Long-acting Polypeptides And Methods Of Producing Same

Abstract

A polypeptide and polynucleotides encoding same comprising one carboxy-terminal peptide (CTP) of chorionic gonadotrophin attached to an amino terminus of a cytokine and two carboxy-terminal peptides (CTP) of chorionic gonadotrophin attached to a carboxy terminus of a cytokine are disclosed. Pharmaceutical compositions comprising the polypeptide and polynucleotides of the invention and methods of using same are also disclosed.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This Application is a divisional of U.S. patent application Ser. No. 14/550,346, filed Nov. 21, 2014 which is a divisional of U.S. patent Ser. No. 13/867,728, filed Apr. 22, 2013 and issued as U.S. Pat. No. 8,999,670 Apr. 7, 2015, which is a divisional of U.S. patent application Ser. No. 13/191,478, filed Jul. 27, 2011 and issued as U.S. Pat. No. 8,426,166 Apr. 23, 2013, which is a divisional of U.S. patent application Ser. No. 12/476,916, filed Jun. 2, 2009 and issued as U.S. Pat. No. 8,048,849 Nov. 1, 2011, all of which are hereby incorporated in their entirety by reference herein.

FIELD OF INVENTION

[0002] A polypeptide and polynucleotides encoding same comprising at least three carboxy-terminal peptides (CTP) of chorionic gonadotrophin attached to a cytokine are disclosed. Pharmaceutical compositions comprising the polypeptide and polynucleotides of the invention and methods of using same are also disclosed.

BACKGROUND OF THE INVENTION

[0003] Polypeptides are susceptible to denaturation or enzymatic degradation in the blood, liver or kidney. Accordingly, polypeptides typically have short circulatory half-lives of several hours. Because of their low stability, peptide drugs are usually delivered in a sustained frequency so as to maintain an effective plasma concentration of the active peptide. Moreover, since peptide drugs are usually administrated by infusion, frequent injection of peptide drugs cause considerable discomfort to a subject. Thus, there is a need for technologies that will prolong the half-lives of therapeutic polypeptides while maintaining a high pharmacological efficacy thereof. Such desirous peptide drugs should also meet the requirements of enhanced serum stability, high activity and a low probability of inducing an undesired immune response when injected into a subject.

[0004] Unfavorable pharmacokinetics, such as a short serum half-life, can prevent the pharmaceutical development of many otherwise promising drug candidates. Serum half-life is an empirical characteristic of a molecule, and must be determined experimentally for each new potential drug. For example, with lower molecular weight polypeptide drugs, physiological clearance mechanisms such as renal filtration can make the maintenance of therapeutic levels of a drug unfeasible because of cost or frequency of the required dosing regimen. Conversely, a long serum half-life is undesirable where a drug or its metabolites have toxic side effects.

SUMMARY OF THE INVENTION

[0005] In one embodiment, the present invention provides a polypeptide comprising a cytokine, one chorionic gonadotrophin carboxy terminal peptide (CTP) attached to an amino terminus of the cytokine, and two chorionic gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the cytokine.

[0006] In another embodiment, the present invention further provides a polynucleotide comprising a coding portion encoding a polypeptide, wherein the polypeptide comprises a cytokine, one chorionic gonadotrophin carboxy terminal peptide (CTP) attached to an amino terminus of the cytokine, and two chorionic gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the cytokine.

[0007] In another embodiment, the present invention further provides a method of reducing a dosing frequency of a cytokine, comprising the step of attaching one chorionic gonadotrophin carboxy terminal peptide to an amino terminus of the cytokine and two chorionic gonadotrophin carboxy terminal peptides to a carboxy terminus of the cytokine, thereby reducing a dosing frequency of a cytokine.

[0008] In another embodiment, the present invention further provides a method of increasing compliance in the use of cytokine therapy, comprising providing to a subject in need thereof, a polypeptide comprising a cytokine, one chorionic gonadotrophin carboxy terminal peptide (CTP) attached to an amino terminus of the cytokine, and two chorionic gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the cytokine, thereby increasing compliance in the use of cytokine therapy.

[0009] In another embodiment, the present invention further provides a method of improving a biological half life of a cytokine, comprising the step of attaching one chorionic gonadotrophin carboxy terminal peptide to an amino terminus of the cytokine and two chorionic gonadotrophin carboxy terminal peptides to a carboxy terminus of the cytokine, thereby improving a biological half life of a cytokine.

[0010] In another embodiment, the present invention further provides a method of improving the area under the curve (AUC) of a cytokine, comprising the step of attaching one chorionic gonadotrophin carboxy terminal peptide to an amino terminus of the cytokine and two chorionic gonadotrophin carboxy terminal peptides to a carboxy terminus of the cytokine, thereby improving the area under the curve (AUC) of a cytokine.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGS. 1A-1F are diagrams illustrating six EPO-CTP constructs.

[0012] FIG. 1A is a diagram of the polypeptide of SEQ ID NO: 1.

[0013] FIG. 1B is a diagram of the polypeptide of SEQ ID NO: 2.

[0014] FIG. 1C is a diagram of the polypeptide of SEQ ID NO: 3.

[0015] FIG. 1D is a diagram of the polypeptide of SEQ ID NO: 4.

[0016] FIG. 1E is a diagram of the polypeptide of SEQ ID NO: 5.

[0017] FIG. 1F is a diagram of the polypeptide of SEQ ID NO: 6.

[0018] FIG. 2 is a photograph illustrating the expression of the EPO-CTP variants from transfected DG44 cells. Final test samples from transfected cells were prepared as described under "sample preparation" and run on SDS/PAGE. Proteins were detected by Western Blot.

[0019] FIG. 3 is a graph illustrating the in vivo bioactivity of recombinant hEPO derivatives and EPO-3 (SEQ ID NO: 3). ICR mice (n=7/group) received a single i.v. injection/week (15 .mu.g/kg) for three weeks of EPO-3, rhEPO-WT (SEQ ID NO: 16), Recormon.RTM. (Commercial EPO) or Recormon.RTM. (5 .mu.g/kg) 3 times a week. Control animals were injected i.v. with PBS. Blood samples were collected three times a week and haematocrit levels were detected. Each point represents the group average of haematocrit (%).+-.SE.

[0020] FIG. 4 is a graph illustrating the in vivo bioactivity of recombinant hEPO derivatives and EPO-1 (SEQ ID NO: 1). ICR mice (n=7/group) received a single i.v. injection/week (15 .mu.g/kg) for three weeks of EPO-1, rhEPO-WT (SEQ ID NO: 16), Recormon.RTM. or Recormon.RTM. (5 .mu.g/kg) 3 times a week. Control animals were injected i.v. with PBS. Blood samples were collected three times a week and haematocrit levels were detected. Each point represents the group average of haematocrit (%).+-.SE.

[0021] FIG. 5 is a graph illustrating the in vivo bioactivity of recombinant hEPO derivatives and EPO-2 (SEQ ID NO: 2). ICR mice (n=7/group) received a single i.v. injection/week (15 .mu.g/kg) for three weeks of EPO-2 (SEQ ID NO: 2), rhEPO-WT (SEQ ID NO: 16), Recormon.RTM. or Recormon.RTM. (5 .mu.g/kg) 3 times a week. Control animals were injected i.v. with PBS. Blood samples were collected three times a week and haematocrit levels were detected. Each point represents the group average of haematocrit (%).+-.SE.

[0022] FIG. 6 is a time graph illustrating the change in reticulocyte level following a single bolus dose of EPO-0 (SEQ ID NO: 16), EPO-3 (SEQ ID NO: 3) and Aranesp.RTM..

[0023] FIG. 7 is a time graph illustrating the change in hemoglobin level (presented as change from baseline) following a single bolus dose of EPO-0 (SEQ ID NO: 16), EPO-3 (SEQ ID NO: 3) and Aranesp.RTM..

[0024] FIG. 8 is a time graph illustrating the change in hematocrit level following a single bolus dose of EPO-0 (SEQ ID NO: 16), EPO-3 (SEQ ID NO: 3) and Aranesp.RTM..

[0025] FIG. 9 is a graph illustrating the change in serum concentration of EPO-0 (SEQ ID NO: 16), EPO-3 (SEQ ID NO: 3) and Aranesp.RTM. post i.v. injection.

[0026] FIG. 10 is a Western blot illustrating the molecular weight & identity of MOD-4020 (SEQ ID NO: 36), MOD-4021 (SEQ ID NO: 37), MOD-4022 (SEQ ID NO: 38), MOD-4023 (SEQ ID NO: 39) and MOD-4024 (SEQ ID NO: 40). PAGE SDS gel was blotted and stained using monoclonal anti-hGH antibodies. The photograph indicates that like commercial and wild type hGH, MOD-7020-4 variants are recognized by anti-hGH antibodies.

[0027] FIG. 11 is a bar graph illustrating the weight gain of hypophysectomized rats following administration of the GH-CTP polypeptides of the present invention.

[0028] FIG. 12 is a Western blot illustrating the molecular weight & identity of Avonex.RTM., MOD-9013 (SEQ ID NO: 56), MOD-9016 (SEQ ID NO: 62), MOD-9015 (SEQ ID NO: 60), MOD-9012 (SEQ ID NO: 54), MOD-9011 (SEQ ID NO: 52) and Mock. PAGE SDS gel was blotted and stained using monoclonal anti-IFN-.beta.1A antibodies (B). The photograph indicates that like commercial Avonex.RTM., MOD-901X variants are recognized by anti IFN-31A antibodies.

[0029] FIG. 13 presents mean plasma IFN-.beta.1a or MOD-901x variant concentrations (ng/ml) following single-dose i.v. administration of IFN-.beta.1a or MOD-901x variants in SD rats (n=3 per dose/route/timepoint). IFN-.beta.1a serum concentrations were determined using commercial ELISA kit.

[0030] FIG. 14 presents the MOD-9010 amino acid sequence (SEQ ID NO: 48) (A) and nucleic acid sequence (SEQ ID NO: 49) (B), MOD-9011 amino acid sequence (SEQ ID NO: 52) (C) and nucleic acid sequence (SEQ ID NO: 53) (D), MOD-9012 amino acid sequence (SEQ ID NO: 54) (E) and nucleic acid sequence (SEQ ID NO: 55) (F), MOD-9013 amino acid sequence (SEQ ID NO: 56) (G) and nucleic acid sequence (SEQ ID NO: 57) (H), MOD-9014 amino acid sequence (SEQ ID NO: 58) (I) and nucleic acid sequence (SEQ ID NO: 59) (J), MOD-9015 amino acid sequence (SEQ ID NO: 60) (K) and nucleic acid sequence (SEQ ID NO: 61) (L), and MOD-9016 amino acid sequence (SEQ ID NO: 62) (M) and nucleic acid sequence (SEQ ID NO: 63) (N) amino acid (AA) sequences followed by DNA sequences. Underline: Signal sequence, Black letters: Mature protein, Italic: CTP unit.

[0031] FIG. 15 are graphs showing the mean plasma concentrations (ng/ml) of Rebif.RTM., MOD-9012, and MOD-9013 following single-dose i.v. or s.c. administration of IFN-.beta.1a or MOD-9012, and MOD-9013 in SD rats (n=3 per dose/route/timepoint). IFN-.beta.1a serum concentrations were determined using a commercial ELISA kit.

[0032] FIG. 16 includes two schemes (1) a map of MOD 4023 pCI-dhfr Plasmid and (2) structural protein formula of MOD-4023.

[0033] FIG. 17 are graphs showing the mean plasma MOD-4023 or GH concentrations (pg/ml) following a single i.v. or s.c. dose of MOD-4023 or GH in rats (n=3-6 per dose/route).

[0034] FIG. 18 is a graph showing the mean incremental weight gain following a single s.c. doses of MOD-4023 (0.4, 0.8 and 4 mg/Kg) in hypophysectomized rats in comparison to daily GH injections (0.1 mg/Kg/Day) (n=10 per dose).

[0035] FIG. 19 is a graph showing the area under the curve following single injection of MOD-4023 correlates with body weight gain in rats.

[0036] FIG. 20 is a graph showing the incremental weight gain following s.c. doses of MOD-4023 (0.4, 0.8 and 4 mg/Kg) 4 days apart in hypophysectomized rats in comparison to daily GH injections (0.1 mg/Kg/Day) (n=10 per dose).

[0037] FIG. 21 is a graph showing hGH serum concentration in hypophysectomized rats following s.c. injection of MOD-4023 and commercial hGH. Single dose of MOD-4023 0.6 or 1.8 mg/Kg and Biotropin.RTM. 0.35 or 1.05 mg/Kg were injected subcutaneously to hypophysectomised rats for determination of PK/PD profile. Serum hGH post injection was measured using specific ELISA kits.

[0038] FIG. 22 is a graph showing IGF-1 serum levels in hypophysectomized rats following an s.c. injection of MOD-4023 or commercial hGH. Single dose of MOD-4023 0.6 or 1.8 mg/Kg and Biotropin.RTM. 0.35 or 1.05 mg/Kg were injected subcutaneously to hypophysectomised rats for determination of PK/PD profile. Serum IGF-1 post injection was measured using specific ELISA kits (Roche Diagnostics).

DETAILED DESCRIPTION OF THE INVENTION

[0039] In one embodiment, the present invention provides long-acting cytokines and methods of producing and using same. In another embodiment, long-acting cytokines comprise carboxy terminal peptide (CTP, also referred to as CTP unit). In another embodiment, long-acting polypeptides comprise carboxy terminal peptide (CTP) of human Chorionic Gonadotropin (hCG). In another embodiment, CTP acts as a protectant against degradation of cytokines or polypeptides of interest. In another embodiment, CTP extends the C.sub.max of cytokines or polypeptides of interest. In another embodiment, CTP extends the T.sub.max of cytokines or polypeptides of interest. In another embodiment, CTP extends circulatory half-lives of cytokines or polypeptides of interest. In some embodiments, CTP enhances the potency of cytokines or polypeptides of interest.

[0040] In other embodiments, engineered cytokines or polypeptides of interest of the invention comprising a single CTP attached to the amino terminus and two CTP peptides attached in tandem to the carboxy terminus are at least equivalent to the non CTP-modified cytokines or polypeptides of interest, in terms of biological activity. In other embodiments, engineered cytokines or polypeptides of interest of the invention comprising a single CTP attached to the amino terminus and two CTP peptides attached in tandem to the carboxy terminus are at least equivalent to the non CTP-modified cytokines or polypeptides of interest, in terms of pharmacological measures such as pharmacokinetics and pharmacodynamics.

[0041] In another embodiment, the present invention provides a polypeptide comprising a cytokine and at least one CTP peptide attached to an amino terminus of the cytokine and at least two chorionic gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the cytokine. In another embodiment, the present invention provides a polypeptide comprising one chorionic gonadotrophin carboxy terminal peptide attached to an amino terminus of a cytokine and two chorionic gonadotrophin carboxy terminal peptides attached to a carboxy terminus of a cytokine.

[0042] In another embodiment, the terms "CTP peptide," "carboxy terminal peptide" and "CTP sequence" are used interchangeably herein. In another embodiment, the carboxy terminal peptide is a full-length CTP. In another embodiment, the carboxy terminal peptide is a truncated CTP. Each possibility represents a separate embodiment of the present invention.

[0043] In another embodiment, "signal sequence" and "signal peptide" are used interchangeably herein. In another embodiment, "sequence" when in reference to a polynucleotide can refer to a coding portion. Each possibility represents a separate embodiment of the present invention.

[0044] In another embodiment, the invention provides a polypeptide consisting a cytokine, a single chorionic gonadotrophin carboxy terminal peptide attached to the amino terminus of the cytokine, and two chorionic gonadotrophin carboxy terminal peptides attached to the carboxy terminus of the cytokine. In another embodiment, the invention provides a polypeptide consisting a cytokine, a single chorionic gonadotrophin carboxy terminal peptide attached to the amino terminus of the cytokine, two chorionic gonadotrophin carboxy terminal peptides attached to the carboxy terminus of the cytokine, and a signal peptide attached to the amino terminus of one chorionic gonadotrophin carboxy terminal peptide.

[0045] In another embodiment, a cytokine is a low molecular weight protein. In another embodiment, a cytokine is a protein secreted by a cell. In another embodiment, a cytokine induces and/or regulates an immune response. In another embodiment, a cytokine has a high affinity binding to a specific receptor or receptors. In another embodiment, cytokines as described herein include mimetics of cytokines that can be used to inhibit or potentiate their effects in vivo. In another embodiment, a cytokine comprises an autocrine activity. In another embodiment, a cytokine comprises a paracrine activity. In another embodiment, a cytokine comprises an endocrine activity.

[0046] In another embodiment, the cytokine is a Hematopoietin cytokine. In another embodiment, the cytokine is an Interferon cytokine. In another embodiment, the cytokine is a chemokine. In another embodiment, the cytokine is a Tumor Necrosis Factor cytokine. In another embodiment, a cytokine as used herein comprises biological activity and clinical efficacy. In another embodiment, a cytokine as used herein is a therapeutic protein.

[0047] In another embodiment, a cytokine comprising CTPs as described herein has enhanced biological activity in vivo compared to the same cytokine without CTPs. In another embodiment, a cytokine comprising at least one CTP attached to its amino terminus and at least two CTPs attached to its carboxy terminus has enhanced biological activity in vivo compared to the same cytokine without CTPs. In another embodiment, a cytokine comprising one CTP attached to its amino terminus and two CTPs attached to its carboxy terminus has enhanced biological in vivo activity compared to the same cytokine in without CTPs.

[0048] In another embodiment, a cytokine modified with CTPs is used to facilitate organ transplantation. In another embodiment, a cytokine modified with CTPs is used to reduce inflammation. In another embodiment, a cytokine modified with CTPs is used to induce erythropoiesis. In another embodiment, a cytokine modified with CTPs is used to induce growth. In another embodiment, a cytokine modified with CTPs is used to induce weight gain.

[0049] In another embodiment, a cytokine modified with CTPs is used in cancer therapy as will be readily understood by one of average skill in the art. In another embodiment, a cytokine modified with CTPs is used to induce an immune response. In another embodiment, a cytokine modified with CTPs is used in infectious disease therapy as will be readily understood by one of average skill in the art. In another embodiment, a cytokine modified with CTPs is used in treating allergy as will be readily understood by one of average skill in the art.

[0050] In another embodiment, a subject is a human subject. In another embodiment, a subject is a pet. In another embodiment, a subject is a mammal. In another embodiment, a subject is a farm animal. In another embodiment, a subject is a monkey. In another embodiment, a subject is a horse. In another embodiment, a subject is a cow. In another embodiment, a subject is a mouse. In another embodiment, a subject is a rat.

[0051] In another embodiment, a CTP-cytokine-CTP-CTP as described herein comprises a cytokine or an active fragment thereof connected via a peptide bond to at least one CTP unit. In another embodiment, a CTP-cytokine-CTP-CTP as described herein comprises a cytokine or an active fragment thereof connected via a peptide bond to at least one CTP unit which is connected to an additional CTP unit via a peptide bond. In another embodiment, a polypeptide as described herein comprising cytokine fragments thereof and CTP units and/or fragments thereof are interconnected via a peptide bond. In another embodiment, one nucleic acid molecule encodes a polypeptide as described herein comprising a cytokine and/or fragments thereof and CTP units and/or fragments thereof.

[0052] In one embodiment, the cytokine is a homologue. In one embodiment, a homologue also refers to a deletion, insertion, or substitution variant, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof.

[0053] In another embodiment, the invention provides a polypeptide consisting of a cytokine antagonist, a single chorionic gonadotrophin carboxy terminal peptide attached to the amino terminus of the cytokine antagonist, and two chorionic gonadotrophin carboxy terminal peptides attached to the carboxy terminus of the cytokine antagonist. In another embodiment, the invention provides a polypeptide consisting of a cytokine antagonist, a single chorionic gonadotrophin carboxy terminal peptide attached to the amino terminus of the cytokine antagonist, two chorionic gonadotrophin carboxy terminal peptides attached to the carboxy terminus of the cytokine antagonist, and a signal peptide attached to the amino terminus of one chorionic gonadotrophin carboxy terminal peptide.

[0054] In another embodiment, a cytokine antagonist modified with CTPs is applied as an anti-cytokine strategy. In another embodiment, a cytokine antagonist modified with CTPs is effective in decreasing an inflammatory response. In another embodiment, a cytokine antagonist modified with CTPs is more effective in decreasing an inflammatory response compared to an unmodified cytokine antagonist. In another embodiment, a cytokine antagonist modified with CTPs is more stable than an unmodified cytokine antagonist. In another embodiment, a cytokine antagonist modified with CTPs is more stable in vivo than an unmodified cytokine antagonist. In another embodiment, a cytokine antagonist modified with CTPs is more bioactive than an unmodified cytokine antagonist.

[0055] In another embodiment, a cytokine antagonist is a cytokine homologue. In another embodiment, a cytokine antagonist is a soluble fragment of a cytokine receptor. In another embodiment, a cytokine antagonist is a chemokine receptor homologue.

[0056] In another embodiment, a cytokine as described herein is involved in cytokine signaling cascade comprising Ras-MAP kinase pathway. In another embodiment, a cytokine as described herein is involved in induction of JNK. In another embodiment, a cytokine as described herein is involved in induction of p38MAP. In another embodiment, a cytokine as described herein induces cell proliferation. In another embodiment, a cytokine as described herein is involved in cytokine signaling cascade comprising the JAK/STAT pathway. In another embodiment, a cytokine as described herein induces cell growth inhibition. In another embodiment, a cytokine as described herein induces differentiation.

[0057] In another embodiment, a cytokine as described herein is a four .alpha.-helix bundle cytokine. In another embodiment, a cytokine as described herein is a long-chain 4-helix bundle cytokine. In another embodiment, a cytokine as described herein is a short-chain 4-helix bundle cytokine.

[0058] In another embodiment, a cytokine as described herein is a beta-trefoil cytokine. In another embodiment, a cytokine as described herein is a beta-sandwich cytokine. In another embodiment, a cytokine as described herein is an EGF-like cytokine. In another embodiment, a cytokine as described herein comprises a Cystine knot dimerization domain. In another embodiment, a cytokine as described herein comprises both alpha and beta chains. In another embodiment, a cytokine as described herein is an alpha superfamily cytokine such as IL-2, IL-4, IL-5, GM-CSF, IL-3, IFN-alpha, or IL-13. In another embodiment, a cytokine as described herein is a dimeric 4-helix bundles cytokine. In another embodiment, a cytokine as described herein is a member of the IL family of cytokines.

[0059] In another embodiment, a cytokine as described herein is a long-chain 4-helix bundle superfamily cytokine such as GH, G-CSF, Myelomonocytic growth factor, IL-6, IL-3, IL-7, LIF, Oncostatin M, Ciliary neurotrophic factor (CNTF), or cholinergic differentiation factor (CDF). In another embodiment, a cytokine as described herein is a short-chain 4-helix bundle superfamily cytokine such as IL-2, IL-4, IL-13, IFN-alpha, IL-5, GM-CSF, IL-3, or Macrophage colony-stimulating factor (M-CSF). In another embodiment, a cytokine as described herein is a dimeric 4-helix bundles such as IFN-Gamma, IL-10, or IFN-Beta.

[0060] In another embodiment, a cytokine as described herein is a Beta-Trefoil cytokine such as IL1-A, IL1-B, or FGF. In another embodiment, a cytokine as described herein is a Beta-sandwich cytokine such as TNF-alpha or TNF-Beta. In another embodiment, a cytokine as described herein is an EGF-like cytokine such as TGF-Alpha. In another embodiment, a cytokine as described herein comprises cystine knot dimerization domains. In another embodiment, Gonadotropin, Nerve Growth Factor (NGF), Platelet-derived growth factor (PDGF), and TGF-Beta2 comprise cystine knot dimerization domains. In another embodiment, a cytokine as described herein comprises both alpha and beta chains. In another embodiment, IL-8, IP10, platelet factor 4 (PF-4), bTG, GRO, 9E3, HLA-A2, macrophage inflammatory protein 1 alpha (MIP-1 alpha), macrophage inflammatory protein 1 beta (MIP-1 beta), and Melanoma growth stimulating activity (MGSA) comprise both alpha and beta chains.

[0061] In another embodiment, a cytokine as described herein binds a hematopoietin-receptor family member (also called the class I cytokine receptor family). In another embodiment, a cytokine as described herein binds a class II cytokine receptor (interferons or interferon-like cytokines). In another embodiment, a cytokine as described herein binds a tumor necrosis factor-receptor (TNFR). In another embodiment, a cytokine as described herein binds a chemokine receptor. In another embodiment, a cytokine as described herein binds a G protein-coupled receptor.

[0062] In another embodiment, a cytokine as described herein is an IL-2 cytokine. In another embodiment, a cytokine as described herein is an interferon. In another embodiment, a cytokine as described herein is an IL-10 cytokine. In another embodiment, a cytokine as described herein is EPO. In another embodiment, a cytokine as described herein is thrombopoietin (THPO). In another embodiment, a cytokine as described herein is IL-1, IL-18, or IL-17. In another embodiment, a cytokine as described herein promotes proliferation of T-cells.

[0063] In another embodiment, a cytokine as described herein is a member of the superfamily of growth hormone (GH)-like cytokines. In another embodiment, a cytokine as described herein is close to the cluster formed by ciliary neurotrophic factor and granulocyte colony-stimulating factor (CSF).

[0064] In another embodiment, a cytokine as described herein enhances cytokine responses, type 1 (IFN-.gamma., TGF-.beta. etc.). In another embodiment, a cytokine as described herein enhances antibody responses, type 2 (IL-4, IL-10, IL-13, etc).

[0065] In another embodiment, a cytokine is a peptide. In another embodiment, the cytokine is glycosylated. In another embodiment, a cytokine is a polypeptide. In another embodiment, a cytokine as described herein is a modified cytokine comprising at least one CTP peptide attached to an amino terminus of said cytokine and at least two chorionic gonadotrophin carboxy terminal peptides attached to a carboxy terminus of said cytokine. In another embodiment, a cytokine as described herein is a modified cytokine consisting of a cytokine, one CTP peptide attached to an amino terminus of the cytokine, and at least two chorionic gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the cytokine. In another embodiment, a cytokine as described herein is a modified cytokine consisting of a cytokine, at least one CTP peptide attached to an amino terminus of the cytokine, and two chorionic gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the cytokine. In another embodiment, a cytokine as described herein is a modified cytokine consisting of a cytokine, one CTP peptide attached to an amino terminus of the cytokine, and two chorionic gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the cytokine.

[0066] In another embodiment, the carboxy-terminal peptide (CTP) is attached to the cytokine via a linker. In another embodiment, the linker which connects the CTP sequence to the cytokine is a covalent bond. In another embodiment, the linker which connects the CTP sequence to the cytokine is a peptide bond. In another embodiment, the linker which connects the CTP sequence to the cytokine is a substituted peptide bond. In another embodiment, the carboxy-terminal peptide (CTP) sequence comprises an amino acid sequence selected from the sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 18.

[0067] In another embodiment, SEQ ID NO: 17 comprise the following amino acid (AA) sequence: DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 17). In another embodiment, SEQ ID NO: 18 comprise the following amino acid (AA) sequence: SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 18).

[0068] In another embodiment, the carboxy terminal peptide (CTP) peptide of the present invention comprises the amino acid sequence from amino acid 112 to position 145 of human chorionic gonadotrophin, as set forth in SEQ ID NO: 17. In another embodiment, the CTP sequence of the present invention comprises the amino acid sequence from amino acid 118 to position 145 of human chorionic gonadotropin, as set forth in SEQ ID NO: 18. In another embodiment, the CTP sequence also commences from any position between positions 112-118 and terminates at position 145 of human chorionic gonadotrophin. In some embodiments, the CTP sequence peptide is 28, 29, 30, 31, 32, 33 or 34 amino acids long and commences at position 112, 113, 114, 115, 116, 117 or 118 of the CTP amino acid sequence.

[0069] In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 1-5 conservative amino acid substitutions as described in U.S. Pat. No. 5,712,122 which is incorporated herein by reference. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 1 conservative amino acid substitution. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 2 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 3 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 4 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 5 conservative amino acid substitutions. In another embodiment, the CTP peptide amino acid sequence of the present invention is at least 70% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence of the present invention is at least 80% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence of the present invention is at least 90% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence of the present invention is at least 95% homologous to the native CTP amino acid sequence or a peptide thereof.

[0070] In another embodiment, the CTP peptide DNA sequence of the present invention is at least 70% homologous to the native human CTP DNA sequence or a peptide thereof. In another embodiment, the CTP peptide DNA sequence of the present invention is at least 80% homologous to the native human CTP DNA sequence or a peptide thereof. In another embodiment, the CTP peptide DNA sequence of the present invention is at least 90% homologous to the native CTP DNA sequence or a peptide thereof. In another embodiment, the CTP peptide DNA sequence of the present invention is at least 95% homologous to the native CTP DNA sequence or a peptide thereof.

[0071] In one embodiment, at least one of the chorionic gonadotrophin CTP amino acid sequences is truncated. In another embodiment, both of the chorionic gonadotrophin CTP amino acid sequences are truncated. In another embodiment, 2 of the chorionic gonadotrophin CTP amino acid sequences are truncated. In another embodiment, 2 or more of the chorionic gonadotrophin CTP amino acid sequences are truncated. In another embodiment, all of the chorionic gonadotrophin CTP amino acid sequences are truncated. In one embodiment, the truncated CTP comprises the first 10 amino acids of SEQ ID NO: 67. In another embodiment, SEQ ID NO: 67 comprises the following amino acid (AA) sequence: SSSSKAPPPSLP.

[0072] In one embodiment, the truncated CTP comprises the first 11 amino acids of SEQ ID NO:43. In one embodiment, the truncated CTP comprises the first 12 amino acids of SEQ ID NO: 67. In one embodiment, the truncated CTP comprises the first 8 amino acids of SEQ ID NO: 67. In one embodiment, the truncated CTP comprises the first 13 amino acids of SEQ ID NO: 43. In one embodiment, the truncated CTP comprises the first 14 amino acids of SEQ ID NO: 67. In one embodiment, the truncated CTP comprises the first 6 amino acids of SEQ ID NO: 67. In one embodiment, the truncated CTP comprises the first 5 amino acids of SEQ ID NO: 67.

[0073] In one embodiment, at least one of the chorionic gonadotrophin CTP amino acid sequences is glycosylated. In another embodiment, both of the chorionic gonadotrophin CTP amino acid sequences are glycosylated. In another embodiment, 2 of the chorionic gonadotrophin CTP amino acid sequences are glycosylated. In another embodiment, 2 or more of the chorionic gonadotrophin CTP amino acid sequences are glycosylated. In another embodiment, all of the chorionic gonadotrophin CTP amino acid sequences are glycosylated. In one embodiment, the CTP sequence of the present invention comprises at least one glycosylation site. In one embodiment, the CTP sequence of the present invention comprises 2 glycosylation sites. In one embodiment, the CTP sequence of the present invention comprises 3 glycosylation sites. In one embodiment, the CTP sequence of the present invention comprises 4 glycosylation sites.

[0074] In another embodiment, at least one carboxy-terminal peptide (CTP) sequence comprises an amino acid sequence selected from the sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 18. In another embodiment, at least one carboxy-terminal peptide (CTP) is truncated.

[0075] In another embodiment, the cytokine further comprises a signal peptide for its secretion. In some embodiments, signal sequences include, but are not limited to the endogenous signal sequence for IFN. In some embodiments, signal sequences include, but are not limited to the endogenous signal sequence of any known cytokine. In another embodiment, the polypeptides and methods of the present invention provide a cytokine having additionally a signal peptide of SEQ ID NO: 64 and at least one CTP peptide on the N-terminus and at least one CTP peptide on the C-terminus. In another embodiment, the polypeptides and methods of the present invention provide a cytokine having additionally on the N-terminus the signal peptide of SEQ ID NO: 64 and at least one CTP peptide on the N-terminus and at least two CTP peptides on the C-terminus. In another embodiment, the polypeptides and methods of the present invention provide a cytokine having additionally on the N-terminus the signal peptide of SEQ ID NO: 64 and a single CTP peptide on the N-terminus and two CTP peptides on the C-terminus. In another embodiment, SEQ ID NO: 64 comprise the following amino acid (AA) sequence: MTNKCLLQIALLLCFSTTALS (SEQ ID NO: 64).

[0076] In some embodiments, CTP sequences are attached at both the amino terminal end of a cytokine and at the carboxy terminal end of the cytokine provide enhanced protection against degradation of a cytokine. In another embodiment, at least one CTP is attached sequence at the amino terminal end of a cytokine and two CTP are attached units at the carboxy terminal end of a cytokine provide enhanced protection against clearance. In another embodiment, at least one CTP is attached sequence at the amino terminal end of a cytokine and two CTP are attached units at the carboxy terminal end of a cytokine provide prolonged clearance time. In another embodiment, at least one CTP sequence is attached at the amino terminal end of a cytokine and two CTP units are attached at the carboxy terminal end of a cytokine enhance C.sub.max of a cytokine.

[0077] In another embodiment, at least one CTP sequence is attached at the amino terminal end of a cytokine and two CTP units are attached at the carboxy terminal end of a cytokine enhance T.sub.max of a cytokine. In another embodiment, at least one CTP sequence is attached at the amino terminal end of a cytokine and two CTP units are attached at the carboxy terminal end of a cytokine enhanced T.sub.1/2.

[0078] In some embodiments, CTP sequences at both the amino terminal end of a cytokine and at the carboxy terminal end of the cytokine extend the half-life of the attached cytokine. In another embodiment, at least a single CTP sequence at the amino terminal end of a cytokine and at least two CTP sequences at the carboxy terminal end of the cytokine provide an extended half-life to the modified cytokine. In another embodiment, a single CTP sequence at the amino terminal end of a cytokine and two CTP sequences at the carboxy terminal end of the cytokine provide an extended half-life to the attached cytokine. In another embodiment, a single CTP sequence at the amino terminal end of a cytokine and two CTP sequences in tandem at the carboxy terminal end of the cytokine provide an extended half-life to the cytokine.

[0079] In some embodiments, a CTP sequence at the amino terminal end of a polypeptide, a CTP sequence at the carboxy terminal end of the cytokine, and at least one additional CTP sequence attached in tandem to the CTP sequence at the carboxy terminus provide enhanced protection against degradation to a cytokine. In some embodiments, a CTP sequence at the amino terminal end of a cytokine, a CTP sequence at the carboxy terminal end of the cytokine, and at least one additional CTP sequence attached in tandem to the CTP sequence at the carboxy terminus extend the half-life of the cytokine. In some embodiments, a CTP sequence at the amino terminal end of a cytokine, a CTP sequence at the carboxy terminal end of the cytokine, and at least one additional CTP sequence attached in tandem to the CTP sequence at the carboxy terminus enhance the biological activity of the cytokine.

[0080] In another embodiment, conjugated cytokines of this invention are used in the same manner as unmodified cytokines. In another embodiment, conjugated cytokines of this invention have an increased circulating half-life and plasma residence time, decreased clearance, and increased clinical activity in vivo. In another embodiment, due to the improved properties of the conjugated cytokines as described herein, these conjugates are administered less frequently than unmodified cytokines. In another embodiment, conjugated cytokines as described herein are administered once a week instead of the three times a week for unmodified cytokines. In another embodiment, decreased frequency of administration will result in improved patient compliance leading to improved treatment outcomes, as well as improved patient quality of life. In another embodiment, compared to conventional conjugates of cytokines linked to poly(ethylene glycol), it has been found that conjugates having the molecular weight and linker structure of the conjugates of this invention have an improved potency, improved stability, elevated AUC levels, and enhanced circulating half-life. In another embodiment, compared to conventional conjugates of cytokines linked to poly(ethylene glycol) it has been found that EPO having the molecular weight and linker structure of the conjugates of this invention have an improved potency, improved stability, elevated AUC levels, enhanced circulating half-life. In another embodiment, a therapeutically effective amount of a conjugated cytokine is the amount of conjugate necessary for the in vivo measurable expected biological activity. In another embodiment, a therapeutically effective amount of a conjugated EPO is the amount of EPO conjugate necessary for the biological activity of inducing bone marrow cells to increase production of reticulocytes and red blood cells. In another embodiment, a therapeutically effective amount of a conjugated cytokine is determined according to factors as the exact type of condition being treated, the condition of the patient being treated, as well as the other ingredients in the composition. In another embodiment, a therapeutically effective amount of a conjugated cytokine is 0.01 to 10 .mu.g per kg body weight administered once a week. In another embodiment, a therapeutically effective amount of a conjugated cytokine is 0.1 to 1 .mu.g per kg body weight, administered once a week. In another embodiment, a pharmaceutical composition comprising a conjugated cytokine is formulated at a strength effective for administration by various means to a human patient.

[0081] In another embodiment, the cytokine is an interferon. In another embodiment, the IFN amino acid sequence of the present invention is at least 60% homologous to an IFN sequence set forth in GenBank Accession No. NP_002167.1, as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. In another embodiment, the IFN amino acid sequence of the present invention is at least 70% homologous to an IFN sequence set forth in GenBank Accession No. NP_002167.1, as determined using BlastP software of the NCBI using default parameters. In another embodiment, the IFN amino acid sequence of the present invention is at least 80% homologous to an IFN sequence set forth in GenBank Accession No. NP_002167.1, as determined using BlastP software of the NCBI using default parameters. In another embodiment, the IFN amino acid sequence of the present invention is at least 90% homologous to an IFN sequence set forth in GenBank Accession No. NP_002167.1, as determined using BlastP software of the NCBI using default parameters.

[0082] In some embodiments, "homology" according to the present invention also encompasses deletions, insertions, or substitution variants, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof. In one embodiment the substitution variant is one, in which the glutamine in position 65 of hGH is substituted by a valine (SEQ ID NO: 23) [Gellerfors et al., J Pharm Biomed Anal 1989, 7:173-83].

[0083] In another embodiment, a cytokine utilized according to the teachings of the present invention exhibits increased potency. In some embodiments, the attachment of CTP sequence to both the amino and carboxy termini of a cytokine results in prolonged in vivo activity.

[0084] In one embodiment, the term "interferon" refers to the mammalian interferon polypeptide (e.g., Type I). In one embodiment, "interferon" refers to the mammalian interferon polypeptide (Type II interferon) which exhibits an interferon activity, e.g. antiviral or antiproliferative activity. In another embodiment, GenBank accession numbers of non-limiting examples of interferons are listed in Table 1 below. An interferon of the present invention also refers in one embodiment, to homologs (e.g., polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95% or more say 100% homologous to interferon sequences listed in Table 1 as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters). In another embodiment, homolog may also refer to a deletion, insertion, or substitution variant, including an amino acid substitution thereof and biologically active polypeptide fragments thereof. In some embodiments, additional suitable interferon polypeptides are as known to those of ordinary skill in the art. In some embodiments, the interferon is a Type I or Type II interferon, including those commonly designated as alpha-interferon. In some embodiments, the interferon is beta-interferon. In some embodiments, the interferon is gamma-interferon. In some embodiments, the interferon is omega-interferon. In another embodiment, the interferon is a subspecies of interferon such as a Type I or Type II interferon. In one embodiment, the subspecies of interferon (IFN) is IFN-.alpha.2a. In one embodiment, the subspecies of interferon (IFN) is IFN-.alpha.2b. In one embodiment, the subspecies of interferon (IFN) is IFN-.beta.1a and. In one embodiment, the interferon (IFN) subspecies is IFN-.beta.1b.

[0085] In some embodiments, GenBank Accession Nos. of non-limiting examples of interferons are listed in Table 1 below.

[0086] In one embodiment, an interferon of the present invention also refers to a homologue. In one embodiment, an interferon amino acid sequence of the present invention is at least 50% homologous to interferon sequences listed in Table 1 as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. In one embodiment, an interferon amino acid sequence of the present invention is at least 60% homologous interferon sequences listed in Table 1 as determined using BlastP software of the NCBI using default parameters. In one embodiment, an interferon amino acid sequence of the present invention is at least 70% homologous interferon sequences listed in Table 1 as determined using BlastP software of the NCBI using default parameters. In one embodiment, an interferon amino acid sequence of the present invention is at least 80% homologous to interferon sequences listed in Table 1 as determined using BlastP software of the NCBI using default parameters. In one embodiment, an interferon amino acid sequence of the present invention is at least 90% homologous to interferon sequences listed in Table 1 as determined using BlastP software of the NCBI using default parameters. In one embodiment, an interferon amino acid sequence of the present invention is at least 95% homologous interferon sequences listed in Table 1 as determined using BlastP software of the NCBI using default parameters. In some embodiments, homology according to the present invention also encompasses deletions, insertions, or substitution variants, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof. In one embodiment the cysteine in position 17 of interferon 3 is substituted by a Serine (SEQ ID NO: 24).

[0087] Table 1 below lists examples of interferons with their respective NCBI sequence numbers.

TABLE-US-00001 TABLE 1 Interferon name NCBI sequence number interferon, .alpha.1 NP_076918.1 interferon, .alpha.10 NP_002162.1 interferon, .alpha.13 NP_008831.2 interferon, .alpha.14 NP_002163.1 interferon, .alpha.16 NP_002164.1 interferon, .alpha.17 NP_067091.1 interferon, .alpha.2 NP_000596.2 interferon, .alpha.21 NP_002166.1 interferon, .alpha.4 NP_066546.1 interferon, .alpha.5 NP_002160.1 interferon, .alpha.6 NP_066282.1 interferon, .alpha.7 NP_066401.2 interferon, .alpha.8 NP_002161.2 interferon, .beta.1 NP_002167.1 interferon, .epsilon.1 NP_795372.1 interferon, .gamma. NP_000610.2 interferon, .epsilon. NP_064509.1 interferon, .OMEGA.1 NP_002168.1

[0088] In another embodiment, a method of treating or reducing a disease treatable or reducible by a cytokine or a pharmaceutical formulation comprising the same, in a subject, comprises the step of administering to a subject a therapeutically effective amount of the polypeptide comprising a cytokine and CTP units as described herein, thereby treating or reducing a disease treatable or reducible by a cytokine in a subject.

[0089] In another embodiment, a method of treating or reducing a disease treatable or reducible by an interferon or a pharmaceutical formulation comprising the same, in a subject, comprises the step of administering to a subject a therapeutically effective amount of the polypeptide comprising IFN protein and CTP units as described herein, thereby treating or reducing a disease treatable or reducible by an interferon in a subject.

[0090] In another embodiment, a disease treatable or reducible by an interferon is Hepatitis C infection, cancer, bacterial infection, viral infection, injury, multiple sclerosis, hairy cell leukemia, malignant melanoma, Kaposi's sarcoma, bladder cancer, chronic myelocytic leukemia, kidney cancer, carcinoid tumors, non-Hodgkin's lymphoma, ovarian cancer, skin chronic Hepatitis C (CHC), condylomata acuminata (CA), chronic Hepatitis B, follicular non-Hodgkin's lymphoma, chronic granulomatous disease, Mycobacterium avium complex (MAC), pulmonary fibrosis osteoarthritis, and osteoporosis.

[0091] In another embodiment, polypeptides of the present invention comprising IFN .alpha.-2a as well as pharmaceutical compositions comprising the same are indicated for hairy cell leukemia (HCL), acquired immune deficiency syndrome(AIDS)-related Kaposi's sarcoma (KS), chronic-phase Philadelphia (Ph) chromosome-positive chronic myelogenous leukemia (CML) and chronic Hepatitis C (CHC). IFN .alpha.-2a dosage varies depending on the indication. In another embodiment, the effectiveness of IFN .alpha.2a as an antineoplastic, immunomodulator and antiviral agent has been established.

[0092] In another embodiment, polypeptides of the present invention comprising IFN .alpha.-2b as well as pharmaceutical compositions comprising the same are indicated for HCL, AIDS-related Kaposi's sarcoma and CHC. It is also indicated for condylomata acuminata (CA), chronic Hepatitis B, malignant melanoma and follicular non-Hodgkin's lymphoma. IFN .alpha.-2b dosage varies depending on its indication of usage.

[0093] In another embodiment, a polypeptide comprising an IFN protein, at least a single CTP attached to its carboxy terminus, and at least a single CTP attached to its amino terminus is used to trigger an immune response. In another embodiment, a polypeptide comprising an IFN protein, a single CTP attached to its amino terminus, and at least two CTP units attached to its carboxy terminus is used to trigger an immune response. In another embodiment, a polypeptide comprising an IFN protein, a single CTP attached to its amino terminus, and two CTP units attached to its carboxy terminus is used to trigger an immune response. In another embodiment, a polypeptide comprising an IFN protein and CTP units is formulated in a pharmaceutical composition that is administered to a subject in need of triggering an immune response.

[0094] In another embodiment, a polypeptide comprising an IFN protein and CTP units as described herein is used to trigger an immune response against a viral infection. In another embodiment, a polypeptide comprising an IFN protein and CTP units is formulated in a pharmaceutical composition that is administered to a subject in need of triggering an immune response against a viral infection.

[0095] In another embodiment, a polypeptide comprising an IFN 3 and CTP units as described herein is used to trigger an immune response via the enhancement of activity of lymphocyte cells. In another embodiment, a polypeptide comprising an IFN 3 and CTP units is formulated in a pharmaceutical composition that is administered to a subject in need of triggering an immune response via the enhancement of activity of lymphocyte cells.

[0096] In another embodiment, a polypeptide comprising a cytokine and CTP units as described herein is used as an anti-tumor agent. In another embodiment, a polypeptide comprising an IFN .alpha. and CTP units as described herein is used as an anti-tumor agent. In another embodiment, a polypeptide comprising an IFN .alpha. and CTP units is formulated in a pharmaceutical composition that is administered to a patient afflicted with cancer.

[0097] In another embodiment, a polypeptide comprising an IFN protein and CTP units as described herein is used equivalently to a regular or a recombinant interferon as known to one of average skill in the art. In another embodiment, a polypeptide comprising an IFN protein and CTP units is formulated equivalently to a regular or a recombinant interferon as known to one of average skill in the art.

[0098] In another embodiment, a polypeptide comprising a cytokine and CTP units as described herein modulates an immune response. In another embodiment, a polypeptide comprising a cytokine and CTP units as described herein modulates a cellular immune response. In another embodiment, a polypeptide comprising a cytokine and CTP units as described herein modulates an antibody immune response. In another embodiment, a polypeptide comprising a cytokine and CTP units as described herein inhibits an immune response as described herein. In another embodiment, a polypeptide comprising a cytokine and CTP units as described herein trigger an immune response as described herein.

[0099] In another embodiment, a polypeptide comprising an IFN inhibits the activity of T-cells, while simultaneously reducing the production cytokines that operate in the inflammatory response to infection and injury. In another embodiment, a polypeptide comprising an IFN protein and CTP units as described herein enhances the activity of T-cells, while simultaneously reducing the production of cytokines that operate in the inflammatory response to infection and injury. In another embodiment, a polypeptide comprising an IFN protein and CTP units is formulated in a pharmaceutical composition that is administered to a patient in need of T-cell activity enhancement. In another embodiment, a polypeptide comprising an IFN protein and CTP units is formulated in a pharmaceutical composition that is administered to a patient afflicted with multiple sclerosis. In another embodiment, a polypeptide comprising an IFN protein and CTP units is formulated in a pharmaceutical composition that is administered to a patient afflicted with a Hepatitis C infection.

[0100] In another embodiment, a cytokine is an interferon (IFN). In another embodiment, a cytokine is a type I interferon. In another embodiment, the interferon (IFN) is IFN-.alpha.. In another embodiment, the interferon (IFN) is IFN-3. In another embodiment, the interferon (IFN) is IFN-.gamma.. In another embodiment, an interferon (IFN) as described herein comprises an amino acid sequence as described herein, including the sequences provided in FIGS. 14A, 14C, 14E, 14G, 14I, 14K and 14M. In another embodiment, a polypeptide of the invention comprising an interferon (IFN) peptide and at least one CTP unit attached to an amino and/or a carboxy terminus of the polypeptide as described herein comprises an amino acid sequence as described herein, including the sequences provided in FIGS. 14A, 14C, 14E, 14G, 14I, 14K and 14M. In another embodiment, an interferon (IFN) peptide as described herein comprises an amino acid sequence set forth in SEQ ID NO: 48. In another embodiment, SEQ ID NO: 48 comprises the following amino acid (AA) sequence:

TABLE-US-00002 (SEQ ID NO: 48, Human Interferon-.beta. 1a-MOD-9010) MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLE YCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGW NETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN.

[0101] In another embodiment, an interferon (IFN) peptide as described herein comprises an amino acid sequence of human interferon .beta.1a (hIFN .beta.1a). In another embodiment, an interferon (IFN) peptide as described herein comprises an amino acid sequence set forth in GenBank Accession No. NP_002167.1

[0102] In another embodiment, an interferon (IFN) as described herein is encoded by a nucleic acid sequence set forth in SEQ ID NO: 49. In another embodiment, SEQ ID NO: 49 comprises the following nucleic acid (NA) sequence: tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgtgcttcagcaccac- cgccctgagcatgagctacaacctg ctgggcttcctgcagaggtccagcaacttccagtgccagaagctgctgtggcagctgaacggcaggctggaat- actgcctgaaggac aggatgaacttcgacatcccagaggaaatcaagcagctgcagcagttccagaaggaggacgccgccctgacca- tctacgagatgct gcagaacatcttcgccatcttcaggcaggacagcagcagcaccggctggaacgagaccatcgtggagaacctg- ctggccaacgtgt accaccagatcaaccacctgaaaaccgtgctggaagagaagctggaaaaggaggacttcaccaggggcaagct- gatgagcagcct gcacctgaagaggtactacggcagaatcctgcactacctgaaggccaaggagtacagccactgcgcctggacc- atcgtgagggtgg agatcctgaggaacttctacttcatcaacaggctgaccggctacctgaggaactgatgagtccgcggccgc (SEQ ID NO: 49, Human Interferon-.beta.1a-MOD-9010). In another embodiment, an interferon (IFN) peptide as described herein is encoded by a nucleic acid (NA) molecule of human interferon .beta.1a (hIFN .beta.1a). In another embodiment, an interferon (IFN) peptide as described herein is encoded by a nucleic acid (NA) molecule comprising a nucleic acid sequence set forth in GenBank Accession No. NM_002176.

[0103] In another embodiment, an interferon (IFN) peptide as described herein comprises an amino acid sequence set forth in SEQ ID NO: 50. In another embodiment, SEQ ID NO: 50 comprises the following amino acid (AA) sequence: TF*LQPFEAFALAQQVVGDT VRVVNMTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYC LKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLAN VYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVR VEILRNFYFINRLTGYLRN (SEQ ID NO: 50).

[0104] In another embodiment, an interferon (IFN) peptide as described herein is encoded by a nucleic acid sequence set forth in SEQ ID NO: 51. In another embodiment, SEQ ID NO: 51 comprises the following nucleic acid (NA) sequence:

TABLE-US-00003 (SEQ ID NO: 51) acattctaactgcaacctttcgaagcctttgctctggcacaacaggtagt aggcgacactgttcgtgttgtcaacatgaccaacaagtgtctcctccaaa ttgctctcctgttgtgcttctccactacagctctttccatgagctacaac ttgcttggattcctacaaagaagcagcaattttcagtgtcagaagctcct gtggcaattgaatgggaggcttgaatactgcctcaaggacaggatgaact ttgacatccctgaggagattaagcagctgcagcagttccagaaggaggac gccgcattgaccatctatgagatgctccagaacatctttgctattttcag acaagattcatctagcactggctggaatgagactattgttgagaacctcc tggctaatgtctatcatcagataaaccatctgaagacagtcctggaagaa aaactggagaaagaagatttcaccaggggaaaactcatgagcagtctgca cctgaaaagatattatgggaggattctgcattacctgaaggccaaggagt acagtcactgtgcctggaccatagtcagagtggaaatcctaaggaacttt tacttcattaacagacttacaggttacctccgaaactga.

[0105] In another embodiment, the cytokine as described herein comprises a cytokine and at least three CTP units. In another embodiment, the polypeptide as described herein comprises an interferon (IFN) peptide and three CTP units. In another embodiment, the cytokine as described herein comprises an interferon (IFN) peptide-CTP polypeptide encoded by an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 52. In another embodiment, SEQ ID NO: 52 comprises the following amino acid (AA) sequence:

TABLE-US-00004 (SEQ ID NO: 52, MOD-9011) MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLE YCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGW NETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNSSSSKAPPPSLPS PSRLPGPSDTPILPQ.

[0106] In another embodiment, the cytokine as described herein comprising an interferon (IFN) peptide- and CTP is encoded by a nucleic acid molecule set forth in SEQ ID NO: 53. In another embodiment, SEQ ID NO: 53 comprises the following acid (NA) sequence: tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgtgcttcagcaccaccgccctgagc- atgagctacaacctg ctgggcttcctgcagaggtccagcaacttccagtgccagaagctgctgtggcagctgaacggcaggctggaat- actgcctgaaggac aggatgaacttcgacatcccagaggaaatcaagcagctgcagcagttccagaaggaggacgccgccctgacca- tctacgagatgct gcagaacatcttcgccatcttcaggcaggacagcagcagcaccggctggaacgagaccatcgtggagaacctg- ctggccaacgtgt accaccagatcaaccacctgaaaaccgtgctggaagagaagctggaaaaggaggacttcaccaggggcaagct- gatgagcagcct gcacctgaagaggtactacggcagaatcctgcactacctgaaggccaaggagtacagccactgcgcctggacc- atcgtgagggtgg agatcctgaggaacttctacttcatcaacaggctgaccggctacctgaggaacagctccagcagcaaggcccc- tccaccttccctgcc cagtccaagccgactccctgggccctccgatacaccaattctgccacagtgatga (SEQ ID NO: 53, MOD-9011).

[0107] In another embodiment, the cytokine as described herein comprises an interferon (IFN) peptide and two CTP units attached to its carboxy terminus. In another embodiment, the polypeptide as described herein comprises an interferon (IFN) peptide-CTP (.times.2) encoded by an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 54. In another embodiment, SEQ ID NO: 54 comprises the following amino acid (AA) sequence MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRM NFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQI NHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILR NFYFINRLTGYLRNSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGP SDTPILPQ (SEQ ID NO: 54, MOD-9012).

[0108] In another embodiment, the cytokine as described herein comprising an interferon (IFN) peptide--and two CTP units attached to its carboxy terminus is encoded by a nucleic acid molecule set forth in SEQ ID NO: 55. In another embodiment, SEQ ID NO: 55 comprises the following nucleic acid (NA) sequence:

TABLE-US-00005 (SEQ ID NO: 55, MOD-9012) ctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgtg cttcagcaccaccgccctgagcatgagctacaacctgctgggcttcctgc agaggtccagcaacttccagtgccagaagctgctgtggcagctgaacggc aggctggaatactgcctgaaggacaggatgaacttcgacatcccagagga aatcaagcagctgcagcagttccagaaggaggacgccgccctgaccatct acgagatgctgcagaacatcttcgccatcttcaggcaggacagcagcagc accggctggaacgagaccatcgtggagaacctgctggccaacgtgtacca ccagatcaaccacctgaaaaccgtgctggaagagaagctggaaaaggagg acttcaccaggggcaagctgatgagcagcctgcacctgaagaggtactac ggcagaatcctgcactacctgaaggccaaggagtacagccactgcgcctg gaccatcgtgagggtggagatcctgaggaacttctacttcatcaacaggc tgaccggctacctgaggaacagctccagcagcaaggcccctccaccttcc ctgcccagtccaagccgactccctgggccctccgacacaccaatcctgcc acagagcagctcctctaaggcccctcctccatccctgccatccccctccc ggctgcctggcccctctgacacccctatcctgcctcagtgatgaaggtct ggatccgcggccgc.

[0109] In another embodiment, the cytokine as described herein comprises an interferon (IFN) peptide, a single CTP unit attached to the IFN's amino terminus, and two CTP units attached to the IFN's carboxy terminus. In another embodiment, the polypeptide as described herein comprises an interferon (IFN) peptide, a single CTP unit attached to the IFN's amino terminus and two CTP units attached in tandem to the IFN's carboxy terminus. In another embodiment, the polypeptide as described herein comprises (from amino to carboxy termini): CTP (.times.1)-interferon (IFN) peptide-CTP (.times.2) comprising an amino acid sequence set forth in SEQ ID NO: 56. In another embodiment, SEQ ID NO: 56 comprises the following amino acid (AA) sequence:

TABLE-US-00006 (SEQ ID NO: 56, MOD-9013) MTNKCLLQIALLLCFSTTALSSSSSKAPPPSLPSPSRLPGPSDTPILPQM SYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQ KEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTV LEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEIL RNFYFINRLTGYLRNSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAP PPSLPSPSRLPGPSDTPILPQ.

[0110] In another embodiment, the cytokine as described herein comprising an interferon (IFN) peptide, a single CTP unit attached to the IFN's amino terminus and two CTP units attached to the IFN's carboxy terminus is encoded by a nucleic acid molecule set forth in SEQ ID NO: 57. In another embodiment, SEQ ID NO: 57 comprises the following acid (NA) sequence:

TABLE-US-00007 (SEQ ID NO: 57, MOD-9013) tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgt gcttcagcaccaccgccctgagcagcagcagctccaaggccccacccccc agcctgcccagccccagcagactgccaggccccagcgacacccccatcct gccccagatgagctacaacctgctgggcttcctgcagaggtccagcaact tccagtgccagaagctgctgtggcagctgaacggcaggctggaatactgc ctgaaggacaggatgaacttcgacatcccagaggaaatcaagcagctgca gcagttccagaaggaggacgccgccctgaccatctacgagatgctgcaga acatcttcgccatcttcaggcaggacagcagcagcaccggctggaacgag accatcgtggagaacctgctggccaacgtgtaccaccagatcaaccacct gaaaaccgtgctggaagagaagctggaaaaggaggacttcaccaggggca agctgatgagcagcctgcacctgaagaggtactacggcagaatcctgcac tacctgaaggccaaggagtacagccactgcgcctggaccatcgtgagggt ggagatcctgaggaacttctacttcatcaacaggctgaccggctacctga ggaacagctccagcagcaaggcccctccaccttccctgcccagtccaagc cgactccctgggccctccgacacaccaatcctgccacagagcagctcctc taaggcccctcctccatccctgccatccccctcccggctgcctggcccct ctgacacccctatcctgcctcagtgatgaaggtctggatccgcggccgc.

[0111] In another embodiment, the cytokine as described herein comprises an interferon (IFN) peptide, a single CTP attached to the IFN's amino terminus, and a single CTP located within an IFN coding sequence. In another embodiment, the polypeptide as described herein comprises (from amino to carboxy termini): CTP (.times.1)-interferon (IFN) peptide (fragment 1)-CTP-interferon (IFN) peptide (fragment 2) comprising an amino acid sequence set forth in SEQ ID NO: 58. In another embodiment, SEQ ID NO: 58 comprises the following amino acid (AA) sequence: MTNKCLLQIALLLCFSTTALSSSSSKAPPPSLPSPSRLPGPSDTPILPQMSYNL LGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEM LQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSL HLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNSSSSKAPPPSLPS PSRLPGPSDTPILPQMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEI KQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVL EEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRL TGYLRN (SEQ ID NO: 58, MOD-9014).

[0112] In another embodiment, the cytokine as described herein comprising an interferon (IFN) peptide, a single CTP unit attached to the IFN's amino terminus, and a single CTP unit located within the IFN coding sequence is encoded by a nucleic acid molecule set forth in SEQ ID NO: 59. In another embodiment, SEQ ID NO: 59 comprises the following nucleic acid (NA) sequence:

TABLE-US-00008 (SEQ ID NO: 59, MOD-9014) tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgt gcttcagcaccaccgccctgagcagcagcagctccaaggccccacccccc agcctgcccagccccagcaggctgccaggccccagcgacacccccatcct gccccagatgagctacaacctgctgggcttcctgcagaggtccagcaact tccagtgccagaaactgctgtggcagctgaacggcaggctggaatactgc ctgaaggaccggatgaacttcgacatccccgaagagatcaagcagctgca gcagttccagaaagaggacgccgccctgaccatctacgagatgctgcaga acatcttcgccatcttcaggcaggacagcagcagcaccggctggaacgag accatcgtggagaacctgctggccaacgtgtaccaccagatcaaccacct gaaaaccgtgctggaagagaagctggaaaaagaggacttcaccaggggca agctgatgagcagcctgcacctgaagaggtactacggcagaatcctgcac tacctgaaggccaaagagtacagccactgcgcctggaccatcgtgagggt ggagatcctgcggaacttctacttcatcaacaggctgaccggctacctga ggaacagctccagcagcaaggcccctccaccctccctgccctccccaagc agactgcccggaccctccgacacaccaattctgccacagatgtcctacaa tctgctcggatttctgcagcgctcctccaactttcagtgtcagaagctcc tctggcagctcaatggccgcctggaatattgtctgaaagacagaatgaat tttgacatcccagaggaaattaaacagctccagcagtttcagaaagaaga tgctgctctcacaatctatgaaatgctccagaatatctttgcaatctttc gccaggacagctcctccaccgggtggaatgagacaattgtcgagaatctg ctcgccaatgtctatcatcagatcaatcacctcaagacagtcctcgaaga aaaactcgaaaaagaagatttcacacgcggcaaactgatgtcctccctgc atctgaagcgctactatgggcgcatcctgcattatctgaaagctaaagaa tactcccactgtgcttggacaattgtgcgcgtcgagatcctgagaaactt ttatttcattaaccgcctgacaggatacctgcgcaactgatgaaggtctg gatgcggccgc.

[0113] In another embodiment, the cytokine as described herein comprises an interferon (IFN) peptide and a single CTP unit attached to its amino terminus. In another embodiment, the polypeptide as described herein comprises an interferon (IFN) peptide-CTP comprising an amino acid sequence set forth in SEQ ID NO: 60. In another embodiment, SEQ ID NO: 60 comprises the following amino acid (AA) sequence:

TABLE-US-00009 (SEQ ID NO: 60, MOD-9015) MTNKCLLQIALLLCFSTTALSSSSSKAPPPSLPSPSRLPGPSDTPILPQM SYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQ KEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTV LEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEIL RNFYFINRLTGYLRN*.

[0114] In another embodiment, the polypeptide as described herein comprising an interferon (IFN) peptide--and a single CTP attached to its amino terminus is encoded by a nucleic acid molecule set forth in SEQ ID NO: 61. In another embodiment, SEQ ID NO: 61 comprises the lo following nucleic acid (NA) sequence: tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgtgcttcagcaccaccgccctgagc- agcag cagctccaaggccccaccccccagcctgcccagccccagcaggctgccaggccccagcgacaccccca- tcctgccccagatgagc tacaacctgctgggcttcctgcagaggtccagcaacttccagtgccagaaactgctgtggcagctgaacggca- ggctggaatactgcc tgaaggaccggatgaacttcgacatccccgaagagatcaagcagctgcagcagttccagaaagaggacgccgc- cctgaccatctac gagatgctgcagaacatcttcgccatcttcaggcaggacagcagcagcaccggctggaacgagaccatcgtgg- agaacctgctggc caacgtgtaccaccagatcaaccacctgaaaaccgtgctggaagagaagctggaaaaagaggacttcaccagg- ggcaagctgatga gcagcctgcacctgaagaggtactacggcagaatcctgcactacctgaaggccaaagagtacagccactgcgc- ctggaccatcgtga gggtggagatcctgcggaacttctacttcatcaacaggctgaccggctacctgaggaactgatgagtccgcgg- ccgc (SEQ ID NO: 61, MOD-9015).

[0115] In another embodiment, the polypeptide as described herein comprises an interferon (IFN) peptide, a single CTP unit attached to its amino terminus, and a single CTP unit attached to its carboxy terminus. In another embodiment, the polypeptide as described herein comprises an interferon (IFN) peptide-CTP comprising an amino acid sequence set forth in SEQ ID NO: 62. In another embodiment, SEQ ID NO: 62 comprises the following amino acid (AA) sequence:

TABLE-US-00010 (SEQ ID NO: 62, MOD-9016) MTNKCLLQIALLLCFSTTALSSSSSKAPPPSLPSPSRLPGPSDTPILPQM SYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQ KEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTV LEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEIL RNFYFINRLTGYLRNSSSSKAPPPSLPSPSRLPGPSDTPILPQ*.

[0116] In another embodiment, the cytokine as described herein comprising an interferon (IFN) peptide, a single CTP unit attached to its amino terminus, and a single CTP unit attached to its carboxy terminus is encoded by a nucleic acid molecule set forth in SEQ ID NO: 63. In another embodiment, SEQ ID NO: 63 comprises the following nucleic acid (NA) sequence: tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgtgcttcagcaccac- cgccctgagcagcagcagctccaa ggccccaccccccagcctgcccagccccagcagactgccaggccccagcgacacccccatcctgccccagatg- agctacaacctg ctgggcttcctgcagaggtccagcaacttccagtgccagaagctgctgtggcagctgaacggcaggctggaat- actgcctgaaggac aggatgaacttcgacatcccagaggaaatcaagcagctgcagcagttccagaaggaggacgccgccctgacca- tctacgagatgct gcagaacatcttcgccatcttcaggcaggacagcagcagcaccggctggaacgagaccatcgtggagaacctg- ctggccaacgtgt accaccagatcaaccacctgaaaaccgtgctggaagagaagctggaaaaggaggacttcaccaggggcaagct- gatgagcagcct gcacctgaagaggtactacggcagaatcctgcactacctgaaggccaaggagtacagccactgcgcctggacc- atcgtgagggtgg agatcctgaggaacttctacttcatcaacaggctgaccggctacctgaggaacagctccagcagcaaggcccc- tccaccttccctgcc cagtccaagccgactccctgggccctccgatacaccaattctgccacagtgatgaaggtctggatgcggccgc (SEQ ID NO: 63, MOD-9016).

[0117] In another embodiment, an interferon .beta. peptide comprises SEQ ID NO: 6 comprising the following amino acid (AA) sequence:

TABLE-US-00011 (SEQ ID NO: 68) MSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQ KEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVL EEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRN FYFINRLTGYLRN.

[0118] In another embodiment, the methods of the present invention provide an interferon beta 1 peptide having additionally one CTP amino acid peptide on the N-terminus and two CTP amino acid peptides on the C-terminus for treating or inhibiting multiple sclerosis. In another embodiment, the methods of the present invention provide an interferon beta 1 protein comprising an interferon beta 1 sequence as provided herein for treating diseases such as, but not limited to, multiple sclerosis, cancer, or viral infections. In another embodiment, the methods of the present invention provide an interferon beta 1 protein comprising an interferon beta 1 sequence as provided herein for treating diseases such as, but not limited to, multiple sclerosis, cancer, or viral infections. In another embodiment, the methods of the present invention provide an interferon beta 1 peptide set forth in SEQ ID NO: 56 for treating diseases such as, but not limited to, multiple sclerosis, cancer, or viral infections. In another embodiment, the methods of the present invention provide an interferon beta 1 peptide set forth in SEQ ID NO: 58 for treating diseases such as, but not limited to, multiple sclerosis, cancer, or viral infections. In another embodiment, the methods of the present invention provide an interferon beta 1 peptide set forth in SEQ ID NO: 60 for treating diseases such as, but not limited to, multiple sclerosis, cancer, or viral infections. In another embodiment, the methods of the present invention provide an interferon beta 1 peptide set forth in SEQ ID NO: 62 for treating diseases such as, but not limited to, multiple sclerosis, cancer, or viral infections.

[0119] As provided herein, attachment of a CTP sequence to both the amino and carboxy termini of the EPO protein results in increased potency at stimulating erythropoiesis (FIGS. 3-5 and Table 6 of Example 4), as compared to recombinant EPO and other combinations of EPO and CTP. In some embodiments, an EPO attached to three CTP sequences does not impair binding to its receptor as evidenced in Table 4 of Example 3, which demonstrates that EPO attached to three CTP sequences is equally effective at stimulating proliferation of TF-1 cells as wild-type EPO.

[0120] In some embodiments, "homology" according to the present invention also encompasses deletions, insertions, or substitution variants, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof. In one embodiment, the substitution variant comprises a substitution of the glycine in position 104 of erythropoietin amino acid sequence with a serine (SEQ ID NO: 22).

[0121] In another embodiment, the methods of the present invention provide an EPO protein having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of anemia. In another embodiment, the methods of the present invention provide an EPO protein having additionally one CTP amino acid peptide on the N-terminus and two CTP amino acid peptides on the C-terminus for the treatment of anemia.

[0122] In another embodiment, the methods of the present invention provide an EPO protein set forth in SEQ ID NO: 1 having additionally at least one CTP amino acid peptide on the N-terminus for the treatment of anemia. In another embodiment, the methods of the present invention provide an EPO protein set forth in SEQ ID NO: 1 having additionally at least one CTP amino acid peptide on the N-terminus and at least one additional CTP amino acid peptide on the C-terminus for the treatment of anemia. In another embodiment, the methods of the present invention provide an EPO protein set forth in SEQ ID NO: 2 having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of anemia. In another embodiment, the methods of the present invention provide an EPO protein set forth in SEQ ID NO: 3 having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of anemia. In another embodiment, the methods of the present invention provide an EPO protein set forth in SEQ ID NO: 4 having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of anemia. In another embodiment, the methods of the present invention provide an EPO protein set forth in SEQ ID NO: 5 having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of anemia. In another embodiment, the methods of the present invention provide an EPO protein set forth in SEQ ID NO: 6 having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of anemia. In another embodiment, the methods of the present invention provide an EPO protein set forth in SEQ ID NO: 16 having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of anemia. In another embodiment, the methods of the present invention provide an EPO protein set forth in SEQ ID NO: 22 having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of anemia.

[0123] In another embodiment, the methods of the present invention provide a nucleic acid sequence encoding an EPO protein having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of anemia. In another embodiment, the methods of the present invention provide a nucleic acid sequence encoding an EPO protein having additionally one CTP amino acid peptide on the N-terminus and two CTP amino acid peptides on the C-terminus for the treatment of anemia. In another embodiment, the methods of the present invention provide a nucleic acid sequence as set forth in SEQ ID NO: 20 encoding an EPO protein and one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of anemia. In another embodiment, the methods of the present invention provide a nucleic acid sequence as set forth in SEQ ID NO: 21 encoding an EPO protein and one CTP amino acid peptide on the N-terminus and two CTP amino acid peptides on the C-terminus for the treatment of anemia.

[0124] In another embodiment, the methods of the present invention provide an EPO protein having additionally one CTP amino acid peptide on the N-terminus and two CTP amino acid peptides on the C-terminus for inhibiting anemia, for treating or inhibiting tumor-associated anemia for treating or inhibiting tumor hypoxia for treating or inhibiting chronic infections such as HIV, inflammatory bowel disease, or septic episodes, for treating fatigue syndrome following cancer chemotherapy, for improving stem cell engraftment, for increasing the survival rate of a patient with aplastic anemia or myelodysplastic syndrome. In another embodiment, the methods of the present invention provide an EPO protein as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16 or SEQ ID NO: 22 having additionally one CTP peptide on the N-terminus and two CTP peptides on the C-terminus for inhibiting anemia, for treating or inhibiting tumor-associated anemia for treating or inhibiting tumor hypoxia for treating or inhibiting chronic infections such as HIV, inflammatory bowel disease, or septic episodes, for treating fatigue syndrome following cancer chemotherapy, for improving stem cell engraftment, for increasing the survival rate of a patient with aplastic anemia or myelodysplastic syndrome.

[0125] In another embodiment, the methods of the present invention provide a nucleic acid sequence encoding an EPO protein having additionally one CTP peptide on the N-terminus and two CTP amino acid peptides on the C-terminus for inhibiting anemia, for treating or inhibiting tumor-associated anemia for treating or inhibiting tumor hypoxia for treating or inhibiting chronic infections such as HIV, inflammatory bowel disease, or septic episodes, for treating fatigue syndrome following cancer chemotherapy, for improving stem cell engraftment, or for increasing the survival rate of a patient with aplastic anemia or myelodysplastic syndrome. In another embodiment, the methods of the present invention provide a nucleic acid sequence as set forth in SEQ ID NO: 20 or SEQ ID NO: 21 encoding an EPO protein and one CTP peptide on the N-terminus and two CTP peptides on the C-terminus for inhibiting anemia, for treating or inhibiting tumor-associated anemia, for treating or inhibiting tumor hypoxia, for treating or inhibiting chronic infections such as HIV, inflammatory bowel disease, or septic episodes, for treating fatigue syndrome following cancer chemotherapy, for improving stem cell engraftment, or for increasing the survival rate of a patient with aplastic anemia or myelodysplastic syndrome.

[0126] In another embodiment, human growth hormone (hGH) is the cytokine as described herein. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention bind adipocytes and stimulates them to break down triglyceride and suppresses their ability to take up and accumulate circulating lipids. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention exert indirect effects mediated primarily by an insulin-like growth factor-I (IGF-I) (as shown in the examples section).

[0127] In another embodiment, CTP-hGH-CTP-CTP constructs of the invention stimulate body growth by stimulating the liver and other tissues to secrete IGF-I. In another embodiment, IGF-I stimulates proliferation of chondrocytes, resulting in bone growth.

[0128] In another embodiment, CTP-hGH-CTP-CTP constructs of the invention induce metabolic effects on protein, lipid and carbohydrate metabolism. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention have a direct effect. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention have an indirect effect through induction of IGF-I.

[0129] In another embodiment, CTP-hGH-CTP-CTP constructs include constructs comprising a leader peptide. In another embodiment, CTP-hGH-CTP-CTP constructs include truncated constructs.

[0130] In another embodiment, CTP-hGH-CTP-CTP constructs of the invention stimulate protein anabolism in a tissue. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention stimulate amino acid uptake, increased protein synthesis, and decreased oxidation of proteins.

[0131] In another embodiment, CTP-hGH-CTP-CTP constructs of the invention stimulate fat metabolism. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention stimulate the utilization of fat by stimulating triglyceride breakdown and oxidation in adipocytes.

[0132] In another embodiment, CTP-hGH-CTP-CTP constructs of the invention stimulate carbohydrate metabolism. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention maintain blood glucose within a normal range. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention comprise an anti-insulin activity. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention suppress the abilities of insulin to stimulate uptake of glucose in peripheral tissues and enhance glucose synthesis in the liver. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention stimulate insulin secretion, leading to hyperinsulinemia.

[0133] In another embodiment, CTP-hGH-CTP-CTP constructs of the invention are used to compensate for limited or no production of growth hormone in a subject. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention compensate for limited or no production of growth hormone-releasing hormone (GHRH). In another embodiment, CTP-hGH-CTP-CTP constructs of the invention compensate for the increased activity of somatostatin. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention compensate for limited or no production of ghrelin.

[0134] In another embodiment, CTP-hGH-CTP-CTP constructs of the invention are used to treat diseases associated with lesions in either the hypothalamus, the pituitary, or in target cells.

[0135] In another embodiment, CTP-hGH constructs of the invention are used to treat diseases associated with reduced target cell's response to the hormone.

[0136] In another embodiment, CTP-hGH-CTP-CTP constructs of the invention are used to treat children with severe growth retardation. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention are used to treat children of pathologically short stature. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention are used to enhance athletic performance. In another embodiment, CTP-hGH-CTP-CTP constructs of the invention are used to treat symptoms of aging. In another embodiment, CTP-hGH constructs of the invention are used to treat cosmetic symptoms of aging.

[0137] In another embodiment, CTP-hGH-CTP-CTP constructs of the invention are used for enhancing milk production in a female subject. In another embodiment, CTP-cowGH-CTP-CTP constructs of the invention are used for enhancing milk production in dairy cattle. In another embodiment, CTP-animal-GH-CTP-CTP constructs of the invention are used in animal agriculture technology. In another embodiment, CTP-farm animal-GH-CTP-CTP constructs of the invention are used for enhancing growth of farm animal such as, but not limited to, pigs.

[0138] In some embodiments, human growth hormone (hGH) is utilized according to the teachings of the present invention. In some embodiments, the attachment of CTP sequences to both the amino and carboxy termini of the hGH protein results in increased potency (FIG. 11).

[0139] In some embodiments, the attachment of CTP sequences to both the amino and carboxy termini of the hGH protein results in prolonged in vivo activity. In one embodiment, CTP-hGH polypeptides of the present invention are set forth in SEQ ID NOs: 39-41.

[0140] In one embodiment, the phrase "human growth hormone" (hGH) refers to a polypeptide, such as that set forth in Genbank Accession No. P01241 (SEQ ID NO: 47), exhibiting hGH activity (i.e. stimulation of growth).

[0141] In one embodiment, "human growth hormone" (hGH) refers to a polypeptide, such as that set forth in Genbank Accession No. P01241, exhibiting hGH activity (i.e. stimulation of growth). In one embodiment, an hGH of the present invention also refers to homologues. In one embodiment, an hGH amino acid sequence of the present invention is at least 50% homologous to an hGH sequence set forth in GenBank Accession No. P01241 as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters). In one embodiment, an hGH amino acid sequence of the present invention is at least 60% homologous to an hGH sequence set forth in GenBank Accession No. P01241 as determined using BlastP software of the NCBI using default parameters. In one embodiment, an hGH amino acid sequence of the present invention is at least 70% homologous to an hGH sequence set forth in GenBank Accession No. P01241 as determined using BlastP software of the NCBI using default parameters. In one embodiment, an hGH amino acid sequence of the present invention is at least 80% homologous to an hGH sequence set forth in GenBank Accession No. P01241 as determined using BlastP software of the NCBI using default parameters.). In one embodiment, an hGH amino acid sequence of the present invention is at least 90% homologous to an hGH sequence set forth in GenBank Accession No. P01241 as determined using BlastP software of the NCBI using default parameters. In one embodiment, an hGH amino acid sequence of the present invention is at least 95% homologous to an hGH sequence set forth in GenBank Accession No. P01241 as determined using BlastP software of the NCBI using default parameters.

[0142] Exemplary CTP-hGH polypeptides of the present invention are set forth in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41.

[0143] In another embodiment, the methods of the present invention provide a CTP-hGH-CTP-CTP construct used for stimulating muscle growth, increasing cardiac function, stimulating bone growth, maintaining muscle integrity, balancing muscle metabolism, inducing muscle buildup, inducing de-novo muscle build-up, enhancing bone load, treating symptoms associated with osteoporosis, treating a wasting disease, increasing lipolysis, improving fluid balance, treating osteoporosis, improving lung function, improving immunity, regrowing a vital organ, increasing sense of well-being, restoring REM sleep, or any combination thereof. In another embodiment, the methods of the present invention provide an hGH protein as set forth in SEQ ID NO: 23 having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for stimulating muscle growth, increasing cardiac function, stimulating bone growth, maintaining muscle integrity, balancing muscle metabolism, inducing muscle buildup, inducing de-novo muscle build-up, enhancing bone load, treating symptoms associated with osteoporosis, treating a wasting disease, increasing lipolysis, improving fluid balance, treating osteoporosis, improving lung function, improving immunity, regrowing a vital organ, increasing sense of well-being, restoring REM sleep, or any combination thereof. In another embodiment, the methods of the present invention provide an hGH protein as set forth in SEQ ID NO: 36 having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for stimulating muscle growth, increasing cardiac function, stimulating bone growth, maintaining muscle integrity, balancing muscle metabolism, inducing muscle buildup, inducing de-novo muscle build-up, enhancing bone load, treating symptoms associated with osteoporosis, treating a wasting disease, increasing lipolysis, improving fluid balance, treating osteoporosis, improving lung function, improving immunity, regrowing a vital organ, increasing sense of well-being, restoring REM sleep, or any combination thereof. In another embodiment, the methods of the present invention provide an hGH protein as set forth in SEQ ID NO: 37 having additionally at least one CTP amino acid peptide on the N-terminus for stimulating muscle growth, increasing cardiac function, stimulating bone growth, maintaining muscle integrity, balancing muscle metabolism, inducing muscle buildup, inducing de-novo muscle build-up, enhancing bone load, treating symptoms associated with osteoporosis, treating a wasting disease, increasing lipolysis, improving fluid balance, treating osteoporosis, improving lung function, improving immunity, regrowing a vital organ, increasing sense of well-being, restoring REM sleep, or any combination thereof. In another embodiment, the methods of the present invention provide an hGH protein as set forth in SEQ ID NO: 38 having additionally at least one CTP amino acid peptide on the N-terminus for stimulating muscle growth, increasing cardiac function, stimulating bone growth, maintaining muscle integrity, balancing muscle metabolism, inducing muscle buildup, inducing de-novo muscle build-up, enhancing bone load, treating symptoms associated with osteoporosis, treating a wasting disease, increasing lipolysis, improving fluid balance, treating osteoporosis, improving lung function, improving immunity, regrowing a vital organ, increasing sense of well-being, restoring REM sleep, or any combination thereof. In another embodiment, the methods of the present invention provide an hGH protein as set forth in SEQ ID NO: 39 for stimulating muscle growth, increasing cardiac function, stimulating bone growth, maintaining muscle integrity, balancing muscle metabolism, inducing muscle buildup, inducing de-novo muscle build-up, enhancing bone load, treating symptoms associated with osteoporosis, treating a wasting disease, increasing lipolysis, improving fluid balance, treating osteoporosis, improving lung function, improving immunity, regrowing a vital organ, increasing sense of well-being, restoring REM sleep, or any combination thereof. In another embodiment, the methods of the present invention provide an hGH protein as set forth in SEQ ID NO: 40 for stimulating muscle growth, increasing cardiac function, stimulating bone growth, maintaining muscle integrity, balancing muscle metabolism, inducing muscle buildup, inducing de-novo muscle build-up, enhancing bone load, treating symptoms associated with osteoporosis, treating a wasting disease, increasing lipolysis, improving fluid balance, treating osteoporosis, improving lung function, improving immunity, regrowing a vital organ, increasing sense of well-being, restoring REM sleep, or any combination thereof. In another embodiment, the methods of the present invention provide an hGH protein as set forth in SEQ ID NO: 41 for stimulating muscle growth, increasing cardiac function, stimulating bone growth, maintaining muscle integrity, balancing muscle metabolism, inducing muscle buildup, inducing de-novo muscle build-up, enhancing bone load, treating symptoms associated with osteoporosis, treating a wasting disease, increasing lipolysis, improving fluid balance, treating osteoporosis, improving lung function, improving immunity, regrowing a vital organ, increasing sense of well-being, restoring REM sleep, or any combination thereof. In another embodiment, the methods of the present invention provide an hGH protein as set forth in SEQ ID NO: 42 having additionally at least one CTP amino acid peptide on the N-terminus for stimulating muscle growth, increasing cardiac function, stimulating bone growth, maintaining muscle integrity, balancing muscle metabolism, inducing muscle buildup, inducing de-novo muscle build-up, enhancing bone load, treating symptoms associated with osteoporosis, treating a wasting disease, increasing lipolysis, improving fluid balance, treating osteoporosis, improving lung function, improving immunity, regrowing a vital organ, increasing sense of well-being, restoring REM sleep, or any combination thereof. In another embodiment, the methods of the present invention provide an hGH protein modified by CTPs as described herein for stimulating muscle growth, increasing cardiac function, stimulating bone growth, maintaining muscle integrity, balancing muscle metabolism, inducing muscle buildup, inducing de-novo muscle build-up, enhancing bone load, treating symptoms associated with osteoporosis, treating a wasting disease, increasing lipolysis, improving fluid balance, treating osteoporosis, improving lung function, improving immunity, regrowing a vital organ, increasing sense of well-being, restoring REM sleep, or any combination thereof.

[0144] In another embodiment, the methods of the present invention provide a nucleic acid sequence encoding an hGH protein as described herein. In another embodiment, the methods of the present invention provides a nucleic acid of SEQ ID NO: 45 encoding an hGH protein comprising one CTP amino acid peptide on the N-terminus and two CTP amino acid peptides on the C-terminus for stimulating muscle growth, increasing cardiac function, stimulating bone growth, maintaining muscle integrity, balancing muscle metabolism, inducing muscle buildup, inducing de-novo muscle build-up, enhancing bone load, treating symptoms associated with osteoporosis, treating a wasting disease, increasing lipolysis, improving fluid balance, treating osteoporosis, improving lung function, improving immunity, regrowing a vital organ, increasing sense of well-being, restoring REM sleep, or any combination thereof. In another embodiment, the methods of the present invention provide a nucleic acid of SEQ ID NO: 46 encoding an hGH protein and one CTP amino acid peptide on the N-terminus and two CTP amino acid peptides on the C-terminus for stimulating muscle growth, increasing cardiac function, stimulating bone, growth, maintaining muscle integrity, balancing muscle metabolism, inducing muscle buildup, inducing de-novo muscle build-up, enhancing bone load, treating symptoms associated with osteoporosis, treating a wasting disease, increasing lipolysis, improving fluid balance, treating osteoporosis, improving lung function, improving immunity, regrowing a vital organ, increasing sense of well-being, restoring REM sleep, or any combination thereof

[0145] In some embodiments, glucagon-like peptide-1 is utilized according to the teachings of the present invention. In some embodiments, the attachment of CTP sequences to both the amino and carboxy termini of a "glucagon-like peptide-1" results in increased potency. In some embodiments, the attachment of CTP to both the amino and carboxy termini of a peptide results in prolonged in vivo activity. In some embodiments, the attachment of CTP to both the amino and carboxy termini of the glucagon-like peptide-results in prolonged in vivo activity.

[0146] In one embodiment, "glucagon-like peptide-1" (GLP-1) refers to a mammalian polypeptide. In one embodiment, "glucagon-like peptide-1" (GLP-1) refers to a human polypeptide. In some embodiments, GLP-1 is cleaved from the glucagon preproprotein (Genbank ID No. NP002045) that has the ability to bind to the GLP-1 receptor and initiate a signal transduction pathway resulting in insulinotropic activity. In one embodiment, "insulinotropic activity" refers to the ability to stimulate insulin secretion in response to elevated glucose levels, thereby causing glucose uptake by cells and decreased plasma glucose levels. In some embodiments, GLP-1 polypeptides include, but are not limited to those described in U.S. Pat. No. 5,118,666; which is incorporated by reference herein.

[0147] In one embodiment, "GLP-1" refers to a polypeptide, such as set forth in SEQ ID NO: 25 as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. In one embodiment, a GLP-1 of the present invention also refers to a GLP-1 homologue. In one embodiment, a GLP-1 amino acid sequence of the present invention is at least 50% homologous to GLP-1 sequences set forth in SEQ ID NO: 25 as determined using BlastP software of NCBI using default parameters. In one embodiment, a GLP-1 amino acid sequence of the present invention is at least 60% homologous to GLP-1 sequences set forth in SEQ ID NO: 25 as determined using BlastP software of NCBI using default parameters. In one embodiment, a GLP-1 amino acid sequence of the present invention is at least 70% homologous to GLP-1 sequences set forth in SEQ ID NO: 25 as determined using BlastP software of NCBI using default parameters. In one embodiment, a GLP-1 amino acid sequence of the present invention is at least 80% homologous to GLP-1 sequences set forth in SEQ ID NO: 25 as determined using BlastP software of NCBI using default parameters. In one embodiment, a GLP-1 amino acid sequence of the present invention is at least 90% homologous to GLP-1 sequences set forth in SEQ ID NO: 25 as determined using BlastP software of NCBI using default parameters. In one embodiment, a GLP-1 amino acid sequence of the present invention is at least 95% homologous to GLP-1 sequences set forth in SEQ ID NO: 25 as determined using BlastP software of NCBI using default parameters.

[0148] In another embodiment, the methods of the present invention provides a GLP-1 peptide having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for treating or inhibiting type II diabetes. In another embodiment, the methods of the present invention provides a GLP-1 peptide having additionally one CTP amino acid peptide on the N-terminus and two CTP amino acid peptides on the C-terminus for treating or inhibiting type II diabetes. In another embodiment, the methods of the present invention provides a GLP-1 peptide set forth in SEQ ID NO: 25 having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for treating or inhibiting type II diabetes.

[0149] In another embodiment, the polypeptide sequence-of-interest is an insulin-like growth factor. In another embodiment, the polypeptide sequence-of-interest is an epidermal growth factor. In another embodiment, the polypeptide sequence-of-interest is an acidic or basic fibroblast growth factor. In another embodiment, the polypeptide sequence-of-interest is a platelet-derived growth factor. In another embodiment, the polypeptide sequence-of-interest is a granulocyte-CSF. In another embodiment, the polypeptide sequence-of-interest is a macrophage-CSF. In another embodiment, the polypeptide sequence-of-interest is an IL-2. In another embodiment, the polypeptide sequence-of-interest is an IL-3. In another embodiment, the polypeptide sequence-of-interest is a tumor necrosis factor. In another embodiment, the polypeptide sequence-of-interest is an LHRH. In another embodiment, the polypeptide sequence-of-interest is an LHRH analog. In another embodiment, the polypeptide sequence-of-interest is a somatostatin. In another embodiment, the polypeptide sequence-of-interest is a growth hormone releasing factor. In another embodiment, the polypeptide sequence-of-interest is an endorphin. In another embodiment, the polypeptide sequence-of-interest is an alveolar surfactant protein. In another embodiment, the polypeptide sequence-of-interest is a natriuretic factor. In another embodiment, the polypeptide sequence-of-interest is an adhesion. In another embodiment, the polypeptide sequence-of-interest is an angiostatin. In another embodiment, the polypeptide sequence-of-interest is an endostatin. In another embodiment, the polypeptide sequence-of-interest is a receptor peptide. In another embodiment, the polypeptide sequence-of-interest is a receptor binding ligand. In another embodiment, the polypeptide sequence-of-interest is an antibody. In another embodiment, the polypeptide sequence-of-interest is an antibody fragment. In another embodiment, the polypeptide sequence-of-interest is a peptide or a protein including any modified form.

[0150] In another embodiment, the polypeptide of the invention comprises a cytokine having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus. In another embodiment, the cytokine having additionally at least one CTP amino acid peptide on the N-terminus and two CTP amino acid peptides on the C-terminus is selected from lymphokines, monokines, chemokine, and interleukin. In another embodiment, the cytokine comprises an autocrine action activity. In another embodiment, the cytokine comprises a paracrine action activity. In another embodiment, the cytokine comprises an endocrine action activity.

[0151] In another embodiment, the methods of the present invention provide insulin having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of diabetes.

[0152] In another embodiment, the methods of the present invention provide albumin having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of hypovolemic shock, hemodialysis or cardiopulmonary bypass.

[0153] In another embodiment, the methods of the present invention provide Activase-altiplase/tPA having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of acute myocardial infarction, acute massive pulmonary embolism, or ischemic stroke.

[0154] In another embodiment, the methods of the present invention provide adenosine deaminase having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of severe combined immunodeficiency disease.

[0155] In another embodiment, the methods of the present invention provide immune globulin having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of transplant recipients.

[0156] In another embodiment, the methods of the present invention provide immune globulin is a CMV immune globulin. In another embodiment, the methods of the present invention provide glucocerebrosidase having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of Gaucher disease.

[0157] In another embodiment, the methods of the present invention provide leukine-sargramostim/GM-CSF having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the stimulation of hematopoietic progenitor cells.

[0158] In another embodiment, the methods of the present invention provide G-CSF having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of neutropenia. In another embodiment, the methods of the present invention provide venoglobulin-S/IgG having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of immunodeficiency diseases.

[0159] In another embodiment, the methods of the present invention provide proleukin-aldesleukin having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of renal carcinoma or metastatic melanoma.

[0160] In another embodiment, the methods of the present invention provide DNase having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of cystic fibrosis.

[0161] In another embodiment, the methods of the present invention provide Factor VIII having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of Hemophilia A.

[0162] In another embodiment, the methods of the present invention provide helixate having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of Hemophilia A.

[0163] In another embodiment, the methods of the present invention provide L-asparaginase having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of acute lymphoblastic leukemia.

[0164] In another embodiment, the methods of the present invention provide WinRho SDF Rh IgG having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of Rh isoimmunization and immune thrombocytopenic purpura.

[0165] In another embodiment, the methods of the present invention provide Retavase retaplase/tPA having additionally at least one CTP amino acid peptide on the N-terminus and one CTP amino acid peptide on the C-terminus for the treatment of acute myocardial infarction.

[0166] In another embodiment, the methods of the present invention provide Factor IX having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of Hemophilia B.

[0167] In another embodiment, the methods of the present invention provide Factor IX having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of Hemophilia B.

[0168] In another embodiment, the methods of the present invention provide FSH having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for stimulation of ovulation during assisted reproduction.

[0169] In another embodiment, the methods of the present invention provide globulin having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the prevention of respiratory syncytial virus disease.

[0170] In another embodiment, the methods of the present invention provide fibrin having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for wound management and hemostasis. In another embodiment, the methods of the present invention provide interleukin-11 having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for chemotherapy-induced thrombocytopenia.

[0171] In another embodiment, the methods of the present invention provide becaplermin/PDGF having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of diabetic foot ulcers.

[0172] In another embodiment, the methods of the present invention provide lepirudin/herudin having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for anticoagulation in heparin-induced thrombocytopenia.

[0173] In another embodiment, the methods of the present invention provide soluble TNF having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of rheumatoid arthritis.

[0174] In another embodiment, the methods of the present invention provide Thymoglobulin having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of organ transplant rejection disease.

[0175] In another embodiment, the methods of the present invention provide factor VIIa having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of hemophilia.

[0176] In another embodiment, the methods of the present invention provide interferon alpha-2a having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of hairy cell leukemia and AIDS-related Kaposi's sarcoma.

[0177] In another embodiment, the methods of the present invention provide interferon alpha-2b having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of Hairy cell leukemia, genital warts, AIDS-related Kaposi's sarcoma, Hepatitis C, Hepatitis B, malignant melanoma, and follicular lymphoma.

[0178] In another embodiment, the methods of the present invention provide interferon alfa-N3 having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of genital warts.

[0179] In another embodiment, the methods of the present invention provide interferon gamma-1b having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of chronic granulomatous disease.

[0180] In another embodiment, the methods of the present invention provide interferon alfa n-1 having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of Hepatitis C infection.

[0181] In another embodiment, the methods of the present invention provide Interleukin-2 having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of renal carcinoma and metastatic melanoma.

[0182] In another embodiment, the methods of the present invention provide interferon beta-1b having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of multiple sclerosis.

[0183] In another embodiment, the methods of the present invention provide hGH having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for the treatment of wasting disease, AIDS, cachexia, or hGH deficiency.

[0184] In another embodiment, the methods of the present invention provide an OKT3 monoclonal antibody having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for organ transplant.

[0185] In another embodiment, the methods of the present invention provide a Reo monoclonal antibody having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for prevention of complications from coronary intervention and angioplasty.

[0186] In another embodiment, the methods of the present invention provide a monoclonal antibody having additionally at least one CTP amino acid peptide on the N-terminus and at least one CTP amino acid peptide on the C-terminus for treating colorectal cancer, Non-Hodgkin's lymphoma, kidney transplant rejection, metastatic breast cancer, or the prevention of respiratory syncytial virus disease.

[0187] In some embodiments, the CTP sequence modification is advantageous in permitting lower dosages to be used.

[0188] In some embodiments, "polypeptide" or "protein" as used herein encompasses native polypeptides (either degradation products, synthetically synthesized polypeptides or recombinant polypeptides) and peptidomimetics (typically, synthetic polypeptides), as well as peptoids and semipeptoids which are polypeptide analogs, which have, in some embodiments, modifications rendering the polypeptides even more stable while in a body or more capable of penetrating into cells.

[0189] In some embodiments, modifications include, but are not limited to N terminus modification, C terminus modification, polypeptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S.dbd.O, O.dbd.C--NH, CH2-O, CH2-CH2, S.dbd.C--NH, CH.dbd.CH or CF.dbd.CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinbelow.

[0190] In some embodiments, polypeptide bonds (--CO--NH--) within the polypeptide are substituted. In some embodiments, the polypeptide bonds are substituted by N-methylated bonds (--N(CH3)-CO--). In some embodiments, the polypeptide bonds are substituted by ester bonds (--C(R)H--C--O--O--C(R)--N--). In some embodiments, the polypeptide bonds are substituted by ketomethylen bonds (--CO-CH2-). In some embodiments, the polypeptide bonds are substituted by .alpha.-aza bonds (--NH--N(R)--CO--), wherein R is any alkyl, e.g., methyl, carba bonds (--CH2-NH--). In some embodiments, the polypeptide bonds are substituted by hydroxyethylene bonds (--CH(OH)--CH2-). In some embodiments, the polypeptide bonds are substituted by thioamide bonds (--CS--NH--). In some embodiments, the polypeptide bonds are substituted by olefinic double bonds (--CH.dbd.CH--). In some embodiments, the polypeptide bonds are substituted by retro amide bonds (--NH--CO--). In some embodiments, the polypeptide bonds are substituted by polypeptide derivatives (--N(R)--CH2-CO--), wherein R is the "normal" side chain, naturally presented on the carbon atom. In some embodiments, these modifications occur at any of the bonds along the polypeptide chain and even at several (2-3 bonds) at the same time.

[0191] In some embodiments, natural aromatic amino acids of the polypeptide such as Trp, Tyr and Phe, are substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. In some embodiments, the polypeptides of the present invention include one or more modified amino acid or one or more non-amino acid monomers (e.g. fatty acid, complex carbohydrates etc).

[0192] In one embodiment, "amino acid" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acid including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. In one embodiment, "amino acid" includes both D- and L-amino acids.

[0193] In some embodiments, the polypeptides of the present invention are utilized in therapeutics which requires the polypeptides to be in a soluble form. In some embodiments, the polypeptides of the present invention include one or more non-natural or natural polar amino acids, including, but not limited to, serine and threonine, which are capable of increasing polypeptide solubility due to their hydroxyl-containing side chain.

[0194] In some embodiments, the cytokines of the present invention are utilized in a linear form, although it will be appreciated by one skilled in the art that in cases where cyclicization does not severely interfere with cytokines characteristics, cyclic forms of the cytokines can also be utilized.

[0195] In some embodiments, the cytokines of present invention are biochemically synthesized such as by using standard solid phase techniques. In some embodiments, these biochemical methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis. In some embodiments, these methods are used when the cytokines are relatively short (about 5-15 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.

[0196] In some embodiments, solid phase cytokines synthesis procedures are well known to one skilled in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). In some embodiments, synthetic polypeptides are purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.], the composition of which can be confirmed via amino acid sequencing by methods known to one skilled in the art.

[0197] In some embodiments, recombinant protein techniques are used to generate the cytokines of the present invention. In some embodiments, recombinant protein techniques are used for generation of relatively long polypeptides (e.g., longer than 18-25 amino acid). In some embodiments, recombinant protein techniques are used for the generation of large amounts of the cytokines of the present invention. In some embodiments, recombinant techniques are described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

[0198] In another embodiment, cytokines of the present invention are synthesized using a polynucleotide encoding a polypeptide of the present invention. In some embodiments, the polynucleotide encoding cytokines of the present invention is ligated into an expression vector, comprising a transcriptional control of a cis-regulatory sequence (e.g., promoter sequence). In some embodiments, the cis-regulatory sequence is suitable for directing constitutive expression of the cytokines of the present invention. In some embodiments, the cis-regulatory sequence is suitable for directing tissue-specific expression of the cytokines of the present invention. In some embodiments, the cis-regulatory sequence is suitable for directing inducible expression of the cytokines of the present invention.

[0199] In some embodiments, tissue-specific promoters suitable for use with the present invention include sequences which are functional in specific cell population. Examples include, but are not limited to promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Inducible promoters suitable for use with the present invention include, for example, the tetracycline-inducible promoter (Srour, M. A., et al., 2003. Thromb. Haemost. 90: 398-405).

[0200] In one embodiment, the phrase "a polynucleotide" refers to a single or double stranded nucleic acid sequence which be 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).

[0201] In one embodiment, "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. In one embodiment, the sequence can be subsequently amplified in vivo or in vitro using a DNA polymerase.

[0202] In one embodiment, "genomic polynucleotide sequence" refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.

[0203] In one embodiment, "composite polynucleotide sequence" refers to a sequence, which is at least partially complementary and at least partially genomic. In one embodiment, a composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing there between. In one embodiment, the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. In one embodiment, intronic sequences include cis acting expression regulatory elements.

[0204] In one embodiment, the polynucleotides of the present invention further comprise a signal sequence encoding a signal peptide for the secretion of the cytokines of the present invention. In some embodiments, signal sequences include, but are not limited to the endogenous signal sequence for EPO as set forth in SEQ ID NO: 19 or the endogenous signal sequence for IFN-.beta.1 as set forth in SEQ ID NO: 64. In another embodiment, the signal sequence is N-terminal to the CTP sequence that is in turn N-terminal to the polypeptide sequence of interest; e.g. the sequence is (a) signal sequence--(b) CTP--(c) sequence-of-interest--(d) optionally, 1 or more additional CTP sequences. In another embodiment, 1 or more CTP sequences is inserted between the signal sequence of a polypeptide sequence of interest and the polypeptide sequence of interest itself, thus interrupting the wild-type sequence of interest. Each possibility represents a separate embodiment of the present invention.

[0205] In one embodiment, following expression and secretion, the signal peptides are cleaved from the precursor proteins resulting in the mature proteins.

[0206] In some embodiments, polynucleotides of the present invention are prepared using PCR techniques as described in Example 1, or any other method or procedure known to one skilled in the art. In some embodiments, the procedure involves the ligation of two different DNA sequences (See, for example, "Current Protocols in Molecular Biology", eds. Ausubel et al., John Wiley & Sons, 1992).

[0207] In one embodiment, polynucleotides of the present invention are inserted into expression vectors (i.e., a nucleic acid construct) to enable expression of the recombinant polypeptide. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector of the present invention includes a shuttle vector which renders this vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, cloning vectors comprise transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation terminators (e.g., polyadenylation signals).

[0208] In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the cytokines of the present invention. In some embodiments, these include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the polypeptide coding sequence.

[0209] In some embodiments, non-bacterial expression systems are used (e.g. mammalian expression systems such as CHO cells) to express the cytokines of the present invention. In one embodiment, the expression vector used to express polynucleotides of the present invention in mammalian cells is pCI-DHFR vector comprising a CMV promoter and a neomycin resistance gene. Construction of the pCI-dhfr vector is described, according to one embodiment, in Example 1.

[0210] In some embodiments, in bacterial systems of the present invention, a number of expression vectors can be advantageously selected depending upon the use intended for the polypeptide expressed. In one embodiment, large quantities of polypeptide are desired. In one embodiment, vectors that direct the expression of high levels of the protein product, possibly as a fusion with a hydrophobic signal sequence, which directs the expressed product into the periplasm of the bacteria or the culture medium where the protein product is readily purified are desired. In one embodiment, certain fusion protein engineered with a specific cleavage site to aid in recovery of the polypeptide. In one embodiment, vectors adaptable to such manipulation include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)].

[0211] In one embodiment, yeast expression systems are used. In one embodiment, a number of vectors containing constitutive or inducible promoters can be used in yeast as disclosed in U.S. Pat. No. 5,932,447. In another embodiment, vectors which promote integration of foreign DNA sequences into the yeast chromosome are used.

[0212] In one embodiment, the expression vector of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.

[0213] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

[0214] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009/A.sup.+, pMTO10/A.sup.+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0215] In some embodiments, recombinant viral vectors are useful for in vivo expression of the cytokines of the present invention, because they offer advantages such as lateral infection and targeting specificity. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[0216] In one embodiment, various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0217] In some embodiments, introduction of nucleic acid by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.

[0218] In one embodiment, it will be appreciated that the cytokines of the present invention can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (i.e., in vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed, and then the modified cells are expanded in culture and returned to the individual (i.e., ex vivo gene therapy).

[0219] In one embodiment, in vivo gene therapy using a cytokine has been attempted in animal models such as rodents [Bohl et al., Blood. 2000; 95:2793-2798], primates [Gao et al., Blood, 2004, Volume 103, Number 9] and has proven successful in human clinical trials for patients with chronic renal failure [Lippin et al Blood 2005, 106, Number 7].

[0220] In one embodiment, plant expression vectors are used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.

[0221] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.

[0222] Various methods, in some embodiments, can be used to introduce the expression vector of the present invention into the host cell system. In some embodiments, such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0223] In some embodiments, transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant polypeptide. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. In one embodiment, an effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide of the present invention. In some embodiments, a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. In some embodiments, culturing conditions are within the expertise of one of ordinary skill in the art.

[0224] In some embodiments, depending on the vector and host system used for production, resultant cytokines of the present invention either remain within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or retained on the outer surface of a cell or viral membrane.

[0225] In one embodiment, following a predetermined time in culture, recovery of the recombinant polypeptide is affected.

[0226] In one embodiment, the phrase "recovering the recombinant polypeptide" used herein refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification.

[0227] In one embodiment, cytokines of the present invention are purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

[0228] In one embodiment, to facilitate recovery, the expressed coding sequence can be engineered to encode the polypeptide of the present invention and fused cleavable moiety. In one embodiment, a fusion protein can be designed so that the polypeptide can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the cleavable moiety. In one embodiment, a cleavage site is engineered between the polypeptide and the cleavable moiety, and the polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].

[0229] In one embodiment, the polypeptide of the present invention is retrieved in "substantially pure" form.

[0230] In one embodiment, the phrase "substantially pure" refers to a purity that allows for the effective use of the protein in the applications described herein.

[0231] In one embodiment, the polypeptide of the present invention can also be synthesized using in vitro expression systems. In one embodiment in vitro synthesis methods are well known in the art and the components of the system are commercially available.

[0232] In one embodiment, production of CTP-cytokine polypeptides using recombinant DNA technology is illustrated in Example 1.

[0233] In some embodiments, the recombinant polypeptides are synthesized and purified; their therapeutic efficacy can be assayed either in vivo or in vitro. In one embodiment, the binding activities of the recombinant cytokines of the present invention can be ascertained using various assays as described in Examples 2-6 and 8-9.

[0234] In another embodiment, in vitro binding activity is ascertained by measuring the ability of the cytokine as described herein as well as pharmaceutical compositions comprising the same to treat diseases such as cancers such as hairy cell leukemia, malignant melanoma, Kaposi's sarcoma, bladder cancer, chronic myelocytic leukemia, kidney cancer, carcinoid tumors, non-Hodgkin's lymphoma, ovarian cancer, and skin cancers (for interferons). In one embodiment, in vivo activity is deduced by analyzing heamatocrit levels (FIGS. 3-5) and/or as a percentage of reticulocytes (for EPO). In another embodiment, in vivo activity is deduced by known measures of the disease that is being treated.

[0235] In some embodiments, the phrase "erythropoietin-associated conditions" refers to any condition associated with below normal, abnormal, or inappropriate modulation of erythropoietin. In some embodiments, levels of erythropoietin associated with such conditions are determined by any measure accepted and utilized by those of skill in the art. In some embodiments, erythropoietin-associated conditions typically include anemic conditions.

[0236] In some embodiments, "anemic conditions" refers to any condition, disease, or disorder associated with anemia. In some embodiments, anemic conditions include, but are not limited to, aplastic anemia, autoimmune hemolytic anemia, bone marrow transplantation, Churg-Strauss syndrome, Diamond Blackfan anemia, Fanconi's anemia, Felty syndrome, graft versus host disease, hematopoietic stem cell transplantation, hemolytic uremic syndrome, myelodysplasic syndrome, nocturnal paroxysmal hemoglobinuria, osteomyelofibrosis, pancytopenia, pure red-cell aplasia, purpura Schoenlein-Henoch, sideroblastic anemia, refractory anemia with excess of blasts, rheumatoid arthritis, Shwachman syndrome, sickle cell disease, thalassemia major, thalassemia minor, thrombocytopenic purpura, etc.

[0237] In one embodiment, the present invention comprises CTP-hGH-CTP-CTP polypeptides. In one embodiment, recombinant DNA technology methods are used for the production of CTP-hGH-CTP-CTP polypeptides as illustrated in Example 7. In one embodiment, the therapeutic efficacy of the CTP-hGH-CTP-CTP polypeptides of the present invention is assayed in vivo. In one embodiment, the therapeutic efficacy of the CTP-hGH-CTP-CTP polypeptides of the present invention is assayed in vitro. In one embodiment, the binding activities of the recombinant hGH polypeptides of the present invention are measured using Nb2 (a prolactin-dependent rat lymphoma cell line (ECACC Cell Bank)) or a FCD-P1 murine cell line, previously transfected with human growth hormone receptor. In one embodiment, binding of hGH to these receptors induces cell proliferation, which in one embodiment, is measured by the levels of MTT cellular stain as a function of hGH activity. In one embodiment, in vivo activity is deduced by measuring weight gain over time in treated growth hormone-deficient animals.

[0238] In some embodiments, human growth hormone polypeptides of the present invention can be used to treat a subject, with conditions related to growth and weight, such as a growth deficiency disorder, AIDS wasting, aging, impaired immune function of HIV-infected subjects, a catabolic illness, surgical recovery, a congestive cardiomyopathy, liver transplantation, liver regeneration after hepatectomy, chronic renal failure, renal osteodystrophy, osteoporosis, achondroplasia/hypochondroplasia, skeletal dysplasia, a chronic inflammatory or nutritional disorder such as Crohn's disease, short bowel syndrome, juvenile chronic arthritis, cystic fibrosis, male infertility, X-linked hypophosphatemic rickets, Down's syndrome, Spina bifida, Noonan Syndrome, obesity, impaired muscle strength and fibromyalgia.

[0239] In some embodiments, interferon polypeptides of the present invention are used to treat a subject, with a variety of conditions such as hairy cell leukemia (HCL), Kaposi's sarcoma (KS), chronic myelogenous leukemia (CML), chronic Hepatitis C (CHC), condylomata acuminata (CA), chronic Hepatitis B, malignant melanoma, follicular non-Hodgkin's lymphoma, multiple sclerosis, chronic granulomatous disease, Mycobacterium avium complex (MAC), pulmonary fibrosis, osteoarthritis, and osteoporosis.

[0240] In another embodiment, polypeptides of the present invention comprising IFN .alpha.-2a as well as pharmaceutical compositions comprising the same are indicated for hairy cell leukemia (HCL), acquired immune deficiency syndrome (AIDS)-related Kaposi's sarcoma (KS), chronic-phase Philadelphia (Ph) chromosome-positive chronic myelogenous leukemia (CML) and chronic Hepatitis C (CHC). IFN .alpha.-2a dosage varies depending on the indication. In another embodiment, the effectiveness of IFN .alpha.-2a as an antineoplastic, immunomodulator and antiviral agent has been established.

[0241] In another embodiment, polypeptides of the present invention comprising IFN .alpha.2b as well as pharmaceutical compositions comprising the same are indicated for HCL, AIDS-related Kaposi's sarcoma and CHC. It is also indicated for condylomata acuminata (CA), chronic Hepatitis B, malignant melanoma and follicular non-Hodgkin's lymphoma. IFN .alpha.-2b dosage varies depending on its indication of usage.

[0242] In another embodiment, polypeptides of the present invention comprising a cytokine are administered in a dose of 1-90 micrograms in 0.1-5 ml solution. In another embodiment, polypeptides of the present invention comprising a cytokine are administered in a dose of 1-50 micrograms in 0.1-5 ml solution. In another embodiment, polypeptides of the present invention comprising a cytokine are administered in a dose of 1-25 micrograms in 0.1-5 ml solution. In another embodiment, polypeptides of the present invention comprising a cytokine are administered in a dose of 50-90 micrograms in 0.1-5 ml solution. In another embodiment, polypeptides of the present invention comprising a cytokine are administered in a dose of 10-50 micrograms in 0.1-5 ml solution.

[0243] In another embodiment, polypeptides of the present invention comprising a cytokine are administered in a dose of 1-90 micrograms in 0.1-5 ml solution by intramuscular (i.m.) injection, subcutaneous (s.c.) injection, or intravenous (i.v.) injection once a week. In another embodiment, polypeptides of the present invention comprising a cytokine are administered in a dose of 1-90 micrograms in 0.1-5 ml solution by intramuscular (i.m.) injection, subcutaneous (s.c.) injection, or intravenous (i.v.) injection twice a week. In another embodiment, polypeptides of the present invention comprising a cytokine are administered in a dose of 1-90 micrograms in 0.1-5 ml solution by intramuscular (i.m.) injection, subcutaneous (s.c.) injection, or intravenous (i.v.) injection three times a week. In another embodiment, polypeptides of the present invention comprising a cytokine are administered in a dose of 1-90 micrograms in 0.1-5 ml solution by intramuscular (i.m.) injection, subcutaneous (s.c.) injection, or intravenous (i.v.) injection once every two weeks. In another embodiment, polypeptides of the present invention comprising a cytokine are administered in a dose of 1-90 micrograms in 0.1-5 ml solution by intramuscular (i.m.) injection, subcutaneous (s.c.) injection, or intravenous (i.v.) injection once every 17 days. In another embodiment, polypeptides of the present invention comprising a cytokine are administered in a dose of 1-90 micrograms in 0.1-5 ml solution by intramuscular (i.m.) injection, subcutaneous (s.c.) injection, or intravenous (i.v.) injection once every 19 days.

[0244] In another embodiment, polypeptides of the present invention comprise recombinant cytokines. In another embodiment, polypeptides of the present invention comprise a recombinant IFN-.beta.. In another embodiment, polypeptides of the present invention comprise a recombinant IFN-.alpha.. In another embodiment, various recombinant IFN are known to one of skill in the art.

[0245] In another embodiment, protein drugs of molecular weight lower than 50,000 daltons, such as interferons, are in general short-lived species in vivo, having short circulatory half-lives of several hours. In another embodiment, the subcutaneous route of administration in general provides slower release into the circulation. In another embodiment, the CTP-modified polypeptide of the invention prolongs the half-life of protein drugs of molecular weight lower than 50,000 daltons, such as interferons. In another embodiment, the CTP-modified polypeptide of the invention enables interferons to exert their beneficial effects for a longer period of time.

[0246] In another embodiment, the immunogenicity of a CTP-modified polypeptide comprising a cytokine is equal to an isolated cytokine. In another embodiment, the immunogenicity of a CTP-modified polypeptide comprising a cytokine is comparable to an isolated cytokine. In another embodiment, modifying a cytokine as described herein with CTP peptides reduces the immunogenicity of the cytokine. In another embodiment, the CTP-modified polypeptide comprising a cytokine is as active as an isolated cytokine protein. In another embodiment, the CTP-modified polypeptide comprising a cytokine is more active than an isolated cytokine. In another embodiment, the CTP-modified polypeptide comprising a cytokine maximizes the cytokine's protective ability against degradation while minimizing reductions in bioactivity.

[0247] In another embodiment, the cytokine of the present invention can be provided to the individual per se. In one embodiment, the cytokine of the present invention can be provided to the individual as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier.

[0248] In another embodiment, 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.

[0249] In another embodiment, "active ingredient" refers to the polypeptide sequence of interest, which is accountable for the biological effect.

[0250] In another embodiment, any of the compositions of this invention will comprise at least two CTP sequences bound to a cytokine of interest, in any form. In one embodiment, the present invention provides combined preparations. In one embodiment, "a combined preparation" defines especially a "kit of parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners i.e., simultaneously, concurrently, separately or sequentially. In some embodiments, the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is, at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners, in some embodiments, can be administered in the combined preparation.

[0251] In one embodiment, the combined preparation can be varied, e.g., in order to cope with the needs of a patient subpopulation to be treated or the needs of the single patient which different needs can be due to a particular disease, severity of a disease, age, sex, or body weight as can be readily made by a person skilled in the art.

[0252] In another embodiment, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which can be used interchangeably, 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. In one embodiment, one of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979).

[0253] In another embodiment, "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. In one embodiment, excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0254] Techniques for formulation and administration of drugs are found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0255] In another embodiment, suitable routes of administration, for example, include oral, rectal, transmucosal, transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0256] In another embodiment, the preparation is administered in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body.

[0257] Various embodiments of dosage ranges are contemplated by this invention. The dosage of the cytokine of the present invention, in one embodiment, is in the range of 0.005-100 mg/day. In another embodiment, the dosage is in the range of 0.005-5 mg/day. In another embodiment, the dosage is in the range of 0.01-50 mg/day. In another embodiment, the dosage is in the range of 0.1-20 mg/day. In another embodiment, the dosage is in the range of 0.1-10 mg/day. In another embodiment, the dosage is in the range of 0.01-5 mg/day. In another embodiment, the dosage is in the range of 0.001-0.01 mg/day. In another embodiment, the dosage is in the range of 0.001-0.1 mg/day. In another embodiment, the dosage is in the range of 0.1-5 mg/day. In another embodiment, the dosage is in the range of 0.5-50 mg/day. In another embodiment, the dosage is in the range of 0.2-15 mg/day. In another embodiment, the dosage is in the range of 0.8-65 mg/day. In another embodiment, the dosage is in the range of 1-50 mg/day. In another embodiment, the dosage is in the range of 5-10 mg/day. In another embodiment, the dosage is in the range of 8-15 mg/day. In another embodiment, the dosage is in a range of 10-20 mg/day. In another embodiment, the dosage is in the range of 20-40 mg/day. In another embodiment, the dosage is in a range of 60-120 mg/day. In another embodiment, the dosage is in the range of 12-40 mg/day. In another embodiment, the dosage is in the range of 40-60 mg/day. In another embodiment, the dosage is in a range of 50-100 mg/day. In another embodiment, the dosage is in a range of 1-60 mg/day. In another embodiment, the dosage is in the range of 15-25 mg/day. In another embodiment, the dosage is in the range of 5-10 mg/day. In another embodiment, the dosage is in the range of 55-65 mg/day.

[0258] In another embodiment, a polypeptide comprising a cytokine and CTP units is formulated in an intranasal dosage form. In another embodiment, a polypeptide comprising a cytokine and CTP units is formulated in an injectable dosage form. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 0.0001 mg to 0.6 mg. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 0.001 mg to 0.005 mg. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 0.005 mg to 0.01 mg. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 0.01 mg to 0.3 mg. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose in a dose ranging from 0.2 mg to 0.6 mg.

[0259] In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 1-100 micrograms. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 10-80 micrograms. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 20-60 micrograms. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 10-50 micrograms. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 40-80 micrograms. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 10-30 micrograms. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 30-60 micrograms.

[0260] In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 0.2 mg to 2 mg. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 2 mg to 6 mg. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 4 mg to 10 mg. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject in a dose ranging from 5 mg to 15 mg.

[0261] In another embodiment, a polypeptide comprising a cytokine and CTP units is injected into the muscle (intramuscular injection). In another embodiment, a polypeptide comprising a cytokine and CTP units is injected below the skin (subcutaneous injection). In another embodiment, a polypeptide comprising an IFN protein and CTP units is injected into the muscle. In another embodiment, a polypeptide comprising an IFN protein and CTP units is injected below the skin.

[0262] In another embodiment, the methods of the invention include increasing the compliance in the use of cytokine therapy, comprising providing to a subject in need thereof, a polypeptide comprising a cytokine, one chorionic gonadotrophin carboxy terminal peptide (CTP) attached to an amino terminus of the cytokine, and two chorionic gonadotrophin carboxy terminal peptides attached to a carboxy terminus of the cytokine, thereby increasing compliance in the use of cytokine therapy.

[0263] In another embodiment, the methods of the invention include increasing the compliance of patients afflicted with chronic illnesses that are in need of a cytokine therapy. In another embodiment, the methods of the invention enable reduction in the dosing frequency of a cytokine by modifying the cytokine with CTPs as described hereinabove. In another embodiment, the term compliance comprises adherence. In another embodiment, the methods of the invention include increasing the compliance of patients in need of a cytokine therapy by reducing the frequency of administration of the cytokine. In another embodiment, reduction in the frequency of administration of the cytokine is achieved due to the CTP modifications which render the CTP-modified cytokine more stable. In another embodiment, reduction in the frequency of administration of the cytokine is achieved as a result of increasing T.sub.1/2 of the cytokine. In another embodiment, reduction in the frequency of administration of the cytokine is achieved as a result of increasing the clearance time of the cytokine. In another embodiment, reduction in the frequency of administration of the cytokine is achieved as a result of increasing the AUC measure of the cytokine.

[0264] In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject once a day. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject once every two days. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject once every three days. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject once every four days. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject once every five days. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject once every six days. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject once every week. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject once every 7-14 days. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject once every 10-20 days. In another embodiment, a polypeptide comprising a cytokine and CTP units is administered to a subject once every 5-15 days. In another embodiment, a polypeptide comprising an a cytokine and CTP units is administered to a subject once every 15-30 days.

[0265] In another embodiment, the dosage is in a range of 50-500 mg/day. In another embodiment, the dosage is in a range of 50-150 mg/day. In another embodiment, the dosage is in a range of 100-200 mg/day. In another embodiment, the dosage is in a range of 150-250 mg/day. In another embodiment, the dosage is in a range of 200-300 mg/day. In another embodiment, the dosage is in a range of 250-400 mg/day. In another embodiment, the dosage is in a range of 300-500 mg/day. In another embodiment, the dosage is in a range of 350-500 mg/day.

[0266] In one embodiment, the dosage is 20 mg/day. In one embodiment, the dosage is 30 mg/day. In one embodiment, the dosage is 40 mg/day. In one embodiment, the dosage is 50 mg/day. In one embodiment, the dosage is 0.01 mg/day. In another embodiment, the dosage is 0.1 mg/day. In another embodiment, the dosage is 1 mg/day. In another embodiment, the dosage is 0.530 mg/day. In another embodiment, the dosage is 10 mg/day. In another embodiment, the dosage is 20-70 mg/day. In another embodiment, the dosage is 5 mg/day.

[0267] In another embodiment, the dosage is 1-90 mg/day. In another embodiment, the dosage is 1-90 mg/2 days. In another embodiment, the dosage is 1-90 mg/3 days. In another embodiment, the dosage is 1-90 mg/4 days. In another embodiment, the dosage is 1-90 mg/5 days. In another embodiment, the dosage is 1-90 mg/6 days. In another embodiment, the dosage is 1-90 mg/week. In another embodiment, the dosage is 1-90 mg/9 days. In another embodiment, the dosage is 1-90 mg/11 days. In another embodiment, the dosage is 1-90 mg/14 days.

[0268] In another embodiment, the cytokine dosage is 10-50 mg/day. In another embodiment, the dosage is 10-50 mg/2 days. In another embodiment, the dosage is 10-50 mg/3 days. In another embodiment, the dosage is 10-50 mg/4 days. In another embodiment, the dosage is 10-50 micrograms mg/5 days. In another embodiment, the dosage is 10-50 mg/6 days. In another embodiment, the dosage is 10-50 mg/week. In another embodiment, the dosage is 10-50 mg/9 days. In another embodiment, the dosage is 10-50 mg/11 days. In another embodiment, the dosage is 10-50 mg/14 days.

[0269] Oral administration, in one embodiment, comprises a unit dosage form comprising tablets, capsules, lozenges, chewable tablets, suspensions, emulsions and the like. Such unit dosage forms comprise a safe and effective amount of the desired cytokine of the invention, each of which is, in one embodiment, from about 0.7 or 3.5 mg to about 280 mg/70 kg, or, in another embodiment, about 0.5 or 10 mg to about 210 mg/70 kg. The pharmaceutically acceptable carriers suitable for the preparation of unit dosage forms for peroral administration are well-known in the art. In some embodiments, tablets typically comprise conventional pharmaceutically-compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrants such as starch, alginic acid and croscarmelose; lubricants such as magnesium stearate, stearic acid and talc. In one embodiment, glidants such as silicon dioxide can be used to improve flow characteristics of the powder-mixture. In one embodiment, coloring agents, such as the FD&C dyes, can be added for appearance. Sweeteners and flavoring agents, such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets. Capsules typically comprise one or more solid diluents disclosed above. In some embodiments, the selection of carrier components depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of this invention, and can be readily made by a person skilled in the art.

[0270] In one embodiment, the oral dosage form comprises predefined release profile. In one embodiment, the oral dosage form of the present invention comprises an extended release tablets, capsules, lozenges or chewable tablets. In one embodiment, the oral dosage form of the present invention comprises a slow release tablets, capsules, lozenges or chewable tablets. In one embodiment, the oral dosage form of the present invention comprises an immediate release tablets, capsules, lozenges or chewable tablets. In one embodiment, the oral dosage form is formulated according to the desired release profile of the pharmaceutical active ingredient as known to one skilled in the art.

[0271] Peroral compositions, in some embodiments, comprise liquid solutions, emulsions, suspensions, and the like. In some embodiments, pharmaceutically acceptable carriers suitable for preparation of such compositions are well known in the art. In some embodiments, liquid oral compositions comprise from about 0.001% to about 0.933% of the desired compound or compounds, or in another embodiment, from about 0.01% to about 10%.

[0272] In some embodiments, compositions for use in the methods of this invention comprise solutions or emulsions, which in some embodiments, are aqueous solutions or emulsions comprising a safe and effective amount of the compounds of the present invention and optionally, other compounds, intended for topical intranasal administration. In some embodiments, the compositions comprise from about 0.001% to about 10.0% w/v of a subject compound, more preferably from about 00.1% to about 2.0, which is used for systemic delivery of the compounds by the intranasal route.

[0273] In another embodiment, the pharmaceutical compositions are administered by intravenous, intra-arterial, or intramuscular injection of a liquid preparation. In some embodiments, liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment, the pharmaceutical compositions are administered intravenously, and are thus formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intra-arterially, and are thus formulated in a form suitable for intra-arterial administration. In another embodiment, the pharmaceutical compositions are administered intramuscularly, and are thus formulated in a form suitable for intramuscular administration.

[0274] Further, in another embodiment, the pharmaceutical compositions are administered topically to body surfaces, and are thus formulated in a form suitable for topical administration. Suitable topical formulations include gels, ointments, creams, lotions, drops and the like. For topical administration, the compounds of the present invention are combined with an additional appropriate therapeutic agent or agents, prepared and applied as solutions, suspensions, or emulsions in a physiologically acceptable diluent with or without a pharmaceutical carrier.

[0275] In one embodiment, pharmaceutical compositions of the present invention are 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.

[0276] In one embodiment, pharmaceutical compositions for use in accordance with the present invention is 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. In one embodiment, formulation is dependent upon the route of administration chosen.

[0277] In one embodiment, injectables of the invention are formulated in aqueous solutions. In one embodiment, injectables of the invention are formulated in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. In some embodiments, for transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0278] In one embodiment, the preparations described herein are formulated for parenteral administration, e.g., by bolus injection or continuous infusion. In some embodiments, formulations for injection are presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. In some embodiments, compositions are suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0279] The compositions also comprise, in some embodiments, preservatives, such as benzalkonium chloride and thimerosal and the like; chelating agents, such as edetate sodium and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol and others; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulfote and others; aromatic agents; viscosity adjustors, such as polymers, including cellulose and derivatives thereof; and polyvinyl alcohol and acid and bases to adjust the pH of these aqueous compositions as needed. The compositions also comprise, in some embodiments, local anesthetics or other actives. The compositions can be used as sprays, mists, drops, and the like.

[0280] In some embodiments, pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients, in some embodiments, are prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include, in some embodiments, fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions contain, in some embodiments, substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. In another embodiment, the suspension also contains suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0281] In another embodiment, the active compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).

[0282] In another embodiment, the pharmaceutical composition delivered in a controlled release system is formulated for intravenous infusion, implantable osmotic pump, transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump is used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990).

[0283] In some embodiments, the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water-based solution, before use. Compositions are formulated, in some embodiments, for atomization and inhalation administration. In another embodiment, compositions are contained in a container with attached atomizing means.

[0284] In one embodiment, the preparation of the present invention is formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[0285] In some embodiments, pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. In some embodiments, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.

[0286] In one embodiment, determination of a therapeutically effective amount is well within the capability of those skilled in the art.

[0287] Some examples of substances which can serve as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the Tween.TM. brand emulsifiers; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions. The choice of a pharmaceutically acceptable carrier to be used in conjunction with the compound is basically determined by the way the compound is to be administered. If the subject compound is to be injected, in one embodiment, the pharmaceutically acceptable carrier is sterile, physiological saline, with a blood-compatible suspending agent, the pH of which has been adjusted to about 7.4.

[0288] In addition, the compositions further comprise binders (e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris-HCl, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents (e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g. aspartame, citric acid), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.

[0289] Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For a suspension, typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, cellulose (e.g. Avicel.TM., RC-591), tragacanth and sodium alginate; typical wetting agents include lecithin and polyethylene oxide sorbitan (e.g. polysorbate 80). Typical preservatives include methyl paraben and sodium benzoate. In another embodiment, peroral liquid compositions also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above.

[0290] The compositions also include incorporation of the active material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.

[0291] Also comprehended by the invention are particulate compositions coated with polymers (e.g. poloxamers or poloxamines) and the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors.

[0292] In some embodiments, compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. In another embodiment, the modified compounds exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds. In one embodiment, modifications also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. In another embodiment, the desired in vivo biological activity is achieved by the administration of such polymer-compound abducts less frequently or in lower doses than with the unmodified compound.

[0293] In some embodiments, preparation of an effective amount or dose can be estimated initially from in vitro assays. In one embodiment, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

[0294] In one embodiment, 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. In one embodiment, 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. In one embodiment, the dosages vary depending upon the dosage form employed and the route of administration utilized. In one embodiment, the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1].

[0295] In one embodiment, depending on the severity and responsiveness of the condition to be treated, 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.

[0296] In one embodiment, the amount of a composition 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.

[0297] In one embodiment, compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0298] In another embodiment, a cytokine as described herein is administered via systemic administration. In another embodiment, a cytokine as described herein is administered by intravenous, intramuscular or subcutaneous injection. In another embodiment, a cytokine as described herein is in a lyophilized (i.e., freeze-dried) preparation in combination with complex organic excipients and stabilizers such as nonionic surface active agents (i.e., surfactants), various sugars, organic polyols and/or human serum albumin. In another embodiment, a pharmaceutical composition comprises a lyophilized cytokine as described in sterile water for injection. In another embodiment, a pharmaceutical composition comprises a lyophilized cytokine as described in sterile PBS for injection. In another embodiment, a pharmaceutical composition comprises a lyophilized cytokine as described in sterile 0.9% NaCl for injection.

[0299] In another embodiment, the pharmaceutical composition comprises a cytokine as described herein and complex carriers such as human serum albumin, polyols, sugars, and anionic surface active stabilizing agents. See, for example, WO 89/10756 (Hara et al.--containing polyol and p-hydroxybenzoate). In another embodiment, the pharmaceutical composition comprises a cytokine as described herein and lactobionic acid and an acetate/glycine buffer. In another embodiment, the pharmaceutical composition comprises a cytokine as described herein and amino acids, such as arginine or glutamate that increase the solubility of interferon compositions in water. In another embodiment, the pharmaceutical composition comprises a lyophilized cytokine as described herein and glycine or human serum albumin (HSA), a buffer (e g. acetate) and an isotonic agnet (e g. NaCl). In another embodiment, the pharmaceutical composition comprises a lyophilized cytokine as described herein and phosphate buffer, glycine and HSA.

[0300] In another embodiment, the pharmaceutical composition comprising a cytokine as described herein is stabilized when placed in buffered solutions having a pH of between about 4 and 7.2. In another embodiment, the pharmaceutical composition comprising a cytokine as described herein is stabilized with an amino acid as a stabilizing agent and in some cases a salt (if the amino acid does not contain a charged side chain).

[0301] In another embodiment, the pharmaceutical composition comprising a cytokine as described herein is a liquid composition comprising a stabilizing agent at between about 0.3% and 5% by weight which is an amino acid.

[0302] In another embodiment, the pharmaceutical composition comprising a cytokine as described herein provides dosing accuracy and product safety. In another embodiment, the pharmaceutical composition comprising a cytokine as described herein provides a biologically active, stable liquid formulation for use in injectable applications. In another embodiment, the pharmaceutical composition comprises a non-lyophilized cytokine as described herein.

[0303] In another embodiment, the pharmaceutical composition comprising a cytokine as described herein provides a liquid formulation permitting storage for a long period of time in a liquid state facilitating storage and shipping prior to administration.

[0304] In another embodiment, the pharmaceutical composition comprising a cytokine as described herein comprises solid lipids as matrix material. In another embodiment, the injectable pharmaceutical composition comprising a cytokine as described herein comprises solid lipids as matrix material. In another embodiment, the production of lipid microparticles by spray congealing was described by Speiser (Speiser and al., Pharm. Res. 8 (1991) 47-54) followed by lipid nanopellets for peroral administration (Speiser EP 0167825 (1990)). In another embodiment, lipids, which are used are well tolerated by the body (e. g. glycerides composed of fatty acids which are present in the emulsions for parenteral nutrition).

[0305] In another embodiment, the pharmaceutical composition comprising a cytokine as described herein is in the form of liposomes (J. E. Diederichs and al., Pharm./nd. 56 (1994) 267-275).

[0306] In another embodiment, the pharmaceutical composition comprising a cytokine as described herein comprises polymeric microparticles. In another embodiment, the injectable pharmaceutical composition comprising a cytokine as described herein comprises polymeric microparticles. In another embodiment, the pharmaceutical composition comprising a cytokine as described herein comprises nanoparticles. In another embodiment, the pharmaceutical composition comprising a cytokine as described herein comprises liposomes. In another embodiment, the pharmaceutical composition comprising a cytokine as described herein comprises lipid emulsion. In another embodiment, the pharmaceutical composition comprising a cytokine as described herein comprises microspheres. In another embodiment, the pharmaceutical composition comprising a cytokine as described herein comprises lipid nanoparticles. In another embodiment, the pharmaceutical composition comprising a cytokine as described herein comprises lipid nanoparticles comprising amphiphilic lipids. In another embodiment, the pharmaceutical composition comprising a cytokine as described herein comprises lipid nanoparticles comprising a drug, a lipid matrix and a surfactant. In another embodiment, the lipid matrix has a monoglyceride content which is at least 50% w/w.

[0307] In one embodiment, compositions of the present invention are presented in a pack or dispenser device, such as an FDA approved kit, which contain one or more unit dosage forms containing the active ingredient. In one embodiment, the pack, for example, comprise metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration. Such notice, in one embodiment, is labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.

[0308] In one embodiment, it will be appreciated that the cytokines of the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself. In another embodiment, measures (e.g., dosing and selection of the complementary agent) are taken to minimize adverse side effects which are associated with combination therapies.

[0309] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

[0310] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique" by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W.H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, Calif. (1990); Marshak et al., "Strategies for Protein Purification and Characterization--A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

Example 1

Generation of EPO Constructs

Materials and Methods:

[0311] Construction of expression vector pCI-dhfr: pCI-neo mammalian expression vector was purchased from Promega (Catalog No. E1841). The vector contains a CMV IE enhancer/promoter and neomycin phosphotransferase gene. The pSV2-dhfr clone was purchased from ATCC (Catalog No. 37146). The plasmid contains the murine dhfr gene. The construction of pCI-dhfr vector was performed as follows:

[0312] The pSV2-dhfr plasmid was digested with restriction enzyme BglII (3' end of the dhfr gene). DNA polymerase I, Large (Klenow) Fragment was used to fill in the 5' overhangs to form blunt ends. The DNA was then digested with restriction enzyme AvrII (5' end of the dhfr gene). The dhfr gene (AvrII-blunt end) fragment was isolated.

[0313] The pCI-neo vector was digested with restriction enzyme BstXI (3' end of the neo gene). DNA polymerase I, Large (Klenow) Fragment was used to remove the 3' overhangs to form blunt ends. The DNA was then digested with restriction enzyme AvrII (5' end of the neo gene). The expression vector (AvrII-blunt end) was isolated.

[0314] The dhfr gene was ligated into pCI vector to form an expression vector containing the dhfr gene (pCI-dhfr).

[0315] Construction of hEPO-CTP Variants:

[0316] A cassette gene containing the C-Terminal Peptide (CTP) of the beta subunit of hCG was fused to the coding sequence of human EPO (NP_000790.2) at different locations. Six EPO-CTP variants were constructed as illustrated in FIGS. 1A-F. The proEPO signal peptide was used for the construction of the secreted EPO-CTP variants. XbaI-NotI fragments containing EPO sequences were ligated into the pCI-dhfr expression vector of the present invention.

[0317] Table 2 hereinbelow summarizes the primer sequences used for constructing the CTP-containing polypeptides of the present invention.

TABLE-US-00012 TABLE 2 Restric- tion Pri- site (un- mer SEQ derlined num- ID in se- ber NO sequence quence) 1 7 5' AATCTAGAGGTCATCATGGGGGTGC 3' XbaI 2 8 5' ATTGCGGCCGCGGATCCAGAAGACCTT NotI TATTG 3' 17.sup.R 9 5' TAAATATTGGGGTGTCCGAGGGCCC 3' SspI 10 10 5' CCAATATTACCACAAGCCCCACCACGCC SspI TCAT 3' 11.sup.R 11 5' TGCGGCCGCGGATCCTTATCTGTCCCCT NotI GTCCTGC 3' 15 12 5' GCCCTGCTGTCGGAAGC 3' 2.sup.R 13 5' ATTGCGGCCGCGGATCCAGAAGACCTTT NotI ATTG 23.sup.R 14 5' CTTTGAGGAAGAGGAGCCCAGGACTGGG AGGC 3' 24 15 5' CCTGGGCTCCTCTTCCTCAAAGGC 3' 38.sup.R 16 5' GCTTCCGACAGCAGGGC 3'

[0318] EPO-1 701-1-p17-6 (Epo-1-SEQ ID NO: 1):

[0319] The XbaI-NotI 702 bp fragment was constructed by PCR using the above primers (SEQ ID NOs: 7-16). Then the XbaI-NotI PCR fragment containing Epo-ctp sequence was ligated into pCI-dhfr expression vector.

[0320] EPO-2 701-2-p24-2 (Epo-2-SEQ ID NO: 2):

[0321] The XbaI/ApaI fragment (hGH-ctp) of pCI-dhfr-401-2-p21-2 (hGH-ctpx2) was replaced by the XbaI/ApaI fragment (EPO-ctp) of 701-1-p17-6 to create an Epo-ctpx2.

[0322] EPO-4-701-4-p42-1(Epo-4-SEQ ID NO: 4):

[0323] Firstly, a fragment from pCI-dhfr-EPO-ctp (701-1-p17-6) was constructed by PCR using primers 1 and 17 followed by XbaI/SspI digestion. This resulted in a fragment containing EPO and partial 5' CTP.

[0324] Secondly, a new fragment was constructed by overlapping PCR, on pGT123-hEpo as a template, using primer 10 and primer 11. SspI/NotI digestion resulted in a fragment containing 3' partial CTP and Epo.

[0325] The two fragments were ligated into pCI-dhfr to construct the p701-4-p42-1 clone.

[0326] EPO-3-p56-6 (Epo-3 SEQ ID NO 3):

[0327] Primers were purchased from Sigma-Genosys. A PCR reaction was performed using primer 15 (SEQ ID NO: 12) and primer 2.sup.R (SEQ ID NO: 13) and plasmid DNA of pCI-dhfr-EPO-ctp .times.2 (701-2-p24-2) as a template. As a result of the PCR amplification, a 486 bp product was formed and ligated into TA cloning vector (Invitrogen, catalog K2000-01). Stu I-NotI fragment containing *Epo-ctp .times.2 sequence was isolated (209 bp).

[0328] Three sequential PCR reactions were performed. The first reaction was conducted with primer 1 (SEQ ID NO: 7) and primer 23.sup.R (SEQ ID NO: 14) and plasmid DNA of pGT123-epo-ctp as a template; as a result of the PCR amplification, an 80 bp product was formed (signal peptide).

[0329] The second reaction was conducted with primer 24 (SEQ ID NO: 15) and primer 11.sup.R (SEQ ID NO: 11) and plasmid DNA of 701-4-p42-1 as a template; as a result of the PCR amplification, a 610 bp product was formed.

[0330] The last reaction was conducted with primers 1 (SEQ ID NO: 7) and 11.sup.R (SEQ ID NO: 11) and a mixture of the products of the previous two reactions as a template; as a result of the PCR amplification, a 700 bp product was formed and the XbaI-StuI fragment was isolated.

[0331] The two fragments (XbaI-StuI and StuI-NotI) were inserted into the eukaryotic expression vector pCI-dhfr (triple ligation) to yield the 701-3-p56-6 clone.

[0332] EPO-5-p91-4 (Epo-5 SEQ ID NO 5-(ctp-Epo):

[0333] Primers were ordered from Sigma-Genosys. A PCR reaction was performed using primer 1 (SEQ ID NO: 7) and primer 11.sup.R (SEQ ID NO: 11) and plasmid DNA of pCI-dhfr-ctp-EPO-ctp .times.2 (701-3-p56-6) as a template; as a result of the PCR amplification, a 670 bp product was formed and ligated into TA cloning vector (Invitrogen, catalog K2000-01). XbaI-NotI fragment containing ctp-Epo sequence was ligated into our eukaryotic expression vector pCI-dhfr to yield the 701-5-p91-4 clone.

[0334] EPO-6-p90-1 (Epo-6 SEQ ID NO: 6--(ctp-Epo-ctp):

[0335] Three PCR reactions were performed. The first reaction was conducted with primer 1 (SEQ ID NO: 7) and primer 38.sup.R(SEQ ID NO: 16) and plasmid DNA of 701-3-p56-6 as a template; as a result of the PCR amplification, a 400 bp product was formed.

[0336] The second reaction was conducted with primer 15 (SEQ ID NO: 12) and primer 2.sup.R (SEQ ID NO: 13) and plasmid DNA of 701-1-p17-6 as a template; as a result of the PCR amplification, a 390 bp product was formed.

[0337] The last reaction was conducted with primers 1 (SEQ ID NO: 7) and 2.sup.R (SEQ ID NO: 13) and a mixture of the products of the previous two reactions as a template; as a result of the PCR amplification, a 787 bp product was formed and ligated into a TA cloning vector (Invitrogen, catalog K2000-01). The XbaI-NotI fragment containing ctp-Epo-ctp sequence was ligated into the eukaryotic expression vector pCI-dhfr to yield the 701-6-p90-1 clone.

Example 2

Expression and Isolation of EPO-CTP Polypeptides

Materials and Methods

[0338] DNA Transfection and Clone Selection:

[0339] DG44 cells were transfected with pCI-DHFR expression vectors containing EPO-CTP variants using FuGENE6 Reagent (FuGENE Transfection Reagent--Roche Cat. 11 815 091 001). 48 hr following transfection, cells were diluted and seeded at 50-200 cells per well in a selective medium (CD DG44 Medium w/o HT (Gibco: Scotland part: #07990111A) Sku num.:ME060027 supplemented with 8 mM L-Glutamine (Biological Industries: Cat: 03-020-1A) and 18 mL/L of 10% Pluronic F-68 solution (Gibco: Cat: 240040-032). Selected clones were screened for highest protein production using commercial ELISA. 3-5 producing clones per each variant were frozen for a backup cell bank. A selected clone for each variant was adapted to growth in larger scale cultures up to 1 L flasks on an orbital shaker platform. Supernatants were collected and analyzed by ELISA, SDS-PAGE and Western Blot. Following the withdrawal of aliquots, the protein-containing supernatants were kept frozen until further use.

[0340] Cell Culture:

[0341] DG44 cells were maintained in DG44 medium with HT (cat#12610-010, Gibco) supplemented with 8 mM L-Glutamine (Biological Industries: Cat: 03-020-1A) and 18 mL/L of 10% Pluronic F-68 solution (Gibco: Cat: 240040-032), at 37.degree. C. in a humidified 8% % CO.sub.2 incubator. Transfected clones were maintained in DG44 basal medium without HT supplement, hypoxanthine and thymidine, with pluronic acid and L-glutamine.

[0342] Sample Preparation:

[0343] Supernatants were collected, filtrated and analyzed by ELISA to determine protein concentration. SDS-PAGE and Western Blot were used to determine purity and identity. Following ELISA, sample concentrations were defined and the solution was dialyzed against PBS. Following the withdrawal of aliquots, the protein-contained supernatants were kept frozen at -20.degree. C. until further use.

[0344] Western Blotting:

[0345] Samples were electrophoresed on nondenaturing 15% SDS-polyacrylamide gels. Gels were allowed to equilibrate for 10 min in 25 mM Tris and 192 mM glycine in 20% (vol/vol) methanol). Proteins were transferred to a 0.2 .mu.m pore size nitrocellulose membrane (Sigma, Saint Louis, Mo.) at 250 mA for 3 h, using a Mini Trans-Blot electrophoresis cell (Biorad Laboratories, Richmond, Calif.). The nitrocellulose membrane was incubated in 5% non-fat dry milk for 2 h at room temperature. The membrane was incubated with EPO anti-serum (1:1000 titer) overnight at 4.degree. C. followed by three consecutive washes in PBS containing 0.1% Tween (10 min/wash). The membrane was incubated with secondary antibody conjugated to Horse Radish Peroxidase (HRP) (Zymed, San Francisco, Calif.) for 2 h at room temperature, followed by three washes. Finally, the nitrocellulose paper was reacted with enhanced chemiluminescent substrate (ECL) (Pierce, Rockford, Ill.) for 5 min, dried with a Whatman sheet, and exposed to X-ray film.

Results

[0346] Table 3 hereinbelow shows the concentrations of the various CTP-modified EPO forms obtained from 5 selected clones and their preparation for further testing.

TABLE-US-00013 TABLE 3 Post dilution in Mock sup. according to Post Stock Titer Epo3 titer ultrafiltration #Version # Clone IU/ml.sup.1 IU/ml.sup.2 IU/ml.sup.3 Epo0 17 3093 102 335 SEQ ID NO: 16 Epo1 47 1049 104 291 SEQ ID NO: 1 Epo2 67 2160 110 303 SEQ ID NO: 2 Epo3 85 105 119 392 SEQ ID NO: 3 Epo4 112 6100 ND 342 SEQ ID NO: 4 .sup.1EPO variants stock concentration were determined by ELISA (Quantikine IVD Epo ELISA, DEP00, R&D Systems) .sup.2Samples EPO-0, 1, 2 and 4 were diluted to 105 IU/ml in mock sup (Adjusted to Epo3 titer). Epo0 = wild type EPO expressed in the same system as the CTP modified EPOs .sup.3All samples were concentrated and dialyzed by ultrafiltration against PBS to a final concentration of 180 IU/ml.

[0347] All proteins were detected by Western blot as illustrated in FIG. 2.

Example 3

Biological Activity of the EPO-CTP Polypeptides of the Present Invention

[0348] The TF-1 bioactivity test represents the ability of the EPO-CTP variant to bind its receptor and then stimulate activity which results in cell proliferation. Therefore, this test was used as a first step in evaluating the biological potency of the EPO-CTP polypeptides of the present invention.

Materials and Methods

[0349] Cell Proliferation Analysis: Proliferation assay was performed with the cell line TF-1, measuring levels of MTT cellular stain as a function of EPO activity (Kitamura et al., Kitamura, T. et al. (1989) Establishment and characterization of a unique human cell line that proliferates; Hammerling U. et al. In vitro bioassay for human erythropoietin based on proliferative stimulation of an erythroid cell line and analysis of carbohydrate-dependent microheterogeneity. Journal of Pharm. Biomed. Analysis 14(11): 1455-1469 (1996)). Exponentially growing TF-1 cells were washed twice, plated at about 10.sup.4 cells/well in microtiter plates, and incubated in basal medium with a titrated dilution series of EPO (Recormon.RTM.), EPO standard (NIBSC standard), rhEPO (MOD-7010), MOD-701 variants (EPO-1, EPO-2, EPO-3 and EPO-4) for 48 hours. 4 hours prior to assaying for cell proliferation, MTT reagent was added to the wells, and absorbance was measured by ELISA reader. A calculated protein concentration value for each variant protein was obtained from Eprex.RTM.'s (Epoetin (EPO)-man-made form of the human hormone) dose-response standard curve.

Results

[0350] The in vitro biological activity of EPO polypeptides was determined with an Epo-dependent cell line, human erythroleukemia TF-1 (DSMZ Cell Bank) [Dong et al., Biochemical and Biophysical Research Communications, Volume 339, Issue 1, 6 Jan. 2006, Pages 380-385]. The MTT assay was performed [Hammerling U. et al. In vitro bioassay for human erythropoietin based on proliferative stimulation of an erythroid cell line and analysis of carbohydrate-dependent microheterogeneity. Journal of Pharm. Biomed. Analysis 14(11): 1455-1469 (1996);], and the laboratory standard of EPO used to generate the standard curve was calibrated against the International Standard (Epo ampoule code 87/684 of NIBSC).

[0351] The results are summarized in Table 4 hereinbelow. The results indicate that the highest potency was achieved by using EPO 3 and EPO 0 in both 2 and 0.5 IU/ml concentrations.

TABLE-US-00014 TABLE 4 TF-1 Bioactivity IU/ml Eprex STD EPO 0 EPO 1 EPO 2 EPO 3 EPO 4 IU/ml SEQ ID NO: 16 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 Recormon .RTM. EPO st 2 4.93 2.32 2.13 6.91 3.55 3.44 7.40 0.5 1.60 0.76 0.53 1.34 0.84 0.87 1.53

[0352] Conclusion

[0353] As summarized in Table 4 hereinabove, different levels of potency were exerted by EPO-CTP polypeptides, indicating differences in receptor binding. EPO-CTP polypeptides differ by the number of CTP cassettes and the location to which they are fused. EPO-1 and EPO-2 contain 1 CTP sequence or 2 CTP sequences at the C-terminal of EPO, while EPO-3 contains 1 CTP at N-terminal and 2 CTP sequences at C-terminal. EPO-4 is a dimer of two EPO molecules linked by CTP sequence. EPO-3 demonstrated potency level quite similar to WT-EPO, while EPO-1 and EPO-4 were about 50% less potent than WT-EPO, and EPO-2 potency was even less than 50%.

Example 4

Evaluation of the EPO-CTP Polypeptides of the Present Invention in a Mouse Model

[0354] The following experiment was performed in order to compare the bio-activity of the EPO-CTP polypeptides of the present invention and commercial EPO.

Materials and Methods

Animals:

[0355] Species/Strain: ICR or CD-1 Mice of either sex about 20-25 g Group Size: n=7 No. Groups: 9 Total No. Animals: n=63

[0356] Experimental design of the study: The experiment was set up as summarized in Table 5 hereinbelow.

TABLE-US-00015 TABLE 5 No. Mice (a) TREATMENT Group per (c) Dose Dosing No. Group (b) Compound Level Regimen 1 n = 7 Vehicle (Control) 0 1x 2 MOCK weekly 3 MOD-7010 15 .mu.g/kg 4 MOD-7011 5 MOD-7012 6 MOD-7013 7 MOD-7014 8 Commercial 15 .mu.g/kg 9 rhEPO 5 .mu.g/kg 3 x

[0357] Animal Treatment:

[0358] All animals were administered with either control or the test EPO polypeptides of the present invention by bolus injection. The injection volume did not exceed 10 ml/kg. The length of the experiment was 22 days. A morbidity and mortality check was performed daily.

[0359] Reticulocyte Count and Hematocrit (hct) Examination:

[0360] Reticulocyte count was carried out in all test animals at day 2 and 14 hrs following the 1st respective vehicle or treatment injection. HCT was determined in all animals once prior to initial treatment ("0" Baseline control) and at 24 hrs after the 1st respective vehicle or treatment injection, and thereafter twice weekly until study termination (Day22).

Results

[0361] The hematocrit results which are illustrated in FIGS. 3-5 show that EPO 3 has the highest hematocrit percentage change from baseline compared to EPO 1, EPO 2, Recormon.RTM. 1, Recormon.RTM. 3, rhEPO, and vehicle. The results demonstrating the percentage of reticulocytes in mice treated with the EPO-CTP polypeptides are summarized in Table 6 hereinbelow. These results show that EPO-3 is the most potent stimulator of erythropoiesis.

TABLE-US-00016 TABLE 6 % reticulocytes Days 2 14 Control 3.72 3.46 1.08 0.8 Mock 3.5 3.64 0.6 1.13 7010 SEQ ID NO: 16 3.5 3.9 0.6 1.54 7011 SEQ ID NO: 1 3.52 1.94 1.38 1.08 7012 SEQ ID NO: 2 3.82 3.0 1.02 0.88 7013 SEQ ID NO: 3 2.66 5.20 0.97 2.96 7014 SEQ ID NO: 4 3.48 3.82 0.71 0.90 Recormon .RTM. 1/W 3.23 3.27 0.73 0.59 Recormon .RTM. 3/w 4.13 4.24 1.21 1.14

[0362] Conclusion

[0363] The in vivo experiment was designed to measure two parameters; the first was to measure erythropoiesis parameters such as percentage of reticulocytes and increase of hemoglobin, RBC and hematocrit levels. The second was to measure the durability of the biological activity of each variant by injecting once weekly doses.

[0364] A superior performance of EPO-3 in its ability to stimulate erythropoiesis was observed in normal mice.

Example 5

Comparison of the EPO-CTP Polypeptides of the Present Invention to Aranesp.RTM.

[0365] The following experiment was performed in order to compare the biological activity of a single bolus dose of some EPO-CTP polypeptides of the present invention, commercial EPO and Aranesp.RTM.. Aranesp.RTM. is a commercial long-acting recombinant erythropoietin in which two site mutations were introduced, resulting in two additional N-glycosylation sites and an increase in the number of incorporated sialic acid residues.

Materials and Methods

Animals:

[0366] Species/Strain: Female CD-1 Mice of either sex about 20-25 g Group Size: n=3

[0367] Experimental design of the study: The experiment was set up as summarized in Table 7 hereinbelow.

TABLE-US-00017 TABLE 7 Dose animals/ Solution Dose group/ Conc. Volume Time-Points * Group # Test Article time-point (.mu.g/mL) (mL/kg) (hours post-administration) 1 MOD-7010 3 1.5 10 0 (Pre-dose), 0.25, 0.5, 1, 2, 6, SEQ ID NO: 11 24, 48, 96, 168, 216, 264 and 336 hr post-dose administration 2 MOD-7013 3 1.5 10 0.25, 0.5, 1, 2, 6, 24, 48, 96, 168, SEQ ID NO: 3 216, 264 and 336 hr post-dose administration 3 Aranesp .RTM. 3 1.5 10 0.25, 0.5, 1, 2, 6, 24, 48, 96, 168, 216, 264 and 336 hr post-dose administration

[0368] Animal Treatment:

[0369] All animals were administered with either control or the test EPO polypeptides of the present invention by bolus injection. The injection volume did not exceed 10 ml/kg. The length of the experiment was 14 days. A morbidity and mortality check was performed daily.

[0370] Reticulocyte Count and Hematocrit (Hct) Examination:

[0371] Reticulocyte count and hematocrit examination were performed as described above.

[0372] Results

[0373] The results are illustrated in FIGS. 6-9. Following a single I.V. injection of 15 .mu.g/kg of EPO 3, all three blood parameters associated with erythropoietin i.e. number of reticulocytes, hemoglobin level and hematocrit, were improved relative to those obtained with similar injected dose of rhEPO or Aranesp.RTM..

Example 6

Comparison of the Pharmacokinetics of EPO-CTP Polypeptides of the Present Invention to Aranesp.RTM.

[0374] The following experiment was performed in order to compare the pharmacokinetics of EPO-CTP polypeptide of the present invention, commercial EPO and Aranesp.RTM..

Materials and Methods

[0375] Serum samples were analyzed in order to determine specific concentration levels for each sample. Concentration and time-point data were processed using WinNonLin noncompartmental analysis. Parameters determined included: AUC, CL, Ke, t.sub.1/2, C.sub.max, T.sub.max, and Vdz.

[0376] Serum concentrations were determined using two ELISA kits in parallel. EPO-3 serum concentration was measured using StemCell ELISA kit in comparison to EPO-0 and Aranesp.RTM. serum concentration which were determined using R&D system ELISA kit.

Results

[0377] The results of the pharmacokinetic analysis are summarized in Table 8, hereinbelow. These results show that EPO 3 exhibited favorable pharmacokinetic measures as indicated for example in AUC measures, t.sub.1/2, and C.sub.max. T.sub.max measures were equal to EPO-0, EPO-3, and Aranesp.RTM..

TABLE-US-00018 TABLE 8 Parameters Units EPO-0 EPO-3 Aranesp .RTM. AUClast hr * mIU/mL 31739 306072 178661 CL{circumflex over ( )} mL/hr/kg 1.1152 0.2188 0.1207 Ke 1/hr 0.157 0.0529 0.0639 t.sub.1/2 hr 4.4139 13.1141 10.84 C.sub.max mIU/mL 10766 16466 13266 T.sub.max Hr 0.25 0.25 0.25 Vdz mL/kg 7.1017 4.1394 1.8877

[0378] The results of the serum concentration analysis are illustrated in FIG. 9. These results show that EPO-3 was still detectable in the serum after about 190 hours. Both EPO-0 and Aranesp.RTM. were not detectable in the serum after about 140 hours and 50 hours, respectively.

Conclusion

[0379] Clearance of EPO-3 (MOD-7013) from the blood of CD-1 mice was significantly slower than that for rhEPO or Aranesp.RTM.. The corresponding calculated half-life times were: rhEPO--4.41 h; Aranesp.RTM.--0.84 h; and MOD-7013--13.11 h.

Example 7

Generation of hGH Constructs

Materials and Methods

[0380] Four hGH clones (variants of 20 kD protein) were synthesized. Xba I-Not I fragments containing hGH sequences from the four variants were ligated into the eukaryotic expression vector pCI-dhfr previously digested with XbaI-NotI. DNA from the 4 clones (401-0, 1, 2, 3 and 4) was prepared. Another partial hGH clone (1-242 bp) from 22 kD protein was also synthesized (0606114). Primers were ordered from Sigma-Genosys. The primer sequences used to generate the hGH-CTP polypeptides of the present invention are summarized in Table 9, hereinbelow.

TABLE-US-00019 TABLE 9 Restric- tion Pri- site (un- mer SEQ derlined num- ID in se- ber NO sequence quence) 25 27 5' CTCTAGAGGACATGGCCAC 3' XbaI 32.sup.R 28 5' ACAGGGAGGTCTGGGGGTTCTGCA 3' 33 29 5' TGCAGAACCCCCAGACCTCCCTGTGC 3' 4.sup.R 30 5' CCAAACTCATCAATGTATCTTA 3' 25 31 5' CTCTAGAGGACATGGCCAC 3' XbaI 35.sup.R 32 5' CGAACTCCTGGTAGGTGTCAAAGGC 3' 34 33 5' GCCTTTGACACCTACCAGGAGTTCG 3' 37.sup.R 34 5' ACGCGGCCGCATCCAGACCTTCATCAC NotI TGAGGC 3' 39.sup.R 35 5' GCGGCCGCGGACTCATCAGAAGCCGCA GCTGCCC 3'

[0381] Construction of 402-0-p69-1 (hGH) SEQ ID NO: 36:

[0382] MOD-4020 is the wild type recombinant human growth hormone (without CTP) which was prepared for use as control in the below described experiments.

[0383] Three PCR reactions were performed. The first reaction was conducted with primer 25 and primer 32.sup.R and plasmid DNA of 0606114 (partial clone of hGH 1-242 bp) as a template; as a result of the PCR amplification, a 245 bp product was formed.

[0384] The second reaction was conducted with primer 33 and primer 4.sup.R and plasmid DNA of 401-0-p57-2 as a template; as a result of the PCR amplification, a 542 bp product was formed.

[0385] The last reaction was conducted with primers 25 and 4.sup.R and a mixture of the products of the previous two reactions as a template; as a result of the PCR amplification, a 705 bp product was formed and ligated into the TA cloning vector (Invitrogen, catalog K2000-01). The XbaI-NotI fragment containing hGH-0 sequence was ligated into the eukaryotic expression vector pCI-dhfr. The vector was transfected into DG-44 CHO cells. Cells were grown in protein-free medium.

[0386] Construction of 402-1-p83-5 (hGH-CTP)--SEQ ID NO: 37 and 402-2-p72-3(hGH-CTP.times.2)--SEQ ID NO:

[0387] 38: MOD-4021 is a recombinant human growth hormone which was fused to 1 copy of the C-terminal peptide of the beta chain of human Chorionic Gonadotropin (CTP). The CTP cassette of MOD-4021 was attached to the C-terminus (one cassette). MOD-4022 is a recombinant human growth hormone which was fused to 2 copies of the C-terminal peptide of the beta chain of human Chorionic Gonadotropin (CTP). The two CTP cassettes of MOD-4022 were attached to the C-terminus (two cassettes).

[0388] Construction of hGH-CTP and hGH-CTP-CTP was performed in the same way as the construction of hGH-0. pCI-dhfr-401-1-p20-1 (hGH*-ctp) and pCI-dhfr-401-2-p21-2 (hGH*-ctp .times.2) were used as templates in the second PCR reaction.

[0389] MOD-4021 and MOD-4022 were expressed in DG-44 CHO cells. Cells were grown in protein-free medium. The molecular weight of MOD-4021 is .about.30.5 Kd since hGH has a MW of 22 Kd while each "CTP cassette" contributes 8.5 Kd to the overall molecular weight (see FIG. 10). The molecular weight of MOD-4022 is .about.39 Kd (see FIG. 10).

[0390] Construction of 402-3-p81-4 (CTP-hGH-CTP-CTP)--SEQ ID NO: 39 and 402-4-p82-9(CTP*hGH-CTP-CTP)--SEQ ID NO: 40:

[0391] MOD-4023 is a recombinant human growth hormone which was fused to 3 copies of the C-terminal peptide of the beta chain of human Chorionic Gonadotropin (CTP). The three CTP cassettes of MOD-4023 were attached to both N-terminus (one cassette) and the C-terminus (two cassettes). MOD-4024 is a recombinant human growth hormone which is fused to 1 truncated and 2 complete copies of the C-terminal peptide of the beta chain of human Chorionic Gonadotropin (CTP). The truncated CTP cassette of MOD-4024 was attached to the N-terminus and two CTP cassettes were attached to the C-terminus (two cassettes).

[0392] Three PCR reactions were performed. The first reaction was conducted with primer 25 and primer 35.sup.R and plasmid DNA of p401-3-p12-5 or 401-4-p22-1 as a template; as a result of the PCR amplification, a 265 or 220 bp product was formed. The second reaction was conducted with primer 34 and primer 37.sup.R and plasmid DNA of TA-hGH-2-q65-1 as a template; as a result of the PCR amplification, a 695 bp product was formed. The last reaction was conducted with primers 25 and 37.sup.R and a mixture of the products of the previous two reactions as a template; as a result of the PCR amplification, a 938 or 891 bp product was formed and ligated into TA cloning vector (Invitrogen, catalog K2000-01). Xba I-Not I fragment containing hGH sequence was ligated into our eukaryotic expression vector pCI-dhfr.

[0393] MOD-4023 and MOD-4024 were expressed in DG-44 CHO cells. Cells were grown in protein-free medium. The molecular weight of MOD-4023 is .about.47.5 Kd (see FIG. 10) and the molecular weight of MOD-4024 is .about.43.25 Kd (see FIG. 10).

[0394] Construction of 402-6-p95a-8 (CTP-hGH-CTP)--SEQ ID NO: 41:

[0395] Construction of hGH-6 was performed in the same way as the construction of hGH-3. pCI-dhfr-402-1-p83-5 (hGH-ctp) was used as a template in the second PCR reaction.

[0396] Construction of 402-5-p96-4 (CTP-hGH)--SEQ ID NO: 42:

[0397] PCR reaction was performed using primer 25 and primer 39.sup.R and plasmid DNA of pCI-dhfr-ctp-EPO-ctp (402-6-p95a-8) as a template; as a result of the PCR amplification, a 763 bp product was formed and ligated into TA cloning vector (Invitrogen, catalog K2000-01). Xba I-Not I fragment containing ctp-hGH sequence was ligated into our eukaryotic expression vector pCI-dhfr to yield the 402-5-p96-4 clone.

Example 8

In Vivo Bioactivity Tests of hGH-CTP Polypeptides of the Present Invention

[0398] The following experiment was performed in order to test the potential long acting biological activity of hGH-CTP polypeptides in comparison with commercial recombinant human GH and MOD-4020.

Materials and Methods

[0399] Female hypophysectomized rats (60-100 g) received a weekly S.C. injection of 21.7 .mu.g hGH-CTP polypeptides or a once daily 5 .mu.g S.C. injection of control commercial rhGH.

[0400] Weight was measured in all animals before treatment, 24 hours following first injection and then every other day until the end of the study on day 21. Each point represents the group's average weight gain percentage ((Weight day 0-weight last day)/Weight day 0). Average weight gain was normalized against once-daily injection of commercial hGH. The treatment schedule is summarized in Table 10.

TABLE-US-00020 TABLE 10 Equi- Accumu- Treat- molar late Dose ment Dose Dosage Vol. No. Drug N Route Schedule (.mu.g/rat) (.mu.g/rat) (ml) 1 Vehicle 7 s.c. days 1, 7 NA NA 0.25 and 13; 1/W 2 Mock 7 s.c. days 1, 7 NA NA 0.25 and 13; 1/W 3 MOD-4020 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID and 13; NO: 36 1/W 4 MOD-4021 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID and 13; NO: 37 1/W 5 MOD-4022 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID and 13; NO: 38 1/W 6 MOD-4023 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID and 13; NO: 39 1/W 7 MOD-4024 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID and 13; NO: 40 1/W 8 Commercial 7 s.c. days 1, 7 21.7 65 0.25 hGH v.1 and 13; 1/W 9 Commercial 7 s.c. days 1-13; 5 65 0.25 hGH v.1 d/W

Results

[0401] Results are summarized in FIG. 11. These results show that MOD-4023 (SEQ ID NO: 39) and MOD-4024 (SEQ ID NO: 40) induced over 120% weight gain compared to commercial rhGH which induced 100% weight gain.

Conclusion

[0402] Three weekly doses (Days of injections; 1, 7, and 13) of 21.7 lag of MOD-4023 (SEQ ID NO: 39) and MOD-4024 (SEQ ID NO: 40) induced a 30% greater weight increase in hypophysectomised rats compared to commercial rhGH injected at the same accumulated dose which was administered once per day at a dose of 5 .mu.g for 13 days.

Example 9

The Superiority hGH-CTP Polypeptides of the Present Invention

[0403] Pharmacokinetic Studies

[0404] Single-dose pharmacokinetic studies were conducted in Sprague-Dawley rats. All animal experimentation was conducted in accordance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and under the supervision and approval of the Institutional Animal Care and Use Committees of Modigene Biotechnology General Ltd. Rats were housed either individually or two per cage in rooms with a 12-h light/dark cycle. Access to water (municipal supply) and noncertified rodent chow was provided ad libitum.

[0405] To compare the pharmacokinetics of MOD-4023 and GH in rats, four groups of Sprague-Dawley rats (270-290 g), three to six male rats per group. The rats were randomly assigned to four treatment groups (see Table 11). Rats were administered a single s.c. or i.v. injection of GH (50 .mu.g/kg i.v: or s.c.) or MOD-4023 (108 .mu.g/kg i.v. or s.c.). With the exception of the predose sample, which was collected under isoflurane anesthesia, blood collection was performed in unanesthetized animals. Blood samples (approximately 0.25 ml) were collected in EDTA-coated microtainers for ELISA analyses of MOD-4023 plasma concentration at the times outlined in Table 11. After each sampling, the blood volume was replaced with an equal volume of sterile 0.9% saline. Samples were stored on wet ice for up to 1 h prior to centrifugation and plasma harvest. Plasma samples were stored at approximately -20.degree. C. prior to analysis.

TABLE-US-00021 TABLE 11 Experimental design of rat pharmacokinetic study No. of Concentration animals/ Dose Injected (.mu.g/ml)/ Trt. Test group/ Dose Level Vol. Total vol. Time-Points * Grp. Article timepoint Route Gender (.mu.g/kg) (.mu.l) (ml) (hours post-dose) 1 Biotropin .RTM. 6# s.c. Male 50 500 .sup. 20/5 0 (Pre-dose) 0.5, 2, 4, 8, 24, 48 2 MOD-4023 6# s.c. Male 108 500 43.2/5 0.5, 2, 4, 8, 24, 48, 72, 96 3 Biotropin .RTM. 6# i.v. Male 50 300 .sup. 20/3 0, 0.12, 2, 4, 8, 24 4 MOD-4023 6# i.v. Male 108 300 43.2/3 0.12, 2, 4, 8, 24, 48, 72 Volume of blood sample/time point - 500 .mu.l Terminal blood samples #3 rats per time point.

[0406] A commercial sandwich ELISA kit specific for detection of human growth hormone (Roche Diagnostics) was used for evaluation of the rat plasma samples. This kit detects human growth hormone in plasma by means of an antibody sandwich ELISA format. This kit was initially used to measure the concentration of MOD-4023 in rat plasma. For these plasma samples, a MOD-4023 standard curve (1.2-100 ng/ml) was used, and the concentrations of MOD-4023 in rat plasma were interpolated from this curve.

[0407] Standard pharmacokinetic parameters, including clearance (CL or CL/F), volume of distribution (Vd or Vd/F), half-life (t.sub.1/2), area under the plasma concentration versus time curve (AUC), maximal observed plasma concentration (C.sub.max) and time to maximal observed plasma concentration (T.sub.max), were obtained from plasma albutropin or GH concentration/time curves by noncompartmental analysis using the modeling program WinNonlin (Pharsight, version 3.1). Plasma MOD-4023 or GH concentration data were uniformly weighted for this analysis. The AUC was calculated using the log-linear trapezoidal analysis for the i.v. data and the linear-up/log-down trapezoidal method for the s.c. data. Plasma concentration profiles for each rat (with the exception of the s.c. albutropin data) or monkey were analyzed individually, and mean.+-.standard error of the mean (S.E.M.) values for the pharmacokinetic parameters are reported in Table 13, and FIG. 17.

[0408] Single Dose/Repeated Dose Weight Gain Assay (WGA)

[0409] Hypophysectomized (interaural method) male rats, 3-4 weeks of age, were obtained from CRL Laboratories. During a post-surgical acclimation period of 3 weeks, rats were examined and weighed twice weekly to eliminate animals deemed to have incomplete hypophysectomy evidenced by weight gain similar to that of sham-operated rats. Those rats with incomplete hypophysectomized were eliminated from the study. The average body weights of the hypophysectomized were 70-90, at the time of the experiment. This is the standard USP and EP bioassay for hGH. Hypophysectomized rats (rats from which the pituitary gland was removed) lose their ability to gain weight. Injections of hGH (and of MOD-4023) to these rats result in weight gain. Based on the measured weight gain along a defined period of time and the amount of hGH injected, the specific activity of hGH (and MOD-4023) is determined. Rats were administered either a single s.c. dose of 0.4, 0.8 and 4 mg/Kg or repeated s.c. doses of 0.6 and 1.8 mg/Kg 4 days apart for 3 weeks. Individual body weights of all animals are determined at randomization, prior to the first dosing, thereafter every two days or in case of decedents at the time of death, and prior to sacrifice.

[0410] Pharmacodynamics/Pharmacokinetics Studies

[0411] Hypophysectomized (interaural method) male rats, 3-4 weeks of age, were obtained from CRL Laboratories. During a post-surgical acclimation period of 3 weeks, rats were examined and weighed twice weekly to eliminate animals deemed to have incomplete hypophysectomy evidenced by weight gain similar to that of sham-operated rats. Those rats with incomplete hypophysectomized were eliminated from the study. The average body weights of the hypophysectomized and sham rats were 70 and 150 g, respectively, at the time of the experiment.

[0412] Rats were administered a single s.c. with MOD-4023, vehicle, human growth hormone MOD-4023 or human growth hormone (20 .mu.g/rat) in an injection volume of 0.2 ml/rat. The dose of GH was 0.35 and 1.05 .mu.g/Kg, a dose of growth hormone that was equimolar with the amount of GH in a corresponding 0.6 and 1.8 .mu.g/Kg dose of MOD-4023. The treatment groups are summarized in Table 12. Following injection, plasma samples for IGF-1 analyses were obtained at the times described in Table 12. Samples were analyzed for IGF-1 concentration using a commercial ELISA (R&D systems).

TABLE-US-00022 TABLE 12 Treatment schedule for hypophysectomized rat study No. of animals/ Eq. Eq. MOD-4023 Dose Trt. Test group/ Dose Dose Dosage Conc. Vol. Time-Points * Grp. Article timepoint Route (mg/rat) (mg/Kg) mg/ml (ml) (hours post-dose) M7 Biotropin .RTM. 9 s.c. 0.032 0.35 0.16 0.2 0 (Pre-dose) 0.5, 2, 4, 8, 24, 48, 72, 96 M8 Biotropin .RTM. 9 s.c. 0.095 1.05 0.475 0.2 0 (Pre-dose) 0.5, 2, 4, 8, 24, 48, 72, 96 M9 EN648-01- 12 s.c. 0.032 0.35 0.275 0.2 1, 2, 4, 8, 24, 48, 72, 96 08-005 (0.055) (0.6) M10 EN648-01- 12 s.c. 0.095 1.05 0.825 0.2 1, 2, 4, 8, 24, 48, 72, 96 08-005 (0.165) (1.8) Volume of blood sample/time point - 500 .mu.l Terminal blood samples

[0413] Carbohydrate Content and Sialic Acid Content

[0414] Analysis of O-glycans is based on a Prozyme kit. O-glycans are chemically and enzymatically cleaved from the protein and separated from peptides using paper chromatography. Sequencing of the O-glycan pool is performed by sequential enzymatic digestions (exo-glycosidases) followed by HPLC analysis compared to standards.

[0415] Glycoprofiling with Sequence Analysis

[0416] Glycoprofiling was performed by Ludger Ltd. Two samples (EN648 and RS0708) were taken through triplicate releases and each release was also analyzed by HPLC in triplicate. Triplicate 300 .mu.g samples of EN648 and RS0708 and a single 100 .mu.l sample of citrate/sodium chloride buffer, plus a positive control fetuin (250 .mu.g) and a 100 .mu.l water negative control, were ultra-filtrated by centrifugation using a molecular weight cut off membrane of 10,000 Da to replace the buffer with water, then taken through hydrazinolysis under O-mode conditions (6 h at 60.degree. C.). Released glycans were re-N-acetylated and cleaned up by LudgerClean CEX cartridges. An aliquot of the released glycans was then labeled with 2-aminobenzamide (2AB), cleaned up with Ludger Clean S cartridges and analyzed by LudgerSep-N2 HILIC-HPLC.

[0417] Monosaccharide Content

[0418] Analysis of neutral monosaccharides requires hydrolysis of glycans to their constituent monosaccharide components. The hydrolysis was performed by Ludger Ltd, on intact glycoprotein samples. Specifically, 50 .mu.g of intact glycoprotein was acid hydrolyzed, 2-AB (2-aminobenzamide) labeled and run on a reverse phase HPLC column. This method hydrolyzes all glycans present on the glycoprotein inclusive of N and O linked types.

[0419] Sialic Acid Profiling

[0420] Two samples (EN648 and RS0708) and a buffer control were analyzed. Sialic acid analysis requires mild acid release of the monosaccharides followed by DMB fluorophore labeling and HPLC analysis on a LudgerSep-R1 column. 50 .mu.g of intact glycoprotein was acid hydrolyzed for each analysis.

Results and Conclusions

[0421] MOD-4023 (CTP-hGH-CTP-CTP) is a single chain protein of 275 amino acids and up to twelve O-linked carbohydrates. The structure consists of modified human Growth Hormone (Somatropin) attached to three copies of the C-terminal peptide (CTP) of the beta chain of human Chorionic Gonadotropin (hCG); one copy at the N-terminus and two copies (in tandem) at the C terminus. Human Growth Hormone is comprised of 191 amino acids. CTP is comprised of 28 amino acids and four O-linked sugar chains.

[0422] Pharmacokinetics of MOD-4023 in SD Rats

[0423] The pharmacokinetics of MOD-4023 was evaluated and compared to that of commercial hGH (Biotropin.RTM.).

[0424] Data Statistical Analysis

[0425] Analysis of serum samples was performed in order to determine specific concentration levels for each sample. Concentration and time-point data were processed using WinNonLin noncompartmental analysis.

[0426] Parameters that were determined included: AUC, MRT, t.sub.1/2, C.sub.max, and T.sub.max. FIG. 17 demonstrates the superior pharmacokinetic profile of MOD-4023 plasma concentration compared to GH concentrations (pg/ml) following a single i.v. or s.c. dose of MOD-4023 or GH in rats (n=3-6 per dose/route).

[0427] Following a single s.c. injection of 50 .mu.g/kg, clearance of MOD-4023 from SD rat's blood was significantly slower than that of MOD-4026 and of Biotropin.RTM.. The corresponding calculated half-life times and AUCs were:

TABLE-US-00023 Biotropin .RTM. T.sub.1/2 1.7 h, AUC 41 hr * ng/mL MOD-4026 T.sub.1/2 8.5 h, AUC 424 hr * ng/mL MOD-4023 T.sub.1/2 9.0 h, AUC 680 hr * ng/mL

Conclusion:

[0428] MOD-4023 was chosen as the final candidate out of 6 other variants. MOD-4023 demonstrated superior performance in terms of biological activity and pharmacokinetics.

[0429] Single Dose and Repeated Dose Weight Gain Assay

[0430] The results comparing whole body growth response following different dosing patterns of MOD-4023 in hypophysectomized rats are demonstrated in FIG. 18. The results demonstrate that a single injection of 0.4 & 0.8 mg/Kg/day doses of hGH-CTP were equivalent to 4 daily injections of 0.1 mg/Kg/day of Biotropin.RTM.. The peak of the hGH-CTP effect was after 2 days.

[0431] FIG. 19 further demonstrates that the area under the curve following a single injection of MOD-4023 correlates with body weight gain in rats. Thus, the collective data demonstrates that body weight gain is closely correlated with cumulative AUC.

[0432] hGH-CTP construct administered 4 days apart promotes the same weight gain as daily injections of Biotropin.RTM. as demonstrated in FIG. 20. Half-life of hGH in humans is expected to be 5.times. better than in rats, indicating a potential peak effect in humans after 10 days for one single injection. These results support administration of hGH-CTP construct, MOD-4023, once weekly or bi-weekly in humans.

[0433] Glyco Analysis of MOD-4023

TABLE-US-00024 TABLE 14 Glycan analysis. Structural assignments and percentage areas of peaks are based upon HPLC and enzyme array digests. Percent from total glycans.sup.e Peak ABS ID.sup.a GU.sup.b Structure.sup.c name Und.sup.d NAN1 ABS BTG 1.sup.f 0.92 ##STR00001## + bgd GaINAc 0.4 0.4 .sup. 0.6 53.0 .sup. 2.sup.f 1.02 ##STR00002## + bgd galactose 1.9 9.7 .sup. 23.8 26.5 .sup. * 1.72 4.3 4.6 .sup. 2.3 3 1.79 ##STR00003## Gal.beta.1-3GalNAc 2.3 67.7 .sup. 69.4 17.1 .sup.h 4.sup.g 2.25 ##STR00004## NeuNAc.alpha.2-3Gal 19.8 13.0 .sup.h * 2.57 1.5 1.9 .sup. 1.1 1.1 .sup. 5 2.90 ##STR00005## NeuNAc.alpha.2-3Gal.beta.1-3 GalNAc 70.6 * 3.58 0.6 0.7 .sup. 0.6 6 3.22 ##STR00006## Gal.beta.1-3[NeuNAc.alpha.2-6] GalNAc 0.9 2.3 .sup. 7 4.42 ##STR00007## NeuNAc.alpha.2-3Gal.beta.1-3 [NeuNAc.alpha.2-6]GalNAc 1.8

[0434] The monosaccharide profiles indicate that the MOD-4023 glycoprotein samples contain predominantly O-link type glycans. The major O-glycan peak is sialylated core 1 (Neu5Ac.alpha.2-3Gal.beta.1-3GalNAc). The major sialic acid is Neu5Ac and there are some minor peaks suggesting the presence of 3-4% of di-acetylated sialic acid N-acetyl-9-O-acetylneuraminic acid (Neu5, 9Ac2) and less than 1% N-glycolylneuraminic acid. There are also small amounts of Neu5Ac.alpha.2-6(Gal.beta.1-3)GalNAc.

[0435] Pharmacokinetics and Pharmacodynamics of MOD-4023 in Hypophysectomized Rats

[0436] Non-compartmental pharmacokinetic analysis was performed on the mean serum concentration versus time curves for each group. MOD-4023 C.sub.max was significantly higher than Biotropin.RTM. C.sub.max. The terminal half-life of MOD-4023 was 6 times higher than Biotropin.RTM..

TABLE-US-00025 TABLE 15 Pharmacokinetic Parameter Estimates of MOD-4023 and Biotropin Following a Single Subcutaneous Injection in hypophysectomized Rats Dose C.sub.max T.sub.max AUC.sub.0-.infin. AUC.sub.0-t CL/F T.sub.1/2 Group mg/kg Gender ng/mL hr ng-hr/mL ng-hr/mL mL/hr/kg hr MOD- 1.8 M 2,150 8 37,713 37,695 0.928 6.86 4023 0.6 M 681 8 11,505 11,489 3.042 6.8 hGH 1.05 M 1,078 0.5 3,541 3,540 9.884 1 0.35 M 439 0.5 1,279 1,279 27.36 1

[0437] The AUC.sub.0-t and the AUC.sub.0-.infin. were very similar suggesting the duration of sampling was adequate to characterize the pharmacokinetic profiles. AUC of MOD-4023 was 10 times higher than of Biotropin.RTM.. Moreover, C.sub.max was generally proportional to dose and for MOD-4023, and twice as high as the C.sub.max of Biotropin.RTM.. However, as shown in FIG. 21, T.sub.max of MOD-4023 was 8 hr as compare to 0.5 hr of Biotropin.RTM. and the terminal half-lives did not appear to vary with dose level. T.sub.1/2 of MOD-4023 was 6.8 times longer than of Biotropin.RTM..

[0438] Indirect effects of GH are mediated primarily by an insulin-like growth factor-I (IGF-I), a hormone that is secreted from the liver and other tissues in response to growth hormone. A majority of the growth-promoting effects of growth hormone is actually due to IGF-I acting on its target cells. Accordingly, the effect of the CTP-hGH construct, MOD-4023, on IGF-1 serum levels in hypophysectomized rats was measured. FIG. 22 presents results of IGF-1 serum levels in hypophysectomized rats following s.c. injection of MOD-4023 and commercial hGH.

[0439] A single dose of MOD-4023 0.6 or 1.8 mg/Kg, or Biotropin.RTM. 0.35 or 1.05 mg/Kg was injected subcutaneously to hypophysectomised rats for the determination of the PK/PD profile. Serum IGF-I post-injection was measured using specific ELISA kits (Roche Diagnostics).

[0440] The cumulative serum levels of IGF-I following injection of MOD-4023 was significantly higher than following injection of Biotropin.RTM.. The C.sub.max was generally proportional to the dose, and for MOD-4023, it was 3-4 times higher than the C.sub.max of Biotropin.RTM.. The T.sub.max of MOD-4023 was 36-48 hr as compared to 20-24 hr of Biotropin.RTM.. In conclusion, higher hGH levels and longer presence in serum result in a significant increase in IGF-1 levels.

[0441] Pharmacokinetic/Toxicokinetic Analysis in Rhesus Monkeys

[0442] Serum concentrations versus time curves were generated for each animal. Non-compartmental analysis was performed with WinNonlin professional version 5.2.1 (Pharsight Corporation, Mt View Calif.). The estimated pharmacokinetic parameters are shown in the table 16 below:

TABLE-US-00026 TABLE 16 Estimates of MOD-4023 Pharmacokinetic Parameters (Mean .+-. SD) from Non-compartmental Analysis Following A Single Subcutaneous Injection in Rhesus Monkeys Parameter 1.8 mg/kg 90 mg/kg C.sub.max (.mu.g/mL) 2.073 .+-. 0.417 108.7 .+-. 46.0 T.sub.max (hr) 4 .+-. 0 11 .+-. 7 AUC.sub.0-t (.mu.g-hr/mL) 38.7 .+-. 7.4 2,444 .+-. 394 AUC.sub.0-.infin. (.mu.g-hr/mL) 39.0 .+-. 7.3 2,472 .+-. 388 CL/F (mL/hr/kg) 47.5 .+-. 9.0 37.04 .+-. 4.78 T.sub.1/2 (hr) 10.00 .+-. 1.47 9.85 .+-. 1.07 Vz/F (mL/kg) 701 .+-. 236 529 .+-. 104

[0443] The AUC.sub.0-t and the AUC.sub.0-.infin. were very similar suggesting the duration of sampling was adequate to characterize the pharmacokinetic profiles. The C.sub.max was proportional to dose. The T.sub.max was later at the higher dose. The T.sub.max was at 4 hours for all animals in the low dose group and was at 8 or 24 hours in the high dose group. The terminal half-lives were similar for the two dose groups.

[0444] The AUC was approximately proportional to dose with a slightly larger than proportional AUC at the higher dose producing a slightly lower estimate for CL/F and Vz./F compared to the lower dose. It is not possible to say if CL and Vz are lower at the higher dose or if F is lower at the lower dose. There was overlap between the groups so it is questionable that this represents a meaningful difference in CL/F and Vz/F.

[0445] Pharmacokinetic parameters estimated by the model were very similar to those from non-compartment analysis. Absorption and elimination half-lives are shown in Table 17 below:

TABLE-US-00027 TABLE 17 Estimates of MOD-4023 Absorption and Elimination Half-lives (Mean .+-. SD) Following Subcutaneous Injection Derived From Pharmacokinetic Modeling in Rhesus Monkeys Dose T.sub.1/2 abs (hr) T.sub.1/2 el (hr) 1.8 mg/kg 1.17 .+-. 0.40 10.41 .+-. 2.36 90 mg/kg 6.49 .+-. 1.87 7.26 .+-. 1.85

[0446] The data indicate that the elimination rates are fairly similar between the groups with a slightly longer T.sub.1/2 el in the lower dose group. The absorption, however, is more than 5-fold slower following subcutaneous administration of 90 mg/kg compared to that following 1.8 mg/kg. As in the case of the non-compartmental analysis, modeling indicated a later T.sub.max at the high dose.

[0447] Although GH supplementation is effective in the treatment of GH deficiency in children and adults, the disadvantages of daily injections over extended periods of time limit its use by physicians in certain patient populations as well as increase the risk of dosing error, the number of care givers, the cost of treatment and noncompliance. Especially important in certain populations, such as children of short stature who may not understand the implications of not following the prescribed GH dosing regimen, is the necessity of compliance to achieve the optimal benefit from GH therapy. The issue of finding a more suitable alternative to daily GH injections and subsequent compliance gains further importance as GH-deficient children transition into adults with a continuing need for GH treatment. The requirement of daily therapy is largely due to recombinant GH's short plasma half-life and has led to the development of a sustained release form of GH (Reiter E O. Attire K M., Mashing T J. Silverman B L. Kemp S F. Neolith R B. Ford K M. and Sanger P. A multimember study of the efficacy and safety of sustained release GH in the treatment of naive pediatric patients with GH deficiency. J. Clin. Endocrinol. Metab. 86 (2001), pp. 4700-4706.).

[0448] MOD-4023, a recombinant human growth hormone-CTP fusion protein, as described herein, has a pharmacokinetic profile in the rat that is longer in duration than that of GH. This unique pharmacokinetic profile allows for intermittent administration of MOD-4023 to achieve pharmacodynamic effects in growth hormone-deficient rat as evidenced by growth and elevations in plasma IGF-1 levels, respectively.

[0449] MOD-4023 offers a superior pharmacokinetic profile compared with that of GH when administered s.c. in the rat. There are substantial differences in plasma clearance of MOD-4023 compared to GH. Specifically, plasma is cleared of MOD-4023 at more than 6 times slower rate than of GH following s.c. dosing. The terminal half-life and mean residence time of MOD-4023 were approximately six times longer than that of GH in rats following s.c. administration. In addition, the Cl/F following s.c. dosing is 10 times lower for MOD-4023 than for GH.

[0450] In an effort to examine whether the pharmacokinetic advantages in the rat translated to a pharmacodynamic benefit, the possibility that MOD-4023 might stimulate growth in GH-deficient hypophysectomized rats with dosing regimens less frequent than daily was tested at equimolar MOD-4023 and GH dose levels. Single s.c. injection of MOD-4023 promoted incremental weight gain which was equal to 4 daily consecutive injections of GH. In addition, the every fourth day administration schedule for MOD-4023 shows enhanced body weight gain over GH.

[0451] Pharmacodynamically, the long circulation time of MOD-4023 relative to GH in the hypophysectomized rats resulted in a prolonged IGF-1 response measured in blood plasma following a single s.c. injection. Subcutaneous administration of a single dose of MOD-4023 increased circulating IGF-1 concentrations in a dose-dependent manner in the hypophysectomized rats. At the highest albutropin dose, IGF-1 concentrations were elevated above baseline for as long as 75 hours after a single administration. Thus, the enhanced circulation time of a single dose of MOD-4023 resulted in substantial pharmacodynamic improvement over a single dose of GH, raising the possibility that MOD-4023 could offer similar growth enhancement with reduced dosing frequency compared with standard GH treatment regimens.

[0452] Single CTP-modified hGH doses of 90 mg/kg in rhesus and 180 mg/kg in rats were well tolerated in both species. The allometric factor between rats and primates is approximately X2 which is based on the anticipated clearance of proteins in these animals. In line with industry-accepted extrapolation models for therapeutic proteins half-life increase between species (FDA Guidance). 90 mg/kg in primates has a PK profile slightly better than 180 mg/kg of CTPs modified hGH in rat. Thus, allometric extrapolation to humans supports weekly or once/2 week injections.

[0453] The present concept utilizing a CTP-GH construct, reduced dosing frequency compared to the commercial GH recombinant product. Nutropin Depot.RTM. is a sustained release formulation of GH approved for use in pediatric populations; however, comparisons to historical controls have revealed that 1- and 2-year growth rates are significantly (p<0.001) lower in children given Nutropin Depot.RTM. (1-year growth rate 8.2.+-.1.8 cm/year) than in children treated with GH (one-year growth rate 10.1.+-.2.8 cm/year) (Silverman B L. Blethen S L. Reiter E O. Attie K M. Neuwirth R B. and Ford K M. A long-acting human growth hormone (Nutropin Depot.RTM.): efficacy and safety following two years of treatment in children with growth hormone deficiency. J. Pediatr. Endocrinol. Metab. 15 (2002), pp. 715-722.). The local effects of subcutaneously administered Nutropin Depot.RTM. include nodules, erythema, pain at the injection site, headache and vomiting. Preclinical toxicology studies in both rat and monkey have shown that s.c. administration of CTP-hGH-CTP-CTP produces no local reactions compared to vehicle. Given the medical need for a less frequently administered form of GH, the pharmacologic properties of MOD-4023 in this study in rats suggest that this product is favorable also in terms of toxicology and patient compliance. The sustained activity of MOD-4023 in the rat support its potential utility as an agent that requires only intermittent administration to attain a therapeutic benefit that is currently achieved with daily dosing.

Example 10

Construction of hIFN Beta-CTP Variants

[0454] Construction of hIFN.beta.-CTP Variants:

[0455] A cassette gene containing the C-Terminal peptide (CTP) of the beta subunit of hCG was fused to the coding sequence of human IFN beta 1a (SEQ ID NO: 49) at different locations. Seven IFN.beta.-CTP variants were constructed as illustrated in FIGS. 14A-14N. The proIFN.beta. signal peptide was used for the construction of the secreted IFN.beta.-CTP variants. XbaI-NotI fragments containing IFN.beta. sequences were ligated into the pCI-dhfr expression vector of the present invention.

[0456] Table 18 hereinbelow summarizes the primer sequences used for constructing the CTP-containing polypeptides of the present invention.

TABLE-US-00028 TABLE 18 Restric- tion Pri- site (un- mer SEQ derlined num- ID in se- ber NO Sequence quence) 40 20 5' GAATTCTAGAGGACATGACCAAC 3' XbaI 41.sup.R 21 5' GCGGCCGCGGACTCATCAGTTCCTCA NotI GGTAGCCG 3'

[0457] IFN.beta.-1 901-1-p107-2 (IFN.beta.-1--SEQ ID NO: 53): The IFN.beta.-ctp clone was synthesized by GeneArt (Geneart No. 0609229).

[0458] Then the XbaI-NotI fragment containing IFN.beta.-ctp sequence was ligated into pCI-dhfr expression vector. The amino acid sequence of this clone is presented in SEQ ID NO: 52.

[0459] IFN.beta.-2 901-2-p113-3 (IFN.beta.-2--SEQ ID NO: 55): The XbaI/ApaI fragment (IFN-ctp) of pCI-dhfr-701-2-p24-2 (IFN-ctpx2) was replaced by the XbaI/ApaI fragment (IFN.beta.ctp) of 901-1-p107-2 to create a IFN.beta.ctpx2 clone. The amino acid sequence of this clone is presented in SEQ ID NO: 54.

[0460] IFN.beta.-4 901-4-p108-16 (IFN.beta.-4--SEQ ID NO: 59): The ctp-IFN.beta.-ctp-IFN.beta. clone was synthesized by GeneArt (Geneart No. 0609227).

[0461] Then the XbaI-NotI fragment containing sequence ctp-IFN.beta.-ctp-IFN.beta. was ligated into pCI-dhfr expression vector. The amino acid sequence of this clone is presented in SEQ ID NO: 11.

[0462] IFN.beta.-6 901-6-p109-3 (IFN.beta.-6 SEQ ID NO:16): The ctp-IFN.beta.-ctp clone was synthesized by GeneArt (Geneart No. 0609228).

[0463] Then the XbaI-NotI fragment containing sequence ctp-IFN.beta.-ctp was ligated into pCI-dhfr expression vector. The amino acid sequence of this clone is presented in SEQ ID NO: 62.

[0464] IFN.beta.-5-p103-10 (IFN.beta.-5 SEQ ID NO: 14--(ctp-IFN.beta.): Primers were ordered from Sigma-Genosys. A PCR reaction was performed using primer 40 (SEQ ID NO: 65) and primer 41 R (SEQ ID NO:21) and plasmid DNA of the synthesized ctp-IFN.beta.-ctp (Geneart No. 0609228) as a template; as a result of the PCR amplification, a 677 bp product was formed. The PCR fragment was digested with XbaI-NotI, and the fragment containing ctp-IFN.beta. sequence was ligated into our eukaryotic expression vector pCI-dhfr to yield the 901-5-p103-10 clone. The amino acid sequence of this clone is presented in SEQ ID NO: 60.

[0465] IFN.beta.-3-3 901-3-p114-5 (IFN.beta.-3 SEQ ID NO: 57 (ctp-IFN-CTP(.times.2)): The XbaI/ApaI fragment (IFN-ctp) of pCI-dhfr-701-2-p24-2 (IFN-ctpx2) was replaced by the XbaI/ApaI fragment (ctp-IFN.beta.-ctp) of 901-6-p109-3 to create a ctp-IFN.beta.ctpx2 clone. The amino acid sequence of this clone is presented in SEQ ID NO: 56.

[0466] IFN.beta.-901-0-p102-1 (IFN.beta.-0 SEQ ID NO: 2--(IFN.beta.): Primers were ordered from Sigma-Genosys. A PCR reaction was performed using primer 40 (SEQ ID NO:20) and primer 41R (SEQ ID NO:21) and plasmid DNA of the synthesized IFN.beta.-ctp (Geneart No. 0609229) as a template; as a result of the PCR amplification, a 599 bp product was formed. The PCR fragment was digested with XbaI-NotI, and the fragment containing IFN.beta. sequence was ligated into our eukaryotic expression vector pCI-dhfr to yield the 901-0-p102-1 clone. The amino acid sequence of this clone is presented in SEQ ID NO: 1.

Example 11

Expression and Isolation of IFN-CTP Polypeptides

Materials and Methods

[0467] DNA Transfection and Clone Selection:

[0468] DG44 cells were transfected with pCI-DHFR expression vectors containing IFN.beta.-CTP variants using FuGENE6 Reagent (FuGENE Transfection Reagent--Roche Cat. 11 815 091 001). Forty-eight hours following transfection, cells were diluted and seeded at 50-200 cells per well in a selective medium (CD DG44 Medium w/o HT (Gibco: Scotland part: #07990111A) Sku num.:ME060027 supplemented with 8 mM L-Glutamine Biological Industries: Cat: 03-020-1A) and 18 mL/L of 10% Pluronic F-68 solution (Gibco: Cat: 240040-032). Selected clones were screened for highest protein production using commercial ELISA. Three to five producing clones per each variant were frozen for a backup cell bank. A selected clone for each variant was adapted to growth in larger scale cultures up to 1 L flasks on an orbital shaker platform. Supernatants were collected and analyzed by ELISA, SDS-PAGE and Western Blot. Following the withdrawal of aliquots, the protein-containing supernatants were kept frozen until further use.

[0469] Cell Culture:

[0470] DG44 cells were maintained in DG44 medium with HT (cat#12610-010, Gibco) supplemented with 8 mM L-Glutamine (Biological Industries: Cat: 03-020-1A) and 18 mL/L of 10% Pluronic F-68 solution (Gibco: Cat: 240040-032), at 37.degree. C. in humidified 8% CO.sub.2 incubator. Transfected clones were maintained in DG44 basal medium without HT supplement, hypoxanthine and thymidine, with pluronic acid and L-glutamine.

[0471] Sample Preparation:

[0472] Supernatants were collected, filtrated and analyzed by ELISA to determine protein concentration. SDS-PAGE and Western Blot were used to determine purity and identity. Following ELISA, sample concentrations were defined and the solution was dialyzed against PBS. Following the withdrawal of aliquots, the protein-contained supernatants were kept frozen at -20.degree. C. until further use.

[0473] Western Blotting:

[0474] Samples were electrophoresed on nondenaturing 15% SDS-polyacrylamide gels. Gels were allowed to equilibrate for 10 min in 25 mM Tris and 192 mM glycine in 20% (vol/vol) methanol). Proteins were transferred to a 0.2 .mu.m pore size nitrocellulose membrane (Sigma, Saint Louis, Mo.) at 250 mA for 3 h, using a Mini Trans-Blot electrophoresis cell (Biorad Laboratories, Richmond, Calif.). The nitrocellulose membrane was incubated in 5% non-fat dry milk for 2 h at room temperature. The membrane was incubated with IFN anti-serum (1:1000 titer) overnight at 4. .degree. C. followed by three consecutive washes in PBS containing 0.1% Tween (10 min/wash). The membrane was incubated with secondary antibody conjugated to Horse Radish Peroxidase (HRP) (Zymed, San Francisco, Calif.) for 2 h at room temperature, followed by three washes. Finally, the nitrocellulose paper was reacted with enhanced chemiluminescent substrate (ECL) (Pierce, Rockford, Ill.) for 5 min, dried with a Whatman sheet, and exposed to X-ray film.

[0475] FIG. 12 indicates that MOD-901X-variants are recognized by anti IFN-.beta.1a antibodies. The SDS PAGE gel was stained using coomassie blue (A) or (B) blotted and stained using monoclonal anti-IFN.beta.1a antibodies.

Example 12

The IFN-CTP Polypeptides are Bioactive

[0476] To determine the bioactivity of MOD-901X variants through its recognition and binding to the IFN receptor, the Daudi cell line (human Burkitt lymphoma) ATCC catalog No. CCL-213 TM (one of the most sensitive cell lines to the anti-proliferative effect of IFN-.beta.1a) were used. Daudi cells, grown in suspension were treated with different concentrations of IFN-.beta.1a (50-1000 pg/ml final concentration) and incubated for 72 hours. The number of viable cells was measured using CellTiter 96.RTM. AQueous One Solution Cell Proliferation Assay kit (Promega G3580) according to manufacturer procedures. The assay's standard curve was prepared using recombinant human IFN-.beta.1a (PtoSpec Tany TechnoGene).

[0477] IFN-.beta.1a is a cytokine that exhibit antiviral activity against a variety of viruses. The potency of IFN-.beta.1a as an antiviral agent can be determined by a viral cytopathic effect (CPE) bioassay that measures the ability of the protein to protect human lung carcinoma A549 cells (grown at 37.degree. C., 5% CO.sub.2) challenged with encephalomyocarditis (ECM) virus. A549 cells were plated into 96 well microtiter plate. Serial dilutions of IFN-.beta.1a standards and test samples were added, and 24 h later, the cells were challenged with ECP virus. Viable cells were quantified two days later.

[0478] The potency (titer) of an IFN-.beta.1a test sample is determined as the reciprocal of the dilution represented in the well in which 50% of the cell monolayer is protected from the CPE virus. The actual potency is calculated by comparing the sample's protective effect with the same effect of a reference standard calibrated in International Units, provided by the National Institute of Allergy and Infectious Diseases (NIH). The results are shown in Table 19.

TABLE-US-00029 TABLE 19 Specific Activity IU/mg .times. 10{circumflex over ( )}8 IC50 Anti-viral Anti-proliferation pg/ml Intl. Standard 2.00 318 IFNb-0 3.90 2.53 251 IFNb-1 4.00 2.41 264 IFNb-2 4.00 1.90 334 IFNb-3 4.00 2.77 230 IFNb-5 4.00 6.24 102 IFNb-6 3.70 1.97 323 *concentration was determined by Elisa assay

[0479] Conclusion: The activity of MOD-901X variants as measured by its antiviral effects were at normal range of the international standard and similar to rhIFN. The same effect was observed in anti-proliferation assay except for MOD-9015 which was 3 times more potent than the other variants. IFNb-0 is SEQ ID NO: 1. IFNb-1 is SEQ ID NO: 52 (MOD-9011). IFNb-2 is SEQ ID NO: 54 (MOD-9012). IFNb-3 is SEQ ID NO: 56 (MOD-9013). IFNb-4 is SEQ ID NO: 58 (MOD-9014). IFNb-5 is SEQ ID NO: 60 (MOD-9015). IFNb-6 is SEQ ID NO: 62 (MOD-9016).

Example 13

Comparative Pharmacokinetics (Mod-901X Variants, Avonex.RTM. and REBIF.RTM.)

[0480] In order to determine the pharmacokinetics of MOD-901x and compare it to that of commercial IFN-.beta.1a (Rebif.RTM., Avonex.RTM.) data, statistical analysis was performed. The analysis included analysis of serum samples that was performed in order to determine specific concentration levels for each sample. Concentration and time-point data were processed using WinNonLin nocomparmental analysis. The following parameters were determined: AUC, CL, Ke, t.sub.1/2, C.sub.max, T.sub.max, and Vdz.

[0481] The experimental design is provided in Table 20.

[0482] FIG. 13 shows the change in serum concentration of IFN-.beta.1a or MOD-901x concentrations (ng/ml) following single-dose i.v. administration of IFN-.beta.1a or MOD-901x in SD rats.

[0483] Table 21 shows the mean pharmacokinetic parameters following single-dose i.v. or Sub-Cutaneous (s.c.) administration of IFN-.beta.1a and MOD-901x in Sprague-Dawley rats.

[0484] In conclusion: IFN-.beta.1a with 3 CTP units has 8 times longer half-life than that of Rebif.RTM. or Avonex.RTM. when injected i.v.

[0485] FIG. 13 shows the mean plasma of Rebif.RTM., MOD-9012, and MOD-9013 concentrations (ng/ml) following single-dose i.v. or s.c. administration of IFN-.beta.1a, MOD-9012 or MOD-9013 in SD rats (n=3 per dose/route/timepoint). IFN-.beta.1a serum concentrations were determined using commercial ELISA kit.

[0486] Table 22 displays the mean pharmacokinetic parameters following single-dose i.v. or s.c. administration of Rebif.RTM., MOD-9012, and MOD-9013 in Spargue-Dawley rats.

[0487] In conclusion, IFN-.beta.1a with 3 CTP units (MOD-9013) has 9.2 times longer half-life than that of Rebif.RTM. when injected i.v. and 6.7 times longer half-life when injected s.c. AUClast of MOD-9013 is 66 times better than Rebif.RTM. when injected s.c. and 24 times better when injected i.v. MRT of MOD-9013 is 5.8 times better when injected s.c. and 9.3 times better when injected i.v.

[0488] The MOD-9013 molecule, which comprises one CTP attached to the N-terminus of IFN-.beta.1a and two CTP attached to its C-terminus, was tested in vitro for its ability to bind to the human IFN receptor and in vivo for its pharmacokinetic performance. The conclusions of these studies can be summarized as follows: (1) The in vitro anti-proliferation activity of MOD-9013 as demonstrated in the Daudi cell assay was similar to the international standard and to that of MOD-9010 (rIFN-.beta.1a expressed by Modigene). (2) The anti-viral protective activity of MOD-9013 shown in Daudi cells was the same as the international standard and as of that of MOD-9010 (rIFN-.beta.1a expressed by Modigene). (3) In terms of its pharmacokinetic features, MOD-9013 was compared in SD rats to Rebif.RTM. and Avonex.RTM.. Following a single i.v./s.c. injection of 38/66 .mu.g/kg, clearance of MOD-9013 from SD rats blood was significantly slower than that for Rebif.RTM. and Avonex.RTM.. The corresponding calculated half-life times and AUCs for i.v. administration were:

TABLE-US-00030 Rebif .RTM. T.sub.1/2 1 h, AUC106 hr * ng/mL MOD-9013 T.sub.1/2 8.4 h, AUC2563 hr * ng/mL

[0489] For s.c. administration, the corresponding calculated half-life times and AUCs were:

TABLE-US-00031 Rebif .RTM. T.sub.1/2 2.1 h, AUC34.8 hr * ng/mL MOD-9013 T.sub.1/2 14.2 h, AUC2299.5 hr * ng/mL

[0490] The superior performance of MOD-9013 to stimulate anti-viral and anti-proliferation activity and to retain long lasting stimulation results from three main reasons: i) Addition of up to 24 sialic acid residues; ii) Stabilizing effect on the IFN-.beta.1a molecule by fusing the CTP cassettes to both N and C termini; and iii) Increase in molecular weight of the whole molecule from .about.31,242-48,000 Daltons.

[0491] As shown hereinabove, different levels of potency were exerted by IFN-CTP polypeptides, indicating differences in receptor binding. IFN-CTP polypeptides differ by the number of CTP cassettes and the location to which they are fused. MOD-9011 and MOD-9012 contain 1 CTP sequence or 2 CTP sequences at the C-terminal of IFN protein, while MOD-9013 contains 1 CTP at N-terminal and 2 CTP sequences at the C-terminal. MOD-9014 is a dimer of two IFN molecules linked by a CTP sequence. MOD-9013 demonstrated unexpected potency.

Sequence CWU 1

1

681221PRTHomo sapiens 1Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser 195 200 205 Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 210 215 220 2249PRTHomo sapiens 2Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser 195 200 205 Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser 210 215 220 Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly 225 230 235 240 Pro Ser Asp Thr Pro Ile Leu Pro Gln 245 3277PRTHomo sapiens 3Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ser Ser Ser Ser Lys 20 25 30 Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser 35 40 45 Asp Thr Pro Ile Leu Pro Gln Ala Pro Pro Arg Leu Ile Cys Asp Ser 50 55 60 Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile 65 70 75 80 Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val 85 90 95 Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly 100 105 110 Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala 115 120 125 Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu 130 135 140 Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu 145 150 155 160 Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro 165 170 175 Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr 180 185 190 Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu 195 200 205 Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg Ser Ser Ser 210 215 220 Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly 225 230 235 240 Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser Ser Lys Ala Pro 245 250 255 Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr 260 265 270 Pro Ile Leu Pro Gln 275 4387PRTHomo sapiens 4Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser 195 200 205 Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ala Pro Pro 210 215 220 Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala 225 230 235 240 Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu 245 250 255 Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp 260 265 270 Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu 275 280 285 Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn 290 295 300 Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val 305 310 315 320 Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln 325 330 335 Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg 340 345 350 Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn 355 360 365 Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr 370 375 380 Gly Asp Arg 385 5221PRTHomo sapiens 5Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ser Ser Ser Ser Lys 20 25 30 Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser 35 40 45 Asp Thr Pro Ile Leu Pro Gln Ala Pro Pro Arg Leu Ile Cys Asp Ser 50 55 60 Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile 65 70 75 80 Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val 85 90 95 Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly 100 105 110 Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala 115 120 125 Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu 130 135 140 Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu 145 150 155 160 Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro 165 170 175 Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr 180 185 190 Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu 195 200 205 Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg 210 215 220 6249PRTHomo sapiens 6Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ser Ser Ser Ser Lys 20 25 30 Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser 35 40 45 Asp Thr Pro Ile Leu Pro Gln Ala Pro Pro Arg Leu Ile Cys Asp Ser 50 55 60 Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile 65 70 75 80 Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val 85 90 95 Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly 100 105 110 Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala 115 120 125 Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu 130 135 140 Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu 145 150 155 160 Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro 165 170 175 Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr 180 185 190 Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu 195 200 205 Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg Ser Ser Ser 210 215 220 Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly 225 230 235 240 Pro Ser Asp Thr Pro Ile Leu Pro Gln 245 725DNAArtificial SequenceForward primer for EPO-CTP constructs 7aatctagagg tcatcatggg ggtgc 25832DNAArtificial SequenceForward primer for NotI CTP containing polypeptide 8attgcggccg cggatccaga agacctttat tg 32925DNAArtificial SequenceReverse primer for SspI CTP containing polypeptide 9taaatattgg ggtgtccgag ggccc 251032DNAArtificial SequenceForward primer for SspI CTP containing polypeptide 10ccaatattac cacaagcccc accacgcctc at 321135DNAArtificial SequenceReverse primer for NotI CTP containing polypeptide 11tgcggccgcg gatccttatc tgtcccctgt cctgc 351217DNAArtificial SequenceForward primer for EPO-CTP constructs 12gccctgctgt cggaagc 171332DNAArtificial SequenceReverse primer for NotI CTP containing polypeptide 13attgcggccg cggatccaga agacctttat tg 321432DNAArtificial SequenceReverse primer for EPO-CTP constructs 14ctttgaggaa gaggagccca ggactgggag gc 321524DNAArtificial SequenceForward primer for EPO-CTP constructs 15cctgggctcc tcttcctcaa aggc 241617DNAArtificial SequenceReverse primer for EPO-CTP constructs 16gcttccgaca gcagggc 171734PRTArtificial SequenceCTP amino acid sequence 17Asp Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser 1 5 10 15 Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu 20 25 30 Pro Gln 1828PRTArtificial SequenceCTP amino acid sequence 18Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg 1 5 10 15 Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 20 25 1927PRTHomo sapiens 19Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly 20 25 20786DNAHomo sapiens 20tctagaggtc atcatggggg tgcacgaatg tcctgcctgg ctgtggcttc tcctgtccct 60tctgtcgctc cctctgggcc tcccagtcct gggctcctct tcctcaaagg cccctccccc 120gagccttcca agtccatccc gactcccggg gccctcggac accccaatat taccacaagc 180cccaccacgc ctcatctgtg acagccgagt cctggagagg tacctcttgg aggccaagga 240ggccgagaat atcacgacgg gctgtgctga acactgcagc ttgaatgaga atatcactgt 300cccagacacc aaagttaatt tctatgcctg gaagaggatg gaggtcgggc agcaggccgt 360agaagtctgg cagggcctgg ccctgctgtc ggaagctgtc ctgcggggcc aggccctgtt 420ggtcaactct tcccagccgt gggagcccct gcagctgcat gtggataaag ccgtcagtgg 480ccttcgcagc ctcaccactc tgcttcgggc tctgggagcc cagaaggaag ccatctcccc 540tccagatgcg gcctcagctg ctccactccg aacaatcact gctgacactt tccgcaaact 600cttccgagtc tactccaatt tcctccgggg aaagctgaag ctgtacacag gggaggcctg 660caggacaggg gacagatcct cttcctcaaa ggcccctccc ccgagccttc caagtccatc 720ccgactcccg gggccctcgg acaccccgat cctcccacaa taaaggtctt ctggatccgc 780ggccgc 78621873DNAHomo sapiens 21tctagaggtc atcatggggg tgcacgaatg tcctgcctgg ctgtggcttc tcctgtccct 60tctgtcgctc cctctgggcc tcccagtcct gggctcctct tcctcaaagg cccctccccc 120gagccttcca agtccatccc gactcccggg gccctcggac accccaatat taccacaagc 180cccaccacgc ctcatctgtg acagccgagt cctggagagg tacctcttgg aggccaagga 240ggccgagaat atcacgacgg gctgtgctga acactgcagc ttgaatgaga atatcactgt 300cccagacacc aaagttaatt tctatgcctg gaagaggatg gaggtcgggc agcaggccgt 360agaagtctgg cagggcctgg ccctgctgtc ggaagctgtc ctgcggggcc aggccctgtt 420ggtcaactct tcccagccgt gggagcccct gcagctgcat gtggataaag ccgtcagtgg 480ccttcgcagc ctcaccactc tgcttcgggc tctgggagcc cagaaggaag ccatctcccc 540tccagatgcg gcctcagctg ctccactccg aacaatcact gctgacactt tccgcaaact 600cttccgagtc tactccaatt tcctccgggg aaagctgaag ctgtacacag gggaggcctg 660caggacaggg gacagatcct cttcctcaaa ggcccctccc ccgagccttc caagtccatc 720ccgactcccg gggccctccg acacaccaat cctgccacag agcagctcct ctaaggcccc 780tcctccatcc ctgccatccc cctcccggct gcctggcccc tctgacaccc ctatcctgcc 840tcagtgatga aggtcttctg gatccgcggc cgc 87322221PRTHomo sapiens 22Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Ser Gln Ala Leu Leu Val Asn Ser Ser 100 105 110 Gln Pro

Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser 195 200 205 Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 210 215 220 23217PRTHomo sapiens 23Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu 20 25 30 Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln 35 40 45 Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys 50 55 60 Val Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe 65 70 75 80 Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys 85 90 95 Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp 100 105 110 Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val 115 120 125 Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu 130 135 140 Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg 145 150 155 160 Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser 165 170 175 His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe 180 185 190 Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys 195 200 205 Arg Ser Val Glu Gly Ser Cys Gly Phe 210 215 24166PRTHomo sapiens 24Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr Leu Arg Asn 165 2530PRTHomo sapiens 25His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 2621PRTHomo sapiens 26Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser 20 2719DNAArtificial SequenceXbaI Forward primer for HGH-CTP constructs 27ctctagagga catggccac 192824DNAArtificial SequenceReverse primer for HGH-CTP constructs 28acagggaggt ctgggggttc tgca 242926DNAArtificial SequenceForward primer for HGH-CTP constructs 29tgcagaaccc ccagacctcc ctgtgc 263022DNAArtificial SequenceReverse primer for HGH-CTP constructs 30ccaaactcat caatgtatct ta 223119DNAArtificial SequenceXbaI Forward primer for HGH-CTP constructs 31ctctagagga catggccac 193225DNAArtificial SequenceReverse primer for HGH-CTP constructs 32cgaactcctg gtaggtgtca aaggc 253325DNAArtificial SequenceForward primer for HGH-CTP constructs 33gcctttgaca cctaccagga gttcg 253433DNAArtificial SequenceNotI Reverse primer for HGH-CTP constructs 34acgcggccgc atccagacct tcatcactga ggc 333534DNAArtificial SequenceReverse primer for HGH-CTP constructs 35gcggccgcgg actcatcaga agccgcagct gccc 3436217PRTHomo sapiens 36Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu 20 25 30 Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln 35 40 45 Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys 50 55 60 Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe 65 70 75 80 Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys 85 90 95 Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp 100 105 110 Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val 115 120 125 Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu 130 135 140 Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg 145 150 155 160 Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser 165 170 175 His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe 180 185 190 Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys 195 200 205 Arg Ser Val Glu Gly Ser Cys Gly Phe 210 215 37245PRTHomo sapiens 37Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu 20 25 30 Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln 35 40 45 Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys 50 55 60 Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe 65 70 75 80 Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys 85 90 95 Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp 100 105 110 Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val 115 120 125 Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu 130 135 140 Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg 145 150 155 160 Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser 165 170 175 His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe 180 185 190 Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys 195 200 205 Arg Ser Val Glu Gly Ser Cys Gly Phe Ser Ser Ser Ser Lys Ala Pro 210 215 220 Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr 225 230 235 240 Pro Ile Leu Pro Gln 245 38273PRTHomo sapiens 38Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu 20 25 30 Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln 35 40 45 Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys 50 55 60 Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe 65 70 75 80 Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys 85 90 95 Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp 100 105 110 Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val 115 120 125 Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu 130 135 140 Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg 145 150 155 160 Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser 165 170 175 His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe 180 185 190 Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys 195 200 205 Arg Ser Val Glu Gly Ser Cys Gly Phe Ser Ser Ser Ser Lys Ala Pro 210 215 220 Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr 225 230 235 240 Pro Ile Leu Pro Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 245 250 255 Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 260 265 270 Gln 39301PRTHomo sapiens 39Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Ser Ser Ser Ser Lys Ala 20 25 30 Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp 35 40 45 Thr Pro Ile Leu Pro Gln Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe 50 55 60 Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp 65 70 75 80 Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr 85 90 95 Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile 100 105 110 Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu 115 120 125 Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val 130 135 140 Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser 145 150 155 160 Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln 165 170 175 Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile 180 185 190 Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp 195 200 205 Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met 210 215 220 Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu 225 230 235 240 Gly Ser Cys Gly Phe Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 245 250 255 Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 260 265 270 Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser 275 280 285 Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 290 295 300 40285PRTHomo sapiens 40Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Ser Ser Ser Ser Lys Ala 20 25 30 Pro Pro Pro Ser Leu Pro Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe 35 40 45 Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp 50 55 60 Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr 65 70 75 80 Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile 85 90 95 Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu 100 105 110 Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val 115 120 125 Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser 130 135 140 Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln 145 150 155 160 Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile 165 170 175 Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp 180 185 190 Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met 195 200 205 Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu 210 215 220 Gly Ser Cys Gly Phe Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 225 230 235 240 Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 245 250 255 Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser 260 265 270 Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 275 280 285 41273PRTHomo sapiens 41Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Ser Ser Ser Ser Lys Ala 20 25 30 Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp 35 40 45 Thr Pro Ile Leu Pro Gln Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe 50 55 60 Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp 65 70 75 80 Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr 85 90 95 Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile 100 105 110 Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu 115 120 125 Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val 130 135 140 Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser 145 150 155 160 Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln 165 170 175 Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile 180 185 190 Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp 195 200 205 Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met 210 215 220 Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu 225 230 235 240 Gly Ser Cys Gly Phe Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 245

250 255 Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 260 265 270 Gln 42245PRTHomo sapiens 42Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Ser Ser Ser Ser Lys Ala 20 25 30 Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp 35 40 45 Thr Pro Ile Leu Pro Gln Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe 50 55 60 Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp 65 70 75 80 Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr 85 90 95 Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile 100 105 110 Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu 115 120 125 Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val 130 135 140 Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser 145 150 155 160 Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln 165 170 175 Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile 180 185 190 Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp 195 200 205 Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met 210 215 220 Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu 225 230 235 240 Gly Ser Cys Gly Phe 245 4312PRTArtificial SequenceCTP amino acid sequence 43Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro 1 5 10 44853DNAHomo sapiens 44tctagaggac atggccaccg gcagcaggac cagcctgctg ctggccttcg gcctgctgtg 60cctgccatgg ctgcaggagg gcagcgccag ctcttcttct aaggctccac ccccatctct 120gcccagcccc agcagactgc cgggccccag cgacacaccc attctgcccc agttccccac 180catccccctg agcaggctgt tcgacaacgc catgctgagg gctcacaggc tgcaccagct 240ggcctttgac acctaccagg agttcgagga agcctacatc cccaaggagc agaagtacag 300cttcctgcag aacccccaga cctccctgtg cttcagcgag agcatcccca cccccagcaa 360cagagaggag acccagcaga agagcaacct ggagctgctg aggatctccc tgctgctgat 420ccagagctgg ctggagcccg tgcagttcct gagaagcgtg ttcgccaaca gcctggtgta 480cggcgccagc gacagcaacg tgtacgacct gctgaaggac ctggaggagg gcatccagac 540cctgatgggc cggctggagg acggcagccc caggaccggc cagatcttca agcagaccta 600cagcaagttc gacaccaaca gccacaacga cgacgccctg ctgaagaact acgggctgct 660gtactgcttc agaaaggaca tggacaaggt ggagaccttc ctgaggatcg tgcagtgcag 720aagcgtggag ggcagctgcg gcttcagctc cagcagcaag gcccctcccc cgagcctgcc 780ctccccaagc aggctgcctg ggccctccga cacaccaatc ctgcctcagt gatgaaggtc 840tggatgcggc cgc 85345937DNAHomo sapiens 45tctagaggac atggccaccg gcagcaggac cagcctgctg ctggccttcg gcctgctgtg 60cctgccatgg ctgcaggagg gcagcgccag ctcttcttct aaggctccac ccccatctct 120gcccagcccc agcagactgc cgggccccag cgacacaccc attctgcccc agttccccac 180catccccctg agcaggctgt tcgacaacgc catgctgagg gctcacaggc tgcaccagct 240ggcctttgac acctaccagg agttcgagga agcctacatc cccaaggagc agaagtacag 300cttcctgcag aacccccaga cctccctgtg cttcagcgag agcatcccca cccccagcaa 360cagagaggag acccagcaga agagcaacct ggagctgctg aggatctccc tgctgctgat 420ccagagctgg ctggagcccg tgcagttcct gagaagcgtg ttcgccaaca gcctggtgta 480cggcgccagc gacagcaacg tgtacgacct gctgaaggac ctggaggagg gcatccagac 540cctgatgggc cggctggagg acggcagccc caggaccggc cagatcttca agcagaccta 600cagcaagttc gacaccaaca gccacaacga cgacgccctg ctgaagaact acgggctgct 660gtactgcttc agaaaggaca tggacaaggt ggagaccttc ctgaggatcg tgcagtgcag 720aagcgtggag ggcagctgcg gcttcagctc cagcagcaag gcccctcccc cgagcctgcc 780ctccccaagc aggctgcctg ggccctccga cacaccaatc ctgccacaga gcagctcctc 840taaggcccct cctccatccc tgccatcccc ctcccggctg cctggcccct ctgacacccc 900tatcctgcct cagtgatgaa ggtctggatg cggccgc 93746889DNAHomo sapiens 46tctagaggac atggccaccg gcagcaggac cagcctgctg ctggccttcg gcctgctgtg 60cctgccatgg ctgcaggagg gcagcgccag ctcttcttct aaggctccac ccccgagcct 120gcccttcccc accatccccc tgagcaggct gttcgacaac gccatgctga gggctcacag 180gctgcaccag ctggcctttg acacctacca ggagttcgag gaagcctaca tccccaagga 240gcagaagtac agcttcctgc agaaccccca gacctccctg tgcttcagcg agagcatccc 300cacccccagc aacagagagg agacccagca gaagagcaac ctggagctgc tgaggatctc 360cctgctgctg atccagagct ggctggagcc cgtgcagttc ctgagaagcg tgttcgccaa 420cagcctggtg tacggcgcca gcgacagcaa cgtgtacgac ctgctgaagg acctggagga 480gggcatccag accctgatgg gccggctgga ggacggcagc cccaggaccg gccagatctt 540caagcagacc tacagcaagt tcgacaccaa cagccacaac gacgacgccc tgctgaagaa 600ctacgggctg ctgtactgct tcagaaagga catggacaag gtggagacct tcctgaggat 660cgtgcagtgc agaagcgtgg agggcagctg cggcttcagc tccagcagca aggcccctcc 720cccgagcctg ccctccccaa gcaggctgcc tgggccctcc gacacaccaa tcctgccaca 780gagcagctcc tctaaggccc ctcctccatc cctgccatcc ccctcccggc tgcctggccc 840ctctgacacc cctatcctgc ctcagtgatg aaggtctgga tgcggccgc 88947217PRTHomo sapiens 47Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu 20 25 30 Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln 35 40 45 Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys 50 55 60 Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe 65 70 75 80 Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys 85 90 95 Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp 100 105 110 Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val 115 120 125 Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu 130 135 140 Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg 145 150 155 160 Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser 165 170 175 His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe 180 185 190 Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys 195 200 205 Arg Ser Val Glu Gly Ser Cys Gly Phe 210 215 48187PRTHomo sapiens 48Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg 20 25 30 Ser Ser Asn Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg 35 40 45 Leu Glu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu 50 55 60 Ile Lys Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile 65 70 75 80 Tyr Glu Met Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser 85 90 95 Ser Thr Gly Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val 100 105 110 Tyr His Gln Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu 115 120 125 Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys 130 135 140 Arg Tyr Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser 145 150 155 160 His Cys Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr 165 170 175 Phe Ile Asn Arg Leu Thr Gly Tyr Leu Arg Asn 180 185 49589DNAHomo sapiens 49tctagaggac atgaccaaca agtgcctgct gcagatcgcc ctgctgctgt gcttcagcac 60caccgccctg agcatgagct acaacctgct gggcttcctg cagaggtcca gcaacttcca 120gtgccagaag ctgctgtggc agctgaacgg caggctggaa tactgcctga aggacaggat 180gaacttcgac atcccagagg aaatcaagca gctgcagcag ttccagaagg aggacgccgc 240cctgaccatc tacgagatgc tgcagaacat cttcgccatc ttcaggcagg acagcagcag 300caccggctgg aacgagacca tcgtggagaa cctgctggcc aacgtgtacc accagatcaa 360ccacctgaaa accgtgctgg aagagaagct ggaaaaggag gacttcacca ggggcaagct 420gatgagcagc ctgcacctga agaggtacta cggcagaatc ctgcactacc tgaaggccaa 480ggagtacagc cactgcgcct ggaccatcgt gagggtggag atcctgagga acttctactt 540catcaacagg ctgaccggct acctgaggaa ctgatgagtc cgcggccgc 58950211PRTHomo sapiens 50Thr Phe Leu Gln Pro Phe Glu Ala Phe Ala Leu Ala Gln Gln Val Val 1 5 10 15 Gly Asp Thr Val Arg Val Val Asn Met Thr Asn Lys Cys Leu Leu Gln 20 25 30 Ile Ala Leu Leu Leu Cys Phe Ser Thr Thr Ala Leu Ser Met Ser Tyr 35 40 45 Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln Cys Gln Lys 50 55 60 Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu Lys Asp Arg 65 70 75 80 Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln Gln Phe Gln 85 90 95 Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln Asn Ile Phe 100 105 110 Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn Glu Thr Ile 115 120 125 Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn His Leu Lys 130 135 140 Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr Arg Gly Lys 145 150 155 160 Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg Ile Leu His 165 170 175 Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr Ile Val Arg 180 185 190 Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu Thr Gly Tyr 195 200 205 Leu Arg Asn 210 51639DNAHomo sapiens 51acattctaac tgcaaccttt cgaagccttt gctctggcac aacaggtagt aggcgacact 60gttcgtgttg tcaacatgac caacaagtgt ctcctccaaa ttgctctcct gttgtgcttc 120tccactacag ctctttccat gagctacaac ttgcttggat tcctacaaag aagcagcaat 180tttcagtgtc agaagctcct gtggcaattg aatgggaggc ttgaatactg cctcaaggac 240aggatgaact ttgacatccc tgaggagatt aagcagctgc agcagttcca gaaggaggac 300gccgcattga ccatctatga gatgctccag aacatctttg ctattttcag acaagattca 360tctagcactg gctggaatga gactattgtt gagaacctcc tggctaatgt ctatcatcag 420ataaaccatc tgaagacagt cctggaagaa aaactggaga aagaagattt caccagggga 480aaactcatga gcagtctgca cctgaaaaga tattatggga ggattctgca ttacctgaag 540gccaaggagt acagtcactg tgcctggacc atagtcagag tggaaatcct aaggaacttt 600tacttcatta acagacttac aggttacctc cgaaactga 63952215PRTHomo sapiens 52Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg 20 25 30 Ser Ser Asn Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg 35 40 45 Leu Glu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu 50 55 60 Ile Lys Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile 65 70 75 80 Tyr Glu Met Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser 85 90 95 Ser Thr Gly Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val 100 105 110 Tyr His Gln Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu 115 120 125 Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys 130 135 140 Arg Tyr Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser 145 150 155 160 His Cys Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr 165 170 175 Phe Ile Asn Arg Leu Thr Gly Tyr Leu Arg Asn Ser Ser Ser Ser Lys 180 185 190 Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser 195 200 205 Asp Thr Pro Ile Leu Pro Gln 210 215 53661DNAHomo sapiens 53tctagaggac atgaccaaca agtgcctgct gcagatcgcc ctgctgctgt gcttcagcac 60caccgccctg agcatgagct acaacctgct gggcttcctg cagaggtcca gcaacttcca 120gtgccagaag ctgctgtggc agctgaacgg caggctggaa tactgcctga aggacaggat 180gaacttcgac atcccagagg aaatcaagca gctgcagcag ttccagaagg aggacgccgc 240cctgaccatc tacgagatgc tgcagaacat cttcgccatc ttcaggcagg acagcagcag 300caccggctgg aacgagacca tcgtggagaa cctgctggcc aacgtgtacc accagatcaa 360ccacctgaaa accgtgctgg aagagaagct ggaaaaggag gacttcacca ggggcaagct 420gatgagcagc ctgcacctga agaggtacta cggcagaatc ctgcactacc tgaaggccaa 480ggagtacagc cactgcgcct ggaccatcgt gagggtggag atcctgagga acttctactt 540catcaacagg ctgaccggct acctgaggaa cagctccagc agcaaggccc ctccaccttc 600cctgcccagt ccaagccgac tccctgggcc ctccgataca ccaattctgc cacagtgatg 660a 66154243PRTHomo sapiens 54Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg 20 25 30 Ser Ser Asn Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg 35 40 45 Leu Glu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu 50 55 60 Ile Lys Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile 65 70 75 80 Tyr Glu Met Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser 85 90 95 Ser Thr Gly Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val 100 105 110 Tyr His Gln Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu 115 120 125 Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys 130 135 140 Arg Tyr Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser 145 150 155 160 His Cys Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr 165 170 175 Phe Ile Asn Arg Leu Thr Gly Tyr Leu Arg Asn Ser Ser Ser Ser Lys 180 185 190 Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser 195 200 205 Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro 210 215 220 Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile 225 230 235 240 Leu Pro Gln 55765DNAHomo sapiens 55tctagaggac atgaccaaca agtgcctgct gcagatcgcc ctgctgctgt gcttcagcac 60caccgccctg agcatgagct acaacctgct gggcttcctg cagaggtcca gcaacttcca 120gtgccagaag ctgctgtggc agctgaacgg caggctggaa tactgcctga aggacaggat 180gaacttcgac atcccagagg aaatcaagca gctgcagcag ttccagaagg aggacgccgc 240cctgaccatc tacgagatgc tgcagaacat cttcgccatc ttcaggcagg acagcagcag 300caccggctgg aacgagacca tcgtggagaa cctgctggcc aacgtgtacc accagatcaa 360ccacctgaaa accgtgctgg aagagaagct ggaaaaggag gacttcacca ggggcaagct 420gatgagcagc ctgcacctga agaggtacta cggcagaatc ctgcactacc tgaaggccaa 480ggagtacagc cactgcgcct ggaccatcgt gagggtggag atcctgagga acttctactt 540catcaacagg ctgaccggct acctgaggaa cagctccagc agcaaggccc ctccaccttc 600cctgcccagt ccaagccgac tccctgggcc ctccgacaca ccaatcctgc cacagagcag 660ctcctctaag gcccctcctc catccctgcc atccccctcc cggctgcctg gcccctctga 720cacccctatc ctgcctcagt gatgaaggtc tggatccgcg gccgc 76556271PRTHomo sapiens 56Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 20 25 30 Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 35 40 45 Gln Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe 50 55

60 Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys 65 70 75 80 Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu 85 90 95 Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu 100 105 110 Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp 115 120 125 Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile 130 135 140 Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe 145 150 155 160 Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly 165 170 175 Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp 180 185 190 Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg 195 200 205 Leu Thr Gly Tyr Leu Arg Asn Ser Ser Ser Ser Lys Ala Pro Pro Pro 210 215 220 Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile 225 230 235 240 Leu Pro Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser 245 250 255 Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 260 265 270 57846DNAHomo sapiens 57tctagaggac atgaccaaca agtgcctgct gcagatcgcc ctgctgctgt gcttcagcac 60caccgccctg agcagcagca gctccaaggc cccacccccc agcctgccca gccccagcag 120actgccaggc cccagcgaca cccccatcct gccccagatg agctacaacc tgctgggctt 180cctgcagagg tccagcaact tccagtgcca gaagctgctg tggcagctga acggcaggct 240ggaatactgc ctgaaggaca ggatgaactt cgacatccca gaggaaatca agcagctgca 300gcagttccag aaggaggacg ccgccctgac catctacgag atgctgcaga acatcttcgc 360catcttcagg caggacagca gcagcaccgg ctggaacgag accatcgtgg agaacctgct 420ggccacgtgt accaccagat caaccacctg aaaaccgtgc tggaagagaa gctggaaaag 480gaggacttca ccaggggcaa gctgatgagc agcctgcacc tgaagaggta ctacggcaga 540atcctgcact acctgaaggc caaggagtac agccactgcg cctggaccat cgtgaggtgg 600agatcctgag gaacttctac ttcatcaaca ggctgaccgg ctacctgagg aacagctcca 660gcagcaaggc ccctccactt ccctgcccag tccaagccga ctccctgggc cctccgacac 720accaatcctg ccacagagca gctcctctaa ggcccctcct ccatccctgc catccccctc 780ccggctgcct ggcccctctg acacccctat cctgcctcag tgatgaaggt ctggatccgc 840ggccgc 84658409PRTHomo sapiens 58Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 20 25 30 Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 35 40 45 Gln Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe 50 55 60 Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys 65 70 75 80 Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu 85 90 95 Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu 100 105 110 Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp 115 120 125 Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile 130 135 140 Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe 145 150 155 160 Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly 165 170 175 Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp 180 185 190 Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg 195 200 205 Leu Thr Gly Tyr Leu Arg Asn Ser Ser Ser Ser Lys Ala Pro Pro Pro 210 215 220 Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile 225 230 235 240 Leu Pro Gln Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser 245 250 255 Asn Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu 260 265 270 Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys 275 280 285 Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu 290 295 300 Met Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr 305 310 315 320 Gly Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His 325 330 335 Gln Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu 340 345 350 Asp Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr 355 360 365 Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys 370 375 380 Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile 385 390 395 400 Asn Arg Leu Thr Gly Tyr Leu Arg Asn 405 591262DNAHomo sapiens 59tctagaggac atgaccaaca agtgcctgct gcagatcgcc ctgctgctgt gcttcagcac 60caccgccctg agcagcagca gctccaaggc cccacccccc agcctgccca gccccagcag 120gctgccaggc cccagcgaca cccccatcct gccccagatg agctacaacc tgctgggctt 180cctgcagagg tccagcaact tccagtgcca gaaactgctg tggcagctga acggcaggct 240ggaatactgc ctgaaggacc ggatgaactt cgacatcccc gaagagatca agcagctgca 300gcagttccag aaagaggacg ccgccctgac catctacgag atgctgcaga acatcttcgc 360catcttcagg caggacagca gcagcaccgg ctggaacgag accatcgtgg agaacctgct 420ggccaacgtg taccaccaga tcaaccacct gaaaaccgtg ctggaagaga agctggaaaa 480agaggacttc accaggggca agctgatgag cagcctgcac ctgaagaggt actacggcag 540aatcctgcac tacctgaagg ccaaagagta cagccactgc gcctggacca tctgtgaggg 600tggagatcct gcggaacttc tacttcatca acaggctgac cggctacctg aggaacagct 660ccagcagcaa ggcccctcca ccctccctgc cctccccaag cagactgccc ggaccctccg 720acacaccaat tctgccacag atgtcctaca atctgctcgg atttctgcag cgctcctcca 780actttcagtg tcagaagctc ctctggcagc tcaatggccg cctggaatat tgtctgaaag 840acagaatgaa ttttgacatc ccagaggaaa ttaaacagct ccagcagttt cagaaagaag 900atgctgctct cacaatctat gaaatgctcc agaatatctt tgcaatcttt cgccaggaca 960gctcctccac cgggtggaat gagacaattg tcgagaatct gctcgccaat gtctatcatc 1020agatcaatca cctcaagaca gtcctcgaag aaaaactcga aaaagaagat ttcacacgcg 1080gcaaactgat gtcctccctg catctgaagc gctactatgg gcgcatcctg cattatctga 1140aagctaaaga atactcccac tgtgcttgga caattgtgcg cgtcgagatc ctgagaaact 1200tttatttcat taaccgcctg acaggatacc tgcgcaactg atgaaggtct ggatgcggcc 1260gc 126260215PRTHomo sapiens 60Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 20 25 30 Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 35 40 45 Gln Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe 50 55 60 Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys 65 70 75 80 Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu 85 90 95 Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu 100 105 110 Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp 115 120 125 Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile 130 135 140 Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe 145 150 155 160 Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly 165 170 175 Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp 180 185 190 Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg 195 200 205 Leu Thr Gly Tyr Leu Arg Asn 210 215 61673DNAHomo sapiens 61tctagaggac atgaccaaca agtgcctgct gcagatcgcc ctgctgctgt gcttcagcac 60caccgccctg agcagcagca gctccaaggc cccacccccc agcctgccca gccccagcag 120gctgccaggc cccagcgaca cccccatcct gccccagatg agctacaacc tgctgggctt 180cctgcagagg tccagcaact tccagtgcca gaaactgctg tggcagctga acggcaggct 240ggaatactgc ctgaaggacc ggatgaactt cgacatcccc gaagagatca agcagctgca 300gcagttccag aaagaggacg ccgccctgac catctacgag atgctgcaga acatcttcgc 360catcttcagg caggacagca gcagcaccgg ctggaacgag accatcgtgg agaacctgct 420ggccaacgtg taccaccaga tcaaccacct gaaaaccgtg ctggaagaga agctggaaaa 480agaggacttc accaggggca agctgatgag cagcctgcac ctgaagaggt actacggcag 540aatcctgcac tacctgaagg ccaaagagta cagccactgc gcctggacca tcgtgagggt 600ggagatcctg cggaacttct acttcatcaa caggctgacc ggctacctga ggaactgatg 660agtccgcggc cgc 67362243PRTHomo sapiens 62Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 20 25 30 Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 35 40 45 Gln Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe 50 55 60 Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys 65 70 75 80 Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu 85 90 95 Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu 100 105 110 Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp 115 120 125 Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile 130 135 140 Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe 145 150 155 160 Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly 165 170 175 Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp 180 185 190 Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg 195 200 205 Leu Thr Gly Tyr Leu Arg Asn Ser Ser Ser Ser Lys Ala Pro Pro Pro 210 215 220 Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile 225 230 235 240 Leu Pro Gln 63763DNAHomo sapiens 63tctagaggac atgaccaaca agtgcctgct gcagatcgcc ctgctgctgt gcttcagcac 60caccgccctg agcagcagca gctccaaggc cccacccccc agcctgccca gccccagcag 120actgccaggc cccagcgaca cccccatcct gccccagatg agctacaacc tgctgggctt 180cctgcagagg tccagcaact tccagtgcca gaagctgctg tggcagctga acggcaggct 240ggaatactgc ctgaaggaca ggatgaactt cgacatccca gaggaaatca agcagctgca 300gcagttccag aaggaggacg ccgccctgac catctacgag atgctgcaga acatcttcgc 360catcttcagg caggacagca gcagcaccgg ctggaacgag accatcgtgg agaacctgct 420ggccaacgtg taccaccaga tcaaccacct gaaaaccgtg ctggaagaga agctggaaaa 480ggaggacttc accaggggca agctgatgag cagcctgcac ctgaagaggt actacggcag 540aatcctgcac tacctgaagg ccaaggagta cagccactgc gcctggacca tcgtgagggt 600ggagatcctg aggaacttct acttcatcaa caggctgacc ggctacctga ggaacagctc 660cagcagcaag gcccctccac cttccctgcc cagtccaagc cgactccctg ggccctccga 720tacaccaatt ctgccacagt gatgaaggtc tggatgcggc cgc 7636421PRTHomo sapiens 64Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser 20 6523DNAHomo sapiens 65gaattctaga ggacatgacc aac 236633DNAHomo sapiens 66gcggccgcgg actcatcagt tcctcaggta gcc 336712PRTHomo sapiens 67Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro 1 5 10 68166PRTHomo sapiens 68Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr Leu Arg Asn 165

Claims

1. A method of treating or ameliorating a disease in a subject in need of cytokine therapy, comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide comprising a cytokine attached to a single chorionic gonadotropin carboxy terminal peptide (CTP) at the amino terminus of said cytokine, and two chorionic gonadotropin carboxy terminal peptides (CTP) attached to the carboxy terminus of said cytokine, thereby treating said subject in need of a cytokine therapy.

2. The method of claim 1, wherein the sequence of at least one CTP consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 17 and SEQ ID NO: 18.

3. The method of claim 1, wherein said at least one CTP is glycosylated.

4. The method of claim 1, wherein said at least one CTP is truncated.

5. The method of claim 1, wherein at least one CTP is optionally attached to said cytokine via a linker.

6. The method of claim 5, wherein the linker is a peptide bond.

7. The method of claim 1, wherein said attaching produces a CTP-modified cytokine polypeptide, wherein when said polypeptide is a precursor polypeptide consisting of a cytokine attached to a single chorionic gonadotropin carboxy terminal peptide (CTP) at the amino terminus of said cytokine, and two chorionic gonadotropin carboxy terminal peptides (CTP) attached to the carboxy terminus of said cytokine, and a signal peptide attached to the amino terminus of said one CTP, and when said polypeptide is a mature polypeptide, said polypeptide consists of a cytokine attached to a single chorionic gonadotropin carboxy terminal peptide (CTP) at the amino terminus of said cytokine, and two chorionic gonadotropin carboxy terminal peptides (CTP) attached to the carboxy terminus of said cytokine and said polypeptide lacks a signal peptide.

8. The method of claim 7, wherein said signal peptide consists of an amino acid sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 64.

9. The method of claim 1, wherein said treating or ameliorating a disease in said subject induces growth, induces weight gain, induces erythropoiesis, treats or ameliorates anemia, provides an anti-viral activity, or provides an anti-proliferative activity, or any combination thereof, in said subject.

10. The method of claim 1, wherein said subject is a human subject.

11. The method of claim 1, wherein said disease is a cancer.

12. The method of claim 11, wherein said cancer is hairy cell leukemia, malignant melanoma, Kaposi's sarcoma, bladder cancer, chronic myelocytic leukemia, kidney cancer, carcinoid tumors, non-Hodgkin's lymphoma, ovarian cancer, or skin cancers.

13. The method of claim 1, wherein said pharmaceutical composition comprises a pharmaceutical formulation comprising a buffer, a tonicity agent, and the polypeptide comprising the cytokine attached to a single chorionic gonadotropin carboxy terminal peptide (CTP) at the amino terminus of said cytokine, and two chorionic gonadotropin carboxy terminal peptides (CTP) attached to the carboxy terminus of said cytokine.

14. The method of claim 13, wherein said formulation comprises a liquid formulation or a topical formulation.

15. The method of claim 13, wherein said buffer is citrate

16. The method of claim 13, wherein said tonicity agent is NaCl.

17. The method of claim 13, wherein said composition is at a pH of about 4-7.2.

18. The method of claim 13, wherein said composition is a liquid.

19. The method of claim 13, wherein said composition is administered once weekly or once bi-weekly.

20. The method of claim 13, wherein said composition is formulated in a form suitable for an intramuscular (IM) injection, a subcutaneous (SC) injection, or an intravenous (IV) injection.