U.S. patent application number 10/444701 was filed with the patent office on 2004-02-19 for modified mature insulin variants and composition containing same.
Invention is credited to Dubaquie, Yves, Lowman, Henry.
Application Number | 20040033952 10/444701 |
Document ID | / |
Family ID | 22358799 |
Filed Date | 2004-02-19 |
United States Patent
Application |
20040033952 |
Kind Code |
A1 |
Dubaquie, Yves ; et
al. |
February 19, 2004 |
Modified mature insulin variants and composition containing
same
Abstract
IGF-I and insulin variants are provided that selectively bind to
IGFBP-1 or IGFBP-3. These agonist variants are useful, for example,
to improve the half-lives of IGF-I and insulin, respectively.
Inventors: |
Dubaquie, Yves; (San
Francisco, CA) ; Lowman, Henry; (El Granada,
CA) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
275 MIDDLEFIELD ROAD
MENLO PARK
CA
94025-3506
US
|
Family ID: |
22358799 |
Appl. No.: |
10/444701 |
Filed: |
May 22, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10444701 |
May 22, 2003 |
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09724198 |
Nov 28, 2000 |
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09724198 |
Nov 28, 2000 |
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09477923 |
Jan 5, 2000 |
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60115010 |
Jan 6, 1999 |
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Current U.S.
Class: |
514/5.9 ;
514/8.6; 514/8.7; 530/399 |
Current CPC
Class: |
A61P 3/04 20180101; A61P
15/00 20180101; A61P 41/00 20180101; A61P 17/02 20180101; A61P 1/14
20180101; A61P 9/04 20180101; A61P 11/16 20180101; A61P 35/00
20180101; A61P 25/28 20180101; A61P 9/10 20180101; A61P 19/10
20180101; A61P 17/00 20180101; A61P 5/44 20180101; A61P 21/00
20180101; A61P 5/38 20180101; A61P 19/04 20180101; A61P 21/04
20180101; A61P 37/04 20180101; A61P 3/10 20180101; A61P 31/18
20180101; A61P 11/00 20180101; A61P 19/00 20180101; A61P 25/02
20180101; A61P 25/24 20180101; A61P 25/18 20180101; A61P 5/02
20180101; A61P 13/12 20180101; A61P 3/08 20180101; A61P 5/48
20180101; A61P 43/00 20180101; C07K 14/65 20130101; A61K 38/00
20130101; A61P 5/50 20180101; A61P 25/00 20180101; A61P 37/00
20180101; A61P 9/00 20180101; A61P 15/08 20180101 |
Class at
Publication: |
514/12 ;
530/399 |
International
Class: |
A61K 038/30; C07K
014/65 |
Claims
What is claimed is:
1. An IGF-I variant wherein an amino acid at position 3, 4, 5, 7,
10, 14, 17, 23, 24, 25, 43, 49 or 63, or any of such amino acids in
combination with an amino acid at position 12 or 16 or both 12 and
16 of native-sequence human IGF-I, or any combination thereof, is
replaced with any amino acid at said position 7 or with an alanine,
a glycine, or a serine residue at any position other than said
position 7.
2. The variant of claim 1 wherein an amino acid at said position 3
is replaced.
3. The variant of claim 1 wherein an amino acid at said position 4
is replaced.
4. The variant of claim 1 wherein an amino acid at said position 5
is replaced.
5. The variant of claim 1 wherein an amino acid at said position 7
is replaced.
6. The variant of claim 1 wherein an amino acid at said position 10
is replaced.
7. The variant of claim 1 wherein an amino acid at said position 14
is replaced.
8. The variant of claim 1 wherein an amino acid at said posi1tion
17 is replaced.
9. The variant of claim 1 wherein an amino acid at said position 23
is replaced.
10. The variant of claim 1 wherein an amino acid at said position
24 is replaced.
11. The variant of claim 1 wherein an amino acid at said position
25 is replaced.
12. The variant of claim 1 wherein an amino acid at said position
43 is replaced.
13. The variant of claim 1 wherein an amino acid at said position
49 is replaced.
14. The variant of claim 1 wherein an amino acid at said position
63 is replaced.
15. The variant of claim 1 wherein amino acids at said positions 16
and 49 are replaced.
16. The variant of claim 1 wherein amino acids at said positions 3
and 7 are replaced.
17. The variant of claim 15 wherein additionally tyrosine at said
position 24 is replaced with leucine or tyrosine at said position
31 is replaced with alanine or both are replaced.
18. The variant of claim 16 wherein additionally tyrosine at said
position 24 is replaced with leucine or tyrosine at said position
31 is replaced with alanine or both are replaced.
19. The variant of claim 17 wherein both tyrosines are
replaced.
20. The variant of claim 18 wherein both tyrosines are
replaced.
21. An IGF-I variant wherein an amino acid at position 3, 4, 5, 7,
10, 14, 17, 23, 24, 25, or 43, or any combination thereof, of
native-sequence human IGF-I is replaced with any amino acid at said
position 7 or with an alanine, a glycine, or a serine residue at
any position other than said position 7.
22. An IGF-I variant wherein an amino acid at position 3, 4, 5, 7,
10, 14, 17, 23, 24, 25, 43, or 49, or any combination thereof, of
native-sequence human IGF-I is replaced with any amino acid at said
position 7 or with an alanine, a glycine, or a serine residue at
any position other than said position 7.
23. An IGF-I variant wherein an amino acid at position 3, 4, 5, 7,
10, 14, 17, 23, 24, 25, 43, or 63, or any combination thereof, of
native-sequence human IGF-I is replaced with any amino acid at said
position 7 or with an alanine, a glycine, or a serine residue at
any position other than said position 7.
24. IGF-like insulin wherein phenylalanine at position 1 of
native-sequence human pro-insulin is deleted, or glutamine at
position 4 of native-sequence human pro-insulin is replaced with
glutamic acid, or leucine at position 17 of native-sequence human
pro-insulin is replaced with phenylalanine, or phenylalanine at
position 25 of native-sequence human pro-insulin is replaced with
tyrosine, or tyrosine at position 26 of native-sequence human
pro-insulin is replaced with phenylalanine, or threonine at
position 73 of native-sequence human pro-insulin is replaced with
phenylalanine, or any combination thereof.
25. The-insulin of claim 24 wherein the amino acid at position 1 is
deleted and the amino acid at said position 25 is replaced.
26. The insulin of claim 24 wherein the amino acid at position 1 is
deleted and the amino acid at said position 26 is replaced.
27. The insulin of claim 24 wherein the amino acid at position 1 is
deleted and the amino acid at said position 73 is replaced.
28. The insulin of claim 24 wherein the amino acid at position 1 is
deleted and the amino acids at said positions 25 and 26 are
replaced.
29. The insulin of claim 24 wherein the amino acid at position 1 is
deleted and the amino acids at said positions 25 and 73 are
replaced.
30. The insulin of claim 24 wherein the amino acid at position 1 is
deleted and the amino acids at said positions 26 and 73 are
replaced.
31. The insulin of claim 24 wherein the amino acid at position 1 is
deleted and the amino acids at said positions 25, 26, and 73 are
replaced.
32. The insulin of claim 24 wherein amino acids at said positions 4
and 17 are replaced.
33. The insulin of claim 24 wherein amino acids at said positions 4
and 26 are replaced.
34. The insulin of claim 24 wherein amino acids at said positions 4
and 73 are replaced.
35. The insulin of claim 24 wherein amino acids at said positions
17 and 26 are replaced.
36. The insulin of claim 24 wherein amino acids at said positions
17 and 73 are replaced.
37. The insulin of claim 24 wherein amino acids at said positions
26 and 73 are replaced.
38. The insulin of claim 24 wherein amino acids at said positions
4, 17, and 26 are replaced.
39. The insulin of claim 24 wherein amino acids at said positions
4, 17, and 73 are replaced.
40. The insulin of claim 24 wherein amino acids at said positions
4, 26, and 73 are replaced.
41. The insulin of claim 24 wherein amino acids at said positions
17, 26, and 73 are replaced.
42. IGF-like insulin wherein phenylalanine at position 1 of
native-sequence human pro-insulin is deleted, or glutamine at
position 4 of native-sequence human pro-insulin is replaced with
glutamic acid, or phenylalanine at position 25 of native-sequence
human pro-insulin is replaced with tyrosine, or tyrosine at
position 26 of native-sequence human pro-insulin is replaced with
phenylalanine, or threonine at position 73 of native-sequence human
pro-insulin is replaced with phenylalanine, or any combination
thereof.
43. IGF-like insulin wherein the phenylalanine at position 1 is
deleted, or glutamine at position 4 of native-sequence human mature
insulin is replaced with glutamic acid, or leucine at position 17
of native-sequence human mature insulin is replaced with
phenylalanine, or phenylalanine at position 25 of native-sequence
human mature insulin is replaced with tyrosine, or tyrosine at
position 26 of native-sequence human mature insulin is replaced
with phenylalanine, or threonine at position 38 of native-sequence
human mature insulin is replaced with phenylalanine, or any
combination thereof.
44. The insulin of claim 43 wherein amino acids at said positions
4, 17, 26, and 38 are replaced.
45. The insulin of claim 43 wherein the amino acid at position 1 is
deleted and amino acids at said positions 25, 26, and 38 are
replaced.
46. IGF-like insulin wherein the phenylalanine at position 1 is
deleted, or glutamine at position 4 of native-sequence human mature
insulin is replaced with glutamic acid, or phenylalanine at
position 25 of native-sequence human mature insulin is replaced
with tyrosine, or tyrosine at position 26 of native-sequence human
mature insulin is replaced with phenylalanine, or threonine at
position 38 of native-sequence human mature insulin is replaced
with phenylalanine, or any combination thereof.
47. A composition comprising the variant of claim 1 in a
carrier.
48. A composition comprising the insulin of claim 24 in a
carrier.
49. A composition comprising the insulin of claim 43 in a carrier.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of Invention
[0002] 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.
[0003] 2. Description of Background and Related Art
[0004] 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
.alpha..sub.2.beta..sub.2-heterotetr- amer closely related to the
insulin receptor (McInnes and Sykes, Biopoly., 43: 339-366 (1998);
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 IGF-I receptor has recently been reported (Garrett et al.,
Nature, 394: 395-399 (1998)).
[0005] 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,
132, 1337-1344 (1993)).
[0006] 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)).
[0007] 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., 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
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.
[0008] Additionally, a variant designated
(1-27,gly.sup.4,38-70)-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 IGFBP's but not to IGF
receptors (Bar et al., Endocrinology, 127: 3243-3245 (1990)).
[0009] 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 IGFBP's. Additionally, an epitope involving
residues 49-51 in binding to IGFBP-1, -2 and-S 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., Biol.
Chem., 271:13948-13952 (1996); U.S. Pat. Nos. 5,077,276;5,164,370;
5,470,828).
[0010] 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.
[0011] 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.sup.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.
[0012] 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.sup.23, Phe.sup.24, Tyr.sup.25)IGF-I (which is equipotent to
human IGF-I in its affinity to the Types 1 and 2 IGF and insulin
receptors), (Leu.sup.24)IGF-I and (Ser.sup.24)IGF-I (which have a
lower affinity than IGF-I to the human placental Type I IGF
receptor, the placental insulin receptor, and the Type 1 IGF
receptor of rat and mouse cells), and desoctapeptide
(Leu.sup.24)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 (Leu.sup.24)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.
[0013] Bayne et al., J. Biol. Chem., 264: 11004-11008 (1988)
discloses three structural analogs of IGF-1: (1-62)IGF-I, which
lacks the carboxyl-terminal 8-amino-acid D region of IGF-I;
(1-27,Gly.sup.4,38-70)I- GF-I, in which residues 28-37 of the C
region of IGF-I are replaced by a four-residue glycine bridge; and
(1-27,Gly.sup.4,38-62)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.sup.4 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.
[0014] 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:
(Ile.sup.41,Glu.sup.45,Gln.sup.46,Thr.sup.49,Ser.sup.50,-
Ile.sup.51,Ser.sup.50,Ile.sup.51,Ser.sup.53,Tyr.sup.55,Gln.sup.56)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;
(Thr.sup.49,Ser.sup.50,Ile.sup.51)IGF-I; and
(Tyr.sup.55,Gln.sup.56)IGF-I- .
[0015] WO 94/04569 discloses a specific binding molecule, other
than a natural IGFBP, that is capable of binding to IGF-I and can
enhance the biological activity of IGF-I. WO98/45427 published Oct.
15, 1998 and Lowman et al., supra, disclose IGF-I agonists
identified by phage display. Also, WO 97/39032 discloses ligand
inhibitors of IGFBP's and methods for their use.
[0016] There are various forms of human insulin on the market that
differ in the duration of action and onset of action, but have the
native human sequence. Jens Brange, Galenics of Insulin, The
Physico-chemical and Pharmaceutical Aspects of Insulin and Insulin
Preparations (Springer-Verlag, New York, 1987), page 17-40. Regular
insulin is a clear neutral solution that contains hexameric
insulin. It is short acting, its onset of action occurs in 0.5 hour
after injection and duration of action is about 6-8 hours. NPH
(Neutral Protamine Hagedorn) insulin, also called Isophane Insulin,
is a crystal suspension of insulin-protamine complex. These
crystals contain approximately 0.9 molecules of protamine and two
zinc atoms per insulin hexamer. Dodd et al., Pharmaceutical
Research, 12: 60-68 (1993). NPH-insulin is an intermediate-acting
insulin; its onset of action occurs in 1.5 hours and its duration
of action is 18-26 hours. 70/30 insulin is composed of 70%
NPH-insulin and 30% Regular insulin. There are also Semilente
insulin (amorphous precipitate of zinc insulin complex), UltraLente
insulin (zinc insulin crystal suspension), and Lente insulin (a 3:7
mixture of amorphous and crystalline insulin particles). Of the
various types of insulins available, NPH-, 70/30, and Regular
insulin are the most widely used insulins, accounting for 36%, 28%,
and 15%, respectively, of the insulin prescriptions in 1996.
[0017] The use of recombinant DNA technology and peptide chemistry
have allowed the generation of insulin analogs with a wide variety
of amino acid substitutions, and IGF-like modifications to insulin
have been made for the purpose of modifying insulin
pharmocokinetics (Brang et al., Nature, 333: 679 (1988); Kang et
al., Diabetes Care, 14: 571 (1991); DiMarchi et al., "Synthesis of
a fast-acting insulin analog based upon structural homology with
insulin-like growth factor-I," in: Peptides: Chemistry and Biology,
Proceedings of the Twelfth American Peptide Symposium, J. A. Smith
and J. E. Rivier, eds. (ESCOM, Leiden, 1992), pp. 26-28; Weiss et
al., Biochemistry, 30: 7373 (1991); Howey et al., Diabetes, 40:
(Supp 1) 423A (1991); Slieker and Sundell, Diabetes, 40: (Supp 1)
168A (1991); Cara et al., J. Biol. Chem., 265: 17820 (1990);
Wolpert et al., Diabetes, 39: (Supp 1) 140A (1990); Bornfeldt et
al., Diabetologia, 34: 307 (1991); Drejer, Diabetes/Metabolism
Reviews, 8: 259 (1992); Slicker et al., Adv. Experimental Med.
Biol., 343: 25-32 (1994)). One example of such an insulin analog is
Humalog.TM. insulin (rapid-acting monomeric insulin solution, as a
result of reversing the Lys (B28) and Pro(B29) amino acids on the
insulin B-chain) that was recently introduced into the market by
Eli Lilly and Company. A review of the recent insulin mutants in
clinical trials and on the market is found in Barnett and Owens,
Lancet, 349: (1997).
[0018] Slicker et al., 1994, supra, describe the binding affinity
of various IGF and insulin variants to IGFBPs, IGF receptor, and
insulin receptor, and in particular sought to confer IGFBP-binding
ability to insulin through several combinations of mutations,
including: (Phe.sup.38, Arg.sup.39, Ser.sup.40) insulin,
(Glu.sup.4, Gln.sup.6, Phe.sup.17) insulin, and (Glu.sup.4,
Gln.sup.16, Phe.sup.17, Phe.sup.38, Arg.sup.39, Ser.sup.40) insulin
(the numbering of mature insulin used herein consists of
consecutive numbering in the B chain (residues 1-30), followed by
consecutive numbering in the A chain (residues 31-51); these
correspond to residues numbered 1-30 and residues 66-86,
respectively of proinsulin; cf. FIG. 4 herein). However, only weak
affinity was found for these variants binding to the IGF binding
proteins and insulin-receptor affinity was reduced as compared with
wild-type insulin (Slieker et al., supra).
[0019] Although earlier reports could not find any affinity of
insulin for the binding proteins, a group has measured a weak
affinity of 251+/-91 nM of insulin for IGFBP-3 by BIAcore.TM.
experiments (Heding et al., supra).
[0020] Despite all these efforts, the view of the IGFBP-binding
epitope on IGF-I has remained diffuse and at low resolution. The
previous studies most often involved insertions of homologous
insulin regions into IGF-I or protein truncations (e.g.
des(1-3)-IGF-I), not differentiating between effects attributed to
misfolding and real binding determinants. Combining the results of
all these studies is further complicated by the fact that different
techniques were used to analyze complex formation of the mutant IGF
forms with the IGFBP's, ranging from radiolabeled ligand binding
assays to biosensor analysis.
[0021] There is a need in the art for molecules that act as IGF or
insulin agonists, and also for molecules that binds to IGF binding
proteins with high affinity and specificity for therapeutic or
diagnostic purposes.
SUMMARY OF THE INVENTION
[0022] Accordingly, in one embodiment, the invention provides an
IGF-I variant wherein an amino acid at position 3, 4, 5, 7, 10, 14,
17, 23, 24, 25, 43, 49 or 63, or any of such amino acids in
combination with an amino acid at position 12 or 16 or both 12 and
16 of native-sequence human IGF-I, or any combination thereof, is
replaced with any amino acid at said position 7 or with an alanine,
a glycine, or a serine residue at any position other than said
position 7.
[0023] In one preferred embodiment, the amino acids at said
positions 16 and 49 are replaced to obtain binders to IGFBP-3.
Another preferred embodiment for obtaining binders to IGFBP-3 is a
variant containing mutations at positions 3 and 7.
[0024] In a still further preferred embodiment, additionally
tyrosine at said position 24 is replaced with leucine or tyrosine
at said position 31 is replaced with alanine or both are replaced,
to disrupt or prevent receptor binding. Most preferably, both
tyrosines at said positions 24 and 31 are replaced.
[0025] In another embodiment, the invention provides a
long-half-life IGF-like insulin wherein phenylalanine at position 1
of native-sequence human pro-insulin is deleted
(des(1)-proinsulin), or glutamine at position 4 of native-sequence
human pro-insulin is replaced with glutamic acid, or leucine at
position 17 of native-sequence human pro-insulin is replaced with
phenylalanine, or phenylalanine at position 25 of native-sequence
human pro-insulin is replaced with tyrosine, or tyrosine at
position 26 of native-sequence human pro-insulin is replaced with
phenylalanine, or threonine at position 73 of native-sequence human
pro-insulin is replaced with phenylalanine, or any combination
thereof.
[0026] Preferably, for the IGF-like insulin, amino acids at said
positions 4, 17, 26, and/or 73 are replaced to generate
IGFBP-1-specific mutants, or the amino acid at position 1 is
deleted and the amino acids at positions 25, 26, and/or 73 are
replaced to generate IGFBP-3-specific mutants.
[0027] In yet another embodiment, the invention provides an
IGF-like insulin wherein the phenylalanine at position 1 is deleted
(des(1)-insulin), or glutamine at position 4 of native-sequence
human mature insulin is replaced with glutamic acid, or leucine at
position 17 of native-sequence human mature insulin is replaced
with phenylalanine, or phenylalanine at position 25 of
native-sequence human mature insulin is replaced with tyrosine, or
tyrosine at position 26 of native-sequence human mature insulin is
replaced with phenylalanine, or threonine at position 38 of
native-sequence human mature insulin is replaced with
phenylalanine, or any combination thereof (Note: the numbering of
mature insulin used here consists of consecutive numbering in the B
chain (residues 1-30), followed by consecutive numbering in the A
chain (residues 31-51)).
[0028] In a preferred embodiment, amino acids of the above mature
insulin at positions 4, 17, 26, and 38 are replaced, to create a
mutant that is IGFBP-I specific.
[0029] In another preferred embodiment, the amino acid at position
1 of the above mature insulin is deleted, and amino acids of the
above mature insulin at positions 25, 26, and 38 are replaced, to
create a mutant that is IGFBP-3 specific.
[0030] Also provided herein is a composition comprising one of the
peptides described above in a carrier, preferably a
pharmaceutically acceptable carrier. Preferably, this composition
is sterile.
[0031] Uses of these peptides include all uses that liberate or
enhance at least one biological activity of exogenous or endogenous
IGFs or insulin. They can be used in treating, inhibiting, or
preventing conditions in which an IGF such as IGF-I or insulin is
useful, i.e., in treating an IGF disorder or an insulin disorder by
administering an effective amount of die peptide to a mammal, as
described below.
[0032] Additionally provided herein is a method for increasing
serum and tissue levels of biologically active IGF or insulin in a
mammal comprising administering to the mammal an effective amount
of a peptide as described above. The mammal is preferably human.
Also preferred is where administering the peptide, if it is
mimicking IGF-I, preferably in an amount effective to produce body
weight gain, causes an increase in anabolism in the mammal.
Additionally preferred is that glycemic control is effected in the
mammal after the peptide is administered.
[0033] The peptide herein can be administered alone or together
with another agent such as GH, a GH-releasing peptide (GHRP), a
GH-releasing factor (GHRF), a GH-releasing hormone (GHRH), a GH
secretagogue, an IGF, an IGF in combination with an IGFBP, an
IGFBP, GH in combination with a GH binding protein (GHBP), insulin,
or a hypoglycemic agent (which includes in the definition below an
insulin-sensitizing agent such as thiazolidinedione).
[0034] In yet another aspect of the invention, a method is provided
for effecting glycemic control in a mammal comprising administering
to the mammal an effective amount of one or more of the above
peptides. Preferably, the peptide also reduces plasma insulin
secretion and blood glucose levels in a mammal. Also preferably,
the mammal has a hyperglycemic disorder such as diabetes. This
method can additionally comprise administering to the mammal an
effective amount of a hypoglycemic agent or insulin.
[0035] Also provided is a method for increasing serum and tissue
levels of biologically active IGF in a mammal, or a method for
increasing anabolism in a mammal, or a method for controlling
glycemia in a mammal comprising administering to the mammal an
effective amount of the composition containing the peptide
herein.
[0036] Also contemplated herein is a kit comprising a container
containing a pharmaceutical composition containing the peptide
herein and instructions directing the user to utilize the
composition. This kit may optionally further comprise a container
containing a GH, a GHRP, a GHRF, a GHRH, a GH secretagogue, an IGF,
an IGF complexed to an IGFBP, an IGFBP, a GH complexed with a GHBP,
insulin, or a hypoglycemic agent.
[0037] For an identification of the peptides herein, human IGF-I
was displayed monovalently on filamentous phagemid particles (U.S.
Pat. Nos. 5,750,373 and 5,821,047), and a complete alanine-scanning
mutagenesis thereof (Cunningham and Wells, Science, 244: 1081-1085
(1989); U.S. Pat. No. 5,834,250) was performed by phage display
("turbo-ala scan") (Cunningham et al., EMBO J., 13: 2508-2515
(1994); Lowman, Methods Mol. Biol., 87: 249-264 (1998)). The mutant
IGF-phagemids were used to map the binding determinants on IGF-I
for IGFBP-1 and IGFBP-3. The alanine scanning reveals specificity
determinants for these binding proteins, so as to generate
binding-protein-specific IGF variants or insulin variants that bind
specifically to IGFBP-1 or IGFBP-3 to modulate their clearance
half-life, improve proteolytic stability, or alter their tissue
distribution in vivo. These mutants should also be useful for
mapping the functional binding site for IGF receptor, whose crystal
structure was recently reported (Garrett et al., supra). In
addition, it may be of interest to map the epitopes of various
IGF-binding antibodies or of other peptides or proteins that bind
to IGF-I.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIGS. 1A and 1B show a phage ELISA of the variant, G1S-A70V
IGF-I, binding to IGFBP-I (FIG. 1A) and IGFBP-3 (FIG. 1B).
Microtiter plates coated with 1 .mu.g/ml IGFBP-I (FIG. 1A) or
IGFBP-3 (FIG. 1B) were incubated with phage particles displaying
G1S-A70V in the presence of the indicated amounts of soluble
competitor protein, IGFBP-1 (FIG. 1A) or IGFBP-3 (FIG. 1B). The
half-maximal inhibitory concentration (IC.sub.50) of competitor,
i.e., the inhibitory concentration of competitor that resulted in
half-maximal binding of the phagemid in that particular experiment,
is denoted for the respective IGFBP.
[0039] FIG. 2 shows the loss or gain of IGFBP affinity for the
IGF-I mutants tested by phage ELISA. Relative IC.sub.50 values
(IC.sub.50mut/IC.sub.50 G1S-A70V) of each IGF-I alanine mutant
(affinity changes of each mutant for the binding proteins with
respect to IGF-I G1S-A70V) are shown for IGFBP-I (filled bars) and
IGFBP-3 (open bars). Data are taken from Table I below. Relative
IC.sub.50 values <1 denote gain of affinity; values >1 denote
loss of affinity. The asterisk indicates that these particular
variants were not displayed on phage, as judged by antibody
binding.
[0040] FIGS. 3A and 3B show binding specificity of the IGF-I
variant F49A displayed on phage to IGFBP-1 and -3, respectively, in
competitive-phage ELISA. Phagemid particles displaying F49A
(squares) were bound to plates coated with IGFBP-3 in the presence
of the indicated amounts of soluble IGFBP-1 (FIG. 3A) or IGFBP-3
(FIG. 3B). The same experiment was carried out in parallel with
phage displaying the wild-type-like IGF-I variant G1S-A70V
(circles). See Tables I and II below for absolute IC.sub.50 values.
Data points are mean.+-.standard deviation, n=2. Immunosorbent
plates were coated with 1 .mu.g/ml IGFBP-3 and ELISA were carried
out as described in the Examples below using wild-type IGF-I phage
(WT, circles) and IGF-F49A phage (F49A, squares) in parallel.
Experiments were carried out in duplicate, and data points are
shown as mean.+-.standard deviation. The IC.sub.50 values of the
actual experiment are indicated in the figure.
[0041] FIG. 4 discloses a sequence alignment of native-sequence
human IGF-I (designated wtIGF) (SEQ ID NO:1), native-sequence human
proinsulin (designated proinsulin) (SEQ ID NO:2), and
native-sequence human insulin (designated insulin (B chain)
followed by insulin (A chain)) (SEQ ID NO:3). The asterisks and
dots indicate sequence identity and sequence similarity,
respectively, at the indicated amino acid positions among the three
sequences.
[0042] FIGS. 5A-5D show a biosensor analysis of IGFBP binding to
immobilized IGF-I variants. Sensor grams are shown for IGFBP-1
(FIGS. 5A, 5C) or IGFBP-3 (FIGS. 5B, 5D) binding to immobilized
wild-type IGF-I (FIGS. 5A, 5B) or F49A IGF variant (FIGS. 5C, 5D).
The concentrations of ligand in each experiment were 1 .mu.M, 500
nM, and 250 nM. See Table II for kinetic parameters.
[0043] FIGS. 6A-6B show a model of the functional binding epitopes
for IGFBP-1 and IGFBP-3, respectively, on the surface of IGF-I.
Amino acid side chains were classified according to their relative
contribution in binding energy (Table I) and colored as follows: no
effect (grey); 2-5 fold loss of apparent affinity (yellow); 5-10
fold (orange); 10-100 fold (bright red); >100 fold (dark red).
If available, numbers from phage ELISA experiments in Table I below
were used. BIAcore.TM. data were used instead for V11A, R36A, and
P39A variants (Table II). The NMR structure of IGF-I (Cooke et al.,
supra) was represented using the program Insight II.TM. (MSI, San
Diego, Calif.). The binding epitope for IGFBP-1 (FIG. 6A) is
located on the "upper" and "lower" face of the N-terminal helix
(residues 8-17), connected by the energetically-important residue
F49. For IGFBP-3 (FIG. 6B), individual IGF-I side chains contribute
very little binding energy. The binding epitope has shifted away
from the N-terminus and newly includes G22, F23, Y24.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0044] A. Definitions
[0045] As used herein, "mammal" for purposes of treatment refers to
any animal classified as a mammal, including humans, domestic, and
farm animals, and zoo, sports, or pet animals, such as dogs,
horses, cats, sheep, pigs, cows, etc. The preferred mammal herein
is a human. The term "non-adult" refers to mammals that are from
perinatal age (such as low-birth-weight infants) up to the age of
puberty, the latter being those that have not yet reached full
growth potential.
[0046] As used herein, "IGF" refers to native insulin-like growth
factor-I and native insulin-like growth factor-II as well as
natural variants thereof such as brain IGF, otherwise known as
des(1-3)IGF-I.
[0047] As used herein, "IGF-I" refers to insulin-like growth
factor-I from any species, including bovine, ovine, porcine,
equine, and human, preferably human, and, if referring to exogenous
administration, from any source, whether natural, synthetic, or
recombinant. "Native-sequence" human IGF-I, the sequence of which
is shown in FIG. 4 (SEQ ID NO:1), is prepared, e.g., by the process
described in EP 230,869 published Aug. 5, 1987; EP 128,733
published Dec. 19, 1984; or EP 288,451 published Oct. 26, 1988.
More preferably, this native-sequence IGF-I is recombinantly
produced and is available from Genentech, Inc., South San
Francisco, Calif. for clinical investigations.
[0048] As used herein, "IGF-II" refers to insulin-like growth
factor-II from any species, including bovine, ovine, porcine,
equine, and human, preferably human, and, if referring to exogenous
administration, from any source, whether natural, synthetic, or
recombinant. It may be prepared by the method described in, e.g.,
EP 128,733.
[0049] An "IGFBP" or an "IGF binding protein" refers to a protein
or polypeptide normally associated with or bound or complexed to
IGF-I or IGF-II, whether or not it is circulatory (i.e., in serum
or tissue). Such binding proteins do not include receptors. This
definition includes IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5,
IGFBP-6, Mac 25 (IGFBP-7), and prostacyclin-stimulating factor
(PSF) or endothelial cell-specific molecule (ESM-1), as well as
other proteins with high homology to IGFBPs. Mac 25 is described,
for example, in Swisshelm et al., Proc. Natl. Acad. Sci. USA, 92:
4472-4476 (1995) and Oh et al., J. Biol. Chem., 271: 30322-30325
(1996). PSF is described in Yamauchi et al., Biochemical Journal,
303: 591-598 (1994). ESM-1 is described in Lassalle et al., J.
Biol. Chem., 271: 20458-20464 (1996). For other identified IGFBPs,
see, e.g., EP 375,438 published Jun. 27, 1990; EP 369,943 published
May 23, 1990; WO 89/09268 published Oct. 5, 1989; Wood et al.,
Molecular Endocrinology, 2: 1176-1185 (1988); Brinkman et al., The
EMBO J. 7: 2417-2423 (1988); Lee et al., Mol. Endocrinol., 2:
404-411 (1988); Brewer et al., BBRC, 152:1289-1297 (1988); EP
294,021 published Dec. 7, 1988; Baxter et al., BBRC, 147: 408-415
(1987); Leung et al., Nature, 330: 537-543 (1987); Martin et al.,
J. Biol. Chem., 261: 8754-8760 (1986); Baxter et al., Comp.
Biochem. Physiol., 91B: 229-235 (1988); WO 89/08667 published Sep.
21, 1989; WO 89/09792 published Oct. 19, 1989; and Binkert et al.,
EMBO J., 8: 2497-2502 (1989).
[0050] The term "body fluid" refers to a biological sample of
liquid from a mammal, preferably from a human. Such fluids include
aqueous fluids such as serum, plasma, lymph fluid, synovial fluid,
follicular fluid, seminal fluid, amniotic fluid, milk, whole blood,
urine, cerebrospinal fluid, saliva, sputum, tears, perspiration,
mucus, tissue culture medium, tissue extracts, and cellular
extracts.
[0051] As used herein, "human IGF receptor" refers to any receptor
for an IGF found in humans and includes the Type 1 and Type 2 IGF
receptors in humans to which both human IGF-I and IGF-II bind, such
as the placental Type 1 IGF-I receptor, etc.
[0052] "Peptides" include an IGF-I agonist, IGF-I variant, insulin
agonist, insulin variant, or IGF-like insulin having at least two
amino acids and include polypeptides having at least about 50 amino
acids. The definition includes peptide derivatives, their salts, or
optical isomers.
[0053] As used herein, "insulin" refers to any form of insulin from
any species, and whether natively or synthetically or recombinantly
derived. It may be formulated, for example, as Regular insulin, NPH
insulin, 70/30 insulin, Semilente insulin, UltraLente insulin, or
Lente insulin. If an insulin is to be administered together with an
IGF-like insulin or IGF-I variant herein, it is preferably Regular
insulin, NPH insulin, 70/30 insulin, or HUMALOG.TM. brand
insulin.
[0054] "Proinsulin" refers to insulin that contains the A, B, and C
peptide, the native sequence of which is shown in FIG. 4 (SEQ ID
NO:2). Conversion of proinsulin to "mature insulin" occurs by
excision of the region from R3 to R65. The resulting amino-terminal
peptide of mature insulin is called B-chain, and the
carboxy-terminal peptide A-chain. The chains are held together by
two inter-chain disulfides. Mature insulin is a soluble protein.
The numbering for mature insulin variants herein consists of
consecutive numbering in the B chain (residues 1-30), followed by
consecutive numbering in the A chain (residues 31-51).
"Native-sequence" human proinsulin has the sequence (SEQ ID NO:2)
shown in FIG. 4, and "native-sequence" human mature insulin has the
sequence (SEQ ID NO:3) shown in FIG. 4.
[0055] "IGF-like insulin" is a peptide that simulates at least one
of the biological activities of IGF-I, including those biological
activities listed under "IGF disorder" and under Modes below.
Preferably, such IGF-like insulin is long-acting.
[0056] An "IGF disorder" is any condition that would benefit from
treatment with an IGF, including but not limited to, for example,
lung diseases, hyperglycemic disorders as set forth below, renal
disorders, such as acute and chronic renal insufficiency, end-stage
chronic renal failure, glomerulonephritis, interstitial nephritis,
pyelonephritis, glomerulosclerosis, e.g., Kimmelstiel-Wilson in
diabetic patients and kidney failure after kidney transplantation,
obesity, GH-insufficiency, Turner's syndrome, Laron's syndrome,
short stature, undesirable symptoms associated with aging such as
obesity and increased fat mass-to-lean ratios, immunological
disorders such as immunodeficiencies including decreased CD4 counts
and decreased immune tolerance or chemotherapy-induced tissue
damage, bone marrow transplantation, diseases or insufficiencies of
cardiac structure or function such as heart disfunctions and
congestive heart failure, neuronal, neurological, or neuromuscular
disorders, e.g., peripheral neuropathy, multiple sclerosis,
muscular dystrophy, or myotonic dystrophy, and catabolic states
associated with wasting caused by any condition, including, e.g.,
trauma or wounding or infection such as with a bacterium or human
virus such as HIV, wounds, skin disorders, gut structure and
function that need restoration, and so forth. The IGF disorder
being treated may be a combination of two or more of the above
disorders. The preferred disorders targeted for treatment herein
are diabetes and obesity, heart dysfunctions, kidney disorders,
neurological disorders, whole body growth disorders, and
immunological disorders.
[0057] An "insulin disorder" is a condition that would benefit from
treatment with an insulin, such as hyperglycemic disorders.
[0058] As used herein, the term "hyperglycemic disorders" refers to
all forms of diabetes and disorders resulting from insulin
resistance, such as Type I and Type II diabetes, as well as severe
insulin resistance, hyperinsulinemia, and hyperlipidemia, e.g.,
obese subjects, and insulin-resistant diabetes, such as
Mendenhall's Syndrome, Werner Syndrome, leprechaunism, lipoatrophic
diabetes, and other lipoatrophies. The preferred hyperglycemic
disorder is diabetes, especially Type 1 and Type II diabetes.
"Diabetes" itself refers to a progressive disease of carbohydrate
metabolism involving inadequate production or utilization of
insulin and is characterized by hyperglycemia and glycosuria.
[0059] As used herein, the term "treating" refers to both
therapeutic treatment and prophylactic or preventative measures.
Those in need of treatment include those already with the disorder
as well as those prone to having the disorder or diagnosed with the
disorder or those in which the disorder is to be prevented.
Consecutive treatment or administration refers to treatment on at
least a daily basis without interruption in treatment by one or
more days. Intermittent treatment or administration, or treatment
or administration in an intermittent fashion, refers to treatment
that is not consecutive, but rather cyclic in nature. The treatment
regime herein can be either consecutive or intermittent.
[0060] As used herein, the term "hypoglycemic agent" refers to
compounds that are useful for regulating glucose metabolism,
preferably oral agents. More preferred herein for human use are
insulin and the sulfonylurea class of oral hypoglycemic agents,
which cause the secretion of insulin by the pancreas. Examples
include glyburide, glipizide, and gliclazide. In addition, agents
that enhance insulin sensitivity or are insulin sensitizing, such
as biguanides (including metformin and phenformin) and
thiazolidenediones such as REZULIN.TM. (troglitazone) brand
insulin-sensitizing agent, and other compounds that bind to the
PPAR.gamma. nuclear receptor, are within this definition, and also
are preferred.
[0061] As used herein, "active" or "biologically active" IGF in the
context of changing serum and tissue levels of endogenous IGF
refers to IGF that binds to its receptor or otherwise causes a
biological activity to occur, such as those biological activities
of endogenous or exogenous IGF referred to herein.
[0062] "Growth-hormone-releasing peptides or factors" ("GHRP" or
"GHRF") are described below, as are secretagogues. A
"growth-hormone-releasing hormone" ("GHRH") can be any hormone that
releases GH from the cells or tissue. "Growth hormone in
combination with a growth hormone binding protein" ("GH" plus
"GHBP") means a GH complexed with or otherwise associated with one
of its binding proteins. Similarly, "IGF in combination with an IGF
binding protein" ("IGF" plus "IGFBP") refers to an IGF complexed
with or otherwise associated with one of its IGFBPs.
[0063] B. Modes for Carrying Out the Invention
[0064] The invention herein relates, in one aspect, to an IGF-I
variant wherein one or more amino acids of native-sequence human
IGF-I at selected positions are replaced. Specifically, one or more
amino acids at positions 3, 4, 5, 7, 10, 14, 17, 23, 24, 25, 43, 49
and/or 63, or one or more amino acids at the above positions along
with one or both amino acids at positions 12 and/or 16, are
replaced. The replacement at position 7 is with any amino acid
residue, and the replacement at any position other than position 7
is with either an alanine, a glycine, or a serine residue.
Preferably, the amino acids in question are replaced by an alanine,
glycine, or serine.
[0065] One preferred variant has the amino acids at positions 16
and 49 replaced. Another preferred variant has amino acids at
positions 3 and 7 replaced. Preferably, the amino acids at
positions 49 and 63 are not singly replaced.
[0066] In another preferred embodiment, the variant additionally
has its tyrosine at position 24 replaced with leucine or its
tyrosine at position 31 replaced with alanine. Most preferably,
both tyrosine residues are replaced.
[0067] The invention additionally provides, in another aspect, two
types of IGF-like insulins. In one such embodiment, the
phenylalanine at position 1 of native-sequence human proinsulin is
deleted, or the glutamine at position 4 of native-sequence human
pro-insulin is replaced with glutamic acid, or leucine at position
17 of native-sequence human pro-insulin is replaced with
phenylalanine, or phenylalanine at position 25 of native-sequence
human proinsulin is replaced with tyrosine, or tyrosine at position
26 of native-sequence human pro-insulin is replaced with
phenylalanine, or threonine at position 73 of native-sequence human
pro-insulin is replaced with phenylalanine, or any combination
thereof is made.
[0068] Preferred combinations are those wherein amino acids at said
positions 4 and 17 are replaced, or wherein amino acids at said
positions 4 and 26 are replaced, or wherein amino acids at said
positions 4 and 73 are replaced, or wherein amino acids at said
positions 17 and 26 are replaced, or wherein amino acids at said
positions 26 and 73 are replaced, or wherein amino acids at said
positions 17 and 73 are replaced, or wherein amino acids at said
positions 4, 17, and 26 are replaced, or wherein amino acids at
said positions 4, 26, and 73 are replaced, or wherein amino acids
at said positions 4, 17, and 73 are replaced, or wherein amino
acids at said positions 17, 26, and 73 are replaced, or wherein the
amino acid at position 1 is deleted and the amino acid at said
position 25 is replaced, or wherein the amino acid at position 1 is
deleted and the amino acid at said position 26 is replaced, or
wherein the amino acid at position 1 is deleted and the amino acid
at said position 73 is replaced, or wherein the amino acid at
position 1 is deleted and the amino acids at said positions 25 and
26 are replaced, or wherein the amino acid at position 1 is deleted
and the amino acids at said positions 25 and 73 are replaced, or
wherein the amino acid at position 1 is deleted and the amino acids
at said positions 26 and 73 are replaced, or wherein the amino acid
at position 1 is deleted and the amino acids at said positions 25,
26, and 73 are replaced.
[0069] Most preferred is the variant wherein amino acids at said
positions 4, 17, 26, and 73 are replaced, to be IGFBP-1-selective,
or wherein the amino acid at position 1 is deleted and the amino
acids at said positions 25, 26, and 73 are replaced, to be
IGFBP-3-selective.
[0070] The other type of IGF-like insulin is based on soluble
mature insulin. In this case the same mutations are made as above
for pro-insulin, but the numbering is changed in certain cases.
Hence, glutamine at position 4 of native-sequence human mature
insulin is replaced with glutamic acid, or leucine at position 17
of native-sequence human mature insulin is replaced with
phenylalanine, or phenylalanine at position 25 of native-sequence
human mature insulin is replaced by tyrosine, or tyrosine at
position 26 of native-sequence human mature insulin is replaced
with phenylalanine, or threonine at position 38 of native-sequence
human mature insulin is replaced with phenylalanine, or any
combination thereof is made.
[0071] For IGFBP-1-selective mutants amino acids at said positions
4, 17, 26, and 38 are replaced, and for IGFBP-3-selective mutants,
the amino acid at position 1 is deleted and the amino acids at said
positions 25, 26, and 38 are replaced.
[0072] The peptides of this invention can be made by chemical
synthesis or by employing recombinant technology. These methods are
known in the art. Chemical synthesis, especially solid phase
synthesis, is preferred for short (e.g., less than 50 residues)
peptides or those containing unnatural or unusual amino acids such
as D-Tyr, Ornithine, amino adipic acid, and the like. Recombinant
procedures are preferred for longer polypeptides. When recombinant
procedures are selected, a synthetic gene may be constructed de
novo or a natural gene may be mutated by, for example, cassette
mutagenesis. Set forth below are exemplary general recombinant
procedures.
[0073] From a purified IGF or insulin and its amino acid sequence,
for example, an IGF or insulin variant that is a peptidyl mutant of
an IGF or insulin parent molecule may be produced using recombinant
DNA techniques. These techniques contemplate, in simplified form,
taking the gene, either natural or synthetic, encoding the peptide;
inserting it into an appropriate vector; inserting the vector into
an appropriate host cell; culturing the host cell to cause
expression of the gene; and recovering or isolating the peptide
produced thereby. Preferably, the recovered peptide is then
purified to a suitable degree.
[0074] Somewhat more particularly, the DNA sequence encoding a
peptidyl IGF or insulin variant is cloned and manipulated so that
it may be expressed in a convenient host. DNA encoding parent
polypeptides can be obtained from a genomic library, from cDNA
derived from mRNA from cells expressing the peptide, or by
synthetically constructing the DNA sequence (Sambrook et al.,
Molecular Cloning: A Laboratory Manual (2d ed.), Cold Spring Harbor
Laboratory, N.Y., 1989).
[0075] The parent DNA is then inserted into an appropriate plasmid
or vector which is used to transform a host cell. In general,
plasmid vectors containing replication and control sequences which
are derived from species compatible with the host cell are used in
connection with those hosts. The vector ordinarily carries a
replication site, as well as sequences which encode proteins or
peptides that are capable of providing phenotypic selection in
transformed cells.
[0076] For example, E. coli may be transformed using pBR322, a
plasmid derived from an E. coli species (Mandel et al., J. Mol.
Biol. 53: 154 (1970)). Plasmid pBR322 contains genes for ampicillin
and tetracycline resistance, and thus provides easy means for
selection. Other vectors include different features such as
different promoters, which are often important in expression. For
example, plasmids pKK223-3, pDR720, and pPL-lambda represent
expression vectors with the tac, trp, or P.sub.L promoters that are
currently available (Pharmacia Biotechnology).
[0077] A preferred vector is pB0475. This vector contains origins
of replication for phage and E. coli that allow it to be shuttled
between such hosts, thereby facilitating both mutagenesis and
expression (Cunningham et al., Science, 243: 1330-1336 (1989); U.S.
Pat. No. 5,580,723). Other preferred vectors are pR1T5 and pR1T2T
(Pharmacia Biotechnology). These vectors contain appropriate
promoters followed by the Z domain of protein A, allowing genes
inserted into the vectors to be expressed as fusion proteins.
[0078] Other preferred vectors can be constructed using standard
techniques by combining the relevant traits of the vectors
described above. Relevant traits include the promoter, the ribosome
binding site, the decorsin or ornatin gene or gene fusion (the Z
domain of protein A and decorsin or ornatin and its linker), the
antibiotic resistance markers, and the appropriate origins of
replication.
[0079] The host cell may be prokaryotic or eukaryotic. Prokaryotes
are preferred for cloning and expressing DNA sequences to produce
parent IGF-I polypeptide, segment-substituted peptides,
residue-substituted peptides, and peptide variants. For example, E.
coli K12 strain 294 (ATCC No. 31446) may be used as well as E. coli
B, E. coli X1776 (ATCC No.31537), and E. coli c600 and c600hf1, E.
coli W3110 (F-, gamma-, prototrophic/ATCC No. 27325), bacilli such
as Bacillus subtilis, and other enterobacteriaceae such as
Salmonella typhimurium or Serratia marcesans, and various
Pseudomonas species. The preferred prokaryote is E. coli W3110
(ATCC 27325). When expressed by prokaryotes the peptides typically
contain an N-terminal methionine or a formyl methionine and are not
glycosylated. In the case of fusion proteins, the N-terminal
methionine or formyl methionine resides on the amino terminus of
the fusion protein or the signal sequence of the fusion protein.
These examples are, of course, intended to be illustrative rather
than limiting.
[0080] In addition to prokaryotes, eukaryotic organisms, such as
yeast cultures, or cells derived from multicellular organisms may
be used. In principle, any such cell culture is workable. However,
interest has been greatest in vertebrate cells, and propagation of
vertebrate cells in culture (tissue culture) has become a
reproducible procedure. Tissue Culture, Academic Press, Kruse and
Patterson, editors (1973). Examples of such useful host cell lines
are VERO and HeLa cells, Chinese Hamster Ovary (CHO) cell lines,
W138, 293, BHK, COS-7 and MDCK cell lines.
[0081] A variation on the above procedures contemplates the use of
gene fusions, wherein the gene encoding the desired peptide is
associated, in the vector, with a gene encoding another protein or
a fragment of another protein. This results in the desired peptide
being produced by the host cell as a fusion with another protein or
peptide. The "other" protein or peptide is often a protein or
peptide which can be secreted by the cell, making it possible to
isolate and purify the desired peptide from the culture medium and
eliminating the necessity of destroying the host cells which arises
when the desired peptide remains inside the cell. Alternatively,
the fusion protein can be expressed intracellularly. It is useful
to use fusion proteins that are highly expressed.
[0082] The use of gene fusions, though not essential, can
facilitate the expression of heterologous peptides in E. coli as
well as the subsequent purification of those gene products (Harris,
in Genetic Engineering, Williamson, R., Ed. (Academic Press,
London, Vol. 4, 1983), p. 127; Ljungquist et al., Eur. J. Biochem.,
186: 557-561 (1989) and Ljungquist et al., Eur. J. Biochem., 186:
563-569 (1989)). Protein A fusions are often used because the
binding of protein A, or more specifically the Z domain of protein
A, to IgG provides an "affinity handle" for the purification of the
fused protein. It has also been shown that many heterologous
proteins are degraded when expressed directly in E. coli, but are
stable when expressed as fusion proteins. Marston, Biochem J. 240:
1 (1986).
[0083] Fusion proteins can be cleaved using chemicals, such as
cyanogen bromide, which cleaves at a methionine, or hydroxylamine,
which cleaves between an Asn and Gly residue. Using standard
recombinant DNA methodology, the nucleotide base pairs encoding
these amino acids may be inserted just prior to the 5' end of the
gene encoding the desired peptide.
[0084] Alternatively, one can employ proteolytic cleavage of fusion
protein (Carter, in Protein Purification: From Molecular Mechanisms
to Large-Scale Processes, Ladisch et al., eds. (American Chemical
Society Symposium Series No. 427, 1990), Ch 13, pages 181-193).
[0085] Proteases such as Factor Xa, thrombin, and subtilisin or its
mutants, and a number of others have been successfully used to
cleave fusion proteins. Typically, a peptide linker that is
amenable to cleavage by the protease used is inserted between the
"other" protein (e.g., the Z domain of protein A) and the desired
peptide. Using recombinant DNA methodology, the nucleotide base
pairs encoding the linker are inserted between the genes or gene
fragments coding for the other proteins. Proteolytic cleavage of
the partially purified fusion protein containing the correct linker
can then be carried out on either the native fusion protein, or the
reduced or denatured fusion protein.
[0086] The peptide may or may not be properly folded when expressed
as a fusion protein. Also, the specific peptide linker containing
the cleavage site may or may not be accessible to the protease.
These factors determine whether the fusion protein must be
denatured and refolded, and if so, whether these procedures are
employed before or after cleavage.
[0087] When denaturing and refolding are needed, typically the
peptide is treated with a chaotrope, such a guanidine HCl, and is
then treated with a redox buffer, containing, for example, reduced
and oxidized dithiothreitol or glutathione at the appropriate
ratios, pH, and temperature, such that the peptide is refolded to
its native structure.
[0088] When peptides are not prepared using recombinant DNA
technology, they are preferably prepared using solid-phase
synthesis, such as that generally described by Merrifield, J. Am
Chem. Soc., 85: 2149 (1963), although other equivalent chemical
syntheses known in the art are employable. Solid-phase synthesis is
initiated from the C-terminus of the peptide by coupling a
protected .alpha.-amino acid to a suitable resin. Such a starting
material can be prepared by attaching an .alpha.-amino-protected
amino acid by an ester linkage to a chloromethylated resin or a
hydroxymethyl resin, or by an amide bond to a BHA resin or MBHA
resin. The preparation of the hydroxymethyl resin is described by
Bodansky et al., Chem. Ind. (London), 38: 1597-1598 (1966).
Chloromethylated resins are commercially available from BioRad
Laboratories, Richmond, Calif. and from Lab. Systems, Inc. The
preparation of such a resin is described by Stewart et al., "Solid
Phase Peptide Synthesis" (Freeman & Co., San Francisco 1969),
Chapter 1, pp. 1-6. BHA and MBHA resin supports are commercially
available and are generally used only when the desired polypeptide
being synthesized has an unsubstituted amide at the C-terminus.
[0089] The amino acids are coupled to the peptide chain using
techniques well known in the art for the formation of peptide
bonds. One method involves converting the amino acid to a
derivative that will render the carboxyl group more susceptible to
reaction with the free N-terminal amino group of the peptide
fragment. For example, the amino acid can be converted to a mixed
anhydride by reaction of a protected amino acid with
ethylchloroformate, phenyl chloroformate, sec-butyl chloroformate,
isobutyl chloroformate, pivaloyl chloride or like acid chlorides.
Alternatively, the amino acid can be converted to an active ester
such as a 2,4,5-trichlorophenyl ester, a pentachlorophenyl ester, a
pentafluorophenyl ester, a p-nitrophenyl ester, a
N-hydroxysuccinimide ester, or an ester formed from
1-hydroxybenzotriazole.
[0090] Another coupling method involves use of a suitable coupling
agent such as N,N'-dicyclohexylcarbodiimide or
N,N'-diisopropyl-carbodiimide. Other appropriate coupling agents,
apparent to those skilled in the art, are disclosed in E. Gross
& J. Meienhofer, The Peptides: Analysis, Structure, Biology,
Vol. I: Major Methods of Peptide Bond Formation (Academic Press,
New York, 1979).
[0091] It should be recognized that the .alpha.-amino group of each
amino acid employed in the peptide synthesis must be protected
during the coupling reaction to prevent side reactions involving
their active .alpha.-amino function. It should also be recognized
that certain amino acids contain reactive side-chain functional
groups (e.g., sulfhydryl, amino, carboxyl, and hydroxyl) and that
such functional groups must also be protected with suitable
protecting groups to prevent a chemical reaction from occurring at
that site during both the initial and subsequent coupling steps.
Suitable protecting groups, known in the art, are described in
Gross and Meienhofer, The Peptides: Analysis, Structure, Biology,
Vol.3: "Protection of Functional Groups in Peptide Synthesis"
(Academic Press, New York, 1981).
[0092] In the selection of a particular side-chain protecting group
to be used in synthesizing the peptides, the following general
rules are followed. An .alpha.-amino protecting group (a) must
render the .alpha.-amino function inert under the conditions
employed in the coupling reaction, (b) must be readily removable
after the coupling reaction under conditions that will not remove
side-chain protecting groups and will not alter the structure of
the peptide fragment, and (c) must eliminate the possibility of
racemization upon activation immediately prior to coupling. A
side-chain protecting group (a) must render the side chain
functional group inert under the conditions employed in the
coupling reaction, (b) must be stable under the conditions employed
in removing the .alpha.-amino protecting group, and (c) must be
readily removable upon completion of the desired amino acid peptide
under reaction conditions that will not alter the structure of the
peptide chain.
[0093] It will be apparent to those skilled in the art that the
protecting groups known to be useful for peptide synthesis will
vary in reactivity with the agents employed for their removal. For
example, certain protecting groups such as triphenylmethyl and
2-(p-biphenylyl)isopropylox- ycarbonyl are very labile and can be
cleaved under mild acid conditions. Other protecting groups, such
as t-butyloxycarbonyl (BOC), t-amyloxycarbonyl,
adamantyl-oxycarbonyl, and p-methoxybenzyloxycarbonyl are less
labile and require moderately strong acids, such as
trifluoroacetic, hydrochloric, or boron trifluoride in acetic acid,
for their removal. Still other protecting groups, such as
benzyloxycarbonyl (CBZ or Z), halobenzyloxycarbonyl,
p-nitrobenzyloxycarbonyl cycloalkyloxycarbonyl, and
isopropyloxycarbonyl, are even less labile and require stronger
acids, such as hydrogen fluoride, hydrogen bromide, or boron
trifluoroacetate in trifluoroacetic acid, for their removal. Among
the classes of useful amino acid protecting groups are
included:
[0094] (1) for an .alpha.-amino group, (a) aromatic urethane-type
protecting groups, such as fluorenylmethyloxycarbonyl (FMOC) CBZ,
and substituted CBZ, such as, e.g., p-chlorobenzyloxycarbonyl,
p-6-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, and
p-methoxybenzyloxycarbonyl, o-chlorobenzyloxycarbonyl,
2,4-dichlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, and
the like; (b) aliphatic urethane-type protecting groups, such as
BOC, t-amyloxycarbonyl, isopropyloxycarbonyl,
2-(p-biphenylyl)-isopropyloxycar- bonyl, allyloxycarbonyl and the
like; (c) cycloalkyl urethane-type protecting groups, such as
cyclopentyloxycarbonyl, adamantyloxycarbonyl, and
cyclohexyloxycarbonyl; and d) allyloxycarbonyl. The preferred
.alpha.-amino protecting groups are BOC or FMOC.
[0095] (2) for the side chain amino group present in Lys,
protection may be by-any of the groups mentioned above in (1) such
as BOC, p-chlorobenzyloxycarbonyl, etc.
[0096] (3) for the guanidino group of Arg, protection may be by
nitro, tosyl, CBZ, adamantyloxycarbonyl,
2,2,5,7,8-pentamethylchroman-6-sulfonyl or
2,3,6-trimethyl-4-methoxyphenylsulfonyl, or BOC.
[0097] (4) for the hydroxyl group of Ser, Thr, or Tyr, protection
may be, for example, by C1-C4 alkyl, such as t-butyl; benzyl (BZL);
substituted BZL, such as p-methoxybenzyl, p-nitrobenzyl,
p-chlorobenzyl, o-chlorobenzyl, and 2,6-dichlorobenzyl.
[0098] (5) for the carboxyl group of Asp or Glu, protection may be,
for example, by esterification using groups such as BZL, t-butyl,
cyclohexyl, cyclopentyl, and the like.
[0099] (6) for the imidazole nitrogen of His, the tosyl moiety is
suitably employed.
[0100] (7) for the phenolic hydroxyl group of Tyr, a protecting
group such as tetrahydropyranyl, tert-butyl, trityl, BZL,
chlorobenzyl, 4-bromobenzyl, or 2,6-dichlorobenzyl is suitably
employed. The preferred protecting group is 2,6-dichlorobenzyl.
[0101] (8) for the side chain amino group of Asn or Gln, xanthyl
(Xan) is preferably employed.
[0102] (9) for Met, the amino acid is preferably left
unprotected.
[0103] (10) for the thio group of Cys, p-methoxybenzyl is typically
employed.
[0104] The C-terminal amino acid, e.g., Lys, is protected at the
N-amino position by an appropriately selected protecting group, in
the case of Lys, BOC. The BOC-Lys-OH can be first coupled to the
benzyhydrylamine or chloromethylated resin according to the
procedure set forth in Horiki et al., Chemistry Letters 165-168
(1978) or using isopropylcarbodiimide at about 25.degree. C. for 2
hours with stirring. Following the coupling of the BOC-protected
amino acid to the resin support, the .alpha.-amino protecting group
is removed, as by using trifluoroacetic acid (TFA) in methylene
chloride or TFA alone. The deprotection is carried out at a
temperature between about 0.degree. C. and room temperature. Other
standard cleaving reagents, such as HCl in dioxane, and conditions
for removal of specific .alpha.-amino protecting groups are
described in the literature.
[0105] After removal of the .alpha.-amino protecting group, the
remaining .alpha.-amino and side-chain protected amino acids are
coupled stepwise within the desired order. As an alternative to
adding each amino acid separately in the synthesis, some may be
coupled to one another prior to addition to the solid-phase
synthesizer. The selection of an appropriate coupling reagent is
within the skill of the art. Particularly suitable as a coupling
reagent is N,N'-dicyclohexyl carbodiimide or
diisopropylcarbodiimide.
[0106] Each protected amino acid or amino acid sequence is
introduced into the solid-phase reactor in excess, and the coupling
is suitably carried out in a medium of dimethylformamide (DMF) or
CH.sub.2Cl.sub.2 or mixtures thereof. If incomplete coupling
occurs, the coupling procedure is repeated before removal of the
N-amino protecting group prior to the coupling of the next amino
acid. The success of the coupling reaction at each stage of the
synthesis may be monitored. A preferred method of monitoring the
synthesis is by the ninhydrin reaction, as described by Kaiser et
al., Anal. Biochem, 34: 595 (1970). The coupling reactions can be
performed automatically using well known methods, for example, a
BIOSEARCH 9500.TM. peptide synthesizer.
[0107] Upon completion of the desired peptide sequence, the
protected peptide must be cleaved from the resin support, and all
protecting groups must be removed. The cleavage reaction and
removal of the protecting groups is suitably accomplished
simultaneously or stepwise. When the resin support is a
chloro-methylated polystyrene resin, the bond anchoring the peptide
to the resin is an ester linkage formed between the free carboxyl
group of the C-terminal residue and one of the many chloromethyl
groups present on the resin matrix. It will be appreciated that the
anchoring bond can be cleaved by reagents that are known to be
capable of breaking an ester linkage and of penetrating the resin
matrix.
[0108] One especially convenient method is by treatment with liquid
anhydrous hydrogen fluoride. This reagent not only will cleave the
peptide from the resin but also will remove all protecting groups.
Hence, use of this reagent will directly afford the fully
deprotected peptide. When the chloromethylated resin is used,
hydrogen fluoride treatment results in the formation of the free
peptide acids. When the benzhydrylamine resin is used, hydrogen
fluoride treatment results directly in the free peptide amines.
Reaction with hydrogen fluoride in the presence of anisole and
dimethylsulfide at 0.degree. C. for one hour will simultaneously
remove the side-chain protecting groups and release the peptide
from the resin.
[0109] When it is desired to cleave the peptide without removing
protecting groups, the protected peptide-resin can undergo
methanolysis to yield the protected peptide in which the C-terminal
carboxyl group is methylated. The methyl ester is then hydrolyzed
under mild alkaline conditions to give the free C-terminal carboxyl
group. The protecting groups on the peptide chain then are removed
by treatment with a strong acid, such as liquid hydrogen fluoride.
A particularly useful technique for methanolysis is that of Moore
et al., Peptides, Proc. Fifth Amer. Pept. Symp., M. Goodman and J.
Meienhofer, Eds., (John Wiley, N.Y., 1977), p. 518-521, in which
the protected peptide-resin is treated with methanol and potassium
cyanide in the presence of crown ether.
[0110] Another method for cleaving the protected peptide from the
resin when the chloromethylated resin is employed is by ammonolysis
or by treatment with hydrazine. If desired, the resulting
C-terminal amide or hydrazide can be hydrolyzed to the free
C-terminal carboxyl moiety, and the protecting groups can be
removed conventionally.
[0111] It will also be recognized that the protecting group present
on the N-terminal .alpha.-amino group may be removed preferentially
either before or after the protected peptide is cleaved from the
support.
[0112] Purification of the polypeptides of the invention is
typically achieved using conventional procedures such as
preparative HPLC (including reversed phase HPLC) or other known
chromatographic techniques such as gel permeation, ion exchange,
partition chromatography, affinity chromatography (including
monoclonal antibody columns) or countercurrent distribution.
[0113] The peptides of this invention may be stabilized by
polymerization. This may be accomplished by crosslinking monomer
chains with polyfunctional crosslinking agents, either directly or
indirectly, through multi-functional polymers. Ordinarily, two
substantially identical polypeptides are crosslinked at their C- or
N-termini using a bifunctional crosslinking agent. The agent is
used to crosslink the terminal amino and/or carboxyl groups.
Generally, both terminal carboxyl groups or both terminal amino
groups are crosslinked to one another, although by selection of the
appropriate crosslinking agent the alpha amino of one polypeptide
is crosslinked to the terminal carboxyl group of the other
polypeptide. Preferably, the polypeptides are substituted at their
C-termini with cysteine. Under conditions well known in the art a
disulfide bond can be formed between the terminal cysteines,
thereby crosslinking the polypeptide chains. For example, disulfide
bridges are conveniently formed by metal-catalyzed oxidation of the
free cysteines or by nucleophilic substitution of a suitably
modified cysteine residue. Selection of the crosslinking agent will
depend upon the identities of the reactive side chains of the amino
acids present in the polypeptides. For example, disulfide
crosslinking would not be preferred if cysteine was present in the
polypeptide at additional sites other than the C-terminus. Also
within the scope hereof are peptides crosslinked with methylene
bridges.
[0114] Suitable crosslinking sites on the peptides, aside from the
N-terminal amino and C-terminal carboxyl groups, include epsilon
amino groups found on lysine residues, as well as amino, imino,
carboxyl, sulfhydryl and hydroxyl groups located on the side chains
of internal residues of the peptides or residues introduced into
flanking sequences. Crosslinking through externally added
crosslinking agents is suitably achieved, e.g., using any of a
number of reagents familiar to those skilled in the art, for
example, via carbodiimide treatment of the polypeptide. Other
examples of suitable multi-functional (ordinarily bifunctional)
crosslinking agents are found in the literature.
[0115] The peptides of this invention also may be conformationally
stabilized by cyclization. The peptides ordinarily are cyclized by
covalently bonding the - and C-terminal domains of one peptide to
the corresponding domain of another peptide of this invention so as
to form cyclo-oligomers containing two or more iterated peptide
sequences, each internal peptide having substantially the same
sequence. Further, cyclized peptides (whether cyclo-oligomers or
cyclo-monomers) are crosslinked to form 1-3 cyclic structures
having from 2 to 6 peptides comprised therein. The peptides
preferably are not covalently bonded through .alpha.-amino and main
chain carboxyl groups (head to tail), but rather are crosslinked
through the side chains of residues located in the - and C-terminal
domains. The linking sites thus generally will be between the side
chains of the residues.
[0116] Many suitable methods per se are known for preparing mono-or
poly-cyclized peptides as contemplated herein. Lys/Asp cyclization
has been accomplished using Na-Boc-amino acids on solid-phase
support with Fmoc/9-fluorenylmethyl (OFm) side-chain protection for
Lys/Asp; the process is completed by piperidine treatment followed
by cyclization.
[0117] Glu and Lys side chains also have been crosslinked in
preparing cyclic or bicyclic peptides: the peptide is synthesized
by solid phase chemistry on a p-methylbenzhydrylamine resin. The
peptide is cleaved from the resin and deprotected. The cyclic
peptide is formed using diphenylphosphorylazide in diluted
methylformamide. For an alternative procedure, see Schiller et al,
Peptide Protein Res., 25: 171-177 (1985). See also U.S. Pat. No.
4,547,489. Disulfide crosslinked or cyclized peptides are generated
by conventional methods. The method of Pelton et al. (J. Med.
Chem., 29: 2370-2375 (1986)) is suitable, except that a greater
proportion of cyclo-oligomers are produced by conducting the
reaction in more concentrated solutions than the dilute reaction
mixture described by Pelton et al., for the production of
cyclo-monomers. The same chemistry is useful for synthesis of
dimers or cyclo-oligomers or cyclo-monomers. Also useful are
thiomethylene bridges. Lebl and Hruby, Tetrahedron Letters, 25:
2067-2068 (1984). See also Cody et al., J. Med. Chem., 28: 583
(1985).
[0118] The desired cyclic or polymeric peptides are purified by gel
filtration followed by reversed-phase high pressure liquid
chromatography or other conventional procedures. The peptides are
sterile filtered and formulated into conventional pharmacologically
acceptable vehicles.
[0119] The starting materials required for the processes described
herein are known in the literature or can be prepared using known
methods and known starting materials.
[0120] If in the peptides being created carbon atoms bonded to four
nonidentical substituents are asymmetric, then the peptides may
exist as diastercoisomers, enantiomers or mixtures thereof. The
syntheses described above may employ racemates, enantiomers or
diastereomers as starting materials or intermediates.
Diastereomeric products resulting from such syntheses may be
separated by chromatographic or crystallization methods. Likewise,
enantiomeric product mixtures may be separated using the same
techniques or by other methods known in the art. Each of the
asymmetric carbon atoms, when present, may be in one of two
configurations {circle over (R)} or S) and both are within the
scope of the present invention.
[0121] The peptides of this invention are shown to bind selectively
to IGFBPs. It is known to those skilled in the art that there are
many uses for IGFs or insulin molecules. Therefore, administration
of the peptides of this invention for purposes of agonizing an IGF
or insulin action can have the same effects or uses as
administration of an exogenous IGF or insulin itself These uses of
IGF and insulin include the following, which may be additional to
or the same as the disorders as defined above: increasing whole
body, bone, and muscle growth rate in normal and hypopituitary
animals; protection of body weight and nitrogen loss during
catabolic states (such as fasting, nitrogen restriction, elevated
corticosteroid levels, and/or diabetes); kidney regeneration;
treating peripheral and central nervous system (CNS) degenerative
disorders and promoting neuroprotection or repair following CNS
damage or injury; treating hypoxia; promotion of wound healing;
cardiac regeneration; reversal of cancer cachexia; inhibition of
angiogenesis; regeneration of the gastrointestinal tract;
stimulation of mammary function; counteracting IGF-I-dependent
actions of GH such as metabolic stress, age-related decreases in GH
activity, and adult GH deficiency; treating maturity-onset
diabetes; and/or treating a specific IGF deficiency.
[0122] Additional and specific disorders for which the peptides
herein are useful include growth disorders such as GH-resistant
short stature, GH-insensitivity syndrome, osteoporosis, and
catabolic states; disorders where treatment requires regeneration
of tissues or cells, for example, peripheral nerves and supporting
cells, central nervous system cells including nerves and glia, and
other cells such as oligodendrocytes, muscle, skin, and bone; heart
disorders, e.g., heart ischemia, cardiac myopathy, and congestive
heart disorders; hyperglycemic disorders such as insulin-dependent
and non-insulin-dependent diabetes mellitus and extreme insulin
resistance; and renal disorders such as renal failure. These also
include stimulation of an anabolic response in elderly humans,
prevention of catabolic side effects of glucocorticoids, treatment
of osteoporosis, stimulation of the immune system, reduction of
obesity, acceleration of wound healing, acceleration of bond
fracture repair, treatment of growth retardation, treatment of
renal failure or insufficiency resulting in growth retardation,
treatment of physiological short stature, including
growth-hormone-deficient children, treating short stature
associated with chronic illness, treatment of obesity and growth
retardation associated with obesity, treatment of growth
retardation associated with Prader-Willi syndrome and Turner's
syndrome, acceleration of the recovery and reduction in the
hospitalization of burn patients, treatment of interuterine growth
retardation, skeletal dysplasia, hypercortisolism, and Cushings
syndrome, induction of pulsatile growth hormone release,
replacement of growth hormone in stressed patients, treatment of
osteochondrodysplasias, Noonans syndrome, schizophrenia,
depression, peripheral neuropathy, ALS, depression, Alzheimer's
disease, diseases of demyelination, multiple sclerosis, and delayed
wound healing, stimulation of the immune system, treatment of
physcosocia depravation, treatment of pulmonary dysfunction and
ventilator dependency, attenuation of protein catabolic response
after a major operation, reduction of cachexia and protein loss due
to chronic illness such as cancer or AIDS, treatment of
hyperinsulinemia including Type II and Type I diabetes, adjuvant
treatment for ovulation induction, stimulation of thymic
development and prevention of the age-related decline of thymic
function, treatment of immunosuppressed patients, treatment of bone
marrow transplanted patients, improvement in muscle strength,
mobility, diseases of muscle function, muscular dystrophy,
maintenance of skin thickness, and metabolic homeostasis,
enhancement of renal function and homeostasis including acute and
chronic renal failure, stimulation of osteoblasts, bone remodeling,
and cartilage growth, stimulation of the immune system, and growth
promotion in livestock. Various IGF-I uses are found, for example,
in WO 94/04569; WO 96/33216; and Bondy, Ann Intern. Med., 120:
593-601 (1994).
[0123] In one example, the peptides can be administered to
commercially important mammals such as swine, cattle, sheep, and
the like to accelerate and increase their rate and extent of growth
and the efficiency of their conversion of feed into body tissue.
The peptides can be administered in vivo to adults and children to
stimulate IGF or insulin action.
[0124] The peptides of this invention may be administered to the
mammal by any suitable technique, including oral, parenteral (e.g.,
intramuscular, intraperitoneal, intravenous, or subcutaneous
injection or infusion, or implant), nasal, pulmonary, vaginal,
rectal, sublingual, or topical routes of administration, and can be
formulated in dosage forms appropriate for each route of
administration. The specific route of administration will depend,
e.g., on the medical history of the patient, including any
perceived or anticipated side effects using the peptide, the type
of peptide being administered, and the particular disorder to be
corrected. Most preferably, the administration is by continuous
infusion (using, e.g., slow-release devices or minipumps such as
osmotic pumps or skin patches), or by injection (using, e.g.,
intravenous or subcutaneous means).
[0125] The peptide to be used in the therapy will be formulated and
dosed in a fashion consistent with good medical practice, taking
into account the clinical condition of the individual patient
(especially the side effects of treatment with the peptide), the
site of delivery, the method of administration, the scheduling of
administration, and other factors known to practitioners. The
"effective amounts" of the peptide for purposes herein are thus
determined by such considerations and must be amounts that result
in bioavailability of the drugs to the mammal and the desired
effect.
[0126] A preferred administration is a chronic administration of
about two times per day for 4-8 weeks to reproduce the effects of
IGF-I or insulin. Although injection is preferred, chronic infusion
may also be employed using an infusion device for continuous
subcutaneous (SC) infusions. An intravenous bag solution may also
be employed. The key factor in selecting an appropriate dose for
diabetes is the result obtained, as measured by decreases in blood
glucose so as to approximate the normal range, or by other criteria
for measuring treatment of diabetes as are deemed appropriate by
the medical practitioner.
[0127] As a general proposition, the total pharmaceutically
effective amount of the peptide administered parenterally per dose
will be in a range that can be measured by a dose-response curve.
For example, IGFs bound to IGFBPs or in the blood can be measured
in body fluids of the mammal to be treated to determine the dosing.
Alternatively, one can administer increasing amounts of the peptide
to the patient and check the serum levels of the patient for IGF-I
and IGF-II. The amount of peptide to be employed can be calculated
on a molar basis based on these serum levels of IGF-I and IGF-II.
See the Example below on displacement of IGF-I tracer from IGFBPs
present in human serum. Specifically, one method for determining
appropriate dosing of the peptide entails measuring IGF levels in a
biological fluid such as a body or blood fluid. Measuring such
levels can be done by any means, including RIA and ELISA. After
measuring IGF levels, the fluid is contacted with the peptide using
single or multiple doses. After this contacting step, the IGF
levels are re-measured in the fluid. If the fluid IGF levels have
fallen by an amount sufficient to produce the desired efficacy for
which the molecule is to be administered, then the dose of the
molecule can be adjusted to produce maximal efficacy. This method
may be carried out in vitro or in vivo. Preferably, this method is
carried out in vivo, i.e., after the fluid is extracted from a
mammal and the IGF levels measured, the peptide herein is
administered to the mammal using single or multiple doses (that is,
the contacting step is achieved by administration to a mammal) and
then the IGF levels are re-measured from fluid extracted from the
mammal.
[0128] Another method for determining dosing is to use antibodies
to the peptide or another detection method for the peptide in the
LIFA format. This would allow detection of endogenous or exogenous
IGFs bound to IGFBP and the amount of peptide bound to the
IGFBP.
[0129] Another method for determining dosing would be to measure
the level of "free" or active IGF in blood. For some uses the level
of "free" IGF would be a suitable marker of efficacy and effective
doses or dosing.
[0130] For example, one method is described for detecting
endogenous or exogenous IGF or insulin bound to an IGF binding
protein or the amount of the peptide herein or detecting the level
of unbound IGF or unbound insulin in a biological fluid. This
method comprises:
[0131] (a) contacting the fluid with 1) a means for detecting the
peptide that is specific for the peptide (such as a first antibody
specific for epitopes on the peptide) attached to a solid-phase
carrier, such that in the presence of the peptide the IGF binding
sites remain available on the peptide for binding to the IGF
binding protein, thereby forming a complex between the means and
the IGF binding protein; and 2) the peptide for a period of time
sufficient to saturate all available IGF binding sites on the IGF
binding protein, thereby forming a saturated complex;
[0132] (b) contacting the saturated complex with a detectably
labeled second means which is specific for the IGF binding protein
(such as a second antibody specific for epitopes on the IGFBP)
which are available for binding when the peptide is bound to the
IGF binding protein; and
[0133] (c) quantitatively analyzing the amount of the labeled means
bound as a measure of the IGFBP in the biological fluid, and
therefore as a measure of the amount of bound peptide and IGF
binding protein, bound IGF or bound insulin and IGF binding
protein, or active IGF or active insulin present in the fluid.
[0134] Given the above methods for determining dosages, in general,
the amount of peptide that may be employed can be estimated, i.e.,
from about 10 .mu.g/kg/day to 200 .mu.g/kg/day might be used, based
on kg of patient body weight, although, as noted above, this will
be subject to a great deal of therapeutic discretion.
[0135] A further method is provided to estimate the distribution of
IGFs on specific IGFBPs, e.g., on IGFBP-1 or IGFBP-3 using the LIFA
format.
[0136] The peptide is suitably administered by a sustained-release
system. Suitable examples of sustained-release compositions include
semi-permeable polymer matrices in the form of shaped articles, eg.
films, or microcapsules. Sustained-release matrices include
polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of
L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al.,
Biopolymers, 22, 547-556 (1983), poly(2-hydroxyethyl methacrylate)
(Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981), and
Langer, Chem. Tech., 12:98-105 (1982), ethylene vinyl acetate
(Langer et al., supra) or poly-D-(-)-3-hydroxybutyric acid (EP
133,988). Sustained-release compositions also include a liposomally
entrapped peptide. Liposomes containing the peptide are prepared by
methods known per se: DE 3,218,121; Epstein et al., Proc. Natl.
Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al., Proc. Natl.
Acad. Sci. U.S.A., 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP
88,046; EP 143,949; EP 142,641; Japanese Pat. Appln. 83-118008;
U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily,
the liposomes are of the small (from or about 200 to 800 Angstroms)
unilamellar type in which the lipid content is greater than about
30 mol. percent cholesterol, the selected proportion being adjusted
for the most efficacious therapy.
[0137] PEGylated peptides having a longer life can also be
employed, based on, e.g., the conjugate technology described in WO
95/32003 published Nov. 30, 1995.
[0138] For parenteral administration, in one embodiment, the
peptide is formulated generally by mixing each at the desired
degree of purity, in a unit dosage injectable form (solution,
suspension, or emulsion), with a pharmaceutically, or parenterally,
acceptable carrier, i.e., one that is non-toxic to recipients at
the dosages and concentrations employed and is compatible with
other ingredients of the formulation. For example, the formulation
preferably does not include oxidizing agents and other peptides
that are known to be deleterious to polypeptides.
[0139] Generally, the formulations are prepared by contacting the
peptide uniformly and intimately with liquid carriers or finely
divided solid carriers or both. Then, if necessary, the product is
shaped into the desired formulation. Preferably the carrier is a
parenteral carrier, more preferably a solution that is isotonic
with the blood of the recipient. Examples of such carrier vehicles
include water, saline, Ringer's solution, a buffered solution, and
dextrose solution. Non-aqueous vehicles such as fixed oils and
ethyl oleate are also useful herein.
[0140] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
glycine; amino acids such as glutamic acid, aspartic acid,
histidine, or arginine; monosaccharides, disaccharides, and other
carbohydrates including cellulose or its derivatives, glucose,
mannose, trehalose, or dextrins; chelating agents such as EDTA;
sugar alcohols such as mannitol or sorbitol; counter-ions such as
sodium; non-ionic surfactants such as polysorbates, poloxamers, or
polyethylene glycol (PEG); and/or neutral salts, e.g., NaCl, KCl,
MgCl.sub.2, CaCl.sub.2, etc.
[0141] The peptide typically formulated in such vehicles at a pH of
from or about 4.5 to 8. It will be understood that use of certain
of the foregoing excipients, carriers, or stabilizers will result
in the formation of salts of the peptide. The final preparation may
be a stable liquid or lyophilized solid.
[0142] Typical formulations of the peptides as pharmaceutical
compositions are discussed below. About 0.5 to 500 mg of the
peptide or mixture of peptides, as the free acid or base form or as
a pharmaceutically acceptable salt, is compounded with a
physiologically acceptable vehicle, carrier, excipient, binder,
preservative, stabilizer, flavor, etc., as called for by accepted
pharmaceutical practice. The amount of active ingredient in these
compositions is such that a suitable dosage in the range indicated
is obtained.
[0143] The peptide to be used for therapeutic administration must
be sterile. Sterility is readily accomplished by filtration through
sterile filtration membranes (e.g., 0.2 micron membranes).
Therapeutic compositions generally are placed into a container
having a sterile access port, for example, an intravenous solution
bag or vial having a stopper pierceable by a hypodermic injection
needle.
[0144] The peptide ordinarily will be stored in unit or multi-dose
containers, for example, sealed ampules or vials, as an aqueous
solution or as a lyophilized formulation for reconstitution. As an
example of a lyophilized formulation, 10-mL vials are filled with 5
mL of sterile-filtered 1% (w/v) aqueous solution of peptide, and
the resulting mixture is lyophilized. The infusion solution is
prepared by reconstituting the lyophilized peptide using
bacteriostatic Water-for-Injection.
[0145] Combination therapy with the peptide herein and one or more
other appropriate reagents that increase total IGF or insulin in
the blood or enhance the effect of the peptide is also part of this
invention. These reagents generally allow the peptide herein to
release the generated IGF or insulin, and include growth-promoting
agents.
[0146] Growth-promoting agents for this purpose include, but are
not limited to, GH secretagogues that promote the release of
endogenous GH in mammals to increase concentrations of the IGF in
the blood. Examples include TRH, diethylstilbestrol, theophylline,
enkephalins, E series prostaglandins, peptides of the
VIP-secretin-glucagon-GRF family, and other GH secretagogues such
as GHRP-6, GHRP-1 as described in U.S. Pat. No. 4,411,890, and
benzo-fused lactams such as those disclosed in U.S. Pat. No.
5,206,235. See also, e.g., WO 96/15148 published May 23, 1996.
Other growth-promoting agents include GHRPs, GHRFs, GH and their
analogs. For example, GHRPs are described in WO 95/17422 and WO
95/17423 both published Jun. 29, 1995; Bowers, J. Pediatr.
Endocrinol., 6: 21-31 (1993); and Schoen et al., Annual Reports in
Medicinal Chemistry, 28: 177-186 (1993). GHRFs and their analogs
are described, for example, in WO 96/37514 published Nov. 28,
1996.
[0147] Additionally, GHRH, any of the IGFBPs, long-acting GH, GH
plus GHBP, insulin, or a hypoglycemic agent can be employed in
conjunction with the peptide herein for this purpose. In addition,
IGF-I or IGF-II or an IGF with an IGFBP such as IGF-I complexed to
IGFBP-3 can also be employed with the peptide herein. For example,
pharmaceutical compositions containing IGF-I and IGFBP in a carrier
as described in WO 94/16723 published Aug. 4, 1994 can be used in
conjunction with the peptide. The entities can be administered
sequentially or simultaneously with the peptide. In addition, other
means of manipulating IGF status, such as regimens of diet or
exercise, are also considered to be combination treatments as part
of this invention.
[0148] If insulin is also administered, it can be any formulation
or type of insulin as noted above. The exact dose of such insulin
to be used is subject to a great deal of therapeutic discretion,
and depends upon, for example, the type of disorder, the clinical
profile of the patient, the type and amount of IGF-I variant or
IGF-like insulin employed, the type of insulin, etc., but generally
is from about 0.5 to 500 units/day of insulin. As an example, for
treatment of diabetes in humans, the dose of NPH insulin is
preferably from about 5 to 50 units/injection (i.e., from about 0.2
to 2 mg) twice a day subcutaneously.
[0149] Furthermore, the formulation is suitably administered along
with an effective amount of a hypoglycemic agent such as a
sulfonylurea. The hypoglycemic agent is administered to the mammal
by any suitable technique including parenterally, intranasally,
orally, or by any other effective route. Most preferably, the
administration is by the oral route. For example, MICRONASE.TM.
tablets (glyburide) marketed by Upjohn in 1.25, 2.5, and 5 mg
tablet concentrations are suitable for oral administration. The
usual maintenance dose for Type II diabetics, placed on this
therapy, is generally in the range of from or about 1.25 to 20 mg
per day, which may be given as a single dose or divided throughout
the day as deemed appropriate. Physician's Desk Reference,
2563-2565 (1995). Other examples of glyburide-based tablets
available for prescription include GLYNASE.TM. brand drug (Upjohn)
and DIABETA.TM. brand drug (Hoechst-Roussel). GLUCOTROL.TM. (Pratt)
is the trademark for a glipizide
(1-cyclohexyl-3-(p-(2-(5-methylpyrazine
carboxamide)ethyl)phenyl)sulfonyl- )urea) tablet available in both
5- and 10-mg strengths and is also prescribed to Type II diabetics
who require hypoglycemic therapy following dietary control or in
patients who have ceased to respond to other sulfonylureas.
Physician's Desk Reference, 1902-1903 (1995). Other hypoglycemic
agents than sulfonylureas, such as the biguanides (e.g., metformin
and phenformin) or thiazolidinediones (e.g., troglitozone), or
other drugs affecting insulin action may also be employed. If a
thiazolidinedione is employed with the peptide, it is used at the
same level as currently used or at somewhat lower levels, which can
be adjusted for effects seen with the peptide alone or together
with the dione. The typical dose of troglitazone (REZULIN.TM.)
employed by itself is about 100-1000 mg per day, more preferably
200-800 mg/day, and this range is applicable herein. See, for
example, Ghazzi et al., Diabetes,46: 433-439 (1997). Other
thiazolidinediones that are stronger insulin-sensitizing agents
than troglitazone would be employed in lower doses. In addition,
the invention contemplates using gene therapy for treating a
mammal, using nucleic acid encoding the peptide, if it is a
peptide. Generally, gene therapy is used to increase (or
overexpress) IGF or insulin levels in the mammal. Nucleic acids
which encode the peptide can be used for this purpose. Once the
amino acid sequence is known, one can generate several nucleic acid
molecules using the degeneracy of the genetic code, and select
which to use for gene therapy.
[0150] There are two major approaches to getting the nucleic acid
(optionally contained in a vector) into the patient's cells for
purposes of gene therapy: in vivo and ex vivo. For in vivo
delivery, the nucleic acid is injected directly into the patient,
usually at the site where the peptide is required. For ex vivo
treatment, the patient's cells are removed, the nucleic acid is
introduced into these isolated cells and the modified cells are
administered to the patient either directly or, for example,
encapsulated within porous membranes which are implanted into the
patient. See, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187.
[0151] There are a variety of techniques available for introducing
nucleic acids into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc. A commonly used vector for ex vivo
delivery of the gene is a retrovirus.
[0152] The currently preferred in vivo nucleic acid transfer
techniques include transfection with viral vectors (such as
adenovirus, Herpes simplex I virus, or adeno-associated virus) and
lipid-based systems (useful lipids for lipid-mediated transfer of
the gene are DOTMA, DOPE and DC-Chol, for example). In some
situations it is desirable to provide the nucleic acid source with
an agent that targets the target cells, such as an antibody
specific for a cell surface membrane protein or the target cell, a
ligand for a receptor on the target cell, etc. Where liposomes are
employed, proteins which bind to a cell surface membrane protein
associated with endocytosis may be used for targeting and/or to
facilitate uptake, e.g., capsid proteins or fragments thereof
tropic for a particular cell type, antibodies for proteins which
undergo internalization in cycling, and proteins that target
intracellular localization and enhance intracellular half-life. The
technique of receptor-mediated endocytosis is described, for
example, by Wu et al., J. Biol. Chem., 262: 4429-4432 (1987); and
Wagner et al., Proc. Natl. Acad. Sci. USA, 87: 3410-3414 (1990).
For review of the currently known gene marking and gene therapy
protocols, see Anderson et al., Science, 256: 808-813 (1992). See
also WO 93/25673 and the references cited therein.
[0153] Kits are also contemplated for this invention. A typical kit
would comprise a container, preferably a vial, for the peptide
formulation comprising peptide in a pharmaceutically acceptable
buffer and instructions, such as a product insert or label,
directing the user to utilize the pharmaceutical formulation. The
kit optionally includes a container, preferably a vial, for a GH, a
GHRP, a GHRH, a GH secretagogue, an IGF, an IGF complexed to an
IGFBP, an IGFBP, a GH complexed with a GHBP; insulin, or a
hypoglycemic agent.
[0154] In another embodiment herein, a method is provided for
directing endogenous IGF or insulin either away from, or towards, a
particular site in a mammal comprising administering to the mammal
an effective amount of the peptide herein that is specific for an
IGFBP that is either prevalent at, or absent from, the site.
"Sites" for this purpose include specific tissues or organs such as
the heart, or such as the brain via brain-specific IGFBPs.
Prevalence at the site indicates that the IGFBP in question is
located at the site and constitutes a substantial or biologically
important portion of the IGFBP at the site. This indication follows
from the specificity for IGFBP-1 versus IGFBP-3 of the peptides
demonstrated herein.
[0155] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention. All literature and patent
citations mentioned herein are expressly incorporated by
reference.
EXAMPLE 1
Alanine-Scanning Mutagenesis of IGF-I and Structural Variants
[0156] Introduction:
[0157] An alanine-scanning mutagenesis approach (Cunningham and
Wells, supra) was used to remove that portion of each side chain of
IGF-I beyond the beta carbon. The contribution of these atoms to
the free energy of binding of the peptide to IGFBP-1 or to IGFBP-3
was then assessed by competitive phage ELISA. In this assay,
IGFBP-1 or IGFBP-3 is used to inhibit IGF-phage mutants from
binding to an IGFBP-1- or IGFBP-3-coated immunosorbant plate. From
a titration series of binding protein, binding (IC.sub.50) can be
calculated. Some mutants were also assessed for direct binding in
BIAcore.TM. assays.
[0158] In the next two sets of examples, common .alpha.-amino acids
may be described by the standard one- or three-letter amino acid
code when referring to intermediates and final products. By common
.alpha.-amino acids is meant those amino acids incorporated into
proteins under mRNA direction. Standard abbreviations are listed in
The Merck Index, 10th Edition, pp Misc-2-Misc-3. Unless otherwise
designated the common .alpha.-amino acids have the natural or
"L"-configuration at the alpha carbon atom. If the code is preceded
by a "D" this signifies the opposite enantiomer of the common
.alpha.-amino acid. Modified or unusual a-amino acids such as
norleucine (Nle) and ornithine (Orn) are designated as described in
U.S. Patent and Trademark Office Official Gazette 1114 TMOG, May
15, 1990.
[0159] Based upon the results of experiments using the IGF mutant
described below, it is predicted that molecules of the type claimed
herein should increase active IGF levels in a subject being
treated.
[0160] Materials and Methods:
[0161] Construction of Phagemid Vector and Mutagenesis
[0162] The gene encoding mature human IGF-I was amplified from
pBKIGF2B (U.S. Pat. No. 5,342,763) using PCR primers 5'-AGC TGC TTT
GAT ATG CAT CTC CCG AAA CTC TGT GCG GT-3' (SEQ ID NO:4) and 5'-GAG
CGA TCT GGG TCT AGA CAG ATT TAG CGG GTT TCA G-3' (SEQ ID NO:5). The
resulting fragment was cut with NsiI and XbaI, and ligated into pH
0753 previously digested with NsiI and XbaI. pH0753 is a derivative
of phGHam-g3 (Lowman et al., Biochemistry, 30: 10832-10838 (1991))
in which the additional XbaI site in the alkaline phosphatase
promoter (PhoA) region has been deleted using the oligonucleotide
5'-AAA AGG GTA TGT AGA GGT TGA GGT-3' (SEQ ID NO:6). The ligated
vector pH0753 containing the IGF-I open reading frame was named
pIGF-g3. It encodes for IGF-I harboring the double mutation
G1S-A70V fused to a fragment of the gene III protein (residues
249-406) from the E. coli bacteriophage M13. Binding of this IGF-I
variant to IGFBP-1 and -3 was found to be indistinguishable from
wild-type IGF-I. Alanine mutagenesis was performed using
single-stranded plasmid pIGF-g3 as template (Kunkel et al., Methods
Enzymol., 204: 125-139 (1991)). All residues of IGF-I with the
exception of cysteines and alanines were singly replaced by
alanine. The resulting constructs were verified by DNA
sequencing.
[0163] Binding of IGF Mutants Displayed on Phage to IGFBP-1 and -3
(Phage ELISA)
[0164] Immunosorbent plates (Nunc, MAXISORP.TM., 96 wells) were
coated with 100 .mu.l/well of 1 .mu.g/mL IGFBP-1 or IGFBP-3 in PBS
buffer pH 7.2 at 4.degree. C. overnight. The plates were then
blocked with 0.5% TWEEN 20.TM./PBS (also used as binding buffer)
for 2 hours at room temperature (proteinaceous blocking agents like
bovine serum albumin were avoided to prevent potential IGF or IGFBP
contamination). E. coli cells (XL1-Blue, Stratagene) freshly
transformed with phagemid vector were grown overnight in 5 mL 2YT
medium (Sambrook et al., supra) in the presence of M13-VCS helper
phage (Stratagene). Phage particles were harvested and resuspended
in PBS buffer as described in Lowman, H. B., "Phage Display of
Peptide Libraries on Protein Scaffolds," in Cabilly, S. (ed.),
Combinatorial Peptide Library Protocols (Humana Press Inc.: Totowa,
N.J., 1998), pp. 249-264. Then phage concentrations were normalized
to yield a maximal ELISA signal of 0.2-0.4 for each mutant (Lowman,
in Cabilly, S. (ed.), supra). Threefold serial dilutions of soluble
competitor were prepared on non-absorbent microtiter plates (Nune,
F, 96 wells) with binding buffer (0.5% TWEEN.TM. 20/PBS) containing
phage at the previously-determined concentrations. The dilution
range of competitor protein extended over six orders of magnitude,
starting at 5 .mu.M for IGFBP-1 and 500 nM for IGFBP-3. After
blocking, the plates containing immobilized target were washed with
0.05% TWEEN.TM./PBS buffer and subsequently incubated with 80
.mu.l/well of the premixed phage-competitor solutions for 1 hour at
room temperature. After washing, bound phage was detected with 80
.mu.l/well of a solution containing a primary rabbit anti-phage
polyclonal antibody and a secondary goat anti-rabbit monoclonal
antibody-horseradish peroxidase conjugate in 0.5% TWEEN20.TM./PBS.
o-Phenylenediamine (Sigma) and tetramethylbenzidine (Kirkegaard and
Perry) were used as chromogenic substrates, resulting in product
detection at 492 and 450 nm, respectively. IC.sub.50 values were
determined by fitting the binding data to a generic saturation
curve (Lowman, in Cabilly, S. (ed.), supra). At least two
individual clones of each IGF-I mutant were assayed. Numbers in
Table I represent mean.+-.standard deviation of individually
assessed IC.sub.50 values.
[0165] Expression and Purification of IGFBP-1 and IGFBP-3
[0166] Human IGFBP-1 was expressed in CHO cells and purified from
the conditioned medium as described by Mortensen et al.,
Endocrinology 138: 2073-2080(1997). Recombinant human IGFBP-3 has
also been cloned and expressed in mammalian cells (Wood et al, Mol.
Endocrinology, 2: 1176-1185 (1988)). Purification from conditioned
medium essentially followed the procedure described for IGFBP-1,
with use of an IGF affinity column (Martin and Baxter, J. Biol.
Chem., 261: 8754-8760 (1986)).
[0167] Expression and Purification of Soluble IGF-I Mutants
[0168] Plasmid pBKIGF2B (U.S. Pat. No. 5,342,763) expresses human
wild-type IGF-I fused to the leader peptide of lamB under the
control of the P.sub.phoA promoter. For ease of site-directed
mutagenesis the phage f1 origin of replication (f1 ori) was
introduced into plasmid pBKIGF2B. For that purpose a 466-bp BamHI
fragment containing the f1 ori was excised from pH0753 (Lowman et
al., supra, 1991), while plasmid pBKIGF2B was linearized with
EcoRI. Vector and fragment were both treated with Klenow enzyme to
fill in restriction-site overhangs prior to blunt-end ligation.
Correct constructs were selected for the ability to produce
single-stranded phagemid DNA in the presence of M13VCS helper
phage. The resulting phagemid vector was named pBKIGF2B-f1-ori and
was used as template to construct the IGF-I ala-mutants of interest
(see Table II) using the procedure of Kunkel et al., Methods
Enzymol., 204:125-139(1991)). Every mutagenesis step was confirmed
by DNA sequencing.
[0169] Expression of IGF-I mutants was as described for the IGF-I
wild-type (Joly et al., Proc. Natl. Acad. Sci. USA, 95: 2773-2777
(1998)), but without transient overexpression of oxidoreductases.
The purification procedure was based on a previous protocol (Chang
and Swartz, "Single-Step Solubilization and Folding of IGF-I
Aggregates from Escherichia coli " In Cleland, J. L. (ed.), Protein
Folding In Vivo and In Vitro (American Chemical Society,
Washington, D.C., 1993), pp.178-188), with minor adaptations.
Typically, 6 g of wet cell paste (equivalent to 2 liters low
phosphate medium grown for 24 hrs) was resuspended in 150 ml of 25
mM Tris-HCl pH 7.5 containing 5 mM EDTA. Cells were lysed in a
microfluidizer (Microfluidics Corp., Newton, Mass.), and refractile
particles containing accumulated IGF-I aggregates were collected by
centrifugation at 12,000.times.g. Refractile particles were washed
twice with lysis buffer, twice with lysis buffer containing 1%
N-lauroyl-sarcosine (Sigma) to extract membrane proteins, and twice
with lysis buffer again. Washed refractile bodies were resuspended
at approximately 2 mg/ml in 50 mM CAPS
(3-(cyclohexylamino)-1-propanesulfoni- c acid; Sigma) buffer pH
10.4 containing 2 M urea, 100 nM NaCl, 20% MeOH, and 2 mM DTT. This
procedure combines solubilization of refractile bodies and
subsequent oxidative refolding of IGF-I mutants (Chang and Swartz,
supra). After 3 hrs at room temperature the refolding solutions
were filtered through microconcentrator membranes (Centricon,
Amicon) with a molecular weight cut off of 50 kDa. The majority of
monomeric IGF-I was recovered in the eluate, while higher molecular
weight contaminants were concentrated in the retentate. At this
point IGF-I fractions were >95% pure, as judged from SDS-PAGE
analysis. To separate correctly disulfide-bonded IGF-I from
IGF-swap (containing two non-native disulfides; Hober et al.,
Biochemistry, 31: 1749-1756 (1992); Miller et al., Biochemistry,
32: 5203-5213 (1993)), refolding solutions were acidified with 5%
acetic acid and loaded on a Dynamax.TM. C18 semi-preparative HPLC
column (Varian; 10.0 mm ID) at 4 ml/min. Buffers were H.sub.2O/0.1%
TFA (A) and acetonitrile/0.1% TFA (B). Separation of the disulfide
isomers was achieved by applying the following gradient: 0-30% B in
20 min, 3045% B in 60 min. The ratio of native IGF-I to IGF-swap
was usually about 2:1 for each mutant, with IGF-swap eluting
earlier in the gradient than native IGF-I. The molecular mass of
each mutant was verified by mass spectrometry. After HPLC
purification, samples were lyophilized and reconstituted at
approximately 1 mg/ml in 100 mM HEPES buffer, pH 7.4.
[0170] Biosensor Kinetic Measurements
[0171] The binding affinities of the IGF variants for IGFBP-1 and
IGFBP-3 were determined using a BIAcore.TM.-2000 real time kinetic
interaction analysis system (Biacore, Inc., Piscataway, N.J.) to
measure association (k.sub.a) and dissociation (k.sub.d) rates.
Carboxymethylated dextran biosensorchips (CM5, BIAcore Inc.) were
activated with EDC (N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride) and NHS(N-hydroxysuccinimide) according to the
supplier's instructions. For immobilization, IGF mutants in 20 mM
sodium acetate, pH 4.8, were injected onto the biosensor chip at a
concentration of 50 .mu.g/ml to yield approximately 450-600 RU's
(resonance-responce units) of covalently-coupled protein. Unreacted
groups were blocked with an injection of 1 M ethanolamine. Kinetic
measurements were carried out by injecting two-fold serial
dilutions (starting at 1 .mu.M) of either IGFBP-1 or IGFBP-3 in
running buffer (PBS, 0.05% Tween 20, 0.1% ovalbumin, 0.1% sodium
azide) at 25.degree. C. using a flow rate of 20 .mu.l/min.
Association rates (k.sub.a) and dissociation rates (k.sub.d) were
calculated separately using a 1:1 Langmuir.TM. association model in
the BIAcore.TM. evaluation software v. 3.0. The equilibrium
dissociation constant (K.sub.D) was calculated as
k.sub.d/k.sub.a.
[0172] Results
[0173] Monovalent Phage Display of IGF-I
[0174] For a rapid and comprehensive alanine scan of the 70 amino
acid residues of IGF-I it was first determined whether the protein
could be monovalently displayed on the surface of phage M13 (Bass
et al., Proteins, 8: 309-314 (1990)). Phage display technology
combines the advantage of rapid single-stranded DNA mutagenesis
with an easy purification of the resulting mutant protein, simply
by isolation of the corresponding phage particles (e.g., Cunningham
et al., 1994, supra). A vector was constructed in which mature
human IGF-I was fused to the carboxy-terminal domain of the M13
gene III product. This construct includes the stII signal sequence
which directs the fusion protein to the periplasmic space of E.
coli and allows monovalent display of the protein (Bass et al.,
supra; Lowman et al., supra, 1991). For cloning purposes the first
and the last amino acids of IGF-I were changed; the resulting
mutant G1S-A70V was used as the template construct for the
subsequent alanine scanning mutagenesis.
[0175] When phage particles displaying IGF-I G1S-A70V were isolated
and assayed in a binding competition phage ELISA for their affinity
to IGFBP's, the IC.sub.50 determined in that experiment were 8.5 nM
for IGFBP-1 and 0.5 nM for IGFBP-3 (FIG. 1). These values are in
good agreement with dissociation constants determined by
BIAcore.TM. experiments using wild-type IGF-I (Heding et al.,
supra). Wild-type IGF-I affinities determined by radioactive
immunoassays (RIA) are .about.2.8 nM for IGFBP-1 and .about.0.8 nM
for IGFBP-3, further supporting the IC.sub.50 values derived from
phage ELISA. Additionally, phage particles displaying IGF-I
G1S-A70V were efficiently captured by 11 independent monoclonal
mouse anti-IGF-I antibodies immobilized on microtiter plates. These
results together suggested that the displayed IGF-variant is folded
correctly and accessible on the surface of the phage particles.
[0176] Ala-Scanning Mutagenesis of IGF-I Binding to IGFBP-1 and
IGFBP-3
[0177] All residues of G1S-A70V IGF-I with the exception of the
four native alanines and six cysteines were singly substituted by
alanine, using the described G1S-A70V IGF-I gIII vector as a
template. Additionally, the single mutants S1G and V70A and the
double-mutation restoring wild-type IGF-I were constructed. Each of
these constructs was expressed in E. coli and displayed on phage.
IC.sub.50 values for binding to IGFBP-1 and IGFBP-3 were determined
by competitive phage ELISA as shown in FIG. 1. At least two
different clones of every mutant were tested. The resulting
IC.sub.50 values are listed in Table 1, and the loss or gain in
IC.sub.50 for each mutant with respect to G1S-A70V is graphed in
FIG. 2.
1TABLE I Apparent Affinities (IC.sub.50) of IGF-I Variants for
IGFBP-1 and IGFBP-3 Determined by Phage Display.sup.a IGFBP-1
IGFBP-3 relative relative relative IGF-I mutant IC.sub.50 (nM)
IC.sub.50 IC.sub.50 (nM) IC.sub.50 specificity S1A 5.2 .+-. 0.9 0.6
0.91 .+-. 0.32 1.2 0.5 P2A 11.0 .+-. 3.7 1.3 0.81 .+-. 0.18 1.1 1.2
E3A 278 .+-. 86 33.9 1.05 .+-. 0.08 1.4 24.2 T4A 19.4 .+-. 6.4 2.4
0.80 .+-. 0.02 1.1 2.2 L5A 55.3 .+-. 11.6 6.7 1.53 .+-. 0.22 2.0
3.3 G7A >1000 >100 4.58 .+-. 0.28 6.1 >16 E9A 8.6 .+-. 0.6
1.0 1.32 .+-. 0.30 1.8 0.6 L10A 311 .+-. 87 37.9 3.55 .+-. 0.33 4.7
8.1 V11A* n.d. -- n.d. -- -- D12A 4.3 .+-. 0.8 0.5 1.49 .+-. 0.38
2.0 0.3 L14A 36.7 .+-. 1.1 4.5 0.90 .+-. 0.04 1.2 3.7 Q15A 13.9
.+-. 0.9 1.7 1.26 .+-. 0.41 1.7 1.0 F16A 57.8 .+-. 20.1 7.0 1.32
.+-. 0.25 1.8 4.0 V17A 42.9 .+-. 3.2 5.2 3.67 .+-. 1.02 4.9 1.1
G19A 11.0 .+-. 2.3 1.3 0.90 .+-. 0.28 1.2 1.1 D20A 8.4 .+-. 4.1 1.0
1.11 .+-. 0.06 1.5 0.7 R21A 7.1 .+-. 1.6 0.9 0.58 .+-. 0.01 0.8 1.1
G22A 15.9 .+-. 2.8 1.9 2.07 .+-. 0.11 2.8 0.7 F23A 10.9 .+-. 1.9
1.3 2.18 .+-. 0.01 2.9 0.5 Y24A 13.3 .+-. 2.9 1.6 2.53 .+-. 0.76
3.4 0.5 F25A 181 .+-. 46 22.1 3.69 .+-. 0.25 4.9 4.5 N26A 9.1 .+-.
1.8 1.1 0.90 .+-. 0.07 1.2 0.9 K27A 12.8 .+-. 0.1 1.6 0.66 .+-.
0.35 0.9 1.8 P28A 9.3 .+-. 1.4 1.1 1.41 .+-. 0.05 1.9 0.6 T29A 7.3
.+-. 2.4 0.9 1.23 .+-. 0.16 1.6 0.5 G30A 7.1 .+-. 1.7 0.9 0.58 .+-.
0.11 0.8 1.1 Y31A 6.8 .+-. 0.5 0.8 0.73 .+-. 0.10 1.0 0.9 G32A 10.9
.+-. 1.3 1.3 0.76 .+-. 0.28 1.0 1.3 S33A 9.1 .+-. 1.0 1.1 1.01 .+-.
0.24 1.3 0.8 S34A 9.5 .+-. 0.7 1.2 1.65 .+-. 0.21 2.2 0.5 S35A 11.7
.+-. 0.6 1.4 0.47 .+-. 0.01 0.6 2.3 R36A* n.d. -- n.d. -- -- R37A
12.3 .+-. 0.1 1.5 0.75 .+-. 0.08 1.00 1.5 P39A* n.d. -- n.d. -- --
Q40A 10.2 .+-. 0.9 1.2 0.56 .+-. 0.03 0.7 1.7 T41A 13.7 .+-. 3.1
1.7 0.43 .+-. 0.06 0.6 2.9 G42A 15.7 .+-. 3.4 1.9 0.53 .+-. 0.20
0.7 2.7 I43A 31.3 .+-. 4.1 3.8 1.17 .+-. 0.07 1.6 2.4 V44A 18.8
.+-. 5.4 2.3 1.03 .+-. 0.06 1.4 1.7 D45A 4.7 .+-. 0.7 0.6 0.69 .+-.
0.21 0.9 0.6 E46A 7.9 .+-. 2.1 1.0 0.94 .+-. 0.28 1.3 0.8 F49A
>1000 >100 2.72 .+-. 1.11 3.6 >28 R50A 16.2 .+-. 1.8 2.0
0.64 .+-. 0.18 0.9 2.3 S51A 13.4 .+-. 0.4 1.6 0.65 .+-. 0.35 0.9
1.9 D53A 15.3 .+-. 2.8 1.9 1.05 .+-. 0.11 1.2 1.6 L54A 23.1 .+-.
12.0 2.8 1.83 .+-. 0.91 2.4 1.2 R55A 9.0 .+-. 2.3 1.1 0.66 .+-.
0.03 0.9 1.2 R56A 13.1 .+-. 1.8 1.6 1.00 .+-. 0.19 1.3 1.2 L57A
21.8 .+-. 5.6 2.7 1.78 .+-. 0.56 2.4 1.1 E58A 11.9 .+-. 1.8 1.5
1.03 .+-. 0.47 1.4 1.1 M59A 13.1 .+-. 1.8 1.6 0.74 .+-. 0.14 1.0
1.6 Y60A 6.6 .+-. 1.8 0.8 0.52 .+-. 0.01 0.7 1.2 P63A >1000
>100 >100 >100 -- L64A 12.1 .+-. 3.3 1.5 0.93 .+-. 0.03
1.2 1.2 K65A 12.4 .+-. 0.6 1.5 0.69 .+-. 0.05 0.9 1.6 P66A 9.4 .+-.
3.2 1.1 0.57 .+-. 0.12 0.8 1.5 K68A 10.5 .+-. 2.8 1.3 0.76 .+-.
0.23 1.0 1.3 S69A 12.8 .+-. 2.3 1.6 0.71 .+-. 0.62 1.2 1.3 V70A
19.1 .+-. 0.7 2.3 0.68 .+-. 0.15 0.9 2.6 S1G 11.2 .+-. 1.1 1.4 0.99
.+-. 0.42 1.3 1.0 IGF-I WT 8.4 .+-. 0.8 1.0 1.01 .+-. 0.42 1.3 0.8
G1S-A70V 8.2 .+-. 1.6 1.0 0.75 .+-. 0.32 1.0 1.0 Ala(1-3)-IGF 90.4
.+-. 9.6 11.0 1.12 .+-. 0.04 1.5 7.3 Des(1-2)- 5.0 .+-. 0.1 0.6
0.53 .+-. 0.03 0.7 0.9 IGF .sup.aThe variants noted with an
asterisk were not successfully displayed on phage (n.d.), as judged
by antibody experiments described in the text. Relative IC.sub.50
is defined as IC.sub.50 mut/IC.sub.50 G1S-A70V. Relative
specificity is defined as relative IC.sub.50 IGFBP-1/relative
IC.sub.50 IGFBP-3 for each variant.
[0178] The majority of the alanine mutants yielded only minor
changes in IC.sub.50 values in the phage ELISA. Importantly,
wild-type IGF-I showed the same affinities for IGFBP-1 and IGFBP-3
as G1S-A70V in which background the alanine substitutions were
performed (Table I, FIG. 2). Only a few residues caused
considerable (>10-fold) losses in affinity when changed to
alanine: E3, G7, L10, V11, F25, R36, P39, F49, and P63 for IGFBP-1
binding; V11, R36, P39, and P63 for IGFBP-3 binding. It has been
noted that ala-substitutions of glycines and prolines can lead to
structural perturbations of the protein backbone (Di Cera, Chem.
Rev., 98: 1563-1591 (1998)).
[0179] Only a few modest improvements in binding affinity were
found by alanine replacements. S1A, D12A, and D45A showed an
approximately 2-fold increase in IGFBP-I binding, while S35A and
T41A showed a similar effect for IGFBP-3. However, 2-fold changes
in IC.sub.50 values are at the limit of precision in these
experiments.
[0180] IGFBP-Specificity Determinants
[0181] E3A, G7A, L10A, F25A, and F49 showed a differential effect
in binding IGFBP-1 versus IGFBP-3. For these five IGF-I single
alanine mutants the relative IC.sub.50 for IGFBP-I differed by more
than 4-fold from the one for IGFBP-3 (FIG. 2; Table 1, relative
specificity). E3A and F49A showed the biggest relative specificity
factors in this group. Alanine substitution of E3 had virtually no
effect on IGFBP-3 affinity (1.4 fold) while binding to IGFBP-1 is
weakened 34-fold. Even more dramatic, the affinity of F49A is down
more than 100-fold for IGFBP-1 but only 3.6-fold for BP-3. This
result was illustrated in a direct comparison by phage ELISA. Phage
particles displaying IGF-I F49A were added to IGFBP-3 coated wells
in the presence of soluble IGFBP-1 (FIG. 3A) or IGFBP-3 (FIG. 3B).
Compared to control phage displaying IGF-I G1S-A70V, the binding
curve of F49A shifted by more than two orders of magnitude in the
IGFBP-1 competition (FIG. 3A). In contrast, the binding curves were
similar in the IGFBP-3 competition, and the IC.sub.50 values
differed by less than a factor of 4 (FIG. 3B). Thus, E3 and F49 are
two major specificity determinants for IGFBP-1 binding in the IGF-I
molecule.
[0182] Residues G7, L10, and F25 appeared to be important for
binding of both IGFBP's, although showing a more pronounced loss of
affinity for IGFBP-1 than for IGFBP-3 when substituted by alanines.
No significant specificity determinant for IGFBP-3 was identified,
such as a mutant binding much tighter to IGFBP-1 than to IGFBP-3.
However, mutations E9A, D12A, F23A, Y24A, T29A, S34A, and D45A had
slightly larger (about 2-fold) effects on IGFBP-3 than on IGFBP-1
binding.
[0183] BIAcore.TM. Measurements of Purified Soluble IGF Mutants
[0184] For validation of the results obtained by phage ELISA,
specific alanine mutants were expressed and purified for kinetic
analysis using a BIAcore.TM. instrument. The dissociation constant
(K.sub.D) of wild-type IGF-I was determined to be 13 nM for IGFBP-1
and 1.5 nM for IGFBP-3 (FIGS. 5A and 5B; Table II). The difference
in affinity for the IGFBP's is due to a 10-fold faster association
rate (k.sub.a) of IGF-I to IGFBP-3 (3.2.times.10.sup.5 versus
3.2.times.10.sup.4 M.sup.-1s.sup.-1). These results correspond well
with the absolute IC.sub.50 values determined by phage ELISA (FIGS.
1A and 1B; Table I). As expected, the double-mutant G1S-A70V showed
kinetic parameters essentially indistinguishable from wild-type
(Table II).
[0185] V11A, R36A, and P39A were tested because these variants had
not been displayed correctly on phage, based upon the antibody
recognition experiments (see above). R36A and P39A showed wild-type
kinetics for both binding proteins, whereas V11A showed a 5-fold
reduction in affinity for both IGFBP-1 and IGFBP-3.
[0186] Furthermore, it was decided to examine the soluble IGF
variant T4A. This residue had been implicated in IGFBP binding in
earlier publications (Bayne et al., supra, J. Biol. Chem., 263;
Clemmons et al., supra, 1990), but had shown modest effects in the
phage assays herein. The increase in the K.sub.D values of T4A
relative to wild-type IGF-I was approximately 2-3-fold higher than
the IC.sub.50 ratios determined by phage ELISA (Table II). A bigger
discrepancy between the results obtained by phage and the biosensor
analysis was seen for F16A. In this case the two methods differed
by a factor of 4.
[0187] It has been shown that mutations in the first
.alpha.-helical region have a destabilizing effect on the
IGF-protein structure (Jansson et al., supra, 1997). Without being
limited to any one theory, it is believed that the g3 fusion
protein on the surface of the phage might be more stable than the
refolded, purified soluble protein. This is supported by the
BIAcore.TM. results obtained for F25A and F49A, two residues
located outside the structurally sensitive N-terminal helix. The
respective changes in K.sub.D and IC.sub.50 values are in excellent
agreement for these two mutants (Table II). The differential effect
of F49A on binding to the IGFBP's was confirmed by the BIAcore.TM.
analysis. A 70-fold decrease in affinity was measured for IGFBP-1
binding (FIG. 5C; Table II), whereas IGFBP-3 binding was reduced
only 4-fold (FIG. 5D; Table II).
2TABLE II Kinetic Parameters for the Interaction of Purified IGF-I
Variants with IGFBP-1 and -3 Determined by BIAcore .TM.
Analysis.sup.a Binding to IGFBP-1 k.sub.a k.sub.d K.sub.D relative
relative (.times.10.sup.4 M.sup.-1s.sup.-1) (.times.10.sup.4
s.sup.-1) (nM) K.sub.D IC.sub.50 IGF-I 3.2 .+-. 0.2 4.1 .+-. 0.2
13.0 .+-. 1.0 1.0 1.0 WT G1S- 3.2 .+-. 0.2 4.5 .+-. 0.01 14.0 .+-.
0.7 1.1 1.0 A70V T4A 1.9 .+-. 0.2 16.7 .+-. 1.6 90.0 .+-. 11.0 6.9
2.4 V11A 1.9 .+-. 0.1 12.3 .+-. 0.6 66.5 .+-. 4.5 5.1 -- F16A 1.9
.+-. 0.6 60.3 .+-. 4.5 321 .+-. 98 25 6.0 F25A 1.5 .+-. 0.5 49.0
.+-. 5.7 323 .+-. 107 25 22 R36A 4.0 .+-. 0.2 5.6 .+-. 0.2 13.9
.+-. 0.8 1.1 -- P39A 3.1 .+-. 0.2 4.2 .+-. 0.1 13.6 .+-. 0.8 1.0 --
F49A 1.26 .+-. 0.8 115 .+-. 1.5 913 .+-. 551 70 >100 Binding to
IGFBP-3 k.sub.a k.sub.d K.sub.D relative relative (.times.10.sup.5
M.sup.-1s.sup.-1) (.times.10.sup.4 s.sup.-1) (nM) K.sub.D IC.sub.50
IGF-I 3.2 .+-. 0.5 4.7 .+-. 0.8 1.5 .+-. 0.3 1.0 1.4 WT G1S- 2.9
.+-. 0.8 6.3 .+-. 0.5 2.2 .+-. 0.6 1.5 1.0 A70V T4A 1.8 .+-. 0.6
5.5 .+-. 0.1 3.1 .+-. 1.0 2.1 1.1 V11A 3.1 .+-. 0.5 20.9 .+-. 2.8
6.7 .+-. 1.3 4.5 -- F16A 1.1 .+-. 0.4 11.4 .+-. 2.7 10.3 .+-. 4.7
6.9 1.8 F25A 1.5 .+-. 0.5 11.8 .+-. 0.1 7.7 .+-. 0.3 5.1 4.9 R36A
4.0 .+-. 0.1 4.7 .+-. 0.2 1.2 .+-. 0.1 0.8 -- P39A 2.7 .+-. 0.2 6.0
.+-. 0.3 2.2 .+-. 0.2 1.5 -- F49A 2.7 .+-. 0.7 17.1 .+-. 0.9 6.3
.+-. 1.7 4.2 3.6 .sup.aThe relative changes in dissociation
constants (K.sub.D mut/K.sub.D wt) are compared to the relative
IC.sub.50 values (IC.sub.50 mut/IC.sub.50 G1S-A70V) determined by
phage display (Table I).
[0188] Role of the N-Terminal IGF-I Residues
[0189] Surprisingly, the IGFBP-3 interaction was generally much
less affected by the alanine substitutions than was the interaction
with IGFBP-1, despite the fact that IGFBP-3 binds IGF-I with
approximately 10-fold higher affinity. Apart from P63 A, no alanine
mutant exhibited a >6-fold reduction in IGFBP-3 affinity (FIG. 2
and Table I).
[0190] It had previously been shown in biosensor experiments that
des(1-3)-IGF-I binds IGFBP-3 with 25-fold reduced affinity (Heding
et al., supra). This naturally-occurring form of IGF-I lacks the
first three N-terminal residues and shows increased mitogenic
potency, presumably due to its reduction in IGFBP-binding (Bagley
et al., supra). Since none of the first three amino acid side
chains seem to contribute any energy to the binding of IGFBP-3
(Table I) but nevertheless des(1-3)-IGF-I is compromised in IGFBP-3
binding, without being limited to any one theory, it is
hypothesized that backbone interactions might be involved.
[0191] This hypothesis was tested by displaying on phage a triple
alanine mutant (Ala(1-3)-IGF-I), substituting the first three
N-terminal amino acids. If the backbone in that region contributes
to the interaction with IGFBP-3 this mutant should be able to bind.
Binding to IGFBP-1, however, should be reduced due to the lack of
the E3 side chain (Table I). As a control the des(1-2)-IGF-I mutant
was generated, testing for any potential backbone interactions with
IGFBP-I at positions 1 and 2. As expected, Ala(1-3)-IGF-I showed a
decreased IGFBP-I affinity similar to E3A but no change in IGFBP-3
affinity (Table I; FIG. 2). For des(1-2)-IGF-I, no difference in
affinity was observed for both binding proteins. Combined with the
observations on des(1-3)-IGF-I (Heding et al., supra), these
results suggest, without limitation to any one theory, that the
peptide backbone between residue 3 and 4 of IGF-I mediates
important interactions with IGFBP-3.
[0192] Discussion
[0193] The functional IGFBP-1 and IGFBP-3 binding epitopes on the
surface of IGF-I have been probed by alanine-scanning mutagenesis.
Both binding epitopes are illustrated in FIG. 6. Individual IGF-I
side-chain interactions play a much more important role for binding
to IGFBP-1 than to IGFBP-3. Two major binding patches are found for
IGFBP-1 (FIG. 6A). One is situated on the upper face of the
N-terminal helix (composed of G7, L10, V11, L14, F25, 143, and V44)
and one the lower face (composed of E3, T4, LS, F16, V17, and L54).
These two binding patches are bridged by F49 and R50. For IGFBP-3,
the binding epitope is more diffuse and has shifted to include G22,
F23, and Y24 (FIG. 6B). Binding of IGFBP-3 is generally much less
sensitive to alanine substitutions. In fact, the biggest reduction
in affinity (apart from P63A, see below) is a 6-fold decrease seen
for G7A. This result is intriguing since IGFBP-3 binds with 10-fold
higher affinity to IGF-I than does IGFBP-1 Most probably, without
limitation to any one theory, interactions originating from the
IGF-I main chain backbone are contributing to the binding of
IGFBP-3. This hypothesis is further substantiated by the
experiments with the Ala(1-3)-IGF mutant. While the single and
triple alanine substitutions have no effect on IGFBP-3 binding,
deletion of the first 3 amino acids resulted in a 25-fold decrease
in affinity (Bagley et al., supra; Clemmons et al., supra, 1992;
Heding et al., supra). In summary, IGF-I uses different binding
modes to associate with IGFBP-1 and IGFBP-3: a few amino acid
side-chain interactions are important for binding to IGFBP-1, while
backbone interactions seem to play a major energetic role for
binding to IGFBP-3.
[0194] A recent publication has investigated the binding epitope on
IGF-I for IGFBP-1 by heteronuclear NMR spectroscopy (Jansson et
al., supra, 1998). The authors found that the IGF-I residues 29,
30, 36, 37, 40, 41, 63, 65, and 66 amongst others experienced
chemical shift perturbations upon complexation with IGFBP-1 at
30.degree. C. Furthermore, Jansson and co-workers identified R36,
R37, and R50 to be part of the functional binding epitope and
tested those alanine mutants in BIAcore.TM. experiments. The
largest change in affinity observed by these authors was a 3-fold
decrease for R50A. However, due to the structural flexibility of
IGF-I already observed in the first NMR study of the hormone (Cooke
et al., supra), Jansson et al. were unable to completely assign
many residues in the NMR spectrum, including F49.
[0195] In similar studies of protein-protein interfaces it was
found that only a few side-chain residues contribute to the bulk of
free-binding energy (Clackson and Wells, Science, 267: 383-386
(1995); Kelley et al., Biochemistry, 34: 10383-10392 (1995)). The
same holds true for the IGF-IGFBP-1 interaction. However, here, as
it was noticed for tissue factor binding to factor VIIa, the
magnitude of the free energy of binding (.DELTA..DELTA.G) values
derived from important side chains is smaller than in the case of
growth hormone (Kelley et al., supra). The residues with
predominant .DELTA..DELTA.G contributions were not clustered on the
IGF-I surface like in the growth hormone-receptor interface
(Clackson and Wells, supra), but still formed a continuous IGFBP-1
binding epitope (FIG. 6A). In contrast, the IGFBP-3 binding epitope
on IGF-I was discontinuous, and side chains contributed very modest
individual binding energies.
[0196] Substitution of P63 by alanine in IGF-I results in a
decreased affinity for both binding proteins that cannot be
measured in the concentration range used in the competition phage
ELISA's. However, residue P63 is located on the opposite side of
the IGF-I molecule with respect to the main binding epitope.
Furthermore, it has been noticed that alanine substitutions of
glycines and prolines can lead to structural changes (Di Cera,
supra). In addition, Jansson et al., 1998, supra, concluded that
the C-terminal part of IGF-I is not involved in direct IGFBP-1
contacts, but rather undergoes indirect conformational changes upon
complex formation. An extensive characterization of antibody
binding sites on IGF-I has been carried out by Mnes et al,
Endocrinology, 138: 905-915 (1997). They showed simultaneous
binding of IGFBP-1 or -3 to IGF-I in complex with antibodies
recognizing the C-terminal D-domain. These results further support
earlier observations that the D-domain, beginning with residue P63,
is not involved in binding of IGFBP-1 or -3 (Bayne et al., supra,
1988).
[0197] The major discrepancy between an IC.sub.50 ratio obtained by
phage ELISA and a BIAcore.TM. result was observed with residue F16.
As already mentioned substitution of this residue by alanine
induced structural changes in the IGF-I molecule (Jansson et al.,
supra, 1997). The same effect was seen with the K.sub.D in the
BIAcore.TM. results, but the affinity decrease was less pronounced
in the phage ELISA experiments (see Table II). Both BIAcore.TM.
measurements used IGF-F16A that had been refolded during the
purification procedure (Jansson et al., supra, 1997). In phage
display, however, the protein of interest is translocated naturally
by the secretion machinery of E. coli. The low protein abundance in
monovalent phage display (<1 molecule per phage particle) may
disfavor aggregation and misfolding. Additionally, fusing IGF-I to
the truncated g3 phage protein might exert a stabilizing effect on
the native structure of the peptide.
[0198] The majority of IGF-I in the circulation is found in complex
with IGFBP-3 and a third protein termed acid-labile subunit (ALS)
(Bach and Rechler, supra; Clemmons, Cytokine Growth Factor Rev.,
8:45-62 (1997); Jones and Clemmons, supra). This ternary complex of
150-kD molecular weight is unable to traverse the vasculature walls
and acts as a circulating reservoir for IGF's. By this mechanism
the half-life of IGF-I is dramatically increased (Simpson et al,
Growth Horm IGF Res, 8: 83-95 (1998)). The levels of IGFBP-3 are
positively regulated by IGF-I. The role of IGFBP-1, in contrast, is
less clear. This class of binding proteins is generally less
abundant than IGFBP-3, and its levels are negatively regulated by
insulin (Bach and Rechler, supra; Clemmons, supra, 1997; Jones and
Clemmons, supra).
[0199] Based on the results herein, IGFBP-specific variants of
IGF-I are obtained: Combination of several alanine mutations
generates a variant that binds IGFBP-1 very weakly while retaining
high-affinity binding of IGFBP-3. The design of IGFBP-1 specific
variants that no longer bind to IGFBP-3, can involve phage display
of IGF-I and the randomization of amino acids at specific positions
(Cunningham et al., 1994, supra; Lowman and Wells, J. Mol. Biol.,
243: 564-578 (1993)).
CONCLUSION
[0200] Residues in IGF-I important for binding to IGFBP-1 and
IGFBP-3 have been identified. Several residues were found that
determine the binding specificity for a particular IGFBP. Recent
publications (Loddick et al., supra; Lowman et al., supra 1998))
have reported animal studies where increased pools of bioavailable
"free" IGF-I were generated by displacing endogenous IGF-I from
binding proteins. IGFBP-specific IGF-I variants may be used
diagnostically and therapeutically as described above.
EXAMPLE 2
IGF-Like Insulins
[0201] It has been reported that insulin has a weak affinity of
251+/-91 nM for IGFBP-3, as measured by BIAcore.TM. experiments
(Heding et al., supra). Thus, compared to the high-affinity complex
with IGF-I (0.23 nM), insulin binds 1000-fold weaker. Hence,
insulin likely presents the correct structural scaffold needed to
bind IGFBP's, and if some correct residues are introduced, binding
will improve.
[0202] Cascieri et al., Endocrinology, supra, report an
approximately 1000-fold reduction in affinity to binding protein
with substitution of the N-terminal region of insulin onto IGF-I,
in contrast to the alanine scanning data herein (the wild-type
affinity of Ala(1-3)IGF-I for IGFBP-3 (Table I)), which suggests
that other substitutions near the N-terminus of IGF-I should allow
IGFBP-3 binding. This is likely due to an additional residue,
Phe.sup.-1, present at the N-terminus of the IGF/insulin hybrid,
(Phe.sup.-1, Val.sup.1, Asn.sup.2, Gln.sup.3, His.sup.9, Ser.sup.8,
His.sup.9, Glu.sup.12, Tyr.sup.15, Leu.sup.16)IGF-I (numbering is
that of Cascieri et al., Endocrinology, supra, for IGF-I). Deletion
of Phe.sup.1 in proinsulin or insulin is expected to improve
binding to IGFBP-3. Based on alanine-scanning results, additional
improvement in binding to IGFBP-3 is obtained by making mutations
(proinsulin numbering) F25Y, Y26F, and T73F, because substitutions
of these side chains in IGF-I affect IGFBP-3 binding (Table I) and
proinsulin (as well as insulin) differs from IGF-I at these sites
(FIG. 4). Binding of insulin or proinsulin to IGFBP-1 is expected
to be improved by mutations Q4E, L17F, Y26F, and T49F because
substitutions of these side chains in IGF-I affect IGFBP-1 binding
(Table I) and proinsulin (as well as insulin) differs from IGF-I at
these sites (FIG. 4).
[0203] Slieker et al., supra, proposed that long-acting analogs of
insulin could be produced by engineering insulin to bind to
endogenous factors. Such complexes, by analogy with IGF-I:IGFBP
complexes (see, e.g., Cascieri et al., Endocrinology, supra) might
be cleared more slowly from the circulation than the free hormone.
However, the insulin variants that they reported had only poor
binding affinity for IGFBP, and reduced affinity for insulin
receptor (Slieker et al., supra). By defining binding determinants
for IGFBP-1 and IGFBP-3 at higher resolution than earlier studies,
different proinsulin and insulin variants are engineered that
retain receptor binding, but achieve significant affinity for
IGFBPs.
[0204] Human pro-insulin has also been displayed on phage.
Therefore, binding affinities of single-site and multiple-site
mutants can be readily measured by the techniques described
above.
[0205] Conversion of pro-insulin to insulin occurs by excision of
the region from R31 to R65 (including the mentioned residues). The
resulting amino-terminal peptide of mature insulin is called
B-chain, and the carboxy-terminal peptide A-chain. The chains are
held together by two inter-chain disulfides. The above numbering
system refers to native-sequence human pro-insulin, the sequence of
which is shown in FIG. 4 compared to the native sequence of human
IGF-I. If pro-insulin mutants displayed on phage successfully bind
to the IGFBP's these mutations are introduced in soluble, mature
insulin.
EXAMPLE 3
Treatment of Humans with Human IGF-I
[0206] This example shows the principle of how an exogenously
administered peptide that binds to one or more of the IGFBPs acts
to displace endogenous IGFs and how to dose a peptide herein for
use in humans.
[0207] In this study human Type II diabetics were administered
recombinant human IGF-I or placebo by twice daily injection at four
doses (10, 20, 40 or 80 .mu.g/kg) for 12 weeks. Blood samples were
drawn, before, every two weeks during, and after (EP) the 12 weeks
of treatment. The concentrations of IGF-I, IGF-II, and IGFBP-3 were
measured in all the samples, with the exception of IGF-II not being
measured in the samples taken from the patients treated with 10
.mu.g/day of IGF-I.
[0208] FIG. 43 of WO 98/45427 shows the concentrations of IGF-I in
the blood of the patients. The unexpected finding was the "plateau"
effect of administering 40 and 80 .mu.g of IGF-I; the same total
blood concentration of IGF-I was reached with these two doses.
[0209] FIG. 44 of WO 98/45427 shows the concentrations of IGF-II in
the blood of the patients. In contrast to the rising levels of
IGF-I, the levels of IGF-II fell in almost a mirror image pattern
to the rise in IGF-I concentrations. As with the plateauing of the
rising IGF-I concentrations, the falling IGF-II concentrations also
reached a plateau.
[0210] FIG. 45 of WO 98/45427 shows the concentrations of IGFBP-3
in the blood of the patients. In contrast to the clear changes in
the patterns of IGF-I and IGF-II in the blood, the concentrations
of IGFBP-3 showed no statistically significant or clear pattern of
change.
[0211] Inspection of FIGS. 43 and 44 of WO 98/45427 reveals that
the total IGF concentrations (IGF-I plus IGF-II) showed little
change with treatment. This was because the rise in the
concentrations of IGF-I closely matched the fall in the
concentrations of IGF-II. Inspection of all three Figures shows
that the dose-related changes in the concentrations of IGF-I and
IGF-II in the blood of the patients were not accompanied by a
reduced IGFBP-3 binding protein capacity (IGFBP-3 is the major
binding protein in blood).
[0212] The obvious explanation for the fall in the concentration of
IGF-II, and the plateauing of IGF-I and IGF-II concentrations, is
that there is a finite amount of IGF binding protein capacity and
in this experiment the doses of IGF-I used caused a dose-related
displacement of IGF-II from the binding proteins.
[0213] It is a logical extension of the observations in this
Example to expect that any molecule with the ability to enhance
levels of active IGF would show activities similar to those shown
for IGF-I in this Example. In addition, from the doses of IGF-I
used and the concentrations of IGFBP and IGF-I and IGF-II
demonstrated, it is simple to calculate how much of a peptide
should be given to increase levels of active endogenous IGF. The
molar size relative to IGF-I, the affinity of the peptide for the
IGFBP, and its bioavailability would be other variables taken into
account to arrive at doses that increased active IGF in a
human.
[0214] The present invention has of necessity been discussed herein
by reference to certain specific methods and materials. It is to be
understood that the discussion of these specific methods and
materials in no way constitutes any limitation on the scope of the
present invention, which extends to any and all alternative
materials and methods suitable for accomplishing the objectives of
the present invention
Sequence CWU 1
1
6 1 70 PRT Homo sapiens 1 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu
Val Asp Ala Leu Gln 1 5 10 15 Phe Val Cys Gly Asp Arg Gly Phe Tyr
Phe Asn Lys Pro Thr Gly 20 25 30 Tyr Gly Ser Ser Ser Arg Arg Ala
Pro Gln Thr Gly Ile Val Asp 35 40 45 Glu Cys Cys Phe Arg Ser Cys
Asp Leu Arg Arg Leu Glu Met Tyr 50 55 60 Cys Ala Pro Leu Lys Pro
Ala Lys Ser Ala 65 70 2 86 PRT Homo sapiens 2 Phe Val Asn Gln His
Leu Cys Gly Ser His Leu Val Glu Ala Leu 1 5 10 15 Tyr Leu Val Cys
Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 20 25 30 Arg Arg Glu
Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly 35 40 45 Gly Gly
Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly 50 55 60 Ser
Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile 65 70 75
Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn 80 85 86 3 51 PRT Homo
sapiens 3 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala
Leu 1 5 10 15 Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro
Lys Thr 20 25 30 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser
Leu Tyr Gln 35 40 45 Leu Glu Asn Tyr Cys Asn 50 51 4 38 DNA
Artificial Artificial 1-38 Synthesized primer 4 agctgctttg
atatgcatct cccgaaactc tgtgcggt 38 5 37 DNA Artificial Artificial
1-37 Synthesized primer 5 gagcgatctg ggtctagaca gatttagcgg gtttcag
37 6 24 DNA Artificial Artificial 1-24 Synthesized oligonucleotide
6 aaaagggtat gtagaggttg aggt 24
* * * * *