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WO2018154584A1 - Chimères de récepteur dr3 soluble et de pro-domaine de tace et leur utilisation - Google Patents

Chimères de récepteur dr3 soluble et de pro-domaine de tace et leur utilisation Download PDF

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Publication number
WO2018154584A1
WO2018154584A1 PCT/IL2018/050214 IL2018050214W WO2018154584A1 WO 2018154584 A1 WO2018154584 A1 WO 2018154584A1 IL 2018050214 W IL2018050214 W IL 2018050214W WO 2018154584 A1 WO2018154584 A1 WO 2018154584A1
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WIPO (PCT)
Prior art keywords
amino acid
moiety
seq
chimeric polypeptide
acid sequence
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PCT/IL2018/050214
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English (en)
Inventor
Amir Aharoni
Irit Sagi
Itay LEVIN
Tomer WEIZMAN
Original Assignee
National Institute For Biotechnology In The Negev Ltd.
Yeda Research And Development Co. Ltd.
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Application filed by National Institute For Biotechnology In The Negev Ltd., Yeda Research And Development Co. Ltd. filed Critical National Institute For Biotechnology In The Negev Ltd.
Publication of WO2018154584A1 publication Critical patent/WO2018154584A1/fr

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    • 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
    • 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
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention is directed to, inter alia, chimeras and use thereof such as in the treatment of autoimmune and/or inflammatory diseases.
  • TNF-like ligand 1A is a newly described member of the TNF superfamily that has been shown to be involved in promoting a range of autoimmune diseases including IBD, rheumatoid arthritis (RA), and asthma through the generation and activation of the type-1 T helper cell (T H I) and type-17 T helper cell (T H 17).
  • TLIA was shown to be expressed by endothelial cells, lymphocytes and monocytes and its expression is enhanced in the intestinal tissues of patients with IBD.
  • TLIA is currently the only known ligand for death receptor 3 (DR3), which is predominantly expressed by activated T cells and endothelial cells. The binding of TLIA to DR3 was shown to boost the secretion of IFN- ⁇ from T cells by acting in synergy with IL-12 and IL-18 and thus direct the immune response toward a T H I like response.
  • DR3 death receptor 3
  • TACE Disintegrin And Metalloproteinase 17
  • ADAM 17 Disintegrin And Metalloproteinase 17
  • T F a Converting Enzyme
  • a chimera comprising a first moiety and a second moiety, said first moiety comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 2 or an analog, or a fragment thereof, wherein said amino acid sequence comprises at least one amino acid substitution at a position selected from the group consisting of: H15, 118, E38, V47, D51, W56, N61, A65, K93, Q101, Q104 and L129; and said second moiety comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 40 or an analog, or a fragment thereof, wherein said amino acid sequence comprises at least one amino acid substitution at a position selected from the group consisting of: R35, K36, R37.
  • the second moiety further comprises at least one amino acid substitution at a position selected from the group consisting of: C163, R190, and R193.
  • the first moiety is capable of binding TL1A and inhibiting or reducing at least one of TL1 A mediated IFN-gamma secretion and TL1 A mediated apoptosis.
  • the second moiety is capable of inhibiting or reducing the catalytic activity of TACE.
  • the first moiety comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 30-38.
  • the second moiety comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 41-46.
  • the second moiety and said first moiety are attached via a first peptide linker.
  • the first peptide linker comprises an amino acid sequence selected from SEQ ID NO: 3 (HHHHHH), SEQ ID NO: 4 (GPQGIAGQHHHHHHDY), and SEQ ID NO: 5 (IEGRMDRS).
  • the chimera further comprising a third moiety capable of facilitating homodimerization of the chimera.
  • the third moiety comprises a peptide of the fragment crystallizable (Fc) region of an antibody.
  • the peptide of the Fc region of an antibody comprises the amino acid sequence as set forth in SEQ ID NO: 6.
  • the third moiety is attached to any one of said first moiety and said second moiety via a second peptide linker.
  • the second peptide linker comprises the amino acid sequence of SEQ ID NO: 5 (IEGRMDRS).
  • the chimera comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 8-29.
  • the chimera is capable of binding at least one of: TL1A and TACE.
  • the invention provides a pharmaceutical composition comprising any of the chimeras of the invention, and a pharmaceutically acceptable carrier.
  • the invention provides a polynucleotide molecule encoding any of the chimeras of the invention.
  • the invention provides an expression vector comprising the polynucleotide molecule of the invention.
  • a cell comprising the expression vector of the invention.
  • the invention provides the expression vector of and a carrier.
  • composition of claim comprising said expression vector and a carrier, thereby treating, ameliorating, reducing and/or preventing inflammation, an immune response, or both, in said subject.
  • the subject is afflicted with a chronic inflammatory disease. In some embodiments, the subject is afflicted with an inflammatory bowel disease.
  • the invention provides any of the chimera of the invention, a pharmaceutical composition comprising same, the expression vector of the invention, or a composition comprising same of the invention for use in the treatment, amelioration, reduction and/or prevention of inflammation in a subject in need thereof.
  • the chimera is for treating a chronic inflammatory disease. In one embodiment, the chimera is for treating an inflammatory bowel disease.
  • Fig. 1A is a schematic representation of the bi-specific DR3-pTACE inhibitor targeting TL1A and TACE;
  • Fig. IB shows an immunoblot of human Fc demonstrating expression of Al and A2 in HEK293F cells
  • Fig. 1C shows an immunoblot of DR3 demonstrating expression of Al and A2 in HEK293F cells
  • Fig. ID is a bar graph showing binding of the bi-specific A2 to immobilized TL1A following expression in HEK293F mammalian cell line as analyzed by ELISA analysis;
  • Fig. IE shows a Coomassie blue staining of A2
  • Fig 2A is a bar graph comparing the binding of the chimera (A2) and TL1A to the binding of the mono -specific H3 protein and TL1A;
  • Fig. 2B is a bar graph demonstrating the inhibition of TACE by the chimera (A2) compared to the inhibition of TACE by the mono-specific proTACE inhibitor;
  • Fig. 3 is a bar graph demonstrating a similar inhibition level of TNF-a release from macrophages following incubation with A2 and the mono- specific proTACE inhibitor;
  • Fig. 4A is a graph showing TLlA-induced secretion of IFN- ⁇ in human CD4+ cells incubated for 24 hours with 100 ng/ml TL1A, 2 ng/ml IL-12 and 50 ng/ml IL-18 and soluble DR3 variant having SEQ ID NO: 2 ( ⁇ 3') compared to soluble DR3 WT;
  • Fig. 4B is a bar graph showing inhibition of TLlA-induced secretion of IFN- ⁇ in human PBL by increased concentration of A2 and H3;
  • Fig. 4C is a bar graph showing inhibition of TLlA-induced apoptosis in TF-1 cells by increased concentration of A2 and H3;
  • Fig. 5 is a bar graph showing that TLlA-induced apoptosis in TF-1 cells is not inhibited by pTACE.
  • Cells were inoculated for six hours with 8 ⁇ g/ml cyclohexamide (CHX) and 75 ng/ml TL1A and the indicated concentration of pTACE;
  • CHX cyclohexamide
  • FIG. 6A shows flow cytometry histogram analysis of gated TF-1 cell population incubated with A2, H3 and antibody control, binding to the cell membrane was analyzed following incubation with allophycocyanin (APC) fluorescent anti -human Fc;
  • APC allophycocyanin
  • Fig. 6B shows flow cytometry histogram analysis of TF-1 cell population incubated with 100 nM A2 in the presence or absence of 15 ⁇ pTACE inhibitor that lacks an Fc region, a decrease in cell labeled population observed in the presence of pTACE indicates that A2 binds cells through endogenous membrane TACE;
  • FIG. 6C shows dot plot analysis of TF-1 cell population incubated with A2 (left), A2+pTACE (middle) and antibody control (without the addition of the A2 or H3, right).
  • a decreased in the gated population of the A2+pTACE labeled cells from 33.3% to 15% indicates that pTACE compete with the A2 for TF-1 cell binding.
  • Fig. 7 A is an exemplary model for TL1A inhibition by the mono-specific soluble DR3-Fc (sDR3) demonstrating that binding of sDR3 to TL1A in a solution may lead to depletion of free TL1A due to a competition with the endogenous DR3 membrane receptor (A);
  • sDR3 mono-specific soluble DR3-Fc
  • Fig. 7B is an exemplary model for TL1A inhibition by the A2 bi-specific sDR3-Ptace (sDR3-pTACE), demonstrating that the sDR3-pTACE bound to cell surface TACE located on the cell membrane leading to a high local concentration of the inhibitor on the cell membrane;
  • sDR3-pTACE A2 bi-specific sDR3-Ptace
  • Fig. 8A is a bar graph showing that the H3 and 06 mutants of DR3 are more potent in inhibiting TLlA-induced secretion of interferon gamma (IFN- ⁇ values presented here were calculated according to IFN- ⁇ calibration curve in human CD4+); and
  • Fig. 8B is a bar graph showing that H3 and 06 mutants are more potent in inhibiting TL1 A- induced apoptosis in TF-1 cells.
  • the present invention provides a chimera comprising an sDR3 mutant ('first moiety') and a pTACE domain ('second moiety') and compositions comprising same.
  • the chimera further comprises an Fc region of an antibody ('third moiety').
  • the invention further provides methods of treatment using said chimera.
  • the chimeras of the invention simultaneously inhibit TL1A mediated IFN- ⁇ secretion and TACE catalytic activity.
  • the chimeras of the invention reduce, ameliorate, and/or inhibit inflammation.
  • the inflammation is an autoimmune inflammatory disease.
  • the inflammation is inflammatory bowel disease.
  • the present invention is based, in part, on the surprising finding that a chimera comprising an sDR3 mutant, a pTACE domain, and optionally a human IgGi Fc region is capable of targeting cells at sites of inflammation and inhibit immunological signals that drive inflammation.
  • the chimera described herein simultaneously inhibits TACE catalytic activity and binds TL1A to inhibit TL1A induced IFN- ⁇ secretion.
  • the chimera of the invention demonstrated strong synergistic effect of up to -80 fold in inhibiting TL1A induced cytokine secretion from T cells or apoptosis in TF-1 cell line.
  • the pTACE domain was further shown to facilitate cell surface binding of the chimera which leads to increased concentration of the chimera on the cell membrane, e.g., on cells expressing membrane TACE.
  • the chimeras of the invention are capable of binding TL1A.
  • the chimeras of the invention have increased affinity for binding TL1A compared to WT DR3.
  • the binding affinity towards TL1A is increased by at least a 1.5- fold, at least a 2-fold, at least a 2.5-fold, at least a 3-fold, at least a 3.5-fold, at least a 4-fold, at least a 4.5-fold, at least a 5-fold, at least a 5.5-fold, at least a 6-fold, at least a 6.5-fold, at least a 7-fold, at least a 7.5-fold, at least a 8-fold, at least a 8.5-fold, at least a 9-fold, at least a 9.5-fold, or at least a 10-fold compared to DR3 wild-type (WT) binging affinity.
  • WT wild-type
  • the chimeras of the invention have increased potency compared to DR3 WT in inhibiting TL1 A induced secretion of IFN- ⁇ .
  • the chimeras of the invention have at least a 1.5-fold, at least a 2-fold, at least a 2.5-fold, at least a 3-fold, at least a 3.5- fold, at least a 4-fold, at least a 4.5-fold, at least a 5-fold, at least a 5.5-fold, at least a 6-fold, at least a 6.5-fold, at least a 7-fold, at least a 7.5-fold, at least a 8-fold, at least a 8.5-fold, at least a 9-fold, at least a 9.5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 30-fold, at least a 35-fold, at least a 40-fold, at least a
  • the chimeras of the invention have increased potency compared to the sDR3 mutant in inhibiting TL1A induced secretion of IFN- ⁇ .
  • the chimeras of the invention have at least a least a 1.5-fold, at least a 2-fold, at least a 2.5-fold, at least a 3-fold, at least a 3.5-fold, at least a 4-fold, at least a 4.5-fold, at least a 5-fold, at least a 5.5-fold, at least a 6-fold, at least a 6.5-fold, at least a 7-fold, at least a 7.5-fold, at least a 8-fold, at least a 8.5-fold, at least a 9-fold, at least a 9.5-fold, or at least a 10-fold increase in the potency compared to the sDR3 mutant of the invention.
  • Each possibility represents a separate embodiment of the present invention.
  • the chimeras of the invention inhibit TACE catalytic activity.
  • the chimeras of the invention are localized to the target cell membrane, such as cell expressing membrane TACE.
  • the invention provides a chimera comprising a first moiety and a second moiety, said first moiety comprises an sDR3 mutant, and said second moiety comprises pTACE.
  • the second moiety is contiguous to the C-terminus and/or the N- terminus of the first moiety. In some embodiments, the second moiety is attached to the first moiety via a covalent bond. In some embodiments, the second moiety is attached to the first moiety via a peptide bond. In some embodiments, the second moiety is attached to the first moiety via a linker.
  • the linker has a length of no more than 20 amino acids, no more than 19 amino acids, no more than 18 amino acids, no more than 17 amino acids, no more than 16 amino acids, no more than 15 amino acids, no more than 14 amino acids, no more than 13 amino acids or no more than 12 amino acids, no more than 11 amino acids, no more than 10 amino acids, no more than 9 amino acids, no more than 8 amino acids, no more than 7 amino acids, no more than 6 amino acids, no more than 5 amino acids, no more than 4 amino acids, no more than 3 amino acids, no more than 2 amino acids or of one amino acid.
  • the linker comprises the amino acid sequence as set forth in SEQ ID NO: 3 (HHHHHH).
  • the linker comprises the amino acid sequence as set forth in SEQ ID NO: 4 (GPQGIAGQHHHHHHDY). In some embodiments, the linker comprises the amino acid sequence as set forth in SEQ ID NO: 5 (IEGRMD).
  • the second moiety is attached to the first moiety via one or more linkers. In some embodiments, the one or more linkers are selected from, but not limited to, SEQ ID NOs: 3, 4, and 5. In some embodiments, the one or more linkers are selected from the group consisting of: SEQ ID NOs: 3, 4, and 5.
  • the chimera is capable of homodimerizing. In some embodiments, the chimera further comprises a third moiety that facilitates homodimerization of the chimera.
  • the third moiety comprises a peptide of the fragment crystallizable (Fc) region of an antibody. In some embodiments, the third moiety comprises a peptide of the fragment crystallizable (Fc) region of human IgGl.
  • said peptide is a commercially available human engineered IgGl Fc region termed hIgGle3-Fcl (InvivoGen) comprising the amino acid sequence as set forth in SEQ ID NO: 6 (RSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VE VHN AKTKPREEQ YNS T YRV VS VLT VLHQD WLNGKE YKC KVS NKGLPS S IEKTIS KAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK).
  • SEQ ID NO: 6 RSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VE VHN AKTKP
  • the third moiety is contiguous to the C-terminus and/or the N- terminus of the first moiety. In some embodiments, the third moiety is contiguous to the C-terminus and/or the N-terminus of the second moiety. In some embodiments, the third moiety is attached to the C-terminus of the first moiety or the C-terminus of the second moiety. In some embodiments, the third moiety is attached to the first moiety or the second moiety via a covalent bond. In some embodiments, the third moiety is attached to the first moiety or the second moiety via a peptide bond. In some embodiments, the third moiety is attached to the first moiety or the second moiety via a linker.
  • Said linker in some embodiments, has a length of no more than 20 amino acids, no more than 19 amino acids, no more than 18 amino acids, no more than 17 amino acids, no more than 16 amino acids, no more than 15 amino acids, no more than 14 amino acids, no more than 13 amino acids or no more than 12 amino acids, no more than 11 amino acids, no more than 10 amino acids, no more than 9 amino acids, no more than 8 amino acids, no more than 7 amino acids, no more than 6 amino acids, no more than 5 amino acids, no more than 4 amino acids, no more than 3 amino acids, no more than 2 amino acids or of one amino acid.
  • the linker having the amino acid sequence as set forth in SEQ ID NO: 5 is attached to the N-terminus of the peptide of the fragment crystallizable (Fc) region of human IgGl having the amino acid sequence as set forth in SEQ ID NO: 6.
  • the chimera further comprises a signal peptide.
  • the signal peptide directs the chimera across or into a cell membrane.
  • the signal peptide directs the chimera of the present invention for secretion from cells.
  • the signal peptides are cleaved from the precursor chimera resulting in the mature chimera.
  • the signal peptide comprises the amino acid sequence as set for in SEQ ID NO: 7 (MGWSIILFLVATAT).
  • chimera and “chimeric polypeptide” are used interchangeably to refer to a polypeptide formed by the joining of two or more peptides through a peptide bond formed between the amino terminus of one peptide and the carboxyl terminus of another peptide.
  • the chimera further comprises an acetyl group.
  • the acetyl group is attached to the N-terminus of the chimera.
  • the chimera may be formed by a chemical coupling of the constituent peptides, or it may be expressed as a single polypeptide fusion protein from a nucleic acid sequence encoding the single contiguous conjugate.
  • peptide As used herein, the terms “peptide”, “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues.
  • the terms “peptide”, “polypeptide” and “protein” as used herein encompass native peptides, pep tidomime tics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogs peptoids and semi-peptoids or any combination thereof.
  • the terms “peptide”, “polypeptide” and “protein” apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analog of a corresponding naturally occurring amino acid.
  • the chimeras may comprise additional amino acids, either at the peptide's N-terminus, at the peptide's C-terminus or both.
  • the peptide has a length of at most 1000, 990, 980, 970, 960, 950, 940, 930, 920, 910, 900, 890, 880, 870, 860, 850, 840, 830, 820, 800, 790, 780, 760, or 750 amino acids. Each possibility represents a separate embodiment of the present invention.
  • the peptide has a length of at most 80 amino acids.
  • the chimera of the invention comprises or consists of an amino acid sequence selected from the amino acid sequences presented in Table 1, or a sequence derived therefrom.
  • Xi is selected from R and A
  • X 2 is selected from K and A
  • X 3 is selected from R and A
  • X 4 is selected from C and A
  • X5 is selected from R and A
  • X 6 is selected from R and G
  • X7 is H or Q
  • X 8 is I, T or Y
  • X9 is E or K
  • X10 is V or P
  • Xn is D or G
  • X12 is W or R
  • Xi 3 is N or E
  • Xi 4 is
  • Xi is selected from R and A
  • X 2 is selected from K and A
  • X3 is selected from R and A
  • X 4 is selected from C and A
  • X5 is selected from R and A
  • X 6 is selected from R and G
  • X 7 is H or Q
  • X 8 is I, T or Y
  • Xio is V or P
  • Xn is D or G
  • Xi 2 is W or
  • LGSCPERCAAVCGWRQMF wherein: Xi is H or Q; X 2 is I, T or Y; X 3 is E or K; X 4 is
  • X 8 is A or T; X9 IS K, E or A; Xio is Q or S; Xn is Q or P; and Xi 2 is L or P
  • EHGDGCVSCPTSTLGSCPERCAAVCGWRQMF wherein: Xi is H or Q; X 2 is I, T or
  • GGTRSPRCDCAGDFXiKKX 2 GLFCCRGCPAGHYLKAPCTX 3 PCGNSTCLX 4 CPQX 5 TFLAX 6 ENHHX 7
  • SECX 8 RCQACDEQASQVALENCSAVADTRCGCX9PGWFVECXi 0VSX11CVSSSPFYCQPCLDCGALHRHTRLX12CSRRDTDCGTCLPGFYEHGDGCV SCPTSTLGSCPERCAAVCGWROMFIEGRMDGPOGIAGOHHHHHHDYGDPGFGP HQRLEKLDSLLSDYDILSLSNIQQHSVXi 3 Xi 4 Xi 5 DLQTSTHVETLLTFSALKRHFK LYLTSSTERFSQNFKVVVVDGKNESEYTVKWQDFFTGHVVGEPDSRVLAHIRD DDVIIRINTDGAEYNIEPLWRFVNDTKDKRMLVYKSEDIKNVSRLQSPKVXieGY LKVDNEELLPKGLV
  • X 2 is I, T or Y; X is E or K; X 4 is V or P; X 5 is D or G; X 6 is W or R; X 7 is N or E; X 8 is A or T; X9 IS K, E or A; Xio is Q or S; Xn is Q or P; Xi 2 is L or P; Xi 3 is R or A; Xi 4 is K or
  • Xi5 is R or A
  • X1 ⁇ 2 is C or A
  • Xi 8 is R or G
  • Xi is H or Q
  • X2 is I, T or Y
  • X3 is E or K
  • X 4 is V or P
  • X5 is D or G
  • X 6 is W or
  • GGTRSPRCDCAGDFXiKKX 2 GLFCCRGCPAGHYLKAPCTX3PCGNSTCLX 4 CPQX5 TFLAX 6 ENHHX 7
  • SECX 8 RCQACDEQASQVALENCSAVADTRCGCX9PGWFVECXi 0VSX11CVSSSPFYCQPCLDCGALHRHTRLX12CSRRDTDCGTCLPGFYEHGDGCV SCPTSTLGSCPERCAAVCGWROMFIEGRMDGPOGIAGOHHHHHHDYGDPGFGP HQRLEKLDS LLS D YDILS LS NIQQHS VRKADLQTS TH VETLLTFS ALKRHFKLYL TSSTERFSQNFKVVVVDGKNESEYTVKWQDFFTGHVVGEPDSRVLAHIRDDDVI IRINTDGAEYNIEPLWRFVNDTKDKRMLVYKSEDIKNVSRLQSPKVAGYLKVDN EELLPKGLVDREPPEELVHAV
  • GGTRSPRCDCAGDFXiKKX 2 GLFCCRGCPAGHYLKAPCTX3PCGNSTCLX 4 CPQX5 TFLAX 6 ENHHX 7
  • SECX 8 RCQACDEQASQVALENCSAVADTRCGCX9PGWFVECXi 0VSX11CVSSSPFYCQPCLDCGALHRHTRLX12CSRRDTDCGTCLPGFYEHGDGCV SCPTSTLGSCPERCAAVCGWROMFIEGRMDGPOGIAGOHHHHHHDYGDPGFGP HQRLEKLDS LLS D YDILS LS NIQQHS VRKADLQTS TH VETLLTFS ALKRHFKLYL TSSTERFSQNFKVVVVDGKNESEYTVKWQDFFTGHVVGEPDSRVLAHIRDDDVI IRINTDGAEYNIEPLWRFVNDTKDKRMLVYKSEDIKNVSRLQSPKVAGYLKVDN EELLPKGLVDREPPEELVHAV
  • SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSPGK wherein: Xi is H or Q; X 2 is I, T or Y; X 3 is E or K; X 4 is V or P; X5 is D or G; X 6 is W or
  • the chimeras and/or each of the moieties of the invention encompass variant thereof.
  • variant refers to a polypeptide or nucleotide sequence which comprises a modification of one or more amino acids or nucleotides as compared to another polypeptide or polynucleotide, respectively.
  • the modifications are substitution, deletion, and/or insertion of one or more amino acids or nucleotides as compared to another polypeptide or polynucleotide, respectively.
  • the number and position of the modification may vary so long as the chimera is capable of displaying the function of disclosed chimera of the invention.
  • the chimera comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-29 with between 1 and 75, 1 and 70, 1 and 65, 1 and 60, 1 and 55, 1 and 50, 1 and 45, 1 and 40, 1 and 35, 1 and 30, 1 and 25, 1 and 20, 1 and 15, 1 and 10, or 1 and 5 amino acid modifications.
  • the chimera comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-29 with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 amino acid modifications.
  • the chimera comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-29 with at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 amino acid modifications.
  • Each possibility represents a separate embodiment of the present invention.
  • the chimera comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 29.
  • SEQ ID NOs: 8 to 29 Each possibility represents a separate embodiment of the present invention.
  • the changes may be of minor nature, such as conservative amino acid substitutions or for nucleotide sequence resulting in conservative amino acid substitutions that do not significantly affect the activity of the polypeptide.
  • conservative amino acid substitutions or for nucleotide sequence resulting in conservative amino acid substitutions that do not significantly affect the activity of the polypeptide.
  • individual substitutions, deletions or additions to a peptide, a polypeptide or a protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a conservatively modified variant where the alteration results in the substitution of an amino acid with a similar charge, size, and/or hydrophobicity characteristics, such as, for example, substitution of a glutamic acid (E) to aspartic acid (D).
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • the chimera comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 29 with one or more conservative substitutions.
  • the chimera of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 29 with at most , 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 conservative substitutions.
  • Each possibility represents a separate embodiment of the present invention.
  • analog includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein.
  • conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
  • the phrase "conservative substitution” also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite function as specified herein.
  • the present invention encompasses derivatives of the polypeptides (chimeras and each of the moieties).
  • derivatives or “chemical derivative” includes any chemical derivative of the polypeptide having one or more residues chemically derivatized by reaction of side chains or functional groups.
  • derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acid residues.
  • 4- hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3- methylhistidine may be substituted for histidine; homoserine may be substituted or serine; and ornithine may be substituted for lysine.
  • a peptide derivative can differ from the natural sequence of the peptides of the invention by chemical modifications including, but are not limited to, terminal-NH2 acylation, acetylation, or thioglycolic acid amidation, and by terminal-carboxlyamidation, e.g., with ammonia, methylamine, and the like.
  • Peptides can be either linear, cyclic or branched and the like, which conformations can be achieved using methods well known in the art.
  • the present invention also encompasses peptide derivatives and analogs in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonylamino groups, carbobenzoxyamino groups, t-butyloxycarbonylamino groups, chloroacetylamino groups or formylamino groups.
  • Free carboxyl groups may be derivatized to form, for example, salts, methyl and ethyl esters or other types of esters or hydrazides.
  • the imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine.
  • salts refers to both salts of carboxyl groups and to acid addition salts of amino or guanido groups of the peptide molecule.
  • Salts of carboxyl groups may be formed by means known in the art and include inorganic salts, for example sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as salts formed for example with amines such as triethanolamine, piperidine, procaine, and the like.
  • Acid addition salts include, for example, salts with mineral acids such as, for example, acetic acid or oxalic acid. Salts describe here also ionic components added to the peptide solution to enhance hydrogel formation and /or mineralization of calcium minerals.
  • the peptide analogs can also contain non-natural amino acids.
  • non-natural amino acids include, but are not limited to, sarcosine (Sar), norleucine, ornithine, citrulline, diaminobutyric acid, homoserine, isopropyl Lys, 3-(2'-naphtyl)-Ala, nicotinyl Lys, amino isobutyric acid, and 3-(3'-pyridyl-Ala).
  • the peptide analogs can contain other derivatized amino acid residues including, but not limited to, methylated amino acids, N-benzylated amino acids, O-benzylated amino acids, N-acetylated amino acids, O-acetylated amino acids, carbobenzoxy-substituted amino acids and the like.
  • Specific examples include, but are not limited to, methyl- Ala (Me Ala), MeTyr, MeArg, MeGlu, MeVal, MeHis, N-acetyl-Lys, O-acetyl-Lys, carbobenzoxy-Lys, Tyr-O-Benzyl, Glu-O-Benzyl, Benzyl-His, Arg-Tosyl, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, and the like.
  • the invention further includes peptide analogs, which can contain one or more D-isomer forms of the amino acids.
  • Production of retro-inverso D-amino acid peptides where at least one amino acid and perhaps all amino acids are D-amino acids is well known in the art.
  • the result is a molecule having the same structural groups being at the same positions as in the L-amino acid form of the molecule.
  • the molecule is more stable to proteolytic degradation and is therefore useful in many of the applications recited herein.
  • Diastereomeric peptides may be highly advantageous over all L- or all D-amino acid peptides having the same amino acid sequence because of their higher water solubility, lower immunogenicity, and lower susceptibility to proteolytic degradation.
  • the term "diastereomeric peptide" as used herein refers to a peptide comprising both L-amino acid residues and D-amino acid residues.
  • the number and position of D-amino acid residues in a diastereomeric peptide of the preset invention may be variable so long as the chimera is capable of displaying the function of disclosed chimera of the invention.
  • the sDR3 mutant is derived from or corresponding to amino acids 26-201 of human DR3 (NCBI Accession No. BAB40663, SEQ ID NO: 1).
  • the term "derived from” or “corresponding to” refers to construction of an amino acid sequence based on the knowledge of a sequence using any one of the suitable means known to one skilled in the art, e.g. chemical synthesis in accordance with standard protocols in the art.
  • a peptide derived from or corresponding to amino acids 26-201 of SEQ ID NO: 1 is a peptide based on SEQ ID NO: 1, or an analog, a variant, a derivative or a fragment thereof.
  • the peptide derived from or corresponding to amino acids 26-201 of SEQ ID NO: 1 has the amino acid sequence as set forth in SEQ ID NO: 2, or an analog, a variant, a derivative or a fragment thereof.
  • sDR3 mutants refers to a soluble variant of the extracellular domain of death receptor 3 comprising the nucleic acid and/or nucleotide sequences of DR3 (SEQ ID NO: l) or a fragment thereof having one or more substitutions.
  • the sDR3 mutant comprises the amino acid sequence of SEQ ID NO: 2 (GGTRSPRCDCAGDFHKKIGLFCCRGCPAGHYLKAPCTEPCGNSTCLVCPQDTFLAWEN HHNSECARCQACDEQASQVALENCSAVADTRCGCKPGWFVECQVSQCVSSSPFYCQPC LDCGALHRHTRLLCSRRDTDCGTCLPGFYEHGDGCVSCPTSTLGSCPERCAAVCGWRQ MF), or an analog, or a derivative or a fragment thereof, wherein said amino acid sequence comprises at least one amino acid substitution at a position selected from the group consisting of: H15, 118, E38, V47, D51, W56, N61, A65, K93, Q101, Q104 and L129.
  • the sDR3 mutant is capable of binding TLA1 with increased affinity compared to WT DR3 (SEQ ID NO: 2).
  • the affinity is increased by at least a 1.5-fold, at least a 2-fold, at least a 2.5-fold, at least a 3-fold, at least a 3.5-fold, at least a 4-fold, at least a 4.5-fold, at least a 5-fold, at least a 5.5-fold, at least a 6-fold, at least a 6.5-fold, at least a 7-fold, at least a 7.5-fold, at least a 8-fold, at least a 8.5-fold, at least a 9-fold, at least a 9.5-fold, or at least a 10-fold.
  • the binding of the first moiety to TLA1 inhibits or reduces TL1A mediated IFN- ⁇ secretion.
  • the DR3 mutant of the invention has increased selectively to TL1A compared to the selectivity of WT DR3.
  • selective refers to having a binding affinity to TL1A that is substantially greater than said binding affinity for wild-type (WT) DR3 to TL1A.
  • substantially greater means at least a 1.5-fold, at least a two-fold, at least a three-fold, at least a four-fold or at least a five-fold increase in the selectivity to a TL1A.
  • the sDR3 mutant comprises or consists of an amino acid sequence selected from the amino acid sequences presented in Table 2 (e.g., SEQ ID NOs: 30-38).
  • the present invention further provides fragments, analogs and chemical modifications of the DR3 mutants of the present invention as long as they are capable of binding TLIA and/or modulating (e.g. reducing or inhibiting) TLIA induced IFN- ⁇ secretion.
  • the sDR3 mutants of the invention encompass truncated forms and/or fragments of any one of SEQ ID NOs: 30-38 as long as they are capable of binding TLIA and/or modulating (e.g. reducing or inhibiting) TLIA induced IFN- ⁇ secretion.
  • the fragments or the truncated forms of sDR3 mutants of the invention comprise at least 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175 or 176 amino acids derived from any one of SEQ ID NOs: 22- 30.
  • Each possibility represents a separate embodiment of the present invention.
  • the fragments or the truncated forms of sDR3 mutants of the invention comprise 100 to 176, 100 to 175, 100 to 174, 100 to 173, 100 to 172, 100 to 171, 100 to 170, 100 to 169, 100 to 168, 100 to 167, 100 to 166, 100 to 165, 100 to 164, 100 to 163, 100 to 162, 100 to 161, 100 to 160, 100 to 159, 100 to 158, 100 to 157, 100 to 156, 100 to 155, 100 to 154, 100 to 153, 100 to 152, 100 to 151, 100 to 150, 120 to 176, 120 to 175, 120 to 174, 120 to 173, 120 to 172, 120 to 171, 120 to 170, 120 to 169, 120 to 168, 120 to 167, 120 to 166, 120 to 165, 120 to 164, 120 to 163, 120 to 162, 120 to 161, 120 to 160, 120 to 159, 120 to 158, 120 to 157, 120 to 156, 120 to 155, 120 to
  • the first moiety comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-38 with one or more conservative substitutions. In some embodiments, the first moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-38 with at most 1, 2, 3, or 4 conservative substitutions. Each possibility represents a separate embodiment of the present invention. According to another embodiment of the invention, the first moiety comprises a sequence homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-38.
  • the first moiety comprises a sequence having greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-38.
  • SEQ ID NOs: 30-38 Each possibility represents a separate embodiment of the present invention.
  • the first moiety comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-38, as long as the variant is capable of binding TL1A and/or modulating (e.g. reducing or inhibiting) TL1A induced IFN- ⁇ secretion.
  • pTACE moiety
  • the second moiety comprises a pTACE mutant having an amino acid sequence derived from or corresponding to a pro-domain of TNF-a converting enzyme (pTACE domain), comprising at least one mutation at the natural furin cleavage site, which renders the moiety resistant to furin degradation.
  • the moiety is devoid of the catalytic domain of TACE.
  • the pTACE mutant is capable of reducing/inhibiting TACE catalytic activity in vitro and/or in vivo.
  • the second moiety comprises an amino acid sequence derived from or corresponding to the pTACE domain of human TACE, or an analog, or a derivative or a fragment thereof.
  • the pTACE mutant comprises an amino acid sequence derived from or corresponding to amino acids 23 to 214 of human TACE (NCBI Reference Sequence: NP_003174.3, SEQ ID NO: 39).
  • the pTACE mutant comprises at most 220, 219, 218, 217, 216, 215,
  • the pTACE mutant is devoid of the catalytic domain of TACE.
  • the second moiety has a length of at most 220, 219, 218, 217, 216,
  • the amino acid sequence derived from or corresponding to amino acids 23 to 214 of human TACE comprises at least one amino acid substitution at a position selected from the group consisting of: R56, K57, R58 of the human TACE (SEQ ID NO: 39). In some embodiments, the amino acid sequence further comprises at least one amino acid substitution at a position selected from the group consisting of: C184, R211, R214 of the human TACE (SEQ ID NO: 39). In some embodiments, the pTACE peptide of the invention is capable of downregulating an activity of human TACE in vivo and in vitro.
  • the pTACE peptide of the invention comprises the amino acid sequence as set forth in SEQ ID NO: 40,
  • amino acid sequence comprises at least one amino acid substitution at a position selected from the group consisting of: R35, K36, and R37.
  • the amino acid sequence comprises at least one amino acid substitution at a position selected from the group consisting of: C163, R190 and R193.
  • the R35, K36, R37 is substituted with any amino acid other than arginine or lysine.
  • the R35, K36, R37 is substituted with an amino acid selected from: Alanine, Aspargine, Aspartic Acid, Cysteine, Glutamine, Glutamic Acid, Glycine, Histidine, Isolucine, Leucine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine or Valine.
  • the pTACE mutant of the invention comprises the amino acid sequence as set forth in SEQ ID NO: 41
  • Xi is selected from R and A
  • X 2 is selected from K and A
  • X 3 is selected from R and A
  • X 4 is selected from C and A
  • X5 is selected from R and A
  • X 6 is selected from R and G, and wherein at least one of the Xi, X 2 and X 3 is an amino acid other than R or K.
  • At least one of the Xi, X 2 and X 3 is selected from: Alanine, Aspargine, Aspartic Acid, Cysteine, Glutamine, Glutamic Acid, Glycine, Histidine, Isolucine, Leucine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine or Valine.
  • the pTACE mutant of the invention comprises the amino acid sequence as set forth in SEQ ID NO: 42
  • the pTACE mutant comprises or consists of an amino acid sequence selected from the amino acid sequences presented in Table 3 (e.g., SEQ ID NOs: 43-46).
  • Xi is selected from R and A
  • X 2 is selected from K and A
  • X3 is A
  • X 4 is selected from C and A
  • X5 is selected from R and A
  • X 6 is selected from R and G.
  • X 4 GYLKVDNEELLPKGLVDREPPEELVHX 5 VKX 6 wherein Xi is A, X 2 is K, X 3 is A, X 4 is selected from C and A, X5 is selected from R and A, and X 6 is selected from R and G.
  • X 4 GYLKVDNEELLPKGLVDREPPEELVHX 5 VKX 6 wherein Xi is A, X 2 is K, X 3 is A, X 4 is A, X5 is A, and X 6 is G.
  • the present invention further provides fragments, analogs and chemical modifications of the pTACE mutants of the present invention as long as they are capable of reducing/inhibiting TACE both in vitro and in vitro.
  • the pTACE mutants of the invention encompass truncated forms and/or fragments of any one of SEQ ID NOs: 41-46 as long as they are capable of e.g. reducing or inhibiting TACE activity.
  • the fragments or the truncated forms of pTACE mutants of the invention comprise at least 192, 191, 190, 189, 188, 187, 186, 185, 184, 183, 182, 181, 180, 179, 178, 177, 176, 175, 174, 173, 172, 171, 170, 169, 168, 167, 166, 165, 164, 163, 162, 161, 160, 159, 158, 157, 156, 155, 154, 153, 152, 151, or 150 amino acids derived from any one of SEQ ID NOs: 41-46. Each possibility represents a separate embodiment of the present invention.
  • the fragments or the truncated forms of pTACE mutants of the invention comprise 100 to 192, 100 to 191, 100 to 190, 100 to 189, 100 to 1886, 100 to 187, 100 to 186, 100 to 185, 100 to 184, 100 to 183, 100 to 182, 100 to 181, 100 to 180, 100 to 179, 100 to 178, 100 to 177, 100 to 176, 100 to 175, 100 to 174, 100 to 173, 100 to 172, 100 to 171, 100 to 170, 100 to 169, 100 to 168, 100 to 167, 100 to 166, 100 to 165, 100 to 164, 100 to 163, 100 to 162, 100 to 161, 100 to 160, 100 to 159, 100 to 158, 100 to 157, 100 to 156, 100 to 155, 100 to 154, 100 to 153, 100 to 152, 100 to 151, 100 to 150, 120 to 192, 120 to 191, 120 to 190, 120 to 189, 120 to 1886, 120 to
  • the second moiety comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-46 with one or more conservative substitutions.
  • the second moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-46 with at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions.
  • the second moiety comprises a sequence homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-38.
  • the second moiety comprises a sequence having greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-38.
  • SEQ ID NOs: 33-38 Each possibility represents a separate embodiment of the present invention.
  • the second moiety comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-46, as long as the variant is capable of is capable of reducing/inhibiting TACE catalytic activity in vivo and/or in vitro.
  • the chimeras of the present invention may be synthesized or prepared by any method and/or technique known in the art for peptide synthesis.
  • the polypeptides may be synthesized by a solid phase peptide synthesis method of Merrifield (see J. Am. Chem. Soc, 85:2149, 1964).
  • the polypeptides of the present invention can be synthesized using standard solution methods well known in the art (see, for example, Bodanszky, M., Principles of Peptide Synthesis, Springer - Verlag, 1984).
  • the synthesis methods comprise sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain bound to a suitable resin.
  • a suitable protecting group either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group.
  • the protected or derivatized amino acid can then be either attached to an inert solid support (resin) or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions conductive for forming the amide linkage.
  • the protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is added, and so forth.
  • any remaining protecting groups are removed sequentially or concurrently, and the peptide chain, if synthesized by the solid phase method, is cleaved from the solid support to afford the final peptide.
  • the alpha-amino group of the amino acid is protected by an acid or base sensitive group.
  • Such protecting groups should have the properties of being stable to the conditions of peptide linkage formation, while being readily removable without destruction of the growing peptide chain.
  • Suitable protecting groups are t-butyloxycarbonyl (BOC), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, (alpha,alpha)-dimethyl-3 ,5 dimethoxybenzyloxycarbonyl, o- nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC) and the like.
  • the C-terminal amino acid is attached to a suitable solid support.
  • Suitable solid supports useful for the above synthesis are those materials, which are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions, as well as being insoluble in the solvent media used. Suitable solid supports are chloromethylpolystyrene-divinylbenzene polymer, hydroxymethyl-polystyrene-divinylbenzene polymer, and the like.
  • the coupling reaction is accomplished in a solvent such as ethanol, acetonitrile, ⁇ , ⁇ -dimethylformamide (DMF), and the like.
  • the coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer as is well known in the art.
  • polypeptides of the invention may be synthesized such that one or more of the bonds, which link the amino acid residues of the peptides are non-peptide bonds.
  • the non-peptide bonds include, but are not limited to, imino, ester, hydrazide, semicarbazide, and azo bonds, which can be formed by reactions well known to one skilled in the art.
  • the invention further encompasses a polynucleotide sequence comprising a nucleic acid encoding any of the chimeras of the invention.
  • the nucleic acid sequence encoding the chimeras of the invention is at least 70%, or alternatively at least 80%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 99% homologous to the nucleic acid sequence encoding the nucleic acid sequence of the chimeras of the invention or a fragment thereof.
  • the invention provides a polynucleotide encoding the chimeras of the invention.
  • the polynucleotide of the present invention is ligated into an expression vector, comprising a transcriptional control of a cis -regulatory sequence (e.g., promoter sequence).
  • a cis -regulatory sequence e.g., promoter sequence
  • the cis-regulatory sequence is suitable for directing constitutive expression of the polypeptide of the present invention.
  • the cis-regulatory sequence is suitable for directing tissue- specific expression of the polypeptide of the present invention.
  • the cis-regulatory sequence is suitable for directing inducible expression of the polypeptide of the present invention.
  • polynucleotide refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide.
  • a polynucleotide refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
  • complementary polynucleotide sequence refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase.
  • the sequence can be subsequently amplified in vivo or in vitro using a DNA polymerase.
  • genomic polynucleotide sequence refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
  • composite polynucleotide sequence refers to a sequence, which is at least partially complementary and at least partially genomic.
  • a composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing there between.
  • the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences.
  • intronic sequences include cis acting expression regulatory elements.
  • polynucleotides of the present invention are prepared using PCR techniques as described in Example 1, or any other method or procedure known to one skilled in the art.
  • the procedure involves the ligation of two different DNA sequences (See, for example, "Current Protocols in Molecular Biology", eds. Ausubel et al., John Wiley & Sons, 1992).
  • polynucleotides of the present invention are inserted into expression vectors (i.e., a nucleic acid construct) to enable expression of the recombinant polypeptide.
  • the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes.
  • the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in eukaryotes.
  • the expression vector of the present invention includes a shuttle vector which renders this vector suitable for replication and integration in both prokaryotes and eukaryotes.
  • cloning vectors comprise transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation terminators (e.g., polyadenylation signals).
  • prokaryotic or eukaryotic cells can be used as host- expression systems to express the polypeptide of the present invention.
  • these include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the polypeptide coding sequence.
  • microorganisms such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence
  • yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence e.g
  • non-bacterial expression systems are used (e.g. mammalian expression systems) to express the polypeptide of the present invention.
  • the expression vector is used to express polynucleotides of the present invention in mammalian cells.
  • a number of expression vectors can be advantageously selected depending upon the use intended for the polypeptide expressed. In one embodiment, large quantities of polypeptide are desired.
  • vectors that direct the expression of high levels of the protein product, possibly as a fusion with a hydrophobic signal sequence, which directs the expressed product into the periplasm of the bacteria or the culture medium where the protein product is readily purified are desired.
  • certain fusion protein engineered with a specific cleavage site to aid in recovery of the polypeptide.
  • vectors adaptable to such manipulation include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)].
  • yeast expression systems are used.
  • a number of vectors containing constitutive or inducible promoters can be used in yeast as disclosed in U.S. Pat. No. 5,932,447.
  • vectors which promote integration of foreign DNA sequences into the yeast chromosome are used.
  • the expression vector of the present invention may further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES).
  • IRES internal ribosome entry site
  • mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+), pGL3, pZeoSV2(+), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention.
  • SV40 vectors include pSVT7 and pMT2.
  • vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205.
  • exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • recombinant viral vectors which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression of the chimera of the present invention.
  • lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells.
  • the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles.
  • viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • polypeptide of the present invention can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (i.e., in vivo gene therapy).
  • the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex vivo gene therapy).
  • in vivo gene therapy using a cytokine has been attempted in animal models such as rodents [Bohl et al., Blood. 2000; 95:2793-2798], primates [Gao et al., Blood, 2004, Volume 103, Number 9] and has proven successful in human clinical trials for patients with chronic renal failure [Lippin et al Blood 2005, 106, Number 7].
  • plant expression vectors are used.
  • the expression of a polypeptide coding sequence is driven by a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511- 514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 3:17-311 (1987)] are used.
  • plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J.
  • constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421- 463 (1988)].
  • Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.
  • the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.
  • transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant polypeptide.
  • effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • an effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide of the present invention.
  • a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates.
  • culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell.
  • culturing conditions are within the expertise of one of ordinary skill in the art.
  • resultant polypeptides of the present invention either remain within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or retained on the outer surface of a cell or viral membrane.
  • recovery of the recombinant polypeptide is affected.
  • the phrase "recovering the recombinant polypeptide" used herein refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification.
  • polypeptides of the present invention are purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • the expressed coding sequence can be engineered to encode the polypeptide of the present invention and fused cleavable moiety.
  • a fusion protein can be designed so that the polypeptide can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the cleavable moiety.
  • a cleavage site is engineered between the polypeptide and the cleavable moiety, and the polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265: 15854-15859 (1990)].
  • polypeptide of the present invention is retrieved in "substantially pure" form that allows for the effective use of the protein in the applications described herein.
  • substantially pure describes a peptide/ polypeptide or other material which has been separated from its native contaminants.
  • a monomeric peptide is substantially pure when at least about 60 to 75% of a sample exhibits a single peptide backbone. Minor variants or chemical modifications typically share the same peptide sequence.
  • a substantially pure peptide can comprise over about 85 to 90% of a peptide sample, and can be over 95% pure, over 97% pure, or over about 99% pure. Purity can be measured on a polyacrylamide gel, with homogeneity determined by staining. Alternatively, for certain purposes high resolution may be necessary and HPLC or a similar means for purification can be used. For most purposes, a simple chromatography column or polyacrylamide gel can be used to determine purity.
  • the term "purified” does not require the material to be present in a form exhibiting absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition.
  • a peptide is in the "purified” state after purification of the starting material or of the natural material by at least one order of magnitude, 2 or 3, or 4 or 5 orders of magnitude.
  • the polypeptides of the present invention are substantially free of naturally-associated host cell components.
  • substantially free of naturally-associated host cell components describes a peptide or other material which is separated from the native contaminants which accompany it in its natural host cell state.
  • a peptide which is chemically synthesized or synthesized in a cellular system different from the host cell from which it naturally originates will be free from its naturally-associated host cell components.
  • polypeptide of the present invention can also be synthesized using in vitro expression systems.
  • in vitro synthesis methods are well known in the art and the components of the system are commercially available.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of the chimera of the present invention, and a pharmaceutically acceptable carrier and/or diluents.
  • the pharmaceutical composition facilitates administration of a compound to an organism.
  • compositions of the invention may be formulated in the form of a pharmaceutically acceptable salt of the polypeptides of the present invention or their analogs, or derivatives thereof.
  • pharmaceutically acceptable salts include those salts formed with free amino groups such as salts derived from nontoxic inorganic or organic acids such as hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those salts formed with free carboxyl groups such as salts derived from non-toxic inorganic or organic bases such as sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.
  • the carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.
  • the term "pharmaceutically acceptable” means suitable for administration to a subject, e.g., a human.
  • pharmaceutically acceptable can mean approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • compositions of the invention take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, gels, creams, ointments, foams, pastes, sustained-release formulations and the like.
  • the compositions of the invention can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in: Remington's Pharmaceutical Sciences" by E.W.
  • compositions will contain a therapeutically effective amount of the polypeptide of the invention, preferably in a substantially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • compositions contain 0.1% - 95% of the chimera(s) of the present invention, derivatives, or analogs thereof.
  • pharmaceutical compositions contain 1% - 70% of the chimera(s) derivatives, or analogs thereof.
  • the composition or formulation to be administered may contain a quantity of chimera(s), derivatives, or analogs thereof, according to embodiments of the invention in an amount effective to treat the condition or disease of the subject being treated.
  • An embodiment of the invention relates to chimeras of the present invention, derivatives, or analogs thereof, presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.
  • the unit dosage form is in the form of a tablet, capsule, lozenge, wafer, patch, ampoule, vial or pre-filled syringe.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the nature of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in-vitro or in-vivo animal model test bioassays or systems.
  • compositions of the present invention are administered in the form of a pharmaceutical composition comprising at least one of the active components of this invention together with a pharmaceutically acceptable carrier or diluent.
  • compositions of this invention can be administered either individually or together in any conventional oral, parenteral or transdermal dosage form.
  • the pharmaceutical composition further comprises at least one anti-inflammatory agent.
  • the pharmaceutical composition is adopted for combined administration with an antiinflammatory agent.
  • administering refers to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect.
  • the chimera of the present invention can be administered in any manner suitable for the provision of the chimeras to cells within the tissue of interest.
  • a composition containing the polypeptides of the present invention can be introduced, for example, into the systemic circulation, which will distribute the peptide to the tissue of interest.
  • a composition can be applied topically to the tissue of interest (e.g., injected, or pumped as a continuous infusion, or as a bolus within a tissue, applied to all or a portion of the surface of the skin, etc.).
  • the pharmaceutical compositions comprising the chimeras are administered via oral, rectal, vaginal, topical, nasal, ophthalmic, transdermal, subcutaneous, intramuscular, intraperitoneal or intravenous routes of administration.
  • the route of administration of the pharmaceutical composition will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injections, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other mode of injection as known in the art.
  • compositions of the invention can be lower than when administered via parenteral injection, by using appropriate formulations it is envisaged that it will be possible to administer the compositions of the invention via transdermal, oral, rectal, vaginal, topical, nasal, inhalation and ocular modes of treatment.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer.
  • a peptide of the present invention, derivative, analog or a fragment thereof can be combined with a pharmaceutically acceptable carrier so that an effective dosage is delivered, based on the desired activity.
  • the carrier can be in the form of, for example, and not by way of limitation, an ointment, cream, gel, paste, foam, aerosol, suppository, pad or gelled stick.
  • the pharmaceutical composition may be in the form of tablets or capsules, which can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; or a glidant such as colloidal silicon dioxide.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose
  • a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate
  • a glidant such as colloidal silicon dioxide.
  • dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier such as fatty oil.
  • dosage unit forms can contain various other materials which modify the physical form
  • solutions in sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water- soluble salts.
  • aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes.
  • the chimeras of the present invention, derivatives, or analogs thereof can be delivered in a controlled release system.
  • an infusion pump can be used to administer the peptide such as the one that is used, for example, for delivering insulin or chemotherapy to specific organs or tumors.
  • the peptides of the invention are administered in combination with a biodegradable, biocompatible polymeric implant, which releases the peptide over a controlled period of time at a selected site.
  • polymeric materials include, but are not limited to, polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, copolymers and blends thereof (See, Medical applications of controlled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla., the contents of which are hereby incorporated by reference in their entirety).
  • a controlled release system can be placed in proximity to a therapeutic target, thus requiring only a fraction of the systemic dose.
  • the presently described chimeras, derivatives, or analogs thereof may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half -life of the peptides or polypeptides in serum.
  • Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • Liposomes for use with the presently described peptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood.
  • compositions also include incorporation of the active material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.)
  • polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc.
  • liposomes such as polylactic acid, polglycolic acid, hydrogels, etc.
  • Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.
  • the present invention provides combined preparations.
  • a combined preparation defines especially a "kit of parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners i.e., simultaneously, concurrently, separately or sequentially.
  • the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts.
  • the ratio of the total amounts of the combination partners in some embodiments, can be administered in the combined preparation.
  • the combined preparation can be varied, e.g., in order to cope with the needs of a patient subpopulation to be treated or the needs of the single patient which different needs can be due to a particular disease, severity of a disease, age, sex, or body weight as can be readily made by a person skilled in the art.
  • the peptides of the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself.
  • measures e.g., dosing and selection of the complementary agent
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is affected or diminution of the disease state is achieved.
  • the chimeras are administered in a therapeutically safe and effective amount.
  • safe and effective amount refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the presently described manner.
  • a therapeutically effective amount of the chimera is the amount of the chimera necessary for the in vivo measurable expected biological effect. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g.
  • preparation of effective amount or dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
  • toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosages vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p. l].
  • compositions containing the presently described polypeptide as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990). See also, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa. (2005).
  • compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier are prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • compositions of the present invention are presented in a pack or dispenser device, such as an FDA approved kit, which contain one or more unit dosages forms containing the active ingredient.
  • the pack for example, comprises metal or plastic foil, such as a blister pack.
  • the pack or dispenser device is accompanied by instructions for administration.
  • the pack or dispenser is accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • Such notice is labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • the invention provides a method for reducing or inhibiting inflammation, an immune response, or both, in a subject in need thereof, comprising the step of administering to said subject a composition comprising an effective amount of the chimera of the invention, thereby reducing or inhibiting inflammation, an immune response, or both in said subject.
  • the method of the invention is useful for treating a T cell-mediated disease, particularly a Thl cell-mediated disease.
  • the inflammatory disease includes but is not limited to inflammatory or allergic diseases such as asthma, hypersensitivity lung diseases, hypersensitivity pneumonitis, delayed-type hypersensitivity, interstitial lung disease (ILD) (e.g., idiopathic pulmonary fibrosis, or ILD associated with rheumatoid arthritis or other inflammatory diseases); scleroderma; psoriasis (including T-cell mediated psoriasis); dermatitis (including atopic dermatitis and eczematous dermatitis), crizis, conjunctivitis, keratoconjunctivitis, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, ILD (ILD) (e.g
  • the chimera of the invention is useful for treating an autoimmune disease, including but not limited to: multiple sclerosis (MS), autoimmune neuritis, systemic lupus erythematosus (SLE), psoriasis, Type I diabetes (IDDM), Sjogren's disease, thyroid disease, myasthenia gravis, sarcoidosis, autoimmune uveitis, inflammatory bowel disease (Crohn's and ulcerative colitis), atherosclerosis, primary biliary cirrhosis (PBC), or autoimmune hepatitis, rheumatoid arthritis.
  • MS multiple sclerosis
  • SLE systemic lupus erythematosus
  • IDDM Type I diabetes
  • Sjogren's disease thyroid disease
  • myasthenia gravis myasthenia gravis
  • sarcoidosis autoimmune uveitis
  • inflammatory bowel disease Crohn's and ulcerative colitis
  • atherosclerosis primary biliary
  • the chimera of the invention is useful for treating graft rejection, including allograft rejection or graft-versus-host disease.
  • said reducing or inhibiting inflammation, an immune response, or both is inhibiting TLlA-mediated disease.
  • said reducing or inhibiting inflammation, an immune response, or both is inhibiting TLlA-induced IFN- ⁇ secretion in said subject.
  • reducing inflammation refer to a statistically significant reduction in inflammation.
  • the subject is afflicted with a chronic inflammatory disease.
  • the subject is afflicted with an inflammatory bowel disease.
  • the subject is afflicted with psoriasis.
  • said subject is afflicted with an autoimmune disease.
  • said subject is afflicted with asthma.
  • said subject is afflicted with arthritis.
  • said subject is afflicted with colitis.
  • a method for treating, ameliorating, reducing and/or preventing inflammation, an immune response, or both in a subject in need thereof comprising the step of: administering to a subject a pharmaceutical composition comprising an effective amount of the chimeras of the invention, thereby treating, ameliorating, reducing and/or preventing an inflammation, an immune response, or both in a subject in need thereof.
  • the method further comprises a step of administering a therapeutically effective amount of at least one anti-inflammatory agent.
  • a method for treating, ameliorating, reducing and/or preventing an inflammation, an immune response, or both in a subject in need thereof comprising administering to the subject any one of:
  • the chimera of the invention or a composition comprising the chimera is for use in treatment, amelioration, reduction, and/or prevention of inflammation, an immune response, or both in a subject in need thereof.
  • composition comprising an effective amount of an amino acid molecule comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 18 for use in the treatment or prevention of an inflammation, an immune response, or both in a subject in need thereof.
  • composition comprising an effective amount of an amino acid molecule comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 29 for use in the treatment or prevention of an inflammation, an immune response, or both in a subject in need thereof.
  • compositions comprising an effective amount of chimera of the invention in the preparation of a medicament for the treatment, amelioration, reduction, or prevention of an inflammation, an immune response, or both in a subject in need thereof.
  • the chimera of the present invention is provided to the subject per se. In one embodiment, the chimera of the present invention is provided to the subject as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier. In one embodiment, one or more of the chimeras of the present invention are provided to the subject as part of a pharmaceutical composition where they are mixed with a pharmaceutically acceptable carrier.
  • subject refers to an animal, more particularly to non-human mammals and human organism.
  • Non-human animal subjects may also include prenatal forms of animals, such as, e.g., embryos or fetuses.
  • Non-limiting examples of non-human animals include: horse, cow, camel, goat, sheep, dog, cat, non-human primate, mouse, rat, rabbit, hamster, guinea pig, pig.
  • the subject is a human.
  • Human subjects may also include fetuses.
  • a subject in need thereof is a subject afflicted with and/or at risk of being afflicted with an inflammation, an immune response, or both.
  • treatment encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured.
  • a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject's quality of life.
  • prevention of a disease, disorder, or condition encompasses the delay, prevention, suppression, or inhibition of the onset of a disease, disorder, or condition.
  • prevention relates to a process of prophylaxis in which a subject is exposed to the presently described peptides prior to the induction or onset of the disease/disorder process. This could be done where an individual has a genetic pedigree indicating a predisposition toward occurrence of the disease/disorder to be prevented. For example, this might be true of an individual whose ancestors show a predisposition toward certain types of, for example, inflammatory disorders.
  • suppression is used to describe a condition wherein the disease/disorder process has already begun but obvious symptoms of the condition have yet to be realized.
  • the cells of an individual may have the disease/disorder, but no outside signs of the disease/disorder have yet been clinically recognized.
  • prophylaxis can be applied to encompass both prevention and suppression.
  • treatment refers to the clinical application of active agents to combat an already existing condition whose clinical presentation has already been realized in a patient.
  • condition includes anatomic and physiological deviations from the normal that constitute an impairment of the normal state of the living animal or one of its parts, that interrupts or modifies the performance of the bodily functions.
  • concentration ranges, percentage range, or ratio range recited herein are to be understood to include concentrations, percentages or ratios of any integer within that range and fractions thereof, such as one tenth and one hundredth of an integer, unless otherwise indicated.
  • the terms "subject” or “individual” or “animal” or “patient” or “mammal,” refers to any subject, particularly a mammalian subject, for whom therapy is desired, for example, a human.
  • each of the verbs, "comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.
  • Other terms as used herein are meant to be defined by their well-known meanings in the art.
  • proTACE and H3 fragments were digested and cloned into pFUSE- hIgGle3-Fc (Invivogen) with Ncol and Bglll to yield an open reading frame coding for a signal peptide followed by H3-linker-6xHis-TACE-Fc (A2) and TACE-linker-6xHis-H3-Fc (Al).
  • H293F were grown in Freestyle media with lx Pen/strep solution (Biological Industries, Beit-Haemek, Israel), at 37 °C, 8% C02 with shaking.
  • PBL were grown at a concentration of 1 x 10 6 /ml in RPMI 1640, with 10% FBS, 2 mM Glutamine, 1 x Pen/strep solution (Biological Industries, Beit-Haemek, Israel) supplemented with 10% heat inactivated FBS (Biological Industries, Beit-Haemek, Israel).
  • TF-1 Cells (ATCC - CRL2003) were grown in RPMI supplemented with 10% FBS (Biological Industries, Beit-Haemek, Israel), 2 mM Glutamine (Biological Industries, Beit-Haemek, Israel), 2 mM GM-CSF (Peprotech), 1 x Pen/step solution (Biological Industries, Beit-Haemek, Israel). The culture was maintained between 3 x 10 4 and 5 x 10 5 viable cells/ml .
  • proTACE Large scale protein expression and purification. Purification of proTACE: pMALC2 containing proTACE3mut was transformed into E. coli Rosetta strain (Novagen) and grown over night on LB agar with 100 ⁇ g/ml ampicillin and 30 ⁇ g/ml chloramphenicol. One colony was used to inoculate a 10 milliliters starter and then transferred into 200 ml LB 100 ⁇ g/ml ampicillin and 30 ⁇ g/ml chloramphenicol, the culture was allowed to grow till it reached absorbance of 0.6 at 600 nm and then IPTG was added to a final concentration of 0.4 mM, and the culture was grown at 15 °C, 250 rpm overnight.
  • the cells were centrifuged at 3,220 rcf for 15 minutes and 100 ml lysis buffer containing 50 mM TRIS pH 8.0, 300 mM sodium chloride, 20 mM imidazole, and 0.05 mg/ml lysozyme, 1,250 U benzonaze, 20 ⁇ protease Inhibitor cocktail II (CalBioChem) was added for 30 minutes incubation on ice. The lysate was then sonicated and centrifuged at 10,000 rcf, 4 °C for 1 hour.
  • the supernatant was then removed and filtered through a 0.45 ⁇ filter (Millipore) and the extract was loaded into a column containing Ni-NTA Agarose resin (Thermo scientific) that was pre-equilibrated with wash buffer containing 50 mM Tris pH 8.0, 300 mM sodium chloride and 20mM imidazole. Subsequently the column was washed with 4 column volume (CV) of wash buffer and the sample was eluted and fractionated by applying 2 CV of 50 mM Tris pH 8.0, 300 mM sodium chloride and 250 mM imidazole. The eluted sample was analyzed by SDS-PAGE, dialyzed twice against 50 mM Tris at pH 8.0 at 4 °C, flash frozen in liquid nitrogen and stored in -80 °C.
  • HEK293F cells were grown in Freestyle serum free media (Invitrogen) and were transiently transfected using Genetran III (Biomiga) as recommended by the manufacturer and the transfected cells were incubated with shaking at 37 °C at 8% C0 2 incubator. Next, 7 days post transfection the cell media was collected, filtered and loaded into a Ni-NTA Agarose resin (Thrmo scientific) column pre-equilibrated with 50 mM Tris pH 8.0, 300 mM NaCl and 20 mM imidazole. The column was then washed with 20 CV of 50 mM Tris pH 8.0, 300 mM NaCl, 20 mM imidazole.
  • the protein was eluted with 3 CV 50 mM Tris pH 8.0, 300 mM NaCl, 500 mM imidazole.
  • the eluted fractions were analyzed by SDS-PAGE, and selected fractions were dialyzed twice against 50 mM Tris pH 8.0, flash frozen in liquid nitrogen and stored at -80 °C.
  • the membrane was washed three times with PBST and incubated for 1 hour with 0.08 ⁇ g/ml Horse Reddish Peroxidase (HRP) conjugated Donkey Anti-Goat or 0.1 ⁇ g/ml HRP conjugated streptavidin (Jackson Immunoresearch).
  • HRP horse Reddish Peroxidase
  • the membrane was developed with EZ-ECL (Biological industries, Israel) and analyzed using FusionFX (Vilber Lourmat).
  • ELISA assay for monitoring TL1A-DR3 interaction ELISA plates (Griener Microlon 96W) were incubated with 100 ⁇ of 0.66 ⁇ / ⁇ monoclonal mouse a-TLlA antibodies (Santa Cruz) for 1 hour, washed with PBST and 100 ⁇ of 1 ⁇ g/ml TL1A (peprotech), were added to the plate for an additional hour. The plates were then washed with PBST and blocked by incubation with 150 ⁇ of PBS supplemented with 3% skim milk for 1 hour.
  • DR3-Fc R&D Systems
  • PBS supplemented with 1% BSA served as a negative control. Plates were then washed with PBST, incubated with 100 ⁇ of 0.05 ⁇ / ⁇ of biotinylated goat polyclonal a-DR3 antibodies (R&D Systems), followed by incubation with secondary peroxidase-conjugated streptavidin (Jackson, 1 : 10,000 dilution).
  • HRP horseradish peroxidase
  • TMB 3,3 ',5,5'-tetramethylbenzidine
  • TACE in-vitro activity assay Recombinant human ADAM 17/T ACE (R&D Systems) was diluted as recommended by the manufacturer to a final concentration of 2.85 nM and the A2 or proTACE were added in various concentrations, the mixture was incubated for 5 minutes at RT. Then 10 ⁇ fluorogenic Peptide Substrate Mca-PLAQAV-Dpa-RSSSR-NH2 (R&D systems) was added, and cleavage of the fluorogenic substrate from the Dpa quencher was monitored in Tecan infinite M200 plate reader with excitation set at 320 nm and emission set at 405 nm.
  • Mouse peritoneal macrophages cell-based assay of TNF-a secretion inhibition Mouse peritoneal macrophage cells were harvest, 4 days post Thioglycollate injection, and incubated overnight in RPMI 1640, with 10% FBS 2 mM Glutamine, 1 x Pen/strep solution (Biological Industries, Beit-Haemek, Israel) supplemented with 10% heat inactivated FBS (Biological Industries, Beit-Haemek, Israel). Macrophages medium was replaced and 2.5 ng/ml LPS and 1 ⁇ A2 or proTACE were added and incubated for three hours at 37 °C. The macrophages medium was collected and the TNF-a in the medium was determined using mouse T F- ⁇ DueSet ELISA kit (R&D systems) as recommended by the manufacturer.
  • PBL cell-based assay for the inhibition of TLlA-induced IFN- ⁇ secretion PBMC were isolated from blood of normal healthy volunteers using Histopaque®-1077 (Sigma) according to the manufactures instructions. The PBL fraction was isolated following incubation of the PBMC in complete RPMI in a flask at 37 °C for overnight and the non-adherent fraction was designated as PBL.
  • TL1A induced IFN- ⁇ secretion PBL cells were incubated in RPMI containing 10% FBS with IL-12 (2 ng/ml), IL-18 (50 ng/ml), TL1A (200 ng/ml) and A2 or H3 at the indicated concentrations for 48 h.
  • the cultured media was collected and the levels of IFN- ⁇ were quantitated using a commercial ELISA kits (PeproTech) according to manufacturer description.
  • TF-1 cell-based assay for the inhibition of TLlA-induced apoptosis TF-1 cells were seeded at 75,000 cells/well (7.5 x 10 5 /ml) in RPMI medium containing 1% FBS (Biological Industries, Beit-Haemek, Israel), in a black 96-well plate with clear bottom (Greiner bio-one) at a final volume of 100 ⁇ . Next, 100 ng/ml TL1A, 10 ⁇ g/ml cycloheximide and A2, H3 or proTACE were added.
  • FBS Biological Industries, Beit-Haemek, Israel
  • lysis buffer 50 mM HEPES pH 7.35, 1 mM EDTA, 1% NP-40 detergent, 25 ⁇ DEVD-AMC
  • cleavage of DEVD-AMC by caspase-3 was monitored for 60 minutes in a Tecan Infinite M200 plate reader with excitation and emission set at 350 nm and 450 nm, respectively.
  • TF-1 cells (1.5 x 10 5 ) in a volume of 150 ⁇ were incubated with the indicated concentrations of A2, H3 or A2 and proTACE in RPMI 1% FBS for 1 hour at 37 °C. Next, the cells were centrifuged at 200 rcf for 5 minutes at 4 °C and washed three times with 200 ⁇ PBS, 1% BSA. The washed cells were re-suspended in 50 ⁇ PBS containing 1% BSA and a final concentration of 10 ⁇ g/ml goat anti-human IgG Allophycocyanin (APC) conjugate (Jackson immuneresearch) research was added.
  • APC goat anti-human IgG Allophycocyanin
  • the cells were incubated for 30 minutes at RT in the dark and then washed three times with PBS containing 1% BSA.
  • the cells where resuspended in 500 ⁇ of PBS containing 1% BSA and analyzed on a Acuri C6 flow cytometer (BD Biosciences).
  • H3 an engineered DR3 mutant, termed H3, which exhibits improved TL1A binding affinity and higher stability relative to the WT soluble DR3 receptor, was used.
  • H3 and 06 DR3 mutants are more potent in inhibiting TLlA-induced apoptosis in TF-1 cells than the WT DR3 (Fig. 8B).
  • cells were inoculated for six hours with 8 mg/ml cyclohexamide (CHX) and 75 ng/ml TL1A and the indicated concentration of soluble DR3 WT, H3 and 06 receptors.
  • lysis buffer containing the caspase 3 fluorescent substrate DEVD-AMC was added and caspase-3 activity was monitored for 10 minutes.
  • a pTACE domain containing mutations at the natural furin cleavage site was used.
  • the generated bi-specific inhibitor consists of three major domains: the pTACE and H3 that specifically bind TACE and TL1A, respectively, and an Fc domain derived from human IgGl.
  • the role of the Fc domain is to extend the serum half-life of the inhibitor and to promote dimerization of the bi- specific proTACE-H3 inhibitor.
  • the supernatant of HEK293F containing the A2 protein was subjected to a TL1A ELISA binding assay (see Materials and Methods for details). It was found that the secreted A2 exhibits high TL1A binding signal that is dose dependent (Fig. ID).
  • the A2 was purified on Ni-NTA beads and the pure A2 migrated on an SDS-PAGE gel as a band of ⁇ 100kDa, higher than the calculated 70 kDa, suggesting that protein undergo significant posttranslational modifications (Fig. IE).
  • TACE activity involves proteolytic shedding of pro-inflammatory cytokines and growth factors located on the cell membrane.
  • macrophages were shown to respond to LPS by activating TACE cleavage of membrane bound proTNF-a leading to the release of soluble TNF-a.
  • A2 to inhibit TACE T F- ⁇ "shedase" activity
  • the levels of TNF- ⁇ released from mouse peritoneal macrophages were tested in the absence or presence of A2 and pTACE.
  • mouse peritoneal macrophages were stimulated for 3 hours with LPS at a concentration of 2.5 ng/ml with or without 1 DM A2 or proTACE. Media supernatant was analyzed by ELISA for detection of TNF-a levels, the TNF-a values were calculated according to TNF-a calibration curve, *P ⁇ 0.05.
  • Results demonstrated that A2 and pTACE exhibit similar reduction in the level of TNF-a released to the medium compared to control cells (Fig. 3).
  • the cell-based assay was utilized to examine the ability of A2 and a specific soluble DR3 variant ( ⁇ 3') to inhibit TLIA induced IFN- ⁇ secretion by competing with the endogenous DR3 cell surface receptor.
  • ⁇ 3' a specific soluble DR3 variant
  • cells were incubated for 72 hours with 200 ng/ml TLIA, 20 ng/ml IL-12 and 50 ng/ml IL-18 and different concentrations of A2 and H3 inhibitors.
  • a 1: 10 diluted cell supernatant was analyzed by ELISA for detection of IFN- ⁇ levels.
  • the IFN- ⁇ values were calculated according to an IFN- ⁇ calibration curve.
  • Results demonstrate that A2 exhibits more than 3-fold higher potency in inhibiting TL1A induced IFN- ⁇ secretion from PBL relative to H3 monospecific inhibitor (Fig. 4A). These results highlight the great potency of A2 in inhibiting TL1A induced IFN- ⁇ secretion from PBL and indicates a possible synergism between the H3 and pTACE in this assay.
  • TL1A was shown to significantly enhance cyclohexamide (CHX) induced apoptosis in TF-1 cells detected by the increase in caspase-3 activity toward the DEVD- AMC fluorescent substrate (Figs. 4B and 5).
  • CHX cyclohexamide
  • this assay was utilized to demonstrate that the addition of increasing concentrations of H3 led to a potent inhibition of TL1A induced apoptosis in TF-1 cells.
  • this cell -based assay was utilized to compare the potency of A2 and H3 in inhibiting TL1A induced apoptosis of TF1 cells.
  • cells were inoculated for six hours with 8 ⁇ g/ml cyclohexamide (CHX) and 75 ng/ml TL1A and the indicated concentration of A2 and H3 receptors. Following six hours of incubation, lysis buffer containing the caspase-3 fluorescent substrate DEVD-AMC was added, and enzyme activity was monitored for 10 minutes.
  • CHX cyclohexamide
  • Results demonstrated that A2 inhibits TL1A induced apoptosis at a significantly lower concentration than the H3 mono-specific inhibitor (Fig. 4B). Surprisingly, it was demonstrated that while H3 completely inhibits TL1A induced apoptosis at a concentration 244 nM, the A2 led to a complete inhibition at a concentration of 3 nM reflecting a more than 80-fold increase in potency (Fig. 4B).
  • the increased potency of the bi-specific A2 inhibitor relative to the H3 indicates a strong synergism between the H3 and the pTACE domains in A2.
  • One possible mechanism for such synergism is the independent activity of the pTACE domain in preventing TL1A induced apoptosis of the TF-1 cells due to direct TACE inhibition.
  • TACE was reported to be involved in inducing apoptosis in germ cells and neutrophils and its chemical inhibition resulted in reduced apoptosis.
  • A2 inhibits apoptosis through direct TACE inhibition
  • the inventors examined the levels of TL1A induced apoptosis following cell incubation with a wide range of the mono- specific pTACE concentrations. It was found that pTACE did not lead to the inhibition of TL1A induced TF-1 apoptosis under the assay conditions suggesting that the high potency of A2 is not due to direct TACE inhibition (Fig. 5).
  • An alternative mechanism for increasing the potency of A2 is by targeting the bi-specific inhibitor to the cell membrane leading to a significantly enhanced local concentration of the inhibitor on the membrane. To test this hypothesis, the inventors examined the binding of A2 to the TF-1 cell membrane following cell incubation with A2 and H3.
  • Binding was analyzed by cell labeling with allophycocyanin (APC) fluorescent anti-human Fc.
  • APC allophycocyanin
  • Flow cytometry analysis of the labeled cells showed that while H3 binds very weakly to the TF-1 cells, A2 binds cells very efficiently providing direct evidence for the targeting of A2 to the cell membrane (Fig. 6A).
  • APC allophycocyanin
  • TF-1 cells were incubated with A2 and pTACE domain that lacks an Fc region.
  • the resulting cell population was labeled with APC-conjugated anti-Fc antibody and analyzed by flow cytometry.
  • a significant decrease in the mean cell fluorescence was observed, suggesting that pTACE domain can compete with A2 for binding to the cells (Figs. 6B and 6C).
  • the results support a model in which the increased potency of A2 in inhibiting TLIA induced TF-1 apoptosis may be, at least partially, attributed to increased local concentration of the bi-specific inhibitor on the cell membrane (Figs. 7A-B). Since TLIA signals through binding to the endogenous DR3 cell surface receptor, enhanced local concentration of the soluble DR3 domain in A2 on the cell membrane can increase the potency of TLIA inhibition.

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Abstract

L'invention concerne un polypeptide chimère comprenant un premier fragment qui comporte un domaine extracellulaire de récepteur DR3 muté soluble; et un second fragment comprenant un pro-domaine de TACE muté. L'invention concerne en outre des méthodes de prévention, de traitement, ou d'amélioration de l'inflammation.
PCT/IL2018/050214 2017-02-26 2018-02-25 Chimères de récepteur dr3 soluble et de pro-domaine de tace et leur utilisation WO2018154584A1 (fr)

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EP4487700A1 (fr) * 2023-07-04 2025-01-08 Hangzhou BIBAU Biotechnology Co., Ltd. Peptide anti-inflammatoire à base de protéine de blanc d'oeuf et son application

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Cited By (1)

* Cited by examiner, † Cited by third party
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EP4487700A1 (fr) * 2023-07-04 2025-01-08 Hangzhou BIBAU Biotechnology Co., Ltd. Peptide anti-inflammatoire à base de protéine de blanc d'oeuf et son application

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