇ te, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone, sodium alginate, tragacanth, micro crystalline cellulose, starch, and zein.
  • Another most preferred filler or binder consists
  • disintegrating agents examples include alginic acid, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxypropylcellulose (low substituted), microcrystalline cellulose, powdered cellulose, colloidal silicon dioxide, sodium croscarmellose, crospovidone, methylcellulose, polacrilin potassium, povidone, sodium alginate, sodium starch glycolate, starch, disodium disulfite, disodium edathamil, disodium edetate, disodiumethylenediaminetetraacetate (EDTA) crosslinked polyvinylpyrollidines, pregelatinized starch, carboxymethyl starch, sodium carboxymethyl starch and microcrystalline cellulose.
  • alginic acid alginic acid
  • carboxymethylcellulose carboxymethylcellulose sodium, hydroxypropylcellulose (low substituted)
  • microcrystalline cellulose powdered cellulose
  • colloidal silicon dioxide sodium croscarmellose
  • crospovidone methylcellulose
  • polacrilin potassium povidone
  • Examples of lubricants include calcium stearate, canola oil, glyceryl palmitostearate, hydrogenated vegetable oil (type I), magnesium oxide, magnesium stearate, mineral oil, poloxamer, polyethylene glycol, sodium lauryl sulfate, sodium stearate fumarate, stearic acid, talc, zinc stearate, glyceryl behapate, magnesium lauryl sulfate, boric acid, sodium benzoate, sodium acetate, sodium benzoate/sodium acetate (in combination) and DL leucine.
  • Examples of silica flow conditioners include colloidal silicon dioxide, magnesium aluminum silicate and guar gum. Another most preferred silica flow conditioner consists of silicon dioxide.
  • stabilizing agents include acacia, albumin, polyvinyl alcohol, alginic acid, bentonite, dicalcium phosphate, carboxymethylcellulose, hydroxypropylcellulose, colloidal silicon dioxide, cyclodextrins, glyceryl monostearate, hydroxypropyl methylcellulose, magnesium trisilicate, magnesium aluminum silicate, propylene glycol, propylene glycol alginate, sodium alginate, carnauba wax, xanthan gum, starch, stearate(s), stearic acid, stearic monoglyceride and stearyl alcohol.
  • the present invention also provides for modifications of peptides or peptide derivatives such that they are more stable once administered to a subject (i.e., once administered it has a longer half- life or longer period of effectiveness as compared to the unmodified form).
  • modifications are well known to those skilled in the art to which this invention pertain (e.g., polyethylene glycol derivatization a.k.a. PEGylation, microencapsulation, etc.).
  • the present invention provides nucleic acid molecules encoding SPINTl and TMPRSS4 proteins or peptides that have the ability to modulate TNF ⁇ activity.
  • the protein or peptides possess the ability to modulate TNF ⁇ /NF- ⁇ B, ILl-R/NF- ⁇ B and to TLR5/NF- ⁇ B signaling pathways.
  • the present application is directed to nucleic acid molecules at least 80%, 85%, 88%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences described herein.
  • nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence encoding the TNF ⁇ modulating polypeptide.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5 ' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the polynucleotides of the present invention is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
  • larger fragments 50, 100, 150, 200, 250, 300, 350, 400, or 450 nt in length are also useful.
  • fragments which include 20 or more contiguous bases from the nucleotide sequences described herein. Generating such DNA fragments would be routine to the skilled artisan. For example, restriction endonuclease cleavage or shearing by sonication could easily be used to generate fragments of various sizes. Alternatively, such fragments could be generated synthetically.
  • nucleic acid molecules having a sequence at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequences disclosed herein will encode a polypeptides of the present invention.
  • degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assays.
  • a reasonable number will also encode a polypeptides having the desired activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g. , replacing one aliphatic amino acid with a second aliphatic amino acid).
  • the present invention also provides for antibodies the SPINTl and TMPRSS4 polypeptides of the present invention. According to some embodiments, the antibodies of the present invention target the transmembrane and extracellular domains of SPINTl and
  • TMPRS S4 or like sequences.
  • Antibodies to the SPINTl and TMPRS S4 polypeptides of the present invention may be obtained or prepared using any conventional method of making antibodies.
  • the SPINTl and TMPRSS4 antibodies may be either monoclonal, polyclonal, or a mixture of monoclonal and/or polyclonal antibodies.
  • the SPINTl and TMPRS S4 antibodies may comprise whole antibody or antigen-binding fragments thereof, such as Fv, F(ab')2 and Fab.
  • Antibody fragments may be prepared by cleavage of the intact protein, e.g. by protease or chemical cleavage. Alternatively, a truncated gene is designed.
  • a chimeric gene encoding a portion of the F(ab')2 fragment would include DNA sequences encoding the CHl domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.
  • SPINTl and TMPRS S4 antibodies can be produced as a single chain, instead of the normal multimeric structure.
  • Single chain antibodies are described in Jost et al., 269 J. BlOL. CHEM. 26267-73 (1994), incorporated herein by reference, and others.
  • DNA sequences encoding the variable region of the heavy chain and the variable region of the light chain are ligated to a spacer encoding at least about 4 amino acids of small neutral amino acids, including glycine or serine.
  • the protein encoded by this fusion allows assembly of a functional variable region that retains the specificity and affinity of the original antibody.
  • SPINTl and TMPRSS4 antibodies can be produced by use of Ig cDNA for construction of chimeric immunoglobulin genes (Liu et al, 84 PROC. NATL. ACAD. SCI. 3439 (1987) and 139 J. IMMUNOL. 3521 (1987), incorporated herein by reference.
  • mRNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA.
  • the cDNA of interest may be amplified by the polymerase chain reaction using specific primers (U.S. Patent 4,683,195 and U.S. Patent 4,683,202). Alternatively, a library is made and screened to isolate the sequence of interest.
  • the DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences.
  • the sequences of human constant regions genes may be found in Kabat et al, "Sequences of Proteins of Immunological Interest" N.I.H. PUBLICATION No. 91-3242 (1991). Human C region genes are readily available from known clones. The chimeric, humanized antibody is then expressed by conventional methods.
  • the SPINTl and TMPRSS4 antibodies may be labeled according to standard methods known in the art.
  • antibodies can be labeled with detectable labels such as enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • Enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, acetylcholinesterase, and combinations thereof.
  • Prosthetic groups include, but are not limited to, streptavidin, biotin, avidin, and combinations thereof.
  • Fluorescent material includes, but is not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, and combinations thereof.
  • Luminescent material includes, but is not limited to, luminol.
  • Bioluminescent material includes, but is not limited to, luciferase, luciferin, aequorin, and combinations thereof.
  • Radioactive material includes, but is not limited to, 125 1, 131 1, 35 S, and 3 H.
  • the present invention provides for methods of screening for compounds ⁇ e.g. , small molecules, macromolecules, etc.) that have TNF ⁇ modulating activity.
  • a method for screening for compounds that have TNF ⁇ modulating activity comprising contacting a cell expressing the SPINTl and TMPRS S4 polypeptides of the present invention with a test agent and detecting whether TNF ⁇ activity is affected.
  • the detecting step comprises detecting whether the TNF ⁇ /NF- ⁇ B, ILl-R/NF- ⁇ B and to TLR5/NF- ⁇ B signaling pathways have been modulated (e.g., activated or inhibited).
  • a method for screening for compounds that have SPINTl and TMPRS S4 polypeptide binding activity comprising contacting a SPINTl and TMPRS S4 polypeptide of the present invention with a test agent and detecting whether the SPINTl and TMPRS S4 polypeptide binds to the test agent.
  • Bio system any vertebrate cell, cell population, tissue, organ or organism.
  • TNF ⁇ signaling any chain of molecular events initiated by TNF ⁇ , including but not limited to the NF- ⁇ B pathway, the MAPK pathway, or cell death signaling.
  • TNF ⁇ function any biological process mediated ⁇ e.g., activated or inhibited) by TNF ⁇ under normal or pathological conditions, including but not limited to immune response to bacterial, fungal, viral and parasitic infection; cell proliferation, differentiation and apoptosis at sites of tissue injury of inflammatory and non-inflammatory origin.
  • TNF ⁇ associated disease any pathological condition in which monitoring or modulation of TNF ⁇ level or activity is used for prognosis, prophylaxis or therapy.
  • TNF ⁇ associated diseases include, but are not limited to, septic shock, adult respiratory distress disorder, gastrointestinal necrosis, acute renal tube necrosis, adrenal hemorrhage and disseminated intravascular coagulation.
  • chronic TNF ⁇ associated diseases include but are not limited to cachexia, anorexia, hepatosplenomegaly, subendocardial inflammation, insulin resistance, rheumatoid arthritis and multiple sclerosis.
  • Gene specific reagents reagents that can be designed or generated by the skilled in the art based on nucleotide or amino acid sequence of a disclosed gene or related nucleotide or amino acid sequences including nucleotide or amino acid sequences of alternative transcript variants of a disclosed gene, single nucleotide polymorphism variants of a disclosed gene and other natural or engineered sequences significantly homologous (orthologous or paralogous) to the sequence of a disclosed gene.
  • gene specific reagents include: nucleotide sequence derived reagents, DNA or RNA oligonucleotides, oligonucleotide probes, siRNA molecules, other recombinant DNA or RNA constructs containing complete or partial fragments of nucleotide sequence of a disclosed gene or related nucleotide sequences as well as other reagents that can interact with (e.g.
  • proteins or peptides comprising complete or partial amino acid sequences encoded by a disclosed gene or related nucleotide sequences; reagents mimicking structure of interaction domains of such proteins or peptides; reagents that can specifically interact with such proteins or peptides including, but not limited to engineered interaction partners of such proteins or peptides: antibodies, peptides or small molecule reagents directly binding disclosed proteins or peptides (e.g. synthetic substrates or inhibitors of proteins that posses enzymatic activities) or physiological interaction partners of disclosed proteins or peptides: e.g. proteins identified through their direct binding to disclosed proteins or peptides that modulate disclosed biological function(s) or natural(physiological) substrates or inhibitors of proteins that posses enzymatic activities.
  • Delivery method any method that can be employed by one skilled in the art to introduce a gene specific reagent into a biological system.
  • delivery methods for gene specific reagents include but are not limited to DNA transfection (lipofection, electroporation, nucleoporation), viral gene delivery methods or direct injection of DNA, RNA or proteins molecules into a biological system.
  • Pharmaceutically acceptable carrier refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients of the compound and which is not toxic for the host (e.g., patient) to whom it is administered.
  • the agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modeling methods such as computer modeling.
  • macromolecules having non-naturally occurring modifications are also within the scope of the term "molecule".
  • peptidomimetics well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by modeling as mentioned above.
  • the polypeptides of the present invention are modified to enhance their stability. It should be understood that in most cases this modification should not alter the biological activity of the polypeptide.
  • Purified refers to a compound or compounds having been separated from a component of the composition in which it was originally contained.
  • a “purified peptide” or a “purified composition of peptides” has been purified to a level not found in nature.
  • a “substantially pure” compound is a compound that is lacking in most other components (e.g., 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% free of contaminants).
  • the term “crude” means compounds that have not been separated from the components of the original composition in which it was present.
  • the units e.g. 66, 67 . . . 81, 82, . . . 91, 92% . . .
  • Short peptide is intended to mean a sequence of about 6-25 amino acids.
  • a composition includes a plurality of such compositions, as well as a single composition
  • a reference to “a therapeutic agent” is a reference to one or more therapeutic and/or pharmaceutical agents and equivalents thereof known to those skilled in the art, and so forth.
  • a reference to “a host cell” includes a plurality of such host cells
  • a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
  • NF- ⁇ B inhibitors were isolated by using a genetic screening technology. Majority of NF- ⁇ B inhibitors encoded by identified cDNA variants represented known NF- KB pathway genes: NFKBIA, NFKBIB, NFKBIE and IKBKG (Table 1). Additional cDNA clones inhibiting TNF ⁇ /NF- ⁇ B signaling coded for Kunitz-type membrane-associated serine protease inhibitor deletion variant (SPINTl, Fig. 2A) and full length trans-membrane serine protease 4 (TMPRSS4, Fig. 2B). Neither of these proteins has been previously known as regulators of TNF ⁇ /NF- ⁇ B pathway (Table 1).
  • Table 1 cDNA variants identified through expression cloning and functional selection in TNF ⁇ /NF- ⁇ B reporter cell line.
  • HEK293kB-EGFP reporter cell line with integrated NF- ⁇ B-EGFP reporter construct thus enabling monitoring of NF- ⁇ B status by FACS-based measurement of EGFP fluorescence in individual cells.
  • the HEK293kB-EGFP cells were stably transduced with recombinant retroviruses containing CMV-cDNA-IRES- puro R expression cassettes co-expressing either SPINTl or TMPRSS4 cDNA variants with puro R gene that facilitates an effective puromycin (2 ug/ml) selection.
  • FIG. 4 show FACS-generated EGFP profiles of HEK293kB-EGFP cell pools (vector control, SPINTl -del, TMPRSS4 and frame-shift SPINTl -del variant) following stimulation with TNF ⁇ .
  • vector control SPINTl -del
  • TMPRSS4 frame-shift SPINTl -del variant
  • NF -KB transcription can be induced by variety of pro-inflammatory stimuli, including stimuli that activate IL-Rl and TLR signaling pathways.
  • pro-inflammatory stimuli including stimuli that activate IL-Rl and TLR signaling pathways.
  • TNF ⁇ /NF- ⁇ B signaling pathway we investigated whether activation of NF- ⁇ B by IL- l ⁇ and flagellin (specific ligand of toll like receptor 5) was also impaired in the cells stably expressing SPINTl and TMPRS S4 cDNA variants.
  • Fig. 6 did not IL-I ⁇ - nor flagellin-dependent NF -KB activation was inhibited by stable expression of SPINTl and TMPSS4 cDNA variants.
  • TMPRS S4 significantly increased NF- ⁇ B activity in cells subjected to IL-I ⁇ or flagellin, suggesting a broader role of this protein in coordinating NF- ⁇ B dependent responses under inflammatory conditions.
  • Example 4 Effect of small molecule inhibitors and protein inhibitors of serine proteases on SPINTl and TMPRSS4 mediated inhibition of TNFa signaling.
  • TNF ⁇ receptor complex may be a target of SPINTl and TMPRSS4 mediated inhibition of TNF ⁇ /NF- ⁇ B pathway: 1. Both SPINTl and TMPRS S4 represent membrane associated proteins containing large extracellular domains with defined biochemical activities. 2. SPINTl and TMPRSS4 mediated NF- ⁇ B inhibition is TNF ⁇ specific since NF -KB pathway is still activated by IL- l ⁇ or flagellin in cells ectopically expressing SPINTl or TMPRSS4.
  • SPINTl a serine proteinase inhibitor
  • TMPRS S4 a serine proteinase
  • proteolytic processing of a component/components of TNF ⁇ receptor complex may represent a mechanism of TMPRS S4 dependent inhibition of TNF ⁇ signaling pathway.
  • SPINTl may block function of TNF ⁇ pathway through an alternative mechanism that presumably does not require proteolysis e.g., via a direct interaction with TNF ⁇ receptor complex.
  • TMPRSS4 as the anti-TNF ⁇ agent, we assessed its effect on the status of TNF ⁇ receptor I, TNFRI. To do so, we prepared protein samples from either control cells or from cells stably expressing TMRSS4, as well as from corresponding aliquots of conditioned growth media. Subsequent Western blot analysis with antibodies against the extracellular part of TNF ⁇ receptor I demonstrated that expression of TMPRSS4 induced release of a ⁇ 30 kDa TNFRI fragment into the growth media, while reducing the amount of TNFRI in the corresponding cell lysates (Figure 14a). Similar effects were observed in the presence of TNF ⁇ stimulation. The anti-TNFRI antibody immunoreactive band was not detectable in the media samples collected from the control PE413 cells, suggesting that the expression of TMPRSS4 was absolutely required to facilitate the release of a soluble form of TNFRI under these experimental conditions.
  • TMPRS S4-dependent anti-TNF ⁇ activity shedding of TNFRI from the cell surface renders TMPRS S4- expressing cells refractory to the TNF ⁇ stimulation.
  • Second, secretion of a soluble TNFRI may lead to the local sequestering of TNF ⁇ molecules (through TNF ⁇ - soluble TNFRI interactions) and preventing TNF ⁇ from binding to functional TNF ⁇ receptor complexes present on the cells, regardless of their TMPRSS4 activity.
  • Example 5 Analysis ofspintl domains involved in inhibition of TNFa signaling.
  • spintl_dl604 represents a C-terminal truncation of the wild type mouse spintl that lacks intracellular domain (ID) but retains complete transmembrane (TM) and extracellular domains.
  • ID intracellular domain
  • TM transmembrane
  • h-spintl dID includes a. a. 1-468 of h-spintl ORF
  • h-spintl dTM includes a.a. 1-443 of h-spintl ORF
  • h-spintl dTM includes a.a. 1-443 of h-spintl ORF
  • Example 6 Identification and validation of SPINTl and TMPRSS4 as novel modulators of TNFA signaling.
  • TMPS S4 mouse Kunitz-type membrane-associated serine protease inhibitor
  • Fig. 16 A deletion variant of mouse Kunitz-type membrane-associated serine protease inhibitor, Spintl -dl 604, and another one encoding full-length human trans-membrane serine protease 4 (TMPRS S4) were confirmed to inhibit the TNFA induced activation of NF-kB reporter (Fig. 16).
  • the inhibitory effects of both cDNA variants were stronger than that of cDNA encoding wild type form of IkBa used as a positive control for NF-KB pathway inhibition.
  • Wild type SPINTl (also known as HAI-I) is produced as a type 1 transmembrane protein.
  • the SPINT 1-d 1604 variant comprises 3 '-terminal deletion removing almost the entire region coding for a short C-terminal intracellular domain of the protein, while retaining the transmembrane and extracellular regions.
  • NF-KB/GFP cells (with integrated NF- ⁇ B-GFP reporter construct) were transduced with recombinant retroviruses containing either SPINT 1-dl 604 or TMPRS S4 expression cassettes, stimulated with TNFA and analyzed by flow cytometry.
  • SPINT 1-dl 604 and TMPRS S4 expression cassettes were transduced with recombinant retroviruses containing either SPINT 1-dl 604 or TMPRS S4 expression cassettes, stimulated with TNFA and analyzed by flow cytometry.
  • both cDNA variants strongly inhibited NF-KB dependent accumulation of GFP positive cells over a broad range of TNFA concentrations ((Fig. 16D-F).
  • TNFA activates transcription factor NF-KB since the primary mechanism whereby TNFA activates transcription factor NF-KB includes phosphorylation and degradation of I ⁇ B ⁇ protein, we examined status of IKBA in SPINT 1-dl 604 and TMPRS S4-expressing cells treated with TNFA. Western blot analysis showed that both phosphorylation and degradation of IKBA was impaired in SPINTl-dl604 and TMPRS S4-expressing cells (Fig. 16C). These data indicated that both SPINT 1-dl 604 and TMPRS S4 blocked TNFA/NF-KB signaling events upstream of IKB kinase (IKK) complex activation.
  • IKK IKB kinase
  • [00142] 1 Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP. A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature. 1997 Feb 20;385(6618):729-33.

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Abstract

L’invention concerne des polypeptides S4 de SPINTl et TMPRS ayant une activité modulatrice de TNFa et leurs compositions et utilisations.
PCT/US2009/045084 2008-05-22 2009-05-22 Modulateurs des voies de signalisation dépendantes de tnfa et leurs utilisations WO2009143493A2 (fr)

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CA2651043A1 (fr) * 2006-05-23 2007-12-06 David C. Tully Composes et compositions inhibant les proteases activatrices de canaux
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