WO1992010576A1 - Fusion polypeptides - Google Patents

Fusion polypeptides Download PDF

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Publication number
WO1992010576A1
WO1992010576A1 PCT/US1991/009152 US9109152W WO9210576A1 WO 1992010576 A1 WO1992010576 A1 WO 1992010576A1 US 9109152 W US9109152 W US 9109152W WO 9210576 A1 WO9210576 A1 WO 9210576A1
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ala
seq
leu
tyr
pro
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PCT/US1991/009152
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English (en)
French (fr)
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Teresa M. Kubiak
Satish K. Sharma
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The Upjohn Company
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Priority to SK60893A priority Critical patent/SK60893A3/sk
Priority to JP4501996A priority patent/JPH06503473A/ja
Priority to AU91165/91A priority patent/AU662508B2/en
Priority to RU93045577A priority patent/RU2114119C1/ru
Priority to CS931093A priority patent/CZ109393A3/cs
Publication of WO1992010576A1 publication Critical patent/WO1992010576A1/en
Priority to NO93932148A priority patent/NO932148L/no

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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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • 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
    • C07K14/575—Hormones
    • C07K14/57509—Corticotropin releasing factor [CRF] (Urotensin)
    • 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
    • C07K14/575—Hormones
    • C07K14/57536—Endothelin, vasoactive intestinal contractor [VIC]
    • 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
    • C07K14/575—Hormones
    • C07K14/60—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
    • 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
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • 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
    • C07K14/575—Hormones
    • C07K14/655—Somatostatins
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • FUSION POLYPEP ⁇ DES FIELD OF THE INVENTION The present invention relates to non-naturally occurring fusion polypeptides containing N-terminal portions cleavable by dipeptidylpeptidase IV (DPP IV). BACKGROUND OF THE INVENTION
  • the techniques of molecular biology, specifically recombinant DNA technology, allow for the production of relatively large quantities of desirable biologically active polypeptides.
  • the genetic information encoding the polypeptides may be modified to produce relatively large quantities of modified polypeptides. Modifications made to the polypeptides are often used to improve their activity or facilitate their production and/or preparation.
  • DPP IV dipeptidylpeptidase IV
  • DPP IV was first reported in Hopsu-Havu, V.K. and G.G. Glenner, Histo. Chemie 3:197-201 (1966) and has been shown to be present in many mammalian tissues. DPP IV is presently commercially available from Enzyme Systems Products (Dublin, California). DPP IV recognizes specific amino acid sequences on the N-terminus of proteins.
  • DPP IV will cleave a dipeptide from the N-terminus when the second amino acid from the N-terminus is proline (Pro), hydroxyproline (Hyp), alanine (Ala), serine (Ser), and threonine (Thr) and any amino acid is at the N-terminus residue position provided if proline or hydroxyproline is not the amino acid residue third from the N-terminus.
  • DPP IV activity is more efficient when proline or alanine is the second amino acid from the N-terminus and is usually most efficient when that position is occupied by proline.
  • the activity of DPP IV in the stepwise cleavage of "PRO" parts of precursors of naturally occurring peptides is widely reported.
  • biologically active proteins can be synthesized using peptide synthesizers or in host cells using recombinant DNA technology.
  • biologically active proteins are administered as drugs.
  • active proteins are used as therapeutics, prophylactics or to enhance or repress traits. Since DPP IV and other proteases degrade proteins, these drugs are susceptible to .degradation.
  • DPP IV and other proteases degrade proteins, these drugs are susceptible to .degradation.
  • a problem of using biologically active polypeptides as drugs is that their sustained presence is diminished and they must therefore be administered more frequently.
  • recombinant DNA technology may be used to provide desired polypeptides in the form of non-naturally occuring proteins which contain a linker peptide that may be used as a ligand or other target for purification means.
  • a linker peptide that may be used as a ligand or other target for purification means.
  • U.S. patent number 4,782,137 relates to synthesis of a non- naturally occuring peptide containing an antigenic linker peptide.
  • the non-naturally occuring protein can be passed through a column containing immobilized antibodies which bind to the antigenic linker, thus isolating the non-naturally occuring protein.
  • Patent 4,569,794 relates to a process of purifying non-naturally occuring proteins which contain N-terminal extensions that have an affinity for immobilized metals.
  • the non-naturally occuring proteins bind to immobilized metal ions in a column.
  • One problem with these methods is that the linker peptide is often undesirable and removal of the linker can be difficult.
  • the present invention relates to non-naturally occurring fusion proteins which comprise a core protein portion and an N-terminal extension which is cleavable by DPP IV.
  • a non-naturally occuring protein is provided wherein the extension attached to the core protein is not an N-terminal extension that occurs in nature attached to the core peptide; hence, non-naturally occurring fusion protein.
  • the present invention relates to prodrugs which are DPP IV cleavable non-naturally occuring proteins wherein the core protein portion is a biologically active protein.
  • the present invention relates to DPP IV cleavable non- naturally occuring proteins useful in purification methods whereby the N-terminal extension provides a feature or property which facilitates purification of the non-naturally occuring protein.
  • the present invention provides non-naturally occuring proteins which have N-terminal extensions that are cleavable by DPP IV such that exposure of the non-naturally occuring protein to DPP IV results in conversion of the non-naturally occuring protein to a desirable protein.
  • the non-naturally occuring protein is processed into a biologically active protein in vivo using DPP IV present in the target species.
  • non-naturally occuring protein can be purified by using its specifically designed N-terminus as a ligand and then processed with DPP IV to remove the N-terminal extension and liberate a desired protein.
  • the present invention allows for the production of a desired protein as a non-naturally occuring protein that is later converted to the desired protein when exposed to DPP IV.
  • Prodrugs are converted to drugs over a course of time using the patients' endogenous DPP IV, thereby achieving sustained presence of the active drug in a patient and reducing the frequency of administration.
  • Pure desired proteins can be isolated using the present invention by producing and purifying non-naturally occuring proteins and then processing the non-naturally occuring proteins in vitro with DPP IV to produce the desired protein.
  • the present invention relates to a non-naturally occurring fusion protein comprising an extension peptide portion covalently linked at its C-terminus to the N-terminus of a core protein portion, said extension peptide portion being of the formula:
  • A is optional and when present is methionine; n is 0-20;
  • X is selected from the group consisting of all naturally occurring amino acid residues;
  • X' is selected from the group consisting of all naturally occurring amino acid residues except proline and hydroxyproline;
  • Y is selected from the group consisting of proline, hydroxyproline, alanine, serine and threonine except when n is 0 then Y is selected from the group consisting of alanine, serine and threonine.
  • the present invention also relates to the use of such non-naturally occuring proteins in medicinal preparations and to a method of purifying desired proteins from a mixture containing such non-naturally occuring proteins and impurities comprising the steps of selectively contacting said non-naturally occuring protein with material which immobilizes said non- naturally occuring protein, removing said impurities, separating said non-naturally occuring proteins from said material, contacting said non-naturally occuring protein with DPP IV, and isolating said desired protein.
  • the invention describes a process of isolating fusion proteins which have biologically active polypeptides at the C- terminal end and an N-terminal extension linker that is a metal ion chelating linker.
  • the fusion peptide has an affinity to immobilized metal ions. Impurities can be removed by passing a mixture containing the fusion protein through a column containing immobilized metal ions. The fusion protein becomes associated with the metal ions and only the impurities are eluted. Upon changing conditions the fusion peptide is liberated from the immobilized metal ions thus resulting in purified fusion protein.
  • U.S. Patent No. 4,782,137 issued November 1, 1988 to Hopp et al. discloses the synthesis of a fusion protein having a highly antigenic N-terminal portion and a desired polypeptide at the C-terminal portion.
  • the fusion proteins are purified from crude supernatant by passing crude supernatent through a column containing immobilized antibodies which recognize the antigenic portion of the fusion protein.
  • the immobilized antibodies keep the protein in the column while the undesired components of the supernatent are eluted.
  • the column conditions can then be changed to cause the antigen- antibody complex to dissociate.
  • the fusion protein is then eluted and collected.
  • European patent application Publication Number 0 220 958 published May 6, 1987 relates to selective chemical removal of N-terminal residues.
  • the invention relates to a process and compounds useful in the process to remove N-terminal residues from desired polypeptides.
  • the desired polypeptide exists as a fusion protein having the desired polypeptide link at the N- terminal to a linker having the formula X-Pro.
  • a linker having the formula X-Pro Upon exposure of the fusion protein to specific buffer conditions a diketopiperazine of the X-Pro portion of the fusion protein is formed and cleaved, thereby producing the desired polypeptide from the fusion precursor.
  • the fusion proteins of EPO 220,958 ('958) is not included in the present invention because according to the present invention, when the N-terminal extension is only a dipeptide, i.e., when A is absent, n is zero and X is a naturally-occuring amino acid, Y is either alanine, serine or threonine. Thus, whenever the extension is a dipeptide, it is X-Ala, X-Ser or X-Thr.
  • the '958 application teaches chemical, not enzymatic, cleavage of the dipeptide X-Pro.
  • the dipeptide X- Ala, X-Ser and X-Thr are not susceptible to the type chemical cleavage taught by the '958 application that cuts the X-Pro extension from the core protein.
  • Australian Patent Application Document No. AU-A-12709/88 discloses fusion proteins which contain affinity peptides useful in immobilized metal affinity chromatography (IMAC).
  • the affinity peptides disclosed contained at least two neighboring histidine residues.
  • the IMAC purification means disclosed requires a special synthetic chemistry for making nitrilo- triacetic acid (NTA) resins.
  • Tallon, M.A., et al., Biochem. 26:7767-7774 (1987) relate to synthesis of extended analogs of the fridecapeptide ⁇ -factor from Saccharomyces cerevisiae.
  • the syntiiesized analog are extended ⁇ -factors, which represent sequences of naturally occurring pro- ⁇ -factor coded fo in the MF ⁇ l structural gene.
  • Promelittin is the main constituent of honeybee venom. In the amino acid sequence of the N- terminal portion of the precursor, every second residue is either proline or alanine.
  • DPP IV dipeptidylpeptidase IV.
  • the N-terminal region of the precursor is cleaved in a stepwise fashion producing the mature protein.
  • Promelittin unlike fusion proteins according to the present invention, is a naturally-occurring protein.
  • the yeast ⁇ -factor unlike fusion proteins according to the present invention, is a naturally-occurrin protein.
  • Mentlein, R., FEB, Vol. 234, No. 2, pp. 251-256 reviews proline residues in the maturation and degradation of peptide hormones and neuropeptides. It is reported that in mammals, proline specific proteases such as DPP IV are not involved in the biosynthesis of regulatory peptides but may play an important role in the degradation of them. Thus, it is concluded that while in vertebrates and lower vertebrates precursor proteins rely on DPP IV to convert precursors to mature forms, the processing of regulatory proteins in mammals generally uses DPP IV as a degradation protease.
  • Kubiak, T.M., et al, Drug Metabolism and Disposition, Vol. 17, No. 4, pp. 393-397 (1989) refer to the in vitro metabolic degradation of bovine growth hormone releasing factor (bGRF) analogs in bovine and porcine plasma and the correlation with plasma DPP IV activity.
  • the bGRF analogs tested had an Ala residue at position 2- of the N-terminus. It is reported that the metabolic degradation of bGRF in plasma is due to the presence of DPP IV in the plasma.
  • Kreil, G., TIBS 15:23-26 reviews of the stepwise cleavage of dipeptides by DPP's in the conversion of precursors to final products.
  • the precursors, described by Kreil are naturally-occurring proteins.
  • the fusion proteins of the present invention are non-naturally- occurring fusion proteins. Boman, et al., J. Biol. Chem. 264:5852-5860 (1989) demonstrated that a dipeptidyl peptidase isolated from cecropia pupae (with similar specificity to DPP IV) was able to remove natural N-terminal sequences of Ala-Pro-Glu-Pro from the N-terminal of synthetic copies of the natural precursors of cecropia A and B.
  • the preprocecropin disclosed by Boman is a naturally- occurring protein. Dalboge, H., et al, Bio/technology, 5:161-164 (February 1987) disclose converting E. coli produced precursor of human growth hormone (hGH) to authentic hGH in vitro. The N- terminal extension of the precursor is removed by dipeptidypeptidase I.
  • non-naturally occurring fusion protein As used herein, the terms “non-naturally occurring fusion protein”, “non-naturally occurring fusion polypeptide”, “fusion polypeptides” and “fusion proteins” refer interchange ⁇ ably to proteins and polypeptides which do not normally occur in nature and which comprise a core protein portion and an extension portion.
  • core protein As used herein "core protein”, “core protein portion” and “polypeptide portion” refers to the portion of a fusion polypeptide which is located at the C-terminus end of the molecule and which, absent the extension portion, would be a desired polypeptide and/or a biologically active protein including naturally occuring biologically active proteins and polypeptides and analogs and mutants thereof.
  • N-terminal extension refers to the first up to about 45 amino acids starting at the N-terminus and which are not part of the core protein.
  • prodrugs refers to fusion proteins wherein the biologically desired portion is a biologically active protein useful as a drug.
  • biologically active protein and “biologically active polypeptides” refer to interchangeable proteins and polypeptides which possess biological activity.
  • verified protein and “desirable protein” refer interchangeable to proteins and polypeptides which are sought in pure form.
  • extension portion refers to the portion of a fusion protein which is an N-terminal extension and which is not part of the biologically desired portion.
  • DPP IV cleavable N-terminal extension portion refers to the extension portion of a fusion protein which has an amino acid sequence that can be removed by the stepwise cleavage by DPP IV.
  • Xaa 29 represents C-terminally amidated Argininyl residue.
  • Xaa 29 represents C-terminally amidated Argininyl residue.
  • Xaa 29 represents C-terminally amidated Argininyl residue.
  • Xaa 29 represents C-terminally amidated Argininyl residue.
  • Xaa 31 represents C-terminally amidated Argininyl residue.
  • Xaa 33 represents C-terminally amidated Argininyl residue.
  • Xaa 39 represents C-terminally amidated Argininyl residue.
  • Xaa 45 represents C-terminally amidated Argininyl residue.
  • Xaa 27 represents C-terminally amidated Argininyl residue.
  • Xaa 31 represents C-terminally amidated Argininyl residue.
  • Li Seq ID No. 26 Xaa 33 represents C-terminally amidated Argininyl residue.
  • Xaa 35 represents C-terminally amidated Argininyl residue.
  • Xaa 37 represents C-terminally amidated Argininyl residue.
  • Xaa 33 represents C-terminally amidated Argininyl residue.
  • Xaa 35 represents C-terminally amidated Argininyl residue.
  • Xaa 37 represents C-termi ⁇ ally amidated Argininyl residue.
  • Xaa 39 represents C-terminally amidated Argininyl residue.
  • Xaa 45 represents C-terminally amidated Argininyl residue.
  • Xaa 43 represents C-terminally amidated Argininyl residue.
  • Xaa 45 represents C-terminally amidated Argininyl residue.
  • Xaa 31 represents C-terminally amidated Argininyl residue.
  • Xaa 31 represents C-terminally amidated Argininyl residue.
  • Xaa 31 represents C-terminally amidated Argininyl residue.
  • Xaa 31 represents C-terminally amidated Argininyl residue.
  • Xaa 33 represents C-terminally amidated Argininyl residue.
  • Xaa 31 represents C-terminally amidated Argininyl residue.
  • Xaa 33 represents C-terminally amidated Argininyl residue.
  • the present invention relates to improved proteins and polypeptides.
  • biologically active polypeptides are first produced as fusion proteins which contain the two portions: a first portion which represents the core protein portion; and, a second portion which is an N-terminal extension portion that is covalently linked at its carboxy terminus to the amino terminus of the first portion.
  • the N-terminal extension portion of the fusion polypeptide possesses an amino acid sequence which renders it subject to cleavage by the dipeptidylpeptidase IV (DPP IV).
  • DPP IV dipeptidylpeptidase IV
  • a fusion protein according to the present invention has the formula: Extension portion - Core protein portion wherem "Extension portion” represents a DPP IV cleavable N-terminal extension; " - " represents a coval.ent peptide bond; and, "core protein portion” represents any desired peptide which is liberated from the Extension portion by DPP IV processing.
  • the Extension portion of a fusion protein according to the present invention has an amino acid sequence according to the formula: A-X-Y(X'-Y) n wherein A is optional, and when present is methionine; n represents the number of sequentially linked X'-Y groups, that number representing from 0 to 20 of such groups, preferably 0 to 10 groups.
  • X is selected from the group consisting of any naturally occuring amino acid
  • Fusion proteins in which (A) is present as methionine (Met) represent sequences useful for the production of biologically active proteins by recombinant DNA methods in E. coli.
  • the Met sequence present in these precursors usually will be processed by the E. coli enzymatic system or some other means which can be performed by a person with ordinary skill in the art.
  • Protein synthesis in E. coli is, under normal circumstances, initiated at the translation initiation codon AUG coding for Met. As a consequence, the newly synthesized polypeptides have a methionine residue as their N-terminal amino acid.
  • coli possesses an enzymatic activity with the capacity to effectively remove N-terminal Met when the Met N-terminal residue is adjacent to an amino acid with a relatively small side chain like Gly, Ala or Ser as well as Pro.
  • Highly specific removal of the N-terminal Met can be accomplished using cyanogen bromide mediated cleavage of Met.
  • the N-terminal Met must be the only Met in the entire protein sequence; otherwise the cleavage will take place after each Met in the sequence.
  • the second amino acid from the N-terminus must be Pro, Gly, Ala or Ser if the Met is to be removed by the E. coli enzymatic system.
  • the present invention relates to: recombinant DNA molecules which comprise DNA sequences that encode the fusion polypeptides; methods of using the recombinant DNA molecules; methods of using the fusion polypeptides including methods of purifying desired polypeptides and methods of delivering drugs which comprise administering prodrugs that are converted from precursor to biologically active forms by stepwise proteolytic removal of the N-terminal extension in vivo.
  • Production of fusion polypeptides can be accomplished using standard peptide synthesis or recombinant DNA techniques both well known to those having ordinary skill in the art.
  • Peptide synthesis is the preferred method of making polypeptides which comprise about 50 amino acids or less. For larger molecules, production in host cells using recombinant DNA technology is preferred.
  • Fusion polypeptides which contain N-terminal portions that are recognized and cleaved by DPP IV are useful and advantageous over unmodified polypeptides comprising only the core protein portion.
  • the present invention describes two areas of particular utility. The first use is to provide fusion polypeptides, termed "prodrugs", which comprise biologically active polypeptides that are useful as drugs covalently linked to DPP IV cleavable N-terminal extensions. These proforms can be converted into biologically active forms upon cleavage by DPP IV in the body of a human or other animal that has been administered the prodrug.
  • the present invention relates to fusion polypeptides useful as prodrugs, use of fusion polypeptides in a medicinal preparation and to a method of delivering biologically active polypeptides to a patient.
  • a second use for fusion polypeptides according to the present invention is in protein purification processes in which the N-terminal extension is the component of the polypeptide which renders it effective in a purification method and which N- terminal extension is then removable by cleavage using DPP IV.
  • the present invention relates to fusion polypeptides useful in purification procedures and to a method of purifying desired polypeptides. These uses serve as examples to illustrate die utility of the present invention and are not meant to limit the invention in any way.
  • the core protein portion of the fusion protein is liberated from the extension portion by DPP IV activity.
  • the core proteins be substrates for DPP IV cleavage. That is, it is preferred that DPP IV not be able to cleave the core protein after the extension portion has been removed.
  • the prodrug can result in sustained presence of the core protein since some of the DPP IV found in vivo (e.g. in plasma, kidney tissue and liver tissue) will be used to process N-terminal extensions and, therefore, delay core protein degradation.
  • extension portion of the fusion protein can act as a substrate for DPP IV and competitive inhibitor, delaying the DPP IV action on the core protein thereby temporarily protecting the core protein.
  • prodrug means a fusion protein which contains a DPP IV cleavable
  • N-terminal extension covalently linked to a core protein portion that is a biologically active polypeptide useful as a drug.
  • Prodrugs according to the present invention can be administered as an individual proform or in combination with other compounds.
  • the preferred embodiment is a well defined individual form of a prodrug. In either case, the proforms are processed by naturally occurring DPP IV normally found in the body.
  • prodrugs can remain active longer than unmodified molecules. Prodrugs can exist in a non-active state until such time elapses that a sufficient portion of the extension portion is degraded and the molecule becomes active. Prodrugs, therefore, can act as a time delayed drug delivery system.
  • N-terminal extensions are degraded at different rates, depending on their length and the specific residues present in their amino acid sequence.
  • Combinations of different forms of prodrugs having a variety of N-terminal extensions can be provided which can provide a sustained, steady level of active drug in a patient over a course of time. Prodrugs, therefore, can act as a time delayed drug delivery system.
  • position one refers to the amino acid residue position at the N-terminus.
  • position two refers to the amino acid residue position which is immediately adjacent to position one and which is second from the N-terminus.
  • position three refers to the amino acid residue position which is immediately adjacent to position two and which is third from the N- terminus.
  • the cleavage which will remove the N-terminal dipeptide occurs between position two and position three provided amino acid three is not proline or hydroxyproline and amino acid two is one of five amino acids: proline (Pro), hydroxyproline (Hyp), alanine (Ala), serine (Ser), or threonine (Thr).
  • DPP IV cleaves the N-terminal residues at a different rate depending upon which of the four amino acid residues is present at position two. In most cases, DPP IV cleaves most efficiently when position two is occupied by Pro and it is next most efficient when position two is occupied by Ala. When position one is occupied by tyrosine, phenylalanine or histidine, DPP IV works at about the same rate when position two is occupied by Pro or Ala. DPP IV is next most efficient when position two is occupied by Ser. It is least efficient when Thr occupies the second position.
  • a medicament can be administered which comprises either a specific prodrug or a combination of prodrug forms.
  • the prodrugs, bearing an assortment of N-terminal extensions, will each be processed at a rate which is dependent upon their amino acid sequences.
  • This combination of prodrug forms can be formulated to comprise a series of prodrugs that are processed into active polypeptides across a spectrum of time.
  • embodiments of the present invention include hormones, receptors, enzymes, storage proteins and blood proteins.
  • Specific examples include: Vasoactive Intestinal Peptide (VIP); human 0-casomorphin; Gastric Inhibitory Peptide (GLP); Gastric Releasing Peptide (GRP); human Peptide HI; human Peptide YY; fragment 7-37 of glucagon-like peptide- 1; glucagon-like peptide-2; substance P; Neuropeptide Y; human Pancreatic Polypeptide; insulin-like growth factor-1 (IGF-1); human growth hormone (hGH); bovine growth hormone (bGH); porcine growth hormone (pGH); prolactin (PRL); human, bovine, porcine or ovine growth hormone releasing factor (GRF); interleukin-1 ?
  • VIP Vasoactive Intestinal Peptide
  • GLP Gastric Inhibitory Peptide
  • GRP Gastric Releasing Peptide
  • human Peptide HI human Peptide YY;
  • Smaller fusion proteins according to the present invention can be synthesized, for example, by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, California) as described in detail in PCT/US90/02923 and 07/368,231.
  • genes encoding desired polypeptides are inserted in expression vectors which are then used to transform or transfect suitable host cells. The inserted gene is then expressed in the host cell and the desired polypeptide is produced.
  • an additional DNA sequence is included in the gene insert. Specifically, DNA encoding the N-terminal extension residues is operably linked to the 5' end of the gene encoding the desired polypeptide.
  • oligonucleotides must be designed which encode the amino acid sequence of the desired N-terminal extension and these oligonucleotides must be operably inserted upstream of the 5' end of the gene encoding the core protein portion, generating a chimeric gene.
  • the techniques to make oligonucleotides and the techniques used to producing a chimeric gene are well known to those having ordinary skill in the art.
  • the present invention relates to the purification and processing of biologically active recombinant polypeptides.
  • the desired biologically active recombinant polypeptides are most preferably produced in a soluble form or secreted from the host.
  • the extension portion of the fusion protein can be recognized by purification means.
  • the fusion protein is purified from the material present in the secretion media or extraction solution it is contained in and then processed to remove the extension portion from the core protein portion, thus producing purified desired protein.
  • desired proteins most suited for processing as fusion proteins according to the present invention are those biologically active polypeptides which are not themselves substrates for DPP IV cleavage.
  • a gene sequence encoding for a desired protein is isolated., synthesized or otherwise obtained and operably linked to a DNA sequence coding an extension portion.
  • the hybrid gene containing the gene for a desired protein operably linked to a DNA sequence encoding an extension portion is referred to as a chimeric gene.
  • the present invention relates to recombinant chimeric genes which encode fusion proteins, expression vectors containing the same, hosts transformed or transfected with these expression vectors, and process for obtaining these genes, expression vectors, and hosts transformed or transfected with said vectors.
  • the present invention may be used to purify any prokaryotic or eukaroytic protein that can be expressed as the product of recombinant DNA technology in a transformed or transfected host cell.
  • These recombinant protein products include hormones, receptors, enzymes, storage proteins, blood proteins, mutant proteins produced by protein engineering techniques, or synthetic proteins.
  • the desired polypeptides produced may include HIV RNase H, tPA, IL-1, JX-1 receptor, CD4, human nerve growth factor, sCD4-PE40, human respiratory syncytial virus (RSV) FG chimeric glycoprotein (See U.S. Patent Application Serial No. 07/543,780, incorporated herein by reference), EGF, IGF-1, IGF-2, glucagon, corticotropin releasing factor (CRF), dynorfin, endothelin, transforming growth factor (TGF- ⁇ ), Pseudomonus endotoxin 40 (PE40), transforming growth factor-/? (TGF-j8), insulin and analogs thereof.
  • HIV RNase H HIV RNase H
  • tPA human nerve growth factor
  • sCD4-PE40 human respiratory syncytial virus
  • RSV respiratory syncytial virus
  • purification means examples include IMAC and immunoaffinity.
  • Other purification means which employ the use of extension peptides that can be removed using DPP IV are within the contemplated scope of the present invention.
  • fusion proteins comprising a biologically active polypeptide portion and an extension portion which is a metal chelating peptide are useful in an immobilized metal affinity chromatography system.
  • Immobilized Metal Ion Affinity Chromatography for fractionating proteins was first disclosed by Porath, J. et al., Nature 258:598-599 (1975). Porath disclosed derivatizing a resin with iminodiacetic acid (LDA) and chelating metal ions to the IDA-derivatized resin. Porath disclosed proteins could be immobilized in a column which contained immobilized metal ions. It involves attaching a commonly used iminodiacetic acid (IDA) to a matrix followed by chelating a metal ion to the IDA-containing resin. The proteins bind to the metal ion(s) through functional groups of amino acid residues capable of donating electrons.
  • LDA iminodiacetic acid
  • IDA iminodiacetic acid
  • IDA iminodiacetic acid
  • the proteins bind to the metal ion(s) through functional groups of amino acid residues capable of donating electrons.
  • Potential electron donating amino acid residues are cysteine, histidines, and tryptophan. Proteins interact with metal ions through one or more of these amino acids with electron donating side chains. Smith et al. discloses in U.S. Patent No. 4,569,794, incorporated by reference herein, that certain amino acids residues are responsible for the binding of the protein to the immobilized metal ions. However, the bound protein can be eluted by lowering the pH or using competitive counter ligands such as imidazole if histidine side chains are involved in the binding. Histidine-containing di- or tripeptides in proteins have been used to show that IMAC is a selective purification technique. Accordingly, Smith et al.
  • a fusion protein comprising a metal chelating peptide covalently liked to a desired polypeptide.
  • the metal chelating peptide is an extension portion that is effectively a handle to the desired polypeptide. This handle can be used in protein purification.
  • Use of IMAC technology with metal chelating peptides having alternating His residues is disclosed in U.S. Patent Application Serial No. 07/506,605, which is incorporated herein by reference.
  • U.S. patent application Serial No. 07/506,605 discloses specific metal chelating peptides which provide unexpectedly superior results in the IMAC purification of a fusion protein when the metal chelating peptide comprises three to six alternating His residues.
  • the extension portion is a metal chelating peptide which can be represented by the formula: A-X-Y(X'-Y) n and further, wherein A is optional, and when present is me ⁇ onine; n represents the number of sequentially linked X'-Y groups, that number representing from 0 to 20 of such groups, preferably 0 to 10 groups.
  • X is selected from the group consisting of any naturally occuring amino acid;
  • X' is selected from the group consisting of any naturally occuring amino acid except proline or hydroxyproline; wherein at least two to three residues designated X' and, optionally, X are Histidine
  • Y is Pro and n is at least 3.
  • the N-terminal extension is cleaved in a stepwise fashion, producing the biologically active polypeptide provided the biologically active polypeptide is not itself a DPP IV substrate.
  • Anomer example of a fusion protein includes an Extension portion having the formula
  • n 3 to 8
  • X is any naturally occurring amino acid
  • Ys are Pro, Hyp
  • N-terminal Met is a consequence of protein synthesis in E.coli and it is known to be processed by the E. coli enzymatic system when the adjacent amino acid is Pro, Gly, Ala or Ser
  • the following extensions represent sequences useful for the IMAC purification and cleavage of biologically active peptides or proteins expressed intracellularly in E. coli by recombinant DNA techniques.
  • the vectors could be designed so as to secrete the protein or polypeptide using an extension portion which facilitate transport, such as:
  • X could be any naturally occurring amino acid compatible with the secretion system from a given host and Ys are Pro, Hyp, Ala, Ser, or Thr.
  • Another protein purification system which uses fusion proteins and which is well suited for DPP IV processing technology is immunoaffinity purification.
  • the fusion proteins are purified from crude supernatent by passing crude supernatent through a column containing immobilized antibodies which recognize the antigenic portion of the fusion protein.
  • the immobilized antibodies keep the protein in the column while the undesired components of the supernatant are eluted.
  • the column conditions can then be changed to cause the antigen- antibody complex to dissociate.
  • the highly antigenic N-terminal portion of the fusion protein is an extension portion which contains DPP IV recognizable residues. After collection as described in the Hopp patent, the fusion protein according to the present invention can be exposed to DPP IV, thereby removing the extension portion.
  • One of ordinary skill in the art could practice the immunoaffinity purification system of Hopp with N-terminal extensions according to the present invention.
  • Contemplated equivalents include fusion proteins which have N-terminal extensions which can be processed by at least one other means such that removal of the extension is due to a combination of means. Contemplated equivalents also include fusion polypeptides comprising chemically modified amino acid residues.
  • Example 1 Synthetic Prodrugs which are Fusion Prodrugs Having Core Proteins that are DPP IV Substrates Fusion polypeptides that can be synthesized and administered as prodrugs have a DPP
  • extension portion represents a DPP IV cleavable N-terminal extension
  • - represents a covalent peptide bond
  • core protein portion represents any desired peptide which is liberated from the extension portion by DPP IV processing.
  • the core protein of the fusion protein is a potential substrate for DPP IV following removal of the extension portion.
  • Synthetic prodrugs can be produced using peptide synthesis techniques well known in the art.
  • the core protein portion is epidermal growth factor (EGF) and the extension portion is Gly-Pro-Phe-Ala:
  • the core protein portion is glucagon and the extension portion is Ala-Pro-Phe-Ala:
  • the core protein portion is [Ala 15 Leu 27 ]-bGRF (1-29)NH 2 (Seq ID 3) and the extension portion is Tyr-Ala: Leu 27 ]-bGRF (1-29)NH 2 ⁇ .
  • Example 2 Synthetic Prodrugs Which are Fusion Proteins Having Core Proteins that are not DPP IV Substrates
  • Fusion polypeptides that can be synthesized and administered as prodrugs have a DPP IV degradable N-terminal extension covalently linked to the N-terminal of the biologically active polypeptide.
  • the formula for these prodrugs can be expressed as the formula: extension portion - core protein drug portion wherein “extension portion” represents a DPP TV cleavable N-terminal extension; " - " represents a covalent peptide bond; and, "core protein portion” represents any desired peptide which is liberated from the extension portion by DPP IV processing.
  • the core protein portion is a bGRF analog, [Val 2 ,Ser 8 ' 28 ,Leu 27 ]- bGRF (l-33)OH (Seq ID 1), and the extension portion is Gly-Pro-Tyr-Ala:
  • the core protein portion is corticotropin releasing factor (CRF) and the extension portion is Gly-Pro-Phe-Ala: Gly ⁇ -Pro ⁇ -Phe ⁇ -Ala ⁇ -tCRF].
  • CRF corticotropin releasing factor
  • the core protein portion is dynorfin and the extension portion is Phe-Pro-Phe-Ala:
  • the core protein portion is somatostatin-28 and the extension portion is Gly-Pro-Phe-Pro:
  • the core protein portion is endothelin and the extension portion is Ala-Pro-Phe-Ala:
  • the core protein portion is a bGRF analog [IIe 2 Ser 8 ' 28 Ala 15
  • Leu 27 J-bGRF (1-40)OH (Seq ID 2) and the extension portion is Phe-Ala: Ser 8 ' 28 Ala 15 Leu 27 ]-bGRF (1-40)OH ⁇ .
  • the core protein portion is [Ile 2 Ala 15 Leu 27 ]-bGRF (1-29) NH 2 (Seq ID 4) and the extension portion is Tyr-Ser: Ala 15 Leu 27 ]-bGRF (1-29)NH 2 ⁇ .
  • Example 3 Sustained Presence of bGRF Analog Leu 27 -bGRF (1-29)NH 2
  • a bGRF analog, Leu 27 -bGRF (1-29)NH 2 can be administered as a medicament comprising the core protein shown in Seq ID 5 and a variety of N-terminally extended prodrugs.
  • Seq ID 5 As the core protein portion. Extension portions for these Seq ID 5-based prodrugs are Ile-Ala, Gly-Pro-Ile-Pro, Seq ID 6, Seq ID 7, Tyr-Ala, Gly-Pro-Tyr-Ala, Seq ID 8, Seq ID 9, Seq ID 10, Seq ID 11, Seq ID 12, Seq ID 13, Tyr-Ala-Tyr-Ala and Val-Ala.
  • Example 4 Sustained Presence of bGRF Analog [Th ⁇ Ala ⁇ Leu ⁇ l-bGRF (1-29)NH 2
  • a bGRF analog, (1-29)NH 2 can be administered as a medicament comprising the core protein portion shown in Seq ID 14 and a variety of N-terminally extended prodrugs.
  • Three versions of the prodrug were made having extension portions of Tyr-Thr, Tyr-Ser, and Tyr-Thr-Tyr-Thr, respectively.
  • a strategy to purify chimeric proteins from recombinant E. coli is described based on metal chelating peptide domains containing alternate histidines, with affinity for an immobilized metal ion.
  • Vectors are constructed to direct the synthesis of fusion proteins using HIV RNase H as the core protein. As shown below, these fusion proteins are designed to possess alternating histidines for purification by immobilized metal ion affinity chromatography (IMAC) and alternating prolines or alternating alanines for DPP IV cleavage to remove the metal chelating peptide (mcp).
  • IMAC immobilized metal ion affinity chromatography
  • mcp metal chelating peptide
  • Fusion protein HIVRH/mcp #1 comprises an N-terminal extension of Seq ID 15 linked to HIV RNase H: Met ⁇ Pro- ⁇ -Ala ⁇ -His ⁇ -Pro ⁇ -His ⁇ -Pro ⁇ -His ⁇ -Pro ⁇ -His ⁇ -Pro ⁇ -His ⁇ -Ala ⁇ - ⁇ HIV
  • Fusion protein HIVRH/mcp #2 comprises an N-terminal extension of Seq ID 16 linked to HIV RNase H:
  • Fusion protein HIVRH/mcp #3 comprises an N-terminal extension of Seq ID 17 linked to HIV RNase H:
  • a chimeric gene is inserted into the final expression vector.
  • Expression vectors containing the chimeric gene constructs are used to transform E. coli by standard techniques. Expression of the genes in E. coli results in the production of the fusion proteins encoded by the chimeric genes. These fusion proteins contain HIV RNase H amino acids and an N-terminal extension which contains alternate histidines (metal chelating peptide) and alternate prolines or alanines.
  • E. coli cell paste Approximately 3 g of E. coli cell paste is suspended in 30 ml of 0.25 M potassium phosphate, pH 7.2 containing 1 mM dithiothreitol (DTT), EDTA, phenylmethylsulfonyl fluoride (PMSF), and benzamidine HCL, 10 mg/liter aprotinin, leupeptin, and bestatin.
  • This suspension is passed through a French Press three times to break the cells. Cell lysates are centrifuged at 12,000 rpm for 1 hr. The supernatant is removed and solid ammonium sulfate added to 70% saturation. After stirring for 1 hr, the suspension is centrifuged at 12,000 rpm for 1 hr.
  • the supernatant is discarded and the pellet is redissolved in 2.25 mis of 50 mM Tris pH 7.5 containing 1 mM DTT, PMSF, and benzamidine.
  • the solution is then dialyzed overnight in 20 mM Tris, 50 mM NaCl, 1 mM DTT, 10% glycerol, and 0.1 mM EDTA pH 7.5 (Buffer A) at 4°C.
  • the dialysate is collected, diluted with one volume of Buffer A, and applied to a 10 ml column of washed DEAE cellulose equilibrated in Buffer A. The run through is collected batchwise and the column further washed with 50 mis of Buffer A.
  • IMAC columns are prepared as follows. Chelating Sepharose Fast Flow from Pharmacia is washed thoroughly with Milli-Q water on a scintered glass filter. The gel is then resuspended in water to form a slurry. The slurry is poured carefully into a glass column (Pharmacia) to a volume of 6 mis (1 x 7 cm). After the gel has settled, the column is washed with 5 volumes of 50 mM EDTA (ethylenediaminetetraacetic acid) pH 8.0. Following this, the column is washed with 5 volumes of 0.2 N NaOH and 5 volumes of Milli-Q water. The column then is charged with 5 volumes of 50 mM NiSo (or ZnCl 2 or Q1SO4).
  • EDTA ethylenediaminetetraacetic acid
  • the column is washed with 5 bed volumes of equilibration buffer.
  • the equilibration buffer is made up of 20 mM Tris pH 8.0, containing 500 mM NaCl, 1 mM PMSF, 1 mM benzamidine, 10 mg/L leupeptin, and 10 mg/L aprotinin.
  • the column has been equilibrated with at least 5 volumes of equilibration buffer. 5-10 mis of crude recombinant E. coli extract are applied to the column by gravity. After all the crude material has entered the column, the column is washed with 10 column volumes of equilibration buffer containing 1.0 M NaCl, instead of 500 mM NaCl, pH 8.0.
  • the column is then eluted with increasing concentrations of imidazole in the equilibration buffer at pH 8.0.
  • a large number of elutions are performed for each experiment to determine the concentration at which the chimeric eluted. Later this elution is simplified and usually just three imidazole concentrations are used: 35 mM, 100 mM, and 300 mM imidazole in the equilibration buffer, pH 8.0.
  • Ten bed volumes of each imidazole buffer are used.
  • the column is washed wi 10 volumes of equilibration buffer.
  • the column is stripped with 5 bed volumes of 50 mM EDTA pH 8.0 to determine if any protein is still bound to the column.
  • the flow rates for the columns are l.O ml/min. 5 ml fractions are collected. The columns are run at room temperature.
  • HIV RNase H activity is determined by the method described in Becerra, S. P. et al,
  • DPP IV purified from human placenta (Enzyme Systems Products, Dublin, Ca.) with a specific activity of 5200 mU per mg protein is used.
  • One U is equivalent to hydrolysis of 1 umole of a synthetic substrate, Ala-Pro-7-amino-4-2 trifluoromethyl coumarin in one minute at pH 7.8.
  • Enzymatic conversion is carried out by incubation of the fusion protein (about 1-100 mg) at a concentration of 1-10 mg/ml with DPP IV at 25 degrees C for 30 minutes at an enzyme to substrate ratio of 1:100 (w/w).
  • the desired polypeptide is recovered from the uncleaved fusion protein by IMAC. The authenticity is confirmed by N-terminal sequence analysis.
  • Example 6 Processing of bGRF Analog prodrugs in bovine plasma in vitro.
  • Table 1 summarizes representative experiments to demonstrate generation of the core peptide,
  • Tyr _4 -Ala" 3 -Tyr- 2 -Ala' 1 - ⁇ [Leu 27 ]-bGRF(l-29)NH 2 ⁇ was sequentially converted over time to Tyr _2 -Ala "1 - ⁇ [Leu 27 ]-bGRF(l-29)NH 2 ⁇ (Seq ID 25), [Leu 27 ]-bGRF(l- 29)NH 2 ( ⁇ re peptide, Seq ID 5) and finally to [Leu 27 ]-bGRF(3-29)NH 2 (Seq. ID 24).
  • Tyr- 2 -Ala- 1 - ⁇ ILeu 27 ]-bGRF(l-29)NH 2 ⁇ was converted to [Leu 27 ]-bGRF(l-
  • GH plasma growth hormone
  • bGRF prodrugs having DPP-IV-cleavable N-terminal extensions were processed successfully to produce the core peptide(s) in bovine plasma in vitro via DPP-IV mediated cleavages;
  • the in vitro half-life of the core peptide generated from the prodrugs was significantly longer and was a function of the N-terminal extension length in the prodrug;
  • the fact that the bGRF prodrugs with very low inherent potency were as effective as the core peptide in the release of GH in vivo indicates that most likely the core peptide was generated in vivo as anticipated.
  • Example 8 Preparation of Gly ⁇ -Pro ⁇ -Ile ⁇ -Pro -1 ⁇ [Leu 27 ] bGRF(l-29)NH 2 ⁇ , trifluoroacetate salt.
  • Example 9 Preparation of Tyr ⁇ -Ala ⁇ -Gly ⁇ -Pro ⁇ -Ile ⁇ -Pr ⁇ - 1 ⁇ [Leu 27 ] bGRF(l-29)NH 2 ⁇ , trifluoroacetate salt.
  • the synthesis of the GRF analog peptide Seq ID 27 which comprises Seq ID 6 as the extension portion and which has the formula: #3H-Tyr-Ala-Gly-Pro-Ile-Pro-Tyr-Ala-Asp-Ala-IIe-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly- Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH 2 (as the CF 3 COOH salt) is conducted in a stepwise manner as in procedure A which is described in published PCT patent application PCT/US90/02923 incorporated herein by reference
  • Example 19 Preparation of Tyr ⁇ -Thr "1 ⁇ [Ala 15 Leu 27 ] bGRF(l-29)NH 2 ⁇ , trifluoroacetate salt.
  • Example 23 Preparation of Tyr ⁇ -Thr ⁇ -Tyr ⁇ -Thr 1 ⁇ [Thr 2 Ala 15 Leu 27 ] bGRF(l-29)NH 2 ⁇ , trifluoroacetate salt.
  • Analysis A includes all steers and Analysis B includes only steers responding to GRF injection and control steers.
  • e Vajygg with different superscripts in a column are significantly different (P ⁇ .05).
  • Analysis A includes all steers and Analysis B includes only steers responding to GRF injection and control steers.
  • b ⁇ ⁇ v a j ues >vith different superscripts in a column are significantly different (P ⁇ .05).
  • ADDRESSEE Upjohn Company - Corp. Patents & Trademarks
  • B STREET: 301 Henrietta Street

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EP1205551A1 (en) * 2000-11-09 2002-05-15 Pfizer Products Inc. Growth hormone and growth hormone releasing hormone compositions
US6759393B1 (en) 1999-04-12 2004-07-06 Pfizer Inc. Growth hormone and growth hormone releasing hormone compositions
US7875587B2 (en) 1999-03-29 2011-01-25 Uutech Limited Peptide analogues of GIP for treatment of diabetes, insulin resistance and obesity
WO2011094617A3 (en) * 2010-01-29 2011-09-22 Archer-Daniels-Midland Company Peptide domains that bind small molecules of industrial significance
US8263545B2 (en) 2005-02-11 2012-09-11 Amylin Pharmaceuticals, Inc. GIP analog and hybrid polypeptides with selectable properties
US8404637B2 (en) 2005-02-11 2013-03-26 Amylin Pharmaceuticals, Llc GIP analog and hybrid polypeptides with selectable properties
US8410047B2 (en) 2004-06-11 2013-04-02 Novo Nordisk A/S Counteracting drug-induced obesity using GLP-1 agonists
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US9938331B2 (en) 2005-09-27 2018-04-10 Amunix Operating Inc. Biologically active proteins having increased in vivo and/or in vitro stability
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US7875587B2 (en) 1999-03-29 2011-01-25 Uutech Limited Peptide analogues of GIP for treatment of diabetes, insulin resistance and obesity
EP1052286A3 (en) * 1999-04-12 2001-07-25 Pfizer Products Inc. Growth hormone and growth hormone releasing hormone compositions
EP1052286A2 (en) * 1999-04-12 2000-11-15 Pfizer Products Inc. Growth hormone and growth hormone releasing hormone compositions
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WO2001036643A1 (en) * 1999-11-19 2001-05-25 Transkaryotic Therapies, Inc. Nucleic acid construct for optimized production of products
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EP1205551A1 (en) * 2000-11-09 2002-05-15 Pfizer Products Inc. Growth hormone and growth hormone releasing hormone compositions
US9486504B2 (en) 2003-12-09 2016-11-08 Novo Nordisk A/S Regulation of food preference using GLP-1 agonists
US8410047B2 (en) 2004-06-11 2013-04-02 Novo Nordisk A/S Counteracting drug-induced obesity using GLP-1 agonists
US8895498B2 (en) 2005-02-11 2014-11-25 Astrazeneca Pharmaceuticals, Lp GIP and exendin hybrid polypeptides
US8263545B2 (en) 2005-02-11 2012-09-11 Amylin Pharmaceuticals, Inc. GIP analog and hybrid polypeptides with selectable properties
US8404637B2 (en) 2005-02-11 2013-03-26 Amylin Pharmaceuticals, Llc GIP analog and hybrid polypeptides with selectable properties
US9133260B2 (en) 2005-02-11 2015-09-15 Amylin Pharmaceuticals, Llc GIP analog and hybrid polypeptides with selectable properties
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TW213923B (ru) 1993-10-01
AU9116591A (en) 1992-07-08
HU9301705D0 (en) 1993-10-28
HUT69963A (en) 1995-09-28
SK60893A3 (en) 1993-10-06
NO932148D0 (no) 1993-06-11
CA2094512A1 (en) 1992-06-14
FI932680A (fi) 1993-06-11
IE914347A1 (en) 1992-06-17
RU2114119C1 (ru) 1998-06-27
NO932148L (no) 1993-08-09
JPH06503473A (ja) 1994-04-21
CZ109393A3 (en) 1994-01-19
FI932680A0 (fi) 1993-06-11
EP0561971A1 (en) 1993-09-29
AU662508B2 (en) 1995-09-07

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