WO2013126919A1 - Protéines de fusion à domaine de liaison de nemo - Google Patents

Protéines de fusion à domaine de liaison de nemo Download PDF

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WO2013126919A1
WO2013126919A1 PCT/US2013/027702 US2013027702W WO2013126919A1 WO 2013126919 A1 WO2013126919 A1 WO 2013126919A1 US 2013027702 W US2013027702 W US 2013027702W WO 2013126919 A1 WO2013126919 A1 WO 2013126919A1
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nbd
fusion protein
mcoti
seq
fragment
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PCT/US2013/027702
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Nima Shiva
Mark NOWAK
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Encode Bio, Inc.
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Priority to EP13751500.3A priority Critical patent/EP2817324A4/fr
Priority to JP2014558936A priority patent/JP2015509949A/ja
Publication of WO2013126919A1 publication Critical patent/WO2013126919A1/fr
Priority to US14/466,807 priority patent/US20150031598A1/en

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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—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
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • NBD nuclear factor kB essential modulator-binding domain
  • IKK IkB kinase
  • NBD In order for NBD to be effective, however, it needs to be delivered to cells.
  • Cell penetrating peptides have therefore been used as delivery vehicles to facilitate the introduction of NBD into cells.
  • Such peptides have been found to have a very narrow therapeutic range (i.e., having little difference between toxic and therapeutic doses), which severely limits their application as therapeutics.
  • known NBD peptides In anti-inflammatory animal models, known NBD peptides begin to demonstrate efficacy with doses ranging from 1-20 mg/kg, but toxicity has been observed in mice at doses greater than 50 mg/kg.
  • FIG. 1 is an illustration of the structure of MCoTI-I and MCoTI-II.
  • FIG. 2 is a graph showing that NBD- MCoTI-II inhibits NF- ⁇ signaling with greater potency than NBD Peptide.
  • FIG. 3 includes two graphs showing the dose-response relationship for NBD- MCoTI-II (graph A) and NBD peptide (graph B) inhibition of NF- ⁇ signaling in a cell- based assay.
  • NBD- MCoTI-II is 10 6 - fold more potent than NBD peptide in this assay.
  • FIG. 5 is another graph showing that NBD- MCoTI-II reduces inflammation in a mouse CPE model.
  • NBD- MCoTI-II is 5000-fold more potent than NBD Peptide.
  • FIG. 6 is a graph showing that NBD- MCoTI-II reduces arthritic scores in a mouse CIA model.
  • FIG. 7 is a graph showing that NBD- MCoTI-II reduces paw swelling to background levels in a mouse CIA model.
  • FIG. 8 is a graph showing that NBD- MCoTI-II prevents arthritic damage in joints in a mouse CIA model.
  • FIG. 9 is a graph showing that NBD- MCoTI-II shows a remarkable reduction in composite histopathology score as compared to existing anti-arthritic drugs.
  • FIG. 10 sets forth sequences of nucleotides and polypeptides comprising NBD and MCoTI-II, with NBD sequences underlined.
  • FIG. 11 sets forth more sequences of nucleotides and polypeptides comprising NBD and MCoTI-II, with NBD sequences underlined.
  • FIG. 12 sets forth even more sequences of nucleotides and polypeptides comprising NBD and MCoTI-II, with NBD sequences underlined and linker residues bolded.
  • Figure 13 sets forth further sequences of nucleotides and polypeptides comprising NBD and MCoTI-II, with NBD sequences underlined.
  • the fusion proteins disclosed comprise NBD or a fragment thereof and MCoTI- I II or a fragment thereof.
  • the fusion protein is formed by replacing loop six of MCoTI- I II with NBD or a fragment thereof.
  • NBD can be represented by SEQ ID NOS. 4, 22, or 23, for example, while the fusion protein can be represented by SEQ ID NOS. 9, 10, or 13-21.
  • the NBD or a fragment thereof is at an N-terminus of the fusion protein and replaces loop 6 of MCoTI- I/II.
  • the fusion protein can be cyclic or linear, and can additionally compromise one or more linkers. It can also be formulated as a pharmaceutical composition in a pharmaceutically acceptable carrier..
  • fusion proteins can be used to treat inflammatory diseases and other medical conditions of a subject.
  • a pharmaceutical composition compromising an NBD-MCoTI-I/II fusion protein can be administered to a subject in need thereof in a therapeutically effective amount.
  • amino acid refers to natural amino acids as well as amino acid analogs (i.e., non-natural, synthetic, and modified amino acids, including D and L optical isomers).
  • Bioactive peptide refers to a peptide, such as NBD, having a pharmaceutical effect.
  • Bioactive peptides may be peptide mimetics.
  • Bioactive peptides may be proteins synthesized in the cell in the form of prepropeptides, which are then cleaved and modified to give active products.
  • Bioactive peptides can be peptides with hormone- or drug-like activity that eventually modulate physiological function through binding interactions to specific receptors on target cells leading to induction of physiological responses. According to their functional properties, bioactive peptides can be classified as antimicrobial, antithrombotic, antihypertensive, opioid, immunomodulatory, mineral binding, and antioxidative, and the like.
  • Cell penetrating peptides refer to polypeptides which are able to translocate across a cell's plasma membrane, together with other moieties.
  • Cell penetrating peptides are generally short peptides that facilitate cellular uptake of other molecules, in particular for therapeutic purposes.
  • CPP's can be associated with the molecules being delivered either through chemical linkage via covalent bonds or through non-covalent interactions.
  • Cyclic peptides are polypeptide chains whose amino and carboxyl termini are themselves linked together with a peptide bond that forms a circular chain.
  • “Fragment” refers to a molecule, in particular an amino acid molecule, having truncations, deletions, and/or modifications with respect to a parent or wild-type molecule. Modifications of amino acid residues/proteins are well known in the art, and include, for example, acylation, methylation, phosphorylation, sulfation, and the like.
  • Fusion protein refers primarily to the combination of NBD or a fragment thereof, and MCoTI-I/II or a fragment thereof. It is to be understood that the present fusion proteins can be produced by any means, such as via bacterial synthesis or automated synthesis.
  • Nucleic acids can be single or double-stranded, linear or circular, and/or natural or synthetic polynucleotides. Additionally, nucleic acids can include methylated nucleotides and/or non-naturally occurring nucleotides, such as nucleotide analogs.
  • Linear in regard to polypeptides, refers to a polypeptide comprising a single linear polymer chain of amino acids bonded together. Linear polypeptides can be formed using automated synthesizers, for example, or through recombinant methods. Linear polypeptides may include disulfide bonds.
  • Loop 6 refers to loop 6 of the MCoTI-MI protein (SEQ ID NO:27).
  • Medical condition refers to both a disease and the symptoms of the disease.
  • MCoTI-1/ ⁇ refers to a molecule or fragment thereof comprising SEQ ID NO:25 or a portion thereof.
  • Amino acid 15 in this sequence can be either lysine or glutamine, and amino acid 16 can be either lysine or arginine.
  • amino acids 15 and 16 are glutamine and arginine, respectively, while in wild- type MCoTI-II amino acids 15 and 16 are lysine and lysine, respectively.
  • Fragments of MCoTI-I/II can comprise a fragment in which loop 6 has been deleted, for example.
  • NBD nuclear factor ⁇ (NF- ⁇ ) essential modulator-binding domain peptide sequence.
  • NBD refers to the full-length sequence of NBD, as represented by SEQ ID NO:4, either with or without a terminal glutamine residue.
  • NBD or fragments thereof refers to either the full-length NBD peptide sequence represented by SEQ ID NO:4, or to pharmaceutically effective fragments of the NBD peptide sequence that can be used to produce the fusion proteins disclosed herein, for example, the NBD fragments represented by SEQ ID NO:22 and SEQ ID NO:23.
  • Fragments of NBD preferably include from about four amino acids to about 10 amino acids of the full-length NBD sequence and can include truncations, amino acid deletions, and/or amino acid modifications of NBD.
  • NBD Peptide refers to a molecule comprising NBD and a CPP (other than MCoTI-I/II or a fragment thereof). NBD Peptides are known to block the activation of the ikB kinase (IKK) kinase complex by preventing the interaction of NEMO with IKKa and ⁇ .
  • IKK ikB kinase
  • “Pharmaceutical” refers to an effect in restoring, correcting or modifying a physiological function of a subject, including the cure, mitigation, treatment or prevention of disease in the subject.
  • compositions having a pharmaceutical effect are compositions having a pharmaceutical effect.
  • Polypeptide “peptide,” and “protein,” may be used interchangeably and refer to polymers of amino acids which compromise four or more amino acids bonded via peptide bonds.
  • a protein can be linear, branched, or cyclic and may comprise naturally occurring amino acids and/or amino acid analogs.
  • “Therapeutically effective amount” refers to the amount of a composition that, when administered to a subject, is sufficient to have an effect in restoring, correcting or modifying a physiological function of a subject, including in the treatment of a disease or other medical condition.
  • “Therapeutic range” refers to the dosage difference between toxic and therapeutic dosages.
  • Treatment includes both prophylactic treatment and the treatment after a subject experiences a disease or other medical condition, such as inflammation and/or an inflammatory disease or condition.
  • prophylactic treatment of inflammation includes administration of a composition before a subject experiences inflammation and/or an inflammatory disease or condition.
  • Treating includes reducing, ameliorating, eliminating, blocking, inhibiting, and the like.
  • a polypeptide consists of an amino acid sequence when the polypeptide does not contain any other amino acids but the recited amino acid sequence.
  • a polypeptide consists essentially of an amino acid sequence when such an amino acid sequence is present together with only a few additional amino acid residues, i.e., from about 1 to about 10 additional residues, more preferably between 1 and 5.
  • a polypeptide "compromises" an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the polypeptide.
  • the present invention is directed to a fusion protein compromising an NBD, or a fragment thereof, fused to MCoTI-I/II or a fragment thereof.
  • NBD is known to block activation of NF- ⁇ by blocking the activation of the ⁇ complex in the NF- ⁇ signaling pathway. Blocking activation of ⁇ prevents activation of NF- ⁇ , thereby preventing it from entering the cytoplasm of the cell and acting as a transcription factor.
  • One such fusion protein is NBD-MCoTI-II, which can be used as a medicament in methods to treat inflammatory diseases and other medical conditions.
  • the NBD used in the present fusion molecule is preferably the 11 amino acid sequence as represented by SEQ ID NO:4.
  • therapeutically active fragments of this 11 amino acid sequence can also be used to form the present fusion proteins.
  • a 6 amino acid NBD sequence (deletion of the first three amino acids at positions 1-3 and deletion of the last two amino acids at positions 10-11 of NBD), as represented by SEQ ID NO:22, can be fused to MCoTI-I/II or a fragment thereof to form a molecule of the present invention.
  • MCoTI-I/II a deletion of amino acids at positions 10-11 of NBD
  • MCoTI-I (SEQ ID NO:24) and MCoTI-II (SEQ ID NO:3) are cystine-knot microproteins (CKMs).
  • these polypeptides (1, shown in Figure 1) comprise a plurality of loops, including loop 1 (11), loop 2 (12), loop 3 (13), loop 5 (15), and loop 6 (16), and disulfide bonds (20).
  • MCoTI-I and MCoTI-II can be found or produced as cyclic peptides (with a peptide bond shown by reference numeral 30), or can be produced as linear constructs.
  • CKMs are small peptides, typically consisting of less than 50 amino acids. They are pharmacologically active substances with a defined structure, generally based on intra-molecular disulfide bonds and a small triple stranded ⁇ -sheet [(see, e.g., Craik DJ (2001) Plant cyclotides: circular, knotted peptide toxins. Toxicon 39: 1809-13, the contents of which are herein incorporated by reference in its entirety)]. Their unique structure is responsible for their incredibly high thermal, chemical and enzymatic stability.
  • CKMs can be boiled, incubated at 65°C for weeks, or even placed in IN HCl or IN NaOH without loss of structural and functional integrity, [(see, e.g., Austin J (2009) Biosynthesis and biological screening of a genetically encoded library based on the cyclotide MCoTI-I. Chembiochem 10:2663-70; Fridman JS, et al., (2010) Selective inhibition of JAK1 and JAK2 is efficacious in rodent models of arthritis: preclinical characterization of INCB028050. J Immunol 184:5298-307; Khaja K et al., (2010) Comparison of Functional Protein Transduction Domains Using the NEMO Binding Domain Peptide.
  • CKMs include the intrathecally administered analgesic drug ziconotide (see, e.g., Doggrell SA (2004) Intrathecal ziconotide for refractory pain. Expert Opin Investig Drugs 13:875-7).
  • MCoTI-I or MCoTI-II form the backbone of the fusion proteins disclosed herein.
  • the sequence of MCoTI-I is shown by SEQ ID NO: 25 ( Figure 13), while that of MCoTI-II is shown by SEQ ID NO:3 ( Figure 10).
  • MCoTI-I and MCoTI-II are members of the trypsin inhibitor CKM subfamily.
  • NBD-MCoTI-I/II as disclosed herein is formed by the fusion of (1) NBD
  • MCoTI-II in linear form can be used to form the present NBD-MCoTI-II fusion protein, and can be represented by SEQ ID NO: 10.
  • the fusion protein is formed by replacing loop 6 of MCoTI- I II (as shown in FIG. 1) with NBD or a fragment thereof. Loop 6 of MCoTI-II is represented by SEQ ID NO: 27.
  • the foregoing amino acid sequence is preferably not present.
  • the fusion can be direct, i.e.
  • the inventive fusion protein can be circular and/or can be both linear and circular.
  • the NBD, or a fragment thereof can be inserted into other locations in the MCoTI-II protein sequence.
  • the inventive fusion proteins disclosed herein can be formed by replacing loop 6 and at least a portion of one or more other loops of MCoTI-II and/or the inventive fusion proteins disclosed herein can be formed by replacing at least a portion of loop 6 and/or the inventive fusion proteins disclosed herein can be formed by replacing at least a portion of a loop other than loop 6 of MCoTI-II.
  • the NBD- MCoTI-II can be represented by SEQ ID NO: 11.
  • the NBD has a three amino acid deletion at positions 1-3, and a two amino acid deletion at positions 10 and 11.
  • This NBD fragment is represented by SEQ ID NO:22.
  • This NBD fragment is preferably located at the N-terminus of the fusion protein.
  • the NBD fragment described above may not be located at the N-terminus of the fusion protein, as represented by, for example, SEQ ID NO: 14.
  • the NBD fragment can follow one or more linkers that are immediately at the N-terminus of the fusion protein.
  • NBD-MCoTI-II can alternatively be represented by SEQ ID NO: 12.
  • the NBD has a two amino acid deletion at positions 10 and 11.
  • This NBD fragment is represented by SEQ ID NO:23.
  • the NBD fragment is located at the N- terminus of the fusion protein.
  • the NBD fragment described above may not be located at the N-terminus of the fusion protein, as represented by, for example, SEQ ID NO: 15.
  • this NBD fragment can follow one or more linkers that are immediately at the N-terminus of the fusion protein.
  • NBD-MCoTI-II can be represented by SEQ ID NO: 13.
  • NBD- MCoTI-II can also be represented by SEQ ID NOS: 16-21, where the NBD (or a fragment thereof) is flanked by one or more linkers.
  • the linkers can be located at or near the N-terminus, and/or at or near the C-terminus, of the fusion protein.
  • Linkers can be used to increase the flexibility of NBD (or a fragment thereof) within the framework of MCoTI-I/II.
  • the linker is Gly Gly Ser.
  • Linkers are well known. Any type and number of linkers can be used. For example, more than 3 amino acid linkers can be used, and/or longer linkers can be used. In other embodiments, linkers can be omitted.
  • the NBD-MCoTI-I/II peptides can contain subsets of the NBD sequence (SEQ ID NO: 11, SEQ ID NO: 12), or in which the NBD sequence is not immediately at the N-terminus (SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15).
  • the NBD sequence can also alternatively contain one (SEQ ID NO: 16, SEQ ID NO: 19), two (SEQ ID NO: 17, SEQ ID NO:20) or three (SEQ ID NO: 18, SEQ ID NO:21) linkers (Gly Gly Ser) at the N- and C-termini of NBD to increase the flexibility of NBD within the framework of the MCoTI-II backbone.
  • the present invention can comprise the use of a linear MCoTI-I/II molecule to deliver a bioactive peptide in vivo.
  • the bioactive peptide is fused (or otherwise covalently linked) to the N terminus of a MCoTI- I II molecule.
  • the MCoTI-I/II moiety also preferably lacks loop 6 of the MCoTI-I/II molecule, and the bioactive peptide is attached in place of loop 6.
  • the present fusion proteins can be manufactured by methods known to the art. For example, automated polypeptide synthesis can be performed to produce the present proteins, such as Fmoc-based solid phase peptide synthesis. Alternatively, the present fusion proteins can be produced by recombinant methods, such as by using bacterial, yeast, insect, or mammalian cells. A variety of cell lines can be used to produce the present proteins recombinantly, such as HEK cells, CHO cells, HeLa cells, and others known to the art.
  • Suitable polynucleotides and vectors are selected when producing the present fusion proteins recombinantly.
  • the isolated polynucleic acid molecule represented by SEQ ID NO:8 can be used for bacterial synthesis.
  • the polynucleic acid can be DNA or RNA.
  • vectors include all of the regulatory elements necessary for efficient transfection as well as efficient expression of proteins. Such vectors are well known in the art and any suitable vector can be selected for this purpose.
  • Transfection and cultivation of recombinant cells is likewise well known in the art.
  • such a recombinant cell as well as any descendant cell thereof includes a vector comprising a nucleotide sequence coding for the preset fusion proteins.
  • Such cell lines can express the fusion proteins described herein (e.g., the NBD- MCoTI-II) continuously or upon induction/activation depending on the vector.
  • Polynucleotides coding for the present fusion proteins can, in an alternative embodiment be directly administered to a subject in order to deliver the fusion proteins to the subject. Such delivery would be by way of gene therapy vectors and protocols known to the art.
  • such proteins can be formulated as a pharmaceutical composition compromising the fusion proteins described herein, i.e., compromising NBD- MCoTI-I/II, and pharmaceutically acceptable excipients.
  • the pharmaceutical composition can include, for example, emulsifiers, buffers, inert/inactive ingredients, sugars, salts, and the like.
  • the pharmaceutical composition can include, for example, emulsifiers, buffers, inert/inactive ingredients, sugars, salts, and the like.
  • the pharmaceutical composition can include, for example, emulsifiers, buffers, inert/inactive ingredients, sugars, salts, and the like.
  • the pharmaceutical composition is administered orally.
  • the pharmaceutical composition may be suitable for administration in any manner, such as, but not limited to, injection, orally, transdermally, and/or topically.
  • the pharmaceutical composition can be formulated to have the following properties: enzymatic resistance, stability in gastric juices and GI membrane permeability.
  • NBD-MCoTI-I/II can be used as a medicament in methods to treat a number of different medical conditions involving the activation of NF- ⁇ , in particular
  • inventive proteins, polynucleic acids, and/or vectors described herein can be used as medicaments to treat inflammatory diseases or conditions.
  • the present invention is directed to methods for the treatment of inflammatory diseases or conditions, wherein the NBD- MCoTI-II, isolated polynucleic acid molecule and/or vector described herein, and/or a pharmaceutical composition according to the invention, is administered to a patient.
  • inventive fusion proteins, polynucleic acids, and/or vectors can be administered alone or in addition to other medicaments that treat inflammatory diseases or conditions and/or other diseases and conditions.
  • inventive fusion proteins, polynucleic acids, and/or vectors can be administered as part of a treatment regimen with other known medicaments.
  • inventive proteins, polynucleic acids, and/or vectors described herein can be used as medicaments to treat any other disease or condition that appears to improve upon the administration of the medicament.
  • inventive proteins, polynucleic acids, and/or vectors described herein can be used to inhibit and/or block the activation of the NF- ⁇ pathway.
  • Inflammatory mediators such as TNF-a, IL-6, and IL-1 are important in the pathogenesis of TNF-a, IL-6, and IL-1.
  • NBD- MCoTI-I/II has been discovered to be 10 6 -fold more potent than known cell-permeable NBD peptides. This improvement in potency has translated into a 5000-fold reduction in dosage required to ameliorate inflammatory injury in vivo (as shown, for example, in the Carrageenan Paw Edema (CPE) mouse model). Therefore, NBD- MCoTI-I/II is a potent, orally bioavailable agent to treat inflammatory diseases.
  • the inflammatory diseases and/or conditions that can be treated using the methods described herein include, but are not limited to, chronic or acute diseases or conditions such as Alzheimer's, ankylosing spondylitis, arthritis (i.e., osteoarthritis, rheumatoid arthritis, psoriatic arthritis), cancer, edema, swelling, asthma, atherosclerosis, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome, systemic lupus erythematous, nephritis, Parkinson's disease, ulcerative colitis, acid reflux/heartburn, acne, asthma, atherosclerosis, bronchitis, cancer, carditis, celiac disease, chronic pan, cirrhosis, dementia, diabetes, dry eyes, edema, emphysema, eczema, gastroenteritis, gingivitis, heart disease, high blood pressure, insulin resistance,
  • inventive fusion proteins, polynucleic acids, and/or vectors disclosed herein can be used to prevent certain diseases or conditions, such as to prevent certain inflammatory diseases or conditions.
  • inventive fusion proteins, polynucleic acids, and/or vectors disclosed herein can be used to prevent arthritic joint damage.
  • NF- ⁇ nuclear factor ⁇
  • MMoTI- jyii a condition in which NF- ⁇ plays a role in pathology
  • musculoskeletal diseases such as muscular dystrophy and cachexia
  • NF- ⁇ nuclear factor ⁇
  • DMD Duchenne muscular dystrophy
  • cardiac conditions such as cardiac hypertrophy, ischemia, and reperfusion injury; spinal cord injury; sepsis; and cancer.
  • constitutive NF- ⁇ activity can be suppressed by NBD- MCoTI- jyii.
  • the NBD sequence (SEQ ID NO:4) was grafted into MCoTI-II (SEQ ID NO:3) by replacing the loop 6, as shown in FIG. 1. Synthesis was accomplished in two ways: 1) expression in bacteria [(see, e.g., Austin J et al., (2009) Biosynthesis and biological screening of a genetically encoded library based on the cyclotide MCoTI-I. Chembiochem 10:2663-70 and Schmoldt HU et al., (2005) A fusion protein system for the recombinant production of short disulfide bond rich cystine knot peptides using barnase as a purification handle.
  • NBD-MCoTI-II was synthesized using both bacterial and automated synthetic approaches. Bacterial synthesis resulted in circular, native-folded NBD-MCoTI-II. For the automated synthesis we generated a linear version of NBD-MCoTI-II. Both approaches are described in more detail below.
  • NBD-MCoTI-II A gene coding for the NBD-MCoTI-II, generated using complimentary sense and anti-sense oligonucleotides, was subcloned into an appropriate plasmid for protein expression in bacteria. When expressed in bacteria, NBD-MCoTI-II was purified in its native folded conformation. However, NBD-MCoTI-II had to be purified away from bacterial proteins using multiple columns/HPLC. Wild-type MCoTI-II-intein-chitin binding domain (CBD) fusion protein was generated as follows.
  • Sense (SEQ ID NO: l) and anti-sense (SEQ ID NO:2) oligonucleotides coding for the protein sequence (SEQ ID NO:3) of MCoTI-II were annealed to generate a double- stranded cassette. 5' Nde I and 3' Sap I sites were included for subcloning the cassette into the vector pTXBl (which contains the intein-CBD coding regions). A Bgl II restriction site was also included after loop 6 to allow for subcloning in cDNA sequences coding for the NEMO Binding Domain sequence (NBD) (SEQ ID NO:4). The cDNA sequence for MCoTI-II was subcloned into pTXBl such that it was immediately upstream and in frame with the cDNA region coding for the intein-CBD coding region.
  • NBD NEMO Binding Domain sequence
  • a NBD-MCoTI-II cassette was generated by annealing sense (SEQ ID NO:5) and anti-sense (SEQ ID NO:6) oligonucleotides coding for the NBD sequence and part of the MCoTI-II peptide (SEQ ID NO:7). 5' Nde I and 3' Bgl II sites were included for subcloning the NBD coding sequence into wildtype MCoTI-II-intein-CBD:pTXBl.
  • NBD-MCoTI-ILpTXBl Nucleotide and protein sequences for expression of NBD-MCoTI-II in bacteria are given in SEQ ID NO:8 and SEQ ID NO:9, respectively.
  • E. coli Bacterial expression of wildtype and NBD-MCoTI-II was carried out in Origami E. coli (EMD Chemicals, Inc.). E. coli transfected with either wildtype MCoTI- ILpTXB 1 or NBD-MCoTI- ILpTXB 1 were grown at 37 °C in LB medium in the presence of ampicillin until log phase growth was reached. After reaching log phase growth, the cells were acclimated to 30 °C, IPTG added (at a final concentration of 0.3 ⁇ ) and grown at 30 °C for two hours. Cells were harvested by centrifugation and stored at - 80 °C.
  • E. coli expressing wildtype or NBD-MCoTI-II were lysed in Bugbuster solution (EMD Chemicals, Inc.) containing protease inhibitors and benzonase at room temperature for 20 minutes. Cleared lysate, obtained by centrifugation (10,000 x g, 4 °C, one hour), was added to chitin beads (available from NEB, Inc.) (washed with 30 volumes of HEPES buffer: 20 mM HEPES, pH 8.0, 500 mM NaCl and 500 MgCl 2 ) and incubated at 4 °C with mixing for one hour.
  • Bugbuster solution EMD Chemicals, Inc.
  • HEPES buffer 20 mM HEPES, pH 8.0, 500 mM NaCl and 500 MgCl 2
  • the active peak was collected, ACN removed by roto-evaporation, and lyophilized.
  • the final NBD- MCoTI-II protein sequence after processing can be represented by SEQ ID NO: 10 (though it is to be understood that the product of the foregoing process is a circular protein, and the designation of N- and C- termini in SEQ ID NO: 10 is arbitrary).
  • NBD-MCoTI-II was chemically synthesized by Fmoc-based solid-phase peptide synthesis followed by optional head to tail cyclization.
  • the peptide may have to be folded to the native conformation [(see, e.g., Aboye TL et al., (2011) Interlocking disulfides in circular proteins: toward efficient oxidative folding of cyclotides. Antioxid Redox Signal 14:77-86; Cemazar M et al., (2006) Knots in rings.
  • the circular knotted protein Momordica cochinchinensis trypsin inhibitor- II folds via a stable two-disulfide intermediate.
  • a cell-permeant NBD Peptide has been demonstrated in in vitro binding and cell- based assays as well as in vivo rodent models to inhibit NF- ⁇ signaling and
  • HEK293 cells were transfected with the plasmid pNiFty-SEAP which encodes for a secreted alkaline phosphatase (SEAP) whose expression is under control of an ELAM-1 composite promoter containing five NF- ⁇ sites.
  • SEAP secreted alkaline phosphatase
  • NBD-MCoTI-II blocked TNFa-mediated SEAP expression with an IC 50 of 60 pM, 10 6 -fold more potent than that of the NBD peptide.
  • WT and NBD-MCoTI-II caused no overt cellular toxicity at concentrations up to ⁇ .
  • NBD-MCoTI-II Anti-inflammatory activity of linear NBD-MCoTI-II was tested at two doses (5 and 25 ⁇ g/kg) in the mouse CPE model.
  • Mice were administered (i.p.) either NBD- MCoTI-II, dexamethasone (10 mg/kg) or saline one hour prior to inducing edema by carrageenan injection into the subplantar region of the left hind foot. Swelling was then measured by water displacement method at 2, 4, 6 and 12 hours post-carrageenan injection.
  • Linear NBD-MCoTI-II significantly reduced swelling at 4, 6 and 12 hours (Fig. 4).
  • 5 and 25 ⁇ g/kg NBD MCoTI-II reduced swelling 30 and 50%, respectively (Fig. 5).
  • NBD-MCoTI-II at 5 ⁇ g/kg had a similar effect as the NBD Peptide at 25 mg/kg (Fig. 4., di Meglio P et al., (2005) Amelioration of acute inflammation by systemic administration of a cell-permeable peptide inhibitor of NF- ⁇ activation. Arthritis Rheum 52:951-8, the contents of which are herein incorporated by reference in its entirety), indicating that the in vivo potency of linear NBD-MCoTI-II is 5000-fold greater than that observed for the NBD Peptide. (Fig. 5).
  • NBD-MCoTI-II reduces inflammatory response in a therapeutic mouse CIA model
  • AI arthritis index
  • mice were separated into three treatment groups and administered (i.p.) daily: saline, 100 ⁇ g/kg NBD-MCoTI-II and 3 mg/kg dexamethasone for 14 days.
  • AI scores, body weights, paw thickness and any signs of distress were monitored daily.
  • NBD-MCoTI-II 100 ⁇ g/kg immediately and significantly (p ⁇ 0.01) halted the progression of arthritis similar to that of the positive control dexamethasone (Fig. 6).
  • NBD-MCoTI-II was at least as effective as Pfizer' s JAK 1/2 inhibitor CP690550 (XeljanzTM) and anti-TNF Ab control (see, Milici AJ et al., (2008) Cartilage preservation by inhibition of Janus kinase 3 in two rodent models of rheumatoid arthritis, Arthritis Res Ther 10:R14), in ameliorating arthritic symptoms in the mouse CIA model. Finally, NBD-MCoTI-II also significantly (p ⁇ 0.01) blocked the increase in paw thickness (Fig. 7) associated with CIA. The effect lasted the study duration.
  • NBD-MCoTI-II To compare the efficacy of NBD-MCoTI-II to existing anti- arthritic drugs we calculated a composite histopathology score for the CIA mice from the individual scores of inflammation, pannus, cartilage damage and bone resorption by adding them. Shown in Figure 9 is a comparison of the composite histopathology scores for NBD-MCoTI-II (100 ⁇ g/kg) to published values for the existing anti-arthritic drugs EnbrelTM (15 mg/kg), Orencia IM (5 mg/kg), Xeljanz IM (10 mg/kg) and INCB028050 (15/mg). Data are presented as a percent of the composite histopathology scores for CIA mice treated with saline.
  • Figure 9 shows that NBD- MCoTI-II exhibits a remarkable reduction in composite histopathology scores as compared to existing anti- arthritic drugs (data for EnbrelTM, OrenicaTM, XeljanzTM and INCB028050 as shown in, e.g., Seeuws S. et al., (2010) A multiparameter approach to monitor disease activity in collagen-induced arthritis. Arthritis Res Ther 12:R160; Milici AJ et al., (2008) Cartilage preservation by inhibition of Janus kinase 3 in two rodent models of rheumatoid arthritis.
  • NBD-MCoTI-II had a composite histopathology score almost zero while the existing anti-arthritic drugs were only capable of reducing the composite histopathology scores -40-60%.
  • linear NBD-MCoTI-II prevented joint damage while the other anti- arthritic drugs were only capable of reducing joint damage.
  • NBD-MCoTI-II treatment significantly (p ⁇ 0.01) reduced the spleen weight to 100+17 mg.
  • NBD-MCoTI-II displayed no obvious signs of toxicity or distress.
  • Mouse body weights were unchanged relative to saline controls.
  • Laboratory blood analysis including general blood chemistry, kidney function tests, liver enzyme levels (ALT, AST) and blood cell counts were within normal ranges.
  • Kidney, liver and intestine weights were within normal ranges and no differences were observed between saline and NBD- MCoTI-II-treated CIA mouse.

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Abstract

L'invention concerne de nouvelles protéines de fusion impliquant le domaine de liaison du modulateur essentiel du facteur nucléaire kB (NEMO) ou un fragment de celui-ci et MCoTI-I/II ou un fragment de celui-ci, et leur utilisation pour le traitement de maladies inflammatoires et d'autres affections médicales.
PCT/US2013/027702 2012-02-23 2013-02-25 Protéines de fusion à domaine de liaison de nemo WO2013126919A1 (fr)

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US20090286736A1 (en) * 2000-05-02 2009-11-19 Yale University Anti-inflammatory compounds and uses thereof

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US20090286736A1 (en) * 2000-05-02 2009-11-19 Yale University Anti-inflammatory compounds and uses thereof

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Title
GREENWOOD KATHRYN.: "The development of the cyclotide MCo-TI-II as a molecular engineering framework in drug design.", INSTITUTE FOR MOLECULAR BIOSCIENCE, October 2008 (2008-10-01), XP008174338, Retrieved from the Internet <URL:http://espace.Iibrary.uq.edu.au/view/UQ:183997> [retrieved on 20130513] *
HEITZ ANNIE ET AL.: "Knottin cyclization: impact on structure and dynamics.", BMC STRUCTURAL BIOLOGY, vol. 8, no. 54, 2008, pages 1 - 19, XP021049534 *
See also references of EP2817324A4 *

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