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Igf binding proteins

Igf binding proteins description/claims

This is a divisional application of non-provisional application Ser. No. 11/525,654, filed Sep. 21, 2006, which is a divisional application of non-provisional application Ser. No. 10/936,059, filed on Sep. 8, 2004, now U.S. Pat. No. 7,192,738, issued Mar. 20, 2007, claiming priority under 35 USC 119(e) to provisional application number 60/508,345 filed Oct. 3, 2003, the contents of which are incorporated herein by reference.

1. Field of Invention

This invention relates to molecules useful in determining minimal functional regions of IGFBP-3 and for antagonizing an IGF-I or IGF-II activity.

2. Description of Background and Related Art

The insulin-like growth factors I and II (IGF-I and IGF-II, respectively) mediate multiple effects in vivo, including cell proliferation, cell differentiation, inhibition of cell death, and insulin-like activity (reviewed in Clark and Robinson, Cytokine Growth Factor Rev., 7: 65-80 (1996); Jones and Clemmons, Endocr. Rev., 16: 3-34 (1995)). Most of these mitogenic and metabolic responses are initiated by activation of the IGF-I receptor, an α2β2-heterotetramer closely related to the insulin receptor (McInnes and Sykes, Biopoly., 43: 339-366 (1998); Ullrich et al., EMBO J., 5: 2503-2512 (1986)). The IGF-I and insulin receptors bind their specific ligands with nanomolar affinity. IGF-I and insulin can cross-react with their respective ron-cognate receptors, albeit at a 100-1000-fold lower affinity (Jones and Clemmons, supra). The crystal structure describing part of the extracellular portion of the IGF-I receptor has been reported (Garrett et al., Nature 394: 395-399 (1998)).

Unlike insulin, the activity and half-life of IGF-I are modulated by six IGF-I binding proteins (IGFBPs 1-6), and perhaps additionally by a more distantly related class of proteins (Jones and Clemmons, supra; Baxter et al., Endocrinology, 139:4036 (1998)). IGFBPs can either inhibitor potentiate IGF activity, depending on whether they are soluble or cell-membrane associated (Bach and Rechler, Diabetes Reviews, 3: 38-61 (1995)). The IGFBPs bind IGF-I and IGF-II with varying affinities and specificities (Jones and Clemmons, supra; Bach and Rechler, supra). For example, IGFBP-3 binds IGF-I and IGF-II with a similar affinity, whereas IGFBP-2 and IGFBP-6 bind IGF-II with a much higher affinity than they bind IGF-I (Bach and Rechler, supra; Oh et al., Endocrinology, 132: 1337-1344 (1993)). WO 01/75064 discloses additional human secreted IGFBP-like polypeptides that are encoded by nucleic acid sequences isolated from cDNA libraries from adrenal gland mRNA and thymus mRNA.

Structurally, IGF-I is a single-chain, 70-amino-acid protein with high homology to proinsulin. Unlike the other members of the insulin superfamily, the C region of -IGF's is not proteolytically removed after translation. The solution NMR structures of IGF-I (Cooke et al., Biochemistry, 30: 5484-5491 (1991); Hua et al., J. Mol. Biol., 259: 297-313 (1996)), mini-IGF-I (an engineered variant lacking the C-chain; DeWolf et al., Protein Science, 5: 2193-2202 (1996)), and IGF-II (Terasawa et al., EMBO J. 13: 5590-5597 (1994); Torres et al., J. Mol. Biol. 248: 385-401 (1995)) have been reported. It is generally accepted that distinct epitopes on IGF-I are used to bind receptor and binding proteins. It has been demonstrated in animal models that receptor-inactive IGF mutants are able to displace endogenous IGF-I from binding proteins and thereby generate a net IGF-I effect in vivo (Loddick et al., Proc. Natl. Acad. Sci. USA, 95: 1894-1898 (1998); Lowman et al., Biochemistry, 37: 8870-8878 (1998); U.S. Pat. Nos. 6,121,416 and 6,251,865). While residues Y24, Y29, Y31, and Y60 are implicated in receptor binding, IGF mutants thereof still bind to IGFBPs (Bayne et al., J. Biol. Chem., 265: 15648-15652 (1990); Bayne et al., J. Biol. Chem., 264: 11004-11008 (1989); Cascieri et al., Biochemistry, 27: 3229-3233 (1988); Lowman et al., supra).

Additionally, a variant designated (1-27, gly4, 38-70)-hIGF-I, wherein residues 28-37 of the C region of human IGF-I are replaced by a four-residue glycine bridge, has been discovered that binds to IGFPB's but not to IGF receptors (Bar et al., Endocrinology, 127: 3243-3245 (1990)).

A multitude of mutagenesis studies have addressed the characterization of the IGFBP-binding epitope on IGF-I (Bagley et al., Biochem. J., 259: 665-671 (1989); Baxter et al., J. Biol. Chem., 267: 60-65 (1992); Bayne et al., J. Biol. Chem., 263: 6233-6239 (1988); Clemmons et al., J. Biol. Chem., 265: 12210-12216 (1990); Clemmons et al., Endocrinology, 131: 890-895 (1992); Oh et al., supra). In summary, the N-terminal residues 3 and 4 and the helical region comprising residues 8-17 were found to be important for binding to the IGFBPs. Additionally, an epitope involving residues 49-51 in binding to IGFBP-1, -2 and -5 has been identified (Clemmons et al., Endocrinology, supra, 1992). Furthermore, a naturally occurring truncated form of IGF-I lacking the first three N-terminal amino acids (called des (1-3)-IGF-I) was demonstrated to bind IGFBP-3 with 25 times lower affinity (Heding et al., J. Biol. Chem., 271: 13948-13952 (1996); U.S. Pat. Nos. 5,077,276; 5,164,370; and 5,470,828).

In an attempt to characterize the binding contributions of exposed amino acid residues in the N-terminal helix, several alanine mutants of IGF-I were constructed (Jansson et al., Biochemistry, 36: 4108-4117 (1997)). However, the circular dichroism spectra of these mutant proteins showed structural changes compared to wild-type IGF-I, making it difficult to clearly assign IGFBP-binding contributions to the mutated side chains. A different approach was taken in a very recent study where the IGFBP-1 binding epitope on IGF-I was probed by heteronuclear NMR spectroscopy (Jansson et al., J. Biol. Chem., 273: 24701-24707 (1998)). The authors additionally identified residues R36, R37 and R50 to be functionally involved in binding to IGFBP-1.

Other IGF-I variants have been disclosed. For example, in the patent literature, WO 96/33216 describes a truncated variant having residues 1-69 of authentic IGF-I. EP 742,228 discloses two-chain IGF-I superagonists that are derivatives of the naturally occurring single-chain IGF-I having an abbreviated C domain. The IGF-I analogs are of the formula: BCn , A wherein B is the B domain of IGF-I or a functional analog thereof, C is the C domain of IGF-I or a functional analog thereof, n is the number of amino acids in the C domain and is from about 6 to about 12, and A is the A domain of IGF-I or a functional analog thereof.

Additionally, Cascieri et al., Biochemistry, 27: 3229-3233 (1988) discloses four mutants of IGF-I, three of which have reduced affinity to the Type 1 IGF receptor. These mutants are: (Phe23, Phe24, Tyr25) IGF-I (which is equipotent to human IGF-I in its affinity to the Types 1 and 2 IGF and insulin receptors), (Leu24) IGF-I and (Ser24) IGF-I (which have a lower affinity than IGF-I to the human placental Type 1 IGF receptor, the placental insulin receptor, and the Type 1 IGF receptor of rat and mouse cells), and desoctapeptide (Leu24) IGF-I (in which the loss of aromaticity at position 24 is combined with the deletion of the carboxyl-terminal D region of hIGF-I, which has lower affinity than (Leu24)IGF-I for the Type I receptor and higher affinity for the insulin receptor). These four mutants have normal affinities for human serum binding proteins.

Bayne et al., J. Biol. Chem., 264: 11004-11008 (1988) discloses three structural analogs of IGF-I: (1-62) IGF-I, which lacks the carboxyl-terminal 8-amino-acid D region of IGF-I; (1-27, Gly4, 38-70) IGF-I, in which residues 28-37 of the C region of IGF-I are replaced by a four-residue glycine bridge; and (1-27, Gly4, 38-62) IGF-I, with a C region glycine replacement and a D region deletion. Peterkofsky et al., Endocrinology, 128: 1769-1779 (1991) discloses data using the Gly mutant of Bayne et al., supra, Vol. 264. U.S. Pat. No. 5,714,460 refers to using IGF-I or a compound that increases the active concentration of IGF-I to treat neural damage.

Cascieri et al., J. Biol. Chem., 264: 2199-2202 (1989) discloses three IGF-I analogs in which specific residues in the A region of IGF-I are replaced with the corresponding residues in the A chain of insulin. The analogs are: (Ile41,Glu45,Gln46,Thr49,Ser50,Ile51,Ser53,Tyr55,Gln56)IGF-I, an A chain mutant in which residue 41 is changed from threonine to isoleucine and residues 42-56 of the A region are replaced; (Thr49,Ser50,Ile51)IGF-I; and (Tyr55,Gln56)IGF-I.

Sliecker et al., Adv. Experimental Med. Biol., 343: 25-32 (1994)) describes the binding affinity of various IGF and insulin variants to IGFBPs, IGF receptor, and insulin receptor.

IGFBPs are secreted by cells in culture and either inhibit or enhance IGF-stimulated functions (Clemmons et al., (1991) In Modern Concepts of Insulin-like Growth Factors. E. M. Spencer, editor. Elsevier, New York, N.Y. 475-486). Known forms of IGFBPs include IGFBP-1, having a molecular weight of approximately 30-40 kDa in humans. See, e.g., WO89/09792, published Oct. 19, 1990, pertaining to cDNA sequences and cloning vectors for IGFBP-1 and IGFBP-2; WO89/08667, published Sep. 21, 1989, relating to an amino acid sequence of IGFBP-1; and WO89/09268, published Oct. 5, 1989, relating to a cDNA sequence of IGFBP-1 and methods of expression for IGFBP-1.

IGFBP-2 has a molecular weight of approximately 33-36 kDa. See, e.g., Binkert et al., The EMBO Journal, 8: 2497-2502 (1989), relating to a nucleotide and deduced amino acid sequence for IGFBP-2.

IGFBP-3 has a non-glycosylated molecular weight of about 28 kDa. See, e.g., Baxter et al., Biochim. Biophys. Res. Com., 139:1256-1261 (1986), pertaining to a glycosylated 53-kDa subunit of IGFBP-3 that was purified from human serum; Wood et al., Mol. Endocrinol., 2:1176-1185 (1988), relating to a full-length amino acid sequence for IGFBP-3 and cellular expression of the cloned IGFBP-3 CDNA in mammalian tissue culture cells; WO 90/00569, published Jan. 25, 1990, relating to isolating from human plasma an acid-labile subunit (ALS) of IGFBP complex and the particular amino acid sequence for ALS pertaining to a subunit of IGFBP-3; and Schmid et al., Biochim. Biophys. Res Com., 179: 579-585 (1991), relating to effects of full-length and truncated IGFBP-3 on two different osteoblastic cell lines.

Although initially some inconsistencies in nomenclature for IGFBP-4, IGFBP-5, and IGFBP-6 existed, in 1991 participants of the 2nd International IGF Symposium agreed upon an accepted IGFBP-4, IGFBP-5, and IGFBP-6 nomenclature. Using accepted terminology, Mohan et al., Proc. Natl. Acad. Sci., 86:8338-8342 (1989) relates to an N-terminal amino acid sequence for an IGFBP-4 isolated from medium conditioned by human osteosarcoma cells, and Shimasaki et al., Mol. Endocrinology, 4:1451-1458 (1990) pertains to IGFBP cDNAs encoding IGFBP-4 from rat and human. WO92/03471 published Mar. 5, 1992, relates to anIGFBP-4 (originally designated therein as IGFBP-5); and W092/03470 published Mar. 5, 1992 relates to genetic material encoding IGFBP-4 (originally designated therein as IGFBP-5).

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