US20160017020A1 - Chimeric polypeptides, polynucleotides encoding same, cells expressing same and methods of producing same - Google Patents

Chimeric polypeptides, polynucleotides encoding same, cells expressing same and methods of producing same Download PDF

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US20160017020A1
US20160017020A1 US14/773,360 US201414773360A US2016017020A1 US 20160017020 A1 US20160017020 A1 US 20160017020A1 US 201414773360 A US201414773360 A US 201414773360A US 2016017020 A1 US2016017020 A1 US 2016017020A1
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plant
domain
polypeptide
cell
tnfα
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Yoseph Shaaltiel
Uri Hanania
Tali Kizhner
Tami ARIEL
Svetlana GINGIS-VELITSKI
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Protalix Ltd
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Protalix Ltd
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Assigned to PROTALIX LTD. reassignment PROTALIX LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KIZHNER, TALI, HANANIA, URI, SHAALTIEL, YOSEPH, GINGIS-VELITSKI, SVETLANA, ARIEL, Tami
Publication of US20160017020A1 publication Critical patent/US20160017020A1/en
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Priority to US16/045,848 priority patent/US10730925B2/en
Assigned to PROTALIX LTD. reassignment PROTALIX LTD. RELEASE BY SECURED PARTY (SEE DOCUMENT FOR DETAILS). Assignors: WILMINGTON SAVINGS FUND SOCIETY, FSB, AS COLLATERAL AGENT
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Definitions

  • the present invention in some embodiments thereof, relates to chimeric polypeptides, polynucleotides encoding same, cells expressing same and methods of producing same.
  • Tumor necrosis factor alpha is an important, pro-inflammatory cytokine mediating the regulation of diverse inflammatory, infectious and immune-related processes and diseases, TNF ⁇ being considered the most important mediator responsible for inflammatory pathology.
  • TNF-alpha is a 17 kD molecular weight protein, initially synthesized as a transmembrane protein arranged in stable trimers, then cleaved by metalloprotease-TNF alpha converting enzyme (TACE) to form the homotrimeric soluble TNF (sTNF) which engages to its cognate receptors (TNFRI, p55 and TNFRII, p75), expressed ubiquitously.
  • TACE metalloprotease-TNF alpha converting enzyme
  • TNF-alpha induces a wide variety of cellular responses, many of which result in deleterious consequences, such as cachexia (loss of fat and whole body protein depletion, leading to anorexia, common in cancer and AIDS patients) and septic shock. Elevated secretion of TNF-alpha has been implicated in a variety of human diseases including diabetes, allograft rejection, sepsis, inflammatory bowel diseases, osteoporosis, in many autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, psoriasis, psoriatic arthritis, hypersensitivity, immune complex diseases, and even in malaria, cancer and lung fibrosis.
  • TNF ⁇ The biological effect of TNF ⁇ is mediated by the two distinct receptors.
  • TNF-alpha receptors when shed from mononuclear cells, lower the TNF-alpha levels by “mopping up” and acting as natural inhibitors
  • Neutralization of TNF ⁇ by specific antibodies and decoy receptors has become a common strategy for regulation of TNF ⁇ mediated toxicity.
  • TNF ⁇ antagonists have been approved by the US FDA for clinical use: Cimzia (Certolizumab pegol), a TNFmAb Fab′ fragment-PEG conjugate; Remicade (Infliximab), a TNF rmAB; Humira (Adalimumab), a TNF rmAB, SimponiTM (Golimumab), aTNF human monoclonal antibody and etanercept, a fusion protein of soluble 75 kDa TNF ⁇ receptors fused to the Fc fragment of human IgG (registered as EnbrelTM) Etanercept is indicated for rheumatoid arthritis (RA) and other arthritic indications such as juvenile idiopathic arthritis (JIA), psoriasis and Ankylosing Spondylitis (AS).
  • RA rheumatoid arthritis
  • JIA juvenile idiopathic arthritis
  • AS Ankylosing Spondylitis
  • RA Rheumatoid arthritis
  • Clinical trials of Enbrel therapy include such diverse indications as adult respiratory distress syndrome, pemphigus, Alzheimer's disease, Behcet's syndrome, HIV, myocardial infarct, knee joint synovitis, lupus nephritis, lichen planus, systemic amyloidosis, sciatica, vitiligo, chronic fatigue syndrome, anorexia, TMJ, asthma, bronchitis, diabetes, myelodysplastic disease and others.
  • Enbrel is currently produced in mammalian cells.
  • the safety of biopharmaceuticals has recently come to the forefront for both patients and health care providers due to outbreaks of emerging pathogens, most notably HIV, HCV, Cruezfeld-Jacob's Disease, West Nile Virus and SARS, in multiple regions of the world, emphasizing the risk of pathogen transmission through the use of human- or animal-derived raw materials, such as blood-derived products (serum, plasma cell medium components, etc) in the manufacture of biopharmaceuticals.
  • blood-derived products serum, plasma cell medium components, etc
  • Biopharmaceuticals including modified human proteins, can be produced in transgenic plants in order to address problems of safety, viral infections, immune reactions, production yield and cost.
  • U.S. Pat. No. 6,391,638 and PCT WO2008/135991 teach bioreactor devices for commercial-scale production of recombinant polypeptides from plant cell culture.
  • U.S. Pat. No. 7,951,557, U.S. patent application Ser. Nos. 10/554,387 and 11/790,991 teach construction and expression of nucleic acid vectors for recombinant expression of human proteins in plant cells.
  • PCT WO2007/010533 teaches the expression of recombinant human polypeptides in plant cells, for enteral administration.
  • Additional background art includes: U.S. Pat. No. 7,915,225 to Finck et al, U.S. patent application Ser. Nos. 13/021,545 and 10/853,479 to Finck et al, U.S. patent application Ser. No. 11/906,600 to Li et al, U.S. patent application Ser. No. 10/115,625 to Warren et al and U.S. patent application Ser. No. 11/784,538 to Gombotz et al.
  • a first domain which comprises a TNF ⁇ binding domain of a TNF receptor
  • a second domain which comprises an Fc domain of an immunoglobulin, wherein the first domain and the second domain are N-terminally to C-terminally respectively sequentially translationally fused and wherein the chimeric polypeptide specifically binds TNF ⁇ .
  • a first domain which comprises a TNF ⁇ binding domain of a TNF receptor
  • a second domain which comprises an Fc domain of an immunoglobulin
  • a third domain comprising an endoplasmic reticulum retention signal; wherein the first domain, second domain and third domain are N-terminally to C-terminally respectively sequentially translationally fused and wherein the chimeric polypeptide specifically binds TNF ⁇ .
  • the polypeptide comprises an additional domain encoding an endoplasmic reticulum signal peptide translationally fused N-terminally to the first domain.
  • the signal peptide is a plant signal peptide.
  • the plant signal peptide is as set forth in SEQ ID NO: 4.
  • the first domain is 200-250 amino acids long.
  • the first domain comprises the amino acid sequence LCAP (SEQ ID NO: 11) and VFCT (SEQ ID NO: 12).
  • the first domain further comprises the amino acid sequence LPAQVAFXPYAPEPGSTC (SEQ ID NO: 13) or LPAQVAFTPYAPEPGSTC (SEQ ID NO: 17).
  • the first domain is as set forth in SEQ ID NO: 2.
  • the immunoglobulin is IgG 1 .
  • the second domain is as set forth in SEQ ID NO: 9.
  • the polypeptide is as set forth in SEQ ID NO: 6.
  • the polypeptide is as set forth in SEQ ID NO: 7, 204 or 205.
  • the polypeptide is purified to at least 98% homogeneity.
  • the polypeptide is capable of inhibiting TNF ⁇ -induced apoptosis.
  • the polypeptide comprises a plant-specific glycan.
  • the plant-specific glycan is selected from the group consisting of a core xylose and a core ⁇ -(1,3) fucose.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding the polypeptide.
  • a codon usage of the nucleic acid sequence is optimized for Nicotinia tabaccum.
  • nucleic acid expression construct comprising a nucleic acid sequence encoding the polynucleotide and a cis-acting regulatory element active in a plant cell.
  • the cis-acting regulatory element is a promoter.
  • a plant cell comprising the nucleic acid construct.
  • the plant cell is a Nicotiana tabacum plant cell.
  • the Nicotiana tabacum L. cv plant cell is a Bright Yellow (BY-2) cell.
  • the plant cell is lyophilized.
  • a plant cell suspension culture comprising the plant cell.
  • a pharmaceutical composition comprising as an active ingredient the polypeptide and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising as an active ingredient the plant cell and a pharmaceutically acceptable carrier.
  • a method of treating a TNF ⁇ -associated medical condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the polypeptide, thereby treating the TNF ⁇ -associated medical condition in the subject.
  • a method of treating a TNF ⁇ -associated medical condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the plant cells, thereby treating the TNF ⁇ -associated medical condition in the subject.
  • the polypeptide for use in treating a TNF ⁇ -associated medical condition in a subject.
  • the plant cells are for use in treating a TNF ⁇ -associated medical condition in a subject.
  • polypeptide in treating a TNF ⁇ -associated medical condition in a subject.
  • the medical condition is an inflammatory disease.
  • the medical condition is an autoimmune disease.
  • the medical condition is selected from the group consisting of rheumatoid arthritis, ankylosing spondyloarthritis, plaque psoriasis and juvenile idiopathic arthritis.
  • the medical condition is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, short bowel syndrome, sepsis, endotoxic shock, AIDS, endometriosis, psoriasis, cardiovascular disease, cancer, vitiligo, arthritis, rheumatoid polyarthritis, psoriatic rheumatism, ankylosing spondyloarthritis, plaque psoriasis, juvenile idiopathic arthritis, polyarticular juvenile idiopathic arthritis, psoriasis arthritis, Wegener's disease (granulomatosis), Crohn's disease, short bowel syndrome, ulcerative cholitis, chronic obstructive pulmonary disease (COPD), Hepatitis C, asthma, cachexia, atopic dermatitis.
  • COPD chronic obstructive pulmonary disease
  • Alzheimer's disease hepatic encephalopathy, ADHD, chronic fatigue syndrome dermatitis herpetiformis (Duhring's disease), contact dermatitis, urticaria (including chronic idiopathic urticaria), autoimmune blistering diseases, including pemphigus vulgaris, bullous pemphigoid, myesthenia gravis, sarcoidosis, including pulmonary sarcoidosis, scleroderma, reactive arthritis, hyper IgE syndrome, multiple sclerosis and idiopathic hypereosinophil syndrome, and allergy.
  • urticaria including chronic idiopathic urticaria
  • autoimmune blistering diseases including pemphigus vulgaris, bullous pemphigoid, myesthenia gravis
  • sarcoidosis including pulmonary sarcoidosis, scleroderma, reactive arthritis, hyper IgE syndrome, multiple sclerosis and idiopathic hypereosinophil syndrome, and allergy.
  • the medical condition is an inflammatory bowel disease.
  • the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
  • the plant cells are formulated for oral administration.
  • the polypeptide is formulated for parenteral administration.
  • the plant cells are formulated for enteral administration and wherein the medical condition is not an obesity, metabolic syndrome, diabetes and a liver disease or disorder.
  • the method further comprises isolating the polypeptide from the cell.
  • the cell is an isolated cell cultured in a plant cell culture medium.
  • the culturing is performed in a disposable bioreactor.
  • FIG. 1 is a schematic illustration of the amino acid sequence of plant recombinant human (prh) TNFR2:Fc (also termed herein PRX-106, SEQ ID NO:6).
  • prh TNFR2:Fc cDNA for expression in BY2 cells was assembled with a signal peptide for targeting the fusion polypeptide composed of the TNF-binding moiety of the TNF receptor and FC protein to the secretory pathway.
  • Colour code for the amino acids sequence the signal peptide is coloured in yellow; the TNF receptor portion is coloured in black (green); the Fc portion of IgG1 is in blue; ER retention signal in red.
  • FIGS. 2A-B show comparison of PRH TNFR2:FC and Commercial Enbrel by Western-blot.
  • prh TNFR2:Fc (lane 1) and commercial Enbrel (lane 2) were analyzed under reducing conditions (panel A) and non-reducing conditions (panel B) by 12% and 8% Tris-Glycine SDS-PAGE, respectively.
  • Membranes were blotted with an anti FC antibody (upper panel) and with an anti TNFR2 antibody (lower panel). Molecular weight marker is shown in right lanes.
  • Lane 2 commercial Enbrel.
  • FIG. 3 is a graph showing TNF ⁇ binding by prh TNFR2:Fc and commercial Enbrel by quantitative non radioactive assay for prh TNFR2:Fc binding activity and molecular integrity.
  • An ELISA plate pre-coated with antibodies against TNF ⁇ was incubated with TNF ⁇ followed by exposure to commercial Enbrel and supernatant from BY2 cells expressing prh TNFR2:Fc. Serial dilutions of both tested items are shown in the X axis. Fc portion of the molecule was detected with Goat anti human IgG Fc HRP.
  • FIG. 4 is an image showing screening of individual cell lines for expression of prh TNFR2:Fc by Western blot analysis.
  • FIGS. 5A-F are images showing TNF ⁇ cytotoxicity in A375 cells in the presence of prh TNFR2:Fc or commercial Enbrel by MTT viability assay.
  • FIG. 5 A untreated Cultured A375 cells
  • FIG. 5 B treated with TNF ⁇
  • FIG. 5 C TNF ⁇ exposed cells treated with prh TNFR2:Fc (3.125 ng/ml)
  • FIG. 5 D TNF ⁇ exposed cells treated with commercial Enbrel (3.125 ng/ml)
  • FIG. 5 E TNF ⁇ exposed cells treated with prh TNFR2:Fc (100 ng/ml)
  • FIG. 5 F TNF ⁇ exposed cells treated with commercial Enbrel (100 ng/ml).
  • FIG. 5G is a bar graph showing TNF ⁇ cytotoxicity in A375 cells in the presence of prh TNFR2:Fc or commercial Enbrel by MTT viability assay.
  • FIGS. 6A-F are images showing TNF ⁇ cytotoxicity in L929 cells in the presence of prh TNFR2:Fc or commercial Enbrel by MTT viability assay.
  • FIG. 6 A untreated Cultured L929 cells
  • FIG. 6 B treated with TNF ⁇
  • FIG. 6 C TNF ⁇ exposed cells treated with prh TNFR2:Fc (3.125 ng/ml)
  • FIG. 6 D TNF ⁇ exposed cells treated with commercial Enbrel (3.125 ng/ml)
  • FIG. 6 E TNF ⁇ exposed cells treated with prh TNFR2:Fc (100 ng/ml)
  • FIG. 6 F TNF ⁇ exposed cells treated with commercial Enbrel (100 ng/ml).
  • FIG. 6G is a bar graph showing TNF ⁇ cytotoxicity in L929 cells in the presence of prh TNFR2:Fc or commercial Enbrel by MTT viability assay.
  • FIGS. 7A-B are graphs showing body weight changes following TNBS challenge. Mice were orally administered with prh TNFR2:Fc 6 hours after TNBS induction, For four consecutive days body weights were determined daily (means ⁇ SE).
  • FIG. 7 A Average weight loss at day four following treatment with TNBS, presented in % loss from original weight.
  • FIG. 7 B Average weight loss during four days following treatment with TNBS, presented in % loss from original weight. Note oral treatment with plant cells expressing recombinant TNFR2:Fc attenuated body weight reduction.
  • FIG. 8 is a bar graph showing that oral administration of plant cells expressing TNFR2:Fc inhibits TNBS-induced colonic shorting.
  • the mice with colitis were orally administered with plant cells expressing TNFR2:Fc for four consecutive days after TNBS induction and the colon lengths were determined at day 4 (means ⁇ SE).
  • FIG. 9 is a bar graph showing that oral administration of plant cells expressing TNFR2:Fc improved the macroscopic futures of TNBS-induced colitis.
  • Total macroscopic inflammation scores (Wallace score) in control and treated rats at the end-point of the experiment (means ⁇ SE).
  • FIGS. 10A-C are bar graphs showing serum cytokine content in mice treated by oral administration of plant cells expressing TNFR2:Fc as measured by a cytokine antibody array.
  • FIGS. 11A-B show serum cytokine content by cytokine Antibody array.
  • FIG. 11 B Cytokine quantification of array. Results indicate that treatment with PRX-106 reduced level of inflammatory mediators like granulocyte colony-stimulating factor G-CSF, macrophage colony-stimulating factor (M-CSF), potentially indicating reduced systemic inflammation by lowering systemic recruitment of bone marrow derived cells from the bloodstream.
  • M-CSF macrophage colony-stimulating factor
  • FIG. 12 is a bar graph showing expansion of splenic Treg population in animals treated with plant cells expressing recombinant TNFR2:Fc. Spleen of Balb/c mice, treated with PRX-106 during TNBS induced colitis were analyzed for the percentages of CD4+CD25+Foxp3+, bars indicate SE.
  • FIGS. 13A-B are graphs showing body weight changes following DSS challenge.
  • the mice with colitis were orally administered with plant cells expressing TNFR2:Fc for seven consecutive days 24 hours after DSS induction and the body weights of mice were determined (means ⁇ SE).
  • FIG. 13 A Average weight loss at day ten following treatment with DSS, presented in % loss from original weight.
  • FIGS. 14A-B show that oral administration of TNFR2:Fc inhibited DSS-induced colonic shorting.
  • the mice with colitis were orally administered with plant cells expressing recombinant TNFR2:Fc for 7 consecutive days after DSS induction and the colon lengths were determined at day 10 (means ⁇ SE).
  • FIG. 14 B Representative photograph of colons, ten days after the induction of DSS colitis.
  • FIGS. 16A-C are graphic presentations of serum cytokine content assayed by cytokine Antibody array.
  • FIGS. 17A-B show that therapeutic treatment with orally administered plant cells expressing recombinant TNFR2:Fc reduces the severity of DSS-induced colitis.
  • FIG. 17 A Representative histological sections were examined microscopically after H&E staining with magnification ⁇ 40 and ⁇ 100. The images are representative of at least seven mice per group.
  • FIG. 17 B The effect of orally administered plant cells expressing recombinant TNFR2:FC on histological colitis score was determined.
  • FIG. 19 is a bar graph showing pharmacokinetics of TNFR2:Fc in rat sera following oral administration of plant cells expressing recombinant TNFR2:Fc by gavage.
  • Negative controls received the same volumes of host BY2( ⁇ ) plant.
  • FIG. 20 shows the PRX-106 sequence (SEQ ID NO: 6) elucidated by mass-spec (green shown 84.8% coverage).
  • the present invention in some embodiments thereof, relates to chimeric polypeptides, polynucleotides encoding same, cells expressing same and methods of producing same.
  • Etanercept is a tumor necrosis factor (TNF) blocker indicated for a number of inflammatory conditions such as rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, plaque psoriasis, psoriatic arthritis and ankylosing spondylitis.
  • Etanercept is produced by recombinant DNA technology in Chinese hamster ovary mammalian cell expression system. The production of recombinant proteins in mammalian cell systems is hampered by cellular fragility and the complex nutritional requirements of cells and the possible contamination of the final product with virus or prions.
  • the present inventors Whilst reducing the present invention to practice, the present inventors have constructed an expression vector for recombinant expression of Enteracept (hereinafter, prh TNFR2:Fc) in plant cells, transformed tobacco cells with the vector, and have isolated catalytically active protein from the cell cultures.
  • the expressed recombinant protein retains its TNF ⁇ binding activity and has shown favorable catalytic activity as evidenced by its apoptosis regulatory activity.
  • prh TNFR2 is biologically active as an anti-inflammatory agent.
  • the present results further support a role for orally administered plant cells expressing recombinant TNFR2:Fc as an anti-inflammatory agent with the capacity to ameliorate IBD.
  • Example 4 show that oral administration of plant cells expressing prh TNFR2:Fc is safe and well tolerated.
  • a plant produced chimeric polypeptide comprising:
  • a first domain which comprises a TNF ⁇ binding domain of a TNF receptor
  • a second domain which comprises an Fc domain of an immunoglobulin, wherein the first domain and the second domain are N-terminally to C-terminally respectively sequentially translationally fused and wherein the chimeric polypeptide specifically binds TNF ⁇ .
  • plant produced refers to the chemical signature associated with plant expression, including, but not limited to, host cell impurities in the preparation which comprises the chimeric polypeptide and glycosylation patterns on the chimeric polypeptide per se.
  • chimeric polypeptide refers to a protein created through the joining of two or more individual coding sequences which originally code for separate proteins. Translation of the synthetic (non-naturally occurring) nucleic acid sequence results in a single chimeric polypeptide with functional properties derived from each of the original proteins. Such recombinant fusion proteins are created artificially by recombinant DNA technology.
  • TNF ⁇ refers to Tumor necrosis factor-alpha (TNF, cachexin, or cachectin) that is a cytokine involved in systemic inflammation and a member of a group of cytokines that stimulate the acute phase reaction.
  • TNF ⁇ is produced primarily by activated macrophages (M1), although it can be produced by many other cell types as CD4+ lymphocytes, NK cells and neurons.
  • M1 activated macrophages
  • the protein is encoded by TNFA gene and has the Ref_seq number: NP — 000585. The protein is known to stimulate an inflammatory response (pro-inflammatory cytokine).
  • TNF receptor or “TNFR” refers to a polypeptide which is capable of binding TNF ⁇ in a specific manner e.g., Kd below 10 ⁇ 5 M. According, to a specific embodiment the TNFR is membrane bound.
  • the first domain is thus composed of at least the TNF binding domain of a TNF receptor (TNFR).
  • the first domain is a soluble protein.
  • the first domain and even the entire chimeric polypeptide are soluble proteins which are not membrane anchored.
  • Soluble forms of TNFRs may include monomers, fusion proteins (also called “chimeric proteins), dimers, trimers or higher order multimers.
  • the soluble TNFR derivative is one that mimics the 75 kDa TNFR or the 55 kDa TNFR and that binds to TNF ⁇ . in vivo.
  • the soluble TNFR mimics of the present invention may be derived from TNFRs p55 or p75 or fragments thereof.
  • TNFRs other than p55 and p75 also are useful for deriving soluble TNFR for treating the various medical disorders described herein, such for example the TNFR that is described in WO 99/04001.
  • Soluble TNFR molecules used to construct TNFR mimics include, for example, analogs or fragments of native TNFRs having at least 20 amino acids, that lack the transmembrane region of the native TNFR, and that are capable of binding TNF ⁇ .
  • Such soluble forms of TNFR compete for TNF ⁇ with the receptors on the cell surface, thus inhibiting TNF ⁇ from binding to cells, thereby preventing it from manifesting its biological activities. Binding of soluble TNFRs to TNF ⁇ can be assayed using ELISA or any other convenient assay.
  • the first domain is derived from TNFR2. (e.g., AAA36755).
  • the first domain is 200-250 amino acids long.
  • the first domain comprises the amino acid sequence LCAP (SEQ ID NO: 11) and VFCT (SEQ ID NO: 12).
  • the first domain comprises the amino acid sequence LPAQVAFXPYAPEPGSTC (SEQ ID NO: 13), or LPAQVAFTPYAPEPGSTC (SEQ ID NO: 17).
  • the first domain is as set forth in SEQ ID NO: 2 (encoded by SEQ ID NO: 1).
  • an Fc domain of an immunoglobulin refers to a region of a heavy chain of an antibody, typically comprising at least 2 constant domains (e.g., CH2 and CH3 domains, as these terms are defined in the art) of the heavy chain.
  • the Fc domain may be obtained, for example, in the form of a dimer, by digestion of an antibody by papain.
  • Fc domains of some classes of antibodies may be in the form of multimers.
  • the Fc domain is optionally monomeric, optionally dimeric and optionally multimeric.
  • the polypeptide described herein is in the form of a dimer, the polypeptide comprising an Fc dimer, or in the form of a multimer, the polypeptide comprising an Fc multimer.
  • the Fc domain may encompass modified forms of a native Fc domain (i.e., a domain which occurs naturally in an antibody), for example, polypeptides having at least 90% homology, optionally at least 95% homology, and optionally at least 98% homology, to a native Fc domain.
  • modified Fc domains are described, for example, in International Patent Applications WO 97/34631 and WO 96/32478.
  • a native Fc is modified so as to remove sites which provide structural features or biological activity that are not required for embodiments of the present invention.
  • sites include residues that affect or are involved in disulfide bond formation, incompatibility with a selected host cell, N-terminal heterogeneity upon expression in a selected host cell, glycosylation, interaction with complement, binding to an Fc receptor (other than a neonatal Fc receptor), and/or antibody-dependent cellular cytotoxicity.
  • the polypeptide according to embodiments of the present invention may also comprise a fragment of an Fc domain.
  • the fragment comprises at least 20%, optionally at least 50%, and optionally at least 80% of an Fc domain, as defined hereinabove.
  • the Fc domain or fragment thereof optionally includes a binding site for a neonatal Fc receptor (FcRn). This is of particular significance when administering the chimeric polypeptide via an enteral route.
  • FcRn neonatal Fc receptor
  • attachment of an Fc domain or a fragment thereof to the first domain results in a polypeptide having a longer half-life in vivo than the first domain per se. This may be due to the long serum half-life of the Fc domain (which may be due to salvage of the Fc via binding to FcRn) and/or due to the greater size of the polypeptide in comparison to the first domain per se, which reduces clearance from the bloodstream by glomerular filtration.
  • the resulting polypeptides have reduced immunogenicity as compared to the first domain per se.
  • the Fc domain or fragment thereof is a human Fc domain (e.g., derived from a human antibody) or fragment thereof.
  • the Fc domain (or fragment thereof) is an IgG (e.g., IgG1) Fc domain (or fragment thereof).
  • the second domain is as set forth in SEQ ID NO: 9 (encoded by SEQ ID NO: 8).
  • the second domain of the chimeric polypeptide comprises at least a portion of a constant immunoglobulin domain, e.g. a constant heavy immunoglobulin domain or a constant light immunoglobulin domain.
  • the second domain comprises at least a portion of a constant heavy immunoglobulin domain.
  • the constant heavy immunoglobulin domain is preferably an Fc fragment comprising the CH2 and CH3 domain and, optionally, at least a part of the hinge region.
  • the immunoglobulin domain may be an IgG, IgM, IgD or IgE immunoglobulin domain or a modified immunoglobulin domain derived, therefrom.
  • the second domain comprises at least a portion of a constant IgG immunoglobulin domain.
  • the IgG immunoglobulin domain may be selected from IgG1, IgG2, IgG3 of IgG4 domains or from modified domains such as are described in U.S. Pat. No. 5,925,734.
  • the immunoglobulin domain may exhibit effector functions. In some embodiments, however, modified immunoglobulin domains having modified, e.g. at least partially deleted, effector functions may be used. Thus for example, the receptor.
  • the chimeric fusion of the first domain and the second domain forms Etanercept (Immunex) having SEQ ID NO: 10.
  • the species origin of the first domain and the second domain is selected according to the treated subject.
  • the first domain and the second domain are of human origin or modified such that they don't incur immunogenic reaction when administered to human subjects.
  • Etanercept and “EnbrelTM” are interchangeably used to designate the commercially available TNFR2:Fc by Immunex Corporation.
  • Etanercept is a dimeric fusion polypeptide consisting of the extracellular ligand-binding portion of the human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1.
  • TNFR tumor necrosis factor receptor
  • the Fc component of etanercept contains the constant heavy 2 (CH2) domain, the constant heavy 3 (CH3) domain and hinge region, but not the constant heavy 1 (CH1) domain of human IgG1.
  • a first domain which comprises a TNF ⁇ binding domain of a TNF receptor
  • a second domain which comprises an Fc domain of an immunoglobulin
  • a third domain comprising an endoplasmic reticulum retention signal; wherein the first domain, second domain and third domain are N-terminally to C-terminally respectively sequentially translationally fused and wherein the chimeric polypeptide specifically binds TNF ⁇ .
  • the chimeric protein is expressed such that it is retained in the endoplasmic reticulum (ER).
  • ER endoplasmic reticulum
  • at least a portion (e.g., 20% or more) of the TNFR2:Fc molecules in the cell are retained in the ER.
  • the term “endoplasmic reticulum retention signal peptide” refers to a peptide sequence which, when present at the N- or C-terminus of a polypeptide, causes the polypeptide to be retrieved from the Golgi apparatus, and retained in the endoplasmic reticulum (see Rayon et al. Journal of Experimental Botany, Vol. 49, No. 326, pp. 1463-1472, 1998; and Neumann, et al Annals of Botany, 2003; 92:167-180).
  • the endoplasmic reticulum retention signal peptide is HDEL (SEQ ID NO: 14), KDEL (SEQ ID NO: 15) or SEKDEL (SEQ ID NO: 16).
  • first domain and second domain are N-terminally to C-terminally respectively sequentially translationally fused.
  • first domain is located N-terminally to the second domain (the carboxy terminus of the first domain is translationally fused to the N-terminus of the second domain)
  • second domain is located N-terminally of the third domain (the carboxy terminus of the second domain is translationally fused to the N-terminus of the third domain).
  • the second domain is practically sandwiched by the first domain at the N-terminus and the third domain at the C-teminus Schematic presentation is as follows: first domain>second domain(>third domain) are orderly oriented from the N-terminus to the C-terminus (see FIG. 1 ).
  • the linkage between the domains may be direct or indirect by the use of linkers such as peptide linkers.
  • the molecule may further comprise an additional domain which encodes for an endoplasmic reticulum signal sequence which is oriented upstream (N-terminally) of the first domain and translationally fused thereto.
  • an endoplasmic reticulum (ER) signal peptide refers to a signal sequence, leader sequence or leader peptide that is a short (e.g., 5-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway.
  • the ER signal peptide is derived (taken) from a plant protein.
  • the endoplasmic reticulum signal peptide is from N. plumbaginifolia Calreticulin protein.
  • the signal peptide from N. plumbaginifolia Calreticulin protein is as set forth in SEQ ID NO: 4 and encoded by the nucleic acid sequence of SEQ ID NO: 3.
  • translationally fused at the N-terminal or “translationally fused at the C-terminal” refers to covalent attachment of the indicated peptide via a peptide bond to the N-terminal or C-terminal amino acid of the respective domain typically as a result of recombinant expression.
  • the chimeric polypeptide is as set forth in SEQ ID NO: 6.
  • the chimeric polypeptide is as set forth in SEQ ID NO: 7, 204 or 205.
  • the recombinant chimeric proteins of the invention are produced in plant cells.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding the chimeric polypeptide as described herein is ligated into a “plant nucleic acid expression construct”.
  • plant nucleic acid expression construct refers to a nucleic acid construct which includes the nucleic acid of some embodiments of the invention and at least one promoter for directing transcription of nucleic acid in a host plant cell. Further details of suitable transformation approaches are provided hereinbelow.
  • nucleic acid expression construct comprising the nucleic acid sequence of the invention, and a promoter for directing transcription of the nucleic acid sequence in a plant host cell.
  • nucleic acid sequence 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. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent 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 therebetween.
  • the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.
  • the nucleic acid sequences encoding the polypeptides of the present invention are optimized for expression in plants.
  • sequence modifications include, but are not limited to, an altered G/C content to more closely approach that typically found in the plant species of interest, and the removal of codons atypically found in the plant species commonly referred to as codon optimization.
  • codon optimization the codon usage of the nucleic acid sequence encoding the chimeric polypeptide is optimized for Nicotiana tabacuum or Nicotiana benthamiana.
  • an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified in order to utilize statistically-preferred or statistically-favored codons within the plant.
  • the nucleotide sequence typically is examined at the DNA level and the coding region optimized for expression in the plant species determined using any suitable procedure, for example as described in Sardana et al. (1996, Plant Cell Reports 15:677-681).
  • the standard deviation of codon usage may be calculated by first finding the squared proportional deviation of usage of each codon of the native gene relative to that of highly expressed plant genes, followed by a calculation of the average squared deviation.
  • a table of codon usage from highly expressed genes of dicotyledonous plants has been compiled using the data of Murray et al. (1989, Nuc Acids Res. 17:477-498).
  • Codon Usage Database contains codon usage tables for a number of different species, with each codon usage table having been statistically determined based on the data present in Genbank.
  • a naturally-occurring nucleotide sequence encoding a protein of interest can be codon optimized for that particular plant species. This is effected by replacing codons that may have a low statistical incidence in the particular species genome with corresponding codons, in regard to an amino acid, that are statistically more favored.
  • one or more less-favored codons may be selected to delete existing restriction sites, to create new ones at potentially useful junctions (5′ and 3′ ends to add signal peptide or termination cassettes, internal sites that might be used to cut and splice segments together to produce a correct full-length sequence), or to eliminate nucleotide sequences that may negatively affect mRNA stability or expression.
  • the desired encoding nucleotide sequence may already, in advance of any modification, contain a number of codons that correspond to a statistically-favored codon in a particular plant species. Therefore, codon optimization of the native nucleotide sequence may comprise determining which codons, within the desired nucleotide sequence, are not statistically-favored with regards to a particular plant, and modifying these codons in accordance with a codon usage table of the particular plant to produce a codon optimized derivative.
  • a modified nucleotide sequence may be fully or partially optimized for plant codon usage provided that the protein encoded by the modified nucleotide sequence is produced at a level higher than the protein encoded by the corresponding naturally occurring or native gene. Construction of synthetic genes by altering the codon usage is described in for example PCT Patent Application 93/07278.
  • Nicotinia tabaccum optimized sequence as set forth in SEQ ID NO: 5.
  • the nucleic acid sequence coding for the chimeric polypeptide is operably linked to a cis-acting regulatory sequence active in plant cells, such as a plant promoter sequence.
  • a coding nucleic acid sequence is “operably linked” to a regulatory sequence (e.g., promoter) if the regulatory sequence is capable of exerting a regulatory effect on (e.g. effect on the expression of) the coding sequence linked thereto.
  • a regulatory sequence e.g., promoter
  • any suitable promoter sequence can be used by the nucleic acid construct of the present invention.
  • the promoter is a constitutive promoter, a tissue-specific, or an inducible promoter.
  • plant-expressible refers to a promoter sequence, including any additional regulatory elements added thereto or contained therein, is at least capable of inducing, conferring, activating or enhancing expression in a plant cell, tissue or organ, preferably a monocotyledonous or dicotyledonous plant cell, tissue, or organ.
  • a promoter can be constitutive, i.e., capable of directing high level of gene expression in a plurality of tissues, tissue specific, i.e., capable of directing gene expression in a particular tissue or tissues, inducible, i.e., capable of directing gene expression under a stimulus, or chimeric, i.e., formed of portions of at least two different promoters.
  • the nucleic acid construct of some embodiments of the invention can further include an appropriate selectable marker and/or an origin of replication.
  • the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible with propagation in cells.
  • the construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
  • the nucleic acid construct of some embodiments of the invention can be utilized to stably or transiently transform plant cells.
  • stable transformation the nucleic acid is integrated into the plant genome and as such it represents a stable and inherited trait.
  • transient transformation the exogenous polynucleotide is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.
  • an isolated cell comprising the nucleic acid construct of the invention.
  • the term “isolated cell” refers to a cell at least partially separated from the natural environment e.g., from a plant.
  • the isolated cell is a plant cell of a whole plant.
  • the isolated cell is a plant cell, for example, a plant cell in culture.
  • plant encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), and plant cells, tissues and organs.
  • the plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores.
  • Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroe
  • the plant or plant cell is a duckweed plant, cell or nodule.
  • Duckweed members of the monocotyledonous family Lemnaceae, or Lemna
  • Duckweed nodule cultures can be efficiently transformed with an expression cassette containing a nucleotide sequence of interest by any one of a number of methods including Agrobacterium -mediated gene transfer, ballistic bombardment, or electroporation.
  • Methods for molecular engineering of duckweed cells and detailed description of duckweed expression systems useful for commercial production of polypeptides are known in the art (see, for example, U.S. Pat. Nos. 6,040,498 and 6,815,184 to Stomp, et al, and U.S. Pat. No. 8,022,270 to Dickey et al, all of which are incorporated fully by reference herein).
  • the plant or plant cell used by the method of the invention is a crop plant or cell of a crop plant such as rice, maize, wheat, barley, peanut, potato, sesame, olive tree, palm oil, banana, soybean, sunflower, canola, sugarcane, alfalfa, millet, leguminosae (bean, pea), flax, lupinus , rapeseed, tobacco, poplar and cotton.
  • a crop plant such as rice, maize, wheat, barley, peanut, potato, sesame, olive tree, palm oil, banana, soybean, sunflower, canola, sugarcane, alfalfa, millet, leguminosae (bean, pea), flax, lupinus , rapeseed, tobacco, poplar and cotton.
  • the plant cells includes tobacco cells, Agrobacterium rhizogenes transformed root cell, celery cell, ginger cell, horseradish cell and carrot cells.
  • the tobacco cells are from a tobacco cell line, such as, but not limited to Nicotiana tabacum L. cv Bright Yellow (BY-2) cells.
  • the plant cells may be grown according to any type of suitable culturing method, including but not limited to, culture on a solid surface (such as a plastic culturing vessel or plate for example) or in suspension. It will be noted that some cells, such as the BY-2 and carrot cells can be cultured and grown in suspension.
  • Suitable devices and methods for culturing plant cells in suspension are known in the art, for example, as described in International Patent Application PCT IL2008/000614.
  • the cells are cells of whole tobacco plants or plant tissues, including, but not limited to Nicotiana benthamiana.
  • the Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. See, e.g., Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.
  • DNA transfer into plant cells There are various methods of direct DNA transfer into plant cells.
  • electroporation the protoplasts are briefly exposed to a strong electric field.
  • microinjection the DNA is mechanically injected directly into the cells using very small micropipettes.
  • microparticle bombardment the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.
  • Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein.
  • the new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant.
  • Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant.
  • the advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.
  • Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages.
  • the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening.
  • stage one initial tissue culturing
  • stage two tissue culture multiplication
  • stage three differentiation and plant formation
  • stage four greenhouse culturing and hardening.
  • stage one initial tissue culturing
  • the tissue culture is established and certified contaminant-free.
  • stage two the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals.
  • stage three the tissue samples grown in stage two are divided and grown into individual plantlets.
  • the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that it can be grown in the natural environment.
  • the transgenic plants are generated by transient transformation of leaf cells, meristematic cells or the whole plant.
  • Transient transformation can be effected by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.
  • Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, Tobacco mosaic virus (TMV), brome mosaic virus (BMV) and Bean Common Mosaic Virus (BV or BCMV). Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (bean golden mosaic virus; BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants are described in WO 87/06261.
  • the virus used for transient transformations is avirulent and thus is incapable of causing severe symptoms such as reduced growth rate, mosaic, ring spots, leaf roll, yellowing, streaking, pox formation, tumor formation and pitting.
  • a suitable avirulent virus may be a naturally occurring avirulent virus or an artificially attenuated virus.
  • Virus attenuation may be effected by using methods well known in the art including, but not limited to, sub-lethal heating, chemical treatment or by directed mutagenesis techniques such as described, for example, by Kurihara and Watanabe (Molecular Plant Pathology 4:259-269, 2003), Gal-on et al. (1992), Atreya et al. (1992) and Huet et al. (1994).
  • Suitable virus strains can be obtained from available sources such as, for example, the American Type culture Collection (ATCC) or by isolation from infected plants. Isolation of viruses from infected plant tissues can be effected by techniques well known in the art such as described, for example by Foster and Tatlor, Eds. “Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)”, Humana Press, 1998. Briefly, tissues of an infected plant believed to contain a high concentration of a suitable virus, preferably young leaves and flower petals, are ground in a buffer solution (e.g., phosphate buffer solution) to produce a virus infected sap which can be used in subsequent inoculations.
  • a buffer solution e.g., phosphate buffer solution
  • the virus When the virus is a DNA virus, suitable modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsidate the viral DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.
  • a plant viral polynucleotide in which the native coat protein coding sequence has been deleted from a viral polynucleotide, a non-native plant viral coat protein coding sequence and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral polynucleotide, and ensuring a systemic infection of the host by the recombinant plant viral polynucleotide, has been inserted.
  • the coat protein gene may be inactivated by insertion of the non-native polynucleotide sequence within it, such that a protein is produced.
  • the recombinant plant viral polynucleotide may contain one or more additional non-native subgenomic promoters.
  • Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or polynucleotide sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters.
  • Non-native (foreign) polynucleotide sequences may be inserted adjacent the native plant viral subgenomic promoter or the native and a non-native plant viral subgenomic promoters if more than one polynucleotide sequence is included.
  • the non-native polynucleotide sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.
  • a recombinant plant viral polynucleotide is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non-native coat protein coding sequence.
  • a recombinant plant viral polynucleotide in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral polynucleotide.
  • the inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters.
  • Non-native polynucleotide sequences may be inserted adjacent the non-native subgenomic plant viral promoters such that the sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.
  • a recombinant plant viral polynucleotide is provided as in the third embodiment except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.
  • the viral vectors are encapsidated by the coat proteins encoded by the recombinant plant viral polynucleotide to produce a recombinant plant virus.
  • the recombinant plant viral polynucleotide or recombinant plant virus is used to infect appropriate host plants.
  • the recombinant plant viral polynucleotide is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) (exogenous polynucleotide) in the host to produce the desired protein.
  • polynucleotide of the present invention can also be introduced into a chloroplast genome thereby enabling chloroplast expression.
  • a technique for introducing exogenous nucleic acid sequences to the genome of the chloroplasts involves the following procedures. First, plant cells are chemically treated so as to reduce the number of chloroplasts per cell to about one. Then, the exogenous polynucleotide is introduced via particle bombardment into the cells with the aim of introducing at least one exogenous polynucleotide molecule into the chloroplasts.
  • the exogenous polynucleotides selected such that it is integratable into the chloroplast's genome via homologous recombination which is readily effected by enzymes inherent to the chloroplast.
  • the nucleic acid sequence includes, in addition to a gene of interest, at least one polynucleotide stretch which is derived from the chloroplast's genome.
  • the exogenous polynucleotide includes a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the chloroplast genomes following such selection will include the exogenous polynucleotide. Further details relating to this technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which are incorporated herein by reference.
  • a polypeptide can thus be produced by the protein expression system of the chloroplast and become integrated into the chloroplast's inner membrane.
  • the method further comprises growing the plant cell expressing the nucleic acid.
  • the plant cells can be any plant cells desired.
  • the plant cells can be cultured cells, cells in cultured tissue or cultured organs, or cells in a plant.
  • the plant cells are cultured cells, or cells in cultured tissue or cultured organs.
  • the plant cells are any type of plant that is used in gene transference.
  • the plant cell can be grown as part of a whole plant, or, alternatively, in plant cell culture.
  • the plant cells are grown in a plant cell suspension culture.
  • the term “suspension culture” refers to the growth of cells separate from the organism. Suspension culture can be facilitated via use of a liquid medium (a “suspension medium”). Suspension culture can refer to the growth of cells in liquid nutrient media.
  • Methods and devices suitable for growing plant cells of the invention in plant cell suspension culture are described in detail in, for example, PCT WO2008/135991, U.S. Pat. No. 6,391,683, U.S. patent application Ser. No. 10/784,295; International Patent Publications PCT Nos. WO2004/091475, WO2005/080544 and WO 2006/040761, all of which are hereby incorporated by reference as if fully set forth herein.
  • the invention encompasses plants or plant cultures expressing the nucleic acid sequences, so as to produce the recombinant chimeric polypeptide of the invention.
  • the level of the chimeric polypeptide encoded by the nucleic acid sequence can be determined by methods well known in the art such as, activity assays, Western blots using antibodies capable of specifically binding the chimeric polypeptide (anti TNFR2, and anti Fc, See Examples section which follows), Enzyme-Linked Immuno Sorbent Assay (ELISA), radio-immuno-assays (RIA), immunohistochemistry, immunocytochemistry, immunofluorescence and the like.
  • RNA-in situ hybridization Methods of determining the level in the plant of the RNA transcribed from the nucleic acid sequence are well known in the art and include, for example, Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR) analysis (including quantitative, semi-quantitative or real-time RT-PCR) and RNA-in situ hybridization.
  • RT-PCR reverse transcription polymerase chain reaction
  • the expressed recombinant chimeric polypeptide of the present invention is glycosylated in the plant cell, resulting in a chimeric polypeptide having one, or two or three or more glycan structures having plant specific glycan residues.
  • the cells expressing the expression vector of the invention produce a chimeric polypeptide having various amounts of glycan structures arranged in one, two, three or more antennae. All structures may contain a core structure of two GlcNAcs and one mannose, and variations of different amounts of mannose, in addition to core alpha (1,3) fucose, beta (1,2) xylose, and/or GlcNAc residues.
  • Structures can be of the high mannose type, having at least one, optionally at least two, optionally at least three or optionally at least four or more mannose residues in addition to the core structure; or complex type having both mannose and other glycan types on each glycan, or of the hybrid type having both high mannose and complex antennae.
  • the cells expressing the expression vector of the invention produce a chimeric polypeptide having at least one, optionally at least two, optionally at least three or optionally at least four or more core xylose residues. In yet other embodiments the cells expressing the expression vector of the invention produce a chimeric polypeptide having at least one, optionally at least two, optionally at least three or optionally at least four or more core ⁇ -(1,3) fucose residues. In one embodiment the cells expressing the expression vector of the invention produce a chimeric polypeptide protein having at least one exposed mannose residue, at least one core xylose residue and at least one ⁇ -(1,3) fucose residue. In yet further embodiments, the cells expressing the expression vector of the invention produce a chimeric polypeptide having at least one, at least two, at least 3 or more terminal N-acetyl glucosamine substitutions on the outer mannose sugars.
  • the chimeric polypeptide lacks sialic acid residues. Yet further according to a specific embodiment, the chimeric polypeptide comprises at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or more complex glycans. According to a specific embodiment, the chimeric polypeptide comprises 40-70% complex glycans.
  • chimeric polypeptide protein is purified to a homogeneity of at least 98%.
  • the purified preparation is characterized by a purity of at least 85%, at least 87%, at least 90%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.1%, at least 93.2%, at least 93.3%, at least 93.4%, at least 93.5%, at least 93.6%, at least 93.7%, at least 93.8%, at least 93.9%, at least 94%, at least 94.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, in a range of at least 95.0-99.8% or 100% purity.
  • purity of the chimeric polypeptide is measured by HPLC.
  • the plant-expressed chimeric polypeptide preparation comprises impurities derived from the plant host cell, such as, but not limited to nucleic acids and polynucleotides, amino acids, oligopeptides and polypeptides, glycans and other carbohydrates, lipids and the like.
  • the host-cell derived impurities comprise biologically active molecules, such as enzymes.
  • the plant-expressed chimeric polypeptide composition comprises plant beta-N-acetylhexosaminidase. Where the host cell is a tobacco cell, or tobacco cell line cell, the plant beta-N-acetylhexosaminidase is a tobacco beta-N-acetylhexosaminidase.
  • the plant beta-N-acetylhexosaminidase is inactivated plant beta-N-acetylhexosaminidase.
  • Inactivation of plant beta-N-acetylhexosaminidase can be effected by physical means, chemical means or biochemical means. Physical inactivation can be performed by heating, freezing, desiccation, etc. Chemical inactivation can be performed by extremes of pH, chemical denaturation, addition or removal of side chains, glycans, amino acids, etc. Biochemical inactivation includes, but is not limited to inhibition by reversible or irreversible inhibitors.
  • Exemplary beta-N-acetylhexosaminidase inhibitors include end-product inhibitors such as N-acetyl-D-glucosamine and beta-methyl-N-acetyl glucosamine, and selective inhibitors such as the compounds disclosed in US Patent Applications US2010016386, US20110237631, US20100087477 and US20120046337. It will be appreciated that preferred methods for inhibition and/or inactivation of the plant beta-N-acetylhexosaminidase are those which also effectively preserve the structural and functional integrity of the plant-expressed chimeric polypeptide.
  • the plant beta-N-acetylhexosaminidase is inactivated by heating the composition comprising the chimeric polypeptide.
  • Suitable temperatures for plant beta-N-acetylhexosaminidase inhibition and/or activation include heating within a range of 37-60° C. for a period of 2 to 5, 10, 20, 30, 40, 50, 60 or more minutes. It will be appreciated that effective inhibition and/or inactivation of the plant beta-N-acetylhexosaminidase is achieved more rapidly at higher temperatures and more slowly at lower temperatures of the range.
  • the plant-expressed chimeric polypeptide composition is heated in the range of 45-55° C. for 2-10 minutes.
  • the inhibition/inactivation results in 20, 30, 40, 50, 60, 70, 80% or greater inactivation of the plant beta-N-acetylhexosaminidase.
  • the chimeric polypeptide of the invention is utilized for the treatment of TNF ⁇ -associated medical conditions.
  • treating refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology.
  • pathology disease, disorder or condition
  • Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
  • the term “preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.
  • a TNF ⁇ -associated medical condition refers to a medical condition in which TNF ⁇ activity is associated with onset, progression of the medical conditions and/or related symptoms in a subject.
  • TNF ⁇ -associated medical condition disease in a cell, tissue, organ, animal, or subject in need thereof including, but not limited to, at least one of obesity, an immune related disease, a cardiovascular disease, an infectious disease, a malignant disease or a neurologic disease (see WO0212502).
  • TNF ⁇ -associated medical condition examples include, but are not limited to, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Wegener's disease (granulomatosis), Crohn's disease, inflammatory bowel disease, short bowel syndrome, cholitis, ulcerative cholitis, chronic obstructive pulmonary disease (COPD), Hepatitis C, endometriosis, asthma, cachexia, psoriasis, and atopic dermatitis.
  • Additional diseases or disorders that can be treated with the chimeric polypeptide of the invention include those described in WO 00/62790, WO 01/62272 and U.S. Patent Application No. 2001/0021380, U.S. Pat. No. 7,648,702, the relevant portions of which are incorporated herein by reference.
  • TNF ⁇ -associated medical conditions include, but are not limited to those disclosed in Kuek et al. Postgrad. Med. J. 2007; 83;251-260, which is herein incorporated by reference in its entirety.
  • exemplary indications include, Sjorgen's syndrome, polymyositis, dermatomyositis, Wegener's vasculitis, Bechet's, giant cell arteritis (GCA), Polymyalgia rheumatic, Takayasu's arteritis, Polyarteritis nodosa, Sarcoidosis, adult onset Still's disease, Kawasaki disease, Cryoglobulinemic vasculitis, relapsing polychondritis, Hidradenitis suppurativa, Coeliac disease, myelodysplastic syndromes, Pyoderma gangrenosum, Erythema nodosum, SAPHO syndrome, graft versus host disease, chronic hepatitis B/C, thrombic/idiopathic thrombocytopenic purpura, refractory asthma, lupus, amyloidosis, Multicentric reticulohistiocytosis, pemphigus
  • TNF ⁇ -associated medical condition is an inflammatory bowel disease.
  • the polypeptide is administered enterally, e.g., orally, such as comprised in the plant cells.
  • the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
  • the polypeptide is administered enterally, e.g., orally, such as comprised in the plant cells.
  • TNF ⁇ -associated medical condition include, but are not limited to, immune related disease, such as rheumatoid arthritis, juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis, gastric ulcer, seronegative arthropathies, osteoarthritis, inflammatory bowel disease, short bowel syndrome, ulcerative colitis, systemic lupus erythematosis, antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/ admireer's granulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures, allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis, transplants, organ transplant rejection, graft-versus
  • the present invention also provides a method for treating at least one cardiovascular disease in a cell, tissue, organ, animal, or patient, including, but not limited to, at least one of cardiac stun syndrome, myocardial infarction, congestive heart failure, stroke, ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis, restenosis, diabetic ateriosclerotic disease, hypertension, arterial hypertension, renovascular hypertension, syncope, shock, syphilis of the cardiovascular system, heart failure, cor pulmonale, primary pulmonary hypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter, atrial fibrillation (sustained or paroxysmal), post perfusion syndrome, cardiopulmonary bypass inflammation response, chaotic or multifocal atrial tachycardia, regular narrow QRS tachycardia, specific arrythmias, ventricular fibrillation, His bundle arrythmias, atrioventricular block, bundle branch block, my
  • TNF ⁇ -associated medical condition include, but are not limited to, infectious diseases, such as acute or chronic bacterial infection, acute and chronic parasitic or infectious processes, including bacterial, viral and fungal infections, HIV infection/HIV neuropathy, meningitis, hepatitis (A, B or C, or the like), septic arthritis, peritonitis, pneumonia, epiglottitis, e.
  • infectious diseases such as acute or chronic bacterial infection, acute and chronic parasitic or infectious processes, including bacterial, viral and fungal infections, HIV infection/HIV neuropathy, meningitis, hepatitis (A, B or C, or the like), septic arthritis, peritonitis, pneumonia, epiglottitis, e.
  • coli 0157:h7 hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, mycobacterium tuberculosis, mycobacterium avium intracellulare, pneumocystis carinii pneumonia, pelvic inflammatory disease, orchitis/epidydimitis, legionella, lyme disease, influenza a, epstein-barr virus, vital-associated hemaphagocytic syndrome, vital encephalitis/aseptic meningitis, and the like.
  • TNF ⁇ -associated medical condition include, but are not limited to, malignant diseases such as leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy, solid tumors, adenocarcinomas, sarcomas, malignant melanoma, heman
  • TNF ⁇ -associated medical condition include, but are not limited to, neurologic diseases, such as neurodegenerative diseases, multiple sclerosis, migraine headache, AIDS dementia complex, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders' such as lesions of the corticospinal system; disorders of the basal ganglia or cerebellar disorders; hyperkinetic movement disorders such as Huntington's Chorea and senile chorea; drug-induced movement disorders, such as those induced by drugs which block CNS dopamine receptors; hypokinetic movement disorders, such as Parkinson's disease; Progressive supranucleo Palsy; structural lesions of the cerebellum; spinocerebellar degenerations, such as spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic disorders (Re
  • the term “subject” includes mammals, e.g., human beings at any age which suffer from the pathology. According to a specific embodiment, this term encompasses individuals who are at risk to develop the pathology.
  • the chimeric polypeptide can be formulated in a pharmaceutical composition for oral or enteral delivery comprising transformed plant cell expressing the chimeric polypeptide and a pharmaceutically acceptable carrier.
  • the transformed plant cells of the pharmaceutical composition are lyophilized plant cells.
  • enteral administration refers to administration through any part of the gastro-intestinal tract, such as rectal administration, colonic administration, intestinal administration (proximal or distal) and gastric administration. In some embodiments, enteral administration refers to oral administration.
  • the cells may be formulated as a solid, formulated as a liquid or formulated as a powder. In some embodiments, the cells are resuspended, lyophilized cells.
  • the chimeric polypeptide can be formulated in a pharmaceutical composition for oral or enteral delivery comprising transformed plant cell expressing the chimeric polypeptide and a pharmaceutically acceptable carrier.
  • the transformed plant cells of the pharmaceutical composition are lyophilized plant cells, although the use of fresh (non-lyophilized cells), plant tissues, plant parts or whole plants is also contemplated herein.
  • the cells Prior to lyophilization the cells may be washed to remove any cell debris that may be present in the growth medium.
  • the cells are being prepared for lyophilization, it is sometimes desirable to incubate the cells in a maintenance medium to reduce the metabolic processes of the cells.
  • Pretreatment can be performed at room temperature or at temperatures in which the plant cells are typically cultured. Pretreatment is performed at about room temperature (20° C.) for ease of handling and as most plant cells are fairly stable at room temperature. Stabilizers can be added directly to the medium and replenished as necessary during the pretreatment process.
  • Pretreatments may also involve incubating cells in the presence of one or more osmotic agents.
  • useful osmotic agents include sugars such as saccharides and saccharide derivatives, amino or imino acids such as proline and proline derivatives, or combinations of these agents.
  • Some of the more useful sugars and sugar derivatives are fructose, glucose, maltose, mannitol, sorbitol, sucrose and trehalose.
  • Osmotic agents are utilized at a concentration that prepares cells for subsequent lyophilization.
  • Lyophilization is directed at reducing the water content of the cells by vacuum evaporation.
  • Vacuum evaporation involves placing the cells in an environment with reduced air pressure.
  • the reduced ambient pressure operating at temperatures of between about ⁇ 30° C. to ⁇ 50° C. may be at 100 torr, 1 torr, 0.01 torr or less.
  • the cells are lyophilized by freezing to ⁇ 40° C. and then applying a vacuum to a pressure of 0.1 mbar. The cells are then heated to ⁇ 10° C. so all the ice content will be sublimated and evaporated. Under conditions of reduced pressure, the rate of water evaporation is increased such that up to 60-95% of the water in a cell can be removed.
  • lyophilization removes over 60%, 70%, 80% or specifically over 90%, 91%, 92%, 93%, 94%, 95% or 98% of the water from the cells.
  • the final water content is about 5-10%, 5-8% or 6-7%.
  • the oral dosage form may be provided as an oral nutritional form (e.g., as long as the protein is not exposed to denaturing conditions which include heating above 37° C. and compression), as a complete meal, as a powder for dissolution, e.g. health drinks, as a solution, as a ready-made drink, optionally low calorie, such as a soft drink, including juices, milk-shake, yoghurt drink, smoothie or soy-based drink, in a bar, or dispersed in foods of any sort, such as baked products, cereal bars, dairy bars, snack-foods, breakfast cereals, muesli, candies, tabs, cookies, biscuits, crackers (such as a rice crackers), chocolate, and dairy products.
  • an oral nutritional form e.g., as long as the protein is not exposed to denaturing conditions which include heating above 37° C. and compression
  • a powder for dissolution e.g. health drinks, as a solution, as a ready-made drink, optionally low calorie, such as
  • prh TNRF2:Fc is a chimeric protein carries an Fc segment of human IgG1.
  • the FcRn receptor transcytoses IgG molecules across by binding to their Fc. Therefore, prh TNRF:Fc can also cross the epithelial barrier to bind its cytokine ligand on the serosal side of the epithelia.
  • the metabolic syndrome is a constellation of interrelated risk factors of metabolic origin—metabolic risk factors—that appear to directly promote the development of atherosclerotic cardiovascular disease (ASCVD). Patients with the metabolic syndrome also are at increased risk for developing type 2 diabetes mellitus.
  • the multiple components and criteria that define the metabolic syndrome have varied somewhat in specific elements, but in general they include a combination of both underlying and metabolic risk factors.
  • the most widely recognized of the metabolic risk factors are atherogenic dyslipidemia, elevated blood pressure, and elevated plasma glucose. Individuals with these characteristics commonly manifest a prothrombotic state and a proinflammatory state as well.
  • Atherogenic dyslipidemia consists of an aggregation of lipoprotein abnormalities including elevated serum triglyceride and apolipoprotein B (apoB), increased small LDL particles, and a reduced level of HDL cholesterol (HDL-C).
  • apoB apolipoprotein B
  • HDL-C HDL cholesterol
  • the liver disease or disorder is selected from the group consisting of hepatitis, liver cirrhosis, liver cancer, hepatotoxicity, chronic liver disease, fatty liver disease and non-alcoholic steatohepatitis (NASH).
  • the medical condition is not a obesity, metabolic syndrome, diabetes and a liver disease or disorder.
  • the hepatotoxicity is induced by a chemical agent selected from the group consisting of acetaminophen, NSAIDS, glucocorticoid, isniazed, arsenic, chemotherapy, carbon tetrachloride and vinyl chloride.
  • a chemical agent selected from the group consisting of acetaminophen, NSAIDS, glucocorticoid, isniazed, arsenic, chemotherapy, carbon tetrachloride and vinyl chloride.
  • the diabetes is selected from the group consisting of type I diabetes, type II diabetes and LADA disease.
  • the chimeric polypeptide of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the chimeric polypeptide or cells expressing same accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
  • compositions of this invention are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly, intravenously, intraperitoneal, intracerebrospinal, intra-articular, intrasynovial, and/or intrathecal.
  • Parenteral administration can be by bolus injection or continuous infusion.
  • Pharmaceutical compositions for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • compositions of the present invention are suitable for administration using these new methods, e.g., Inject-easeTM, GenjectTM, injector pens such as GenPenTM, and needleless devices such as MediJectorTM and BioJectorTM.
  • the pharmaceutical composition can also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the formulations may be modified with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (chimeric polypeptide) effective to prevent, alleviate or ameliorate symptoms of a disorder or prolong the survival of the subject being treated.
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. 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 dosage may 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, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).
  • Dosage amount and interval may be adjusted individually to provide the chimeric polypeptide (the target tissue) levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • 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 effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • the effective chimeric polypeptide amount per adult dose ranges from about 1-500 mg/m 2 , or from about 1-200 mg/m 2 , or from about 1-40 mg/m 2 or about 5-25 mg/m 2 .
  • a flat dose may be administered, whose amount may range from 2-500 mg/dose, 2-100 mg/dose or from about 10-80 mg/dose.
  • the effective chimeric polypeptide amount per adult dose is about 1-500 mg/m 2 , or about 1-200 mg/m 2 , or about 1-40 mg/m 2 or about 5-25 mg/m 2 .
  • a flat dose may be administered, whose amount may range about 2-500 mg/dose, 2-100 mg/dose or from about 10-80 mg/dose.
  • the effective chimeric polypeptide amount per adult dose range is about 0.0002 mg/kg to 2 mg/kg, about 0.002-2 mg/kg, about 0.02-2 mg/kg, about 0.2-2 mg/kg, about 0.002-0.2 mg/kg, about 0.0002-1 mg/kg, about 0.002-0.1 mg/kg, about 0.002-0.02 mg/kg, about 0.002-0.01 mg/kg, about 0.002-0.008 mg/kg, about 0.02-0.1 mg/kg, about 0.001-0.05 mg/kg, about 0.001-0.01 mg/kg, about 0.01-1 mg/kg, about 0.01-15 mg/kg, about 0.005-1 mg/kg, about 0.01-5 mg/kg, about 0.005-0.01 mg/kg or about 0.05-0.1 mg/kg.
  • these dose ranges are used for oral administration such as of plant cells expressing the chimeric protein.
  • the effective chimeric polypeptide amount per adult dose ranges about 0.002-0.2 mg/kg. According to a specific embodiment, this dose range is used for oral administration such as of plant cells expressing the chimeric protein.
  • a flat dose may be administered, whose amount may range about 2-500 mg/dose, 2-100 mg/dose or from 10-80 mg/dose. According to a specific embodiment, this dose range is used for oral administration such as of plant cells expressing the chimeric protein.
  • a flat dose of 0.01-100 mg, 0.1-100 mg, 0.1-50 mg, 0.1-20 mg, 0.1-10 mg, 0.1-5 mg is administered.
  • this dose range is used for oral administration such as of plant cells expressing the chimeric protein.
  • the flat dose is about 0.1-10 mg. According to a specific embodiment, this dose range is used for oral administration such as of plant cells expressing the chimeric protein.
  • the oral dose is administered daily.
  • the dose may be divided for a number of administrations during the day (say 2-4 times a day).
  • the dose can also be administered every two days, two times a week, three times a week, biweekly, weekly doses, or separated by several weeks (for example 2 to 8).
  • an exemplary dose range is the same as the foregoing described dose ranges or lower and administered two or more times per week (e.g., 25-100 mg/dose).
  • an acceptable dose for administration by injection contains 80-100 mg/dose, or alternatively, containing 80 mg per dose.
  • the dose may be modified for children and infants.
  • the dose can be administered at biweekly, weekly doses, or separated by several weeks (for example 2 to 8).
  • the chimeric polypeptide is generally administered at 25 mg by a single subcutaneous (SC) injection.
  • an improvement in a patient's condition will be obtained by a dose of up to about 100 mg of the pharmaceutical composition one to three times per week over a period of at least three weeks, though treatment for longer periods may be necessary to induce the desired degree of improvement.
  • the regimen may be continued indefinitely.
  • a suitable regimen involves a dose of 0.4 mg/kg to 5 mg/kg of the chimeric polypeptides of the invention by injection, administered one or more times per week.
  • the pharmaceutical formulation of the invention is prepared in a bulk formulation and as such, the components of the pharmaceutical composition are adjusted so that it is higher than would be required for administration and diluted appropriately prior to administration.
  • compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be 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.
  • Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • concentration of the polypeptide in the aqueous pharmaceutical composition can vary over a wide range, but is generally within the range of from about 0.05 to about 20,000 micrograms per milliliter ⁇ /ml) of aqueous formulation.
  • dosage forms which comprise the plant cells may include additives such as one or more of calcium, magnesium, iron, zinc, phosphorus, vitamin D and vitamin K.
  • a suitable daily amount is 0.1 mg to 3.6 g calcium, preferably 320 to 530 mg.
  • the daily dosage of vitamins and minerals in the nutritional formulation or medicament of the invention is 25-100% by weight of the dosages recommended by the health authorities.
  • Dietary fiber may also be a component of the compositions of the invention.
  • Further components of the supplement may include any bioactive compounds or extracts which are known to have health benefits, especially for improving physical performance.
  • the unit dosage form may further comprise an antioxidant (exemplary embodiments are provided above.
  • the antioxidant is a pharmaceutically acceptable antioxidant.
  • the antioxidant is selected from the group consisting of vitamin E, superoxide dismutase (SOD), omega-3, and beta-carotene.
  • the unit dosage form further comprises an enhancer of the biologically active protein or peptide. In another embodiment, the unit dosage form further comprises a cofactor of the biologically active protein or peptide.
  • a unit dosage form of the present invention further comprises pharmaceutical-grade surfactant.
  • surfactants are well known in the art, and are described, inter alia, in the Handbook of Pharmaceutical Excipients (eds. Raymond C Rowe, Paul J Sheskey, and Sian C Owen, copyright Pharmaceutical Press, 2005).
  • the surfactant is any other surfactant known in the art.
  • a unit dosage form of the present invention further comprises pharmaceutical-grade emulsifier or emulgator (emollient).
  • Emulsifiers and emulgators are well known in the art, and are described, inter alia, in the Handbook of Pharmaceutical Excipients (ibid).
  • Non-limiting examples of emulsifiers and emulgators are eumulgin, Eumulgin B1 PH, Eumulgin B2 PH, hydrogenated castor oil cetostearyl alcohol, and cetyl alcohol.
  • the emulsifier or emulgator is any other emulsifier or emulgator known in the art.
  • a unit dosage form of the present invention further comprises pharmaceutical-grade stabilizer.
  • Stabilizers are well known in the art, and are described, inter alia, in the Handbook of Pharmaceutical Excipients (ibid).
  • the stabilizer is any other stabilizer known in the art.
  • a unit dosage form of the present invention further comprises an amino acid selected from the group consisting of arginine, lysine, aspartate, glutamate, and histidine.
  • analogues and modified versions of arginine, lysine, aspartate, glutamate and histidine are included in the terms “arginine,” “lysine,” “aspartate”, “glutamate” and “histidine,” respectively.
  • the amino acid provides additional protection of ribonuclease or other active molecules.
  • the amino acid promotes interaction of biologically active protein or peptide with a target cell.
  • the amino acid is contained in an oil component of the unit dosage form.
  • a unit dosage form of the present invention further comprises one or more pharmaceutically acceptable excipients, into which the matrix carrier unit dosage form is mixed.
  • the excipients include one or more additional polysaccharides.
  • the excipients include one or more waxes.
  • the excipients provide a desired taste to the unit dosage form.
  • the excipients influence the drug consistency, and the final dosage form such as a gel capsule or a hard gelatin capsule.
  • Non limiting examples of excipients include: Antifoaming agents (dimethicone, simethicone); Antimicrobial preservatives (benzalkonium chloride, benzelthonium chloride, butylparaben, cetylpyridinium chloride, chlorobutanol, chlorocresol, cresol, ethylparaben, methylparaben, methylparaben sodium, phenol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric nitrate, potassium benzoate, potassium sorbate, propylparaben, propylparaben sodium, sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic acid, thimerosal, thymol); Chelating agents (edetate disodium, ethylenediaminetetraacetic acid and salts, edetic acid); Coating agents (sodium carboxymethyl-cellulose, cellulose acetate,
  • compositions of the invention may be included in the compositions of the invention, including any of those selected from preservatives, chelating agents, effervescing agents, natural or artificial sweeteners, flavoring agents, coloring agents, taste masking agents, acidulants, emulsifiers, thickening agents, suspending agents, dispersing or wetting agents, antioxidants, and the like.
  • Flavoring agents can be added to the compositions of the invention to aid in compliance with a dosing regimen.
  • Typical flavoring agents include, but are not limited to natural or synthetic essences, oils and/or extracts of pineapple, orange, lemon, mint, berry, chocolate, vanilla and melon.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • cDNA encoding prh TNFR2:Fc was optimized and synthesized by GENEART AG (Regensburg, Germany). The codon usage was adapted to the codon bias of Nicotiana tabacum genes.
  • the IgG1 portion was cloned from Fc IgG1 heavy chain constant region [ Homo sapiens ] ACCESSION AEV43323.
  • RNA instability elements (“Killer motifs”)
  • Repeat sequences and RNA secondary structures Repeat sequences and RNA secondary structures, splice donor (cryptic) and acceptor sites, branch points.
  • regions of very high (>80%) or very low ( ⁇ 30%) GC content were avoided.
  • the resultant DNA sequence is as set forth in SEQ ID NO: 1.
  • the encoded polypeptide is as set forth in SEQ ID NO: 2.
  • a signal peptide e.g. endoplasmic reticulum target signal peptide
  • plumbaginifolia Calreticulin protein was added to the N′ terminus of the gene, allowing efficient targeting of prh TNFR2:Fc to the secretory pathway and is then cleaved from the polypeptide, by signal peptidase, once the protein has been translocated into the endoplasmic reticulum (SEQ ID NO: 3, SEQ ID NO: 4, representing the DNA and peptide sequences of the ER signal peptide, respectively).
  • an ER retention signal SEKDEL was added to the C′ terminus of the gene. This signal allows protein retrieval from the Golgi apparatus to the ER, and localization in the ER.
  • the entire coding sequence (signal peptide-prh TNFR2:Fc-SEKDEL) is as set forth in SEQ ID NO: 5 and the encoded polypeptide is as set forth in SEQ ID NO: 6.
  • the resultant protein following cleavage is as set forth in SEQ ID NO: 7, 204 or 205 (prh TNFR2:Fc-SEKDEL).
  • Agrobacterium mediated transformation is widely used to introduce foreign genes into a plant cell genome. Using this approach, a T-DNA molecule consisting of a foreign gene and its regulatory elements is randomly introduced into the plant genome. Since the site of integration, as well as the copy number of the gene insertions cannot be controlled, the transformation process results in a highly heterogeneous transgenic ‘pool’ composed of cells with various levels of transgene expression. The transgenic ‘pool’ is subsequently used for clone isolation. The transformation process, results in establishment of numerous single cell lines, each representing an individual transformation event, from which the clone with the highest expression level of the foreign gene is selected.
  • prh TNFR2:Fc the transformation was conducted with a plasmid carrying the prh TNFR2:Fc cassette ( FIG. 1 SEQ ID NOs: 7 and 8).
  • the recombinant protein is targeted to the Endoplasmic reticulum (ER) of the cells.
  • BY2 Bright Yellow 2 suspension culture was co-cultivated, for 48 hours, with the Agrobacterium tumefaciens strain carrying the vector harboring the prhTNFR2:FC-gene and the neomycin phosphotransferase (NPTII) selection gene. Subsequently, cells were kept in media supplemented with 50 mg/L of Kanamaycin and 250 mg/L Cefotaxime. The NPTII gene confers resistance to Kanamycin, thus only NPTII positive BY2 cells survive in this selection media. The Cefotaxime was used to selectively kill the agrobacterium , the plant cells being resistant to this antibiotic.
  • NPTII neomycin phosphotransferase
  • SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
  • the native extraction supernatant was mixed with non-reducing sample buffer prior to analysis. Electrophoresis was performed using CriterionTM cell vertical electrophoresis apparatus (Bio-Rad Lab.) with premixed electrophoresis Tris-Glycine-SDS running buffer (Bio-Rad Laboratories). Following electrophoresis, the proteins were transferred from the Polyacrylamide gel to a protein binding nitrocellulose membrane (iBlotTM). Membranes were blocked for 1 hr at RT with 5% milk buffer containing 0.1% Tween 20. For identification of the Fc portion of the molecule, Goat anti human IgG conjugated to HRP (cat #109-035-098, Jackson.) was used.
  • TNFR2 detection For TNFR2 detection, a Rabbit Anti-TNFRII (ID: ab109853, Abcam) followed by Goat anti Rabbit HRP (cat #111-035-003, Jackson) were employed. Detection was carried out with ECL detection kit (Pierce). The immunoreactivity of prh TNFR2:FC was compared to that of commercial Enbrel (Entanercept; Wyeth). Bands were detected using the Molecular Imager Gel Doc XR System (Bio-Rad Laboratories).
  • prhTNFR2:FC is sent for sequencing analysis at the Smoler Proteomics Center at the Technion—Israel Institute of Technology (Haifa, Israel).
  • the protein is extracted from the gel, reduced with 2.8 mM DTT (60° C. for 30 min), modified with 8.8 mM iodoacetamide in 100 mM ammonium bicarbonate (in the dark, room temperature for 30 min) and digested in 10% ACN and 10 mM ammonium bicarbonate with modified Trypsin (Promega) or with ChymoTrypsin overnight at 37° C. in a 1:50 enzyme-to-substrate ratio.
  • 3% of the resulting peptides are resolved by reverse-phase chromatography on 0.075 ⁇ 200-mm fused silica capillaries (J&W) packed with Reprosil reversed phase material (Dr Maisch GmbH, Germany).
  • the peptides are eluted with linear 60 minutes gradients of 5 to 45% and 15 minutes at 95% acetonitrile with 0.1% formic acid in water at flow rates of 0.25 ⁇ l/min.
  • mass spectrometry is performed by an ion-trap mass spectrometer (Orbitrap, Thermo) in a positive mode using repetitively full MS scan followed by collision induces dissociation (CID) of the 7 most dominant ion selected from the first MS scan.
  • CID dissociation
  • the mass spectrometry data is analyzed using the Sequest 3.31 software (J. Eng and J. Yates, University of Washington and Finnigan, San Jose) vs a specific sequence.
  • glycoproteins produced in Chinese Hamster Ovary (CHO) cell and plant cell systems are glycosylation profile and glycan structure.
  • Preliminary analysis has been performed to characterize the various N-linked glycan structures attached to the protein. These results are compared to results of the N-glycosylation profile found in commercial Enbrel. The presence of O-linked glycans, and glycan site analysis is determined.
  • the released glycans are extracted, cleaned and then labeled with the fluorescent reagent anthranilamide (2-aminobenzamide, 2AB) followed by removal of excess 2AB.
  • the analytical method includes separation of the glycans on a Waters HPLC system with a normal phase amide-based column (Tosoh TSK Amide-80 column), coupled with a fluorescence detector (330 nm excitation, 420 nm emission).
  • Sequencing of the labeled glycan pool is achieved by sequential digestion with various exoglycosidases followed by additional HPLC analysis. Using sequential digestion with various exoglycosidases provides additional information on the profile of the glycans structures and their relative amounts.
  • the exoglycosidase digestions that are carried out for the glycans released from prh TNFR2:FC are with JBH (Jack bean beta-N-Acetylhexosaminidase) that removes beta 1-2, 3, 4 and 6 N-acetylglucosamine (GlcNAc), with JBM (Jack bean mannosidase) that removes mannose alpha 1-2, 6>3 mannose and with BKF (Bovine testis fucosidase) that removes alpha 1-6 and alpha 1-3 core fucose.
  • JBH Jack bean beta-N-Acetylhexosaminidase
  • GlcNAc N-acetylglucosamine
  • JBM Jack bean mannosidase
  • BKF Bovine testis fucosidase
  • the glycans are then separated according to unique glycan linkages and in order of increasing size using a gradient solvent flow consisting of ammonium formate and acetonitrile. Retention time of individual glycans is compared to the retention times of a standard mix of partially hydrolysed dextran fragments, giving a ladder of glucose units (GU).
  • the glycans are assigned to peaks according to their GU values, based on standards and a comparison to an external data base (www.glycobasedotnibrtdotie:8080/database/show_glycobasedotaction). The final assignment and relative peak areas are calculated from the chromatogram of the PNGase A digestion.
  • TNF ⁇ binding ELISA is a combination of a commercial TNF ⁇ detection ELISA kit (Human TNF- ⁇ ; Hycult Biotech Inc. #HK307) and a commercial anti human IgG antibody (Goat anti human IgG FC specific HRP; Sigma).
  • the assay is a quantitative non radioactive assay for prhTNFR2:FC binding activity. This binding ELISA enables to detect functional (capable of binding TNF ⁇ ) molecules comprising both the TNFR and IgG domains.
  • An ELISA plate pre-coated with antibodies against TNF ⁇ was incubated with TNF ⁇ (60 ng/ml, Sigma) for 1 hour at room temperature. Between each ELISA step the plate was washed three times with commercial wash buffer. Commercial Enbrel and supernatant from BY2 cells expressing prh TNFR2:FC (serial dilutions) were incubated on ELISA plate for 2 hr at RT. Goat anti human IgG Fc HRP was diluted 1:10,000 and incubated on plate for 1 hr at RT. TMB was used as substrate for HRP. The colorimetric reaction was stopped with 10% HCL and absorbance determined at 450 nm.
  • A375 cells human melanoma cells were grown in suspension in culture medium (ATCC, #30-2002, supplemented with 10% FBS). 10 4 /well cells were plated in 96-well assay plates and incubated overnight in assay medium (ATCC, #30-2002, supplemented with 5% FBS). Recombinant TNF ⁇ (2 ng/ml, ProSpec, Rehovot, Israel) was incubated for 2 hr at 37° C. in the presence of different concentrations (1.562-100 ng/ml) of prhTNFR2:FC or commercial Enbrel (Entanercept; Wyeth).
  • the mixed solution was added to A375 cells in the presence of actinomycin-D (0.8 ⁇ g/ml), incubated for further 24 hr at 37° C., 5% CO 2 in a humidified incubator and quantification of apoptosis was determined by MTT assay (Sigma Cat. No. M5655). The plate was read at 570-650 nm and the inhibition of TNF- ⁇ induced cytotoxicity (%) was calculated.
  • prhTNFR2:FC was analyzed under reducing ( FIG. 2A ) and non-reducing conditions (native extraction in the FIG. 2B ).
  • prhTNFR2:FC (Lane 1) and commercial Enbrel (lane 2) were detected using anti Fc antibody (upper panel) and anti TNFR2 antibody (lower panel).
  • the two proteins demonstrate a slight difference in migration characteristics, presumably due to differences in glycosylation patterns between the plant and mammalian cell-expressed enzymes.
  • TNF ⁇ binding by both commercial Enbrel and prh TNFR2:FC was examined by comparing serial dilutions of lysates of BY2 cells expressing prh TNFR2:Fc (PRX-106) to commercial Enbrel.
  • prh TNFR2:FC serial dilutions demonstrate a dose response binding pattern similar to the commercial protein (see FIG. 3 ).
  • the selection of transgenic cell lines according to protein expression was done by Western blotting. Thus, to allow for the selection of individual cell lines, aliquots of highly diluted cell suspension were spread on solid BY-2 medium. The cells were then grown until small calli developed. Each callus was then re-suspended in liquid culture.
  • TNF ⁇ activity can be measured by its ability to induce cell death of certain cell lines in the presence of the transcriptional inhibitor, actinomycin D.
  • Pre-incubation with a neutralizing protein of TNF ⁇ prevents binding to the receptors (TNF-R1 and TNF-R2), thereby inhibiting the cytokine effect and preventing TNF ⁇ induced cell death.
  • Quantification of cell viability by MTT assay provides an in-cell activity assay for TNF ⁇ cytotoxicity. The results are shown in FIGS. 5A-G on melanoma cells A375 and in FIGS. 6A-G on L929 fibroblasts.
  • IBD Inflammatory bowel disease
  • GIT Gastro-intestinal Tract
  • CD Crohn's disease
  • UC Ulcerative colitis
  • PRX106 is a soluble receptor for a cytokine overproduced in inflammation involving among others, the TH1 immune cell response. PRX106 was shown to be very effective when injected IV in other models of inflammation (Rheumatoid Arthritis). PRX106 is overexpressed in Protalix's ProCellExTM system, in BY2 plant cells. Being a plant cell, BY2 has a cell wall that can help protect PRX106 while moving through the stomach and small intestine. In the colon, where the polysaccharides are digested, the plant cell releases its content and hence PRX106 is free to bind its cytokine ligand. Moreover, PRX106 is a chimeric protein carrying an Fc segment of human IgG1.
  • PRX106 can also cross the epithelial barrier to bind its cytokine ligand on the serosal side of the epithelia.
  • IBD models are classified into five major groups: chemically induced model, cell-transfer model, spontaneous model, congenital (spontaneous gene mutation) model, and genetically engineered model.
  • chemically induced models colitis is induced by intrarectal administration of the covalently reactive reagents TNBS/oxazolone, which are believed to induce a T-cell-mediated response against hapten-modified autologous proteins/luminal antigens.
  • DSS dexazolone
  • the disease severity is evaluated by scoring 3 major clinical signs (weight loss, diarrhea, and rectal bleeding).
  • the mouse models TNBS (Example 3A) and DSS (Example 3B) are used to determine the therapeutic efficacy of the plant cells expressing the chimeric polypeptide in vivo.
  • mice Male Balb/c mice, 8-9 weeks old were used in all experiments. Each experimental group included 5 to 10 mice. The mice were purchased from Harlan Laboratories, Israel. All mice were moved to SPF-free room (natural bacterial flora) several days before starting the experiment.
  • TNBS was induced in mice by rectal installation of TNBS [M. F. Neurath, I. Fuss et al: j exp. Med 182′ 1281-1290 (1995)]. Mice were sensitized by painting 100 ⁇ L of 1% TNBS in ethanol onto the shaved skin of their abdomens 7 days before challenge. On the day of challenge, the mice were given 120 ⁇ L of 1 TNBS (Sigma Aldrich) slowly injected into the lumen of the colon via a catheter.
  • 1 TNBS Sigma Aldrich
  • mice were treated per os (PO) daily from day 0 to day 4 with BY-2 cells expressing PRX-106, equivalent to 5 ⁇ g protein (Dose I) and 30 ⁇ g protein (Dose II); BY-2( ⁇ ) control cells in the same orally administered volumes of the PRX-106 expressing cells; and saline.
  • TNBS control mice received PBS alone. The animals were monitored once daily for weight. Weight loss was calculated by subtracting the weight on each day from the weight on day 0. After the experiment, the animals were sacrificed and dissected. On day 5, blood samples were collected by cardiac puncture, and were left to clot and then centrifuged to obtain serum for determination of serum cytokine levels. (Cury et al. Cell Immunol. 2013, 282 (1); 66-70. Experiments were performed on 5-15 mice per group in three separate experiments; results followed the same pattern in all experiments.
  • Oral administration of plant cells expressing recombinant TNFR2:Fc was initiated 6 hours after administration of TNBS. Mice received plant cells expressing recombinant TNFR2:Fc, resuspended in 350-500 ⁇ L. Negative controls received the same orally administered volumes of host Mock plant cells, instead of the plant cells expressing recombinant TNFR2:Fc. Oral administration was performed by gavage. Two more controls were untreated mice and TNBS treated mice that received saline.
  • Serum levels of cytokines TNF- ⁇ , and IL-10 were determined using ELISA kits following the manufacturer's instructions (R&D Systems, Minneapolis, Minn., USA).
  • Serum qualitative measurement of cytokine content was performed using the Mouse Cytokine Antibody Array (R&D Systems, Minneapolis, Minn., USA), according to the manufacturer's manual.
  • Paraffin-embedded colonic tissue sections (5 ⁇ m) were deparaffinized, rehydrated, washed and incubated in 3% H 2 O 2 and blocked (Bar Sela et al 2006). Slides were incubated with IkB-alpha pSer32/Ser36 antibodies (Abcam) Color was developed using the DAB substrate kit (Thermo Scientific) or Zymed AEC substrate kit (Zymed Laboratories), followed by counterstaining with Mayer's hematoxylin. Controls without addition of primary antibody showed low or no background staining in all cases. Blocking was performed according to Bar-Sela et al. Histopathology. 2006; 49:188-193.
  • Spleens were harvested from mice in RPMI 1640 medium.
  • Cell suspensions were prepared by dicing spleens with a razor blade, followed by passage through a 40 ⁇ M Nylon filter (BD Falcon).
  • Splenocytes were incubated with anti-mouse CD4 (R&D Systems, Minneapolis, Minn., USA) and anti-mouse CD25 (R&D Systems, Minneapolis, Minn., USA).
  • Cells were then fixed and permeabilized for 20 min at 4° C. and then incubated with anti-mouse Foxp3 (Mouse Regulatory T cell 3-Color Flow kit, R&D Systems, Minneapolis, Minn., USA) diluted in permeabilization buffer for 30 min
  • Ten thousand CD4 + cells were analyzed by FACS.
  • Oral administration of plant cells expressing recombinant TNFR2:Fc improved TNBS-Induced body weight loss as monitored 4 days after initiation of TNBS administration ( FIGS. 7A-B ).
  • Colon lengths were measured as morphological indicators of colon inflammation in TNBS-treated mice; short colon indicating an inflammatory state. As shown in FIG. 8 , the colon length of mice treated with TNBS was significantly shortened compared to the control mice. The length of colon of the treated group (oral administration of cells expressing prTNFR2:Fc) was significantly longer than that in the TNBS-treated group. Oral administration of plant cells expressing recombinant TNFR2:Fc also improved the macroscopic features of TNBS-induced colitis. Macroscopic examination of colons, showed reduced colon damage severity compared with the non treated colons ( FIG. 9 ).
  • TNFR2:Fc Oral administration of plant cells expressing recombinant TNFR2:Fc reduced the expression of proinflammatory cytokines in mice with TNBS-induced colitis ( FIGS. 10A-C ).
  • FIGS. 11A-B showed that treatment with oral administration of plant cells expressing recombinant TNFR2:Fc reduced level of inflammatory mediators like granulocyte colony-stimulating factor G-CSF, macrophage colony-stimulating factor (M-CSF), potentially indicating reduced systemic inflammation by lowering systemic recruitment of bone marrow derived cells from the bloodstream.
  • G-CSF granulocyte colony-stimulating factor
  • M-CSF macrophage colony-stimulating factor
  • Treg cells also known as CD4 + CD25 + , FOXP3 + , are involved in the maintenance of peripheral tolerance and in controlling the immune response by initiating suppressive effects on activated immune cells.
  • the present analysis shows that oral administration of plant cells expressing TNFR2:Fc expands population of functional regulatory T (T reg) cells in the spleen ( FIG. 12 ).
  • Dextran Sulfate Sodium-induced (DSS-induced) mouse model of IBD is used for compounds for efficacy in Inflammatory Bowel Disease.
  • This is an experimental acute Ulcerative Colitis model with symptoms similar to those observed in human UC, such as diarrhea, bloody feces, body weight loss, mucosal ulceration and shortening of the large intestine.
  • mice Male C67/B1 mice, 8-9 weeks old were used in all experiments. Each experimental group included 10 mice. The mice were purchased from Harlan Laboratories, Israel. All mice were moved to SPF-free room (natural bacterial flora) several days before starting the experiment.
  • Daily treatment with orally administered plant cells expressing recombinant TNFR2:Fc; Mock cells comprising vector alone or control treatment (saline) began 24 hours following DSS induction, for a period of 7 days.
  • Colonic inflammation was assessed 5 days after DSS treatment by punch biopsies and histological score. The animals were monitored once daily for weight, weight loss was calculated by subtracting the daily weight from the weight on day 0.
  • Oral administration of plant cells expressing recombinant TNFR2:Fc was initiated 24 hours following DSS administration. Mice received plant cells expressing recombinant TNFR2:Fc (comprising 30 ⁇ g of protein), suspended in 500 ⁇ l saline. Negative controls received the equivalent volumes of host Mock plant cells, to the plant cells expressing recombinant TNFR:Fc. Two more control groups were DSS treated mice administered with saline and untreated mice. Oral administration was performed by gavage.
  • Paraffin-embedded colon tissue sections were stained with hematoxylin and eosin for light microscopic examination to assess colon injury and inflammation. Samples from entire colon were analyzed pathologically by a pathologist blinded to treatment conditions. A scoring system including degree of inflammation, crypt damage, percentage of area involved by inflammation and depth of inflammation was used.
  • Mouse colons were flushed 3 times with PBS containing antibiotics and opened along a longitudinal axis. Thereafter, 4-mm 2 punch biopsies were obtained and incubated for 24 hours in RPMI-1640 medium supplemented with antibiotics. Supernatants were collected and kept in ⁇ 20° C. until assessed for cytokine expression. Qualitative measurement of cytokine content in medium conditioned by colonic explants was performed using the Magnetic Luminex Screening Assay according to the manufacturer's manual R&D Systems, Minneapolis, Minn., USA).
  • Serum levels of cytokines TNF- ⁇ , IL-6 and IL-10 were determined using Magnetic Luminex Screening Assay following the manufacturer's instructions (R&D Systems, Minneapolis, Minn., USA).
  • FIGS. 13A-B Body weight was monitored each day following DSS administration. As can be seen, treatment of mice orally with plant cells expressing recombinant TNFR2:Fc attenuated the weight loss induced by DSS.
  • Colon lengths were measured as it is well established that a short colon can be used as a morphological indicator of colon inflammation in DSS-treated mice. As shown in FIGS. 14A-B , the colon length of mice treated with DSS was significantly shortened compared with the control mice. The length of colon in the oral administration of plant cells expressing recombinant TNFR2:Fc group was significantly longer than that in the DSS-treated group.
  • FIG. 15 shows a statistically significant decrease in gut proinflammatory cytokines following oral treatment with plant cells expressing recombinant TNFR2:Fc.
  • colons exhibited transmural inflammation and intense infiltration of inflammatory cells. This cell influx associated with ulceration, loss of goblet cells and marked disruption in the crypts throughout the colon.
  • oral treatment with plant cells expressing TNFR2:Fc markedly improved the histological features of DSS-induced colitis. Histological examination of colons, showed reduced colon damage severity in colons of orally administered plant cells expressing recombinant TNFR2:Fc treated mice, compared with the DSS and Mock treated colons.
  • the present study supports a role for orally administered plant cells expressing recombinant TNFR2:Fc as an anti-inflammatory agent with the capacity to ameliorate IBD.
  • Plasma samples were collected in the each time point (e.g., 0, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h), left to clot and then centrifuged to obtain serum. Serum levels of human TNFRII were determined using ELISA kits following the manufacturer's instructions (R&D Systems, Minneapolis, Minn., USA).
  • TNFR2:Fc level in serum is shown in FIG. 18 .
  • Results demonstrate the elevation of TNFR2:Fc level in plasma following oral administration of plant cells expressing the protein.
  • TNFR2:Fc level in serum was detected at 8 h, and was still detectable at 24 hours.
  • TNFR2:Fc level in rat's serum fed with host BY2( ⁇ ) was not detectable.
  • TNFR2:Fc protein pharmacokinetic profile in the suckling rat plasma various time points post feeding of plant cells expressing TNFR2:Fc.
  • TNFR2:Fc level in serum is shown in FIG. 19 .
  • Results demonstrate the significant elevation of TNFR2:Fc level in plasma following oral administration of TNFR2:Fc.
  • TNFR2:Fc level in plasma peaked at 4 h.
  • increased level of TNFR2:Fc in serum of suckling rats relative to adult rat was due to expression of FcNR in intestine of suckling rats.
  • TNFR2:Fc level in rat's serum fed with host BY2( ⁇ ) was not detectable.
  • 3 dosing groups comprising 12 rats per group (6 males and 6 females) and a control group comprising 6 rats per group (3 males and 3 females), were assigned.
  • the control group received dilution buffer (0.2 M mannitol) and three treated groups received cells expressing TNFR2:Fc at dose levels of 0.1, 0.5 and 1 mg TNFR2:Fc/Kg body weight. Cells were alliquoted in accordance with requested expressed protein amount. Each aliquot was mixed with 30 grams powder of commercial rodent diet and dilution buffer, to create a pellet. The control pellet was made with dilution buffer and commercial rodent diet powder alone. All animals were daily orally fed with the pellets for 14 days.
  • the procedure determines the N-terminal amino acid sequence of proteins and peptides by the Edman degradation chemistry.
  • the Edman degradation is a cyclic procedure where amino acid residues are cleaved off one at a time and identified by chromatography.
  • 3 steps in the cyclic procedure In step 1, the PITC reagent is coupled to the N-terminal amino group under alkaline conditions.
  • the N-terminal residue is cleaved in acidic media.
  • the PITC coupled residue is transferred to a flask, converted to a PTH-residue and identified by HPLC chromatography. The next cycle is then started for identification of the next N-terminal residue.
  • the sequence was determined to be LPAQV (SEQ ID NO: 18).
  • Sequencing was performed at the Smoler Proteomics Center (Technion-Israel Institute of Technology, Haifa, Israel). Analyses were carried out using reverse-phase HPLC coupled to a mass spectrometry detector.
  • the analyzed samples were resuspended in 8 M Urea, 100 mM ammonium bicabonate (ABC) followed by reduction with 2.8 mM DTT (60° C. for 30 min) and modified with 8.8 mM iodoacetamide in 100 mM ABC in the dark, at ambient temperature for an additional 30 mM
  • the proteins were digested overnight at 37° C. using modified trypsin (Promega) at a 1:50 enzyme-to-substrate ratio in 2 M Urea, 25 mM ABC.
  • the tryptic or chymotryptic peptides were desalted using stage tips (home-made C18), the residual buffer was evaporated and the pellet was resuspended in 0.1% (v/v) formic acid. Twenty nanogram of the resulting peptides were resolved by reversed-phase liquid chromatography on a 0.075 ⁇ 200-mm fused silica capillaries (J and W) packed with Reprosil reversed phase material (Dr Maisch GmbH, Germany). Peptides were eluted with a linear 60 minutes gradient of 5 to 45% followed by 15 minutes at 95% acetonitrile with 0.1% formic acid in water at flow rates of 0.25 ⁇ L/min.
  • On-line mass spectrometry was performed on an ion-trap mass spectrometer (Orbitrap, Thermo) in a positive mode using repetitively full MS scan followed by collision induced dissociation (CID) of the 7 most dominant ions selected from the first MS scan.
  • the mass spectrometry data was analyzed using the Discoverer software version 1.3 software using a specific protein derived database.
  • the sequence was compared to the peptide sequence of the Etanercept sequence.
  • the identified sequences are presented in Table V, below. Presented is 84.8% coverage of the reference sequence (see green color, FIG. 20 ).

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10730925B2 (en) 2013-03-06 2020-08-04 Protalix Ltd. Chimeric polypeptides, polynucleotides encoding same, cells expressing same and methods of producing same

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2616728T3 (es) 2008-05-23 2017-06-14 Siwa Corporation Procedimientos y composiciones para facilitar la regeneración
US8721571B2 (en) 2010-11-22 2014-05-13 Siwa Corporation Selective removal of cells having accumulated agents
EP2964669B1 (en) 2013-03-06 2018-09-12 Protalix Ltd. Use of plant cells expressing a tnfalpha polypeptide inhibitor in therapy
US10000551B2 (en) 2014-09-11 2018-06-19 Protalix Ltd. Chimeric polypeptides, polynucleotides encoding same, cells expressing same and methods of producing same
IL251210B2 (en) * 2014-09-19 2023-12-01 Siwa Corp Anti-aging antibodies for the treatment of inflammation and autoimmune disorders
US9993535B2 (en) 2014-12-18 2018-06-12 Siwa Corporation Method and composition for treating sarcopenia
US10358502B2 (en) 2014-12-18 2019-07-23 Siwa Corporation Product and method for treating sarcopenia
CN109071675A (zh) 2016-02-19 2018-12-21 Siwa有限公司 使用高级糖化终产物(age)的抗体治疗癌症、杀死转移性癌细胞和预防癌症转移的方法和组合物
CA3057829A1 (en) 2016-04-15 2017-10-19 Siwa Corporation Anti-age antibodies for treating neurodegenerative disorders
EP3475306A1 (en) 2016-06-23 2019-05-01 Siwa Corporation Vaccines for use in treating various diseases and disorders
US10925937B1 (en) 2017-01-06 2021-02-23 Siwa Corporation Vaccines for use in treating juvenile disorders associated with inflammation
US10858449B1 (en) 2017-01-06 2020-12-08 Siwa Corporation Methods and compositions for treating osteoarthritis
US10961321B1 (en) 2017-01-06 2021-03-30 Siwa Corporation Methods and compositions for treating pain associated with inflammation
US10995151B1 (en) 2017-01-06 2021-05-04 Siwa Corporation Methods and compositions for treating disease-related cachexia
US10919957B2 (en) 2017-04-13 2021-02-16 Siwa Corporation Humanized monoclonal advanced glycation end-product antibody
US11518801B1 (en) 2017-12-22 2022-12-06 Siwa Corporation Methods and compositions for treating diabetes and diabetic complications
EP4661892A1 (en) * 2023-02-08 2025-12-17 Navigen, Inc. Tnf-alpha binding agents and methods of using the same

Family Cites Families (75)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL154600B (nl) 1971-02-10 1977-09-15 Organon Nv Werkwijze voor het aantonen en bepalen van specifiek bindende eiwitten en hun corresponderende bindbare stoffen.
NL154598B (nl) 1970-11-10 1977-09-15 Organon Nv Werkwijze voor het aantonen en bepalen van laagmoleculire verbindingen en van eiwitten die deze verbindingen specifiek kunnen binden, alsmede testverpakking.
NL154599B (nl) 1970-12-28 1977-09-15 Organon Nv Werkwijze voor het aantonen en bepalen van specifiek bindende eiwitten en hun corresponderende bindbare stoffen, alsmede testverpakking.
US3901654A (en) 1971-06-21 1975-08-26 Biological Developments Receptor assays of biologically active compounds employing biologically specific receptors
US3853987A (en) 1971-09-01 1974-12-10 W Dreyer Immunological reagent and radioimmuno assay
US3867517A (en) 1971-12-21 1975-02-18 Abbott Lab Direct radioimmunoassay for antigens and their antibodies
NL171930C (nl) 1972-05-11 1983-06-01 Akzo Nv Werkwijze voor het aantonen en bepalen van haptenen, alsmede testverpakkingen.
US3850578A (en) 1973-03-12 1974-11-26 H Mcconnell Process for assaying for biologically active molecules
US3935074A (en) 1973-12-17 1976-01-27 Syva Company Antibody steric hindrance immunoassay with two antibodies
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4034074A (en) 1974-09-19 1977-07-05 The Board Of Trustees Of Leland Stanford Junior University Universal reagent 2-site immunoradiometric assay using labelled anti (IgG)
US3984533A (en) 1975-11-13 1976-10-05 General Electric Company Electrophoretic method of detecting antigen-antibody reaction
US4098876A (en) 1976-10-26 1978-07-04 Corning Glass Works Reverse sandwich immunoassay
US4879219A (en) 1980-09-19 1989-11-07 General Hospital Corporation Immunoassay utilizing monoclonal high affinity IgM antibodies
CA1192510A (en) 1981-05-27 1985-08-27 Lawrence E. Pelcher Rna plant virus vector or portion thereof, a method of construction thereof, and a method of producing a gene derived product therefrom
JPS6054684A (ja) 1983-09-05 1985-03-29 Teijin Ltd 新規ｄνａ及びハイブリツドｄνａ
US5011771A (en) 1984-04-12 1991-04-30 The General Hospital Corporation Multiepitopic immunometric assay
US4666828A (en) 1984-08-15 1987-05-19 The General Hospital Corporation Test for Huntington's disease
US4945050A (en) 1984-11-13 1990-07-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues and apparatus therefor
CA1288073C (en) 1985-03-07 1991-08-27 Paul G. Ahlquist Rna transformation vector
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4801531A (en) 1985-04-17 1989-01-31 Biotechnology Research Partners, Ltd. Apo AI/CIII genomic polymorphisms predictive of atherosclerosis
GB8608850D0 (en) 1986-04-11 1986-05-14 Diatech Ltd Packaging system
JPS6314693A (ja) 1986-07-04 1988-01-21 Sumitomo Chem Co Ltd 植物ウイルスｒｎａベクタ−
ES2060646T3 (es) 1987-02-09 1994-12-01 Lubrizol Genetics Inc Virus rna hibrido.
US5316931A (en) 1988-02-26 1994-05-31 Biosource Genetics Corp. Plant viral vectors having heterologous subgenomic promoters for systemic expression of foreign genes
US5693507A (en) 1988-09-26 1997-12-02 Auburn University Genetic engineering of plant chloroplasts
US5272057A (en) 1988-10-14 1993-12-21 Georgetown University Method of detecting a predisposition to cancer by the use of restriction fragment length polymorphism of the gene for human poly (ADP-ribose) polymerase
US5302523A (en) 1989-06-21 1994-04-12 Zeneca Limited Transformation of plant cells
US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
UA48104C2 (uk) 1991-10-04 2002-08-15 Новартіс Аг Фрагмент днк, який містить послідовність,що кодує інсектицидний протеїн, оптимізовану для кукурудзи,фрагмент днк, який забезпечує направлену бажану для серцевини стебла експресію зв'язаного з нею структурного гена в рослині, фрагмент днк, який забезпечує специфічну для пилку експресію зв`язаного з нею структурного гена в рослині, рекомбінантна молекула днк, спосіб одержання оптимізованої для кукурудзи кодуючої послідовності інсектицидного протеїну, спосіб захисту рослин кукурудзи щонайменше від однієї комахи-шкідника
US5281521A (en) 1992-07-20 1994-01-25 The Trustees Of The University Of Pennsylvania Modified avidin-biotin technique
IL113484A0 (en) 1994-04-28 1995-07-31 Immunex Corp Viral proteins pharmaceutical compositions containing them their preparation and use
US6096871A (en) 1995-04-14 2000-08-01 Genentech, Inc. Polypeptides altered to contain an epitope from the Fc region of an IgG molecule for increased half-life
WO1997034631A1 (en) 1996-03-18 1997-09-25 Board Of Regents, The University Of Texas System Immunoglobin-like domains with increased half lives
IL155588A0 (en) 2003-04-27 2003-11-23 Metabogal Ltd Methods for expression of enzymatically active recombinant lysosomal enzymes in transgenic plant root cells and vectors used thereby
IL119310A (en) 1996-09-26 1999-07-14 Metabogal Ltd Cell/tissue culturing device and method
US20050032211A1 (en) 1996-09-26 2005-02-10 Metabogal Ltd. Cell/tissue culturing device, system and method
US7361331B2 (en) * 1996-10-18 2008-04-22 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Plant bioreactors
AU9013998A (en) 1997-07-21 1999-02-10 Zymogenetics Inc. Tumor necrosis factor receptor ztnfr-5
US6040498A (en) 1998-08-11 2000-03-21 North Caroline State University Genetically engineered duckweed
DE69941261D1 (de) 1998-06-22 2009-09-24 Ca Minister Agriculture & Food Herstellung von il-10 in einen pflanzenbioreaktor aus pflanzen, die nicht zur ernährung verwendet werden
US20010021380A1 (en) 1999-04-19 2001-09-13 Pluenneke John D. Soluble tumor necrosis factor receptor treatment of medical disorders
US20040220103A1 (en) 1999-04-19 2004-11-04 Immunex Corporation Soluble tumor necrosis factor receptor treatment of medical disorders
WO2000062790A2 (en) 1999-04-19 2000-10-26 Immunex Corporation Soluble tumor necrosis factor receptor treatment of medical disorders
US7115733B2 (en) * 2003-07-03 2006-10-03 Unicrop Ltd Rubisco promoters and uses thereof
US6391683B1 (en) 2000-06-21 2002-05-21 Siliconware Precision Industries Co., Ltd. Flip-chip semiconductor package structure and process for fabricating the same
US7449308B2 (en) * 2000-06-28 2008-11-11 Glycofi, Inc. Combinatorial DNA library for producing modified N-glycans in lower eukaryotes
NZ523912A (en) 2000-07-31 2005-03-24 Biolex Inc Expression of biologically active polypeptides in duckweed
US8022270B2 (en) 2000-07-31 2011-09-20 Biolex Therapeutics, Inc. Expression of biologically active polypeptides in duckweed
UA81743C2 (uk) 2000-08-07 2008-02-11 Центокор, Инк. МОНОКЛОНАЛЬНЕ АНТИТІЛО ЛЮДИНИ, ЩО СПЕЦИФІЧНО ЗВ'ЯЗУЄТЬСЯ З ФАКТОРОМ НЕКРОЗУ ПУХЛИН АЛЬФА (ФНПα), ФАРМАЦЕВТИЧНА КОМПОЗИЦІЯ, ЩО ЙОГО МІСТИТЬ, ТА СПОСІБ ЛІКУВАННЯ РЕВМАТОЇДНОГО АРТРИТУ
WO2002080847A2 (en) 2001-04-06 2002-10-17 Immunex Corporation Use of tumor necrosis factor inhibitors to treat cardiovascular disease
CA2450019A1 (en) * 2001-06-08 2002-12-19 Icon Genetics, Inc. Production of proteins in plants
US20030180287A1 (en) 2002-02-27 2003-09-25 Immunex Corporation Polypeptide formulation
KR20050083691A (ko) * 2002-09-13 2005-08-26 더 유니버시티 오브 퀸스랜드 코돈 번역 효율에 기초한 유전자 발현 시스템
EP1613264A4 (en) 2003-04-16 2007-08-15 Yeda Res & Dev MEDICAMENT FOR THE TREATMENT OF GAUCHER DISEASE AND METHOD FOR IDENTIFYING IT
US7951557B2 (en) 2003-04-27 2011-05-31 Protalix Ltd. Human lysosomal proteins from plant cell culture
EP1644507B1 (en) 2003-07-03 2010-03-17 Unicrop Oy Novel rubisco promoters and uses thereof
WO2006040761A2 (en) 2004-10-15 2006-04-20 Oren Halpern A system and a method for improving the captured images of digital still cameras
BRPI0616012A2 (pt) 2005-07-18 2011-05-31 Protalix Ltd administração via mucosa ou intestino de macromoléculas biologicamente ativas
US7919521B2 (en) 2005-12-05 2011-04-05 Academia Sinica Five-membered iminocyclitol derivatives as selective and potent glycosidase inhibitors: new structures for antivirals and osteoarthritis therapeutics
US8007790B2 (en) 2006-04-03 2011-08-30 Stowers Institute For Medical Research Methods for treating polycystic kidney disease (PKD) or other cyst forming diseases
JP5380293B2 (ja) 2006-08-31 2014-01-08 サイモン・フレーザー・ユニバーシティ 選択的グリコシダーゼ阻害剤およびその使用
GB0622702D0 (en) 2006-11-15 2006-12-27 Univ Dundee Selective glycosidase inhibitors
BRPI0810343B1 (pt) 2007-05-07 2021-03-30 Protalix Ltd Dispositivo descartável para o cultivo e colheita de tecidos vegetais e/ou células, método de cultivo e colheita de um tecido vegetal e/ou células vegetais em um volume maior do que 400 litros e sistema de cultivo da célula vegetal
WO2010012107A1 (en) 2008-08-01 2010-02-04 Simon Fraser University Selective glycosidase inhibitors and uses thereof
MX2012004993A (es) * 2009-10-30 2012-06-12 Merck Sharp & Dohme Metodos para la produccion de proteinas recombinantes con eficiencias de secrecion mejoradas.
ES2774190T3 (es) * 2011-01-20 2020-07-17 Protalix Ltd Composiciones de alfa-galactosidasa
US10160789B2 (en) * 2011-03-14 2018-12-25 University Of Louisville Research Foundation, Inc. Polypeptides having immunoactivating activity and methods of producing the same
EP2718328A4 (en) * 2011-06-08 2014-12-24 Acceleron Pharma Inc COMPOSITIONS AND METHODS FOR INCREASING THE HALF TIME OF SERUM
WO2013087911A1 (en) 2011-12-16 2013-06-20 Synthon Biopharmaceuticals B.V. Compounds and methods for treating inflammatory diseases
EP2964669B1 (en) 2013-03-06 2018-09-12 Protalix Ltd. Use of plant cells expressing a tnfalpha polypeptide inhibitor in therapy
BR112015021708A2 (pt) 2013-03-06 2017-08-29 Protalix Ltd Inibidor de polipetídeo do tnfa, polinucleotídeo isolado, estrutura de expressão de ácido nucleico, célula de planta, composição farmacêutica e método de produção de um inibidor de polipeptídeo do tnfa
US10000551B2 (en) 2014-09-11 2018-06-19 Protalix Ltd. Chimeric polypeptides, polynucleotides encoding same, cells expressing same and methods of producing same
US11562502B2 (en) 2015-11-09 2023-01-24 Cognex Corporation System and method for calibrating a plurality of 3D sensors with respect to a motion conveyance

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
Bluml et al. Targeting TNF receptors in rheumatoid arthritis. Int Immunity 24(5): 275-281, 2012 *
by Ilan, Y. A novel method for anti-TNF based-oral immunotherapy: oral administration of a plant cell-expressed recombinant anti-TNF fusion protein for treating of fatty liver disease. AASLD LiverLearning Nov 8, 2014; 60709, Abstract 663. *
Chan et al. Plant-made oral vaccines against human infectious diseases--Are we there yet? Plant Biotechnol 13: 1056-1070, 2015 *
Kwon et al. Oral delivery of protein drugs bioencapsulated in plant cells. Mol Therapy 24(8): 1342-1350, 2016 *
MacEwan, D. TNF ligands and receptors-- a matter of life and death. Brit J Pharmacol 135: 855-875, 2002 *
No author. "Protalix releases preclinical data on anti-TNF follow-on biologic arthritis drug". Health and Beauty Close-Up. 29 September 2009 *
Rosales-Mendoza et al. Immunological aspects of using plant cells as delivery vehicles for oral vaccines. Exp Rev Vaccines 13(6): 737-749, 2014 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10730925B2 (en) 2013-03-06 2020-08-04 Protalix Ltd. Chimeric polypeptides, polynucleotides encoding same, cells expressing same and methods of producing same

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