WO1992014834A1 - Proteine de liaison du facteur de croissance proche de l'insuline - Google Patents

Proteine de liaison du facteur de croissance proche de l'insuline Download PDF

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Publication number
WO1992014834A1
WO1992014834A1 PCT/US1992/001196 US9201196W WO9214834A1 WO 1992014834 A1 WO1992014834 A1 WO 1992014834A1 US 9201196 W US9201196 W US 9201196W WO 9214834 A1 WO9214834 A1 WO 9214834A1
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Prior art keywords
glu
gly
igfbp
pro
protein
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PCT/US1992/001196
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English (en)
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Shunichi Shimaski
Nicholas Chai-Kwan Ling
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The Whittier Institute For Diabetes And Endocrinology
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Publication of WO1992014834A1 publication Critical patent/WO1992014834A1/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
    • C07K14/46—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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 controlling the effects of insulin-like growth factors (IGFs) in mammals and more particularly to novel insulin-like growth factor binding proteins which can be employed to complex IGFs and thereby modulate IGF actions.
  • IGFs insulin-like growth factors
  • IGF-I and IGF-II insulin-like growth factors
  • IGF-II insulin-like growth factors
  • the topical use of IGF-II for wound-healing is taught in U.S. Patent No. 4,885,163 (December 5, 1989). It is also reported that IGFs have a particular effect upon the growth of cells of mesodermal origin and on their
  • IGF-I may serve to stimulate collagen synthesis in human fibroblasts, whereas studies report that IGF-II may have a predominant role in undifferentiated cell
  • IGFBPs insulin-like growth factor binding proteins
  • IGF-I and IGF-II Insulin-like growth factors are synthesized by multiple tissues and circulate in plasma to modulate the growth of various cell types.
  • IGFBPs do not exist in the blood as free hormones but are bound to carriers in the form of IGFBPs.
  • IGFBPs IGFBPs
  • four distinct classes of IGFBPs have been characterized, based on their complete primary structure having been obtained by molecular cloning, all of which are able to bind both IGF-I and IGF-II and to modulate IGF actions either in an inhibitory or a stimulatory manner.
  • IGFBP-1 the first BP class whose complete primary structure was deduced has been named IGFBP-1; its structure was deduced from cDNA clones identified in the libraries prepared from a human HEP-G2 hepatoma cell line, from human placenta and from both human and rat decidua.
  • a human genomic clone encoding IGFBP-1 was also isolated and characterized (Brinkman, A., et al., B.B.R.C. 157, 898- 907 (1989)), the gene locus of which is mapped at
  • This protein exhibits a molecular weight (M r ) of 28-30 kDa on SDS/PAGE under non- reducing conditions and has almost equal binding affinity for IGF-I and IGF-II. It contains no potential N-linked glycosylation sites, but it has at least five potential 0-linked glycosylation sites, which may account for a reported 4.3% carbohydrate content of the protein.
  • M r molecular weight
  • the second BP class is one for which the complete primary structure was deduced from cDNAs
  • IGFBP-2 isolated from a rat BRL-3A cell library as well as from adult rat liver and human fetal liver libraries; it has been named IGFBP-2.
  • This BP having M r of about 33-35 kDa on SDS/PAGE under non-reducing conditions, exhibits equal affinity for IGF-I and IGF-II when IGF-I is used as a radioligand, but it shows a marked preference for IGF-II when the radioligand is IGF-II.
  • the level of IGFBP-2 in rat serum is high in fetus but decreases in adult.
  • the physiological role of IGFBP-2 is not well known, but
  • FSH pituitary follicle-stimulating hormone
  • the third BP class is a high molecular weight
  • IGFBP within the 150 kDa IGF-binding complex found in plasma. Its complete primary structure was deduced from human, porcine and rat cDNAs, and it has been named
  • IGFBP-3 see Shimasaki, S., et al., B.B.R.C., 165, 907-912 (1989).
  • This 150 kDa complex consists of three components, an IGFBP-3 of 53 kDa bound to an IGF and an acid-labile 80 kDa protein which can only bind to IGFBP-3 in association with IGF under neutral conditions. Both the 53 kDa IGFBP-3 and the 80 kDa acid-labile subunits are glycosylated. Moreover, the circulating level of the complex is dependent on growth hormone (GH). This protein has recently been isolated from ovarian
  • the fourth class of BP was isolated from human bone cell-conditioned medium, Mohan et al., P.N.A.S. 86, 8338-8342 (1989) and adult rat serum, Shimonaka et al., B.B.R.C. 165, 189-195 (1989). Its complete primary structure was subsequently deduced from a rat liver and a human placenta library and named IGFBP-4, see Shimasaki et al., Mol. Endocrinol. 4 , 1451-58 (1990). Unlike the other three IGFBPs, this BP contains two extra cysteines in the midportion of the molecule in addition to the 18 homologous cysteines found in the other BPs. Moreover, it contains one potential Asn-linked glycosylation site.
  • IGFBPs Two additional novel IGFBPs have been isolated from rat serum, purified to homogeneity and partially sequenced beginning at the N-terminus of each.
  • the cDNAs encoding the complete primary structure of one of these proteins have been isolated and characterized from both the rat and the human species.
  • Homologous cDNAs from other mammalian species can be likewise obtained and amino acid sequences of the IGFBP deduced.
  • homologous is meant having at least about 80% identity on a nucleotide base level. The deduced amino acid sequences of the cDNAs reveal a mature
  • polypeptide of 252 amino acids for both the rat protein and the human protein which are highly homologous, i.e. at least about 90% identity on an amino acid level, and they contain what are believed to be signal sequences of 19-20 amino acid residues.
  • IGFBP-5 rat and human proteins which are hereinafter referred to as IGFBP-5, bind to both IGF-I and IGF-II and can be administered as anti- neoplastic agents along with an appropriate
  • IGFBP-5 can be used to combat breast and bone cancers and other tissues having a high IGF
  • IGFBP-5 and the other protein, herein referred to as IGFBP-6, were isolated and purified from adult rat serum using techniques generally similar to those
  • the retentate was applied onto an IGF-II-coupled Affi-Gel 15 column at 20 ml/hr through a peristaltic pump in a cold room. After all the sample had been pumped through the column, the gel bed was washed with 200 ml PBS buffer, containing 0.5 M NaCl, at the same flow rate. The adsorbed proteins were eluted with 0.5 M acetic acid, pH 3.0, at 3.5 ml/hr, and 1 ml fractions were collected. The IGFBPs in the eluate fractions were located by UV absorbance at 280 nm.
  • the recovered IGFBPs from the affinity column were pooled and, after dilution with an equal volume of water, were pumped directly onto an 0.7 ⁇ 25 cm Aquapore RP-300, 10 ⁇ m particle size, C 8 column (Applied
  • porcine follicular fluid resulting in recovered IGFBPs including porcine IGFBP-6.
  • pFF porcine follicular fluid
  • amino acids are referred to herein using either the standard 3-letter or 1-letter
  • a cDNA library or an expression library is often produced in a conventional manner by reverse transcription from messenger RNA (mRNA) from a mammalian cell line or tissue.
  • mRNA messenger RNA
  • a hybridization probe obtained by PCR technology or mixed probes which accommodate the degeneracy of the genetic code and correspond to a selected portion of the target protein are produced
  • Screening of such an expression library with antibodies to all or a portion of the protein may also be used, either alone or in conjunction with hybridization probing, to identify or confirm the presence of cDNA library clones which are expressing the target protein.
  • PCR polymerase chain reaction
  • two synthetic oligonucleotide mixture primers for PCR were designed which incorporated all possible codon combinations encoding (a) a 7-amino acid residue sequence near the N-terminus selected from SEQ ID NO:1, namely, Phe-Val-His-Cys-Glu-Pro-Cys- and (b) the 7- amino acid residue sequence which appears at the carboxy- terminus of this 36-residue SEQ ID NO:1, namely Glu-Pro- Gly-Cys-Gly-Cys-Cys.
  • the synthetic primer mixtures are as follows:
  • PCR was performed by a TwinBlockTM system (Ericomp, San Diego, CA) with GeneAmpTM DNA Amplification Reagent Kit (Perkin-Elmer Cetus, Norwalk, CT) using a PMSG-stimulated rat ovary cDNA library as a template.
  • Annealing reactions were performed at 60°C. for 30 sec, followed by a 30 sec. extension at 72°C. and 15 sec.
  • PCR-amplified cDNA fragment was determined by the double- stranded dideoxy-chain-termination method using Sequenase (United States Biochem. Co., Cleveland, OH); its deduced amino acid sequence matched with that obtained by protein sequencing.
  • This PCR-amplified cDNA fragment having the expected 98 bp length obtained from the rat ovary cDNA library was thereafter labeled by a random priming method using 32 P-dCTP and used as a probe to screen the same rat ovary cDNA library to isolate cDNA clones encoding rat IGFBP-5. Upon screening with this probe, six positive clones were obtained from one million independent clones. Each of these clones was sequenced, and the results revealed that all six of them contained the complete coding region of rat IGFBP-5 including the signal
  • the complete DNA sequence of one clone is shown in TABLE 1 wherein an open reading frame encodes a protein of 271 amino acid residues with the predicted amino acid sequence being shown below each codon.
  • the amino-terminal residue of the 252-residue mature protein is denoted by +1 so as to be in agreement with the amino-terminal residue of the purified rat IGFBP-5.
  • the preceding 19 amino acid sequence leading to the amino-terminal residue of the mature protein fits a typical signal peptide sequence, terminating in a neutral residue with a small side-chain which, in this case, is Gly at position -1.
  • the complete 271-residue sequence is set forth as follows as SEQ ID NO:3:
  • TABLE 1 shows the nucleotide sequence (SEQ ID NO: 6) and deduced amino acid sequence of rat IGFBP-5 as determined by sequencing a cDNA clone.
  • nucleotides are numbered at the right, and the amino acids, in one-letter code, are numbered throughout.
  • TABLE 2 shows the nucleotide sequence (SEQ ID NO: 5) and deduced amino acid sequence of human IGFBP-5 as determined by sequencing a cDNA clone.
  • nucleotides are numbered at the right, and the amino acids in one-letter code are numbered throughout, as in TABLE 1.
  • the proteins are highly homologous, about 97%, with most of the amino acid substitutions between the two species being located in the middle of the molecule.
  • One gap at position -15 in the signal sequence is inserted in the rat sequence to allow maximal homology alignment.
  • the location of all the cysteines are conserved, and no potential N-linked glycosylation site appears in either molecule.
  • SEQ ID NO: 10 Glu-Gly-Gln-Gln-Cys-Gly-Val-Tyr-Thr-Pro-Asn-Cys-Ala-Pro-Gly-Leu-Gln-Cys-Gln-Pro-Pro-Glu-Glu-Asp-Gln-Ala-Pro-Leu-Arg.
  • SEQ ID NO: 10 was chosen for use to prepare DNA probes using PCR and using pig genomic DNA as a template. A PCR-amplified DNA fragment was obtained and was labeled as set forth hereinbefore and used to screen the PMSG- primed rat ovary cDNA library and also the human placenta library. Positive clones were located from both
  • Antibodies to these IGFBP-5 proteins of either monoclonal or polyclonal form can be produced using techniques presently known in the art, and antibodies which are effective to counteract the effects of IGFBP-5 can be elicited using only the synthetic N-terminal segment of the rat protein. For example, antibodies raised in rabbits against a synthetic peptide,
  • Amino terminal-directed antibodies to IGFBP-5 may be obtained, for example, by immunizing three month old male and female white New Zealand rabbits with the synthetic peptide to which Tyr has been added at the C-terminus in order to couple it, as an antigen, to BSA by a
  • the animals are boosted by injections of
  • antiserum should show its capacity to bind radioiodinated antigen peptide prepared by the chloramine-T method, and then it would be purified by CMC-ion exchange column chromatography.
  • a radioimmunoassay can then be established with the antisera and serum from subsequent bleeds from the same rabbits.
  • the native protein should be recognized by the antibodies on an equimolar basis as compared to the synthetic peptide antigen.
  • Immunoaffinity or affinity chromatography can also be applied to achieve the purification of IGFBP-5 from serum or biological materials; likewise IGFBP-5 can be used in affinity chromatography to purify IGF-I or IGF-II.
  • Antibodies to IGFBP-5 can be used in assays for detecting the levels of IGFBP-5 in mammals, particularly humans.
  • the antibodies can also be used for treatment to neutralize the effect of IGFBP-5 in mammals and are useful for diagnostic test kits and the like.
  • Mammalian IGFBP-5 polypeptides produced by recombinant DNA techniques are considered to be inherently biologically active, and the three-dimensional structure which the IGFBP-5 assumes within cells is likely the structure recognized by the receptor.
  • the three-dimensional structure which the molecule assumes through natural folding and through hydrophobic and hydrophilic interactions with aqueous media may also promote desired bonding or non-bonding between cysteine residues.
  • enzymatic regulatory mechanisms within cells may help to ensure desired disulfide bonding or non-bonding, either by preventing bonding or by directing disulfide bonding between
  • Enzymes might also cleave "incorrect” bonding to enable the molecule to reorientate itself and assume the correct natural structure.
  • Cysteine residues that are not internally bonded may be disulfide-bonded to free cysteine moieties.
  • three-dimensional structure of the molecule may also be such that random bonding or non-bonding of cysteine residues, either with each other or to free cysteines, does not substantially affect the biological structure of the protein molecule.
  • IGFBP-5 amino acid residue sequence e.g. human IGFBP-5 (SEQ ID NO:4) by recombinant DNA
  • a double-stranded DNA chain which encodes IGFBP-5 might be synthetically constructed.
  • PCR techniques would be method of choice to produce DNA chains
  • a DNA chain encoding IGFBP-5 could be designed using certain particular codons that are more efficient for polypeptide expression in a certain type of organism, i.e. selection might employ those codons which are most efficient for expression in the type of organism which is to serve as the host for the recombinant vector.
  • Codon selection may also depend upon vector construction considerations; for example, it may be necessary to avoid placing a particular restriction site in the DNA chain if, subsequent to inserting the synthetic DNA chain, the vector is to be manipulated using the restriction enzyme that cleaves at such a site. Also, one should avoid placing restriction sites in the DNA chain if the host organism, which is to be transformed with the recombinant vector containing the DNA chain, is known to produce a restriction enzyme that would cleave at such a site within the DNA chain.
  • a synthetic IGFBP-5-en ⁇ oding DNA chain could be assembled by constructing oligonucleotides by conventional procedures, such as those described in T. Maniatis et al., Cold Spring Harbor Laboratory Manual. Cold Spring Harbor, New York (1982) (hereinafter, CSHLM).
  • CSHLM oligonucleotide chain
  • Sense and antisense oligonucleotide chains up to about 70 nucleotide residues long, are synthesized, preferably on automated synthesizers, such as the Applied Biosystem Inc. Model 380A DNA synthesizer.
  • the oligonucleotide chains are usually constructed so that portions of the sense and antisense oligonucleotides overlap, associating with each other through hydrogen bonding between
  • IGFBP-5 that was cloned to deduce the complete structure of IGFBP-5 is conveniently used.
  • an IGFBP-5 that was cloned to deduce the complete structure of IGFBP-5 is conveniently used.
  • an IGFBP-5 that was cloned to deduce the complete structure of IGFBP-5 is conveniently used.
  • an IGFBP-5 that was cloned to deduce the complete structure of IGFBP-5 is conveniently used.
  • rat IGFBP-5 DNA sequence encoding the 252-residue mature protein plus the signal sequence might be employed, with the entire 1630 bp nucleic acid sequence being set forth as SEQ ID NO: 6 in the appended SEQUENCE LISTING.
  • a DNA chain should contain additional sequences depending upon vector construction considerations.
  • a synthesized DNA chain has linkers at its ends to
  • a DNA chain may be constructed so as to encode the IGFBP-5 amino acid sequences as a portion of a fusion polypeptide; and if so, it will generally contain terminal sequences that encode amino acid residue
  • IGFBP-5 polypeptide may be proteolytically cleaved from the remainder of the fusion polypeptide.
  • the terminal portions of the synthetic DNA chain may also contain appropriate start and stop signals.
  • a double-stranded IGFBP-5-encoding DNA chain is constructed or modified with appropriate linkers for its insertion into a particular appropriate cloning vector.
  • the DNA chain recombined to incorporate the DNA chain is selected appropriate to its viability and expression in a host organism or cell line, and the manner of insertion of the DNA chain depends upon factors particular to the host. For example, if the DNA chain is to be inserted into a vector for insertion into a prokaryotic cell, such as E. coli, the DNA chain will be inserted 3' of a promoter sequence, a Shine-Delgarno sequence (or ribosome binding site) that is within a 5' non-translated portion and an ATG start codon. The ATG start codon is appropriately spaced from the Shine-Delgarno sequence, and the encoding sequence is placed in correct reading frame with the ATG start codon.
  • the cloning vector also provides a 3' non-translated region and a translation termination site.
  • a eukaryotic cell such as a yeast cell or a cell line obtained from a higher animal
  • the IGFBP-5-encoding oligonucleotide sequence is
  • the cloning vector also provides a 3' non-translated region and a translation termination site.
  • Prokaryotic transformation vectors such as pBR322, pMB9, Col E1, pCR1, RP4 and lambda-phage, are available for inserting a DNA chain of the length which encodes IGFBP-5 with substantial assurance of at least some expression of the encoded polypeptide.
  • such vectors are constructed or modified to have one or more unique restriction sites appropriately positioned relative to a promoter, such as the lac promoter.
  • the DNA chain may be inserted with appropriate linkers into such a restriction site, with substantial assurance of production of IGFBP-5 in a prokaryotic cell line
  • linkers of various lengths may be provided at the ends of the IGFBP-5-encoding sequences.
  • cassettes which include sequences, such as the 5' region of the lac Z gene (including the
  • promoter promoter
  • ribosome binding site and translation initiator a fusion gene containing these two promoters
  • the trp-lac or commonly called the Tac promoter are available into which the synthetic DNA chain may be conveniently inserted and then the cassette inserted into a cloning vector of choice.
  • eukaryotic transformation vectors such as, the cloned bovine papilloma virus genome, the cloned genomes of the murine retroviruses, and eukaryotic cassettes, such as the pSV-2 gpt system (described by Mulligan and Berg, Nature 277. 108-114, 1979), the
  • a convenient way to ensure production of IGFBP-5 or a protein of a similar length is to produce the protein initially as a segment of a gene-encoded fusion protein.
  • the DNA chain is constructed so that the expressed protein has enzymatic processing sites flanking the IGFBP-5 amino acid residue sequences.
  • An IGFBP-5-encoding DNA chain may be inserted, for example, into the beta-galactosidase gene for insertion into E. coli. in which case, the expressed fusion protein is subsequently cleaved with proteolytic enzymes to release the IGFBP-5 from
  • beta-galactosidase peptide sequences An advantage of inserting the IGFBP-5-encoding sequence so that the
  • IGFBP-5 sequence is expressed as a cleavable segment of a fusion protein, e.g. as the IGFBP-5 sequence fused within the beta-galactosidase peptide sequence, is that the endogenous protein into which the IGFBP-5 sequence is inserted is generally rendered non-functional, thereby facilitating selection for vectors encoding the fusion protein.
  • the IGFBP-5 protein may also be reproduced in yeast using known recombinant DNA techniques.
  • plasmid pIGFBP-5 amplified in a pIGFBP- 5-producing E. coli clone, is isolated and cleaved with Eco RI and Sal I. This digested plasmid is
  • the insert is inserted into the plasmid pYEp, a shuttle vector which can be used to transform both E. coli and Saccharomyces cerevisiae yeast. Insertion of the
  • the shuttle vector is used to transform URA3, a strain of S. cerevisiae yeast from which the oratate monophosphate decarboxylase gene is deleted.
  • the transformed yeast is grown in medium to attain log growth.
  • the yeast is separated from its culture medium, and cell lysates are prepared. Pooled cell lysates are determined by RIA to be reactive with antibody raised against IGFBP-5, demonstrating that a protein containing IGFBP-5 protein segment is expressed within the yeast cells.
  • the production of IGFBP-5 can be carried out in both prokaryotic and eukaryotic cell lines to provide protein for biological and therapeutic use. While IGFBP-5 synthesis is easily demonstrated using either bacteria or yeast cell lines, the synthetic genes should be insertable for expression in cells of higher animals, such as mammalian tumor cells. Such mammalian cells may be grown, for example, as peritoneal tumors in host animals, and IGFBP-5 harvested from the peritoneal fluid.
  • IGFBP-5 can be synthesized through recombinant DNA techniques, the examples do not purport to have maximized IGFBP-5 production. It is expected that subsequent selection of more efficient cloning vectors and host cell lines will increase the yield of IGFBP-5.
  • prokaryotic cells may be used to increase production of IGFBP-5.
  • Secretion of the gene-encoded protein from the host cell line into the culture medium is also considered to be an important factor in obtaining synthetic IGFBP-5 in large quantities.
  • IGFBP-5 proteins permit their use to complex and neutralize IGFs and these proteins should be useful in the treatment of conditions which are caused by an overabundance of IGFs, for example, certain types of breast or bone cancer.
  • Substantially pure IGFBP-5 protein can be routinely obtained having significantly higher purity than IGFBP-5 that is extracted from mammalian serum.
  • IGFBP-5 proteins constitute only minor constituents of normal mammalian serum, being present in only very impure form, relative to other native proteins also present.
  • Recombinant DNA techniques for example, can be used to generate organisms or cell lines that produce the
  • heterologous protein in significantly higher proportions relative to total protein, in the cellular material and/or the secretions thereof, than the proportions at which native IGFBP-5 are present. Because the starting material from which such synthetic IGFBP-5 proteins are isolated has a substantially greater concentration of the heterologous protein, purification techniques can fairly simply produce more highly purified IGFBP-5 fractions. Using appropriate isolation techniques, it is possible to routinely obtain IGFBP-5 proteins which are at least about 95% pure (by weight of total proteins) and which is herein referred to as substantially pure.
  • the protein should be administered under the guidance of a physician, and pharmaceutical compositions will usually contain the protein in conjunction with a conventional, pharmaceutically-acceptable carrier.
  • pharmaceutical compositions will usually contain the protein in conjunction with a conventional, pharmaceutically-acceptable carrier.
  • substantially pure synthetic IGFBP-5 or the nontoxic salts thereof, combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition are administered to mammals, including humans, either intravenously, subcutaneously, intramuscularly or orally.
  • the required dosage will vary with the particular
  • Such protein may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g., with zinc, iron or the like (which are broadly considered as salts for purposes of this application).
  • acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate,
  • the tablet may contain a binder, such as
  • tragacanth corn starch or gelatin
  • a disintegrating agent such as alginic acid
  • a lubricant such as magnesium stearate.
  • sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.
  • a dosage form may contain a pharmaceutically acceptable non-toxic salt of the
  • a relatively insoluble salt may also be formulated in a gel, for example, an aluminum stearate gel.
  • a suitable slow release depot formulation for injection may also contain IGFBP-5 or a salt thereof dispersed or encapsulated in a slow
  • mammalian IGFBP-5 proteins should be considered to constitute proteins having the amino acid residue sequences set forth hereinbefore as well as naturally occurring amino acid sequence variants of other mammalian species and fragments of the foregoing having equivalent biological activity. Unless otherwise stated hereinbefore, all percentages are volume percents.
  • CTTTTTTTTC CTCTCCCTTG ATTTCAACAT TTTCCCGATC TTCTTGGCGC CGCCGACGCC 60
  • CTCTTGGCCC CTTTCTCCAC ACACTCTCGC TCTCCTGCCC CGCCCCGAGG TAAAGCCAGA 540
  • AAG CTG ACC CAG TCT AAG TTT GTG GGG GGC GCG
  • GAG AAC ACT GCC CAC 1069 Lys Leu Thr Gln Ser Lys Phe Val Gly Gly Ala Glu Asn Thr Ala His

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Abstract

L'invention se rapporte à deux protéines de liaison du facteur de croissance proche de l'insuline, qui sont isolées à partir de sérum de rat, purifiées jusqu'à l'homogénéité et partiellement divisées en séquences. En utilisant des sondes de nucléotides basées sur la séquence des terminaisons amino de l'une des protéines isolées, on déduit la séquence complète pour la protéine de rat du résidu 252 mature, appelée IGFBP-5. On déduit ensuite séparément la séquence du résidu 252 hautement homologue de la protéine humaine. Ces trois protéines sont utiles pour inhiber la différenciation et/ou la prolifération cellulaire nécessitant la présence du facteur de croissance proche de l'insuline et elles sont particulièrement adaptées pour combattre les cancers du sein et des os. Des anticorps contre ces protéines peuvent être utilisés dans des analyses diagnostiques, dans la purification de la protéine et dans la modulation de la croissance osseuse.
PCT/US1992/001196 1991-02-14 1992-02-13 Proteine de liaison du facteur de croissance proche de l'insuline WO1992014834A1 (fr)

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Cited By (8)

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Publication number Priority date Publication date Assignee Title
EP0556344A1 (fr) * 1991-01-08 1993-08-25 Chiron Corporation Nouvelle proteine de fixation du facteur de croissance proche de l'insuline (igfbp-6)
WO1994010207A2 (fr) * 1992-11-04 1994-05-11 Chiron Corporation Proteines tronquees de liaison au facteur de croissance insulinoide a activite mitogene
WO1995014034A1 (fr) * 1993-11-16 1995-05-26 Pharmacia Ab Utilisation de la proteine de liaison du facteur de croissance de substances apparentees a l'insuline pour le repliement de ce facteur de croissance
WO1996012735A1 (fr) * 1994-10-19 1996-05-02 Genetics Institute, Inc. Nouveaux ligands proteiques au domaine de mort du recepteur du tnf et inhibiteurs de fixation des ligands
US5712381A (en) * 1994-10-19 1998-01-27 Genetics Institute, Inc. MADD, a TNF receptor death domain ligand protein
US5847099A (en) * 1994-10-19 1998-12-08 Genetics Institute, Inc. TNF receptor death domain ligand proteins
US5849501A (en) * 1994-10-19 1998-12-15 Genetics Institute, Inc. TNF receptor death domain ligand proteins and method to identify inhibitors of ligand binding
US7192738B2 (en) 2003-10-03 2007-03-20 Genentech, Inc. IGF binding proteins

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEM. BIOPHYS. RES. COMMUN., Vol. 176, No. 1, issued 15 April 1991, M.C. KIEFER et al., "Molecular cloning of a new human insulin-like growth factor binding protein", pages 219-225. *
J. BIOL. CHEM., Vol. 266, No. 15, issued 15 May 1991, M.C. KIEFER et al., "Identification and molecular cloning of two new 30-kD insulin-like growth factor binding proteins isolated from adult human serum", pages 9043-9049. *
J. BIOL. CHEM., Vol. 266, No. 16, issued 05 June 1991, S. SHIMASAKI et al., Identification of five different Insulin-like growth factor binding proteins (IGFBPs) from adult rat serum and molecular cloning of a novel IGFBP-5 in rat and human", pages 10646-10653. *

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* Cited by examiner, † Cited by third party
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EP0556344A4 (en) * 1991-01-08 1994-08-24 Chiron Corp New insulin-like growth factor binding protein (igfbp-6)
EP0556344A1 (fr) * 1991-01-08 1993-08-25 Chiron Corporation Nouvelle proteine de fixation du facteur de croissance proche de l'insuline (igfbp-6)
US6906029B2 (en) 1991-01-08 2005-06-14 Chiron Corporation Insulin-like growth factor binding protein (IGFBP-5)
US6500635B1 (en) 1991-01-08 2002-12-31 Chiron Corporation Insulin-like growth factor binding protein (IGFBF-5)
US6369029B1 (en) 1992-11-04 2002-04-09 Chiron Corporation Method of stimulating osteogenesis using a c-terminally truncated insulin-like growth factor binding protein-5 (IGFBP-5)
WO1994010207A2 (fr) * 1992-11-04 1994-05-11 Chiron Corporation Proteines tronquees de liaison au facteur de croissance insulinoide a activite mitogene
WO1994010207A3 (fr) * 1992-11-04 1994-07-07 Chiron Corp Proteines tronquees de liaison au facteur de croissance insulinoide a activite mitogene
US6489294B1 (en) 1992-11-04 2002-12-03 Chiron Corporation Truncated insulin-like growth factor binding protein-5 having mitogenic activity
US6391588B1 (en) 1992-11-04 2002-05-21 Chiron Corporation Nucleic acid molecules encoding truncated insulin-like growth factor binding-5 proteins
WO1995014034A1 (fr) * 1993-11-16 1995-05-26 Pharmacia Ab Utilisation de la proteine de liaison du facteur de croissance de substances apparentees a l'insuline pour le repliement de ce facteur de croissance
US5683980A (en) * 1993-11-16 1997-11-04 Pharmacia & Upjohn Ab Use of IGF-BP for refolding of IGF
WO1996012735A1 (fr) * 1994-10-19 1996-05-02 Genetics Institute, Inc. Nouveaux ligands proteiques au domaine de mort du recepteur du tnf et inhibiteurs de fixation des ligands
US5891675A (en) * 1994-10-19 1999-04-06 Genetics Institute, Inc. TNF receptor death domain ligand proteins
US5948638A (en) * 1994-10-19 1999-09-07 Genetics Institute, Inc. TNF receptor death domain ligand proteins
US5852173A (en) * 1994-10-19 1998-12-22 Genetics Institute, Inc. TNF receptor death ligand proteins and inhibitors of ligand binding
US5849501A (en) * 1994-10-19 1998-12-15 Genetics Institute, Inc. TNF receptor death domain ligand proteins and method to identify inhibitors of ligand binding
US5847099A (en) * 1994-10-19 1998-12-08 Genetics Institute, Inc. TNF receptor death domain ligand proteins
US5843675A (en) * 1994-10-19 1998-12-01 Genetics Institute, Inc. TNF receptor death domain ligand proteins and inhibitors of ligand binding
US5712381A (en) * 1994-10-19 1998-01-27 Genetics Institute, Inc. MADD, a TNF receptor death domain ligand protein
US7109307B2 (en) 1994-10-19 2006-09-19 Genetics Institute, Llc TNF receptor death domain ligand proteins and inhibitors of ligand binding
US7192738B2 (en) 2003-10-03 2007-03-20 Genentech, Inc. IGF binding proteins
US7348154B2 (en) 2003-10-03 2008-03-25 Genentech, Inc. IGF binding proteins
US7351545B2 (en) 2003-10-03 2008-04-01 Genentech, Inc. IGF binding proteins

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