WO2008052043A2 - Protéines de fusion agonistes du récepteur opioïde - Google Patents

Protéines de fusion agonistes du récepteur opioïde Download PDF

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WO2008052043A2
WO2008052043A2 PCT/US2007/082363 US2007082363W WO2008052043A2 WO 2008052043 A2 WO2008052043 A2 WO 2008052043A2 US 2007082363 W US2007082363 W US 2007082363W WO 2008052043 A2 WO2008052043 A2 WO 2008052043A2
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seq
protein
opioid receptor
receptor agonist
sequence
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PCT/US2007/082363
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WO2008052043A9 (fr
WO2008052043A3 (fr
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Craig A. Rosen
Adam C. Bell
Indrajit Sanyal
David Lafleur
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Cogenesys, Inc.
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Publication of WO2008052043A3 publication Critical patent/WO2008052043A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates generally to therapeutic fusion proteins that have opioid receptor agonist activity.
  • the agonist generally may be fused to a serum protein such as albumin or fragments or variants of albumin; a protein such as alpha- fetoprotein (AFP) or fragments or variants of AFP; or an albumin- AFP hybrid protein.
  • the invention encompasses polynucleotides encoding opioid receptor agonist fusion proteins, opioid receptor agonist fusion proteins, compositions, pharmaceutical compositions, formulations and kits.
  • Host cells transformed with the polynucleotides encoding opioid receptor agonist fusion proteins are also encompassed by the invention, as are methods of making the opioid receptor agonist fusion proteins of the invention using these polynucleotides, and/or host cells.
  • Albumin is responsible for a significant proportion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands.
  • Human serum albumin (HSA, or HA)
  • HSA Human serum albumin
  • HA Human serum albumin
  • HA is a protein of 585 amino acids in its mature form (as shown in FIG. IA-D).
  • HA for clinical use is produced by extraction from human blood.
  • the production of recombinant HA (rHA) in microorganisms has been disclosed in EP 330 451 and EP 361 991.
  • Alpha- fetoprotein a protein of 609 amino acids, is synthesized in the embryonic liver and is found in fetal serum. AFP is not normally detectable after birth in significant levels. High levels of this protein are found in the developing fetus, but low levels exist in the amniotic fluid and maternal serum.
  • Therapeutic proteins in their native state or when recombinantly produced, such as opioid receptor agonist proteins, are typically labile molecules exhibiting short shelf- lives, particularly when formulated in aqueous solutions. The instability in these molecules when formulated for administration dictates that many of the molecules must be lyophilized and refrigerated at all times during storage, thereby rendering the molecules difficult to transport and/or store. Storage problems are particularly acute when pharmaceutical formulations must be stored and dispensed outside of the hospital environment.
  • Recombinantly produced therapeutic proteins such as opioid receptor agonist proteins also may exhibit a short half-life in vivo, for example after being administered to a patient.
  • Therapeutic proteins such as opioid receptor agonist proteins which exhibit a longer-lived activity in vivo are desirable because they permit longer time periods between dosing in a patient.
  • Opioid receptors are present in the peripheral tissue as well as in the central nervous system, including the brain. Opioid receptor agonists can effect an analgesic or antinociceptive response by binding to peripheral opioid receptors. In contrast, many undesirable side effects of opioid receptor agonists ⁇ e.g., addiction and nausea) are thought to result from the agonist binding to opioid receptors of the central nervous system (CNS) including those of the brain.
  • CNS central nervous system
  • Transport of molecules into the brain across the blood-brain barrier is selective and may occur by a number of mechanisms including (1) simple diffusion and (2) active transport through a protein carrier. Simple diffusion may occur paracellularly (i.e., between cells) or transcellularly (i.e., across cells). Paracellular diffusion is limited by the presence of "tight junctions," which are cellular junctions present in the brain that generally permit diffusion only of molecules having a molecular weight less than approximately 500 daltons. Transcellular diffusion occurs only to a great extent for small, highly lipophilic molecules. In general, opioid peptides have a molecular weight greater than 500 daltons and are not highly lipophilic. Nonetheless, paracellular or transcellular diffusion of an opioid peptide across the blood-brain barrier might occur infrequently and could be further inhibited by increasing the molecular weight of the opioid peptide.
  • opioid peptides have been shown to be transported across the blood- brain barrier by active transport.
  • active transport See, e.g., Banks and Kastin, "Peptide transport systems for opiates across the blood-brain barrier," AM J. PHYSIOL. ENDOCRINOL. METAB. 1990 259:E1-E1O; Banks et al, "Endogenous peptide Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1) is transported from the brain to the blood by peptide transport system- 1," J. NEUROSCI. RES.
  • PTS peptide transport systems
  • PTS-I peptide transport systems
  • Tyr-MIF-1 i.e., Tyr-Pro-Leu-Gly-NH2
  • Modification of opioid peptides may inhibit their active transport across the blood-brain barrier where fragments of Tyr-MIF-1 or variants of Tyr-MIF-1 having iodo-Tyr or D-Tyr modifications were not observed to be transported by PTS-I.
  • opioid receptors agonist proteins that exhibit increased or extended stabilibity and which are not transported efficiently across the blood-brain barrier.
  • these opioid receptor agonist proteins should exhibit an analgesic effect or antinociceptive effect.
  • Fusion proteins of opioid receptor agonist peptides may achieve these goals.
  • opioid receptor agonist peptides are relatively small, a fusion protein may not present an opioid receptor agonist peptide portion properly to function as an agonist.
  • the opioid receptor agonist peptide portion should not effect transport of the fusion protein across the blood-brain barrier. Designing fusion proteins that achieve these goals is not trivial.
  • the present invention encompasses fusion proteins comprising a therapeutic protein portion having opioid receptor agonist activity.
  • the present invention also encompasses polynucleotides comprising, or alternatively consisting of, nucleic acid molecules encoding a therapeutic fusion protein having opioid receptor agonist activity.
  • the fusion protein may include an opioid receptor agonist (either a prohormone or mature form) fused to a serum protein such as albumin, alpha- fetoprotein (AFP), or a hybrid protein of albumin and AFP (e.g., a hybrid protein comprising about a 1-50 amino acid sequence of albumin).
  • the opioid receptor agonist may be fused to a fragment (portion), variant, or analog of the serum protein (e.g., albumin, AFP, or albumin- AFP hybrid).
  • the opioid receptor agonist may be fused to the N-terminus, the C-terminus, or both termini of the serum protein (e.g., albumin, AFP, or albumin-AFP hybrid).
  • the opioid receptor agonist may be fused directly to the serum protein (e.g., albumin, AFP, or albumin-AFP hybrid) or may be fused via a linking sequence (e.g., a linking sequence that does not include a dibasic amino acid sequence such as KK, KR, or RR).
  • the present invention also encompasses polynucleotides, comprising, or alternatively consisting of, nucleic acid molecules encoding proteins comprising a therapeutic fusion protein having opioid receptor agonist activity.
  • the opioid receptor agonist fusion proteins exhibit prolonged biological activity relative to an opioid receptor agonist protein that is not fused to a serum protein such as albumin or AFP.
  • the prolonged activity may be observed in vitro and/or in vivo.
  • the serum protein may stabilize or prolong the therapeutic activity of the protein so that the shelf life of the opioid receptor agonist protein portion of the fusion protein is prolonged or extended compared to the shelf-life of a non- fused opioid receptor agonist protein.
  • Host cells transformed with polynucleotides that encode opioid receptor agonist fusion proteins are also encompassed by the invention, and methods of making the opioid receptor agonist fusion proteins of the invention and using these polynucleotides of the invention and/or host cells.
  • the opioid receptor agonist fusion proteins may include multiple copies of the opioid receptor agonist and/or the serum protein ⁇ e.g., tandem repeats).
  • the opioid receptor agonist fusion proteins disclosed herein are genetic fusions and therefore exhibit higher stability as compared to conjugate proteins which are prepared by chemical conjugation. In addition, because the opioid receptor agonist fusion proteins are genetic fusions, the stoichiometry of the opioid receptor agonist portion and the serum protein portion in the fusion protein can be more precisely determined as contrasted with conjugate proteins.
  • the opioid receptor agonist fusion proteins described herein generally demonstrate opioid receptor agonist activity. For example, the opioid receptor agonist fusion proteins selectively bind and activate one or more opioid receptors ⁇ e.g. , mu- opioid, delta-opioid, and kappa-opioid receptors).
  • the effect of an opioid receptor agonist selectively binding to an opioid receptor may be transduced by G-proteins ⁇ e.g., G 1 or G 0 or both).
  • the fusion proteins binds to an opioid receptor present on a cell ⁇ e.g. , mu-opioid, delta-opioid, and/or kappa-opioid receptors present on nerve cells) and subsequently a G-protein ⁇ e.g. , G 1 or G 0 or both) transduces an effect that may include one or more of the following: a decrease or increase in the concentration of cAMP in the cells, a decrease or increase in activity of calcium channels of the cell; and a decrease or increase in activity of potassium channels of the cell.
  • the fusion proteins disclosed herein cause an analgesic effect or an antinociceptive effect in an animal.
  • Peptide opioid receptor agonists include any peptide that selectively binds and activates an opioid receptor (e.g., mu-opioid receptor, delta-opioid receptor, and/or kappa-opioid receptors).
  • opioid receptor e.g., mu-opioid receptor, delta-opioid receptor, and/or kappa-opioid receptors.
  • Peptide opioid receptor agonists may include opioid peptides such as endorphins (e.g., alpha-endorphrin and beta-endorphin), dynorphins (e.g., dynorphin A, dynorphin B, alpha-neoendorphin, and beta-neoendorphin), enkephalins, endomorphins (e.g., endomorphin-1 and endomorphin-2), deltorphins, xen-dorphins, dermorphins, orphanins, and variants or derivatives thereof.
  • opioid peptides such as endorphins (e.g., alpha-endorphrin and beta-endorphin), dynorphins (e.g., dynorphin A, dynorphin B, alpha-neoendorphin, and beta-neoendorphin), enkephalins, endomorphins (e.
  • Peptide opioid receptor agonists may include polypeptides present in animal venoms (e.g., snake or frog venoms).
  • Preferred snake venoms may include those of snakes belonging to the families Colubridae, Elapidae, Viperidae and Crotalidae such as species of the genera Naja, Dendroaspis, Bungarus, Pseudechis, Ophiophagus and Hemachatus.
  • Particularly preferred snake venoms may include those of snakes belonging to the family Elapidae such as but not limited to King cobra (Ophiohagus hannah); True cobras (Naja spp); Asian or Indian cobra (N.
  • Suitable snake venoms may include Crotalus durissus terrificus venom, Trimeresurus flavoviridis (Habu snake) venom, and Vipera russelli russelli (Russell's viper) venom including beta-RTX.
  • Suitable venoms from frogs may include venoms isolated form Phyllomedusa frogs (e.g., dermorphin, which is present on frog skin).
  • the opioid receptor agonist protein portion of the fusion proteins described herein may comprise one or more of the following polypeptide sequences: YGGFL (SEQ ID NO:1, i.e., [Leu 5 ]enkephalin); YGGFM (SEQ ID NO:2, i.e., [Met 5 ]enkephalin); YGGFLRRIRPKLKWDNQ (SEQ ID NO:3, i.e., Dynorphin A); YGGFLRRQFKVVT (SEQ ID NO:4, i.e., Dynorphin B); YGGFLRKYPK (SEQ IN NO:5, i.e., alpha-Neoendorphin); YGGFLRKYP (SEQ ID NO:6, i.e., beta-Neoendorphin); YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE (SEQ IN NO:7, i.e., beta-Endorphin);
  • the opioid receptor agonist protein portion of the fusion proteins described herein may comprise variants of the above -references polypeptide sequences.
  • the opioid receptor agonist protein portion of the fusion proteins described herein may comprise a variant having one or more deletions, additions, or substitutions in reference to a wild-type opioid receptor agonist protein.
  • the variants may include substitutions where non-basic amino acids ⁇ e.g., G or Q) replace basic amino acids ⁇ e.g., K or R) to remove dibasic sequences in the fusion protein.
  • the opioid receptor agonist protein portion of the fusion proteins described herein may comprise multiple copies of one or more opioid receptor agonist proteins, variants, or fragments thereof ⁇ i.e., multiple copies of the same or different opioid receptor agonist proteins, variants, or fragments thereof).
  • the opioid receptor agonist protein portion of a fusion protein may comprise two or more tandem copies of an opioid receptor agonist protein, variant, or fragment thereof.
  • the opioid receptor agonist protein portion of the fusion proteins may comprise a chimera of two different opioid receptor agonist proteins.
  • the chimera may include the N-terminal portion of a first opioid receptor agonist protein ⁇ e.g., Dynorphin-A (1-10)) and a C-terminal portion of a second, different opioid receptor agonist protein ⁇ e.g., beta-Endorphin (11-31)).
  • the chimera may bind to an opioid receptor ⁇ e.g., mu-, kappa-, and/or delta) that is specific for the first opioid receptor agonist protein, the second opioid receptor agonist protein, or both.
  • the chimera may exhibit a different affinity (i.e., greater affinity or lesser affinity) for an opioid receptor, relative to the first opioid receptor agonist protein or the second opioid receptor agonist protein.
  • the opioid receptor agonist protein portion of the fusion proteins described herein may comprise multiple copies of the same opioid receptor agonist protein.
  • the opioid receptor agonist protein portion of the fusion proteins described herein may comprise multiple copies of enkephalin or an enkaphalin variant (e.g., multiple copies of [Leu 5 ]enkephalin or [Met 5 ] enkephalin such as are present in YGGFMYGGFMYGGFMYGGFM (SEQ ID NO: 12), [Met 5 ] enkephalin 4x).
  • the opioid receptor agonist protein portion of the fusion proteins described herein may comprise a variant of beta-Endorphin, such as: YGGFMTSEKSQTPLVTLFKNAIIKNAYQQGE (SEQ ID NO: 13, beta-Endorphin (K28Q/K28Q)); YGGFMTSEKSQTPLVTLFKNAIIKNAY (SEQ ID NO: 14, beta- Endorphin (1-27)); YGGFMTSEKSQTPLVTLFKNAII (SEQ ID NO: 15, beta- Endorphin (1-23); YGGFMTSEKSQTPLVTLF (SEQ ID NO: 16, beta-Endorphin (1- 18)); YGGFMTSEKSQTPLVTLFKNAIIKNAYKQGE (SEQ ID NO: 17, beta- Endorphin (K29Q)); and YGGFMTSEKSQTPLVTLFKNAIIKNAYQKGE (SEQ ID NO: 18, beta-End
  • the opioid receptor agonist protein portion of the fusion proteins described herein may comprise a variant of Dynorphin-A, such as: YGGFLRRIRPKLKWDNQYGGFLRRIRPKLKWDNQ (SEQ ID NO: 19, Dynorphin-A (2* WT)); YGGFLRQIRPKLKWDNQYGGFLRQIRPKLKWDNQ (SEQ ID NO:20, Dynorphin-A (2* R7Q));
  • YGGFLGRYGGFLGRYGGFLGRYGGFLGRYGGFLGR (SEQ ID NO:21, Dynorphin-A (1-7 R6Q (4x)); and YGGFLGRIRPKLKWDNQYGGFLGRIRPKLKWDNQ (SEQ ID NO:22, Dynorphin-A (2* R6G)).
  • the opioid receptor agonist protein portion of the fusion proteins described herein may comprise a variant of Dynorphin-B, such as: YGGFLRQQFKVVTYGGFLRQQFKVVT (SEQ ID NO:23, Dynorphin-B (2* RJQ)); and YGGFLRRQFKVVTRSQEDPNAYSGELFDA (SEQ ID NO:24,
  • the opioid receptor agonist protein portion of the fusion proteins described herein may comprise a variant of Prohanin such as:
  • NPFPTWRQRPGNPFPTWRQRPG SEQ ID NO:26, Prohanin (2* K8Q)
  • NPFPTRKRP SEQ ID NO:28, Prohanin having deletions of the tryptophan at position 6 and the glycine at position 11;
  • NPFPTWRKRP (SEQ ID NO:29, Prohanin having a deletion of the glycine at position 10); and NPTPTWKRKH (SEQ ID NO:30).
  • the opioid receptor agonist fusion proteins may be selected or modified to include or omit certain amino acids, either throughout or at selected positions.
  • the opioid receptor agonist fusion proteins may be selected or modified to omit a sequence of two basic amino acid residues in succession selected from the group consisting of KK, KR, RK, and RR.
  • the opioid receptor agonist portion of the fusion protein does not include or is modified not to include two basic amino acids residues in succession.
  • the fusion protein includes a linking sequence
  • the linking sequence does not include two basic amino acids residues in succession.
  • the opioid receptor agonist fusion proteins may include an opioid receptor agonist fused to the N-terminus, the C-terminus, or both the N-terminus and C- terminus of a serum protein (e.g., albumin, AFP, or an albumin- AFP hybrid).
  • a serum protein e.g., albumin, AFP, or an albumin- AFP hybrid.
  • the opioid receptor agonist may be inserted within an exposed loop of albumin or AFP.
  • an albumin-opioid receptor agonist fusion proteins may have a formula L-ALB-X n -ORA, where "L” is an optional N-terminal leader or signal sequence, “ALB” is amino acids 1-585 of mature human albumin or a fragment thereof, "X n " is an optional linking sequence where "X” is an amino acid and "n” is 0-
  • ORA is an opioid receptor agonist
  • An AFP-opioid receptor agonist fusion protein may include AFP fused to the
  • the AFP-opioid receptor agonist fusion proteins may have a formula L-AFP-X n -ORA, where "L” is an optional N-terminal leader or signal sequence, "AFP” is amino acids 1-609 of alpha- fetoprotein or a fragment thereof, "X n " is an optional linking sequence where "X” is an amino acid and "n” is 0-20, and "ORA" is an opioid receptor agonist.
  • the opioid receptor agonist fusion proteins may include an albumin- AFP hybrid protein fused to the N-terminus, the C-terminus, or both the N-terminus and C- terminus of the opioid receptor agonist.
  • the opioid receptor agonist fusion proteins may have a formula L-HYB-X n -ORA, where "L” is an optional N- terminal leader or signal sequence, "HYB” is an albumin-AFP hybrid comprising at least a fragment of albumin and alpha- fetoprotein, "X n " is an optional linking sequence where "X” is an amino acid and "n” is 0-20, and "ORA" is an opioid receptor agonist.
  • the albumin-AFP hybrid may include about 1-50 amino acids of albumin fused to AFP, an AFP fragment, an AFP variant or an AFP derivative.
  • N-terminal leader or signal sequences include albumin leader sequence, MPIF-I signal sequence, stanniocalcin signal sequence, invertase signal sequence, yeast mating factor alpha signal sequence, K. lactis killer toxin leader sequence, immunoglobulin Ig signal sequence, Fibulin B precursor signal sequence, clusterin precursor signal sequence, insulin-like growth factor-binding protein 4 signal sequence, acid phosphatase (PHO5) leader, pre-sequence of MFoz-1, pre-sequence of 0 glucanase (BGL2), S.
  • albumin leader sequence MPIF-I signal sequence
  • stanniocalcin signal sequence invertase signal sequence
  • yeast mating factor alpha signal sequence K. lactis killer toxin leader sequence
  • immunoglobulin Ig signal sequence Fibulin B precursor signal sequence
  • S. carlsbergensis ⁇ -galactosidase (MELl) secretion leader sequence S. carlsbergensis ⁇ -galactosidase (MELl) secretion leader sequence
  • MELl S. carlsbergensis ⁇ -galactosidase
  • Candida glucoarnylase leader sequence Candida glucoarnylase leader sequence
  • gp67 signal sequence for use in a baculovirus expression system and S. cerevisiae invertase (SUC2) leader.
  • the invention also encompasses pharmaceutical formulations comprising an opioid receptor agonist fusion protein of the invention and a pharmaceutically acceptable diluent or carrier.
  • Such formulations may be in a kit or container.
  • kit or container may be packaged with instructions pertaining to the extended shelf life of the opioid receptor agonist fusion protein.
  • Such formulations may be used in methods of treating, preventing, ameliorating or diagnosing a disease or disease symptom in a patient, preferably a mammal, most preferably a human, comprising the step of administering the pharmaceutical formulation to the patient.
  • the pharmaceutical formulation may be administered in any suitable manner (e.g., directly to the bloodstream of the patient by injection).
  • the pharmaceutical compositions may be administered to any patient in need thereof including human patients and non-human patients (e.g., dogs, cats, horses).
  • the patient may be experiencing pain or may be at risk for developing pain.
  • the patient may be undergoing or recovering from surgery.
  • the patient may have a disease or disorder associated with inflammatory pain (e.g., cancer or arthritis).
  • the pharmaceutical formulation may be administered to achieve an analgesic effect or an antinociceptive effect.
  • the opioid receptor agonist fusion protein alleviates pain and does not cause undesirable side effects.
  • the opioid receptor agonist fusion protein may have one or more of the following characteristics: may not be addicting, may not cause tolerance, may have fewer side effects than morphine (e.g. , nausea, cardiac depressions, and drowsiness), may have a longer half- life than the unfused opioid receptor agonist fusion protein, and may not effectively traverse the blood-brain barrier.
  • the fusion protein may exhibit extended activity in vivo or in vitro and may have a molecular weight substantially greater than 500 daltons.
  • the opioid receptor agonist fusion proteins may not bind efficiently to and/or may not be transported effectively by transport systems at the blood-brain barrier such as peptide transport systems (e.g., PTS-I) and/or P- glycoprotein.
  • opioid receptor agonist fusion proteins as disclosed herein may have an affinity (Kd) for the PTS which is lower than the affinity of the opioid receptor agonist peptide portion of the fusion protein for the PTS.
  • the affinity of the fusion protein for the PTS may be no more than about 50% of the affinity of the opioid receptor agonist peptide portion of the fusion protein for the PTS (preferably no more than about 20%, 10%, 5%, 1%, or 0.1%).
  • opioid receptor agonist fusion proteins as described herein are not transported as efficiently across the blood-brain barrier in comparison to the opioid receptor agonist peptide portion of the fusion proteins.
  • the fusion proteins may be transported across the blood-brain barrier no more than 50% as efficiently as the opioid receptor agonist peptide portion of the fusion proteins (preferably no more than about 20%, 10%, 5%, 1%, or 0.1%).
  • the pharmaceutical compositions may be administered to treat or prevent pain or any disease or disorder for which activation of opioid receptors is beneficial.
  • the pharmaceutical compositions may be administered to treat or prevent pain or any disease or disorder for which activation of peripheral opioid receptors is beneficial, and optionally, for which activation of opioid receptors of the brain is undesirable.
  • compositions typically include fusion proteins as disclosed herein as an active agent and may include an additional active agent.
  • additional active agents may include additional analgesics and/or antinociceptives.
  • Additional active agents may include agents for treating side effects of opioid receptor agonists such as nausea, cardiac depressions, and/or drowsiness.
  • FIGS. IA-D shows the amino acid sequence of the mature form of human albumin (amino acids 1-585 or amino acids 25-609 of proalbumin) and a polynucleotide encoding it.
  • FIG. 2 shows the restriction map of the pPPC0005 cloning vector ATCC deposit PTA-3278.
  • FIG. 3 shows the restriction map of the pSAC35 yeast S. cerevisiae expression vector (Sleep et al., BioTechnology 8:42 (1990)).
  • FIG. 4 shows the nucleotide sequence of human alpha- fetoprotein (GenBank
  • FIG. 5 shows the amino acid sequence of human alpha-fetoprotein.
  • FIG. 6 shows the annotated amino acid and nucleotide sequences of the engineered SPCON2.AFP ORF.
  • FIG. 7 shows the full ORF (nucleotide sequence) with silent restriction sites for the engineered SPcon2.AFP full ORF.
  • FIG. 8 shows the amino acid sequence of SPcon2.AFP with the signal peptide underlined.
  • FIG. 9 shows the amino acid sequence of SMIIIa-HSA.
  • FIG. 10 shows the amino acid sequence of HSA.contulakin-G.
  • FIG. 11 shows the amino acid sequence of contulakin-G.HSA.
  • FIG. 12 shows four graphs illustrating the effect of forskolin and either ⁇ - endorphin peptide, or enkephalin-HSA fusion peptide on cAMP activiation.
  • the first three graphs illustrate the effect in HEK293 cells over expressing kappa-, delta- or mu opioid receptors.
  • the fourth graph illustrates activation in the SK-N-SH neuroblastoma cell line. In this experiment the enkephalin peptide fused to HSA was
  • YGGFMYGGFMYGGFMYGGFM (4X SEQ ID NO: 2).
  • the grey circles and lines represent ⁇ -endorphin peptide; the black circles and lines represent enkephalin.HSA.
  • FIG. 13 shows two graphs illustrating the effect of different opioid peptides on cAMP activation using HEK 293 cells over expressing the human opioid delta 1 receptor.
  • the first graph illustrates the effect of the enkaphalin peptide YGGFM
  • EC 50 values are nanomolar.
  • FIG. 14 shows two bar graphs illustrating the results of hot plate testing on mice injected subcutaneously with the designated peptide, or with the vehicle control.
  • FIG. 15 shows two bar graphs illustrating the results of tail flick testing in mice injected subcutaneously with the designated peptide or with the vehicle control.
  • FIG. 16 shows two bar graphs illustrating the results of hot plate testing (top graph) and tail flick testing (bottom graph) on mice injected subcutaneously with the designated peptide, oxycodone, or with the vehicle control. Testing was performed 60 minutes after injection.
  • polynucleotide refers to a nucleic acid molecule having a nucleotide sequence encoding a protein.
  • a polynucleotide may encode fusion protein comprising, or alternatively consisting of, at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one therapeutic protein having opioid receptor agonist activity.
  • albumin fusion construct refers to a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least one molecule of a therapeutic protein having opioid receptor agonist activity; a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least one molecule of a therapeutic protein having opioid receptor agonist activity (or fragment or variant thereof) generated as described in the Examples; or a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one polynucleot
  • the polynucleotide encoding the therapeutic protein having opioid receptor agonist activity and albumin protein, once part of the albumin fusion construct, may each be referred to as a "portion,” “region” or “moiety” of the albumin fusion construct.
  • the present invention relates generally to polynucleotides encoding albumin fusion proteins; albumin fusion proteins; and methods of treating, preventing, or ameliorating diseases or disorders using albumin fusion proteins or polynucleotides encoding albumin fusion proteins.
  • albumin fusion protein refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a therapeutic protein having opioid receptor agonist activity (or fragment or variant thereof).
  • An albumin fusion protein of the invention comprises at least a fragment or variant of a therapeutic protein having opioid receptor agonist activity and at least a fragment or variant of human serum albumin, which are associated with one another by genetic fusion (i.e., the albumin fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a therapeutic protein having opioid receptor agonist activity is joined in- frame with a polynucleotide encoding all or a portion of albumin).
  • an albumin fusion protein of the invention comprises at least one molecule of a therapeutic protein having opioid receptor agonist activity or fragment or variant of thereof (including, but not limited to a mature form of the therapeutic protein having opioid receptor agonist activity) and at least one molecule of albumin or fragment or variant thereof (including but not limited to a mature form of albumin).
  • AFP fusion construct refers to a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of alpha-fetoprotein (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least one molecule of a therapeutic protein having opioid receptor agonist activity; a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of AFP (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least one molecule of a therapeutic protein having opioid receptor agonist activity (or fragment or variant thereof) generated as described in the Examples; or a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of AFP (or a fragment or variant thereof) joined in frame to at least one polyn
  • the polynucleotide encoding the therapeutic protein having opioid receptor agonist activity and AFP protein, once part of the AFP fusion construct, may each be referred to as a "portion,” “region” or “moiety” of the AFP fusion construct.
  • the present invention relates generally to polynucleotides encoding AFP fusion proteins; AFP fusion proteins; and methods of treating, preventing, or ameliorating diseases or disorders using AFP fusion proteins or polynucleotides encoding AFP fusion proteins.
  • AFP fusion protein refers to a protein formed by the fusion of at least one molecule of AFP (or a fragment or variant thereof) to at least one molecule of a therapeutic protein having opioid receptor agonist activity (or fragment or variant thereof).
  • An AFP fusion protein of the invention comprises at least a fragment or variant of a therapeutic protein having opioid receptor agonist activity and at least a fragment or variant of human serum AFP, which are associated with one another by genetic fusion (i.e., the AFP fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a therapeutic protein having opioid receptor agonist activity is joined in-frame with a polynucleotide encoding all or a portion of AFP).
  • an AFP fusion protein of the invention comprises at least one molecule of a therapeutic protein having opioid receptor agonist activity or fragment or variant of thereof (including, but not limited to a mature form of the therapeutic protein having opioid receptor agonist activity) and at least one molecule of AFP or fragment or variant thereof (including but not limited to a mature form of AFP).
  • an fusion protein of the invention is processed by a host cell and secreted into the surrounding culture medium. Processing of the nascent fusion protein that occurs in the secretory pathways of the host used for expression may include, but is not limited to signal peptide cleavage; formation of disulfide bonds; proper folding; addition and processing of carbohydrates (such as for example, N- and O-linked glycosylation); specific proteolytic cleavages; and assembly into multimeric proteins.
  • a fusion protein of the invention is preferably in the processed form.
  • the "processed form of a fusion protein" refers to a fusion protein product which has undergone N-terminal signal peptide cleavage, herein also referred to as a "mature fusion protein".
  • the invention provides a fusion protein comprising, or alternatively consisting of, a therapeutic protein having opioid receptor agonist activity, and a biologically active and/or therapeutically active fragment of serum a serum protein (e.g., albumin, AFP or an albumin- AFP hybrid).
  • a fusion protein comprising, or alternatively consisting of, a therapeutic protein having opioid receptor agonist activity (e.g., either the prohormone or mature form) and a biologically active and/or therapeutically active variant of a serum protein (e.g., albumin, AFP, or an albumin- AFP hybrid).
  • the therapeutic protein having opioid receptor agonist activity portion of the fusion protein is the mature portion of the therapeutic protein having opioid receptor agonist activity.
  • the therapeutic protein having opioid receptor agonist activity portion of the fusion protein is the extracellular soluble domain of the therapeutic protein having opioid receptor agonist activity.
  • the therapeutic protein having opioid receptor agonist activity portion of the fusion protein is the active form of the therapeutic protein having opioid receptor agonist activity.
  • the invention further encompasses polynucleotides encoding these fusion proteins.
  • the invention provides an fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment or variant of a therapeutic protein having opioid receptor agonist activity and a biologically active and/or therapeutically active fragment or variant of a serum protein (e.g., albumin, AFP, or an albumin-AFP hybrid).
  • a serum protein e.g., albumin, AFP, or an albumin-AFP hybrid.
  • the invention provides a fusion protein comprising, or alternatively consisting of, the mature portion of a therapeutic protein having opioid receptor agonist activity and the mature portion of serum albumin or AFP.
  • the invention further encompasses polynucleotides encoding these fusion proteins.
  • opioid receptor agonist fusion proteins disclosed herein may not bind efficiently to and/or may not be transported effectively by transport systems at the blood-brain barrier such as peptide transport systems (e.g., PTS-I) and/or P- glycoprotein.
  • opioid receptor agonist fusion proteins as disclosed herein may have an affinity (Kd) for the PTS which is lower than the affinity of the opioid receptor agonist peptide portion of the fusion protein for the PTS.
  • opioid receptor agonist fusion proteins as described herein may not be transported as efficiently across the blood-brain barrier in comparison to the opioid receptor agonist peptide portion of the fusion proteins.
  • Methods for measuring binding at and/or transport across the blood-brain barrier are disclosed herein and known in the art and may include perfusion systems for determining permeability coefficients (see Murakami et ah, "Comparison of blood-brain barrier permeability in mice and rats using in situ brain perfusion technique," AM J. PHYSIOL. HEART CIRC. PHYSIOL. 2000 279:H1022-1028), and intravenous injection techniques (see Spranger et ah, "Adiponectin does not cross the blood-brain barrier but modifies cytokine expression of brain endothelial cells," DIABETES 2006 55:141-147). [0064] Therapeutic Proteins
  • a polynucleotide of the invention encodes a protein comprising or alternatively consisting of, at least a fragment or variant of a therapeutic protein and at least a fragment or variant of a serum protein ⁇ e.g. , human serum albumin, human AFP, or a hybrid of human serum albumin and human AFP), which are associated with one another, preferably by genetic fusion.
  • the preferred therapeutic protein is a opioid receptor agonist protein, which may include wild-type opioid receptor agonist proteins or variants or fragments thereof having opioid receptor agonist protein activity.
  • a therapeutic protein such as an opioid receptor agonist protein may comprises a chimeric protein.
  • a "chimeric protein” includes the N-terminal sequence of a first protein and a C-terminal sequence of a second protein.
  • the chimeric protein includes at least 10 amino acids of a N-terminal sequence of a first protein and at least 10 amino acids of a C-terminal sequence of a second protein.
  • the N-terminal sequence and the C-terminal sequence may be fused directly, or indirectly via a linking sequence.
  • therapeutic protein having opioid receptor agonist activity refers to an opioid receptor agonist polypeptide, or fragment or variant thereof, or an opioid receptor agonist analog. Other natural sequences may include modifications (e.g., one or more amino acid substitutions, additions or deletions).
  • a "therapeutic protein having opioid receptor agonist activity” may include a human or non-human opioid receptor agonist polypeptide (e.g. , from snake, frog, monkey, dog, cat, or horse), or fragment or variant thereof, or an opioid receptor agonist analog (e.g., from snake, frog, monkey, dog, cat, or horse).
  • the terms peptides, proteins, and polypeptides are used interchangeably.
  • a protein having opioid receptor agonist activity encompasses fragments, variants, and analogs of the opioid receptor agonist.
  • a protein of the invention may contain at least a fragment, variant, or an analog of an opioid receptor agonist.
  • a polypeptide displaying a "therapeutic activity” or a protein that is “therapeutically active” is meant a polypeptide that possesses one or more known biological and/or therapeutic activities associated with a therapeutic protein such as one or more of the opioid receptor agonists described herein or otherwise known in the art.
  • a "therapeutic opioid receptor agonist fusion protein” is a protein that is useful to treat, prevent or ameliorate a disease, condition or disorder associated with pain.
  • a "therapeutic opioid receptor agonist fusion protein” may be one that binds specifically to an opioid receptor on a cell (e.g., a nerve cell) and stimulates a G-protein response (e.g., G 0 or G 1 ) that may result in an one or more of the following: a decrease or increase in the concentration of cAMP; a decrease or increase in calcium channel activity; and a decrease or increase in potassium channel activity.
  • a "therapeutic opioid receptor agonist fusion protein” may be one that binds specifically to an opioid receptor and stimulates an antinociceptive and/or analgesic response.
  • therapeutic activity may refer to an activity whose effect is consistent with a desirable therapeutic outcome in humans, or to desired effects in non-human mammals or in other species or organisms.
  • Therapeutic activity may be measured in vivo or in vitro. For example, a desirable effect may be assayed in cell culture.
  • an opioid receptor agonist or analog is the therapeutic protein
  • the effects of the opioid receptor agonist on G-protein activity as described in the Examples may be used as the endpoint for which therapeutic activity is measured.
  • Such in vitro or cell culture assays are commonly available for G-protein coupled receptors.
  • a therapeutic effect may include an analgesic or antinociceptive effect which may be assessed in animals by tests known in the art (e.g., the writhing test, the hot-plate test, and tail-flick test).
  • tests known in the art e.g., the writhing test, the hot-plate test, and tail-flick test.
  • Vectors for expressing proteins are known in the art, and are available commercially or described elsewhere.
  • an "expression cassette" comprising, or alternatively consisting of, one or more of (1) a polynucleotide encoding a given fusion protein, (2) a leader sequence, (3) a promoter region, and (4) a transcriptional terminator, may be assembled in a convenient cloning vector and subsequently be moved into an alternative vector, such as, for example, an expression vector including, for example, a yeast expression vector or a mammalian expression vector.
  • an expression cassette comprising, or alternatively consisting of, a nucleic acid molecule encoding a fusion protein is cloned into pSAC35.
  • an expression cassette comprising, or alternatively consisting of, a nucleic acid molecule encoding a fusion protein is cloned into pC4.
  • a polynucleotide comprising or alternatively consisting of a nucleic acid molecule encoding the therapeutic protein portion of a fusion protein is cloned into pC4:HSA.
  • an expression cassette comprising, or alternatively consisting of, a nucleic acid molecule encoding a fusion protein is cloned into pEE12.
  • an "expression cassette" comprising, or alternatively consisting of one or more of (1) a polynucleotide encoding a given fusion protein, (2) a leader sequence, (3) a promoter region, and (4) a transcriptional terminator can be moved or "subcloned" from one vector into another. Fragments to be subcloned may be generated by methods well known in the art, such as, for example, PCR amplification and/or restriction enzyme digestion.
  • the opioid receptor agonist fusion proteins of the invention are capable of a therapeutic activity and/or biologic activity corresponding to the opioid receptor agonist activity of the opioid receptor agonist portion of the fusion protein.
  • the fusion proteins of the invention may comprise one or more serum proteins of a non-human animal species, fused in tandem and in-frame either at the N-terminus or the C -terminus to one or more opioid receptor agonist proteins of the same or different non-human animal species.
  • Non-human serum albumin, non-human serum AFP, and non-human opioid receptor agonist proteins are well known in the art and available in public databases.
  • an albumin fusion protein of the invention comprises one or more Bos taurus serum albumin proteins, fused in tandem and in-frame either at the N-terminus or the C- terminus to one or more opioid receptor agonist proteins.
  • Fusion proteins comprising fragments or variants of non-human serum albumin, such as, for example, the mature form of serum albumin, are also encompassed by the invention. Fusion proteins comprising fragments, variants, or analogs of non-human opioid receptor agonist proteins are also encompassed by the invention. Preferably, the fragment, variant or analog retains opioid receptor agonist activity (i.e., binds and activates the opioid receptor).
  • Polynucleotides of the invention comprise, or alternatively consist of, one or more nucleic acid molecules encoding a human or non-human fusion protein described above.
  • non-human fusion proteins are encompassed by the invention, as are host cells and vectors containing these polynucleotides.
  • a non-human fusion protein encoded by a polynucleotide as described above has an extended shelf life.
  • a non-human fusion protein encoded by a polynucleotide described above has a longer activity and/or stabilized activity in solution (or in a pharmaceutical composition) in vitro and/or in vivo than the corresponding unfused therapeutic molecule.
  • the fused opioid receptor agonist protein may exhibit a longer shelf-life related to a non- fused protein opioid receptor agonist protein.
  • Non-human serum albumin may include:
  • Bos taurus ABBOS CAA76847, P02769, CAA41735, 229552, AAF28806,
  • the present invention is further directed to fragments of the therapeutic proteins (e.g., opioid receptor agonists, variants, or analogs thereof), albumin, and AFP.
  • the present invention is also directed to polynucleotides encoding fragments of the therapeutic proteins (e.g., opioid receptor agonists, variants, or analogs thereof), albumin, and AFP.
  • opioid receptor agonists, variants, or analogs thereof corresponding to a therapeutic protein portion of a fusion protein of the invention, include the full length protein as well as polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the reference polypeptide.
  • fragments of serum albumin polypeptides corresponding to an albumin protein portion of an albumin fusion protein of the invention include the full length protein as well as polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the reference polypeptide.
  • fragments of albumin fusion proteins of the invention include the full length albumin fusion protein as well as polypeptides having one or more residues deleted from the amino terminus of the albumin fusion protein.
  • fragments of serum AFP polypeptides corresponding to an AFP portion of an AFP fusion protein of the invention include the full length protein as well as polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the reference polypeptide.
  • a reference polypeptide e.g., a therapeutic protein; serum albumin protein; or serum AFP protein
  • other functional activities e.g., biological activities, ability to multimerize, ability to bind a ligand
  • therapeutic activities e.g., biological activities, ability to multimerize, ability to bind a ligand
  • therapeutic activities e.g., biological activities, ability to multimerize, ability to bind a ligand
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of a therapeutic protein (e.g., opioid receptor agonists, variants, or analogs thereof) corresponding to a therapeutic protein portion of fusion protein of the invention.
  • a therapeutic protein e.g., opioid receptor agonists, variants, or analogs thereof
  • the present invention provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of an albumin protein corresponding to an albumin protein portion of an albumin fusion protein of the invention.
  • the present invention provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of an AFP protein corresponding to an AFP protein portion of an AFP fusion protein of the invention.
  • any of the above described N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted reference polypeptide.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the present application is also directed to variants proteins containing polypeptides having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to a reference polypeptide sequence (e.g., an opioid receptor agonist, or a therapeutic protein portion of an opioid receptor agonist fusion protein of the invention, or an albumin protein portion of an albumin-opioid receptor agonist fusion protein of the invention, or an AFP protein portion of an AFP-opioid receptor agonist fusion proteion of the invention) as set forth herein, or fragments thereof.
  • a reference polypeptide sequence e.g., an opioid receptor agonist, or a therapeutic protein portion of an opioid receptor agonist fusion protein of the invention, or an albumin protein portion of an albumin-opioid receptor agonist fusion protein of the invention, or an AFP protein portion of an AFP-opioid receptor agonist fusion proteion of the invention
  • the application is directed to variant proteins comprising polypeptides having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to a reference polypeptide having the amino acid sequence of N- and C-terminal deletions as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention. Variant proteins preferably have the biological function of the reference polypeptide (e.g., opioid receptor agonist activity).
  • Preferred polypeptide fragments of the invention are fragments comprising, or alternatively, consisting of, an amino acid sequence that displays a therapeutic activity and/or functional activity (e.g., biological activity) of the polypeptide sequence of the therapeutic protein or serum protein (e.g., serum albumin or serum AFP) of which the amino acid sequence is a fragment.
  • a preferred fragment of an opioid receptor agonist has opioid receptor binding activity.
  • a preferred fragment of albumin or AFP may function to increase the half- life of a therapeutic protein when fused to the therapeutic protein.
  • Other preferred polypeptide fragments are biologically active fragments.
  • Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention.
  • the biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
  • an albumin fragment or variant will be at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 310, at least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440, at least about 450, at least about 460, at least about 470, at least about 480, at least about 490, at least about 500, at least about 510, at least
  • the albumin portion of a fusion protein comprises at least half of the full length or mature albumin protein.
  • such fragments may be of about 10 or more amino acids in length or may include about 15, about 20, about 25, about 30, about 50, about 70, about 90, about 110, about 130, about 150, about 170, about 190, about 210, about 230, about 250, about 270, about 290, about 310, about 330, about 350, about 370, about 390, about 410, about 430, about 450, about 470, about 490, about 510, about 530, about 550, about 570, or about 585 contiguous amino acids from the full-length or mature sequence or may include part or all of specific domains of albumin.
  • an alpha-fetoprotein fragment or variant will be at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 310, at least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440, at least about 450, at least about 460, at least about 470, at least about 480, at least about 490, at least about 500, at least about
  • the AFP portion of a fusion protein comprises at least half of the full length AFP protein.
  • such fragments may be of about 10 or more amino acids in length or may include about 15, about 20, about 25, about 30, about 50, about 70, about 90, about 110, about 130, about 150, about 170, about 190, about 210, about 230, about 250, about 270, about 290, about 310, about 330, about 350, about 370, about 390, about 410, about 430, about 450, about 470, about 490, about 510, about 530, about 550, about 570, about 590, about 600, about 605 or more contiguous amino acids from the alpha-fetoprotein sequence or may include part or all of specific domains of alpha-fetoprotein.
  • Variants refers to a polynucleotide or nucleic acid differing from a reference nucleic acid or polypeptide, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the reference nucleic acid or polypeptide.
  • variant refers to a therapeutic protein portion of a fusion protein of the invention, an albumin portion of an albumin-fusion protein of the invention, or an AFP portion of an AFP-fusion protein, which differ in sequence from a therapeutic protein, albumin protein, or AFP, respectively, but retaining at least one functional and/or therapeutic property thereof as described elsewhere herein or otherwise known in the art.
  • a preferred variant of an opioid receptor agonist has opioid receptor binding activity.
  • variants are overall very similar, and, in many regions, identical to the amino acid sequence of the therapeutic protein corresponding to a therapeutic protein portion, an albumin protein portion, or an AFP portion. Nucleic acids encoding these variants are also encompassed by the invention.
  • the present invention is also directed to proteins which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, the amino acid sequence of a therapeutic protein corresponding to a therapeutic protein portion of a fusion protein of the invention; or the amino acid sequence of a therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide fusion construct, or fragments or variants thereof); albumin proteins corresponding to an albumin protein portion of an albumin fusion protein of the invention (e.g., the amino acid sequence of an albumin protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct; the amino acid sequence shown in Figures IA-D; or fragments or variants thereof); AFP corresponding to AFP portion of an AFP fusion protein of the invention (e.g., the amino acid sequence of an AFP portion of an AFP fusion protein encoded
  • polypeptides encompassed by the invention are polypeptides encoded by polynucleotides which hybridize to the complement of a nucleic acid molecule encoding a fusion protein of the invention under stringent hybridization conditions (e.g., hybridization to filter bound DNA in 6 ⁇ Sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2 ⁇ SSC, 0.1% SDS at about 50-65 degrees Celsius), under highly stringent conditions (e.g., hybridization to filter bound DNA in 6 ⁇ sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.1 ⁇ SSC, 0.2% SDS at about 68 degrees Celsius), or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F.
  • stringent hybridization conditions e.g., hybridization to filter bound DNA in 6 ⁇ Sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius
  • a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
  • the amino acid sequence of the subject polypeptide may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
  • up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid.
  • These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of a fusion protein of the invention or a fragment thereof (such as a therapeutic protein portion of the fusion protein, an albumin portion of an albumin fusion protein, or an AFP protion of an AFP fusion protein), can be determined conventionally using known computer programs.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.
  • the query and subject sequences are either both nucleotide sequences or both amino acid sequences.
  • the result of said global sequence alignment is expressed as percent identity.
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment.
  • This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
  • This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.
  • a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity.
  • the deletion occurs at the N- terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
  • a 90 residue subject sequence is compared with a 100 residue query sequence.
  • the variant will usually have at least 75% (preferably at least about 80%, 90%, 95% or 99%) sequence identity with a length of normal HA or therapeutic protein which is the same length as the variant.
  • BLAST Basic Local Alignment Search Tool
  • blastp, blastn, blastx, tblastn and tblastx Karlin et al, Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and Altschul, J. MoI. Evol. 36: 290-300 (1993), fully incorporated by reference
  • the approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance.
  • the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively.
  • M i.e., the reward score for a pair of matching residues
  • N i.e., the penalty score for mismatching residues
  • Q IO (gap creation penalty)
  • R IO (gap extension penalty)
  • the polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred.
  • polypeptide variants in which less than 50, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred.
  • Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host, such as, yeast or E. coli).
  • a polynucleotide of the invention encoding a fusion protein may be optimized for expression in a host cell (e.g., a yeast cell, a bacterial cell, or a mammalian cell).
  • a host cell e.g., a yeast cell, a bacterial cell, or a mammalian cell.
  • a polynucleotide of the invention which encodes the therapeutic protein portion of a fusion protein may be optimized for expression in yeast or mammalian cells.
  • the polynucleotide of the invention which encodes an albumin portion of an albumin fusion protein may be optimized for expression in yeast or mammalian cells.
  • the polynucleotide of the invention which encodes an AFP portion of an AFP fusion protein may be optimized for expression in yeast or mammalian cells.
  • a codon-optimized polynucleotide which encodes a therapeutic protein portion of a fusion protein does not hybridize to the wild type polynucleotide encoding the therapeutic protein under stringent hybridization conditions as described herein.
  • a codon-optimized polynucleotide which encodes an albumin portion of an albumin fusion protein does not hybridize to the wild type polynucleotide encoding the albumin protein under stringent hybridization conditions as described herein.
  • a codon-optimized polynucleotide which encodes an AFP fusion protein does not hybridize to the wild type polynucleotide encoding the AFP protein portion under stringent hybridization conditions as described herein.
  • a polynucleotide which encodes a therapeutic protein portion of a fusion protein does not comprise, or alternatively consist of, the naturally occurring sequence of that therapeutic protein.
  • a polynucleotide which encodes an albumin protein portion of an albumin fusion protein does not comprise, or alternatively consist of, the naturally occurring sequence of albumin protein.
  • a polynucleotide which encodes an AFP fusion protein does not comprise, or alternatively consist of, the naturally occurring sequence of AFP.
  • Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis. [0118] Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention.
  • one or more amino acids can be deleted from the N-terminus or C-terminus of the polypeptide of the present invention without substantial loss of biological function.
  • Ron et al. J. Biol. Chem. 268: 2984-2988 (1993)
  • J. Biol. Chem. 268: 2984-2988 (1993) reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues.
  • Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)
  • the invention further includes polypeptide variants which have a functional activity (e.g., biological activity and/or therapeutic activity).
  • the invention provides variants of fusion proteins that have a functional activity (e.g., biological activity and/or therapeutic activity) that corresponds to one or more biological and/or therapeutic activities of the therapeutic protein corresponding to the therapeutic protein portion of the fusion protein.
  • Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity. Polynucleotides encoding such variants are also encompassed by the invention.
  • the variants of the invention have conservative substitutions.
  • substitutions is intended swaps within groups such as replacement of the aliphatic or hydrophobic amino acids Ala, VaI, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and GIu; replacement of the amide residues Asn and GIn, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and GIy.
  • the first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
  • the second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. See Cunningham and Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity. [0126] As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein.
  • variants of the present invention include (i) polypeptides containing substitutions of one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) polypeptides containing substitutions of one or more of the amino acid residues having a substituent group, or (iii) polypeptides which have been fused with or chemically conjugated to another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), (iv) polypeptide containing additional amino acids, such as, for example, an IgG Fc fusion region peptide.
  • polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).
  • the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of the amino acid sequence of a therapeutic protein and/or human serum albumin and/or AFP, wherein the fragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, amino acid residue additions, substitutions, and/or deletions when compared to a reference polypeptide sequence.
  • the amino acid substitutions are conservative.
  • Nucleic acids encoding these polypeptides are also encompassed by the invention.
  • the fragments or variants have one or more functional activities of the reference polypeptide.
  • the polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
  • the polypeptides may be modified by either natural processes, such as post- translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • the amino acids of the fusion proteins described herein may include one or more naturally occurring or non-naturally occurring amino acids.
  • the fusion proteins may include D-amino acids.
  • a polypeptide having functional activity refers to a polypeptide capable of displaying one or more known functional activities associated with the full-length, pro-protein, and/or mature form of a therapeutic protein.
  • Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide for binding) to an anti -polypeptide antibody], immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.
  • a polypeptide having biological activity refers to a polypeptide exhibiting activity similar to, but not necessarily identical to, an activity of a therapeutic protein of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25 -fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).
  • a fusion protein of the invention has at least one biological and/or therapeutic activity associated with the therapeutic protein portion (or fragment or variant thereof) when it is not fused to a serum polypeptide (e.g., albumin or AFP).
  • a serum polypeptide e.g., albumin or AFP
  • the fusion proteins of the invention can be assayed for functional activity (e.g., biological activity) using or routinely modifying assays known in the art, as well as assays described herein. Additionally, one of skill in the art may routinely assay fragments of a therapeutic protein corresponding to a therapeutic protein portion of a fusion protein. Further, one of skill in the art may routinely assay fragments of an albumin protein corresponding to an albumin protein portion of an albumin fusion protein, for activity using assays known in the art and/or as described in the Examples section below.
  • binding partner e.g., a receptor or a ligand
  • binding to that binding partner by a fusion protein which comprises that therapeutic protein as the therapeutic protein portion of the fusion can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., Microbiol. Rev.
  • the ability of physiological correlates of a fusion protein to bind to a substrate(s) of the therapeutic polypeptide corresponding to the therapeutic protein portion of the fusion can be routinely assayed using techniques known in the art.
  • association with other components of the multimer can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., supra.
  • the binding affinity of a fusion protein to a receptor for the therapeutic protein and the off-rate of a fusion protein-receptor interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled fusion protein (e.g., H or 125 I) with a receptor for the therapeutic protein in the presence of increasing amounts of unlabeled fusion protein, and the detection of the bound fusion protein to the receptor.
  • labeled fusion protein e.g., H or 125 I
  • the affinity of the fusion protein for a specific receptor and the binding off- rates can be determined from the data by Scatchard plot analysis.
  • Competition with a second protein that binds the same receptor as the fusion protein can also be determined using radioimmunoassays.
  • the protein, antigen or epitope is incubated with a fusion protein conjugated to a labeled compound (e.g., . 3 H or 125 I) in the presence of increasing amounts of an unlabeled second protein that binds the same protein, antigen, or epitope as the fusion protein of the invention.
  • the second protein may include an unlabeled opiate receptor agonist.
  • BIAcore kinetic analysis is used to determine the binding on and off rates of fusion proteins of the invention to a protein, antigen or epitope.
  • BIAcore kinetic analysis comprises analyzing the binding and dissociation of fusion proteins, or specific polypeptides, antigens or epitopes from chips with immobilized specific polypeptides, antigens or epitopes or fusion proteins, respectively, on their surface.
  • an albumin fusion protein of the invention comprises at least a fragment or variant of a therapeutic protein and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion.
  • An additional embodiment comprises at least a fragment or variant of a therapeutic protein and at least a fragment or variant of human serum albumin, which are linked to one another by chemical conjugation.
  • HSA human serum albumin
  • HA human albumin
  • albumin and HA are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).
  • albumin refers collectively to albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin.
  • albumin refers to human albumin or fragments thereof (see for example, EP 201 239, EP 322 094 WO
  • FIG. IA-D or albumin from other vertebrates or fragments thereof, or analogs or variants of these molecules or fragments thereof.
  • the human serum albumin protein used in the albumin fusion proteins of the invention contains one or both of the following sets of point mutations with reference to the sequence of albumin (FIG. IA-D): Leu-407 to Ala, Leu-408 to VaI, Val-409 to Ala, and Arg-410 to Ala; or Arg-410 to A, Lys-413 to GIn, and Lys-414 to GIn (see, e.g., International Publication No. WO95/23857, hereby incorporated in its entirety by reference herein).
  • albumin fusion proteins of the invention that contain one or both of above-described sets of point mutations have improved stability/resistance to yeast Yap3p proteolytic cleavage, allowing increased production of recombinant albumin fusion proteins expressed in yeast host cells.
  • a portion of albumin sufficient to prolong the therapeutic activity or shelf-life of the therapeutic protein refers to a portion of albumin sufficient in length or structure to stabilize or prolong the therapeutic activity of the protein so that the shelf life of the therapeutic protein portion of the albumin fusion protein is prolonged or extended compared to the shelf-life in the non-fusion state.
  • the albumin portion of the albumin fusion proteins may comprise the full length of the HA sequence as described above, or may include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity. Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the HA sequence or may include part or all of specific domains of HA. For instance, one or more fragments of HA spanning the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HA fragment is the mature form of HA.
  • the albumin portion of the albumin fusion proteins of the invention may be a variant of normal HA.
  • the therapeutic protein portion of the albumin fusion proteins of the invention may also be variants of the therapeutic proteins as described herein.
  • variants includes insertions, deletions and substitutions, either conservative or non conservative, where such changes do not substantially alter one or more of the oncotic, useful ligand-binding and non-immunogenic properties of albumin, or the active site, or active domain which confers the therapeutic activities of the therapeutic proteins.
  • the albumin fusion proteins of the invention may include naturally occurring polymorphic variants of human albumin and fragments of human albumin, for example those fragments disclosed in EP 322 094 (namely HA (Pn), where n is 369 to 419).
  • the albumin may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig.
  • Non-mammalian albumins include, but are not limited to, hen and salmon.
  • the albumin portion of the albumin fusion protein may be from a different animal than the therapeutic protein portion.
  • an HA fragment or variant will be at least 100 amino acids long, preferably at least 150 amino acids long.
  • the HA variant may consist of or alternatively comprise at least one whole domain of HA, for example domains 1 (amino acids 1-194 of albumin (FIG. IA-D)), domain 2 (amino acids 195-387 of albumin (FIG IA-D)), domain 3 (amino acids 388-585 of albumin (FIG IA-D)), domains 1 and 2 (1-387 of albumin (FIG IA-D)), domains 2 and 3 (195-585 of albumin (FIG IA-D)) or domains 1 and 3 (amino acids 1-194 of albumin (FIG IA-D) and amino acids 388-585 of albumin (FIG IA-D)).
  • Each domain is itself made up of two homologous subdomains namely 1-105, 120-194, 195-291, 316-387, 388-491 and 512-585, with flexible inter-subdomain linker regions comprising residues LyslO6 to Glul 19, Glu292 to VaB 15 and Glu492 to Ala511.
  • the albumin portion of an albumin fusion protein of the invention comprises at least one subdomain or domain of HA or conservative modifications thereof. If the fusion is based on subdomains, some or all of the adjacent linker is preferably used to link to the therapeutic protein moiety.
  • an alpha-fetoprotein fusion protein of the invention comprises at least a fragment or variant of a therapeutic protein and at least a fragment or variant of alpha-fetoprotein, which are associated with one another, preferably by genetic fusion.
  • An additional embodiment comprises at least a fragment or variant of a therapeutic protein and at least a fragment or variant of alpha-fetoprotein, which are linked to one another by chemical conjugation.
  • alpha-fetoprotein refers collectively to alpha-fetoprotein or amino acid sequence, or an alpha-fetoprotein fragment or variant, having one or more functional activities (e.g., biological activities) of alpha-fetoprotein.
  • alpha-fetoprotein refers to alpha-fetoprotein or fragments thereof as shown in FIG. 4, or alpha-fetoprotein from other vertebrates or fragments thereof, or analogs or variants of these molecules or fragments thereof.
  • alpha-fetoprotein will provide considerable benefit in the invention.
  • Alpha-fetoprotein is a naturally occurring molecule in human development and, consequently, the immunological tolerance for this protein enables the present invention to have considerable advantage over other carrier proteins that may cause an immunological response when introduced to the body.
  • the high tolerance to alpha-fetoprotein makes it unlikely that adults will raise antibodies to alpha-fetoprotein. This is particularly beneficial for fusion proteins of the invention.
  • the invention relates both to the alpha-fetoprotein fusion proteins, and vectors, constructs, and organisms comprising the polynucleotides encoding these proteins.
  • the vectors of the invention may comprise expression cassettes so that portions of the cassette may be easily changed (e.g., modular portions). This benefit will be useful in, for example, the design of vectors wherein the coding sequence for an opioid receptor agonist may be inserted or replaced with the sequence of another opioid receptor agonist.
  • a portion of alpha-fetoprotein sufficient to prolong the therapeutic activity or shelf- life of the therapeutic protein refers to a portion of alpha- fetoprotein sufficient in length or structure to stabilize or prolong the therapeutic activity of the protein so that the shelf life of the therapeutic protein portion of the alpha-fetoprotein fusion protein is prolonged or extended compared to the shelf-life in the non- fusion state.
  • the alpha-fetoprotein portion of the alpha-fetoprotein fusion proteins may comprise the full length of the alpha-fetoprotein sequence as described above, or may include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity.
  • the invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention.
  • the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects).
  • the subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
  • Various delivery systems are known and can be used to administer a compound of the invention.
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • the pharmaceutical compounds or compositions of the invention may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • care must be taken to use materials to which the protein does not absorb.
  • the compound or composition can be delivered in a controlled release system.
  • a pump may be 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)).
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FIa.
  • a controlled release system can be placed in proximity of the therapeutic target, e.g., 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)).
  • compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency-of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
  • Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the compounds of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a therapeutic protein can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the present invention relates generally to opioid receptor agonist fusion proteins which may include an opioid receptor agonist fused to albumin, a fragment, variant, or analog thereof (i.e., an "albumin fusion protein"), and methods of treating, preventing, or ameliorating diseases or disorders by administering albumin fusion proteins.
  • albumin fusion protein refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of an opioid receptor agonist protein (or fragment or variant thereof).
  • An albumin fusion protein of the invention comprises at least a fragment or variant of an opioid receptor agonist protein and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion (i.e., the albumin fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of an opioid receptor agonist protein is joined in- frame with a polynucleotide encoding all or a portion of albumin) or to one another.
  • the opioid receptor agonist protein and albumin protein, once part of the albumin fusion protein may each be referred to as a "portion", "region” or "moiety" of the albumin fusion protein.
  • the invention provides an albumin fusion protein encoded by a polynucleotide or albumin fusion construct. Polynucleotides encoding these albumin fusion proteins are also encompassed by the invention.
  • Preferred albumin fusion proteins of the invention include, but are not limited to, albumin fusion proteins encoded by a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least one molecule of an opioid receptor agonist protein (or fragment or variant thereof); a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least one molecule of an opioid receptor agonist protein (or
  • the invention provides an albumin fusion protein comprising, or alternatively consisting of, an opioid receptor agonist protein (e.g., an opioid receptor agonist, variant, or analog thereof) and a serum albumin protein.
  • an opioid receptor agonist protein e.g., an opioid receptor agonist, variant, or analog thereof
  • the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment of an opioid receptor agonist protein and a serum albumin protein.
  • the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active variant of an opioid receptor agonist protein and a serum albumin protein.
  • the serum albumin protein component of the albumin fusion protein is the mature portion of serum albumin.
  • the invention provides an albumin fusion protein comprising, or alternatively consisting of, an opioid receptor agonist protein, and a biologically active and/or therapeutically active fragment of serum albumin.
  • the invention provides an albumin fusion protein comprising, or alternatively consisting of, an opioid receptor agonist protein and a biologically active and/or therapeutically active variant of serum albumin.
  • the opioid receptor agonist protein portion of the albumin fusion protein is the mature portion of the opioid receptor agonist protein.
  • the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment or variant of an opioid receptor agonist protein and a biologically active and/or therapeutically active fragment or variant of serum albumin.
  • the invention provides an albumin fusion protein comprising, or alternatively consisting of, the mature portion of an opioid receptor agonist protein and the mature portion of serum albumin.
  • the albumin fusion protein comprises HA as the N-terminal portion, and an opioid receptor agonist protein as the C-terminal portion.
  • an albumin fusion protein comprising HA as the C-terminal portion, and an opioid receptor agonist protein as the N-terminal portion may also be used.
  • the albumin fusion protein has an opioid receptor agonist protein fused to both the N-terminus and the C-terminus of albumin.
  • the opioid receptor agonists fused at the N- and C-termini are the same opioid receptor agonist proteins.
  • the opioid receptor agonists fused at the N- and C-termini are different opioid receptor agonist proteins.
  • the opioid receptor agonist proteins fused at the N- and C-termini are different opioid receptor agonist proteins which may be used to treat or prevent the same or a related disease, disorder, or condition (e.g., pain).
  • the opioid receptor agonist proteins fused at the N- and C-termini are different opioid receptor agonist proteins which may be used to treat, ameliorate, or prevent diseases or disorders which are known in the art to commonly occur in patients simultaneously, concurrently, or consecutively, or which commonly occur in patients in association with one another (e.g., different forms of pain).
  • Albumin fusion proteins of the invention encompass proteins containing one, two, three, four, or more molecules of a given opioid receptor agonist protein X or variant thereof fused to the N- or C-terminus of an albumin fusion protein of the invention, and/or to the N- and/or C-terminus of albumin or variant thereof.
  • Molecules of a given opioid receptor agonist protein X or variants thereof may be in any number of orientations, including, but not limited to, a "head to head” orientation (e.g., wherein the N-terminus of one molecule of an opioid receptor agonist protein X is fused to the N-terminus of another molecule of the opioid receptor agonist protein X), or a "head to tail” orientation (e.g., wherein the C-terminus of one molecule of an opioid receptor agonist protein X is fused to the N-terminus of another molecule of opioid receptor agonist protein X).
  • a "head to head” orientation e.g., wherein the N-terminus of one molecule of an opioid receptor agonist protein X is fused to the N-terminus of another molecule of the opioid receptor agonist protein X
  • head to tail orientation e.g., wherein the C-terminus of one molecule of an opioid receptor agonist protein X is fused to the N-terminus of another molecule of
  • albumin fusion proteins of the invention further encompass proteins containing one, two, three, four, or more molecules of a given opioid receptor agonist protein having opioid receptor agonist activity or variant thereof fused to the N- or C- terminus of an albumin fusion protein of the invention, and/or to the N- and/or C- terminus of albumin or variant thereof, wherein the molecules are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference).
  • Albumin fusion proteins comprising multiple opioid receptor agonist proteins having opioid receptor agonist activity separated by peptide linkers may be produced using conventional recombinant DNA technology. Linkers are particularly important when fusing a small peptide to the large HSA molecule.
  • the peptide itself can be a linker by fusing tandem copies of the peptide or other known linkers can be used. Constructs that incorporate linkers are described in the Examples. In one embodiment, one, two, three, four, five, or more tandemly oriented therapeutic molecules having opioid receptor agonist activity are fused to the N- or C-terminus of albumin or variant thereof.
  • albumin fusion proteins of the invention may also be produced by fusing an opioid receptor agonist protein X or variants thereof to the N-terminal and/or C-terminal of albumin or variants thereof in such a way as to allow the formation of intramolecular and/or intermolecular multimeric forms.
  • albumin fusion proteins may be in monomeric or multimeric forms (i.e., dimers, trimers, tetramers and higher multimers).
  • the opioid receptor agonist protein portion of an albumin fusion protein may be in monomeric form- or multimeric form (i.e., dimers, trimers, tetramers and higher multimers).
  • the opioid receptor agonist protein portion of an albumin fusion protein is in multimeric form (i.e., dimers, trimers, tetramers and higher multimers), and the albumin protein portion is in monomeric form.
  • albumin fusion proteins of the invention may also be produced by inserting the opioid receptor agonist protein or peptide of interest into an internal region of HA. For instance, within the protein sequence of the HA molecule a number of loops or turns exist between the end and beginning of ⁇ -helices, which are stabilized by disulphide bonds. The loops, as determined from the crystal structure of HA (PDB identifiers 1AO6, 1BJ5, IBKE, IBMO, 1E7E to 1E71 and IUOR) for the most part extend away from the body of the molecule. These loops are useful for the insertion, or internal fusion, of therapeutically active peptides, particularly those requiring a secondary structure to be functional, or opioid receptor agonist proteins, to essentially generate an albumin molecule with specific biological activity.
  • Loops in human albumin structure into which peptides or polypeptides may be inserted to generate albumin fusion proteins of the invention include: Val54- Asn ⁇ l, Thr76-Asp89, Ala92-Glul00, Glnl70-Alal76, His 247-Glu252, Glu266- Glu277, GIu 280-His288, Ala362-Glu368, Lys439-Pro447, Val462-Lys475, Thr478- Pro486, and Lys560-Thr566.
  • peptides or polypeptides are inserted into the Val54-Asn61, Glnl70-Alal76, and/or Lys560- Thr566 loops of mature human albumin ((FIG IA-D).
  • Peptides to be inserted may be derived from either phage display or synthetic peptide libraries screened for specific biological activity or from the active portions of a molecule with the desired function. Additionally, random peptide libraries may be generated within particular loops or by insertions of randomized peptides into particular loops of the HA molecule and in which all possible combinations of amino acids are represented.
  • Such library(s) could be generated on HA or domain fragments of HA by one of the following methods: randomized mutation of amino acids within one or more peptide loops of HA or HA domain fragments. Either one, more or all the residues within a loop could be mutated in this manner; replacement of, or insertion into one or more loops of HA or HA domain fragments (i.e., internal fusion) of a randomized peptide(s) of length X n (where X is an amino acid and n is the number of residues; N-, C- or N- and C-terminal peptide/protein fusions in addition to (a) and/or (b).
  • the HA or HA domain fragment may also be made multifunctional by grafting the peptides derived from different screens of different loops against different targets into the same HA or HA domain fragment.
  • peptides inserted into a loop of human serum albumin are peptide fragments or peptide variants of the opioid receptor agonist proteins. More particularly, the invention encompasses albumin fusion proteins which comprise peptide fragments or peptide variants at least 7 at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids in length inserted into a loop of human serum albumin.
  • the invention also encompasses albumin fusion proteins which comprise peptide fragments or peptide variants at least 7 at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids fused to the N-terminus of human serum albumin.
  • the invention also encompasses albumin fusion proteins which comprise peptide fragments or peptide variants at least 7 at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids fused to the C-terminus of human serum albumin.
  • the albumin fusion proteins of the invention may have one HA- derived region and one opioid receptor agonist protein-derived region. Multiple regions of each protein, however, may be used to make an albumin fusion protein of the invention. Similarly, more than one opioid receptor agonist protein may be used to make an albumin fusion protein of the invention. For instance, an opioid receptor agonist protein may be fused to both the N- and C-terminal ends of the HA. In such a configuration, the opioid receptor agonist protein portions may be the same or different opioid receptor agonist protein molecules.
  • the structure of bifunctional albumin fusion proteins may be represented as: X-HA-Y or Y-HA-X.
  • Bi- or multi-functional albumin fusion proteins may also be prepared to target the opioid receptor agonist protein portion of a fusion to a target organ or cell type via protein or peptide at the opposite terminus of HA.
  • the peptides could be obtained by screening libraries constructed as fusions to the N-, C- or N- and C-termini of HA, or domain fragment of HA, of typically 6, 8, 12, 20 or 25 or X n (where X is an amino acid (aa) and n equals the number of residues) randomized amino acids, and in which all possible combinations of amino acids were represented.
  • peptides may be selected in situ on the HA molecule and the properties of the peptide would therefore be as selected for rather than, potentially, modified as might be the case for a peptide derived by any other method then being attached to HA.
  • the albumin fusion proteins of the invention may include a linker peptide between the fused portions to provide greater physical separation between the moieties and thus maximize the accessibility of the opioid receptor agonist protein portion, for instance, for binding to its cognate receptor.
  • the linker peptide may consist of amino acids such that it is flexible or more rigid.
  • the linker sequence may be cleavable by a protease or chemically to yield the therapeutic molecule related moiety.
  • the protease is one which is produced naturally by the host, for example the S. cerevisiae protease kex2 or equivalent proteases.
  • the albumin fusion proteins of the invention may have the following formula R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, wherein Rl is at least one opioid receptor agonist protein, peptide or polypeptide sequence, and not necessarily the same opioid receptor agonist protein, L is a linker and R2 is a serum albumin sequence.
  • Rl may include an opioid receptor agonist, an analog, fragment or variant thereof having opioid receptor agonist activity.
  • Albumin fusion proteins of the invention comprising an opioid receptor agonist protein have extended shelf life compared to the shelf life the same opioid receptor agonist protein when not fused to albumin.
  • Shelf-life typically refers to the time period over which the therapeutic activity of an opioid receptor agonist protein in solution or in some other storage formulation, is stable without undue loss of therapeutic activity. Many of the opioid receptor agonist proteins are highly labile in their unfused state. As described below, the typical shelf- life of these opioid receptor agonist proteins is markedly prolonged upon incorporation into the albumin fusion protein of the invention. [0192] Albumin fusion proteins of the invention with "prolonged" or “extended” shelf-life exhibit greater therapeutic activity relative to a standard that has been subjected to the same storage and handling conditions. The standard may be the unfused full-length opioid receptor agonist protein.
  • an albumin fusion protein of the invention may retain greater than about 100% of the therapeutic activity, or greater than about 105%, 110%, 120%, 130%, 150% or 200% of the therapeutic activity of a standard when subjected to the same storage and handling conditions as the standard when compared at a given time point. [0193] Shelf-life may also be assessed in terms of therapeutic activity remaining after storage, normalized to therapeutic activity when storage began.
  • Albumin fusion proteins of the invention with prolonged or extended shelf- life as exhibited by prolonged or extended therapeutic activity may retain greater than about 50% of the therapeutic activity, about 60%, 70%, 80%, or 90% or more of the therapeutic activity of the equivalent unfused opioid receptor agonist protein when subjected to the same conditions.
  • an albumin fusion protein of the invention comprising hGH fused to the full length HA sequence may retain about 80% or more of its original activity in solution for periods of up to 5 weeks or more under various temperature conditions.
  • the present invention relates generally to opioid receptor agonist fusion proteins which may include an opioid receptor agonist protein fused to AFP, a fragment, variant, or analog thereof (i.e., an "AFP fusion protein"), and methods of treating, preventing, or ameliorating diseases or disorders by administering AFP fusion proteins.
  • AFP fusion protein refers to a protein formed by the fusion of at least one molecule of alpha-fetoprotein (or a fragment or variant thereof) to at least one molecule of an opioid receptor agonist protein (or fragment or variant thereof).
  • An alpha- fetoprotein fusion protein of the invention comprises at least a fragment or variant of an opioid receptor agonist protein and at least a fragment or variant of alpha-fetoprotein, which are associated with one another, preferably by genetic fusion (i.e., the alpha-fetoprotein fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of an opioid receptor agonist protein is joined in- frame with a polynucleotide encoding all or a portion of alpha-fetoprotein) or to one another.
  • the opioid receptor agonist protein and alpha-fetoprotein protein, once part of the alpha-fetoprotein fusion protein, may each be referred to as a "portion", "region” or “moiety” of the alpha- fetoprotein fusion protein.
  • alpha-fetoprotein fusion proteins of the invention include, but are not limited to, alpha-fetoprotein fusion proteins encoded by a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of alpha-fetoprotein (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least one molecule of an opioid receptor agonist protein (or fragment or variant thereof); a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of alpha- fetoprotein (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least one molecule of an opioid receptor agonist protein (or fragment or variant thereof); or a nucleic acid molecule comprising a polynucleotide encoding at least one molecule of alpha-fetoprotein (
  • the invention provides an alpha-fetoprotein fusion protein comprising an opioid receptor agonist protein and an alpha-fetoprotein protein. In other embodiments, the invention provides an alpha-fetoprotein fusion protein comprising a biologically active and/or therapeutically active fragment of an opioid receptor agonist protein and an alpha-fetoprotein protein. In other embodiments, the invention provides an alpha-fetoprotein fusion protein comprising a biologically active and/or therapeutically active variant of an opioid receptor agonist protein and an alpha-fetoprotein protein. In preferred embodiments, the alpha- fetoprotein protein component of the alpha-fetoprotein fusion protein is the mature portion of alpha-fetoprotein.
  • the invention provides an alpha-fetoprotein fusion protein comprising an opioid receptor agonist protein, and a biologically active and/or therapeutically active fragment of alpha-fetoprotein.
  • the invention provides an alpha-fetoprotein fusion protein comprising an opioid receptor agonist protein and a biologically active and/or therapeutically active variant of alpha- fetoprotein.
  • the invention provides an alpha-fetoprotein fusion protein comprising a biologically active and/or therapeutically active fragment or variant of an opioid receptor agonist protein and a biologically active and/or therapeutically active fragment or variant of alpha-fetoprotein.
  • the invention provides an alpha-fetoprotein fusion protein comprising the mature portion of an opioid receptor agonist protein and the mature portion of alpha-fetoprotein.
  • the alpha-fetoprotein fusion protein comprises alpha- fetoprotein as the N-terminal portion, and an opioid receptor agonist protein as the C- terminal portion.
  • an alpha-fetoprotein fusion protein comprising alpha- fetoprotein as the C -terminal portion, and an opioid receptor agonist protein as the N- terminal portion may also be used.
  • the alpha-fetoprotein fusion protein has an opioid receptor agonist protein fused to both the N-terminus and the C-terminus of alpha- fetoprotein.
  • the opioid receptor agonist proteins fused at the N- and C -termini are the same opioid receptor agonist proteins.
  • the opioid receptor agonist proteins fused at the N- and C-termini are different opioid receptor agonist proteins.
  • the opioid receptor agonist proteins fused at the N- and C-termini are different opioid receptor agonist proteins which may be used to treat or prevent the same or a related disease, disorder, or condition (e.g., pain).
  • the opioid receptor agonist proteins fused at the N- and C-termini are different opioid receptor agonist proteins which may be used to treat, ameliorate, or prevent diseases or disorders which are known in the art to commonly occur in patients simultaneously, concurrently, or consecutively, or which commonly occur in patients in association with one another (e.g., the same or different forms of pain).
  • an opioid receptor agonist protein may be attached to the N or C terminal region of alpha-fetoprotein, and a different opioid receptor agonist protein may be attached to the other end of alpha-fetoprotein.
  • Alpha-fetoprotein fusion proteins of the invention encompass proteins containing one, two, three, four, or more molecules of a given opioid receptor agonist protein or variant thereof fused to the N- or C-terminus of an alpha-fetoprotein fusion protein of the invention, and/or to the N- and/or C-terminus of alpha-fetoprotein or variant thereof.
  • Molecules of a given opioid receptor agonist protein or variants thereof may be in any number of orientations, including, but not limited to, a "head to head” orientation (e.g., wherein the N-terminus of one molecule of an opioid receptor agonist protein is fused to the N-terminus of another molecule of the opioid receptor agonist protein), or a "head to tail” orientation (e.g., wherein the C-terminus of one molecule of an opioid receptor agonist protein is fused to the N-terminus of another molecule of the opioid receptor agonist protein).
  • a "head to head” orientation e.g., wherein the N-terminus of one molecule of an opioid receptor agonist protein is fused to the N-terminus of another molecule of the opioid receptor agonist protein
  • head to tail orientation e.g., wherein the C-terminus of one molecule of an opioid receptor agonist protein is fused to the N-terminus of another molecule of the opioid receptor agonist protein.
  • one, two, three, or more tandemly oriented opioid receptor agonist polypeptides are fused to the N- or C-terminus of an alpha-fetoprotein fusion protein of the invention, and/or to the N- and/or C-terminus of alpha-fetoprotein or variant thereof.
  • Alpha-fetoprotein fusion proteins of the invention further encompass proteins containing one, two, three, four, or more molecules of a given opioid receptor agonist protein or variant thereof fused to the N- or C-terminus of an alpha- fetoprotein fusion protein of the invention, and/or to the N- and/or C-terminus of alpha- fetoprotein or variant thereof, wherein the molecules are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627. Alpha- fetoprotein fusion proteins comprising multiple opioid receptor agonist polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.
  • Linkers are particularly important when fusing a small peptide to the alpha- fetoprotein molecule.
  • the peptide itself can be a linker by fusing tandem copies of the peptide or other known linkers can be used.
  • alpha- fetoprotein fusion proteins of the invention may also be produced by fusing an opioid receptor agonist protein or variants thereof to the N- terminal and/or C-terminal of alpha- fetoprotein or variants thereof in such a way as to allow the formation of intramolecular and/or intermolecular multimeric forms.
  • alpha- fetoprotein fusion proteins may be in monomeric or multimeric forms (i.e., dimers, trimers, tetramers and higher multimers).
  • the opioid receptor agonist protein portion of an alpha-fetoprotein fusion protein may be in monomeric form or multimeric form (i.e., dimers, trimers, tetramers and higher multimers).
  • the opioid receptor agonist protein portion of an alpha-fetoprotein fusion protein is in multimeric form (i.e., dimers, trimers, tetramers and higher multimers), and the alpha- fetoprotein portion is in monomeric form.
  • alpha-fetoprotein fusion proteins of the invention may also be produced by inserting the opioid receptor agonist protein of interest into an internal region of alpha-fetoprotein. For instance, within the protein sequence of the alpha-fetoprotein molecule a number of loops or turns exist between the end and beginning of .alpha.- helices, which are stabilized by disulphide bonds.
  • Peptides to be inserted may be derived from either phage display or synthetic peptide libraries screened for specific biological activity or from the active portions of a molecule with the desired function. Additionally, random peptide libraries may be generated within particular loops or by insertions of randomized peptides into particular loops of the alpha-fetoprotein molecule and in which all possible combinations of amino acids are represented.
  • Such library(s) could be generated on alpha-fetoprotein or domain fragments of alpha-fetoprotein by one of the following methods: randomized mutation of amino acids within one or more peptide loops of alpha-fetoprotein or alpha-fetoprotein domain fragments. Either one, more or all the residues within a loop could be mutated in this manner; replacement of, or insertion into one or more loops of alpha- fetoprotein or alpha-fetoprotein domain fragments (i.e., internal fusion) of a randomized peptide(s).
  • the alpha-fetoprotein or alpha-fetoprotein domain fragment may also be made multifunctional by grafting the peptides derived from different screens of different loops against different targets into the same alpha-fetoprotein or alpha- fetoprotein domain fragment.
  • the alpha-fetoprotein fusion proteins of the invention may have one alpha-fetoprotein-derived region and one opioid receptor agonist protein-derived region. Multiple regions of each protein, however, may be used to make an alpha- fetoprotein fusion protein of the invention. Similarly, more than one opioid receptor agonist protein may be used to make an alpha-fetoprotein fusion protein of the invention. For instance, an opioid receptor agonist protein may be fused to both the N- and C-terminal ends of the alpha-fetoprotein. In such a configuration, the opioid receptor agonist protein portions may be the same or different opioid receptor agonist protein molecules.
  • bifunctional alpha-fetoprotein fusion proteins may be represented as: X-alpha-fetoprotein-Y or Y-alpha-fetoprotein-X.
  • Bi- or multi-functional alpha-fetoprotein fusion proteins may also be prepared to target the opioid receptor agonist protein portion of a fusion to a target organ or cell type via protein or peptide at the opposite terminus of alpha-fetoprotein.
  • the peptides could be obtained by screening libraries constructed as fusions to the N-, C- or N- and C-termini of alpha-fetoprotein, or domain fragment of alpha-fetoprotein, of typically 6, 8, 12, 20 or 25 or X (where X is an amino acid (aa) and n equals the number of residues) randomized amino acids, and in which all possible combinations of amino acids were represented).
  • peptides may be selected in situ on the alpha-fetoprotein molecule and the properties of the peptide would therefore be as selected for rather than, potentially, modified as might be the case for a peptide derived by any other method then being attached to alpha-fetoprotein.
  • the alpha-fetoprotein fusion proteins of the invention may include a linker peptide between the fused portions to provide greater physical separation between the moieties and thus maximize the accessibility of the opioid receptor agonist protein portion, for instance, for binding to its cognate receptor.
  • the linker peptide may consist of amino acids such that it is flexible or more rigid.
  • the linker sequence may be cleavable by a protease or chemically to yield the growth hormone related moiety.
  • the protease is one which is produced naturally by the host, for example the S. cerevisiae protease kex2 or equivalent proteases.
  • the alpha-fetoprotein fusion proteins of the invention may have the following formula R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, wherein Rl is at least one opioid receptor agonist protein, peptide or polypeptide sequence, and not necessarily the same opioid receptor agonist protein, L is a linker and R2 is a alpha-fetoprotein sequence.
  • Alpha-fetoprotein fusion proteins of the invention comprising an opioid receptor agonist protein have extended shelf life compared to the shelf life the same opioid receptor agonist protein when not fused to alpha- fetoprotein. Shelf-life typically refers to the time period over which the therapeutic activity of an opioid receptor agonist protein in solution or in some other storage formulation, is stable without undue loss of therapeutic activity. Many of the opioid receptor agonist proteins are highly labile in their unfused state. As described below, the typical shelf-life of these opioid receptor agonist proteins is markedly prolonged upon incorporation into the alpha-fetoprotein fusion protein of the invention.
  • Alpha-fetoprotein fusion proteins of the invention with "prolonged” or “extended” shelf-life exhibit greater therapeutic activity relative to a standard that has been subjected to the same storage and handling conditions.
  • the standard may be the unf ⁇ ised full-length opioid receptor agonist protein.
  • the opioid receptor agonist protein portion of the alpha-fetoprotein fusion protein is an analog, a variant, or is otherwise altered or does not include the complete sequence for that protein, the prolongation of therapeutic activity may alternatively be compared to the unfused equivalent of that analog, variant, altered peptide or incomplete sequence.
  • an alpha-fetoprotein fusion protein of the invention may retain greater than about 100% of the therapeutic activity, or greater than about 105%, about 110%, about 120%, about 130%, about 150% or about 200% of the therapeutic activity of a standard when subjected to the same storage and handling conditions as the standard when compared at a given time point.
  • Shelf-life may also be assessed in terms of therapeutic activity remaining after storage, normalized to therapeutic activity when storage began.
  • Alpha- fetoprotein fusion proteins of the invention with prolonged or extended shelf-life as exhibited by prolonged or extended therapeutic activity may retain greater than about 50% of the therapeutic activity, about 60%, about 70%, about 80%, or about 90% or more of the therapeutic activity of the equivalent unfused opioid receptor agonist protein when subjected to the same conditions.
  • the fusion proteins of the invention may be produced as recombinant molecules by secretion from yeast, a microorganism such as a bacterium, or a human or animal cell line.
  • the polypeptide is secreted from the host cells.
  • a particular embodiment of the invention comprises a DNA construct encoding a signal sequence effective for directing secretion in yeast, particularly a yeast-derived signal sequence (especially one which is homologous to the yeast host), and the fused molecule of the first aspect of the invention, there being no yeast- derived pro sequence between the signal and the mature polypeptide.
  • yeast-derived signal sequence especially one which is homologous to the yeast host
  • the Saccharomyces cerevisiae invertase signal is a preferred example of a yeast-derived signal sequence.
  • the present invention also includes a cell, preferably a yeast cell transformed to express a fusion protein of the invention.
  • a culture of those cells preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.
  • the medium will contain the polypeptide, with the cells, or without the cells if they have been filtered or centrifuged away.
  • Many expression systems are known and may be used, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris, filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.
  • bacteria for example E. coli and Bacillus subtilis
  • yeasts for example Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris
  • filamentous fungi for example Aspergillus
  • plant cells animal cells and insect cells.
  • Preferred yeast strains to be used in the production of fusion proteins are D88, DXYl and BXPlO.
  • D88 [leu2-3, Ieu2-122, canl, pral, ubc4] is a derivative of parent strain AH22his + (also known as DBl; see, e.g., Sleep et al. Biotechnology 8:42-46 (1990)).
  • the strain contains a Ieu2 mutation which allows for auxotropic selection of 2 micron-based plasmids that contain the LEU2 gene.
  • D88 also exhibits a derepression of PRBl in glucose excess.
  • the PRBl promoter is normally controlled by two checkpoints that monitor glucose levels and growth stage. The promoter is activated in wild type yeast upon glucose depletion and entry into stationary phase. Strain D88 exhibits the repression by glucose but maintains the induction upon entry into stationary phase.
  • the PRAl gene encodes a yeast vacuolar protease, YscA endoprotease A, that is localized in the ER.
  • the UBC4 gene is in the ubiquitination pathway and is involved in targeting short lived and abnormal proteins for ubiquitin dependant degradation. Isolation of this ubc4 mutation was found to increase the copy number of an expression plasmid in the cell and cause an increased level of expression of a desired protein expressed from the plasmid (see, e.g., International Publication No. WO99/00504, hereby incorporated in its entirety by reference herein).
  • DXYl a derivative of D88, has the following genotype: [leu2-3, Ieu2-122, canl, pral, ubc4, ura3::yap3].
  • this strain also has a knockout of the YAP3 protease. This protease causes cleavage of mostly di-basic residues (RR, RK, KR, KK) but can also promote cleavage at single basic residues in proteins. Isolation of this yap3 mutation resulted in higher levels of full length HSA production (see, e.g., U.S. Pat. No.
  • BXPlO has the following genotype: leu2-3, Ieu2-122, canl, pral, ubc4, ura3, yap3::URA3, Iys2, hspl50::LYS2, pmtl ::URA3.
  • this strain also has a knockout of the PMTl gene and the HSP150 gene.
  • the PMTl gene is a member of the evolutionarily conserved family of dolichyl- phosphate-D-mannose protein 0-mannosyltransferases (Pmts).
  • the transmembrane topology of Pmtlp suggests that it is an integral membrane protein of the endoplasmic reticulum with a role in 0-linked glycosylation.
  • This mutation serves to reduce/eliminate 0-linked glycosylation of HSA fusions (see, e.g., International Publication No. WO00/44772, hereby incorporated in its entirety by reference herein).
  • Studies revealed that the Hspl50 protein is inefficiently separated from rHA by ion exchange chromatography.
  • the mutation in the HSP 150 gene removes a potential contaminant that has proven difficult to remove by standard purification techniques. See, e.g., U.S. Pat. No. 5,783,423, hereby incorporated in its entirety by reference herein.
  • the desired protein is produced in conventional ways, for example from a coding sequence inserted in the host chromosome or on a free plasmid.
  • the yeasts are transformed with a coding sequence for the desired protein in any of the usual ways, for example electroporation. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.
  • Successfully transformed cells i.e., cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct can be grown to produce the desired polypeptide.
  • Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. MoI. Biol. 98, 503 or Berent et al. (1985) Biotech. 3, 208.
  • the presence of the protein in the supernatant can be detected using antibodies.
  • Useful yeast plasmid vectors include pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.
  • Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, 7RPl, LEU2 and URA3.
  • Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).
  • Preferred vectors for making fusion proteins for expression in yeast include pPPC0005, pScCHSA, pScNHSA, and pC4:HSA which are described in detail in Example 1.
  • FIG. 2 shows a map of the pPPC0005 plasmid that can be used as the base vector into which polynucleotides encoding opioid receptor agonist proteins may be cloned to form HA-fusions. It contains a PRBl S. cerevisiae promoter (PRBIp), a Fusion leader sequence (FL), DNA encoding HA (rHA) and an ADHl S. cerevisiae terminator sequence.
  • PRBIp PRBl S. cerevisiae promoter
  • FL Fusion leader sequence
  • DNA encoding HA rHA
  • ADHl S. cerevisiae terminator sequence an ADHl S. cerevisiae terminator sequence.
  • the sequence of the fusion leader sequence consists of the first 19 amino acids of the signal peptide of human serum albumin and the last five amino acids of the mating factor alpha 1 promoter (SLDKR, see EP-A-387 319 which is hereby incorporated by reference in its entirety).
  • plasmids pPPC0005, pScCHSA, pScNHSA, and pC4:HSA were deposited on Apr. 11, 2001 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 and given accession numbers ATCC PTA-3278, PTA-3276, PTA-3279, and PTA-3277, respectively.
  • Another vector useful for expressing a fusion protein in yeast the pSAC35 vector which is described in Sleep et al., BioTechnology 8:42 (1990) which is hereby incorporated by reference in its entirety.
  • Another yeast promoter that can be used to express the fusion protein is the MET25 promoter. See, for example, Dominik Mumburg, Rolf Muller and Martin Funk. Nucleic Acids Research, 1994, Vol. 22, No. 25, pp. 5767-5768.
  • the Met25 promoter is 383 bases long (bases-382 to -1) and the genes expressed by this promoter are also known as Metl5, Metl7, and YLR303W.
  • a preferred embodiment uses the sequence below including a Not I site for cloning at the 5' end, and an ATG start codon at the 3' end: 5 GCGGCCGCCGGATGCAAGGGTTCGAATCCCTTAG CTCTCATTATTTTTTGCTTTTTCTCTTGAGGTCACATGATCGCAAAATGGCA AATGGCACGTGAAGCTGTCGATATTGGGGAACTGTGGTGGTTGGCAAATG ACTAATTAAGTTAGTCAAGGCGCCATCCTCATGAAAACTGTGTAACATAA TAACCGAAGTGTCGAAAAGGTGGCACCTTGTCCAATTGAACACGCTCGAT GAAAAAAATAAGATATATATAAGGTTAAGTAAAGCGTCTGTTAGAAAGG
  • a variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
  • Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors.
  • the DNA segment, generated by endonuclease restriction digestion, is treated with bacteriophage T4
  • DNA polymerase or E. coli DNA polymerase I enzymes that remove protruding, gamma-single-stranded termini with their 3' 5'-exonucleolytic activities, and fill in recessed 3 '-ends with their polymerizing activities.
  • the combination of these activities therefore generates blunt-ended DNA segments.
  • the blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • the products of the reaction are DNA segments carrying polymeric linker sequences at their ends.
  • These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
  • a desirable way to modify the DNA in accordance with the invention is to use the polymerase chain reaction as disclosed by Saiki et al. (1988) Science 239, 487-491.
  • the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA.
  • the specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
  • Exemplary genera of yeast contemplated to be useful in the practice of the present invention as hosts for expressing the fusion proteins are Pichia (Hansenula), Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, and the like.
  • Preferred genera are those selected from the group consisting of Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia and Torulaspora.
  • Saccharomyces spp. are S. cerevisiae, S. italicus and S. rouxii.
  • Kluyveromyces spp. are K. fragilis, K. lactis and K. marxianus.
  • a suitable Torulaspora species is T. delbrueckii.
  • Pichia (Hansenula) spp. are P. angusta (formerly H. polymorpha), P. anomala (formerly H. anomala) and P. pastoris.
  • Methods for the transformation of S. cerevisiae are taught generally in EP 251 744, EP 258 067 and WO 90/01063, all of which are incorporated herein by reference.
  • Preferred exemplary species of Saccharomyces include S. cerevisiae, S. italicus, S. diastaticus, and Zygosaccharomyces rouxii.
  • Preferred exemplary species of Kluyveromyces include K. fragilis and K. lactis.
  • Preferred exemplary species of Hansenula include H. polymorpha (now Pichia angusta), H. anomala (now Pichia anomala), and Pichia capsulata. Additional preferred exemplary species of Pichia include P. pastoris.
  • Preferred exemplary species of Aspergillus include A. niger and A. nidulans.
  • Preferred exemplary species of Yarrowia include Y. lipolytica.
  • yeast species are available from the ATCC.
  • the following preferred yeast species are available from the ATCC and are useful in the expression of fusion proteins: Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 yap3 mutant (ATCC Accession No. 4022731); Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 hspl50 mutant (ATCC Accession No. 4021266); Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 pmtl mutant (ATCC Accession No. 4023792); Saccharomyces cerevisiae Hansen, teleomorph (ATCC Accession Nos.
  • Suitable promoters for S. cerevisiae include those associated with the PGKI gene, GALl or GALlO genes, CYCI, PHO5, TRPI, ADHI, ADH2, the genes for glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, triose phosphate isomerase, phosphoglucose isomerase, glucokinase, alpha-mating factor pheromone, [a mating factor pheromone], the PRBI promoter, the GUT2 promoter, the GPDI promoter, and hybrid promoters involving hybrids of parts of 5' regulatory regions with parts of 5' regulatory regions of other promoters or with upstream activation sites (e.g.
  • Convenient regulatable promoters for use in Schizosaccharomyces pombe are the thiamine-repressible promoter from the nmt gene as described by Maundrell (1990) J. Biol. Chem. 265, 10857-10864 and the glucose repressible jbpl gene promoter as described by Hoffman & Winston (1990) Genetics 124, 807-816.
  • Methods of transforming Pichia for expression of foreign genes are taught in, for example, Cregg et al. (1993), and various Phillips patents (e.g. U.S. Pat. No.
  • Pichia expression kits are commercially available from Invitrogen BV, Leek, Netherlands, and Invitrogen Corp., San Diego, Calif.
  • Suitable promoters include AOXI and AOX2.
  • Gleeson et al. (1986) J. Gen. Microbiol. 132, 3459-3465 include information on Hansenula vectors and transformation, suitable promoters being MOXl and FMDl; whilst EP 361 991, Fleer et al. (1991) and other-publications from Rhone-Poulenc Rorer teach how to express foreign proteins in Kluyveromyces spp., a suitable promoter being PGKI.
  • the transcription termination signal is preferably the 3' flanking sequence of a eukaryotic gene which contains proper signals for transcription termination and polyadenylation.
  • Suitable 3' flanking sequences may, for example, be those of the gene naturally linked to the expression control sequence used, i.e. may correspond to the promoter. Alternatively, they may be different in which case the termination signal of the S. cerevisiae ADHI gene is preferred.
  • the desired fusion protein may be initially expressed with a secretion leader sequence, which may be any leader effective in the yeast chosen.
  • Leaders useful in yeast include any of the following:
  • MKWVTFISLLFLFSSAYS SEQ ID NO:34 or variants thereof, such as, for example, MKWVSFISLLFLFSSAYS (SEQ ID NO:35)
  • invertase signal sequence e.g., MLLQ AFLFLLAGF AAKISA (SEQ ID NO: 1)
  • yeast mating factor alpha signal sequence e.g., the yeast mating factor alpha signal sequence
  • a hybrid signal sequence e.g., MKWVSFISLLFLFSSAYSRSLEKR
  • HSA/MF ⁇ -1 hybrid signal sequence also known as HSA/kex2
  • insulin-like growth factor-binding protein 4 signal sequence e.g., the insulin-like growth factor-binding protein 4 signal sequence
  • MKWVTFISLLFLFAGVLG SEQ ID NO:47
  • MKWVTFISLLFLFSGVLG SEQ ID NO:47
  • HSA MKWVTFISLLFLFAGVSG SEQ ID NO:50
  • K. lactis killer toxin prepro 29 amino acids; 16 amino acids of pre and 13 amino acids of pro
  • MNIFYIFLFLLSFVQGLEHTHRRGSLDKR SEQ ID NO:58
  • gp67 signal sequence in conjunction with baculoviral expression systems (e.g., amino acids 1-19 of GenBank Accession Number AAA72759) or
  • the present invention also relates to vectors containing a polynucleotide encoding a fusion protein of the present invention, host cells, and the production of fusion proteins by synthetic and recombinant techniques.
  • the vector may be, for example, a phage, plasmid, viral, or retroviral vector.
  • Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • the polynucleotides encoding fusion proteins of the invention may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid.
  • the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • the polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E.
  • the expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • Such markers include dihydro folate reductase, G418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells
  • plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc.
  • preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlbad, Calif).
  • polynucleotides encoding a fusion protein of the invention may be fused to signal sequences which will direct the localization of a protein of the invention to particular compartments of a prokaryotic or eukaryotic cell and/or direct the secretion of a protein of the invention from a prokaryotic or eukaryotic cell.
  • signal sequences which will direct the localization of a protein of the invention to particular compartments of a prokaryotic or eukaryotic cell and/or direct the secretion of a protein of the invention from a prokaryotic or eukaryotic cell.
  • E. coli one may wish to direct the expression of the protein to the periplasmic space.
  • Examples of signal sequences or proteins (or fragments thereof) to which the fusion proteins of the invention may be fused in order to direct the expression of the polypeptide to the periplasmic space of bacteria include, but are not limited to, the pelB signal sequence, the maltose binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit, and the signal sequence of alkaline phosphatase.
  • MBP maltose binding protein
  • ompA signal sequence the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit
  • alkaline phosphatase Several vectors are commercially available for the construction of fusion proteins which will direct the localization of a protein, such as the pMAL series of vectors (particularly the pMAL-p series) available from New England Biolabs.
  • polynucleotides encoding fusion proteins of the invention may be fused to the pelB pectate lyase signal sequence to increase the efficiency of expression and purification of such polypeptides in Gram-negative bacteria. See, U.S. Pat. Nos. 5,576,195 and 5,846,818, the contents of which are herein incorporated by reference in their entireties.
  • signal peptides that may be fused to a fusion protein of the invention in order to direct its secretion in mammalian cells include, but are not limited to:
  • MPIF-I signal sequence e.g., amino acids 1-21 of GenBank Accession number AAB51134
  • MKVSVAALSCLMLVT ALGSQA SEQ ID NO:31
  • stanniocalcin signal sequence MLQNSAVLLILLVISASA (SEQ ID NO:32)
  • the pre-pro region of the HSA signal sequence e.g., MKWVTFISLLFLFSSAYSRGVFRR (SEQ ID NO:33)
  • the pre region of the HSA signal sequence e.g., MKWVTFISLLFLFSSAYS (SEQ ID NO:34)
  • the invertase signal sequence e.g., MLLQ AFLFLLAGF AAKISA (SEQ ID NO:35)
  • yeast mating factor alpha signal sequence e.g., the yeast mating factor alpha signal sequence
  • a hybrid signal sequence e.g., MKWVSFISLLFLFSSAYSRSLEKR
  • HSA/MF ⁇ -1 hybrid signal sequence also known as HSA/kex2
  • a K. lactis killer/MF ⁇ -1 fusion leader sequence (e.g.,
  • MKWVTFISLLFLFAGVLG SEQ ID NO:47
  • MKWVTFISLLFLFSGVLG SEQ ID NO:47
  • MKWVTFISLLFLFSGVSG SEQ ID NO:53
  • Modified HSA G14
  • MKWVTFISLLFLFGGVSG SEQ ID NO:54
  • K. lactis killer toxin prepro 29 amino acids; 16 amino acids of pre and 13 amino acids of pro
  • MNIFYIFLFLLSFVQGLEHTHRRGSLDKR SEQ ID NO:58
  • gp67 signal sequence in conjunction with baculoviral expression systems (e.g., amino acids 1-19 of GenBank Accession Number AAA72759) or
  • Vectors which use glutamine synthase (GS) or DBFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively.
  • An advantage of glutamine synthase based vectors are the availability of cell lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase negative.
  • Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene.
  • glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657, which are hereby incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors can be obtained from Lonza Biologies, Inc. (Portsmouth, N.H.). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog. 11 :1 (1995) which are herein incorporated by reference.
  • the present invention also relates to host cells containing the above- described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • a host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired.
  • Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled.
  • different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
  • nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many Standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
  • the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence corresponding to an opioid receptor agonist protein may be replaced with a fusion protein corresponding to the opioid receptor agonist protein), and/or to include genetic material (e.g., heterologous polynucleotide sequences such as for example, a fusion protein of the invention corresponding to the opioid receptor agonist protein may be included).
  • endogenous genetic material e.g., the coding sequence corresponding to an opioid receptor agonist protein may be replaced with a fusion protein corresponding to the opioid receptor agonist protein
  • genetic material e.g., heterologous polynucleotide sequences such as for example, a fusion protein of the invention corresponding to the opioid receptor agonist protein may be included.
  • the genetic material operably associated with the endogenous polynucleotide may activate, alter, and/or amplify endogenous polynucleotides.
  • techniques known in the art may be used to operably associate heterologous polynucleotides (e.g., polynucleotides encoding an albumin protein, AFP or a fragment or variant thereof) and/or heterologous control regions (e.g., promoter and/or enhancer) with endogenous polynucleotide sequences encoding an opioid receptor agonist protein via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun.
  • Fusion proteins of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, hydrophobic charge interaction chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • the fusion proteins of the invention are purified using Anion Exchange Chromatography including, but not limited to, chromatography on Q-sepharose, DEAE sepharose, poros HQ, poros DEAE, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/Source Q and DEAE, Fractogel Q and DEAE columns.
  • Anion Exchange Chromatography including, but not limited to, chromatography on Q-sepharose, DEAE sepharose, poros HQ, poros DEAE, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/Source Q and DEAE, Fractogel Q and DEAE columns.
  • the fusion proteins of the invention are purified using Cation Exchange Chromatography including, but not limited to, SP-sepharose, CM sepharose, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource/Source S and CM, Fractogel S and CM columns and their equivalents and comparables.
  • the fusion proteins of the invention are purified using Hydrophobic Interaction Chromatography including, but not limited to, Phenyl, Butyl, Methyl, Octyl, Hexyl-sepharose, poros Phenyl, Butyl, Methyl, Octyl, Hexyl, Toyopearl Phenyl, Butyl, Methyl, Octyl, Hexyl Resource/Source Phenyl, Butyl, Methyl, Octyl, Hexyl, Fractogel Phenyl, Butyl, Methyl, Octyl, Hexyl columns and their equivalents and comparables.
  • Hydrophobic Interaction Chromatography including, but not limited to, Phenyl, Butyl, Methyl, Octyl, Hexyl-sepharose, poros Phenyl, Butyl, Methyl, Octyl, Hexyl, Toyopearl Phenyl, Butyl, Me
  • the fusion proteins of the invention are purified using Size Exclusion Chromatography including, but not limited to, sepharose SlOO, S200, S300, superdex resin columns and their equivalents and comparables.
  • Size Exclusion Chromatography including, but not limited to, sepharose SlOO, S200, S300, superdex resin columns and their equivalents and comparables.
  • Affinity Chromatography including, but not limited to, Mimetic Dye affinity, peptide affinity and antibody affinity columns that are selective for either the HSA or the "fusion target" molecules.
  • fusion proteins of the invention are purified using one or more Chromatography methods listed above. In other preferred embodiments, fusion proteins of the invention are purified using one or more of the following Chromatography columns, Q sepharose FF column, SP Sepharose FF column, Q Sepharose High Performance Column, Blue Sepharose FF column, Blue Column, Phenyl Sepharose FF column, DEAE Sepharose FF, or Methyl Column. [0332] Additionally, fusion proteins of the invention may be purified using the process described in PCT International Publication WO 00/44772 which is herein incorporated by reference in its entirety. One of skill in the art could easily modify the process described therein for use in the purification of fusion proteins of the invention.
  • Fusion proteins of the present invention may be recovered from: products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, fusion proteins of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N- terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
  • the yeast Pichia pastoris is used to express fusion proteins of the invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source.
  • a main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O 2 .
  • This reaction is catalyzed by the enzyme alcohol oxidase.
  • Pichia pastoris In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O 2 . Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOXl) is highly active. In the presence of methanol, alcohol oxidase produced from the AOXl gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris.
  • a heterologous coding sequence such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOXI regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
  • the plasmid vector pPIC9K is used to express DNA encoding fusion protein of the invention, as set forth herein, in a Pichea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology," D. R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N. J., 1998.
  • This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong AOXl promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
  • PHO alkaline phosphatase
  • yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.
  • high-level expression of a heterologous coding sequence such as, for example, a polynucleotide encoding a fusion protein of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.
  • fusion proteins of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)).
  • a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence.
  • Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4- diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3- amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b- methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be
  • the invention encompasses fusion proteins of the present invention which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 ; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
  • Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N- terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
  • the fusion proteins may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include iodine ( 121 I, 123 I, 125 I, 131 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 111 In, 112 In, 113 mln, 11 W), technetium ( 99 T
  • fusion proteins of the present invention or fragments or variants thereof are attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, 177 Lu, 90 Y, 166 Ho, and 153 Sm, to polypeptides.
  • the radiometal ion associated with the macrocyclic chelators is 111 In.
  • the radiometal ion associated with the macrocyclic chelator is 90 Y.
  • the macrocyclic chelator is 1,4,7,10-tetraazacyclod- odecane-N,N',N",N'"-tetraacetic acid (DOTA).
  • DOTA is attached to an antibody of the invention or fragment thereof via linker molecule.
  • linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art—see, for example, DeNardo et al, Clin Cancer Res. 4(10):2483-90 (1998); Peterson et al, Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby incorporated by reference in their entirety.
  • the fusion proteins of the invention may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art.
  • Polypeptides of the invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • Fusion proteins of the invention and antibodies that bind a therapeutic protein or fragments or variants thereof can be fused to marker sequences, such as a peptide to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif, 91311), among others, many of which are commercially available.
  • pQE vector QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif, 91311
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag.
  • a fusion protein of the invention may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
  • the conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, B-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al, Int.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
  • a protein such as tumor necrosis factor, alpha-interferon, B-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apopt
  • VEGI See, International Publication No. WO 99/23105
  • a thrombotic agent or an anti-angiogenic agent e.g., angiostatin or endostatin
  • biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-I”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • Techniques for conjugating such therapeutic moiety to proteins are well known in the art.
  • Fusion proteins may also be attached to solid supports, which are particularly useful for immunoassays or purification of polypeptides that are bound by, that bind to, or associate with fusion proteins of the invention.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • fusion proteins of the invention may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337).
  • the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
  • the fusion proteins may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
  • Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
  • the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
  • the polyethylene glycol may have a branched structure.
  • Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
  • polyethylene glycol molecules should be attached to the protein with consideration of effects on functional or antigenic domains of the protein.
  • attachment methods available to those skilled in the art, such as, for example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), herein incorporated by reference; see also Malik et al., Exp. Hematol. 20:1028-1035 (1992), reporting pegylation of GM-CSF using tresyl chloride.
  • polyethylene glycol may be covalently bound through amino acid residues via reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C -terminal amino acid residue.
  • Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
  • polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues.
  • polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues.
  • One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
  • polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
  • the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
  • Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
  • pegylation of the fusion proteins of the invention may be accomplished by any number of means.
  • polyethylene glycol may be attached to the fusion protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al, Crit. Rev. Thera. Drug Carrier Sys.
  • One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresyl chloride (CISO 2 CH 2 CF 3 ).
  • MPEG monmethoxy polyethylene glycol
  • CISO 2 CH 2 CF 3 tresyl chloride
  • polyethylene glycol is directly attached to amine groups of the protein.
  • the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2- trifluoreothane sulphonyl group.
  • Polyethylene glycol can also be attached to proteins using a number of different intervening linkers.
  • U.S. Pat. No. 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins.
  • Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG- succinimidylsuccinate, MPEG activated with l,l'-carbonyldiimidazole, MPEG-2,4,5- trichloropenylca- rbonate, MPEG-p-nitrophenolcarbonate, and various MPEG- succinate derivatives.
  • polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in International Publication No. WO 98/32466, the entire disclosure of which is incorporated herein by reference.
  • Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.
  • the number of polyethylene glycol moieties attached to each fusion protein of the invention i.e., the degree of substitution
  • the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules.
  • the average degree of substitution within ranges such as 1-3,2-4, 3-5,4-6, 5-7,6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule.
  • Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249- 304 (1992).
  • polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
  • HPLC high performance liquid chromatography
  • the presence and quantity of fusion proteins of the invention may be determined using ELISA, a well known immunoassay known in the art.
  • ELISA protocol that would be useful for detecting/quantifying albumin fusion proteins of the invention, comprises the steps of coating an ELISA plate with an anti-human serum albumin antibody, blocking the plate to prevent non-specific binding, washing the ELISA plate, adding a solution containing the albumin fusion protein of the invention (at one or more different concentrations), adding a secondary anti-therapeutic protein specific antibody (e.g., an anti-opioid receptor agonist antibody) coupled to a detectable label (as described herein or otherwise known in the art), and detecting the presence of the secondary antibody.
  • a secondary anti-therapeutic protein specific antibody e.g., an anti-opioid receptor agonist antibody
  • the ELISA plate might be coated with an anti-therapeutic protein specific antibody (e.g., an anti-opioid receptor agonist antibody) and the labeled secondary reagent might be the anti-human albumin specific antibody.
  • the anti-human serum albumin antibody may be replaced with an anti-AFP antibody.
  • polynucleotides of the present invention are useful to produce the fusion proteins of the invention.
  • polynucleotides of the invention encoding fusion proteins
  • polynucleotides of the invention may be used in recombinant DNA methods useful in genetic engineering to make cells, cell lines, or tissues that express the fusion protein encoded by the polynucleotides encoding fusion proteins of the invention.
  • Polynucleotides of the present invention are also useful in gene therapy.
  • One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect.
  • the polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner.
  • Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. Additional non-limiting examples of gene therapy methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the sections labeled "Gene Therapy", and Examples 63 and 64).
  • the fusion proteins of the present invention are useful for diagnosis, treatment, prevention and/or prognosis of various disorders in mammals, preferably humans.
  • disorders include, but are not limited to, those described herein under the section heading "Biological Activities,” below.
  • the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression level of a certain polypeptide in cells or body fluid of an individual using a fusion protein of the invention; and (b) comparing the assayed polypeptide expression level with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide expression level compared to the standard expression level is indicative of a disorder.
  • a diagnostic method of a disorder involves (a) assaying the expression level of a certain polypeptide in cells or body fluid of an individual using a fusion protein of the invention; and (b) comparing the assayed polypeptide expression level with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide expression level compared to the standard expression level is indicative of a disorder.
  • the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease
  • fusion proteins of the present invention can be used to treat or prevent diseases or conditions such as, for example, neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions.
  • patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., opioid receptor agonist), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).
  • a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., opioid receptor agonist), to supplement absent or decreased levels of a different polypeptide (e.g., hemo
  • Transgenic organisms that express the fusion proteins of the invention are also included in the invention.
  • Transgenic organisms are genetically modified organisms into which recombinant, exogenous or cloned genetic material has been transferred. Such genetic material is often referred to as a transgene.
  • the nucleic acid sequence of the transgene may include one or more transcriptional regulatory sequences and other nucleic acid sequences such as introns, that may be necessary for optimal expression and secretion of the encoded protein.
  • the transgene may be designed to direct the expression of the encoded protein in a manner that facilitates its recovery from the organism or from a product produced by the organism, e.g. from the milk, blood, urine, eggs, hair or seeds of the organism.
  • the transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.
  • the transgene may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene.
  • the term "germ cell line transgenic organism” refers to a transgenic organism in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic organism to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic organisms.
  • a transgenic organism may be a transgenic animal or a transgenic plant.
  • Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,602,307; Mullins et al. (1993) Hypertension 22(4):630-633; Brenin et al.
  • a number of recombinant or transgenic mice have been produced, including those which-express an activated oncogene sequence (U.S. Pat. No. 4,736,866); express simian SV40 T-antigen (U.S. Pat. No. 5,728,915); lack the expression of interferon regulatory factor 1 (IRF-I) (U.S. Pat. No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Pat. No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Pat. No. 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Pat. No.
  • mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species.
  • Transgenic procedures have been successfully utilized in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al. (1997) MoI. Reprod. Dev. 46(4):515-526; Houdebine (1995) Reprod. Nutr. Dev. 35(6):609-617; Petters (1994) Reprod. Fertil. Dev. 6(5):643-645; Schnieke et al.
  • transgene-encoded protein of the invention may be put under the control of a promoter that is preferentially activated in mammary epithelial cells. Promoters that control the genes encoding milk proteins are preferred, for example the promoter for casein, beta lactoglobulin, whey acid protein, or lactalbumin (see, e.g., DiTullio (1992) BioTechnology 10:74-77; Clark et al. (1989) BioTechnology 7:487-492; Gorton et al.
  • a fusion protein of the invention can also be expressed in a transgenic plant, e.g. a plant in which the DNA transgene is inserted into the nuclear or plastidic genome. Plant transformation procedures used to introduce foreign nucleic acids into plant cells or protoplasts are known in the art. See, in general, Methods in Enzymology Vol. 153 ("Recombinant DNA Part D") 1987, Wu and Grossman Eds., Academic Press and European Patent Application EP 693554.
  • fusion proteins of the invention or formulations thereof may be administered by any conventional method including parenteral (e.g. subcutaneous or intramuscular) injection or intravenous infusion.
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • a fusion protein of the invention While it is possible for a fusion protein of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the fusion protein and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free. Fusion proteins of the invention are particularly well suited to formulation in aqueous carriers such as sterile pyrogen free water, saline or other isotonic solutions because of their extended shelf- life in solution.
  • pharmaceutical compositions of the invention may be formulated well in advance in aqueous form, for instance, weeks or months or longer time periods before being dispensed.
  • formulations containing the fusion protein may be prepared taking into account the extended shelf- life of the fusion protein in aqueous formulations. As discussed above, the shelf-life of many of these therapeutic proteins are markedly increased or prolonged after fusion to HA.
  • the fusion proteins of the invention can be formulated as aerosols using standard procedures.
  • aerosol includes any gas-borne suspended phase of a fusion protein of the instant invention which is capable of being inhaled into the bronchioles or nasal passages.
  • aerosol includes a gas-borne suspension of droplets of a fusion protein of the instant invention, as may be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition of a compound of the instant invention suspended in air or other carrier gas, which may be delivered by insufflation from an inhaler device, for example.
  • compositions may include aerosols which contain fusion proteins as described herein. Aerosols can be defined as very finely divided liquid droplets or dry particles dispersed in and surrounded by a gas. Both liquid and dry powder aerosol compositions are contemplated.
  • the aerosols may be administered by a dry-powder inhalers ("PDI”) or a metered-dose inhaler ("MDI”) which optionally may be pressurized (“pMDI").
  • PDI dry-powder inhalers
  • MDI metered-dose inhaler
  • pMDI pressurized
  • the formulations of the invention are also typically non-immunogenic, in part, because of the use of the components of the fusion protein being derived from the proper species.
  • both the opioid receptor agonist and serum protein (e.g., albumin or AFP) portions of the fusion protein will typically be human.
  • serum protein e.g., albumin or AFP
  • that component may be humanized by substitution of key amino acids so that specific epitopes appear to the human immune system to be human in nature rather than foreign.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the fusion protein with the carrier that constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation appropriate for the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit- dose or multi-dose containers, for example sealed ampules, vials or syringes, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders.
  • Dosage formulations may contain the therapeutic protein portion at a lower molar concentration or lower dosage compared to the non- fused standard formulation for the therapeutic protein given the extended serum half-life exhibited by many of the fusion proteins of the invention.
  • Formulations or compositions of the invention may be packaged together with, or included in a kit with, instructions or a package insert referring to the extended shelf- life of the fusion protein component.
  • instructions or package inserts may address recommended storage conditions, such as time, temperature and light, taking into account the extended or prolonged shelf- life of the fusion proteins of the invention.
  • Such instructions or package inserts may also address the particular advantages of the fusion proteins of the inventions, such as the ease of storage for formulations that may require use in the field, outside of controlled hospital, clinic or office conditions.
  • formulations of the invention may be in aqueous form and may be stored under less than ideal circumstances without significant loss of therapeutic activity.
  • Fusion proteins of the invention can also be included in nutraceuticals.
  • certain fusion proteins of the invention may be administered in natural products, including milk or milk product obtained from a transgenic mammal which expresses fusion protein.
  • Such compositions can also include plant or plant products obtained from a transgenic plant which expresses the fusion protein.
  • the fusion protein can also be provided in powder or tablet form, with or without other known additives, carriers, fillers and diluents. Nutraceuticals are described in Scott Hegenhart, Food Product Design, December 1993.
  • the invention also provides methods of treatment and/or prevention of diseases or disorders (such as, for example, any one or more of the diseases or disorders disclosed herein) by administration to a subject of an effective amount of a fusion protein of the invention or a polynucleotide encoding a fusion protein of the invention ("a fusion polynucleotide") in a pharmaceutically acceptable carrier.
  • a fusion polynucleotide encoding a fusion protein of the invention
  • the fusion protein and/or polynucleotide will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the fusion protein and/or polynucleotide alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners.
  • the "effective amount" for purposes herein is thus determined by such considerations.
  • the total pharmaceutically effective amount of the fusion protein administered parenterally per dose will be in the range of about lug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone.
  • the fusion protein is typically administered at a dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.
  • Fusion proteins and/or polynucleotides can be are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray.
  • “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any.
  • parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • Fusion proteins and/or polynucleotides of the invention are also suitably administered by sustained-release systems.
  • sustained-release fusion proteins and/or polynucleotides are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray.
  • “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • sustained-release fusion proteins and/or polynucleotides include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).
  • Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
  • polylactides U.S. Pat. No. 3,773,919, EP 58,481
  • copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al.,
  • Sustained-release fusion proteins and/or polynucleotides also may include liposomally entrapped fusion proteins and/or polynucleotides of the invention (see generally, 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. 317-327 and 353-365 (1989)).
  • Liposomes containing the fusion protein and/or polynucleotide are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci.
  • the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal therapeutic.
  • the fusion proteins and/or polynucleotides of the invention are delivered by way of a pump (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)).
  • a pump 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)).
  • Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
  • the fusion protein and/or polynucleotide is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • a pharmaceutically acceptable carrier i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the therapeutic.
  • the formulations are prepared by contacting the fusion protein and/or polynucleotide uniformly and intimately with liquid carriers or finely divided solid carriers or both.
  • the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient.
  • carrier vehicles include water, saline, Ringer's solution, and dextrose solution.
  • Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
  • the carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability.
  • Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.
  • buffers such as phosphate, cit
  • the fusion protein is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.
  • Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes).
  • Fusion proteins and/or polynucleotides generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • Fusion proteins and/or polynucleotides ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
  • a lyophilized formulation 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous albumin fusion protein and/or polynucleotide solution, and the resulting mixture is lyophilized.
  • the infusion solution is prepared by reconstituting the lyophilized fusion protein and/or polynucleotide using bacteriostatic Water- for-Injection.
  • the fusion protein formulations comprises 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromole sodium octanoate/milligram of fusion protein, 15 micrograms/milliliter polysorbate 80, pH 7.2.
  • the fusion protein formulations consists 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromole sodium octanoate/milligram of fusion protein, 15 micrograms/milliliter polysorbate 80, pH 7.2.
  • the pH and buffer are chosen to match physiological conditions and the salt is added as a tonicifier.
  • Sodium octanoate has-been chosen due to its reported ability to increase the thermal stability of the protein in solution.
  • polysorbate has been added as a generic surfactant, which lowers the surface tension of the solution and lowers non-specific adsorption of the fusion protein to the container closure system.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the fusion proteins and/or polynucleotides of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the fusion proteins and/or polynucleotides of the invention.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the fusion proteins and/or polynucleotides may be employed in conjunction with other therapeutic compounds.
  • fusion proteins and/or polynucleotides of the invention may be administered alone or in combination with other therapeutic agents.
  • fusion proteins and/or polynucleotides of the invention are administered alone or in combination with one or more intravenous immune globulin preparations.
  • Intravenous immune globulin preparations that may be administered with the fusion proteins and/or polynucleotides of the invention include, but not limited to, GAMMARTM, IVEEGAMTM, SANDOGLOBULINTM, GAMMAGARD S/DTM, ATGAMTM (antithymocyte glubulin), and GAMIMUNETM.
  • fusion proteins and/or polynucleotides of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).
  • Fusion proteins of the present invention can be administered to any animal, preferably to mammals and birds.
  • Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly preferred.
  • Fusion proteins and/or polynucleotides encoding fusion proteins of the present invention can be used in assays to test for one or more biological activities. If an fusion protein and/or polynucleotide exhibits an activity in a particular assay, it is likely that the therapeutic protein corresponding to the fusion portein may be involved in the diseases associated with the biological activity. Thus, the fusion protein could be used to treat the associated disease.
  • the present invention encompasses a method of treating a disease or disorder associated with pain comprising administering to a patient in which such treatment, prevention or amelioration is desired a fusion protein of the invention that comprises a therapeutic protein portion corresponding to a fusion protein in an amount effective to treat, prevent or ameliorate pain.
  • the present invention encompasses a method of treating a disease or disorder associated with pain comprising administering to a patient in which such treatment, prevention or amelioration is desired a fusion protein of the invention that comprises a therapeutic protein portion corresponding to the therapeutic protein for which the indications in the Examples are related in an amount effective to treat, prevent or ameliorate pain.
  • the above-recited applications have uses in a wide variety of hosts.
  • Such hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non- human primate, and human.
  • the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat.
  • the host is a mammal.
  • the host is a human.
  • PTA-3278 are used as cloning vectors into which polynucleotides encoding a therapeutic protein or fragment or variant thereof is inserted adjacent to and in translation frame with polynucleotides encoding human serum albumin "HSA".
  • pScCHSA may be used for generating therapeutic protein-HSA fusions
  • pScNHSA may be used to generate HSA-therapeutic protein fusions.
  • a vector to facilitate cloning DNA encoding a therapeutic protein N-terminal to DNA encoding the mature albumin protein was made by altering the nucleic acid sequence that encodes the chimeric HSA signal peptide in pPPC0005 to include the
  • ADHl terminator sequence were eliminated by digesting pPPC0005 with Xho I and
  • the primer comprised a nucleic acid sequence that encodes part of the signal peptide sequence of HSA, a kex2 site from the mating factor alpha leader sequence, and part of the amino-terminus of the mature form of HSA.
  • Four point mutations were introduced in the sequence, creating the Xho I and CIa I sites found at the junction of the chimeric signal peptide and the mature form of HSA as provided in the sequence shown below.
  • the nucleotides at these four positions from 5' to 3' are T, G, T, and G. 5'- GCCTCGAGAAAAGAGATGCACACAAGAGTGAGGTTGCTCATCGATTTAAA - GAT TTGGG-3' and 5'-
  • a second round of PCR was then performed with an upstream flanking primer, 5'-TACAAACTTAAGAGTCCAATT- AGC-3' a downstream flanking primer 5'-CACTTCTCTAGAGTGGTTTCATATGTCTT-S'.
  • the resulting PCR product was then purified and digested with AfI II and Xba I and ligated into the same sites in pPPCOOO ⁇ creating pScCHSA.
  • the resulting plasmid has Xho I and CIa I sites engineered into the signal sequence. The presence of the Xho I site creates a single amino acid change in the end of the signal sequence from LDKR to LEKR.
  • the D to E change will not be present in the final albumin fusion protein expression plasmid when a nucleic acid sequence comprising a polynucleotide encoding the therapeutic portion of the albumin fusion protein with a 5' Sal I site (which is compatible with the Xho I site) and a 3' CIa I site is ligated into the Xho I and CIa I sites of pScCHSA. Ligation of Sal I to Xho I restores the original amino acid sequence of the signal peptide sequence.
  • DNA encoding the therapeutic portion of the albumin fusion protein may be inserted after the Kex2 site (Kex2 cleaves after the dibasic amino acid sequence KR at the end of the signal peptide) and prior to the CIa I site.
  • a vector to facilitate cloning DNA encoding a therapeutic protein portion C- terminal to DNA encoding the mature albumin protein was made by adding three, eight-base-pair restriction sites to pScCHSA.
  • the Asc I, Fse I, and Pme I restriction sites were added in between the Bsu36 I and Hind III sites at the end of the nucleic acid sequence encoding the mature HSA protein. This was accomplished through the use of two complementary synthetic primers containing the Asc I, Fse I, and Pme I restriction sites. 5'-
  • the vectors pScNHSA and pScCHSA may be used as cloning vectors into which polynucleotides encoding a therapeutic protein or fragment or variant thereof is inserted adjacent to polynucleotides encoding mature human serum albumin "HSA".
  • pScCHSA is used for generating therapeutic protein-HSA fusions
  • pScNHSA may be used to generate HSA-therapeutic protein fusions.
  • DNA encoding an opioid receptor agonist protein, analog or variant may be PCR amplified using the primers which facilitate the generation of a fusion construct (e.g., by adding restriction sites, encoding seamless fusions, encoding linker sequences, etc.)
  • a fusion construct e.g., by adding restriction sites, encoding seamless fusions, encoding linker sequences, etc.
  • one skilled in the art could design a 5' primer that adds polynucleotides encoding the last four amino acids of the mature form of HSA (and containing the Bsu36I site) onto the 5' end of DNA encoding a therapeutic protein; and a 3' primer that adds a STOP codon and appropriate cloning sites onto the 3' end of the therapeutic protein coding sequence.
  • the forward primer used to amplify DNA encoding a therapeutic protein might have the sequence, 5'- aagctGCCTTAGGCTTA(N)i5-3', including a Bsu36I site, where the upper case nucleotides encode the last four amino acids of the mature HSA protein (ALGL) and (N) 15 is identical to the first 15 nucleotides encoding the therapetic protein of interest.
  • the reverse primer used to amplify DNA encoding a therapeutic protein might have the sequence, 5'-GCGCGCGTTTAAACGGCCGGCCGGC- GCGCC(N) 15 -3', including a Pme I site, an Fse I site, an Asc I site, the reverse complement of two tandem stop codons, and (N) 1S is identical to the reverse complement of the last 15 nucleotides encoding the therapeutic protein of interest.
  • the PCR product may be cut with Bsu36I and one of (Asc I, Fse I, or Pme I) and ligated into pScNHSA.
  • DNA encoding a therapeutic protein may be PCR amplified using the following primers: A 5' primer that adds polynucleotides containing a Sail site and encoding the last three amino acids of the HSA leader sequence, DKR, onto the 5' end of DNA encoding a therapeutic protein; and a 3' primer that adds polynucleotides encoding the first few amino acids of the mature HSA containing a CIa I site onto the 3' end of DNA encoding a therapeutic protein.
  • the forward primer used to amplify the DNA encoding a therapeutic protein might have the sequence, 5'-aggagcgtcGACAAAAGA(N) 1 5-3', including a Sal I site, where the upper case nucleotides encode the last three amino acids of the HSA leader sequence (DKR), and (N) 1S is identical to the first 15 nucleotides encoding the therapetic protein of interest.
  • the reverse primer used to amplify the DNA encoding a therapeutic protein might have the sequence, 5'- CTTTAAATCGATGAGCAACCTCACTCTTGTGTGCATC(N) 15 -3', including a CIa I site, the reverse complement of the DNA encoding the first 9 amino acids of the mature form of HSA (DAHKSEVAH), and (N) 15 is identical to the reverse complement of the last 15 nucleotides encoding the therapeutic protein of interest.
  • the PCR product Once the PCR product is amplified it may be cut with Sal I and CIa I and ligated into pScCHSA digested with Xho I and CIa I.
  • a different signal or leader sequence may be desired, for example, invertase "INV” (Swiss-Prot Accession P00724), mating factor alpha “MAF”(Genbank Accession AAAl 8405), MPIF (Geneseq AAF82936), Fibulin B (Swiss-Prot Accession P23142), Clusterin (Swiss-Prot Accession P10909), Insulin-Like Growth Factor-Binding Protein 4 (Swiss-Prot Accession P22692), and permutations of the HSA leader sequence can be subcloned into the appropriate vector by means of standard methods known in the art.
  • An expression vector compatible with yeast expression can be transformed into yeast S. cerevisiae by lithium acetate transformation, electroporation, or other methods known in the art and or as described in part in Sambrook, Fritsch, and Maniatis. 1989. "Molecular Cloning: A Laboratory Manual, 2nd edition", volumes 1- 3, and in Ausubel et al. 2000. Massachusetts General Hospital and Harvard Medical School “Current Protocols in Molecular Biology", volumes 1-4.
  • the expression vectors are introduced into S. cerevisiae strains DXYl, D88, or BXPlO by transformation, individual transformants can be grown, for example, for 3 days at 30° C.
  • YEPD 1% w/v yeast extract, 2% w/v, peptone, 2% w/v, dextrose
  • Supernatants are collected by clarifying cells at 3000 g for 10 minutes.
  • pSAC35 (Sleep et al., 1990, Biotechnology 8:42 and see FIG. 3) comprises, in addition to the LEU2 selectable marker, the entire yeast 2 ⁇ m plasmid to provide replication functions, the PRBl promoter, and the ADHl termination signal.
  • albumin fusion proteins of the invention comprise the mature form of HSA fused to either the N- or C-terminus of the mature form of a therapeutic protein or portions thereof (e.g. , an opioid receptor agonist, or an analog or a variant thereof).
  • albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression.
  • the signal peptide encoded by the signal sequence is removed, and the mature albumin fusion protein is secreted directly into the culture medium.
  • Albumin fusion proteins of the invention preferably comprise heterologous signal sequences (e.g., the non-native signal sequence of a particular therapeutic protein) including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art.
  • the fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
  • Albumin fusion proteins expressed in yeast as described above can be purified on a small-scale over a Dyax peptide affinity column as follows. Supernatants from yeast expressing an albumin fusion protein is diafiltrated against 3 mM phosphate buffer pH 6.2, 20 mM NaCl and 0.01% Tween 20 to reduce the volume and to remove the pigments. The solution is then filtered through a 0.22 ⁇ m device. The filtrate is loaded onto a Dyax peptide affinity column. The column is eluted with 100 mM Tris/HCl, pH 8.2 buffer. The peak fractions containing protein are collected and analyzed on SDS-PAGE after concentrating 5-fold.
  • the following method can be utilized.
  • the supernatant in excess of 2 L is diafiltered and concentrated to 500 mL in 20 mM Tris/HCl pH 8.0.
  • the concentrated protein solution is loaded onto a pre-equilibrated 50 mL DEAE-Sepharose Fast Flow column, the column is washed, and the protein is eluted with a linear gradient of NaCl from 0 to 0.4 M NaCl in 20 mM Tris/HCl, pH 8.0. Those fractions containing the protein are pooled, adjusted to pH 6.8 with 0.5 M sodium phosphate (NaH 2 PO 4 ).
  • a final concentration of 0.9 M (NH 4 ) 2 SO 4 is added to the protein solution and the whole solution is loaded onto a pre-equilibrated 50 mL Butyl650S column.
  • the protein is eluted with a linear gradient of ammonium sulfate (0.9 to 0 M (NH 4 ) 2 SO 4 ).
  • Those fractions with the albumin fusion are again pooled, diafiltered against 10 mM Na 2 HPO 4 /citric acid buffer pH 5.75, and loaded onto a 50 niL pre-equilibrated SP-Sepharose Fast Flow column.
  • the protein is eluted with a NaCl linear gradient from 0 to 0.5 M.
  • the fractions containing the protein of interest are combined, the buffer is changed to 10 mM Na 2 HPO 4 /citric acid pH 6.25 with an Amicon concentrator, the conductivity is ⁇ 2.5 mS/cm.
  • This protein solution is loaded onto a 15 mL pre-equilibrated Q-Sepharose high performance column, the column is washed, and the protein is eluted with a NaCl linear gradient from 0 to 0.15 M NaCl.
  • the purified protein can then be formulated into a specific buffer composition by buffer exchange.
  • Albumin fusion constructs can be generated in expression vectors for use in mammalian cell culture systems. DNA encoding a therapeutic protein can be cloned
  • N-terminus or C-terminus to HSA in a mammalian expression vector by standard methods known in the art (e.g., PCR amplification, restriction digestion, and ligation).
  • Suitable vectors are known in the art including, but not limited to, for example, the pC4 vector, and/or vectors available from Lonza
  • DHFR DiHydroFolate Reductase
  • the pC4:HSA vector is suitable for expression of albumin fusion proteins in
  • CHO cells For expression, in other mammalian cell culture systems, it may be desirable to subclone a fragment comprising, or alternatively consisting of, DNA which encodes for an albumin fusion protein into an alternative expression vector.
  • a fragment comprising, or alternatively consisting, of DNA which encodes for a mature albumin fusion protein may be subcloned into another expression vector including, but not limited to, any of the mammalian expression vectors described herein.
  • DNA encoding an albumin fusion construct is subcloned into vectors provided by Lonza Biologies, Inc. (Portsmouth, N. H.) by procedures known in the art for expression in NSO cells.
  • albumin fusion constructs can be generated in which the therapeutic protein portion is C terminal to the mature albumin sequence.
  • the same primer design used to clone into the yeast vector system may be employed (see Example 2).
  • albumin fusion constructs can be generated in which a therapeutic protein portion is cloned N terminal to the mature albumin sequence.
  • a therapeutic protein portion is cloned N terminal to the mature albumin sequence.
  • Bam HI or Hind III
  • CIa I sites of pC4:HSA When cloning into either the Bam HI or Hind III site, it is preferrable to include a Kozak sequence (CCGCCACCATG) prior to the translational start codon of the DNA encoding the therapeutic protein.
  • DNA encoding that therapeutic protein may be cloned in between the Xho I and CIa I sites of pC4:HSA.
  • the following 5' and 3' exemplary PCR primers may be used: 5'-
  • the 5' primer includes a Xho I site; and the Xho I site and the DNA following the Xho I site code for the last seven amino acids of the leader sequence of natural human serum albumin.
  • “(N)i8 M designates DNA identical to the first 18 nucleotides encoding the therapeutic protein of interest.
  • the 3' primer includes a CIa I site; and the CIa I site and the DNA following it are the reverse complement of the DNA encoding the first 10 amino acids of the mature HSA protein.
  • (N) 1 S” designates the reverse complement of DNA encoding the last 18 nucleotides encoding the therapeutic protein of interest.
  • the native albumin leader sequence can be replaced with the chimeric albumin leader, i.e., the HSA-kex2 signal peptide, or an alternative leader by standard methods known in the art. (For example, one skilled in the art could routinely PCR amplify an alternate leader and subclone the PCR product into an albumin fusion construct in place of the albumin leader while maintaining the reading frame).
  • An albumin fusion construct generated in an expression vector compatible with expression in mammalian cell-lines can be transfected into appropriate cell-lines by calcium phosphate precipitation, lipofectamine, electroporation, or other transfection methods known in the art and/or as described in Sambrook, Fritsch, and Maniatis. 1989. "Molecular Cloning: A Laboratory Manual, 2 nd edition” and in Ausubel et al. 2000. Massachusetts General Hospital and Harvard Medical School “Current Protocols in Molecular Biology", volumes 1-4. The transfected cells are then selected for by the presence of a selecting agent determined by the selectable marker in the expression vector.
  • the pC4 expression vector (ATCC Accession No. 209646) is a derivative of the plasmid pSV2-DHFR (ATCC Accession No. 37146).
  • pC4 contains the strong promoter Long Terminal Repeats "LTR" of the Rous Sarcoma Virus (Cullen et al., March 1985, Molecular and Cellular Biology, 438-447) and a fragment of the CytoMegalo Virus "CMV"-enhancer (Boshart et al., 1985, Cell 41 : 521-530).
  • the vector also contains the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40 early promoter.
  • the pEE12.1 expression vector is provided by Lonza Biologies, Inc. (Portsmouth, N.H.) and is a derivative of pEE6 (Stephens and Cockett, 1989, Nucl. Acids Res. 17: 7110).
  • This vector comprises a promoter, enhancer and complete 5'- untranslated region of the Major Immediate Early gene of the human CytoMegalo Virus, "hCMV-MIE" (International Publication # WO89/01036), upstream of a sequence of interest, and a Glutamine Synthetase gene (Murphy et al., 1991, Biochem J.
  • Stable CHO and NSO cell-lines transfected with albumin fusion constructs are generated by methods known in the art (e.g., lipofectamine transfection) and selected, for example, with 100 nM methotrexate for vectors having the DiHydroFolate Reductase "DHFR" gene as a selectable marker or through growth in the absence of glutamine.
  • Expression levels can be examined for example, by immunoblotting, primarily, with an anti-HSA serum as the primary antibody, or, secondarily, with serum containing antibodies directed to the therapeutic protein portion of a given albumin fusion protein as the primary antibody.
  • Expression levels are examined by immunoblot detection with anti-HSA serum as the primary antibody.
  • the specific productivity rates are determined via ELISA in which the capture antibody can be a monoclonal antibody towards the therapeutic protein portion of the albumin fusion and the detecting antibody can be the monoclonal anti-HSA-biotinylated antibody (or vice versa), followed by horseradish peroxidase/streptavidin binding and analysis according to the manufacturer's protocol.
  • albumin fusion proteins of the invention comprise the mature form of HSA fused to either the N- or C-terminus of the mature form of a therapeutic protein or portions thereof (e.g. , an opioid receptor agonist, or an analog or variant thereof).
  • albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression.
  • the signal peptide encoded by the signal sequence is removed, and the mature albumin fusion protein is secreted directly into the culture medium.
  • Albumin fusion proteins of the invention preferably comprise heterologous signal sequences (e.g., the non-native signal sequence of a particular therapeutic protein) including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art.
  • the fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
  • Albumin fusion proteins from mammalian cell-line supernatants are purified according to different protocols depending on the expression system used.
  • Purification of an albumin fusion protein from CHO cell supernatant or from transiently transfected 293T cell supernatant may involve initial capture with an anionic HQ resin using a sodium phosphate buffer and a phosphate gradient elution, followed by affinity chromatography on a Blue Sepharose FF column using a salt gradient elution. Blue Sepharose FF removes the main BSA/fetuin contaminants. Further purification over the Poros PI 50 resin with a phosphate gradient may remove and lower endotoxin contamination as well as concentrate the albumin fusion protein.
  • Purification from NSO Cell-Line Purification from NSO Cell-Line.
  • Purification of an albumin- fusion protein from NSO cell supernatant may involve Q-Sepharose anion exchange chromatography, followed by SP-sepharose purification with a step elution, followed by Phenyl-650M purification with a step elution, and, ultimately, diafiltration.
  • the purified protein may then be formulated by buffer exchange.
  • albumin fusion constructs of the invention have been deposited with the ATCC as shown in Table 3.
  • the albumin fusion constructs may comprise any one of the following expression vectors: the yeast S. cerevisiae expression vector pSAC35, the mammalian expression vector pC4, or the mammalian expression vector pEE12.1.
  • vectors comprise an ampicillin resistance gene for growth in bacterial cells.
  • These vectors and/or an albumin fusion construct comprising them can be transformed into an E. coli strain such as Stratagene XL-I Blue (Stratagene Cloning
  • the deposited material in the sample assigned the ATCC Deposit Number cited in Table 3 for any given albumin fusion construct also may contain one or more additional albumin fusion constructs, each encoding different albumin fusion proteins. Thus, deposits sharing the same ATCC Deposit Number contain at least an albumin fusion construct identified in the corresponding row of Table 3.
  • Two approaches can be used to isolate a particular albumin fusion construct from the deposited sample of plasmid DNAs cited for that albumin fusion construct in Table 3.
  • an albumin fusion construct may be directly isolated by screening the sample of deposited plasmid DNAs using a polynucleotide probe corresponding to the sequence of the construct using methods known in the art.
  • a polynucleotide probe corresponding to the sequence of the construct using methods known in the art.
  • a specific polynucleotide with 30-40 nucleotides may be synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported.
  • the oligonucleotide can be labeled, for instance, with 32 P-.gamma.-ATP using T4 polynucleotide kinase and purified according to routine methods.
  • the albumin fusion construct from a given ATCC deposit is transformed into a suitable host, as indicated above (such as XL-I Blue (Stratagene)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above.
  • the transformants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transformants (colonies) per plate.
  • DNA encoding a given albumin fusion protein may be amplified from a sample of a deposited albumin fusion construct, for example, by using two primers of 17-20 nucleotides that hybridize to the deposited albumin fusion construct 5' and 3' to the DNA encoding a given albumin fusion protein.
  • the polymerase chain reaction is carried out under routine conditions, for instance, in 25 ⁇ l of reaction mixture with 0.5 ug of the above cDNA template.
  • a convenient reaction mixture is 1.5-5 mM MgCl 2 , 0.01% (w/v) gelatin, 20 ⁇ M each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase.
  • Thirty five cycles of PCR denaturation at 94° C. for 1 min; annealing at 55° C. for 1 min; elongation at 72° C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler.
  • the amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified.
  • the PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.
  • RNA oligonucleotide is ligated to the 5' ends of a population of RNA presumably containing full-length gene RNA transcripts.
  • a primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5' portion of the desired full-length gene. This amplified product may then be sequenced and used to generate the full length gene.
  • RNA isolation starts with total RNA isolated from the desired source, although poly-A+ RNA can be used.
  • the RNA preparation can then be treated with phosphatase if necessary to eliminate 5' phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step.
  • the phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5' ends of messenger RNAs.
  • This reaction leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.
  • This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide.
  • the first strand synthesis reaction is used as a template for PCR amplification of the desired 5' end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest.
  • the resultant product is then sequenced and analyzed to confirm that the 5' end sequence belongs to the desired gene.
  • the cDNA for the opioid receptor agonist of interest may be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods.
  • the nucleotide sequences for all of these proteins are known and available, for instance, in public databases such as GenBank.
  • the cDNA can be tailored at the 5' and 3' ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the cDNA for HA.
  • the hormone cDNA is cloned into a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast.
  • the albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity.
  • the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 1). This expression cassette is then excised and inserted into a plasmid suitable for the trans fection of mammalian cell lines.
  • a polynucleotide encoding an albumin fusion protein of the present invention comprising a bacterial signal sequence is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' ends of the DNA sequence, to synthesize insertion fragments.
  • the primers used to amplify the polynucleotide encoding insert should preferably contain restriction sites, such as BamHI and Xbal, at the 5' end of the primers in order to clone the amplified product into the expression vector.
  • restriction sites such as BamHI and Xbal correspond to the restriction enzyme sites on the bacterial expression vector pQE-9.
  • This plasmid vector encodes antibiotic resistance (Amp 1 ), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.
  • Amp 1 antibiotic resistance
  • P/O IPTG-regulatable promoter/operator
  • RBS ribosome binding site
  • 6-His 6-histidine tag
  • coli strain M15/rep4 which contains multiple copies of the plasmid pREP4, which expresses the lad repressor and also confers kanamycin resistance (Kan 1 ). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.
  • Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml).
  • the O/N culture is used to inoculate a large culture at a ratio of 1 : 100 to 1 :250.
  • the cells are grown to an optical density 600 (O.D. 600 ) of between 0.4 and 0.6.
  • IPTG Isopropyl-B-D-thiogalacto pyranoside
  • IPTG induces by inactivating the lad repressor, clearing the P/O leading to increased gene expression.
  • Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000 ⁇ g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl or preferably in 8 M urea and concentrations greater than 0.14 M 2-mercaptoethanol by stirring for 3-4 hours at 4° C. (see, e.g., Burton et al, Eur. J. Biochem. 179:379-387 (1989)).
  • the chaotropic agent 6 Molar Guanidine HCl or preferably in 8 M urea and concentrations greater than 0.14 M 2-mercaptoethanol by stirring for 3-4 hours at 4° C.
  • Ni-NTA nickel- nitrilo-tri-acetic acid
  • the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8.
  • the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.
  • the purified protein is then renatured by dialyzing it against phosphate- buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl.
  • PBS phosphate- buffered saline
  • the protein can be successfully refolded while immobilized on the Ni- NTA column.
  • Exemplary conditions are as follows: renature using a linear 6M- IM urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors.
  • the renaturation should be performed over a period of 1.5 hours or more.
  • the proteins are eluted by the addition of 250 mM immidazole. Immidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl.
  • the purified protein is stored at 4° C. or frozen at -80°
  • the present invention further includes an expression vector, called pHE4a (ATCC Accession Number 209645, deposited on Feb. 25, 1998) which contains phage operator and promoter elements operative Iy linked to a polynucleotide encoding an albumin fusion protein of the present invention, called pHE4a. (ATCC Accession Number 209645, deposited on Feb. 25, 1998.)
  • This vector contains: 1) a neomycinphosphotransferase gene as a selection marker, 2) an E.
  • the origin of replication is derived from pU19 (LTI, Gaithersburg, Md.). The promoter and operator sequences are made synthetically.
  • DNA can be inserted into the pHE4a by restricting the vector with Ndel and
  • the DNA insert is generated according to PCR protocols described herein or otherwise known in the art, using PCR primers having restriction sites for Ndel (5' primer) and Xbal,
  • the PCR insert is gel purified and restricted with compatible enzymes.
  • the insert and vector are ligated according to standard protocols.
  • the engineered vector may be substituted in the above protocol to express protein in a bacterial system.
  • albumin fusion proteins of the present invention can be expressed in a mammalian cell.
  • a typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing.
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as, pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0.
  • Mammalian host cells that could be used include, but are not limited to, human HeIa, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CVl, quail QCl-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
  • the albumin fusion protein can be expressed in stable cell lines containing the polynucleotide encoding the albumin fusion protein integrated into a chromosome.
  • the co-transfection with a selectable marker such as DHFR, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.
  • the transfected polynucleotide encoding the fusion protein can also be amplified to express large amounts of the encoded fusion protein.
  • the DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin et al., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page et al., Biotechnology 9:64-68 (1991)).
  • Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.
  • Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession No.209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41 :521-530 (1985)).
  • LTR strong promoter
  • CMV-enhancer Boshart et al., Cell 41 :521-530 (1985)
  • the vectors also contain the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40 early promoter.
  • the plasmid pC6, for example, is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphates by procedures known in the art.
  • the vector is then isolated from a 1% agarose gel.
  • a polynucleotide encoding an albumin fusion protein of the present invention is generated using techniques known in the art and this polynucleotide is amplified using PCR technology known in the art. If a naturally occurring signal sequence is used to produce the fusion protein of the present invention, the vector does not need a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector can be-modified to include a heterologous signal sequence.
  • the amplified fragment encoding the fusion protein of the invention is isolated from a 1% agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., La Jo 11a, Calif). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
  • the amplified fragment encoding the albumin fusion protein of the invention is then digested with the same restriction enzyme and purified on a 1% agarose gel.
  • the isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase.
  • E. coli HBlOl or XL-I Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.
  • Chinese hamster ovary cells lacking an active DHFR gene is used for transfection.
  • Five ⁇ g of the expression plasmid pC6 or pC4 is cotransfected with 0.5 vg of the plasmid pSVneo using lipofectin (Feigner et al., supra).
  • the plasmid pSV2- neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
  • the cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418.
  • the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).
  • Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 ⁇ M, 2 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 ⁇ M.
  • Expression of the desired fusion protein is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.
  • the albumin fusion proteins of the invention can also be expressed in transgenic animals.
  • Animals of any species including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals.
  • techniques described herein or otherwise known in the art are used to express fusion proteins of the invention in humans, as part of a gene therapy protocol.
  • Any technique known in the art may be used to introduce the polynucleotides encoding the albumin fusion proteins of the invention into animals to produce the founder lines of transgenic animals.
  • Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11 :1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc.
  • transgenic clones containing polynucleotides encoding albumin fusion proteins of the invention for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).
  • the present invention provides for transgenic animals that carry the polynucleotides encoding the albumin fusion proteins of the invention in all their cells, as well as animals which carry these polynucleotides in some, but not all their cells, i.e., mosaic animals or chimeric.
  • the transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
  • the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci.
  • vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene.
  • the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)).
  • the regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the polynucleotide encoding the fsuion protien of the invention has taken place. The level of mRNA expression of the polynucleotide encoding the fusion protein of the invention in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR).
  • rt-PCR reverse transcriptase-PCR
  • Samples of fusion protein- expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the fusion protein.
  • founder animals Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal.
  • Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene (i.e., polynucleotide encoding an albumin fusion protein of the invention) on a distinct background that is appropriate for an experimental model of interest.
  • transgene i.e., polynucleotide encoding an albumin fusion protein of the invention
  • Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of fusion proteins of the invention and the therapeutic protein and/or albumin component of the fusion protein of the invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
  • the opioid receptor is expressed in a eukaryotic cell line such as CHO (Chinese Hamster Ovary) or HEK (Human Embryonic Kidney) 293 which are known to express G- coupled receptors and which contain a wide range of G-proteins allowing for functional coupling of the expressed opioid receptor to downstream effectors.
  • the transformed cells are assayed for activation of the expressed receptors in the presence of opioid receptor agonist fusion proteins.
  • Activity is measured by changes in intracellular second messengers, such as cyclic AMP (cAMP) or Ca 2+ . These may be measured directly using standard methods well known in the art, or by the use of reporter gene assays in which a luminescent protein (e.g.
  • firefly luciferase or green fluorescent protein is under the transcriptional control of a promoter responsive to the stimulation of protein kinase C by the activated receptor.
  • Assay technologies are available for both of these second messenger systems (cAMP and Ca ) to allow high throughput readout in multi-well plate format, such as the adenylyl cyclase activation FlashPlate Assay (NEN Life Sciences Products), or fluorescent Ca + indicators such as Fluo-4 AM (Molecular Probes) in combination with the FLIPR fluorimetric plate reading system (Molecular Devices).
  • opioid receptors may be expressed in engineered yeast systems which lack endogenous G-protein coupled receptors, thus providing the advantage of a null background for activation screening. These yeast systems substitute the opioid receptor and G-protein for the corresponding components of the endogenous yeast pheromone receptor pathway. Downstream signaling pathways are also modified so that the normal yeast-response to the signal is converted to positive growth on selective media or to reporter gene expression. (See, e.g., Broach, J. R. and J. Thorner (1996) Nature 384 (supp.): 14-16). The opioid receptors are screened against opioid receptor agonist fusion proteins or other naturally occurring bioactive molecules. Biological extracts from tissues, biological fluids and cell supernatants are also screened.
  • Antinociceptive effects can be assessed using a test that measures, for example, inflammatory pain, allodynia as a model for chronic pain, pain threshold, sensitivity to drug-induced analgesia, mechanical pain, chemical pain, hyperalgesia, thermal pain, and shock sensitivity.
  • Tests may include the allodynia/place avoidance, calibrated von Frey hairs for mechanical pain, conditioned suppression, formalin paw assay, Hargreaves test, cold plate test, cold water tail immersion test, hot plate test, hot water tail immersion test, tail flick test, tail pressure test, and the writhing test, paw pressure test, paw withdrawal, plantar test, susceptibility to actue administration of pharmacological agents in antinociception tests, and susceptibility to chronic administration of pharmacological agents in antinociception tests.
  • Antinociception effects may be assessed by placing an animal on a 55 0 C hot plate.
  • the latency to an observed hind limb response is measured in the presence or absence of an administered antinociceptive agent.
  • This assay is useful for measuring an animal's general analgesic response to a thermal stimulus, and is used to detect an antinociceptive effect.
  • the formalin paw assay measures the response to a noxious chemical injected into the hindpaw. Licking and biting of the hindpaw is quantitated as the amount of response time. Two phases of responses are typically observed with the first phase representing an acute pain response and the second phase representing a hyperalgesic response. Alterations in this normal biphasic display may be used to detect an antinociceptive effect on various forms of pain and chronic pain disorders. (See, e.g., Abbott et al., Pain 60:91- 102, 1995).
  • Forskolin-induced cyclic- AMP (cAMP) activation in the cells can be assayed by using a competitive imrnuno assay such as the CatchPoint cyclic-AMP
  • the cells used in this experiment were either the neuroblastoma cell line,
  • SK-N-SH, or HEK293 cells transiently transfected with an DNA vector (pcDNA3.1) encoding the expression of one of the full-length, human opioid receptors (Kappa,
  • the transient trans fections were performed using a lipofecamine reagent, such as Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA), one to three days prior to treatment with opioids.
  • the cells were seeded at 5xlO 4 /well into
  • the output for this assay is a measure of relative fluorescence units (RFL), where greater fluorescence is associated with higher concentrations of cAMP.
  • RRL relative fluorescence units
  • Inhibition of cAMP by opioid peptides or HSA fusion proteins was determined by comparison to cells treated with forskolin alone. Results are shown in Figures 12 and 13.
  • Figure 12 shows that enkephalin-HSA activates the Kappa-, Delta- and Mu opioid receptors in 293 cells over expressing the receptors.
  • Figure 13 shows that ⁇ - endorphin-HSA activates the opioid delta 1 receptor in 293 cells.
  • opioid peptides and peptide fusions (collectively referred to as "opioid peptides" in this example) in vivo.
  • HSA fusion proteins were purified by methods known in the art. Briefly, HSA fusion proteins were purified using blue sepharose affinity chromatogarphy. For injection, HSA fusion proteins were dissolved in vehicle (phosphate buffered saline at pH 7.2). The diluted proteins were injected subcutaneously in the midscapular region on C57BL/6 mice.
  • Oxycodone was purchased from Shady Grove Pharmacy, and was prepared by disolving in phoshpate buffered saline. Oxycodone was administered orally.
  • mice were injected subcutaneously with an amount of opioid peptide as described above in table 1, at approximately
  • the vehicle carrier
  • oxycodone was dissolved in the vehicle and was administered at 10 mg/kg in a volume of 5 ml/kg.
  • Hot plate tests and tail flick tests were performed by standard methods, well known in the art. Results are shown in Figures 14-16.

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Abstract

L'invention concerne des protéines de fusion qui comprennent une protéine agoniste du récepteur opioïde, ou un fragment, une variante ou un analogue de celle-ci fusionnée avec une protéine de sérum. L'invention concerne également des protéines de sérum convenant aux protéines de fusion dans l'albumine et aux protéines hybrides d'alpha-féroprotéine (AFP) et d'albumine-AFP. Les protéines de fusion présentent une activité agoniste du récepteur opioïde. Généralement, les protéines de fusion peuvent présenter une activité in vivo ou in vitro agoniste du récepteur opioïde étendue ou stabilisée par rapport à un agoniste du récepteur opioïde qui n'est pas fusionné avec la protéine de sérum.
PCT/US2007/082363 2006-10-24 2007-10-24 Protéines de fusion agonistes du récepteur opioïde WO2008052043A2 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009040067A2 (fr) * 2007-09-11 2009-04-02 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent thérapeutique
WO2009040018A2 (fr) * 2007-09-11 2009-04-02 Mondobiotech Laboratories Ag Utilisation d'un peptide comme agent thérapeutique
WO2009039983A3 (fr) * 2007-09-11 2009-05-28 Mondobiotech Lab Ag Utilisation d'un peptide en tant qu'agent thérapeutique
EP2476410A1 (fr) * 2011-01-13 2012-07-18 Theralpha Composition analgésique contenant un peptide pour l'administration à voie transbuccale
WO2012095523A1 (fr) * 2011-01-13 2012-07-19 Theralpha Composition analgésique pour une administration transbuccale
WO2014081864A1 (fr) * 2012-11-20 2014-05-30 Eumederis Pharmaceuticals, Inc. Agents pharmaceutiques peptidiques améliorés
US10577405B2 (en) 2014-05-28 2020-03-03 Mederis Diabetes Llc Peptide pharmaceuticals for insulin resistance
EP3592762A4 (fr) * 2017-03-10 2020-12-30 Bolt Threads, Inc. Compositions et procédés de production de rendements sécrétés élevés de protéines recombinées
EP3592800A4 (fr) * 2017-03-10 2021-01-06 Bolt Threads, Inc. Compositions et procédés de production de rendements sécrétés élevés de protéines recombinées
US11065304B2 (en) 2012-11-20 2021-07-20 Mederis Diabetes, Llc Peptide pharmaceuticals for insulin resistance
US11377482B2 (en) 2017-11-14 2022-07-05 Arcellx, Inc. D-domain containing polypeptides and uses thereof
US11464803B2 (en) 2017-11-14 2022-10-11 Arcellx, Inc. D-domain containing polypeptides and uses thereof
US11541028B2 (en) 2018-01-03 2023-01-03 Altimmune Inc. Peptide pharmaceuticals for treatment of NASH and other disorders
US11730763B2 (en) 2017-11-14 2023-08-22 Arcellx, Inc. Multifunctional immune cell therapies

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107964047B (zh) * 2017-12-18 2018-11-02 哈尔滨工业大学 基于内吗啡肽-1和神经降压素(8-13)的嵌合肽及其合成方法和应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4469631A (en) * 1980-03-10 1984-09-04 The Regents Of The University Of California Fusion peptides comprising an expressible bacterial gene and β-endorphin
WO2000069900A2 (fr) * 1999-05-17 2000-11-23 Conjuchem, Inc. Protection de peptides therapeutiques endogenes contre l'activite peptidase par conjugaison de composants sanguins
US20030022308A1 (en) * 1992-01-31 2003-01-30 Aventis Behring L.L.C. Fusion polypeptides of human serum albumin and a therapeutically active polypeptide
WO2005077042A2 (fr) * 2004-02-09 2005-08-25 Human Genome Sciences, Inc. Proteines hybrides d'albumine

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4469631A (en) * 1980-03-10 1984-09-04 The Regents Of The University Of California Fusion peptides comprising an expressible bacterial gene and β-endorphin
US20030022308A1 (en) * 1992-01-31 2003-01-30 Aventis Behring L.L.C. Fusion polypeptides of human serum albumin and a therapeutically active polypeptide
WO2000069900A2 (fr) * 1999-05-17 2000-11-23 Conjuchem, Inc. Protection de peptides therapeutiques endogenes contre l'activite peptidase par conjugaison de composants sanguins
WO2005077042A2 (fr) * 2004-02-09 2005-08-25 Human Genome Sciences, Inc. Proteines hybrides d'albumine

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BAGGIO L L ET AL: "A Recombinant Human Glucagon-Like Peptide (GLP)-1-Albumin Protein (Albugon) Mimics Peptidergic Activation of GLP-1 Receptor-Dependent Pathways Coupled With Satiety, Gastrointestinal Motility, and Glucose Homeostasis" DIABETES, NEW YORK, NY, US, vol. 53, 1 September 2004 (2004-09-01), pages 2492-2500, XP003004469 ISSN: 0012-1797 *
HUGHES J ET AL: "Identification of two related pentapeptides from the brain with potent opiate agonist activity." NATURE 18 DEC 1975, vol. 258, no. 5536, 18 December 1975 (1975-12-18), pages 577-580, XP009104778 ISSN: 0028-0836 *
KELLAM BARRIE ET AL: "Synthesis and in vitro evaluation of lipoamino acid and carbohydrate-modified enkephalins as potential antinociceptive agents" INTERNATIONAL JOURNAL OF PHARMACEUTICS (AMSTERDAM), vol. 161, no. 1, 9 February 1998 (1998-02-09), pages 55-64, XP002492914 ISSN: 0378-5173 *
OSBORN B L ET AL: "Pharmacokinetic and Pharmacodynamic Studies of a Human Serum Albumin-Interferon-alpha Fusion Protein in Cynomolgus Monkeys" JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, EXPERIMENTAL THERAPEUTICS, BALTIMORE, MD, vol. 303, no. 2, 1 January 2002 (2002-01-01), pages 540-548, XP003006693 ISSN: 0022-3565 *
SCHLECHTINGEN GEORG ET AL: "Structure-activity relationships of dynorphin a analogues modified in the address sequence." JOURNAL OF MEDICINAL CHEMISTRY 22 MAY 2003, vol. 46, no. 11, 22 May 2003 (2003-05-22), pages 2104-2109, XP002492913 ISSN: 0022-2623 *
VARGA E V ET AL: "The third extracellular loop of the human delta-opioid receptor determines the selectivity of delta-opioid agonists" MOLECULAR PHARMACOLOGY, BALTIMORE, MD, US, vol. 50, no. 6, 1 December 1996 (1996-12-01), pages 1619-1624, XP009104735 ISSN: 0026-895X *

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Publication number Priority date Publication date Assignee Title
WO2009040018A2 (fr) * 2007-09-11 2009-04-02 Mondobiotech Laboratories Ag Utilisation d'un peptide comme agent thérapeutique
WO2009039983A3 (fr) * 2007-09-11 2009-05-28 Mondobiotech Lab Ag Utilisation d'un peptide en tant qu'agent thérapeutique
WO2009040018A3 (fr) * 2007-09-11 2009-09-03 Mondobiotech Laboratories Ag Utilisation d'un peptide comme agent thérapeutique
WO2009040067A3 (fr) * 2007-09-11 2009-09-24 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent thérapeutique
WO2009040067A2 (fr) * 2007-09-11 2009-04-02 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent thérapeutique
EP2476410A1 (fr) * 2011-01-13 2012-07-18 Theralpha Composition analgésique contenant un peptide pour l'administration à voie transbuccale
WO2012095523A1 (fr) * 2011-01-13 2012-07-19 Theralpha Composition analgésique pour une administration transbuccale
US11065304B2 (en) 2012-11-20 2021-07-20 Mederis Diabetes, Llc Peptide pharmaceuticals for insulin resistance
WO2014081864A1 (fr) * 2012-11-20 2014-05-30 Eumederis Pharmaceuticals, Inc. Agents pharmaceutiques peptidiques améliorés
US11911447B2 (en) 2012-11-20 2024-02-27 Mederis Diabetes Llc Peptide pharmaceuticals for insulin resistance
US10577405B2 (en) 2014-05-28 2020-03-03 Mederis Diabetes Llc Peptide pharmaceuticals for insulin resistance
EP3592800A4 (fr) * 2017-03-10 2021-01-06 Bolt Threads, Inc. Compositions et procédés de production de rendements sécrétés élevés de protéines recombinées
US11306127B2 (en) 2017-03-10 2022-04-19 Bolt Threads, Inc. Compositions and methods for producing high secreted yields of recombinant proteins
US11370815B2 (en) 2017-03-10 2022-06-28 Bolt Threads, Inc. Compositions and methods for producing high secreted yields of recombinant proteins
US11725030B2 (en) 2017-03-10 2023-08-15 Bolt Threads, Inc. Compositions and methods for producing high secreted yields of recombinant proteins
EP3592762A4 (fr) * 2017-03-10 2020-12-30 Bolt Threads, Inc. Compositions et procédés de production de rendements sécrétés élevés de protéines recombinées
US11377482B2 (en) 2017-11-14 2022-07-05 Arcellx, Inc. D-domain containing polypeptides and uses thereof
US11464803B2 (en) 2017-11-14 2022-10-11 Arcellx, Inc. D-domain containing polypeptides and uses thereof
US11730763B2 (en) 2017-11-14 2023-08-22 Arcellx, Inc. Multifunctional immune cell therapies
US11541028B2 (en) 2018-01-03 2023-01-03 Altimmune Inc. Peptide pharmaceuticals for treatment of NASH and other disorders

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