WO1995032003A1 - Facteurs de croissance proches de l'insuline modifies - Google Patents

Facteurs de croissance proches de l'insuline modifies Download PDF

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Publication number
WO1995032003A1
WO1995032003A1 PCT/US1995/006540 US9506540W WO9532003A1 WO 1995032003 A1 WO1995032003 A1 WO 1995032003A1 US 9506540 W US9506540 W US 9506540W WO 9532003 A1 WO9532003 A1 WO 9532003A1
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Prior art keywords
igf
mutein
peg
muteins
conjugate
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PCT/US1995/006540
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English (en)
Inventor
George N. Cox
Martin J. Mcdermott
Christine Ko
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Amgen Boulder Inc.
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Priority to JP7530514A priority Critical patent/JPH10500693A/ja
Priority to EP95920629A priority patent/EP0756494A1/fr
Priority to AU26018/95A priority patent/AU2601895A/en
Publication of WO1995032003A1 publication Critical patent/WO1995032003A1/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
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

Definitions

  • This invention relates to the modification of polypeptides, and more particularly to the modification of insulin-like growth factors and to methods of making and using such modified polypeptides.
  • the insulin gene family comprised of insulin, relaxin, insulin-like growth factors 1 and 2, and possibly the beta subunit of 7S nerve growth factor, represents a group of structurally related polypeptides whose biological functions have diverged as reported in Dull, et al., Nature 310:777-781 (1984).
  • IGF-1 and IGF-2 Insulin-like growth factors 1 and 2
  • IGF-1 and IGF-2 are about 7-8 kilodalton proteins that are structurally related to each other and to insulin.
  • IGF-1 and IGF-2 share about 70% amino acid identity with each other and about 30% amino acid identity with insulin.
  • IGF-1 and IGF-2 are believed to have related tertiary structures as reported in PCT Application Publication No. WO 90/00569, published on January 25, 1990.
  • the structural similarity between IGF-1 and IGF-2 permits both to bind to IGF receptors.
  • Two IGF receptors are known to exist. IGF-1 and IGF-2 bind to the IGF type I receptor, while insulin binds with less affinity to this receptor.
  • the type I receptor preferentially binds IGF-1 and is believed to transduce the mitogenic effects of IGF-1 and IGF-2.
  • IGF-2 binds to the type I receptor with a 10-fold lower affinity than IGF-1.
  • the second or type ⁇ IGF receptor preferentially binds IGF-2. Receptor binding is believed to be necessary for the biological activities of IGF-1 and IGF-2.
  • IGF-1 and IGF-2 are mitogenic for a large number of cell types, including fibroblasts, keratinocytes, endothelial cells and osteoblasts (bone-forming cells). IGF-1 and IGF-2 also stimulate differentiation of many cell types, e.g., synthesis and secretion of collagens by osteoblasts. IGF-1 and IGF-2 exert their mitogenic and cell differentiating effects by binding to the specific IGF cell surface receptors. IGF-1 also has been shown to inhibit protein catabolism in vivo, stimulate glucose uptake by cells and to promote survival of isolated neurons in culture. These properties have led to IGF-1 being tested as a therapeutic agent for a variety of disease indications as reported in Froesch et al.
  • IGFBP-1 IGF binding proteins
  • IGFBP-2 IGF binding proteins
  • IGF When IGF is bound to binding proteins, it is unable to bind to the IGF receptors and is therefore, no longer active in the body. Decreased affinity to binding proteins allows more of the IGF to be active in the body. Situations when this decreased affinity to binding proteins may be useful include, for example, cachexia, osteoporosis, and peripheral neuropathies.
  • IGF binding proteins can vary greatly, depending upon the disease state. For example, IGFBP-1 levels are very high in diabetes patients, whereas they are nearly undetectable in normal patients as reported in Brismar et al., J. of Endocrinological Investigation. 11:599-602 (1988); Suikkari et al., J. of Clinical Endocrinology and Metabolism. 66:266-273 (1988); and Unterman et al., Biochem. Biophys. Res. Comm., 163:882-887 (1989).
  • IGFBP-3 levels are reduced in severely ill patients such as those that have undergone major surgery as reported in Davies et al., . Endocrinology. 130:469-473 (1991); Davenport et al., J. Clin. Endocrin. Metab.. 75:590- 595 (1992).
  • the reduced levels of IGFBP-3, and consequent shorter circulating half-life of IGF-1, may contribute to the cachexia (weight loss) seen in these patients.
  • IGF-1 Insulin-like growth factor 1
  • somatomedin C also known as somatomedin C
  • IGF-1 insulin-deficient diabetic rats by administration of recombinantly produced human IGF-1 is reported in Scheiwiller et al., Nature. 323:169 (1986).
  • IGF-1 and (desl-3)IGF-l enhance growth in rats after gut resection, as reported in Lemmey et al., Am. J. Phvsiol.. 260 (Endocrinol. Metab. 23) E213-E219 (1991).
  • (desl-3)IGF-l when given by continual subcutaneous infiision, is more potent than IGF-1 in stimulating a number of anabolic functions, such as growth, reported in Cascieri et al., J. Endocrinology. 123:373- 381 (1988) and Gillespie et al., J. Endocrinology. 127:401-405 (1990).
  • the enhanced properties of (desl-3)IGF-l are believed to result from its reduced affimty for IGF binding proteins, reported in Carlsson-Skwirut et al., Biochim. Biophys. Acta. 1101:192-197 (1989).
  • (desl-3)IGF-l The reduced affinity of (desl-3)IGF-l for IGF binding proteins results in (desl- 3)IGF-1 having a shorter circulating half-life than wild type IGF-1 as reported in Cascieri et al., J. Endocrinology. 123:373-381 (1988). Therefore, (desl-3)IGF-l must be administered by continual infiision or by multiple daily injections in order to effect its enhanced potency.
  • IGF- 1 and (desl-3) IGF-1 are limited by their short circulating half-lives to situations when the proteins can be administered by continual infiision or by multiple daily injections to achieve their maximum therapeutic potential.
  • Woodall et al. Horm. Metab. Res.. 23: 581-584 (1991) reports that the same total dose of IGF- 1 administered four times daily by subcutaneous injection was far superior in stimulating growth in mutant lit/lit mice (growth hormone deficient mice) than was the same total dose administered once daily.
  • IGF-1 insulin growth factor-1
  • injectable drugs patient compliance is expected to be higher for drugs that can be administered once a day rather than several times a day.
  • IGF-1 In order for IGF-1 to be therapeutically effective when given once a day or once every few days, methods must be developed to increase its circulating half-life.
  • the present invention satisfies this need by increasing the molecular weight of the IGF. This is accomplished by providing muteins of IGF suitable for thiol-specific attachment of PEG to IGF and PEG conjugates formed from such muteins.
  • the invention relates to various modified forms of IGF.
  • modified IGF One type of modified IGF, referred to as muteins, is produced by replacing cysteine residues for specific amino acids in the wild type molecule, or by inserting cysteine residues adjacent to or between amino acids in the wild type molecule. Cysteine residues which are not involved in intramolecular disulfide bonds are considered to be "free". Cysteine residues can be substituted or inserted in regions of the IGF molecule that are exposed on the protein's surface. For example, the non-native cysteine can be inserted or substituted within the first or last twenty amino acids of wild-type IGF-1.
  • This non-native cysteine is expected to be free and to serve as the attachment site for the polyethylene glycol (PEG) molecules to IGF, resulting in PEGylated molecules.
  • PEG polyethylene glycol
  • the non-native cysteine can become involved in a disulfide bond and thereby free a native cysteine for PEGylation.
  • Attachment of the PEG molecule to a mutein creates a further modified form of IGF, or IGF-PEG conjugate of defined structure, where the PEG is attached to the IGF at a free cysteine residue.
  • the present invention is directed to a polyethylene glycol (PEG) conjugate comprising PEG and a mutein of IGF, and particularly IGF-1 , where the PEG is attached to the mutein at a non-native cysteine.
  • PEG can be attached to the free cysteine through a thiol-specific activating group including, for example, maleimide, sulfhydryl, thiol, triflate, tresylate, aziridine, exirane and 5-pyridyl.
  • a suitable PEG can have a molecular weight of 5 kDa, 8.5 kDa, 10 kDa or 20 kDa.
  • the PEG conjugate of the present invention can also contain a second protein to form a dumbbell. Methods of making the PEG conjugates are also provided.
  • IGF is known to increase the circulating half-life of a protein in the body.
  • IGF can be administered to patients less frequently with equal or better effectiveness than in the past.
  • the present invention is further directed to muteins of IGF particularly those having a non-native cysteine in the N-terminal or C-terminal region of the mutein.
  • the muteins can be produced by recombinant methods.
  • compositions comprising the IGF-PEG conjugate and methods of using the IGF-PEG conjugate to treat a patient having or potentially having an IGF associated condition.
  • the present invention is directed to modified forms of insulin-like growth factors
  • IGF insulin growth factor
  • modified forms of IGF include muteins of these growth factors, containing at least one free cysteine.
  • Conjugates containing the IGF muteins attached to polyethylene glycol (PEG) are also considered modified forms of IGF.
  • IGF refers to any polypeptide that binds to the IGF type I Receptor, including, for example, IGF-1, IGF-2, (desl-3)IGF-l, (R3)IGF-1 which is a mutein having a non-native arginine at residue number 3, and insulin.
  • IGF-1 IGF-1
  • IGF-2 IGF-2
  • R3IGF-1 a mutein having a non-native arginine at residue number 3
  • insulin insulin.
  • This hormone family is described in Blundell and Humbel, Nature. 287:781-787 (1980). Due to this common receptor binding, the teachings of the present invention which are described with respect to IGF-1 are intended to encompass IGF-2, des(l-3) IGF-1, (R3)IGF-1, and insulin also.
  • wild type IGF refers to the unmodified or naturally occurring IGF. This term is used interchangeably with “IGF,” “naturally occurring IGF,” or “native IGF.” The term “wild type IGF” also refers to native IGF to which a methionine residue has been added at the N-terminus.
  • wild type IGF-1 refers to the unmodified or naturally-occurring 70 amino acid form of IGF-1. This term is used interchangeably with “IGF-1,” “naturally occurring IGF-1,” and “native IGF-1.” The term “wild type IGF-1” also refers to native IGF-1 to which a methionine residue has been added at the N-terminus.
  • non-native refers to that which is not found in the native molecule.
  • IGF-PEG conjugate refers to an IGF molecule attached to a polyethylene glycol molecule. This is also referred to as a "Peg conjugate”.
  • N-terminal region refers to approximately the first twenty amino acids from the N-terminus of IGF or an IGF mutein, and up to twelve amino acids preceding the first amino acid of the N-terminus of IGF.
  • N-terminus refers to the first amino acid at the N-terminal region in the sequence of wild-type IGF, for example, glycine in IGF-1.
  • C-terminal region refers to aproximately the last twenty amino acids from the C-terminus of IGF or an IGF mutein and up to twelve amino acids following the last amino acid of the C-terminus of IGF.
  • C-terminus refers to the last amino acid at the C-terminal region in the sequence of wild-type IGF, for example, alanine in IGF-1.
  • mutant refers to a modified form of IGF, which has been modified to contain a non-native cysteine.
  • retain biological activity refers to having at least 10% of the mitogenic activity of wild type recombinant IGF as measured by the relative amount of 3 H-thymidine incorporated into UMR106 rat osteosarcoma cells, in the absence of IGF binding protein- 1, using the assay described herein.
  • the muteins and the conjugates of the present invention retain biological activity.
  • activating group refers to a site on the PEG molecule which attaches to the mutein.
  • pharmaceutically acceptable carrier refers to a physiologically- compatible, aqueous or non-aqueous solvent.
  • IGF associated condition refers to an existing or potential adverse physiological condition which results from an over-production or underproduction of IGF, IGF binding protein or IGF receptor, inappropriate or inadequate binding of IGF to binding proteins or receptors and any disease in which IGF administration alleviates disease symptoms.
  • An IGF associated condition also refers to a condition in which administration of IGF to a normal patient has a desired effect.
  • patient refers to any human or animal in need of treatment for an IGF associated condition.
  • the IGF muteins of the present invention are produced by modifying wild-type IGF, particularly at the N-terminal or C-terminal region of the protein. Such modifications can be substitutions or additions of at least one cysteine residue.
  • An IGF mutein can be produced by replacing a specific amino acid with a cysteine, such as, for example, substituting one of the first or last four amino acids of IGF-1 with a cysteine residue.
  • the amino acid sequence of wild type IGF-1 starting from the N-terminal end is: G P E T L CGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTG IVDECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO.1).
  • modifications include, for example, adding at least one cysteine residue in front of the first or after the last amino acid of IGF.
  • a cysteine residue can be inserted in front of and adjacent to the first amino acid of IGF.
  • the non-native cysteine can appear between Met and the first amino acid of IGF.
  • a free cysteine residue can also appear in a group of about twelve or less amino acids inserted before the first or after the last amino acid of IGF to form a longer IGF mutein.
  • the non-native cysteine residues are located in regions of the IGF-1 molecule exposed to the protein's surface.
  • the N-terminal region for example, is involved in the binding of the IGF to binding proteins, but is not involved in binding of IGF to cell surface IGF receptors.
  • An IGF-1 mutein of the present invention is also referred to as "a cysteine mutein of IGF-1.”
  • the non-native cysteine residue can act as the attachment site for covalent linkage of the activating group on the polyethylene glycol.
  • the non-native cysteine can become involved in disulfide bonding thereby freeing a native cysteine residue for thiol-specific attachment to PEG.
  • the newly created molecule comprising the cysteine mutein of IGF with the PEG attached is referred to as a "PEG conjugate of IGF".
  • the IGF muteins of the present invention can be prepared by methods well known to one skilled in the art.
  • Such methods include mutagenic techniques for replacement or insertion of an amino acid residue, as described, for example, in U.S. Patent 4,518,584, incorporated herein by reference.
  • the muteins produced by mutagenic techmques can then be expressed as recombinant products according to procedures known to those skilled in the art.
  • the muteins can alternatively be synthesized by conventional methods known in the art.
  • the IGF muteins can also be prepared according to the methods and techniques described in the examples set forth below.
  • the present invention also provides IGF-PEG conjugates and methods of making such conjugates by attaching the IGF muteins to polyethylene glycol to increase the circulating half-life of the molecule in the body as well as decrease its affinity to IGF binding proteins.
  • long chain polymer units of polyethylene glycol are bonded to the mutein via a covalent attachment to the sulfhydryl group of a free cysteine residue on the IGF mutein.
  • PEG polyethylene glycol
  • Various PEG polymers with different molecular weights 5.0 kDa (PEG 5000 ), 8.5 kDa (PEG 85(X> ), 10 kDa (PEG 10000 ), and 20 kDa (PEG 20000 ) can be used to make the IGF-PEG conjugates.
  • functionalized polymer units that will react specifically with sulfhydryl or thiol groups.
  • the functional or reactive group attached to the long chain polyethylene glycol polymer is the activating group to which the IGF mutein attaches at a free cysteine site.
  • Useful activating groups include, for example, maleimide, sulfhydryl, thiol, triflate, tresylate, aziridine, exirane, or 5-pyridyl.
  • polyethylene glycol (PEG) polymers containing two activating groups can be used to create "dumbbell” type molecules containing two IGF muteins attached to one molecule of PEG at each end of the PEG molecule.
  • PEG bis-malemide a polyethylene glycol polymer containing a maleimide activating group on each end of the PEG molecule
  • dumbbell molecules can also comprise a single IGF mutein covalently attached via PEG to a second protein or peptide of different structure.
  • the second protein or peptide can be, for example, a growth factor such as platelet-derived growth factor, or fibroblast growth factor.
  • One skilled in the art can readily determine the appropriate pH, concentration of protein, and ratio of protein: PEG necessary to produce a useful yield of either mono- pegylatedIGF-1 (IGF-PEG), or dumbbell IGF- 1 (IGF-PEG-IGF, IGF-PEG-PDGF, or IGF- PEG-FGF) using conventional methods known to one skilled in the art for making these determinations.
  • IGF-PEG mono- pegylatedIGF-1
  • dumbbell IGF- 1 IGF-PEG-IGF, IGF-PEG-PDGF, or IGF- PEG-FGF
  • the invention present also includes pharmaceutical compositions.
  • the IGF muteins and PEG conjugates can be in a pharmaceutically-acceptable carrier to form the pharmaceutical compositions of the present invention.
  • pharmaceutically acceptable carrier means a non-toxic, generally inert vehicle for the active ingredient, which does not adversely affect the ingredient or the patient to whom the composition is administered. Suitable vehicles or carriers can be found in standard pharmaceutical texts, for example, in Remington's Pharmaceutical Sciences. 16th ed. , Mack
  • Such carriers include, for example, aqueous solutions such as bicarbonate buffers, phosphate buffers, Ringer's solution and physiological saline.
  • the carrier can contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
  • the pharmaceutical compositions can be prepared by methods known in the art, including, by way of an example, the simple mixing of reagents. Those skilled in the art will know that the choice of the pharmaceutical carrier and the appropriate preparation of the composition depend on the intended use and mode of administration.
  • the carrier and the IGF mutein or conjugate constitutes a physiologically-compatible, slow-release formulation.
  • the primary solvent in such a carrier can be either aqueous or non-aqueous in nature.
  • the carrier can contain other pharmacologically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
  • the carrier can contain still other pharmacologically-acceptable excipients for modifying or maintaining the stability, rate of dissolution, release, or absorption of the IGF mutein or conjugate.
  • excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dose or multi-dose form.
  • the pharmaceutical composition can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder.
  • Such formulations may be stored either in a ready to use form or requiring reconstitution immediately prior to administration.
  • the preferred storage of such formulations is at temperatures at least as low as 4°C and preferably at -70°C. It is also preferred that such formulations containing the IGF mutein or conjugate are stored and administered at or near physiological pH. It is presently believed that administration in a formulation at a high pH (i.e. greater than 8) or at a low pH (i.e. less than 5) is undesirable.
  • the manner of administering the formulations containing the IGF mutein or conjugate for systemic delivery can be via subcutaneous, intramuscular, intravenous, oral, intranasal, or vaginal or rectal suppository.
  • the manner of administration of the formulations containing the IGF muteins or conjugates for local delivery is via intraarticular, intratracheal, or instillation or inhalations to the respiratory tract.
  • the IGF muteins or conjugates are encapsulated.
  • the encapsulated IGF muteins or conjugates may be formulated with or without pharmaceutically-acceptable carriers customarily used in the compounding of solid dosage forms.
  • the capsule is designed so that the active portion of the formulation is released at that point in the gastro-intestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional excipients may be included to facilitate absorption of the IGF muteins or conjugates. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.
  • the specific dose is calculated according to the approximate body weight of the patient.
  • Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, route of administration and the age, sex and medical condition of the pateint.
  • the dosage and administration is designed to create a preselected concentration range of the IGF muteins or conjugates in the patient's blood stream. It is believed that the maintenance of circulating concentrations of the IGF muteins or conjugates of less than 0.01 ng per ml of plasma may not be an effective composition, while the prolonged maintenance of circulating levels in excess of 100 ⁇ g per ml may have undesirable side effects.
  • IGF mutein and conjugate pharmaceutical compositions described herein may be used for veterinary as well as human applications and that the term "patient” should not be construed in a limiting manner. In the case of veterinary applications, the dosage ranges should be the same as specified above.
  • the pharmaceutical composition of the present invention can be used to treat a patient having or potentially having an IGF associated condition. Some of these conditions can include, for example, dwarfism, diabetes, periodontal disease and osteoporosis.
  • the pharmaceutical composition of the present invention can also be used to treat a condition in which administration of IGF to a normal patient has a desired effect; for example, using
  • IGF-1 to enhance growth of a patient of normal stature.
  • the IGF-1 gene was assembled in two stages. Initially, the DNA sequence encoding
  • IGF-1 was joined to DNA sequences encoding the secretory leader sequence of the E. coli
  • OMP A protein (ompAL). This gene fusion was constructed in order to determine whether
  • IGF-1 could be efficiently secreted from E. coli.
  • This DNA fragment was mixed with BamHI + Pstl-digested PUC18 DNA (commercially available from Boehringer Mannhein Biochemicals, Indianapolis, IN) and the two synthetic oligonucleotides [IGF-1 (1-14) U + L] 5'CCGGTCCGGAGACTCTGTGCGGCGCAGAACTGGTTGAC GCTCTGCA3' (SEQ ID NO. 6) and 5'GAGCGTCAACCAGTTCTGCGCCGC ACAGAGTCTCCGGACCGG3' (SEQ ID NO. 7) were Iigated together.
  • the ligation mixture was used to transform E. coli strain JM109 (commercially available from New England Biolabs, Beverly, MA) and individual colonies isolated.
  • These plasmids have a translational start signal followed by DNA sequences encoding the OmpA signal sequence and the first 14 amino acids of IGF-1.
  • IGF-1 was created by Iigating together the two complementary pairs of oligonucleotides (IGF1U + IL and IGF2U + 2L) 5'GTTCGTATGCGGCGACCGTGGCTTC TACTTCAACAAACCGACTGGCTACGGTTCCAGCTCTCGTCGTGCACCGCAG ACTGGTATC3' (SEQ ID NO. 8) and 5'TTCGTCAACGATACCAGTCTGCGGTGC ACGACGAGAGCTGGAACCGTAGCCAGTCGGTTTGTTGAAGTAGAAGCCACG
  • GTCGCCGCATACGAACTGCA3' (SEQ ID NO. 9) and cloning the DNA fragment into PstI + Hindm-digested Ml 3 mpl9 DNA (commercially available from New England Biolabs, Beverly, MA). Double-stranded DNA was purified from a phage clone and the
  • the Bamffl/Hindm fragment containing the IGF-1 gene fused to the OmpAL sequence was isolated and cloned into the BamHI 4- HindlQ generated site of piasmid pT3XI-2 (described in PCT Application publication WO 91/08285 published on June 13, 1991).
  • the completed piasmid containing the ompAL-IGF-1 gene fusion is called pT3XI-2 ⁇ lOC(TC3)ompALIGF-l .
  • the BamHI/Hindi ⁇ fragment containing the OmpAL-IGF-1 gene fusion described above was purified from piasmid pT3XI-2 ⁇ lOC(TC3)ompALIGF-l and digested with Hinfl.
  • the approximate 200 bp Hinfl/Hindm DNA fragment was mixed with the annealed, complementary synthetic oligonucleotides (MetlGFlU + IL) 5'GATCCGATCGT
  • piasmid pT3XI2 DNA Iigated with BamHI + HindlH-digested piasmid pT3XI2 DNA, and used to transform E. coli JM107.
  • the completed piasmid construct is called ⁇ lOC(TC3)IGF-lpT3XI-2 and contains an extra alanine residue in between the initiator methionine and the beginning of the IGF-1 sequence.
  • the BamHI/Hindi ⁇ fragment containing the mutant IGF-1 gene was isolated and Iigated into the BamHI + Hindi ⁇ generated site of piasmid pT5T (described in Nature, Vol. 343, No. 6256, pp. 341-346, 1990).
  • the ligation mixture was used to transform E. coli BL21/DE3 described in US Patent 4,952,496 and the resulting individual colonies were isolated. This construct was named ⁇ l0C(TC3)IGF-lpT5T.
  • the oiigonucleotide used for mutagenesis had the sequence:
  • the mutagenesis reaction product was used to transform E. coli strain JM109 and individual plaques picked.
  • Double-stranded replicative form DNA from individual phages was isolated, digested with BamHI + HindlH and the 200 bp fragment containing the IGF-1 gene purified.
  • the purified DNA was cloned into the BamHI + HindlH generated site of piasmid pT5T and used to transform E. coli strain BL21/DE3.
  • One bacterial colony with the correct piasmid was named ⁇ l0(TC3)mutIGF-lpT5T.
  • Several isolates were sequenced, and all were correct.
  • IGF-1 insulin growth factor-1
  • Three of the muteins replaced each of the first three amino acids of IGF-1 with a cysteine residue. These muteins are referred to as Cl, C2, and C3, respectively.
  • a fourth mutein introduced a cysteine residue between the N-terminal methionine residue and the first amino acid of IGF-1. This mutein is referred to as -IC.
  • the -IC, Cl, C2 and C3 muteins of IGF- 1 were made using the polymerase chain reaction (PCR) technique as described below.
  • the starting piasmid used for the mutagenesis experiments was ⁇ lO(TC3)mutIGF-lpT5T, which is described in Example 1.
  • This piasmid contains DNA sequences encoding an initiator methionine followed by the sequence of the natural human IGF-1 protein.
  • Mutant IGF-1 DNA sequences were amplified from this gene using a 5' mutagenesis oiigonucleotide that contained the desired mutation and a 3' oiigonucleotide of correct sequence.
  • the 5' mutagenesis oligonucleotides were designed so that they incorporated the first methionine of the gene as part of an Nde I restriction enzyme cleavage site (CATATG).
  • Each mutagenesis oiigonucleotide contained the desired mutation followed by 15 to 21 nucleotides that were a perfect match to the IGF-1 gene sequences in piasmid ⁇ lO(TC3)mutIGF-lpT5T.
  • the 3' oiigonucleotide was 25 nucleotides long and was designed to encode the last 6 codons of IGF-1 and to contain the Hind HI site that follows the stop codon.
  • a wild type clone was made in the same manner using the Ndel site of pT5T (described above) as the first methionine.
  • This wild type clone is designated 85p-ll.
  • the oligonucleotides used to construct the -IC, Cl , C2 and C3 muteins and the 85p-l 1 wild type clone are set forth in Table 1.
  • IGF(C2p)31 5'GGGCATATGGGTTGTGAGACTCTGTGCGGCG3'
  • IGF(C3p)33 5'GGGCATATGGGTCCGTGCACTCTGTGCGGCGCA3' 3' oligo (for all of the above muteins) IGF(262p)25 5 ' CCC AAGCTTAAGCGCTTTTAGCCGG3 '
  • PCR Polymerise chain reaction
  • 20 mM Tris pH 8.8, 10 mM KC1, 6 mM (NH4)2SO4, 1.5 mM MgC12, 0.1 % Triton X-100 using 20 pmole of each oligo and approximately lng of piasmid ⁇ l0(TC3)mutIGF-lpT5T as template DNA.
  • 0.5ul (1.25 units) of Pfu polymerase (Stratagene, San Diego, CA) was added after the first denaturation step with the tubes held at 65 °C.
  • the reactions were overlayed with 2 drops of mineral oil at that time. The reactions were cycled 30 times for 1 min. at 95°C, 1 min.
  • Piasmid pT5T is described in Example 1.
  • the ligation reactions were used to transform E. coli strain BL21/DE3 and colonies selected on LB agar plates containing 50ug/ml of ampicillin. Mini piasmid DNA preps were made from several colonies from the transformation plates. The DNAs were digested with Eco RV and Hind
  • Plasmids containing IGF-1 DNA inserts were sequenced to verify that the inserts were correctly mutated (the entire IGF gene was sequenced for each mutein).
  • Isopropyl-beta-D-thiogalactopyranoside IPTG was added to 1 mM to induce expression of T7 polymerase and the subsequent transcription and translation of the IGF muteins.
  • IPTG Isopropyl-beta-D-thiogalactopyranoside
  • Approximately 0.1 OD unit of cells were lysed in SDS sample buffer by boiling for two minutes and electrophoresed on a 16% polyacrylamide SDS gel. The gel was stained with Coomassie blue.
  • Muteins in which cysteine was substituted for the amino acids at residues 11, 12, 15, 16, 55, 64, 65, 67 and 69 of the wild type IGF-1 were also prepared using PCR.
  • the PCR template was either the full-length piasmid from clone 85p-ll or the Ndel-HindHI fragment from clone 85p-ll containing the wild type IGF-I gene.
  • Cll, C12, C15 and C16 5' oligonucleotides were synthesized that incorporated the first methionine as part of the Ndel site (CATATG) and each contained the mutation desired followed by 21-24 nucleotides that were a perfect match to clone 85p- 11.
  • IGF(262p)25 a 25-mer oiigonucleotide that matched the last 6 codons of IGF-I plus the Hindi ⁇ site following the stop codon was used.
  • IGF(262p)25 is set forth in Table 1.
  • IGF(C12)S-69 5'GGGCATATGGGTCCGGAGACTCTGTGCGGCGCAGAACTGG1TTGCGCTCTGCAGTTCGTATGCGGCGAC3'
  • C65 ...YCAPLC... IGF(C65)AS-55 5'CCCAAGCTTAAGCGCTTTTAGCCGGGCACAGCGGAGCGCAGTACATTTCCAGACG3'
  • PCR Polymerase chain reaction
  • 20 mM Tris pH 8.8, 10 mM KC1, 6 mM (NH4)2SO4, 1.5 mM MgC12, 0.1 % Triton X-100 using 20 pmole of each oligo, approximately lng of template DNA, 200uM each of dATP, dCTP, dGTP, TTP, 20pmole of each oligo primer, and lul (2.5U) of Pfu polymerase (Stratagene, San Diego, CA).
  • the reactions were cycled 30 times for 1 min. at 95°C, 1 min. at 65°C and 1 min. at 72°C in a GeneA p PCR System 9600 (Perkin Elmer Cetus). After the last cycle the reactions were held at 72°C for 10 min.
  • reaction mixtures were purified by passing through ChromaSpin 100 columns (Clontech Lab. Inc., Palo Alto, CA). Purified PCR fragments were digested with Ndel and HindlH and the ⁇ 210bp bands eluted from 1.5% agarose gel using NA45 paper
  • a correct construct was selected for each mutein (clones Cll-1, C12-3, C15- 1, C16-4, C55-1, C64-1, C65-1, C67-2, C69-1) and transformed into E. Coli strain BL21/DE3 for expression.
  • Preliminary expression studies of the muteins were performed by growing two representative transformants for each mutein in LB + tetracycline (12ug/ml) to an approximate OD 600 Of about 0.6-0.8.
  • IPTG was added to a final concentration of ImM and the cells allowed to grow for an additional 2 hrs. The IPTG induces the expression of T7 polymerase and the subsequent transcription and translation of the IGF muteins.
  • Approximately 0.1 OD unit of the cells were lysed in
  • E. coli cells expressing the IGF-1 C3 mutein were suspended in Buffer A (50 mM Tris, pH 7.5, 20 mM NaCI and 1 mM dithiothreitol (DTT) at a concentration of 40 ml Buffer A to 10 g cell paste, and disrupted at 1800 psi using a French pressure cell (SLM Instruments, Inc., Urbana IL). The suspension was centrifuged at 20,000 X g for 30 min, and aiiquots of the pellet and supernatant analyzed by SDS-PAGE. A major band corresponding to the IGF-1 C3 mutein was present in the pellet, but not the supernatant.
  • Buffer A 50 mM Tris, pH 7.5, 20 mM NaCI and 1 mM dithiothreitol (DTT) at a concentration of 40 ml Buffer A to 10 g cell paste, and disrupted at 1800 psi using a French pressure cell (SLM
  • the pellet was suspended in Buffer A at a concentration of 40 ml Buffer A to 10 g cell paste, and re-centrifuged at 20,000 X g for 30 min. This wash procedure was repeated 2 times.
  • the final pellet containing the IGF-1 C3 mutein was suspended in 6 M guanidine, 50 mM Tris, pH 7.5, 6 mM DTT at a concentration of 25 ml to 10 g cells using a ground glass homogenizer. The suspension was incubated at room temperature for 15 min. The undissolved protein was removed by centrifugation at 20,000 X g for 30 min. The final concentration of the C3 mutein was 1.0 mg/ml. SDS-PAGE analysis of the pellet and supernatant following the procedure of Example 2 showed that IGF-1 C3 mutein was present in the supernatant only.
  • the denatured and reduced IGF-1 mutein was subjected to the following three-step refolding procedure:
  • Oxidized glutathione the mixed-disulfide producing reagent (GSSG) was added to the supernatant to a final concentration of 25 mM, and incubated at room temperature for 15 min.
  • Aiiquots (50ul) of the supernatant were diluted to 200ul with Buffer B (0.05% TFA), injected onto a reverse phase column (RP-4, 1 x 250mm, Synchrom, Lafayette, IN), and eluted with 80% acetonitrile, 0.042 % TFA (Buffer C) using a linear gradient (increase of 2% Buffer C/min) at a flow rate of 0.25 ml/min.
  • N-terminal sequence analysis of IGF- 1 C3 mutein eluting at 26.6 min gave the sequence: M G P C T L C (SEQ ID NO. 29) confirming that a cysteine residue has been substituted for glutamic acid in the 3 position of the N-terminal sequence of natural human IGF-1. An extra methionine residue is present at the N-terminus of the recombinant protein expressed by E coli.
  • N-terminal sequence analysis of IGF-1 C2 mutein eluting at 26.0 min gave the sequence: G C E T L C (SEQ ID NO.
  • the earlier eluting peak observed in the WT refold has been shown to be an isomeric form of IGF-I with S-S assignments C6-C47, C48- C52, and C18-C61; whereas the later eluting peak is correctly refolded with S-S assignments C6-C48, C47-C52, C18-C61 (See, Raschdorf et al., Biomedical & Environmental Mass Spectroscopy. Vol. 16, pp.3-8 (1988), specifically incorporated herein by reference).
  • the retention time of the refolded muteins shifted to 27 min after being completely reduced and denatured in 5 M guanidine, 50 mM Tris pH 7.5, 100 mM DTT. This demonstrates that the refolded forms "collapse" to a single polypeptide after reduction of disulfides.
  • the RP4 peaks therefore, represent distinct forms of IGF muteins with different disulfide bonds.
  • EXAMPLE 4 Isolation of Refolded IGF-1 Mutein
  • Refold mixtures (435 ml) prepared from 20g of E. coli paste containing either the refolded C3 or C2 mutein of Example 3 were concentrated to 100ml, acidified to pH 5.5 with 5M HCl, dialyzed against 20 mM sodium acetate, pH 5.5, and loaded onto an S- Sepharose (Pharmacia/LKB, Piscataway, NJ) column (1.6 X 15 cm) previously equilibrated with the sodium acetate buffer described above. The bound protein was eluted with a 300 ml linear gradient from 0 to 0.5M NaCI. Three ml fractions were collected.
  • the C12 & C15 monomer fractions from Superdex 75 also contained significant amounts of apparently mis-folded material eluting after PH (21.5 - 23.0 min).
  • the Cll, C15 and C16 monomer fractions contained multiple (3-6) peaks when analyzed by RP-4. The monomer peaks were collected separately and assayed for bioactivity and subjected to mass analysis (see below).
  • the masses of the reduced muteins match, within experimental error, the expected masses for a polypeptide with the indicated cysteine mutation.
  • the masses of the refolded monomers match the expected mass of polypeptides having 3 intramolecular disulfide bonds and a single mixed disulfide of either cysteine - glutathione (Cys-S-S-Glutathione) or cysteine - cysteine (Cys-S-S-Cys).
  • the mixed disulfides form during refolding and remain intact because there is no other cysteine residue present in the molecule available to form an intramolecular disulfide.
  • Buffer B Aiiquots (50 ul) of each peak were analyzed by reverse phase (RP-4, 1 X 250 mm). Fractions containing predominantly correctly refolded (determined from RP-4 analysis) C69, eluting at ⁇ 32-38 % Buffer B were pooled. Reverse phase analysis showed the correctly refolded C69 pool was 95 % or more homogeneous. This material was assayed for bioactivity (see below).
  • the C3, C2 and C69 muteins were covalently joined to an 8.5 kDa polyethylene glycol (8.5 kDa PEG) or an 20 kDa polyethylene glycol (20 kDa PEG) having a maleimide activating group in a two step process: 1)
  • the purified IGF-1 muteins were partially reduced with DTT in a 15 ml reaction mixture containing 2.3 mg (296 nmoles) IGF-1 mutein, 170 ug DTT (1110 nmoles) in 14 mM sodium acetate, 33 mM sodium phosphate, pH 7.0.
  • the final concentration of protein was 10 ug/ml, and the molar ratio of DTT:protein was 3.75: 1.
  • 91ug DTT (592 nmoles) was used and the molar ratio of DTT:protein was 2:1.
  • the reaction mixture was incubated at room temperature for 3 hours (5 hours for reaction with the C69 mutein) and terminated by the addition of 1.0 ml of IM sodium acetate, pH 5.5.
  • the reaction mixture was dialyzed at 4°C overnight against 20 mM sodium acetate pH 5.5.
  • the partially reduced IGF-1 mutein was reacted with either the 8.5 kDa PEG or the 20 kDa PEG in a 20 ml reaction mixture containing 2.3 mg (296 nmoles) of protein, 9.985 mg (1174 nmoles) 8.5 kDa PEG in 15 mM sodium acetate, 26 mM sodium phosphate, pH 7.0.
  • the final concentration of protein was 112 mg/1.
  • the molar ratio of 8.5 kDa PEG: protein was 4:1; for reaction with the C69 mutein the molar ratio of 20kDa PEG: protein was 4:1.
  • the reaction mixture was incubated at room temperature for 3 hours, and terminated by placing at 4oC or freezing at -20°C. SDS-PAGE analysis of the reaction mixture following the procedure of Example 2 showed that approximately 50% of the partially reduced PEGylated C2 and C3 mutein was converted to a mono-PEGylated species.
  • the C3 and C2 20kDa-PEG conjugates migrated at a relative molecular weight of approximately 60kDa on SDS PAGE;
  • the C3 and C2 8.5kDa-Peg conjugates migrated at a relative molecular weight of about 23 kDa on SDS PAGE.
  • Approximately 20% of the partially reduced PEGylated C69 mutein was converted to a mono-PEGylated species migrating at a relative molecular weight of 67 kDa on SDS PAGE.
  • the pegylated C2 or C3 mutein reaction mixtures (containing approximately 100-200 mg protein) were dialyzed extensively at 4°C against 5 mM citric acid, pH 2.6.
  • the pegylated mutein was separated from the unPEGylated mutein using an S-Sepharose
  • the C69-PEG was separated from the unpegylated C69 mutein by Q-Sepharose anion exchange chromatography .
  • 100 ml of the reaction mixture containing 11 mg of total protein was loaded onto a Q-Sepharose anion exchange column column (Pharmacia/LKB, Piscataway, NJ) column (2.6 X 100 cm) previously equilibrated with 20mM Tris, pH 9.0 (buffer A).
  • the bound protein was eluted with a linear gradient of 20mM tris Ph 9.0, IM
  • IGF-BP1 insulin-like growth factor binding protein 1
  • the relative mitogenic (growth stimulating) activities of the C3 and C2 muteins and pegylated C3 and C2 muteins were compared to that of wild type IGF-1 by measuring the relative amount of 3 H-thymidine incorporated into rat osteosarcoma cells when varying amounts of the proteins were present under serum free conditions.
  • the rat osteosarcoma cells (the UMR106 cell line; obtained from American Type Culture Collection, Accession No.
  • CRL-1661 Rockville, Maryland
  • CRL-1661 Rockville, Maryland
  • X 104 cells were plated at 5-6 X 104 cells in 0.5 ml of Ham's F12 Media (Mediatech, Herndon, VA) containing 7% fetal bovine serum, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin and 2 mM L-glutamine per well in 48-well tissue culture plates (Costar, Cambridge, MA). After incubating for 72 hours at 37°C when the cells were confluent, the cells were washed twice with phosphate buffered saline (PBS) and pre-incubated in serum-free Ham's F12 medium containing 100 U/ml penicillin and 100 mg/ml streptomycin and 2 mM L-glutamine for 24 hours.
  • PBS phosphate buffered saline
  • 3H-thymidine was quantitated by liquid scintillation counting. All assays were performed in triplicate.
  • the C3 and C2 muteins and pegylated C3 and C2 muteins were found to stimulate the same maximal level of 3H-thymidine incorporation into DNA as recombinant IGF-1.
  • the potencies of the C3 and C2 muteins and the pegylated C3 and C2 muteins were about
  • ED50 dose required for half maximal activity
  • recombinant IGF-1 was 5-10 ng/ml compared with 30-40 ng/ml for unpegylated C3 and C2 muteins, and the pegylated C3 and C2 muteins.
  • Peaks H of the C12, C16, C55, C67 and C69 mutein monomers was not significantly different from the mitogenic activity of correctly refolded WT rIGF-I (Peak H).
  • the ED 50 of wild type rIGF-I was 4-6 ng/ml compared with 5-8 ng/ml for these unPEGylated mutein monomers.
  • Table 5 shows that Peaks I of the various muteins had lower (5-30 fold) bioactivity than correctly refolded WT rIGF-I similar to the bioactivity of WT rIGF-I peak I.
  • the C69-PEG conjugate, synthesized from peak H of C69 had the same bioactivity as C69 peak H and correctly refolded WT rIGF-I.
  • IGF-1 by measuring the ability of IGF-BPl to inhibit the mitogenic activities of the proteins on rat osteosarcoma cells.
  • the rat UMR106 osteosarcoma cells were plated at 5-6 X 104 cells in 0.5 ml of Ham's F12 containing 7% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin and 2 mM L-glutamine per well in 48-well tissue culture plates.
  • the cells were washed twice with PBS and pre-incubated in serum-free Ham's F12 medium containing 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine for 24 hours. After the pre-incubation, 0.5 ml of F12 serum-free medium containing either 50 ng/ml or 200ng/ml of rIGF-1, C3 or C2 mutein, or pegylated C3 or C2 mutein were incubated separately with varying amounts of IGF-BPl (100 ng/ml - 1 X 104 ng/ml) for an additional 20-24 hours.
  • IGF-BPl 100 ng/ml - 1 X 104 ng/ml
  • the mitogenic activity of rIGF-1 50 ng/ml was reduced 80%; however, the mitogenic activities of the same concentrations of the unpegylated C3 mutein and pegylated C3 mutein were reduced 35% and 0%, respectively.
  • pegylated C3 mutein has greatly reduced affinity for IGFBPl when compared to IGF-1.
  • the mitogemc activity of the pegylated C3 mutein will not be inhibited by IGF binding proteins under conditions where the mitogemc activity of IGF-1 will be inhibited.
  • the affinity of pegylated C2 and C69 muteins for IGFBPl is the same as the affinity of wild type IGF-1.
  • the mitogemc activity of pegylated C2 and C69 muteins will be inhibited by IGF binding proteins under the same conditions where the mitogenic activity of IGF-1 will be inhibited.
  • EXAMPLE 8 Animal Tests Animal studies were performed to compare the pharmacodynamic properties of the muteins and PEG conjugates of the present invention to the pharmacodynamic properties of wild type IGF- 1.
  • the rats were maintained in cages with lighting controlled over a 12 h-light/12 h-dark cycle.
  • the animals had continuous access to water and food. Five animals were housed per cage. The weights of the rats were monitored daily and only rats with weight gains of less than 2 grams per week during the 2-3 weeks after arrival were considered to be successivefully hypophysectomized and used for the experiments.
  • mice (10 Hypox rats per group) were injected every third day (ETD) subcutaneously (sc) with WT rIGF-I (160 mg, 320mg), unpegylated C2 (320mg), unpegylated C3 (320mg), pegylated C2 0(C2-PEG, 320mg) or Pegylated C3 (C3- PEG,320mg) dissolved in 0.2 ml of binding buffer (0.1 M HEPES-0.05 M NaH 2 PO 4 ).
  • a separate group of 10 animals received 0.2ml vehicle. Injections were given between 0700 hours and 0800 hours and body weights were recorded daily between 1600 h and 1700 h. The weights of rats on the day after the last injection were taken as the final weight.
  • mice (9 Hypox rats per group) were injected every third day sub- cutaneously with WT rIGF-I (320mg, single injection daily, SID; 320mg ETD; 640mg ETD), or C3-PEG (320mg ETD, 640mg ETD, 960mg ETD). dissolved in 0.2 ml of binding buffer (0.1 M HEPES-0.05 M NaH 2 PO 4 ). A separate group of 9 animals received 0.2ml vehicle. Injections were given between 0700 h and 0800 h and body weight were recorded daily between 1600 h and 1700 h. The weights of rats on the day after the last injection were taken as the final weight.
  • WT rIGF-I 320mg, single injection daily, SID; 320mg ETD; 640mg ETD
  • C3-PEG 320mg ETD, 640mg ETD, 960mg ETD
  • mice (10 Hypox rats) were injected every third day sub- cutaneously with C3-PEG (160mg ETD, 320mg ETD), dissolved in 0.2 ml vehicle (0.1 M HEPES-0.05 M NaH 2 PO 4 ). A separate group of 10 animals received 0.2ml vehicle.
  • Injections were given between 0700 h and 0800 h and body weight were recorded daily between 1600-1700 h. The weights of rats on the day after the last injection were taken as the final weight.
  • Experiment H demonstrates that C3-PEG administered sc ETD exhibits greater potency than WT IGF-I administered sc ETD. All doses of C3-PEG stimulated greater mean weight gain than animals given 320mg WT IGF-I SID. The enhanced pharmacodynamics of C3-PEG make it more potent than WT IGF-I in the animal model described.
  • Experiment HI Rats treated with sc injections of C3-PEG 160 mg and 320 mg gained 8.3g ⁇ 0.7g and 9.0g ⁇ 0.6g, respectively (Table 4). Vehicle gained 4.2g + 0.3g. The weight gained induced by C3-PEG was statistically greater than animals given vehicle. Similarly, the tibial epiphyseal widths of rats receiving C3-PEG were statistically greater than rats receiving vehicle (Table 8).
  • Experiment HI demonstrates that C3-PEG stimulates not only weight gain, but also bone growth in HYPOX rats. This indicates that C3-PEG may be a useful pharmaceutical for the induction of bone formation.

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Abstract

L'invention concerne des formes modifiées du facteur de croissance proche de l'insuline (IGF), qui présentent des propriétés biologiques et pharmacologiques améliorées. Ces modifications comprennent des mutéines de l'IGF produits par substitution ou addition d'une cystéine dans la séquence d'acides aminés de l'IGF natif, ainsi que des mutéines fixées à du polyéthylène-glycol (PEG) au niveau du site de cystéine libre. La présente invention concerne, en outre, des procédés de production de ces formes modifiées. Ces conjugués de PEG-IGF peuvent être formulés pour être transformés en compositions pharmaceutiques et utilisés pour le traitement des conditions associées à l'IGF.
PCT/US1995/006540 1994-05-24 1995-05-24 Facteurs de croissance proches de l'insuline modifies WO1995032003A1 (fr)

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AU26018/95A AU2601895A (en) 1994-05-24 1995-05-24 Modified insulin-like growth factors

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Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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US7005504B2 (en) 1998-01-22 2006-02-28 Genentech, Inc. Antibody fragment-peg conjugates
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US9125880B2 (en) 2002-12-26 2015-09-08 Mountain View Pharmaceuticals, Inc. Polymer conjugates of interferon-beta with enhanced biological potency
US9175061B2 (en) 2006-07-07 2015-11-03 Novo Nordisk Health Care Ag Protein conjugates and methods for their preparation
US9718892B2 (en) 2010-05-21 2017-08-01 Merrimack Pharmaceuticals, Inc. Method of treating myocardial infarction by administering a bi-specific fusion protein
US10040840B2 (en) 2015-10-02 2018-08-07 Silver Creek Pharmaceuticals, Inc. Bi-specific annexin A5/IGF-1 proteins and methods of use thereof to promote regeneration and survival of tissue
WO2020206375A1 (fr) * 2019-04-03 2020-10-08 Technical University Of Denmark Conjugués de protéine de facteur neurotrophique et modes de réalisation associés

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990012874A2 (fr) * 1989-04-21 1990-11-01 Genetics Institute, Inc. Variantes de polypeptides par addition de cysteine, et leurs modifications chimiques
WO1992016221A1 (fr) * 1991-03-15 1992-10-01 Synergen, Inc. Pegylation de polypeptides
WO1994012219A2 (fr) * 1992-11-25 1994-06-09 Amgen Boulder Inc. Facteur de croissance insulinoide modifie
WO1994022466A1 (fr) * 1993-04-07 1994-10-13 Synergen, Inc. Procedes d'utilisation de proteines de liaison de facteurs de croissance semblables a l'insuline

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990012874A2 (fr) * 1989-04-21 1990-11-01 Genetics Institute, Inc. Variantes de polypeptides par addition de cysteine, et leurs modifications chimiques
WO1992016221A1 (fr) * 1991-03-15 1992-10-01 Synergen, Inc. Pegylation de polypeptides
WO1994012219A2 (fr) * 1992-11-25 1994-06-09 Amgen Boulder Inc. Facteur de croissance insulinoide modifie
WO1994022466A1 (fr) * 1993-04-07 1994-10-13 Synergen, Inc. Procedes d'utilisation de proteines de liaison de facteurs de croissance semblables a l'insuline

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GOODSON, ROBERT J. ET AL: "Site-directed PEGylation of recombinant interleukin-2 at its glycosylation site", BIO/TECHNOLOGY (1990), 8(4), 343-6 CODEN: BTCHDA;ISSN: 0733-222X *
KUAN, CHIEN TSUN ET AL: "Pseudomonas exotoxin A mutants. Replacement of surface exposed residues in domain II with cysteine residues that can be modified with polyethylene glycol in a site-specific manner", J. BIOL. CHEM. (1994), 269(10), 7610-16 CODEN: JBCHA3;ISSN: 0021-9258 *
TANG, WEI ET AL: "Preparation of a new PEGylation reagent for sulfhydryl-containing polypeptide", TETRAHEDRON LETT. (1994), 35(35), 6515-16 CODEN: TELEAY;ISSN: 0040-4039 *

Cited By (92)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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US6608183B1 (en) 1997-07-14 2003-08-19 Bolder Biotechnology, Inc. Derivatives of growth hormone and related proteins
US9637530B2 (en) 1997-07-14 2017-05-02 Bolder Biotechnology, Inc. Methods of preparing cysteine variants of human recombinant IL-11
US8748392B2 (en) 1997-07-14 2014-06-10 Bolder Biotechnology Inc. Methods of treatment using cysteine variants of interleukin-11
US7314921B2 (en) 1997-07-14 2008-01-01 Bolder Biotechnology, Inc. Cysteine variants of erythropoietin
US8618256B2 (en) 1997-07-14 2013-12-31 Bolder Biotechnology Cysteine variants of interferon gamma
US8148500B2 (en) 1997-07-14 2012-04-03 Bolder Biotechnology, Inc. Method of preparing cysteine mutants of human recombinant GM-CSF
US7964184B2 (en) 1997-07-14 2011-06-21 Bolder Biotechnology, Inc. Cysteine variants of interferon-gamma
US7309781B2 (en) 1997-07-14 2007-12-18 Bolder Biotechnology, Inc. Cysteine variants of granulocyte colony-stimulating factor
US7148333B2 (en) 1997-07-14 2006-12-12 Bolder Biotechnology, Inc. Cysteine variants of granulocyte-macrophage colony-stimulating factor
US7153943B2 (en) 1997-07-14 2006-12-26 Bolder Biotechnology, Inc. Derivatives of growth hormone and related proteins, and methods of use thereof
US7214779B2 (en) 1997-07-14 2007-05-08 Bolder Biotechnology Inc. Termini cysteine-added variants of granulocyte-macrophage colony stimulating factor
US8133480B2 (en) 1997-07-14 2012-03-13 Bolder Biotechnology, Inc. Cysteine variants of interleukin-11
US7824669B2 (en) 1997-07-14 2010-11-02 Bolder Biotechnology, Inc. In vivo stimulation of peripheral blood progenitor cells by granulocyte-macrophage colony stimulating factor (GM-CSF) cysteine muteins and their PEGylated variants
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US7253267B2 (en) 1997-07-14 2007-08-07 Bolder Biotechnology Inc. Cysteine variants of interleukin-11
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US7005504B2 (en) 1998-01-22 2006-02-28 Genentech, Inc. Antibody fragment-peg conjugates
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US7141545B2 (en) 1998-04-03 2006-11-28 Novartis Vaccines And Diagnostics, Inc. Compositions and methods for treating articular cartilage disorders
WO2000042175A1 (fr) * 1999-01-14 2000-07-20 Bolder Biotechnology Inc. Techniques permettant de produire des proteines contenant des residus cysteine libres
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US7399839B2 (en) 1999-01-14 2008-07-15 Bolder Biotechnology, Inc. Monopegylated growth hormone proteins
KR100731826B1 (ko) * 1999-01-14 2007-06-22 볼더 바이오테크놀로지 인코퍼레이티드 유리 시스테인 잔기를 함유하는 단백질의 제조 방법
US20160244798A1 (en) * 1999-01-14 2016-08-25 Bolder Biotechnology, Inc. Methods for making proteins containing free cysteine residues
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US6753165B1 (en) 1999-01-14 2004-06-22 Bolder Biotechnology, Inc. Methods for making proteins containing free cysteine residues
US7842789B2 (en) 1999-01-21 2010-11-30 Genentech, Inc. Antibody fragment-polymer conjugates and uses of same
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US7507405B2 (en) 1999-01-21 2009-03-24 Genentech, Inc. Antibody fragment-polymer conjugates and uses of same
EP1172372A1 (fr) * 1999-04-02 2002-01-16 Ajinomoto Co., Inc. Procede de production de sous-unite de peptide issue d'une proteine de polymere
EP1172372A4 (fr) * 1999-04-02 2003-05-14 Ajinomoto Kk Procede de production de sous-unite de peptide issue d'une proteine de polymere
US7270972B1 (en) 1999-04-02 2007-09-18 Ajinomoto Co., Inc. Process for producing subunit peptide originating in polymer protein
JP4670150B2 (ja) * 1999-04-02 2011-04-13 味の素株式会社 多量体蛋白質に由来するサブユニットペプチドの製造法
US7306931B2 (en) 2000-05-16 2007-12-11 Bolder Biotechnology, Inc. Method for refolding proteins containing free cysteine residues
WO2001087925A3 (fr) * 2000-05-16 2002-08-01 Bolder Biotechnology Inc Procede de repliement de proteines renfermant des residus de cysteine libre
WO2001087925A2 (fr) * 2000-05-16 2001-11-22 Bolder Biotechnology, Inc. Procede de repliement de proteines renfermant des residus de cysteine libre
US8932828B2 (en) 2000-05-16 2015-01-13 Bolder Biotechnology, Inc. Method for preparing recombinant granulocyte colony stimulating factor cysteine muteins
CN1318443C (zh) * 2000-05-16 2007-05-30 博尔德生物技术公司 含游离半胱氨酸残基的蛋白重折叠的方法
EP2382982A2 (fr) 2001-08-24 2011-11-02 Neuren Pharmaceuticals Limited Peptides de régéneration neurale et procédés d'utilisation de ces derniers dans le traitement des lesions cerebrales
US8129330B2 (en) 2002-09-30 2012-03-06 Mountain View Pharmaceuticals, Inc. Polymer conjugates with decreased antigenicity, methods of preparation and uses thereof
US9125880B2 (en) 2002-12-26 2015-09-08 Mountain View Pharmaceuticals, Inc. Polymer conjugates of interferon-beta with enhanced biological potency
WO2006066891A3 (fr) * 2004-12-22 2007-05-31 Hoffmann La Roche Conjugues du facteur de croissance 1 analogue a l'insuline et du poly(ethylene glycol)
KR101106860B1 (ko) * 2004-12-22 2012-01-19 에프. 호프만-라 로슈 아게 인슐린-유사 성장 인자-1(ｉｇｆ-1) 및 폴리(에틸렌 글라이콜)의 접합체
US20150099699A1 (en) * 2004-12-22 2015-04-09 Hoffmann-La Roche Inc. Conjugates of insulin-like growth factor-1 and poly(ethylene glycol)
WO2006066891A2 (fr) * 2004-12-22 2006-06-29 F. Hoffmann-La Roche Ag Conjugues du facteur de croissance 1 analogue a l'insuline et du poly(ethylene glycol)
US9724425B2 (en) 2004-12-22 2017-08-08 Hoffmann-La Roche Inc. Conjugates of insulin-like growth factor-1 and poly(ethylene glycol)
KR100915278B1 (ko) * 2004-12-22 2009-09-03 에프. 호프만-라 로슈 아게 인슐린-유사 성장 인자-1(ｉｇｆ-1) 및 폴리(에틸렌글라이콜)의 접합체
EP1674113A1 (fr) * 2004-12-22 2006-06-28 F. Hoffmann-La Roche Ag Conjugués de l'IGF-1 et du poly(éthylène glycol)
EA011284B1 (ru) * 2004-12-22 2009-02-27 Ф.Хоффманн-Ля Рош Аг Конъюгаты инсулиноподобного фактора роста i (ифр-i) и полиэтиленгликоля
US8293708B2 (en) 2005-08-30 2012-10-23 Novo Nordisk Health Care A/G Liquid formulations N-terminal serine of pegylated growth hormone
US8329866B2 (en) 2005-10-03 2012-12-11 Bolder Biotechnology, Inc. Long acting VEGF inhibitors and methods of use
US8343918B2 (en) 2006-06-09 2013-01-01 Novartis Ag Stabilized insulin-like growth factor polypeptides
US8722621B2 (en) 2006-06-09 2014-05-13 Novartis Ag Stabilized insulin-like growth factor polypeptides
US9175061B2 (en) 2006-07-07 2015-11-03 Novo Nordisk Health Care Ag Protein conjugates and methods for their preparation
US8476232B2 (en) 2006-08-31 2013-07-02 Hoffman-La Roche Inc. Method for the production of conjugates of insulin-like growth factor-1 and poly(ethylene glycol)
US8552158B2 (en) 2006-08-31 2013-10-08 Hoffmann-La Roche Inc. Method for the production of insulin-like growth factor-1
US7625996B2 (en) 2006-08-31 2009-12-01 Hoffmann-La Roche Inc. Method for the production of conjugates of insulin-like growth factor-1 and poly(ethylene glycol)
US8617531B2 (en) 2006-12-14 2013-12-31 Bolder Biotechnology, Inc. Methods of making proteins and peptides containing a single free cysteine
US10508131B2 (en) 2006-12-14 2019-12-17 Bolder Biotechnology, Inc. Cysteine analogs of exendin-4
US9296804B2 (en) 2006-12-14 2016-03-29 Bolder Biotechnology, Inc. NH2-terminal glutamine modified cysteine variants of interferon gamma
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WO2009121759A3 (fr) * 2008-04-03 2010-03-25 F. Hoffmann-La Roche Ag Utilisation de variants de igf-i pegylés pour traiter des troubles neuromusculaires
WO2009121759A2 (fr) * 2008-04-03 2009-10-08 F. Hoffmann-La Roche Ag Utilisation de variants de igf-i pegylés pour traiter des troubles neuromusculaires
WO2011098400A1 (fr) * 2010-02-11 2011-08-18 F. Hoffmann-La Roche Ag Conjugués de l'igf-i et du poly(éthylène glycol)
US9587006B2 (en) 2010-02-11 2017-03-07 Hoffmann-La Roche Inc. IGF-I poly (ethylene glycol) conjugates
CN102740896A (zh) * 2010-02-11 2012-10-17 霍夫曼-拉罗奇有限公司 Igf-i聚乙二醇缀合物
US20120309679A1 (en) * 2010-02-11 2012-12-06 Friederike Hesse Igf-i poly (ethylene glycol) conjugates
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US10407512B2 (en) 2010-05-21 2019-09-10 Silver Creek Pharmaceuticals, Inc. Bi-specific fusion proteins
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WO2012102832A1 (fr) 2011-01-27 2012-08-02 Neuren Pharmaceuticals Limited Traitement de troubles du spectre autistique en utilisant l'acide glycyl-l-2-méthylprolyl-l-glutamique
US10040840B2 (en) 2015-10-02 2018-08-07 Silver Creek Pharmaceuticals, Inc. Bi-specific annexin A5/IGF-1 proteins and methods of use thereof to promote regeneration and survival of tissue
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US11155593B2 (en) 2015-10-02 2021-10-26 Silver Creek Pharmaceuticals, Inc. Method of inhibiting apoptosis or promoting cell survival by providing a bi-specific protein comprising insulin-like growth factor IGF-1 and Annexin A5
US11879002B2 (en) 2015-10-02 2024-01-23 Silver Creek Pharmaceuticals, Inc. Bi-specific therapeutic proteins, in vivo methods of use thereof and encoding nucleic acids thereof
WO2020206375A1 (fr) * 2019-04-03 2020-10-08 Technical University Of Denmark Conjugués de protéine de facteur neurotrophique et modes de réalisation associés

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