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WO1994022466A1 - Procedes d'utilisation de proteines de liaison de facteurs de croissance semblables a l'insuline - Google Patents

Procedes d'utilisation de proteines de liaison de facteurs de croissance semblables a l'insuline Download PDF

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Publication number
WO1994022466A1
WO1994022466A1 PCT/US1994/003755 US9403755W WO9422466A1 WO 1994022466 A1 WO1994022466 A1 WO 1994022466A1 US 9403755 W US9403755 W US 9403755W WO 9422466 A1 WO9422466 A1 WO 9422466A1
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igfbp
igf
growth
modified form
polymer
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PCT/US1994/003755
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English (en)
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George N. Cox
Deborah A. Russell
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Synergen, Inc.
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Priority to AU66267/94A priority Critical patent/AU6626794A/en
Priority to JP6522477A priority patent/JPH08508296A/ja
Priority to EP94914048A priority patent/EP0708655A1/fr
Publication of WO1994022466A1 publication Critical patent/WO1994022466A1/fr
Priority to FI954805A priority patent/FI954805L/fi

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4743—Insulin-like growth factor binding protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to uses of insulin-like growth factor binding protein 1 (IGFBP-1 or BP-1) as a therapeutic agent.
  • IGFBP-1 or BP-1 insulin-like growth factor binding protein 1
  • IGF-I and IGF-II Circulating insulin-like growth factors I and II are 7 kDa proteins that are related in structure to each other and to insulin. IGF-I and IGF-II
  • IGF-I growth and differentiation factors for most cells in the body and are present at high concentrations in serum (about 300 ng/ml for IGF-I and 1000 ng/ml for IGF-II). 15 Circulating levels of IGF-I are determined primarily by growth hormone (GH) , which stimulates the liver to make IGF-I. Most of the growth-promoting effects of growth hormone are believed to be mediated by IGF-I.
  • GH growth hormone
  • Tissue IGF-I has a larger apparent molecular mass (approximately 26 kDa), as determined by gel chromatography, than circulating IGF. 20 Rom, W.N. et al., J. Clin. Invest., 82: 1685-1693 (1988).
  • IGF-I and IGF-II have been shown to play a role in a large number of disease conditions. These include, for example, breast cancer, colon cancer, lung cancer, ovarian cancer, osteosarcoma, glioma, liver cancer, prostate cancer, rhabdomyosarcomas, restenosis, acromegaly, obesity, tumor-induced hypoglycemia,
  • IGF-I and IGF- ⁇ are believed to function as autocrine or paracrine growth factors for a variety of human cancers, including breast cancers, colon cancers, lung cancers, ovarian cancers, osteosarcomas, neuroblastomas, gliomas, Wilm's tumors, and rhabdomyosarcomas as reported in Cullen et al., Cancer Investigation. 9:443-454 (1991).
  • IGF-H overexpression correlated with the degree of invasion of the bowel wall.
  • IGF-I can mediate autocrine proliferation of human small cell lung cancer cell lines NCI-H345 and NCI-N417, as reported in Nakanishi, et al. , J. Clin. Invest.. 82:354-359 (1988).
  • Ovarian cancer cell lines OVCAR-3, OVCAR-7 and PEO4 express IGF-I mRNA.
  • Primary and metastatic ovarian cancer tissues also express IGF-I mRNA as well as Type I IGF receptor mRNA, as reported in Yee, et al, Cancer Res.. 51:5107-5112 (1991).
  • IGF-H mRNA expression is developmentally regulated in liver tissue. Increased IGF-H mRNA levels have been detected in liver cancers of woodchucks, humans and rats, as reported in Cariani, et al., J. Hepatology. 13:220-226 (1990).
  • IGF-H mRNA Elevated levels of IGF-H mRNA and have been reported in rhabdomyosarcoma tumors as described in El-Badry, et al., Cell Growth & Differentiation. 1:325-331 (1990). As indicated above, the insulin-like growth factors have been associated with certain non-cancerous disorders such as acromegaly and restenosis, for example.
  • GH growth hormone
  • IGF-I gene expression is induced 9-fold in the denuded artery following angioplasty surgery, as reported in Cercek et al Circulation Research. 66: 1755-1760 (1990). The timing and level of IGF-I gene expression closely parallels that of smooth muscle cell proliferation. Hybridization studies indicate that the dividing smooth muscle cells are the cells that exhibit increased IGF-I gene expression, as reported in Khorsandi et al, J. Clin. Invest.. 90:1926-1931 (1992).
  • the fibroblasts secrete an extracellular collagenous matrix.
  • the fibroblasts and the matrix secretions cause the alveolar walls to thicken and cause a loss of alveolar- capillary units.
  • Activated alveolar macrophages have been shown to release an IGF-1 that signals replication of fibroblasts. Rom, W.N., et al., J. Clinic. Invest., 82: 1685-1693 (1988).
  • Alveolar macrophages from patients with interstitial lung disorders have been shown to spontaneously secrete this IGF-I. Id.; Bitterman, P.B. et al., J. Clin. Invest., 72: 1801-1813 (1983).
  • Current treatments for the cancerous and non-cancerous disorders include surgery, radiation, chemotherapy and hormone therapy. For example, various cancers, such as breast, lung, ovarian, colon and osteosarcomas are treated with surgery, radiation and chemotherapeutic agents.
  • Chemotherapeutic agents used for treating these cancers include fluoropyrimidines and alkylating agents. Both of these groups exhibit significant toxicities, including, for example, myelosuppression, immunosuppression, neutropenia, gastrointestinal toxicity, renal toxicity and peripheral neuropathies. Additionally, there is no known effective chemotherapeutic agent for treatment of liver cancer. Surgery is highly invasive and unpredictable, while radiation is non-specific at the point of localization. Hormone therapy has undesirable side effects such as unwanted hair growth and mood
  • Antibodies to the type I IGF-I receptor have been shown to block growth of
  • IGF-I-responsive carcinoma cell lines in vitro. Studies have shown that antibodies to the IGF-I receptor block growth of certain breast and lung carcinomas transplanted into immunodeficient nude mice as reported in Arteaga et al., J. Clin. Invest.. 84: 1418-1423, (1989); and Zia et al., Proc. Amer. Assoc. for Cancer Research. 33:270, Abstract 1616 (1992).
  • Antisense sequences to the IGF-I gene have been shown to block growth of a malignant rat glioma cell line transplanted into rats as described in Trojan et al. , Proceedings of the National Academy of Science. 89:4874-4878 (1992). However, using antisense sequences for gene therapy is still in the early development stage. Thus, a need exists for an agent which would inhibit the action of IGFs in the above cancers and disease conditions.
  • the present invention provides such an IGF inhibitory agent, namely IGFBP- 1 for the inhibition of the inappropriate action of IGFs in these disease conditions.
  • IGFBPs insulin-like growth factor binding proteins
  • the binding protein is IGFBP- 1, also termed "BP-1.”
  • IGFBP- 1 insulin-like growth factor binding proteins
  • modified forms of IGFBP- 1 include IGFBP- 1 attached to a polymer or 2 or more IGFBP- 1 molecules attached to a polymer.
  • the methods involve administering IGFBPs, including for example, IGFBP- 1 or a modified form of IGFBP- 1 to a patient having an IGF associated condition sufficient to cause a therapeutic effect.
  • IGFBP- 1 For IGFBP- 1, it is contemplated that a therapeutic effect can be achieved when circulating levels of IGFBP- 1 range from about 0.1 ⁇ g to about 300 ⁇ g per ml in the bloodstream of the patient. Examples of conditions wherein administration of IGFBPs, in particular IGFBP-1 may
  • breast cancer colon cancer
  • lung cancer a malignant neoplasm
  • This invention also relates to pharmaceutical compositions containing IGFBPs, and in particular, IGFBP-1 or a modified form of IGFBP- 1 and methods of treating or preventing IGF associated conditions using the pharmaceutical compositions.
  • IGFBPs Inappropriate expression or utilization of IGF-I or IGF-H is a contributing factor in many disease conditions. It is contemplated that administration of IGFBPs, and in particular IGFBP- 1, may be a useful therapeutic in disease conditions which are associated with inappropriate expression or utilization of IGFs, particularly IGF-I or IGF-H.
  • the present invention is directed to methods of treating a patient having an IGF associated condition or of preventing an IGF-associated condition by administering an amount of IGFBP, including IGFBP- 1 or a modified form of IGFBP- 1, sufficient to cause a therapeutic effect.
  • IGFBP including IGFBP- 1 or a modified form of IGFBP- 1
  • IGF-I refers to a protein containing the same amino acid sequence as naturally occurring IGF-I, or a protein containing the same amino acid sequence as naturally occurring IGF-I with the addition of an N-terminal methionine (met-IGF- I), unless otherwise specified.
  • IGF refers to any polypeptide that binds to the IGF type I Receptor, including, for example, IGF-I, IGF-H, (desl-3)IGF-I, met-IGF-I, insulin, and any active fragments which bind to the type I Receptor. This hormone family is described in Blundell and Humbel, Nature. 287:781-787 (1980).
  • IGF associated condition refers to an existing or potential adverse physiological condition which results from or is associated with an overproduction 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
  • IGFBP administration alleviates or reduces disease symptoms.
  • An IGF associated condition also refers to a condition in which administration of IGFBPs including IGFBP- 1 to a normal patient has a desired effect.
  • IGF associated conditions include breast cancer, colon cancer, osteosarcoma, glioma, lung cancers, rhabdomyosarcomas, ovarian cancer, liver cancer, acromegaly, obesity, tumor-induced hypoglycemia, pulmonary fibrosis, restenosis, diabetic nephropathy and diabetic retinopathy.
  • the term "patient” refers to any animal, including humans, in need of treatment for an IGF associated condition.
  • IGFBP refers to any of the six known IGF binding proteins or to fragments of these binding proteins which bind to IGF.
  • IGF-I and IGF-II circulate in blood bound to specific binding proteins of which six are now known. The binding proteins bind 95% or more of the IGFs in blood.
  • One theory is that when bound by binding proteins, IGF-I and IGF-II are prevented from interacting with certain cell surface receptors mediating their biological functions.
  • IGF binding protein- 1 is a 23 kDa IGF binding protein. IGFBP- 1 is expressed in vivo during periods of growth arrest (e.g., starvation and diabetes), suggesting
  • IGFBP-1 acts as an IGF-I inhibitor.
  • IGFBP-1 acts as an IGF-I inhibitor.
  • Amniotic fluid is a natural source of IGFBP- 1 and contains both phosphorylated and non-phosphorylated forms of this binding protein. Jones, J.I. et al., Proc. Natl. Acad. Sci., 88: 7481-7485 (1991). Phosphorylated BP-1 has a higher affinity for IGF-1 than the non-phosphorylated form. Jones, J.I. et al., J. Biol.
  • non-phosphorylated IGFBP- 1 can also be inhibitory to cell growth in vitro and in vivo. Specifically, it has been found that bacterially-derived recombinant BP-I inhibits the damaging cell growth which attends certain IGF-associated conditions. Accordingly, the IGFBP- 1 useful in the methods of the present invention can be phosphorylated or non-phosphorylated. Thus, the BP-1 useful in the present invention can be purified from natural sources such as amniotic fluid, or can be produced in accordance with recombinant procedures well known in the art.
  • the amino amino acid sequence of mature IGFBP- 1 is:
  • amino acid sequence of the signal sequence is: Ser-Glu-Val-Pro-Val-Ala- Arg-Val-Trr *Leu-Nal-I ⁇ u-Leu-I-eu-I ⁇ u-Thr-Val-Gln-Val-Gly-Val-Thr-Ala-Gly (SEQ ⁇ o.:2).
  • SEQ ID NO.: 1 one skilled in the art can chemically synthesize a DNA encoding IGFBP- 1.
  • oligonucleotide probes based upon SEQ TD NO. : 1 to isolate a genomic DNA or mRNA and generate a cDNA.
  • the DNA encoding the IGBBP-1 can be used to transform a host for recombinant production.
  • BP-1 can be expressed in E. coli BL21/DE3 using the T7 promoter system as an insoluble protein in inclusion bodies.
  • BL21/DE3 is described by Studier, F.W., and Moffatt, B., J. Mol. Biol, 189: 113-30 (1986).
  • a TAC promoter system can be employed.
  • E. Coli the recombinantly expressed
  • BP-1 is contained in the isoluble fraction.
  • the insoluble protein is improperly folded and inactive.
  • BP-1 can be denatured and folded into its proper conformation by dissolving the protein in 6M guanidine and a reducing agent, diluting the mixture 10- fold and allowing the BP-1 to refold overnight.
  • IGFBP- 1 contains 18 cysteine residues, all of which are believed to participate in forming disulfide bridges.
  • the protein refolds to a single major species.
  • the refolded protein can be purified using sequential Q-sepharose and butyl-sepharose columns.
  • the yield of purified BP-1 per 10L fermenter run is about 1.5 g.
  • IGFBP- 1 can also be expressed in mammalian expression systems as set forth in Jones, J.I. et al., Proc. Natl. Acad. Sci.. 88: 7481-7485 (1991) and in Jones, J.I. etal, J. Biol. Chem.. 268, 2: 1125-1131 (1993), both specifically incorporated herein by reference.
  • a DNA encoding both the mature protein and the signal sequence should be employed.
  • One skilled in the art can choose any appropriate vector and expression system as desired.
  • IGFBPs including IGFBP- 1
  • the therapeutic utility of IGFBPs may be enhanced by increasing their circulating half-lives.
  • Increasing the molecular weight of a protein for example by covalently bonding an inert polymer chain such as polyethylene glycol (PEG) to the protein, is known to increase the circulating half-life of the protein in the body.
  • PEG polyethylene glycol
  • Covalent attachment of PEG to protein is termed herein "PEGylation.”
  • PEGylated means conjugated to polymer.
  • One useful method of PEGylation involves creation of a mutein having a cysteine residue available for attachment to a polymer activated with a thiol-specific reactive group.
  • Muteins can be prepared using mutagenesis techniques well known in the art. For example, the IGFBP-l mutein is created by replacing one or more specific amino acids with cysteine residues, or by inserting a cysteine residue between amino acids or at the N or C terminus. It is expected that such non-native cysteine residues will be "free,” i.e. not involved in intramolecular disulfide bonds.
  • Non- native cysteine residues can be substituted or inserted in regions of the IGFBP-l molecule that are exposed on the protein's surface, and which are not involved in receptor binding or binding to IGF.
  • One site for insertion or substitution of the cysteine may occur in the middle of the BP-1 protein. It is believed that cysteine can be substituted or inserted from amino acid 60 to 180, with the residue numbering based upon SEQ ID NO.:l.
  • Particularly useful muteins include the substitution of a cysteine residue at positions 98 and 101 for the naturally occurring serine found at those locations.
  • Attachment of the inert polymer chain molecule to one or more IGFBP-l molecules creates a further modified form of IGFBP-l, an IGFBP-l -polymer conjugate also called "PEGylated IGFBP-l". Coupling of thiol-specific reactive groups to polymers is discussed PCT Application Publication No. WO92/ 16221, incorporated herein by reference. If a cysteine mutein is coupled to the polymer via a thiol-specific reactive group, the conjugate formed is expected to be attached to the protein at the non-native cysteine residue.
  • the non-native cysteine might become involved in a disulfide bond and thereby free a native cysteine for PEGylation.
  • the polymer is attached at that native cysteine residue. Using peptide mapping, one can determine the specific PEGylation site.
  • the present invention further provides a pharmaceutical composition containing the IGFBPs, including of IGFBP-l, in an acceptable pharmaceutical carrier.
  • a pharmaceutical carrier is physiological saline solution, but it is contemplated that other acceptable pharmaceutical carriers may also be used.
  • the carrier and the IGFBP-l constitute a physiologically-compatible, slow-release formulation.
  • the primary solvent in such a carrier may be either aqueous or non-aqueous in nature.
  • the carrier may 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 may contain still other pharmacologically- acceptable excipients for modifying or maintaining the stability, rate of dissolution, release, or absorption of the IGFBP-l.
  • excipients are those substances usually and customarily employed to formulate dosages for administration in either unit dose
  • the therapeutic composition may 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
  • 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 IGFBP-l are stored and administered at or near physiological pH. It is presently believed that storage and administration in a formulation at a high pH (i.e. greater than 9) or at a low pH (i.e. less than 4) is undesirable.
  • compositions of the present invention can be administered by intravenous, parenteral, intramuscular, subcutaneous, intraarticular injection or infusion, inhalant mists, orally active formulations, or suppositories.
  • intravenous, parenteral, intramuscular, subcutaneous, intraarticular injection or infusion, inhalant mists, orally active formulations, or suppositories may be administered.
  • This method is intended to create a preselected concentration range of IGFBP-l in the patient's blood stream. It is believed that the maintenance of circulating concentrations of IGFBP-l of 0.1 ⁇ g to 300 ⁇ g per ml in the bloodstream may be effective in treating an IGF associated condition.
  • the frequency of dosing will depend on pharmacokinetic parameters of the IGFBP-l in the formulation used.
  • IGFBP-l blocks the mitogenic effects of IGF-I and IGF-II on breast cancer, colon and osteosarcoma cancer cells in vitro. Estrogen stimulates growth of breast cancers at least in part by causing the cells to secrete IGF-I or IGF-II.
  • BP-1 Concentrations of BP-1 of 1-10 ⁇ g/ml were required to inhibit growth of the colon cancers.
  • One of the colon carcinoma cell lines grew in serum free media, presumably because it produced its own growth factors. Growth of this cell line in serum free media was inhibited at least 50% by high concentrations of BP-1.
  • IGFBP-l inhibited the mitogenic effect of 50 ng/ml IGF-I by 50% on rat osteosarcoma cells.
  • IGFBP-l was also shown to inhibit the proliferative response of smooth muscle cells to IGF-I.
  • IGFBP-l administered after angioplasty in rats significantly inhibited the intimal thickening that results from smooth muscle proliferation and extracellular matrix deposition. These results indicate that IGFBP-l is useful in the treatment or prevention of restenosis.
  • IGFBP-l In hypophysectomized rats, IGFBP-l inhibited the growth promoting effects of IGF-I and of growth hormone. In addition, IGFBP-l, its muteins, and PEGylated IGFBP-l inhibited IGF-I stimulation of growth of mouse 3T3 fibroblast cells.
  • the following examples are intended to illustrate but not limit the present invention.
  • Example 1 A Purification & Refolding of IGFBP-l
  • E. coli cells expressing the IGFBP-l were suspended in Buffer A (50 mM
  • Tris pH 7.5, 20 mM NaCl and 1 mM DTT
  • concentration 40 ml/10 g cell paste
  • the suspension was centrifuged 20,000 x g for 30 minutes, and aliquots of the pellet & supernatant were analyzed by SDS-PAGE. A major band corresponding to the IGFBP-l was present in the pellet, but not the supernatant.
  • the pellet was suspended in Buffer A
  • the final pellet containing the IGFBP-l was suspended in 6M guanidine, 50 mM Tris, pH 7.5, 6 mM DTT (25 ml/10 g cells) using a ground glass homogenizer. The suspension was incubated at room temperature for 15 minutes. The undissolved protein was removed by centrifugation at 20,000 x g for 30 minutes. Final concentration of the IGFBP-l was 1.0 mg/ml. SDS-PAGE analysis of the pellet and supernatant showed that IGFBP-l was present in the supernatant only.
  • the denatured and reduced IGFBP-l was subjected to a 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 minutes.
  • GSSG mixed-disulfide producing reagent
  • the solution was then diluted 10 fold gradually with 50 mM tris. pH 9.7 and phenylmethylsulfonylfluoride was added to final concentration of ImM. Final concentration of protein was lOO ⁇ g/ml.
  • the refolding mixture was incubated overnight at 4°C, and then centrifuged at 20,000 x g for 15 minutes. SDS-PAGE analysis of the pellet and supernatant showed that the supernatant was composed of relatively homogeneous IGFBP-l.
  • the refold mixture (15000 ml) prepared from 590g of E. coli paste containing the correctly refolded IGFBP-l was concentrated to 1800 ml, dialyzed against 20 mM sodium phosphate, pH 6.0, centrifuged at 10,000 x g for 30 minutes to remove precipitated E. coli proteins and loaded onto an Q-Sepharose (Pharmacia/LKB, Piscataway, NJ) column (5.0 x 60 cm) previously equilibrated with the same buffer. The bound protein was eluted with a 5000 ml linear gradient to 0.5M NaCl at a flow rate of 20 ml minute. 25 ml fractions were collected.
  • the protein was eluted with a 1500 ml linear gradient to 20 mM HEPES, pH 7.5 at a flow rate of 40 ml/minute. A single broad peak eluted at 5 - 15% ethanol. Aliquots (10 ⁇ l) of each peak fraction were analyzed by RP-4 reverse phase chromatography and SDS-PAGE. Fractions containing pure (95 %) correctly refolded IGFBP-l were pooled, concentrated to 6-8 mg/ml and assayed for bioactivity. In all of the following experiments using IGFBP-l, the recombinant E. Coli.- expressed IGFBP-l was used.
  • Example 2 Example 2
  • IGFBP-l inhibits the growth promoting effects of IGF-I and growth hormone in rats
  • Hypophysectomy removes the source of growth hormone from the body and results in growth cessation.
  • Hypophysectomized animals can be stimulated to grow by injection of exogenous growth hormone or IGF-I, as described in Schoenle et al., Nature. 296: 252-253 (1982).
  • the effects of subcutaneously administered IGFBP-l were tested on IGF-I and growth hormone- stimulated growth in this model. Growth was assessed by measuring body weight gain and tibial epiphyseal width.
  • the body weights of the rats were monitored for two to three weeks before the beginning of the experiments in order to verify completeness of the hypophysectomy. Rats gaining more than 2 grams of body weight per week were excluded from the study. Rats were injected subcutaneously twice a day at the nape of the neck for eight consecutive days with 0.2 ml of vehicle solution (40 mM HEPES, 100 mM NaCl), IGF-I (80 ⁇ g) alone, IGFBP-l alone, or IGF-I (80 ⁇ g) combined with various molar ratios of BP-1. The molar ratios of IGFBP-l :IGF-I tested ranged from 0.04:1 to 5:1. Body weights were determined daily.
  • the rats were sacrificed 12 hours after receiving their final injection. Their right and left tibias were removed, fixed with formalin, split at their proximal ends in a sagittal plane and stained with silver nitrate, as described in Greenspan, Endocrinology. 45:455-463 (1949). The calcified tissue was stained dark brown and the proliferating zone of cartilage appeared as a clearly defined white band. The cartilaginous epiphyseal plate was measured with a stereomicroscope equipped with a calibrated micrometer eyepiece. Approximately ten individual readings were made across each epiphysis. The mean of the combined readings from the right and left tibias was calculated for each rat.
  • IGFBP-l IGF-I group showed no significant weight gain, indicating that excess IGFBP-l blocked the growth-promoting effects of IGF-I in this model.
  • the administration of IGFBP-l at molar ratios of 1: 1 and 0.2: 1 relative to IGF-I caused 50 to 75 % inhibition of IGF-I-stimulated growth. No enhancement of growth above that stimulated by IGF-I alone was measured in any group receiving IGFBP-l. In addition, the administration of IGFBP-l alone had no significant growth promoting effects.
  • IGF-I* IGFBP-1* IGFBP-1 INCREASE IN TIBIAL
  • ** Values are means ⁇ standard errors of the mean for 8 to 15 rats per group.
  • the design of this experiment was the same as the IGF-I experiments except that rats received injections of growth hormone rather than IGF-I.
  • Growth hormone and IGFBP-l were injected subcutaneously at separate injection sites.
  • IGFBP-l was administered twice a day at 10 mg/kg per injection, a dose that was equivalent to that administered in the 5:1 molar excess ratio in the experiments described above.
  • Human pituitary-derived growth hormone (Sigma Chemical Company, St. Louis, , MO.) was administered twice daily at 15 mU per injection. This dose of growth hormone stimulated a stronger growth response in the rats than did the dose of IGF-I used in the previous experiment.
  • the growth hormone-treated rats gained 12 grams per rat on average during the six day administration period (Table 2).
  • Weight gain was inhibited by about 75 % in the IGFBP-l -treated rats. Growth hormone stimulated an approximate two-fold increase in tibial epiphyseal width relative to vehicle-treated animals (Table 2). The growth hormone-stimulated increase in tibial epiphyseal width was inhibited by about 75 % by co-administration of IGFBP-l (Table 2).
  • ** Values are means ⁇ standard errors of the means for 5 rats per group.
  • IGFBP-l inhibits growth of a human breast carcinoma cell line in vitro
  • the biological effects of IGF-I, IGF-II and IGFBP-l were determined on a human breast cancer cell line.
  • the human breast carcinoma cell line, MCF7 was obtained from the American Type Culture Collection located in Rockville,MD (catalogue number HTB 22). The cells were maintained in Eagle's minimal essential medium (available from Mediatech, Herndon,VA) containing 10% fetal bovine serum,
  • Varying dilutions of IGF-I, IGF-II or IGFBP-l were added to the wells in a final volume of 200 ⁇ l. After 4 days at 37°C, 20 ⁇ l of a 5 mg/ml solution of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; available from Sigma Chemical Company, catalogue # M5655) was added to each well. The cells were incubated for an additional 6 hours at 37°C. The cells and the hydrolyzed MTT were solubilized by the addition of 50 ⁇ l of a solution of 50% dimethyl formamide, 20% sodium dodecyl sulfate, pH 4.7.
  • MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
  • the hydrolyzed MTT was quantitated by measuring the optical density of the liquid at 570 nm and subtracting the 650 nm optical density background with a VMAX kinetic microplate reader (Molecular Devices Corporation, Palo Alto,CA ).
  • IGF-I and IGF-II caused proliferation of MCF7 cells, as evidenced by an increase in the optical densities of the cell cultures (Table 3).
  • IGF-I was about 5 times more potent than IGF-II at stimulating proliferation of MCF7 cells.
  • MCF7 cell proliferation occurred at IGF-I concentrations ranging from about 1 to 120 ng/ml and at IGF- ⁇ concentrations ranging from about 10 to 1200 ng/ml.
  • IGFBP-l inhibited IGF-I and IGF-II stimulated proliferation of MCF7 cells in a dose dependent manner (Tables 4 and 5).
  • IGFBP-l concentrations tested ranged from 6 to 13,600 ng/ml. Approximately 50% growth inhibition occurred at a IGFBP-l concentration of about 180 ng/ml, which corresponds to about a 1: 1 molar ratio of IGF-I:IGFBP-1 (Table
  • the IGFBP-l concentrations tested for IGF-H ranged from about 30 ng/ml to about 23,000 ng/ml. Approximately 50% inhibition of the IGF-II growth response occurred at a IGFBP-l concentration of about 840 ng/ml, which is slightly greater than a 1:1 molar ratio of IGF-I:IGFBP-1 (Table 5). Essentially complete growth inhibition occurred at IGFBP-l concentrations exceeding 22,000 ng/ml, which corresponds to slightly greater than a 20-fold molar excess of IGFBP-l. TABLE 3
  • IGFBP-l inhibits growth of human colon carcinoma cell lines in vitro
  • IGF-I and IGFBP-l were tested on a number of human colon cancer cell lines.
  • Six human colon carcinoma cell lines were obtained from the American Type Culture Collection located in Rockville, MD. These cell lines were SK-CO-1 (HTB 39), LS 174T (CL 188), DLD-1 (CC1 221), HT-29 (HTB 38), COLO-205 (CCL 222) and Caco-2 (HTB 37). The designations within parentheses refer to the ATCC catalog number. These cell lines were selected because they all form tumors in nude mice according to the descriptions provided in the American Type Culture Collection catalogue. The cells were maintained in Eagle's Minimal Essential Medium (Mediatech, Herndon, VA) containing 10% fetal bovine serum, 2 mM glutamine, 100 Units/ml penicillin and 100 ug/ml streptomycin.
  • Mediatech Mediatech, Herndon, VA
  • IGF-I insulin growth factor-I
  • the cells were detached from the plates by treating them briefly with a trypsin/EDTA solution.
  • the cells were washed several times, counted and resuspended in serum-free media (Eagle's Minimal Essential Medium containing 2 mM glutamine, 100 units/ml penicillin and 100 ⁇ g/ml streptomycin) at a concentration of lxlOVml.
  • serum-free media Eagle's Minimal Essential Medium containing 2 mM glutamine, 100 units/ml penicillin and 100 ⁇ g/ml streptomycin
  • 100 ⁇ l of the cell suspension was added per well of a 96 well tissue culture plate (Corning Glass Works, Rochester, NY).
  • IGF-I* (ng/ml) SK-CO-1 CaCO-2 DLD-1 COLO-205 LS 174T
  • IGF-I concentrations rounded to the nearest whole number.
  • IGFBP-l The effect of IGFBP-l on growth of these cell lines was then determined. Rather than determine the effect of IGFBP-l on IGF-I-stimulated growth of the cells, it was determined whether IGFBP-l could inhibit growth of the cells in the presence of serum, which may be more representative of the in vivo situation.
  • Cells were detached from plates by brief trypsin/EDTA treatment, washed, resuspended in Eagle's Minimal Essential Medium containing 4% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin and 100 ⁇ g/ml streptomycin at a concentration of 1 x lOVml and 100 ⁇ l of the cell suspension added per well of a 96 well tissue culture plate.
  • IGFBP-l was diluted to varying concentrations in serum-free Eagle's Minimal Essential media containing 2 mM glutamine, 100 , units/ml penicillin and 100 ⁇ g/ml streptomycin and 100 ⁇ l of the mixture added per well of the 96 well plate. The cell cultures were mixed by gentle pipetting and incubated for 3 days at 37°C. The final serum concentration was 2%. Cell number was quantitated at this time using the crystal violet dye assay described above. IGFBP-l caused significant inhibition of the growth response of four of the cell lines (Caco-2, COLO-205, HT-29 and SK-CO-1) in the presence of 2% serum (Tables 8 and 9). Little growth inhibition was seen until IGFBP-l levels reached several hundred ng/ml. The maximum growth inhibition observed was between 30% and 100% (at IGFBP-l levels of 10-20 ⁇ g/ml). IGFBP-l had little effect on
  • Example 5 IGFBP-l inhibits growth of a human osteosarcoma cell line in vitro
  • BP-1 The ability of BP-1 to inhibit IGF-I stimulated growth of an osteosarcoma was determined using a rat osteosarcoma cell line, UMR-106 (CRL 1661), obtained from the American Type Culture Collection (Rockville, MD). The cells were maintained in Ham's F12 medium (available from Mediatech, Herndon, VA) containing 7% fetal bovine serum, 100 Units/ml penicillin, 100 ⁇ g/ml streptomycin and 2 mM glutamine. The cells proliferate in response to IGF-I. well of a 48 well tissue culture plate (Costar Corporation, Cambridge, MA).
  • the cells were washed twice with phosphate buffered saline (PBS) and pre-incubated for 24 hours in the above medium lacking fetal bovine serum. After the pre-incubation, the medium was removed and replaced by 0.5 ml of serum-free Ham's F12 medium containing serial dilutions of IGF-I (1 to 1,000 ng/ml). The plates were incubated for an additional 20-24 hours at 37°C. Each well was then pulsed with 0.5 ⁇ Ci of 3 H-thymidine (NEN research products, Dupont Co., Boston,NA) for 4 hours at 37°C, then washed three times with cold PBS. DNA was precipitated by adding cold 7% trichloroacetic acid (J.T.Baker Inc., Phillipsburg,NJ) to the cells.
  • PBS phosphate buffered saline
  • IGF-I caused a dose-dependent increase in 3 H- thymidine incorporated into DNA.
  • the maximal response was about six times over the level seen in the absence of IGF-I.
  • the ED 50 for IGF-I ranged from 4-20 ng/ ml in different experiments.
  • the cells were pulsed with 0.5 ⁇ Ci of 3 H-thymidine for 4 hours, rinsed three times with cold PBS, and DNA precipitated with cold 7% trichloroacetic acid. The cells were rinsed with
  • IGFBP-l (8,000 -16,000 ng/ml). The amount of IGFBP-l required to inhibit effects of IGF-I in these experiments is greater than that observed in other experiments and with other cell lines. This probably is due to the fact that 50 ng/ml IGF-I gave a maximal mitogenic response in this experiment.
  • IGF-I IGFBP-1 (ng/ml) (ng/ml) cpms* % INHIBITION
  • IGFBP-l was tested to determine if it could inhibit the proliferative response of smooth muscle cells to IGF-I.
  • a rat smooth muscle cell-like cell line, A10 was obtained from the American Type Culture Collection located in Rockville, MD (catalogue # CRL 1476). The A10 cell line has been characterized by B.W. Kimes and B.L. Brandt, Experimental Cell Research, 98:349-366 (1976). The cells were maintained in DMEM medium (Dulbecco's Modification of Eagle's Medium, available from Mediatech, Inc. Herndon, VA) containing 10% fetal bovine serum, 2 mM glutamine, 100 Units/ml penicillin and 100 ⁇ g/ml streptomycin.
  • DMEM medium Dulbecco's Modification of Eagle's Medium, available from Mediatech, Inc. Herndon, VA
  • the cells were detached from plates by brief treatment with a trypsin/EDTA solution, washed once with serum containing medium, twice with serum-free medium and counted using a hemocytometer. The cells were resuspended at a concentration of 2xl0 5 /ml in serum-free DMEM medium containing 2 mM glutamine, 100 Units/ ml penicillin and 100 ⁇ g/ml streptomycin. 100 ⁇ l of the cell suspension was aliquoted per well of a 96 well tissue culture plate (Corning Glass Works, Rochester, NY).
  • IGF-I caused a dose-dependent increase in cell number, as measured by an increase in the optical density of the wells (Table 12). The maximum proliferative response occurred at an IGF-I concentration of 100 ng/ml.
  • the effect of recombinant IGFBP-l on IGF-I-stimulated growth of A-10 cells was determined using the above cell proliferation assay.
  • the assay was performed in an identical manner except that the test wells contained 100 ng/ml IGF-I. Some wells also contained IGFBP-l at concentrations ranging from 1- 10,000 ng/ml.
  • IGFBP-l caused a dose-dependent decrease in cell number as evidenced by a decrease in the optical densities of the cell cultures (Table 13). At a concentration of 1000 ng/ml, IGFBP-l reduced the cell number to that seen without any exogenous IGF-I (refened to as baseline proliferation). At a concentration of 10 ⁇ g/ml, IGFBP-l reduced the cell number to below that seen in serum-free media, suggesting that rat A- 10 cells produce endogenous IGF-I or IGF-II. These data also indicate that IGFBP-l inhibits the proliferative response of rat smooth muscle cells to IGF-I.
  • IGFBP-l muteins Two IGFBP-l muteins, C98 and ClOl, were constructed by mutagenesis of the IGFBP-l DNA sequence contained in plasmid pJU1021, ATCC Accession No. 67730.
  • C98 mutein the serine at position 98 of the mature protein sequence has been changed to a cysteine residue.
  • ClOl mutein the serine at position 101 of the mature protein sequence has been changed to a cysteine residue.
  • Residue numbering is based upon SEQ ID No.: 1. Mutagenesis was done utilizing the polymerase chain reaction (PCR) technique.
  • Plasmid pT88:IGFBP-l contains the wild type IGFBP-l coding sequence in plasmid pT88IQ.
  • the expression vector pT88IQ is a derivative of the expression vector pT3XI-2.
  • the vector pT3XI-2 was constructed in the following manner.
  • the starting plasmid for this construction was plasmid pKK223-3 purchased from
  • Plasmid pKK223-3 carries a partial gene for tetracycline resistance. This nonfunctional gene was replaced by a complete tetracycline resistance gene carried on plasmid pBR322. Plasmid pKK223-3 was digested completely with
  • the plasmid vector so obtained is designated pCJXI-1.
  • the tetracycline resistance gene was replaced with a similar gene which had the recognition sites for restriction enzymes Hindlll, BamHl, and Sail destroyed by bisulfite mutagenesis.
  • the following procedure was used to mutate the tetracycline resistance gene of pBR322. Plasmid pBR322 was cut with Hind ⁇ l, then mutagenized with sodium bisulfite (Shortle and Botstein, 1983). The mutagenized DNA was ligated to form circular DNA, then cut with Hindlll to linearize any plasmid that escaped mutagenesis. This digestion mixture was used to transform E.
  • coli JM109 (Yanisch-Perron et al.. 1985). Tetracycline-resistant colonies were isolated and checked for loss of the Hindlll site in the tetracycline resistance gene of the plasmid. A successfully mutated plasmid was designated pTl. A similar procedure was followed to mutagenize the BamHl site in pTl, yielding plasmid pT2. Plasmid pT2 in turn was mutagenized to remove the Sail site, forming plasmid pT3. A Clal-Styl fragment of pT3 carrying the mutated tetracycline resistance gene was isolated and used to replace the homologous fragment of pCJXI-1 to form pT3XI-2.
  • the mutated tetracycline resistance gene still encodes for a functional protein. Downstream of the tac promoter region, a polylinker was introduced which contains, among other sites, BamHl and Kpnl restriction sites useful for cloning genes for expression in E. coli as described below. As in pT3XI-2, the expression of the cloned gene containing the pT88IQ vector is driven by the tac promoter. Translation starts at the ATG of the unique Ndel recognition sequence CATATG (a downstream Ndel site was eliminated so that this start site Ndel sequence would be unique). There is a polylinker downstream of the Ndel site to facilitate insertion of the desired gene.
  • the Xhol fragment containing the lad region is replaced by a truncated fragment which eliminates the lacZ promoter and the operator region which is a binding site for the lac repressor.
  • the lad region in the replacement also carries the laclq mutation — a single base substitution which results in an increase in lac repressor production (Muller-Hill et al., Proc. Nat'l Acad. Sci. OJ.S.A.') 59:1259-1264 (1968)).
  • the specific differences between pT3XI-2 and pT88IQ are as follows:
  • This sequence contains an Ndel site (underlined) at the start codon for expression and a polylinker containing recognition sites for BamHl, Xmal, Kpnl, Sail, Sad, BstBI, Spel and SacII.
  • Ndel site in pT3XI-2 about 2.4 Kb downstream of the cloning region. This site was eliminated so that the Ndel site at the start codon as described above was unique in pT88IQ. The site was changed from 5' > CATATG > 3' to 5' > CATATATG > 3', eliminating the Ndel recognition sequence.
  • laclq region The region in pT3XI-2 between the two Xhol sites containing the lad region was replaced by the 1230 base sequence shown below: laclq sequence of pT88IQ (1230 BP) (SEQ ED NO:7) CCATGGCTGG TGCCTAATGA GTGAGCTAAC TCACATTAAT TGCGTTGCGCTCACTGCCCG CTTTCCAGTC GGGAAACCTG TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGGGGA
  • GCTTCCACAG CAATGGCATC CTGGTCATCC AGCGGATAGT TAATGATCAG CCCACTGACG CGTTGCGCGA GAAGATTGTG CACCGCCGCT TTACAGGCTT CGACGCCGCT TCGTTCTACC ATCGACACCA CCACGCTGGC ACCCAGTTGA TCGGCGCGAG ATTTAATCGC CGCGACAATT TGCGACGGCG CGTGCAGGGC
  • CAGACTGGAG GTGGCAACGC CAATCAGCAA CGACTGTTTG CCCGCCAGTT GTTGTGCCAC GCGGTTGGGA ATGTAATTCA GCTCCGCCAT CGCCGCTTCC ACTTTTTCCC GCGTTTTCGC AGAAACGTGG CTGGCCTGGT TCACCACGCG GGAAACGGTC TGATAAGAGA CACCGGCATA CTCTGCGACA TCGTATAACG
  • This substituted region eliminates the lacZ promoter and the operator region which is a binding site for the lac repressor. It also contains the laclq mutation which causes an increase in lac repressor synthesis (Muller-Hill et al. , supra. 1 ).
  • the IGFBP-l DNA sequence was isolated from plasmid pJU1021 by digestion with the restriction enzymes Xba I and Hindlll and purified by agarose gel electrophoresis using NA-45 paper (Schleicher and Schuell, Keene, NH), following the manufacturer's instructions.
  • the isolated IGFBP-l DNA fragment was cloned into plasmid pT88IQ that had been digested with Xbal and Hindlll and gel-purified as above.
  • a conectly reconstructed plasmid was named pT88IQ:IGFBP-l.
  • the 5' oligonucleotide primer (IGFBP-1-5') used in the PCR mutagenesis reaction has the sequence 5'
  • the 3' oligonucleotide primer (IGFBP- 1-C98) has the sequence 5'
  • the PCR was performed in a 50 ⁇ l reaction mixture containing 10 mM Tris-HCl pH 8.3, 50 mM KCL, 2.5 mM MgCl 2 , 0.001 % gelatin, 500 ⁇ M each of dATP, dCTP, dGTP and TTP, 30 picomoles each of the IGFBP-1-5' and IGFBP-1-C98 primers, 1-10 ng pT88IQ:IGFBP-l plasmid DNA and 5 Units "AmpliTaq" Taq DNA polymerase (Perkin-Elmer Cetus, marketed by Roche Molecular Systems, Inc., Branchburg, NJ).
  • the PCR conditions were an initial 3 min incubation at 96°C, 35 cycles of (96°C for 1 min, 66°C for 1 min, 72°C for 1.5 min) and
  • the ClOl mutein was made using an agarose gel-purified DNA fragment containing the wild type IGFBP-l coding sequence as the starting template DNA.
  • the IGFBP-l coding sequence was obtained by digesting plasmid pJU1020 with Ndel and HindHI and purifying the approximate 0.8 kb IGFBP-l -coding DNA fragment by agarose gel electrophoresis.
  • the 5' oligonucleotide primer used in the PCR mutagenesis reaction was the same as was used to construct the S98C mutein (IGFBP-1-5').
  • the 3' oligonucleotide primer (IGFBP-1-ClOl) has the sequence 5' CCCGAGCTCCTCCTCAGTTATCTCCGTGCACTCTGGGCTTTCAGGGCTCC CTGC 3' (SEQ ID NO.: 10)
  • the PCR was performed in a 100 ul reaction mixture containing 10 mM Tris-HCl pH 8.3, 50 mM KC1, 1.5 mM MgCl 2 , 0.001 % gelatin, 200 uM each of dATP, dCTP, dGTP and TTP, 20 picomoles of the IGFBP-1-5' and IGFBP-1-ClOl primers and 2.5 Units of "AmpliTaq" Taq DNA polymerase.
  • the PCR conditions were 30 cycles of (95°C for 1 min, 50°C for 1 min, 72°C for 1 min) followed by a 10 min incubation at 72°C.
  • the reaction mixtures were passed through ChromaSpin 100 columns (ClonTech,Palo Alto,CA, catalogue number K1332-2) to remove nucleotides and unincorporated DNA primers.
  • the DNA fragments were digested with Xbal and SacI and bands of the conect size (approximately 0.43 kb) purified by agarose gel electrophoresis as described above.
  • the purified DNA fragments were ligated to Xbal + SacI digested pT88IQ:IGFBP-l plasmid DNA.
  • the ligation mixtures were used to transform E. coli strain DH5 alpha (available form
  • Plasmid DNAs were prepared from several colonies resulting from each transformation and sequenced on both strands across the inserted region. A plasmid with the conect sequence was selected for each mutein. They are named clones C101-3 (ClOl mutein) and C98-12 (C98 mutein).
  • the mutated IGFBP-l genes were then transfened back into plasmid pT5T (Eisenberg, S.P. et al., Nature. 343: 341-346 (1990). This was done by digesting plasmid DNA from clones C101-3 and C98-12 with Ndel and Hindlll, gel- purifying the approximate 800 bp band containing the mutant IGFBP-l gene, and ligating them to pT5T plasmid DNA that had been digested with the same restriction enzymes. The ligation mixtures were used to transform E. coli strain BL21/DE3 and plated on LB agar plates containing 50 ug/ml ampicillin.
  • Plasmid DNAs were prepared from several colonies resulting from each transformation. Clones with the conect sequences were named pT5T:IGFBP-l-C98 (C98 mutein) and pT5T:IGFBP-l-C101 (ClOl mutein).
  • E. Coli cells expressing either the C98 or ClOl mutein were grown in a 10 liter fermenter. Cells obtained from the fermenter were resuspended in breaking buffer (50 mM Tris, 25 mM NaCl, 1 mM Dithiothreitol ("DTT"), pH 7.5) at a ratio of 6ml buffer / g cells. The cells were disrupted at 10,000 PSI using a breaking buffer (50 mM Tris, 25 mM NaCl, 1 mM Dithiothreitol ("DTT”), pH 7.5) at a ratio of 6ml buffer / g cells. The cells were disrupted at 10,000 PSI using a
  • Each mutein was first denatured by solubilizing the pellet in 6M Guanidine HC1, 50 mM Tris, 6mM DTT, pH 7.5 (lg pellet for every 10 ml buffer) using a cell homogenizer. Refolding was initiated by adding oxidized glutathione ("GSSG") to the solubilized pellet to a final concentration of 23 mM. The solution was incubated at room temperature for 15 minutes and then diluted gradually with 50 mM Tris, pH 9.7 to a final protein concentration of 100 ⁇ g/ml and a final guanidine concentration of 0.6M. The protein concentration was determined by the Coomassie Blue protein assay. (Pierce, Rockford, IL). Cysteine and phenyl- methanesulfonyl fluoride were then added to final concentrations of 5.6mM and ImM, respectively. The refold solution was incubated at 4°C overnight.
  • Refolding was monitored by analyzing a 100 ⁇ l aliquot of the refold solution on a C4 reverse phase column (RP-4 lX250mm, Synchrom, Lafayette, In).
  • the C4 column was equilibrated with 2% acetonitrile (CH 3 CN), 0.05% trifluoracetic acid ("TFA").
  • the lOO ⁇ l aliquot of the refold solution was injected onto the equilibrated column and eluted using a flow rate of 0.25 ml/min with a linear gradient to 60% CH 3 CN, 0.05% TFA, changing buffer B at 2%/min.
  • the refolded protein eluted as a sharp peak approximately 2 minutes earlier than the reduced denatured but non-refolded proteins.
  • the refold solution was concentrated approximately 10 fold with an Amicon S10Y3 membrane which has a 3kDa cutoff (Amicon division of WR Grace and Co., Beverly Massachusetts) and dialyzed into 20 mM sodium phosphate, pH 6.0.
  • the dialyzed solution was centrifuged at 17,700 x g for 30 minutes and the supernatant was filtered through a 0.2 micron filter.
  • the filtered protein was loaded at 20 ml/minute onto a Q-Sepharose anion exchange column (5x30cm, Pharmacia Biotech,Piscataway, NJ) previously equilibrated with 20 mM Sodium Phosphate pH 6.0.
  • the bound protein was eluted with a linear gradient (5 column volumes) to 20 mM Sodium phosphate, 0.5M NaCl, pH 6.0 at a flow rate of 20 ml/min. 25ml fractions were collected. Each mutein eluted at approximately 0.2-0.25M NaCl. Fractions were analyzed on a C4 reverse phase column (RP-4 lX250mm, Synchrom, Lafayette, IN) using the same conditions described above for monitoring refolding or by SDS-PAGE.
  • Fractions containing the refolded mutein fractions eluting at 0.2 - 0.25M NaCl were pooled and dialyzed into 20 mM Tris HC1, 1M NaCl, pH 7.5. The dialyzed material was loaded at 25 ml/minute onto a Butyl Sepharose (Supelco,
  • the C98 and ClOl muteins were PEGylated using monomethoxy-PEG, with an average molecular weight of 20kDa, having a thiol-specific maleimide reactive group attached (CH 3 O-(CH 2 CH 2 O) n -NHCOCH 2 CH 2 -N ), where n is the number of monomeric units.
  • a thiol-specific maleimide reactive group attached CH 3 O-(CH 2 CH 2 O) n -NHCOCH 2 CH 2 -N .
  • the substituted cysteine residue (CYS 98 or CYS 101 in C98 and ClOl, respectively) can form mixed disulfides with glutathione, to form Cys-S-S-GSH, or with cysteine, to form Cys-S-S-Cys. Accordingly, there may be no free thiol available for reaction with the PEG reagent. Therefore, the purified muteins were partially reduced prior to reaction with the PEG reagent.
  • the partial reduction was accomplished by reacting the purified mutein (0.45mg/ml) with DTT at a molar ratio of DTT to protein of 5.625 to 1 in 20 mM Tris, pH 7.4, 250 mM NaCl for 2 hours at room temperature. Reduction was stopped by acidification to pH 5.5. The DTT was removed by dialysis into 10 mM sodium acetate, pH 5.5.
  • the partially reduced muteins were each reacted with the PEG reagent, at a 4 to 1 molar ratio of PEG to protein (final protein concentration of 0.33 mg/ml) in 15mM sodium acetate, 26mM sodium phosphate, pH 7.0, 120mM NaCl for 4 hours at room temperature. SDS-PAGE analysis of the reaction mixture showed approximately 50% of the partially reduced muteins were converted to a mono-
  • PEGylated species C98-PEG, ClOl -PEG having an approximate apparent molecular weight of about 67 kDa.
  • the large apparent molecular weight was due in part to PEG interactions with the gel.
  • reaction mixture was adjusted to 20 mM sodium phosphate pH 6.5.
  • Q-A Q Sepharose anion exchange column (5 x 10 cm, Pharmacia Biotech,
  • Piscataway, NJ was equilibrated with 20 mM sodium phosphate pH 6.5 and the reaction mixture was loaded at at 20 ml/minute.
  • the bound protein was eluted with a linear gradient (10 column volumes) to 20 mM sodium phosphate, 1M NaCl, pH 6.5 at a flow rate of 20 ml/min.
  • Each of the C98 and ClOl PEGylated muteins eluted at approximately 0.2 M NaCl. 25 ml fractions were collected and aliquots were analyzed by SDS-PAGE.
  • IGFBP-l and its muteins inhibit IGF-1 stimulated growth of 3T3 cells
  • a crystal violet dye assay was used to measure cell proliferation. Assays were performed in 96 well gelatin-coated plates. Balb/c 3T3 fibroblasts were plated at 25,000 cells/well in 200 ⁇ l of serum-free DMEM (Dulbecco's modification of Eagle's media, Mediatech, Herndon, VA) and 0 - 850 ng/ml IGF- 1. Cells were incubated for 72 hours at 37°C . At this time, the media was replaced with 150 ⁇ l of 0.2% crystal violet, 10% formaldehyde, 10 mM potassium phosphate pH 7.0.
  • Table 13 show that recombinant IGF-1 stimulates proliferation of 3T3 fibroblast cells in a dose dependent manner. Maximal proliferation occuned at a IGF-1 concentration of about 20-60 ng/ml. The ED 50 was approximately 5-20 ng/ml. The effect of IGFBP-l, the C98 and ClOl muteins, and the PEGylated muteins on IGF- 1 -stimulated proliferation of 3T3 fibroblasts was determined by co-incubating the cells with a set amount of IGF-1 and increasing amounts of the binding proteins.
  • Balb/c 3T3 fibroblasts were plated at 25,000 cells/ well in 200 ⁇ l of serum-free DMEM containing 21ng/ml IGF-1, and varying amounts of IGFBP- 1 and the various muteins (0 ng/ml - 11,520 ng/ml). The cells were incubated for an additional 72 hours and processed as described above.
  • the concentration of IGFBP-l required to inhibit 50% of the activity of 21ng/ml IGF-I (IC 50 ) under the conditions described is approximately 200 - 300 ng/ml for wild type IGFBP-l, the IGFBP-l muteins, and for PEGylated IGFBP-l.
  • Absorbance values are the mean of triplicate samples. SE represents standard enor of the mean.
  • Absorbance values are the mean of triplicate samples. SE represents standard enor of the mean.
  • Plasma IGFBP-l ng/ml
  • Plasma PEG IGFBP-l ng/ml
  • wild type PEGylated parameter* IGFBP-l IGFBP-l initial distribution volume L/kg 0.095 0.063 steady-state distribution volume, L/kg 0.19 0.074 plasma clearance, mL/min/kg 6.2 0.15 fast (initial) half-life, min 2.9 intermediate half-life, hr 0.24 3.9 slow (terminal) half-life, hr 1.1 13 plasma mean residence time, hr 0.52 8.2
  • Example 11 IGFBP-l inhibits restenosis in the rat
  • IGFBP-l was tested for capacity to modify the proliferative response in carotid arteries of rats following balloon angioplasty.
  • the tethered infusion system is set forth in Francis, P.C., et al., "Continuous Intravenous Infusion in Fisher 344 rats for Six Months: A Feasibility Study," Toxicology Methods. Vol. 2, pp.1-13 (1992), specifically incorporated herein by reference.
  • Blood samples were collected from the tail vein at 3, 9 and 14 days post angioplasty for determination of plasma levels of IGFBP-l.
  • concentration of IGFBP-l in the plasma samples was determined by ELISA using the Medix Biochemica (Kauniainen, Finland) IGFbp-1 test kit, Catalog No. 10831ETMB. Infusions were discontinued at the time of blood collection (approximately 15 to 30 minutes).
  • the effects of treating the animals with IGFBP-l were determined by gross scoring, and by measurement of neointima ( ⁇ m and pixels), neointima plus media (pixels), and media (pixels). The ratio of neointima (pixels) to media (pixels) was also calculated. Those scores and measurements appear in Table 22.
  • Wilcoxon Rank-Sum (Mann- Whitney U) tests were performed for each of those six response measures and p-values determined using the table set forth in Natrella, M.G., Experimental Statistics. National Bureau of Standards Handbook 91, p. T-80 (1967). The p-values from the Wilcoxon Rank-Sum (Mann- Whitney U) tests are reported below.
  • the mean (+ standard enor) scores from treated and control animals were 1.5 ⁇ 0.27 and 2.67 ⁇ 0.24, respectively. These values were found to be statistically significant (p ⁇ 0.05). In this gross assessment, the inhibition of thickening in the IGFBP-l treated animals was 44%.
  • the neointima was also measured. The distance from the medial side of the neointima to the luminal side of the neointima was measured at 4 points opposite and perpendicular to each other and the average of 3 sections determined. The mean ⁇ SE of neointima in ⁇ for treated and control animals was 78.50 ⁇ 10.68 and 139.33
  • Image analysis using the Image 1 System (available from S & M Microscopy, Colorado Springs, CO) was used to determine the area of neointima plus media, neointima alone, and media alone for treated and control animals (3 sections/animal).
  • the area (mean ⁇ SE) of neointima plus media in pixels for treated and control animals was 25,160.30 ⁇ 1817.42 and 35,271.11 ⁇ 1,403.16, respectively (p ⁇ 0.01).
  • treatment with IGFBP-l reduced the neointimal thickness by 29% .
  • the mean area in pixels of neointima alone was 14,015.40 ⁇ 1,834.24 for the treated animals and 23,119.11 ⁇ 1,200.39 for the control animals or 39% inhibition of thickening in the IGFBP-l treated animals (p ⁇ 0.01).
  • the ratio of neointima to media was calculated. This parameter also demonstrated the beneficial effect of treatment with IGFBP-l. The ratio in treated animals was 1.25 ⁇ 0.17 and 1.91 ⁇ 0.13 in the control animals. Using this ratio, inhibition of restenosis was 35% (p ⁇ 0.05). The ratio of neointima to media is generally the most accepted method of evaluating treatment-related effects in this rat model. Edelman and Kamovsky, Circulation. Vol. 89, No. 2 (1994). TABLE 22

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Abstract

L'invention concerne des procédés d'utilisation de protéines de liaison de facteurs de croissance semblables à l'insuline ('IGFBPs'), incluant IGFBP-1 ou des formes modifiées de IGFBP-1 en tant qu'agents thérapeutiques. Des formes modifiées comprennent IGFBP-1 attaché à un polymère ou deux molécules IGFBP-1 attachées aux extrémités opposées d'un polymère. Les procédés consistent à administrer des protéines IGFBPs, y compris IGFBP-1 ou une forme modifiée de IGFBP-1 à un patient se trouvant dans un état pathologique associé à IGF, en quantité suffisante pour provoquer un effet thérapeutique. L'invention concerne également des IGFBPs non phosphorylées utiles dans ces procédés.
PCT/US1994/003755 1993-04-07 1994-04-06 Procedes d'utilisation de proteines de liaison de facteurs de croissance semblables a l'insuline WO1994022466A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU66267/94A AU6626794A (en) 1993-04-07 1994-04-06 Methods of using insulin-like growth factor binding proteins
JP6522477A JPH08508296A (ja) 1993-04-07 1994-04-06 インシュリン様成長因子結合タンパク質の使用方法
EP94914048A EP0708655A1 (fr) 1993-04-07 1994-04-06 Procedes d'utilisation de proteines de liaison de facteurs de croissance semblables a l'insuline
FI954805A FI954805L (fi) 1993-04-07 1995-10-09 Menetelmiä insuliininkaltaisen kasvutekijän sitojaproteiinien käyttämiseksi

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WO1995032003A1 (fr) * 1994-05-24 1995-11-30 Amgen Boulder Inc. Facteurs de croissance proches de l'insuline modifies
WO2000042175A1 (fr) 1999-01-14 2000-07-20 Bolder Biotechnology Inc. Techniques permettant de produire des proteines contenant des residus cysteine libres
WO2000023469A3 (fr) * 1998-10-16 2000-08-24 Musc Found For Res Dev Fragments du facteur de croissance proche de l'insuline et de la proteine de fixation du facteur de croissance proche de l'insuline, et utilisations de ces fragments
WO2000050067A1 (fr) * 1999-02-26 2000-08-31 Saltech I Göteborg Ab Procede et composition pour la regulation de la production hepatique et extra hepatique du facteur de croissance insulinoide 1
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
US7192738B2 (en) 2003-10-03 2007-03-20 Genentech, Inc. IGF binding proteins
WO2008019491A1 (fr) * 2006-08-16 2008-02-21 National Research Council Of Canada Procédé d'inhibition de l'angiogenèse, de la tumorigenèse et de l'activité de la cathepsine à l'aide d'une protéine de liaison au facteur de croissance de type insuline
US7399839B2 (en) 1999-01-14 2008-07-15 Bolder Biotechnology, Inc. Monopegylated growth hormone proteins
US7495087B2 (en) 1997-07-14 2009-02-24 Bolder Biotechnology, Inc. Cysteine muteins in the C-D loop of human interleukin-11
EP2348043A1 (fr) 2001-10-02 2011-07-27 Genentech, Inc. Variantes du ligand APO-2 et leurs utilisations
EP2385064A2 (fr) * 2000-09-22 2011-11-09 Queensland University of Technology Agents qui séparent un complexe du facteur de croissance
EP2500032A1 (fr) 2002-06-24 2012-09-19 Genentech, Inc. Variantes du ligand/Trail APO-2 et leurs utilisations
US8329866B2 (en) 2005-10-03 2012-12-11 Bolder Biotechnology, Inc. Long acting VEGF inhibitors and methods of use
US8859497B2 (en) 1997-07-14 2014-10-14 Bolder Biotechnology, Inc. Method of treatment using cysteine mutants of beta interferon
US8957023B2 (en) 1999-01-14 2015-02-17 Bolder Biotechnology Inc. Methods for making proteins containing free cysteine residues
WO2017075173A2 (fr) 2015-10-30 2017-05-04 Genentech, Inc. Anticorps et conjugués anti-facteur d
US10093978B2 (en) 2013-08-12 2018-10-09 Genentech, Inc. Compositions for detecting complement factor H (CFH) and complement factor I (CFI) polymorphisms
US10179821B2 (en) 2014-05-01 2019-01-15 Genentech, Inc. Anti-factor D antibodies
US10654932B2 (en) 2015-10-30 2020-05-19 Genentech, Inc. Anti-factor D antibody variant conjugates and uses thereof
WO2021194913A1 (fr) 2020-03-24 2021-09-30 Genentech, Inc. Agents de liaison à tie2 et leurs procédés d'utilisation

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EP3534932B1 (fr) * 2016-11-01 2024-05-22 Virginia Commonwealth University Traitement de maladies associées à l'igfb3 et à son récepteur
CN116144667B (zh) * 2022-12-29 2024-03-12 海南大学 卵形鲳鲹胰岛素样生长因子结合蛋白1基因、蛋白及应用

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WO1995032003A1 (fr) * 1994-05-24 1995-11-30 Amgen Boulder Inc. Facteurs de croissance proches de l'insuline modifies
US8859497B2 (en) 1997-07-14 2014-10-14 Bolder Biotechnology, Inc. Method of treatment using cysteine mutants of beta interferon
US8148500B2 (en) 1997-07-14 2012-04-03 Bolder Biotechnology, Inc. Method of preparing cysteine mutants of human recombinant GM-CSF
US9637530B2 (en) 1997-07-14 2017-05-02 Bolder Biotechnology, Inc. Methods of preparing cysteine variants of human recombinant IL-11
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
US10329337B2 (en) 1997-07-14 2019-06-25 Bolder Biotechnology, Inc. Method to increase the number of circulating platelets by administering PEGylated cysteine variants of IL-11
US7253267B2 (en) 1997-07-14 2007-08-07 Bolder Biotechnology Inc. Cysteine variants of interleukin-11
US7959909B2 (en) 1997-07-14 2011-06-14 Bolder Biotechnology, Inc. Cysteine variants of interferon gamma
US7964184B2 (en) 1997-07-14 2011-06-21 Bolder Biotechnology, Inc. Cysteine variants of interferon-gamma
US8748392B2 (en) 1997-07-14 2014-06-10 Bolder Biotechnology Inc. Methods of treatment using cysteine variants of interleukin-11
US8618256B2 (en) 1997-07-14 2013-12-31 Bolder Biotechnology Cysteine variants of interferon gamma
US7495087B2 (en) 1997-07-14 2009-02-24 Bolder Biotechnology, Inc. Cysteine muteins in the C-D loop of human interleukin-11
US7560101B2 (en) 1997-07-14 2009-07-14 Bolder Biotechnology, Inc. Method of treatment of an infection in an animal with GM-CSF cysteine muteins in the proximal region to helix A
US8133480B2 (en) 1997-07-14 2012-03-13 Bolder Biotechnology, Inc. Cysteine variants of interleukin-11
US7732572B2 (en) 1997-07-14 2010-06-08 Bolder Biotechnology, Inc. Cysteine variants of alpha interferon-2
US7795396B2 (en) 1997-07-14 2010-09-14 Bolder Biotechnology, Inc. COOH-terminally added cysteine variant of the beta interferon
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
WO2000023469A3 (fr) * 1998-10-16 2000-08-24 Musc Found For Res Dev Fragments du facteur de croissance proche de l'insuline et de la proteine de fixation du facteur de croissance proche de l'insuline, et utilisations de ces fragments
US8957023B2 (en) 1999-01-14 2015-02-17 Bolder Biotechnology Inc. Methods for making proteins containing free cysteine residues
US7629314B2 (en) 1999-01-14 2009-12-08 Bolder Biotechnology, Inc. Methods for making proteins containing free cysteine residues
US7399839B2 (en) 1999-01-14 2008-07-15 Bolder Biotechnology, Inc. Monopegylated growth hormone proteins
EP2305804A1 (fr) 1999-01-14 2011-04-06 Bolder Biotechnology, Inc. Hormone de croissance humaine monoPEGylée
WO2000042175A1 (fr) 1999-01-14 2000-07-20 Bolder Biotechnology Inc. Techniques permettant de produire des proteines contenant des residus cysteine libres
WO2000050067A1 (fr) * 1999-02-26 2000-08-31 Saltech I Göteborg Ab Procede et composition pour la regulation de la production hepatique et extra hepatique du facteur de croissance insulinoide 1
EP2385064A2 (fr) * 2000-09-22 2011-11-09 Queensland University of Technology Agents qui séparent un complexe du facteur de croissance
EP2348043A1 (fr) 2001-10-02 2011-07-27 Genentech, Inc. Variantes du ligand APO-2 et leurs utilisations
EP2500032A1 (fr) 2002-06-24 2012-09-19 Genentech, Inc. Variantes du ligand/Trail APO-2 et leurs utilisations
US7192738B2 (en) 2003-10-03 2007-03-20 Genentech, Inc. IGF binding proteins
US7351545B2 (en) 2003-10-03 2008-04-01 Genentech, Inc. IGF binding proteins
US7348154B2 (en) 2003-10-03 2008-03-25 Genentech, Inc. IGF binding proteins
US8329866B2 (en) 2005-10-03 2012-12-11 Bolder Biotechnology, Inc. Long acting VEGF inhibitors and methods of use
WO2008019491A1 (fr) * 2006-08-16 2008-02-21 National Research Council Of Canada Procédé d'inhibition de l'angiogenèse, de la tumorigenèse et de l'activité de la cathepsine à l'aide d'une protéine de liaison au facteur de croissance de type insuline
US10947591B2 (en) 2013-08-12 2021-03-16 Genentech, Inc. Compositions and method for treating complement-associated conditions
US10093978B2 (en) 2013-08-12 2018-10-09 Genentech, Inc. Compositions for detecting complement factor H (CFH) and complement factor I (CFI) polymorphisms
US10179821B2 (en) 2014-05-01 2019-01-15 Genentech, Inc. Anti-factor D antibodies
US10407510B2 (en) 2015-10-30 2019-09-10 Genentech, Inc. Anti-factor D antibodies and conjugates
US10654932B2 (en) 2015-10-30 2020-05-19 Genentech, Inc. Anti-factor D antibody variant conjugates and uses thereof
WO2017075173A2 (fr) 2015-10-30 2017-05-04 Genentech, Inc. Anticorps et conjugués anti-facteur d
US10961313B2 (en) 2015-10-30 2021-03-30 Genentech, Inc. Anti-factor D antibody variant conjugates and uses thereof
WO2021194913A1 (fr) 2020-03-24 2021-09-30 Genentech, Inc. Agents de liaison à tie2 et leurs procédés d'utilisation

Also Published As

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AU6626794A (en) 1994-10-24
FI954805A0 (fi) 1995-10-09
CA2160154A1 (fr) 1994-10-13
CN1134111A (zh) 1996-10-23
EP0708655A1 (fr) 1996-05-01
FI954805L (fi) 1995-11-15
JPH08508296A (ja) 1996-09-03

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