Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Insulin; related peptides
Reexamination Certificate
2000-11-28
2003-01-14
Kunz, Gary L. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Insulin; related peptides
C530S300000, C530S350000, C514S003100, C435S069100
Reexamination Certificate
active
06506874
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of Invention
This invention relates to molecules useful as agonists of the insulin-like growth factors (IGFs), as well as IGF-like insulin molecules. More particularly, these molecules inhibit the interaction of an IGF or insulin with one or more of the IGF binding proteins. Such molecules can be used, for example, in any methods where the IGFs or insulins are used, for example, in treating hyperglycemic, obesity-related, neurological, cardiac, renal, immunologic, and anabolic disorders.
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 &agr;
2
&bgr;
2
-heterotetramer closely related to the insulin receptor (McInnes and Sykes,
Biopoly
., 43; 339-366 (1997); Ullrich et al.,
EMBO J
., 5: 2503-2512 (1986)). Both proteins are members of the tyrosine kinase receptor superfamily and share common intracellular signaling cascades (Jones and Clemmons, supra). IGF-insulin hybrid receptors have been isolated, but their function is unknown. The IGF-I and insulin receptors bind their specific ligands with nanomolar affinity. IGF-I and insulin can cross-react with their respective non-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 TGF-I receptor has recently 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 (IGFBP's 1-6), and perhaps additionally by a more distantly-related class of proteins (Jones and Clemmons, supra; Baxter et al.,
Endocrinology
, 139: 4036 (1998)). IGFBP's can either inhibit or 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
, 32, 1337-1344 (1993)).
The classical IGFBP's have a molecular mass ranging from 22-31 kDa and contain a total of 16-20 cysteines in their conserved amino- and carboxy-terminal domains (Bach and Rechler,supra; Clemmons,
Cytokine Growth Factor Rev
., 8: 45-62 (1997); Martin and Baxter,
Curr. Op. Endocrinol. Diab
., 16-21 (1994)). The central domain connecting both cysteine-rich regions is only weakly conserved and contains the cleavage sites for IGFBP-specific proteases (Chernausek et al.,
J. Biol. Chem
., 270: 11377-11382 (1995); Clemmons, supra; Conover,
Prog. Growth Factor Res
., 6: 301-309 (1995)). Further regulation of the IGFBP's may be achieved by phosphorylation and glycosylation (Bach and Rechler supra; Clemmons, supra). There is no high-resolution structure available for any intact member of the IGFBP family. However, the NMR structures of two N-terminal fragments from IGFBP-5 that retain IGF-binding activity have recently been reported (Kalus et al.,
EMBO J
. 17: 6558-6572 (1998)).
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 the 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
., 10 259: 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.,
EMBOJ
., 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 hereby 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)). 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,gly
4
,38-70)hIGF-I, wherein residues 28-37 of the C region human IGF-I are replaced by a four-residue glycine bridge, has been discovered that binds to IGFBP'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); 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 IGFBP's. 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; 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 which are derivatives of the naturally occurring single-chain IGF-I having an abbreviated C domain. The IGF-I analogs are of the formula: BC
n
,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: (Phe
23
, Phe
24
, Tyr
25
)IGF-I (which is equipotent to human IGF-I in its affinity to the Types 1 and 2 IGF and insulin receptors), (Leu
24
)IGF-I and (Ser
24
)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 mou
Dubaquie Yves
Lowman Henry
DeBerry Regina M.
Genentech Inc.
Hasak Janet E.
Kunz Gary L.
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