U.S. patent application number 12/019212 was filed with the patent office on 2008-08-21 for igf binding proteins.
Invention is credited to Samantha Lien, Henry Lowman.
Application Number | 20080199886 12/019212 |
Document ID | / |
Family ID | 34421720 |
Filed Date | 2008-08-21 |
United States Patent
Application |
20080199886 |
Kind Code |
A1 |
Lowman; Henry ; et
al. |
August 21, 2008 |
IGF BINDING PROTEINS
Abstract
IGFBP-3 fusion proteins are provided that are useful, for
example, in cell-based assays, as IGF antagonists, and in mapping
IGF-I and IGF-II binding sites on other molecules such as wild-type
IGFBP-3 and IGF agonist peptides identified by phage display.
Methods for making such fusion proteins are also provided.
Inventors: |
Lowman; Henry; (El Granada,
CA) ; Lien; Samantha; (San Francisco, CA) |
Correspondence
Address: |
HELLER EHRMAN LLP
4350 La Jolla Village Drive, 7th Floor
San Diego
CA
92122
US
|
Family ID: |
34421720 |
Appl. No.: |
12/019212 |
Filed: |
January 24, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11525654 |
Sep 21, 2006 |
7351545 |
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12019212 |
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10936059 |
Sep 8, 2004 |
7192738 |
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11525654 |
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60508345 |
Oct 3, 2003 |
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Current U.S.
Class: |
435/7.8 |
Current CPC
Class: |
G01N 2333/4745 20130101;
C07K 14/71 20130101; G01N 2510/00 20130101; C12N 15/62 20130101;
C07K 14/65 20130101 |
Class at
Publication: |
435/7.8 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Claims
1. A method for determining a biological activity of an agonist of
a native-sequence human insulin-like growth factor-I (IGF-I) in a
cell-based assay comprising contacting cells with a fusion protein
comprising a peptide linked to an IGFBP-3 fragment that binds to
IGF-I, rather than with native-sequence human insulin-like growth
factor binding protein-3 (IGFPB-3), and determining if a biological
activity attributable to an agonist of said native-sequence human
IGF-I is observed.
2. The method of claim 1 wherein the peptide of the fusion protein
is SEQ ID NO:10.
3. The method of claim 1 wherein the IGFBP-3 fragment is a fragment
of residues 47 to 99 of native-sequence human IGFBP-3 (SEQ ID
NO:1).
4. The method of claim 1 wherein the biological activity is
apoptosis of native-sequence human IGFBP-3 that is independent of
IGF-I.
5. The method of claim 1 wherein the method is an IGF-dependent
KIRA phosphorylation method.
6. The method of claim 1 wherein the biological activity is
inhibition of binding of radiolabeled IGF-I to the cells.
Description
RELATED APPLICATIONS
[0001] This is a divisional application of non-provisional
application Ser. No. 11/525,654, filed Sep. 21, 2006, which is a
divisional application of non-provisional application Ser. No.
10/936,059, filed on Sep. 8, 2004, now U.S. Pat. No. 7,192,738,
issued Mar. 20, 2007, claiming priority under 35 USC 119(e) to
provisional application number 60/508,345 filed Oct. 3, 2003, the
contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of Invention
[0003] This invention relates to molecules useful in determining
minimal functional regions of IGFBP-3 and for antagonizing an IGF-I
or IGF-II activity.
[0004] 2. Description of Background and Related Art
[0005] 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-heterotetramer closely related to the
insulin receptor (McInnes and Sykes, Biopoly., 43: 339-366 (1998);
Ullrich et al., EMBO J., 5: 2503-2512 (1986)). The IGF-I and
insulin receptors bind their specific ligands with nanomolar
affinity. IGF-I and insulin can cross-react with their respective
ron-cognate receptors, albeit at a 100-1000-fold lower affinity
(Jones and Clemmons, supra). The crystal structure describing part
of the extracellular portion of the IGF-I receptor has been
reported (Garrett et al., Nature 394: 395-399 (1998)).
[0006] Unlike insulin, the activity and half-life of IGF-I are
modulated by six IGF-I binding proteins (IGFBPs 1-6), and perhaps
additionally by a more distantly related class of proteins (Jones
and Clemmons, supra; Baxter et al., Endocrinology, 139:4036
(1998)). IGFBPs can either inhibitor potentiate IGF activity,
depending on whether they are soluble or cell-membrane associated
(Bach and Rechler, Diabetes Reviews, 3: 38-61 (1995)). The IGFBPs
bind IGF-I and IGF-II with varying affinities and specificities
(Jones and Clemmons, supra; Bach and Rechler, supra). For example,
IGFBP-3 binds IGF-I and IGF-II with a similar affinity, whereas
IGFBP-2 and IGFBP-6 bind IGF-II with a much higher affinity than
they bind IGF-I (Bach and Rechler, supra; Oh et al., Endocrinology,
132: 1337-1344 (1993)). WO 01/75064 discloses additional human
secreted IGFBP-like polypeptides that are encoded by nucleic acid
sequences isolated from cDNA libraries from adrenal gland mRNA and
thymus mRNA.
[0007] Structurally, IGF-I is a single-chain, 70-amino-acid protein
with high homology to proinsulin. Unlike the other members of the
insulin superfamily, the C region of -IGF's is not proteolytically
removed after translation. The solution NMR structures of IGF-I
(Cooke et al., Biochemistry, 30: 5484-5491 (1991); Hua et al., J.
Mol. Biol., 259: 297-313 (1996)), mini-IGF-I (an engineered variant
lacking the C-chain; DeWolf et al., Protein Science, 5: 2193-2202
(1996)), and IGF-II (Terasawa et al., EMBO J. 13: 5590-5597 (1994);
Torres et al., J. Mol. Biol. 248: 385-401 (1995)) have been
reported. It is generally accepted that distinct epitopes on IGF-I
are used to bind receptor and binding proteins. It has been
demonstrated in animal models that receptor-inactive IGF mutants
are able to displace endogenous IGF-I from binding proteins and
thereby generate a net IGF-I effect in vivo (Loddick et al., Proc.
Natl. Acad. Sci. USA, 95: 1894-1898 (1998); Lowman et al.,
Biochemistry, 37: 8870-8878 (1998); U.S. Pat. Nos. 6,121,416 and
6,251,865). While residues Y24, Y29, Y31, and Y60 are implicated in
receptor binding, IGF mutants thereof still bind to IGFBPs (Bayne
et al., J. Biol. Chem., 265: 15648-15652 (1990); Bayne et al., J.
Biol. Chem., 264: 11004-11008 (1989); Cascieri et al.,
Biochemistry, 27: 3229-3233 (1988); Lowman et al., supra).
[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 IGFPB'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 IGFBPs. Additionally, an epitope involving
residues 49-51 in binding to IGFBP-1, -2 and -5 has been identified
(Clemmons et al., Endocrinology, supra, 1992). Furthermore, a
naturally occurring truncated form of IGF-I lacking the first three
N-terminal amino acids (called des (1-3)-IGF-I) was demonstrated to
bind IGFBP-3 with 25 times lower affinity (Heding et al., J. Biol.
Chem., 271: 13948-13952 (1996); U.S. Pat. Nos. 5,077,276;
5,164,370; and 5,470,828).
[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 that are derivatives of the naturally
occurring single-chain IGF-I having an abbreviated C domain. The
IGF-I analogs are of the formula: BCn , A wherein B is the B domain
of IGF-I or a functional analog thereof, C is the C domain of IGF-I
or a functional analog thereof, n is the number of amino acids in
the C domain and is from about 6 to about 12, and A is the A domain
of IGF-I or a functional analog thereof.
[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 1 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-I: (1-62) IGF-I, which
lacks the carboxyl-terminal 8-amino-acid D region of IGF-I; (1-27,
Gly.sup.4, 38-70) IGF-I, in which residues 28-37 of the C region of
IGF-I are replaced by a four-residue glycine bridge; and (1-27,
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 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.su-
p.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] Sliecker et al., Adv. Experimental Med. Biol., 343: 25-32
(1994)) describes the binding affinity of various IGF and insulin
variants to IGFBPs, IGF receptor, and insulin receptor.
[0016] IGFBPs are secreted by cells in culture and either inhibit
or enhance IGF-stimulated functions (Clemmons et al., (1991) In
Modern Concepts of Insulin-like Growth Factors. E. M. Spencer,
editor. Elsevier, New York, N.Y. 475-486). Known forms of IGFBPs
include IGFBP-1, having a molecular weight of approximately 30-40
kDa in humans. See, e.g., WO89/09792, published Oct. 19, 1990,
pertaining to cDNA sequences and cloning vectors for IGFBP-1 and
IGFBP-2; WO89/08667, published Sep. 21, 1989, relating to an amino
acid sequence of IGFBP-1; and WO89/09268, published Oct. 5, 1989,
relating to a cDNA sequence of IGFBP-1 and methods of expression
for IGFBP-1.
[0017] IGFBP-2 has a molecular weight of approximately 33-36 kDa.
See, e.g., Binkert et al., The EMBO Journal, 8: 2497-2502 (1989),
relating to a nucleotide and deduced amino acid sequence for
IGFBP-2.
[0018] IGFBP-3 has a non-glycosylated molecular weight of about 28
kDa. See, e.g., Baxter et al., Biochim. Biophys. Res. Com.,
139:1256-1261 (1986), pertaining to a glycosylated 53-kDa subunit
of IGFBP-3 that was purified from human serum; Wood et al., Mol.
Endocrinol., 2:1176-1185 (1988), relating to a full-length amino
acid sequence for IGFBP-3 and cellular expression of the cloned
IGFBP-3 CDNA in mammalian tissue culture cells; WO 90/00569,
published Jan. 25, 1990, relating to isolating from human plasma an
acid-labile subunit (ALS) of IGFBP complex and the particular amino
acid sequence for ALS pertaining to a subunit of IGFBP-3; and
Schmid et al., Biochim. Biophys. Res Com., 179: 579-585 (1991),
relating to effects of full-length and truncated IGFBP-3 on two
different osteoblastic cell lines.
[0019] Although initially some inconsistencies in nomenclature for
IGFBP-4, IGFBP-5, and IGFBP-6 existed, in 1991 participants of the
2nd International IGF Symposium agreed upon an accepted IGFBP-4,
IGFBP-5, and IGFBP-6 nomenclature. Using accepted terminology,
Mohan et al., Proc. Natl. Acad. Sci., 86:8338-8342 (1989) relates
to an N-terminal amino acid sequence for an IGFBP-4 isolated from
medium conditioned by human osteosarcoma cells, and Shimasaki et
al., Mol. Endocrinology, 4:1451-1458 (1990) pertains to IGFBP cDNAs
encoding IGFBP-4 from rat and human. WO92/03471 published Mar. 5,
1992, relates to anIGFBP-4 (originally designated therein as
IGFBP-5); and W092/03470 published Mar. 5, 1992 relates to genetic
material encoding IGFBP-4 (originally designated therein as
IGFBP-5).
[0020] WO92/12243 published Jul. 23, 1992, relates to IGFBP-5
(originally designated therein as IGFBP-6). Andress and Birnbaum,
Biochim. Biophys. Res Com., 176: 213-218 (1991) relates to the
modulation of cellular action of a mixture of affinity-purified
IGFBPs from U-2-cell-conditioned media on IGFs. WO92/03469
published Mar. 5, 1992, relates to genetic material encoding
IGFBP-6 (originally designated therein as IGFBP-4); and WO92/03152
published Mar. 5, 1992, relates to an IGFBP-6 (originally
designated therein as IGFBP-4). See also US Appln. No. 2003/0082744
as well as U.S. Pat. Nos. 6,025,465 and 5,212,074, which disclose
IGFBP-6 and its fragments.
[0021] Zapf et al., J. Biol. Chem., 265:14892-14898 (1990) pertains
to four IGFBPs (IGFBP-2, IGFBP-3, a truncated form of IGFBP-3, and
IGFBP-4 ) isolated from adult human serum by insulin-like growth
factor (IGF) affinity chromatography and high-performance liquid
chromatography. Shimasaki et al., 2nd International IGF Symposium
Abstract (January 1991) discusses amino-terminal amino acids for
IGFBP-4, IGFBP-5, and IGFBP-6.
[0022] When administered alone, i.e., without any IGF, the IGFBPs
may also be therapeutically useful for blocking the adverse effects
of IGFs, such as those that occur when IGFs are produced in excess,
e.g. free IGFs secreted by certain cancer cells such as
hormone-producing cancer cells such as breast or kidney cancer
cells. More recently, it was demonstrated that U-2 human
osteosarcoma cells secrete IGFBP-5 and IGFBP-6 (Andress and
Birnbaum, supra; Shimasaki et al., J. Biol. Chem. 266: 10646-10653
(1991); Shimasaki et al., Mol. Endocrinol., 5: 938-948 (1991)).
Although affinity-purified IGF-binding proteins derived from
U-2-conditioned medium clearly enhanced IGF-I stimulated
mitogenesis (Andress and Birnbaum, supra), it was unclear from
those studies which protein was responsible for this effect. Mohan
et al. demonstrated that IGFBP-4, purified from TE-89 human
osteosarcoma cells, inhibits IGF-stimulated osteoblast mitogenesis
(Mohan et al., Proc. Natl. Acad. Sci. (U.S.A.), 86: 8338-8342
(1989); see also LaTour et al., Mol. Endocrinol., 4: 1806-1814
(1990)). WO 03/068160 discloses use of IGFBP-3 for inhibiting tumor
growth. WO 03/006029 discloses a method of inducing apoptosis in a
cancer cell comprising increasing the expression of IGFBP-5 by the
cell to an apoptosis-inducing amount. A method of killing cancer
cells, a method of sensitizing cancer cells to agents that induce
apoptosis, and a method of treating cancer in a patient are also
described. U.S. Pat. No. 6,410,335 discloses a method of predicting
risk for prostate cancer, and US Application 2001/0018190 published
Aug. 30, 2001 discloses a method of treating prostate cancer with,
inter alia, IGFBPs, including IGFBP-3. U.S. Pat. No. 5,840,673 and
EP 871,475 disclose a method of inhibiting growth of p53-related
tumors by administering IGFBP-3 or a modulator of IGFBP-3 that
upregulates IGFBP-3 expression or activity. WO 00/35473 discloses
use of IGFBP-6 to treat inflammatory diseases including tumor
angiogenesis. WO 94/22466 discloses use of any IGFBP-3 to treat
cancer, including prostate cancer. WO 92/14834 discloses two IGFBPs
isolated from rat serum, one identified as IGFBP-5 useful in
treating cancer.
[0023] Exploitation of the interaction between IGF and IGFBP in
screening, preventing, or treating disease has been limited,
however, because of a lack of specific antagonists. The application
of an IGF-1/IGF-2 antagonist as a potential therapeutic adjunct in
the treatment of cancer is described by Pietrzkowski et al., Cancer
Res., 52: 6447-6451 (1992). In that report, a peptide corresponding
to the D-region of IGF-1 was synthesized for use as an IGF-1/2
antagonist. This peptide exhibited questionable inhibitory activity
against IGF-1. The basis for the observed inhibition is unclear, as
the D-region does not play a significant role in IGF-1 receptor
(IGF-1R) binding but rather, in IGF-1 binding to the insulin
receptor (Cooke et al., Biochem., 30: 5484-5491 (1991); Bayne et
al., J. Biol. Chem., 264: 11004-11008 (1988); Yee et al., Cell
Growth and Different., 5: 73-77 (1994)). IGF antagonists whose
mechanism of action is via blockade of interactions at the IGF-1
receptor interface may also significantly alter insulin action at
the insulin receptor, a disadvantage of such antagonists.
[0024] Certain IGF-1 antagonists have also been described by WO
00/23469, which discloses the portions of IGFBP and IGF peptides
that account for IGF-1 and IGFBP binding, i.e., an isolated IGF
binding domain of an IGFBP or modification thereof that binds IGF
with at least about the same binding affinity as the full-length
IGFBP. The patent publication also discloses an IGF antagonist that
reduces binding of IGF to an IGF receptor, and/or binds to a
binding domain of IGFBP.
[0025] Additionally, EP 639981 discloses pharmaceutical
compositions comprising short peptides that function as IGF-1
receptor antagonists. The peptides used in the pharmaceutical
compositions consist of less than 25 amino acids, comprise at least
a portion of the C or D region from IGF-1, and inhibit
IGF-1-induced autophosphorylation of IGF-1 receptors. Methods of
inhibiting cell proliferation and of treating individuals suspected
of suffering from or susceptible to diseases associated with
undesirable cell proliferation such as cancer, restenosis, and
asthma are disclosed.
[0026] Generation of specific IGF-1 antagonists has been
restricted, at least in part, because of difficulties in studying
the structure of IGF and IGFBP. Due to the inability to obtain
crystals of IGF-1 suitable for diffraction studies, for example, an
extrapolation of IGF-1 structure based on the crystal structure of
porcine insulin was the most important structural road map for
IGF-1 available (Blundell et al., Proc. Natl. Acad. Sci. USA,
75:180-184 (1978)). See also Blundell et al., Fed Proc., 42: 2592
(1983), which discloses tertiary structures, receptor binding, and
antigenicity of IGFs. Based on studies of chemically modified and
mutated IGF-1, a number of common residues between IGF-1 and
insulin have been identified as being part of the IGF-1R-insulin
receptor contact site, in particular the aromatic residues at
positions 23-25. Using NMR and restrained molecular dynamics, the
solution structure of IGF-1 was recently reported (Cooke et al.,
supra). The resulting minimized structure was shown to better fit
the experimental findings on modified IGF-1, as well as the
extrapolations made from the structure-activity studies of insulin.
Further, De Wolf et al., Protein Sci., 5: 2193 (1996) discloses the
solution structure of a mini-IGF-1. Sato et al., Int. J. Pept., 41:
433 (1993) discloses the three-dimensional structure of IGF-1
determined by 1 H-NMR and distance geometry. Torres et al., J. Mol.
Biol., 248: 385 (1995) discloses the solution structure of human
IGF-2 and its relationship to receptor and binding protein
interactions. Laajoki et al., J. Biol. Chem., 275: 10009 (2000)
discloses the solution structure and backbone dynamics of
long-(Arg(3)) IGF-1.
[0027] Peptide sequences capable of binding to insulin and/or
insulin-like growth factor receptors with either agonist or
antagonist activity and identified from various peptide libraries
are described in WO 01/72771 published Oct. 4, 2001.
[0028] US Application No. 2003/0092631 published May 15, 2003
discloses peptides that antagonize the interaction of IGF-1 with
its binding proteins, insulin receptor, and IGF receptor. These IGF
antagonist peptides are useful in treating disorders involving
IGF-1 as a causative agent, such as, for example, various
cancers.
[0029] Regarding the structural information on the classical
IGFBPs, they have a molecular mass ranging from 22 to 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 IGFBPs may he 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. U.S. Pat. No. 6,500,635
discloses IGFBP-5 and its variants. U.S. Pat. Nos. 6,391,588 and
6,489,294 disclose truncated C-terminal IGFBP-5 fragments with
reduced affinity for IGF-I as compared to full-length IGFBP-5. U.S.
Pat. No. 6,369,029 discloses stimulating osteogenesis with
C-terminal-truncated IGFBP-5. These compounds may be used for
stimulating bone cell growth, for treating a bone disorder, or for
stimulating mitogenic activity.
[0030] The NMR structures of two N-terminal fragments from IGFBP-5
that retain IGF-binding activity have recently been reported,
showing that residues 40-92 of IGFBP-5 comprise the IGF binding
site in the N-terminal domain of that protein (Kalus et al., EMBO
J., 17: 6558-6572 (1998)). Other studies have found that N-terminal
fragments (residues 1-88 and 1-97) of IGFBP-3 are also able to bind
IGFs (Galanis et al., Journal of Endocrinology, 169 (1): 123-133
(2001); Vorwerk et al., Endocrinology, 143 (5): 1677-1685
(2002)).
[0031] In particular, Galanis et al. have synthesized both the
amino-terminal (residues 1-88; N-88) and carboxyl-terminal
(residues 165-264; C-165) domains of human IGFBP-3 in bacteria, as
fusion proteins with a carboxyl-terminal FLAG peptide. Although
only C-165 showed binding to IGF-I and -II by solution-binding
assays, both N-88 and C-165 demonstrated binding to IGF-I and -II
by biosensor analysis albeit with reduced affinities compared with
full-length IGFBP-3. Only the carboxyl-terminal fragment (C-165)
was able to form hetero-trimeric complexes with IGF-I and the
acid-labile subunit (ALS).
[0032] Vorwerk et al. measured the binding of IGF-I and IGF-II to
recombinant human N-terminal (residues 1-97; N-97) and C-terminal
(residues 98-264; C-98) IGFBP-3 fragments and compared it with IGF
binding to intact IGFBP-3 using biosensor analysis. Experiments
were carried out either with binding protein or fragment
immobilized or with IGF immobilized. These experiments showed that
IGF-I and IGF-II bind to IGFBP-3 with affinities of
4-5.times.10.sup.-9 M and similar binding kinetics. The affinities
of both N-97 and C-98 for IGF proteins were approximately three
orders of magnitude less than that of full-length IGFBP-3.
[0033] US Application No. 2003/0161829A1 published Aug. 28, 2003
and WO03/025121 disclose fragments of IGFBP-3 that do not bind
IGF-I to treat conditions characterized by immune stimulation
rather than deficiency. The peptides target the CD74-homology
domain sequence at the C-terminus of IGFBP-3 and activities
localized to that region, having unique antigenicity. Peptides made
to sequences in this region have previously been shown to interfere
with the binding of IGFBP-3 to a number of its known ligands,
including RXR-alpha, transferrin, ALS, plasminogen, fibrinogen and
pre-kallikrein (Liu et al., J. Biol. Chem., 275: 33607-33613
(2000); Weinzimer et al., J. Clin. Endocrinol. Metab., 86: 1806-13
(2001); Campbell et al., Am. J. Physiol., 275: E321-E231 (1998);
Campbell et al., J. Biol. Chem., 274: 30215-30221 (1999); Firth, et
al., J. Biol. Chem. 273: 2631-2638, (1998)).
[0034] The IGFBP-3-derived metal-binding domain peptides differ
from previously disclosed IGFBP-3-derived molecules including in
their inability to bind IGF-I, their unique antigenicity, and the
absence of the IGFBP-3 putative death receptor (P4. 33) interaction
domain of IGFBP-3 (so-called "mid-region"; amino acids 88-148). The
P4.33 putative death receptor is described in WO 01/87238 (Genbank
Accession Number BC031217; gi:21411477). For example, WO 02/34916
teaches the use of point mutants of IGFBP-3 in which the binding to
IGF-I is impaired. However, the described molecules contain the
mid-region of IGFBP-3 and would be expected to exert biological
effects by interacting with the P4.33 putative receptor. WO
01/87238 teaches the use of P4.33 modulators for treating disease.
The metal-binding peptides do not include the P4.33 putative
interaction domain (mid-region of IGFBP-3).
[0035] U.S. Pat. No. 6,417,330, WO 99/63086, and US application no.
2002/0072589 disclose IGFBP-3 variants modified to be resistant to
hydrolysis. Also disclosed are variant IGFBP-3s where the nuclear
localization signal (NLS) in native IGFBP-3 is altered.
Additionally, amino-terminally extended IGFBP-3s are disclosed that
include a variety of N-terminal extensions, including peptide and
nucleotide binding domains, specific binding members such as
ligand-binding domains from receptors or antigen-binding domains
from immunoglobins, and peptide and protein hormones and growth
factors. N-terminally extended IGFBP-3s may comprise
hydrolysis-resistant or NLS-variant IGFBP-3s.
[0036] Some recent publications have described the use of IGFBP-3
peptides to treat cells in culture. The only peptides found to be
active on breast cancer cells are derived from the mid-region of
IGFBP-3 (McCaig et al., Br. J. Cancer, 86: 1963-1969 (2002); Perks
et al., Biochim. Biophys. Res. Comm. 294: 988-994 (2002)).
[0037] US Application No. 2003/0059430 published Mar. 27, 2003
discloses that the IGF-binding protein-derived peptides described
above, including short peptides containing just 12-22 amino acids
from the C-terminal domain of IGFBP-3, can mimic the full
molecule's co-apoptotic, cell-penetrating, and metal-binding
properties.
[0038] WO 03/052079 discloses mutants of IGFBP-3 that can inhibit
DNA synthesis, can induce apoptosis, bind to neither human IGF-I
nor human IGF-II, and comprise a mutation at Y57.
[0039] WO 02/098914 discloses a crystal suitable for X-ray
diffraction, comprising a complex of IGF-I or -II and a polypeptide
consisting of the amino acids 39-91 of IGFBP-1, the amino acids
55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino
acids 39-91 of IGFBP-4, the amino acids 40-92 of IGFBP-5, or the
amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at
least of the 9th to 12th cysteine of IGFBP-1, IGFBP-2, IGFBP-3,
IGFBP-4, or IGFBP-5 or at least of the 7th to 10th cysteine of
IGFBP-6; methods for the determination of the atomic coordinates of
such a crystal; IGFBP mutants with enhanced binding affinity for
IGF-I and/or IGF-II, and methods to identify and optimize small
molecules that displace IGFs from their binding proteins.
[0040] WO 02/34916 discloses mutant IGFBP-3 polypeptides and
fragments thereof that have either no binding, or diminished
binding to IGFs, yet retain their ability to bind to the human
IGFBP-3 receptor ("P4.33"). The fragments are N-deletion fragments
with 87 to 264 amino acids. The fragment 1-87 binds poorly to
IGF-I, and the other fragments (1-46, 1-75, and 1-80) do not bind
at all.
[0041] WO 00/23469 discloses IGFBP fragments that account for
IGF-IGFBP binding. It provides an isolated IGF binding domain of an
IGFBP or modifications thereof, which binds IGF with at least about
the same binding affinity as the full-length IGFBP. It also
provides an IGF antagonist that reduces binding of IGF to an IGF
receptor. It especially relates to IGFBP-2 fragments, but also
provides the isolated IGF binding domains of IGFBP-1, IGFBP-3,
IGFBP-4, IGFBP-5, and IGFBP-6. As with the IGF binding domain of
IGFBP-2, the amino acid sequences comprising the IGF binding domain
of the other IGFBPs could include modified forms so long as the
binding affinity of the binding domain is about the same as that of
the comparable native full-length IGFBP.
[0042] WO 99/32620 discloses IGFBP fragments and utilization
thereof, i.e., peptides that are characterized in that the amino
acid sequence parts thereof correspond to the amino acid sequence
of IGFBP. The invention also relates to cyclic, glycosylated,
phosphorylated, acetylated, amidated and/or sulfatized derivatives.
These include C-terminal domains of IGFBP-3.
[0043] 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)).
[0044] 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.
[0045] Alternatively, one can employ proteolytic cleavage of fusion
proteins (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)).
[0046] There is a continuing need in the art for a molecule that
can be used to elucidate binding epitopes on IGFBP-3 and other
ligands, acts as an IGF antagonist to control the levels of
circulating IGF as well as receptor response, for therapeutic or
diagnostic uses, and can be used for other therapeutic, diagnostic,
or assay purposes.
SUMMARY OF THE INVENTION
[0047] Accordingly, the invention is as claimed. In one embodiment,
this invention provides a fusion protein comprising an IGFBP-3
fragment (i.e., a truncated IGFBP-3) consisting of residues 47 to
99 of native-sequence human IGFBP-3 (SEQ ID NO:1 below) linked to
the synthesized Z domain of protein A from Staphylococcus aureus,
which has the sequence:
TABLE-US-00001 (SEQ ID NO:10)
VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKK LNDAQAPK.
[0048] In one such embodiment, this fusion protein is displayed on
phage. In another embodiment, the fragment is linked to the Z
domain by means of a cleavable linker peptide. Such cleavable
linker peptide preferably comprises one of the sequences: DLVD (SEQ
ID NO:2), DEMD (SEQ ID NO:3), DAVD (SEQ ID NO:4), EFGGDDDK (SEQ ID
NO;5), EFGGLVPRGS (SEQ ID NO:6), EFGGDLVD (SEQ ID NO:7), EFGGDEMD
(SEQ ID NO:8), or EFGGDAVD (SEQ ID NO:9). In another embodiment, an
ASA sequence is at the N-terminus of the Z domain. In yet another
embodiment, the fragment is affinity matured.
[0049] Also provided herein is a composition comprising the fusion
protein described above in a carrier, preferably a pharmaceutically
acceptable carrier. Preferably, this composition is sterile.
[0050] Further provided is a nucleic acid molecule encoding the
fusion protein, a vector comprising the nucleic acid, a host cell
comprising the nucleic acid, and a method of producing an IGFBP-3
fusion protein comprising culturing the host cells under suitable
conditions to express the fusion protein and recovering the fusion
protein from the host cell culture. Preferred is that the host
cells are prokaryotic, more preferably bacterial cells such as E.
coli.
[0051] These fusion proteins can be used in many indications,
including assays for determining if ligands have an IGFBP-3 binding
site. The fusion proteins herein containing an IGF-I or IGF-II
binding site are further useful for clarification and mapping of
the IGF binding sites and binding sites of IGF-I agonist ligands
other than IGF-I (e.g., peptides isolated by phage panning
experiments such as bp 15 described in Lowman et al., supra) on
native-sequence human IGFBP-3 in the absence of any structural
data.
[0052] In yet another embodiment, the invention provides a method
for determining a biological activity of native-sequence human
IGFBP-3, of native-sequence human IGF-I, or of native-sequence
human IGF-II, or of an agonist of said IGF-I or said IGF-II in a
cell-based assay comprising contacting cells with a fusion protein
comprising a peptide linked to an IGFBP-3 fragment that binds to
IGF-I or IGF-II, rather than with native-sequence human IGFBP-3,
and determining if a biological activity attributable to
native-sequence human IGFBP-3, native-sequence human IGF-I, or
native-sequence human IGF-II, or an agonist of said IGF-I or said
IGF-II is observed.
[0053] In one embodiment, the biological activity is apoptosis of
native-sequence human IGFBP-3 that is independent of IGF-I.
[0054] In another embodiment, the assay is an IGF-dependent KIRA
phosphorylation assay. This assay is a direct activity assay for
the human Type 1 receptor. When a receptor in the tyrosine kinase
family, such as the Type 1 IGF receptor, is activated, it is
phosphorylated on tyrosine residues. In this assay cells containing
the Type 1 IGF receptor are activated in vitro, then disrupted, and
antibodies against the receptor are used to precipitate the IGF
receptor. Next, an anti-phosphotyrosine antibody is used to assay
the amount of Type 1 IGF receptor that is phosphorylated. If a
fixed number of cells are used, then the amount of receptor that is
phosphorylated is a direct measure of the activity of a molecule on
the Type 1 IGF receptor. In this KIRA assay, cells such as a breast
cancer cell line are treated with IGF-I or IGF-II plus the fusion
protein and a biological activity of the fusion protein is
determined by the amount of receptor that is phosphorylated.
[0055] In yet another embodiment, the biological activity is
inhibition of binding of radiolabeled IGF-I or IGF-I to the
cells.
[0056] In a further aspect, the invention provides a method for
determining enhancement of apoptosis comprising pre-treating breast
cancer cells with an IGFBP-3 fragment or fusion protein thereof
that binds IGF-I or IGF-II for at least about 24 hours prior to
treating the cells with an apoptotic factor, such as, for example,
paclitaxel or doxorubicin, and native-sequence human IGFBP-3 or
said IGFBP-3 fragment or fusion protein, and determining if the
pre-treatment or treatment enhances the apoptosis induced by the
treatment with the apoptotic factor, or if the amounts of
pre-treatment or treatment are effective for that purpose.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 shows a schematic of the miniBP-3 fusion protein.
This is the fusion of a Z domain (SEQ ID NO:10) to miniBP-3
(residues 47-99 of native-sequence human IGFBP-3 (SEQ ID NO:1))
using a cleavable linker having SEQ ID NO:7 (including the
caspase-3 cleavage site (SEQ ID NO:2), which is underlined).
[0058] FIG. 2 shows a SDS-PAGE analysis, using Coomassie blue
staining, of various chromatographic fractions of the miniBP-3
fusion protein and the cleaved protein miniBP-3.
[0059] FIG. 3 shows a biosensor analysis of native-sequence human
IGF-I binding to immobilized native-sequence human IGFBP-3.
[0060] FIG. 4 shows a biosensor analysis of native-sequence human
IGF-II binding to immobilized native-sequence human IGFBP-3.
[0061] FIG. 5 shows a biosensor analysis of native-sequence human
IGF-I binding to immobilized miniBP-3 fusion protein.
[0062] FIG. 6 shows a biosensor analysis of native-sequence human
IGF-II binding to immobilized miniBP-3 fusion protein.
[0063] FIG. 7 shows the results of peptide bp 15 (SEEVCWPVAEWYLCN)
(SEQ ID NO:11) competition experiments performed on the BIAcore.TM.
analyzer. A total of 15 nM to 100:M bp15 peptide and 20 nM
native-sequence human IGFBP-3 were incubated for 1 hour at room
temperature before injection over immobilized biotinylated
native-sequence human IGF-I and -II.
[0064] FIG. 8 shows the results of peptide bp15 (SEQ ID NO:11)
competition experiments performed on the BIAcore.TM. analyzer. A
total of 1.17:M to 300:M bp15 peptide and 1:M miniBP-3 fusion
protein was incubated for 1 hour at room temperature before
injection over immobilized biotinylated native-sequence human
IGF-I.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
A. Definitions
[0065] 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.
[0066] 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 from any source, whether
natural, synthetic, or recombinant. This may be 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. "Native-sequence human IGF-I" or "wild-type IGF-I" is
wild-type human IGF-I.
[0067] 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 from any source, whether
natural, synthetic, or recombinant. It may be prepared by the
method described in, e.g., EP 128,733. "Native-sequence human
IGF-II" or "wild-type IGF-II" is wild-type human IGF-II.
[0068] An AIGFBP@ 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
(19%). 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 27 Jun. 1990; EP 369,943 published
23 May 1990; WO 89/09268 published 5 Oct. 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 7 Dec. 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 21
Sep. 1989; WO 89/09792 published 19 Oct. 1989; Binkert et al., EMBO
J., 8: 2497-2502 (1989); EP 369,943B1; US Pat. No. 5,973,115; EP
1,295,939; and U.S. Pat. No. 6,004,775 and EP 546053.
[0069] "IGFBP-3" or "insulin-like growth factor binding protein-3"
refers to IGFBP-3 or BP53 from any species, including bovine,
ovine, porcine, equine, and human, preferably human, and from any
source, whether natural, synthetic, or recombinant, as described in
U.S. Pat. Nos. 5,258,287 and 5,328,891. "Native-sequence human
IGFBP-3, "wild-type IGFBP-3," and "full-length IGFBP-3" is
wild-type human IGFBP-3 or BP-53 as described in U.S. Pat. Nos.
5,258,287 and 5,328,891 with a glycine at position 5, i.e., SEQ ID
NO:1, or an alanine at position 5, i.e., SEQ ID NO:12.
TABLE-US-00002 SEQ ID NO:1 is the sequence
GASSGGLGPVVRCEPCDARALAQCAPPPAVCAELVREPGCGCCLTCALSE
GQPCGIYTERCGSGLRCQPSPDEARPLQALLDGRGLCVNASAVSRLRAYL
LPAPPAPGNASESEEDRSAGSVESPSVSSTHRVSDPKFHPLHSKIIIIKK
GHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFL
NVLSPRGVHIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTT KGKEDVHCYSMQSK
SEQ ID NO:12 is the sequence:
GASSAGLGPVVRCEPCDARALAQCAPPPAVCAELVREPGCGCCLTCALSE
GQPCGIYTERCGSGLRCQPSPDEARPLQALLDGRGLCVNASAVSRLRAYL
LPAPPAPGNASESEEDRSAGSVESPSVSSTHRVSDPKFHPLHSKIIIIKK
GHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFL
NVLSPRGVHIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTT
KGKEDVHCYSMQSK
[0070] "IGFBP-3 fragment" refers to native-sequence human IGFBP-3
(SEQ ID NO:1 or 12) lacking at least one amino acid. Preferably,
the fragment is an N-terminal fragment such as, for example, the
peptide having (or corresponding to) residues 1 to 46, residues 1
to 88, residues 1 to 89, residues 1 to 90, residues 1 to 91,
residues 1 to 92, residues 1 to 93, residues 1 to 94, residues 1 to
95, residues 1 to 96, residues 1 to 97, residues 1 to 98, residues
1 to 99, residues 1 to 185, or residues 47 to 99 of native-sequence
human IGFBP-3 (SEQ ID NO:1 or 12), or a C-terminal fragment such
as, for example, the peptide having residues 98 to 264, residues
100 to 264, residues 165 to 264, or residues 185 to 264 of
native-sequence human IGFBP-3 (SEQ ID NO:1). More preferably, the
IGFBP-3 fragment is selected from the group consisting of a peptide
with residues 1 to 46, residues 1 to 88, residues 1 to 97, residues
1 to 99, residues 47 to 99, residues 1 to 185, residues 98 to 264,
residues 100 to 264, residues 165 to 264, and residues 185 to 264
of native-sequence human IGFBP-3 (SEQ ID NO:1 or 12), and most
preferably a peptide with residues 47 to 99 of native-sequence
human IGFBP-3 (SEQ ID NO:1).
[0071] A "fusion protein" is a protein having two separate peptides
linked together, either directly or through a linker (i.e., "linker
peptide" or "linking peptide"), preferably through such a linker.
For example, one fusion protein is a protein comprising the Z
domain of Protein A fused to an IGFBP-3 fragment, which Z domain
and fragment may be directly linked if no cleavage is desired, or
may be linked via a cleavage site such as a caspase-3 proteolytic
site if cleavage is desired.
[0072] As used herein, "Z domain of protein A" refers to an
IgG-binding domain of Staphylococcal protein A as described, for
example, in EP 230,869 B1 as the synthetic Z-region in FIG. 3, and
in Samuelsson et al., Bio/Technology, 9: 363 (1991), and Nilsson et
al., Protein Eng., 1(2): 107-113 (1987). "Peptides" are molecules
having at least two amino acids and include polypeptides having at
least about 50 amino acids. The definition includes peptide
fragments, derivatives, their salts, or optical isomers, as well as
linkers. Preferably, the peptides herein have from about 35 to 200
amino acids, more preferably, about 40 to 170 amino acids.
[0073] An "apoptotic factor" as used herein is a molecule that
induces apoptosis or cell death. Such molecules include
chemotherapeutic agents, anti-hormonal agents, cytotoxic agents and
other agents some of which are defined below that induce cell
death. Preferably, such factor is a chemotherapeutic agent, and
more preferably doxorubicin or paclitaxel, most preferably
paclitaxel.
[0074] An "affinity-matured" IGFBP-3 peptide fragment or fusion
protein is one having one or more alterations within the peptide
fragment or fusion protein that result in an improvement in the
affinity of the peptide fragment or fusion protein for IGF-I or
IGF-II, compared to a corresponding parent peptide fragment or
fusion protein that does not possess those alteration(s). Preferred
affinity-matured IGFBP-3 peptide fragment or fusion protein will
have nanomolar or even picomolar affinities for the target IGF.
Affinity-matured peptide fragments and fusion proteins are produced
by procedures known in the art, including phage display (Lowman and
Wells, J. Mol. Biol., 234 (3): 564-578 (1993); Lowman et al.,
Biochemistry, 30 (45): 10832-10838 (1991)); rational mutagenesis
(Lowman et al., J. Biol. Chem., 261 (17): 10982-10988 (1991);
Hawkins et al., J. Mol. Biol., 234 (4): 958-964 (1993)); random
mutagenesis (Fiedler et al., Protein Eng., 15 (11): 931-941
(2002)); and DNA shuffling and phage display (Huls et al., Cancer
Immunol. Immunother., 50 (3): 163-171 (2001); van den Beucken et
al., J. Mol. Biol., 310(3): 591-601 (2001)).
[0075] 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.
[0076] 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.
[0077] As used herein, "IGF antagonist" refers to a peptide that
blocks or inhibits one or more biological activities of IGF-I or
IGF-II such as its anabolic effects.
[0078] 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 an anabolic effect.
[0079] The term "effective amount" refers to an amount of a peptide
effective to treat a disease or disorder in a mammal. In the case
of cancer, the effective amount of the peptide may reduce the
number of cancercells; reduce the tumor size; inhibit (i.e., slow
to some extent and preferably stop) cancer cell infiltration into
peripheral organs; inhibit (i.e., slow to some extent and
preferably stop) tumor metastasis; inhibit, to some extent, tumor
growth; and/or relieve to some extent one or more of the symptoms
associated with the disorder. To the extent the peptide may prevent
growth and/or kill existing cancer cells, it may be cytostatic
and/or cytotoxic. For cancer therapy, efficacy in vivo can, for
example, be measured by assessing the time to disease progression
(TTP) and/or determining the response rates (RR).
[0080] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include, but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
More particular examples of such cancers include squamous cell
cancer, lung cancer (including small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung, and squamous
carcinoma of the lung), cancer of the peritoneum, hepatocellular
cancer, gastric or stomach cancer (including gastrointestinal
cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney or renal cancer, liver cancer,
prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma
and various types of head and neck cancer, as well as B-cell
lymphoma (including low grade/follicular non-Hodgkin's lymphoma
(NHL); small lymphocytic (SL) NHL; intermediate grade/follicular
NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL;
high grade lymphoblastic NHL; high grade small non-cleaved cell
NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related
lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic
leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell
leukemia; chronic myeloblastic leukemia; and post-transplant
lymphoproliferative disorder (PTLD). Preferably, the cancer
comprises a tumor that expresses an IGF receptor, more preferably
breast cancer, lung cancer, colorectal cancer, or prostate cancer,
and most preferably breast or prostate cancer.
[0081] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. At211, I131, I125, Y90, Re 186, Re188,
Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic
agents, and toxins such as small-molecule toxins or enzymatically
active toxins of bacterial, fungal, plant or animal origin,
including fragments and/or variants thereof.
[0082] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone, metdopa,
and uredopa; ethylenimines and methylamelarines including
altretamine, triethylenemelamine, trietylenephosphoramide,
triethiylenethiophosphoramide and trimethylolomelamine; acetogenins
(especially bullatacin and bullatacinone); a camptothecin
(including the synthetic analogue topotecan); bryostatin;
callystatin; CC-1065 (including its adozelesin, carzelesin and
bizelesin synthetic analogues); cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CBI-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlomaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, and uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e. g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew,
Chem Intl. Ed. Enl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antiobiotic chromophores, aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
carnminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolinodoxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine, diaziquone; elfornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofiran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.TM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin and carboplatin;
vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;
vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide;
edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-1;
topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO);
retinoids such as retinoic acid; capecitabine; and pharmaceutically
acceptable salts, acids or derivatives of any of the above.
[0083] Also included in this definition are anti-hormonal agents
that act to regulate or inhibit hormone action on tumors such as
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone, and FARESTON
toremifene; aromatase inhibitors that inhibit the enzyme aromatase,
which regulates estrogen production in the adrenal glands, such as,
for example, 4(5)-imidazoles, aminoglutethimide, MEGASE.RTM.
megestrol acetate, AROMASIN.RTM. exemestane, formestanie,
fadrozole, RIVISOR.RTM. vorozole, FEMARA.RTM. letrozole, and
ARIMIDEX.RTM. anastrozole; and anti-androgens such as flutamide,
nilutamide, bicalutamide, leuprolide, and goserelin; as well as
troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
antisense oligonucleotides, particularly those that inhibit
expression of genes in signaling pathways implicated in abherant
cell proliferation, such as, for example, PKC-alpha, Ralf and
H-Ras; ribozymes such as a VEGF expression inhibitor (e.g.,
ANGIOZYME.RTM. ribozyme) and a HER2 expression inhibitor; vaccines
such as gene therapy vaccines, for example, ALLOVECTIN.RTM.
vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine;
PROLEUKIN.RTM. rIL-2; LURTOTECAN.RTM. topoisomerase I inhibitor;
ABARELIX.RTM. rmRH; and pharmaceutically acceptable salts, acids or
derivatives of any of the above.
[0084] A "growth-inhibitory agent" when used herein refers to a
compound or composition that inhibits growth of a cell in vitro
and/or in vivo. Thus, the growth-inhibitory agent may he one that
significantly reduces the percentage of cells in S phase. Examples
of growth inhibitory agents include agents that block cell cycle
progression (at a place other than S phase), such as agents that
induce GI arrest and M-phase arrest. Classical M-phase blockers
include the vincas (vincristine and vinblastine), TAXOL.RTM.
paclitaxel, and topo II inhibitors such as doxorubicin, epirubicin,
daunorubicin, etoposide, and bleomycin. Those agents that arrest GI
also spill over into S-phase arrest, for example, DNA alkylating
agents such as tanoxifen, prednisone, dacarbazine, mechlorethamine,
cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further
information can be found in The Molecular Basis of Cancer,
Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle
regulation, oncogenes, and antieioplastic drugs" by Murakaini et
al. (W B Saunders: Philadelphia, 1995), especially p. 13.
[0085] Examples of "growth-inhibitory" anti-HER2 antibodies are
those that bind to HER2 and inhibit the growth of cancer cells
overexpressing HER2. Preferred growth-inhibitory anti-HER2
antibodies inhibit growth of SKBR3 breast tumor cells in cell
culture by greater than 20%, and preferably greater than 50% (e.g.,
from about 50% to about 100%) at an antibody concentration of about
0.5 to 30 .mu.g/ml, where the growth inhibition is determined six
days after exposure of the SKBR3 cells to the antibody (see U.S.
Pat. No. 5,677,171 issued Oct. 14, 1997).
[0086] An antibody that "induces cell death" is one that causes a
viable cell to become nonviable. The cell is generally one that
expresses the antigen to which the antibody binds, especially where
the cell overexpresses the antigen. Preferably, the cell is a
cancer cell, e.g., a breast, ovarian, stomach, endometrial,
salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder
cell. In vitro, the cell may be a SKBR3, BT474, Calu 3, MDA-MB453,
MDA-MB-361 or SKOV3 cell. Cell death in vitro may be determined in
the absence of complement and immune effector cells to distinguish
cell death induced by antibody dependent cell-mediated cytotoxicity
(ADCC) or complement dependent cytotoxicity (CDC). Thus, the assay
for cell death may be performed using heat-inactivated serum (i.e.,
in the absence of complement) and in the absence of immune effector
cells. To determine whether the antibody is able to induce cell
death, loss of membrane integrity as evaluated by uptake of
propidium iodide (PI), trypan blue (see Moore et al.
Cytotechnology, 17:1-11 (1995)) or 7AAD can be assessed relative to
untreated cells.
[0087] An antibody that "induces apoptosis" is one that induces
programmed cell death as determined by binding of annexin V,
fragmentation of DNA, cell shrinkage, dilation of endoplasmic
reticulum, cell fragmentation, and/or formation of membrane
vesicles (called apoptotic bodies). The cell is one that expresses
the antigen to which the antibody binds and may be one that
overexpresses the antigen. The cell may be a tumor cell, e.g., a
breast, ovarian, stomach, endometrial, salivary gland, lung,
kidney, colon, thyroid, pancreatic or bladder cell. In vitro, the
cell may be a SKBR3, BT474, Calu 3 cell, MDA-MB453, MDA-MB-361 or
SKOV3 cell. Various methods are available for evaluating the
cellular events associated with apoptosis. For example,
phosphatidyl serine (PS) translocation can be measured by annexin
binding; DNA fragmentation can be evaluated through DNA laddering;
and nuclear/chromatin condensation along with DNA fragmentation can
be evaluated by any increase in hypodiploid cells. Preferably, the
antibody that induces apoptosis is one that results in about 2- to
50-fold, preferably about 5- to 50-fold, and most preferably about
10- to 50-fold, induction of annexin binding relative to untreated
cell in an annexin-binding assay using cells expressing the antigen
to which the antibody binds.
[0088] Examples of antibodies that induce apoptosis include the
anti-HER2 monoclonal antibodies 7F3 (ATCC HB-12216), and 7C2 (ATCC
HB 12215), including humanized and/or affinity-matured variants
thereof; the anti-DR5 antibodies 3F1 1.39.7 (ATCC HB-12456);
3H3.14.5 (ATCC HB-12534); 3D5. 1.10 (ATCC HB-12536); and 3H3. 14.5
(ATCC HB-12534), including humanized and/or affinity-matured
variants thereof; the human anti-DR5 receptor antibodies 16E2 and
20E6, including affinity-matured variants thereof (WO98/51793,
expressly incorporated herein by reference); and the anti-DR4
antibodies 4E7.24.3 (ATCC HB-12454); 4H6.17.8 (ATCC HB-12455);
1H5.25.9 (ATCC HB-12695); 4G7.18.8 (ATCC PTA-99); and 5G I1.17.1
(ATCC HB-12694), including humanized and/or affinity-matured
variants thereof.
[0089] In order to screen for antibodies that bind to an epitope on
an antigen bound by an antibody of interest, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, eds. Harlow and Lane (New York: Cold Spring
Harbor Laboratory, 1988) can he performed.
B. Modes for carrying Out the Invention
[0090] The invention herein relates, in one aspect, to a fusion
protein comprising an IGFBP-3 peptide fragment containing only
residues 47-99 of native-sequence human IGFBP-3 (SEQ ID NO:1),
referred to herein as miniBP-3, linked to the Z domain of Protein
A, whether linked directly or through a linker, which can be
cleaved by an enzyme so as to release the fragment. Such linker or
linking peptide may be, for example, a cleavable linker, e.g., a
caspase-3 cleavable linker containing the proteolytic site DLVD
(SEQ ID NO:2), DEMD (SEQ ID NO:3) or DAVD (SEQ ID NO:4). Examples
of such linking peptides are EFGGDLVD (SEQ ID NO:7), EFGGDEMD (SEQ
ID NO:8), or EFGGDAVD (SEQ ID NO:9). Other examples of linking
peptides are the enterokinase-cleavable linker EFGGDDDK (SEQ ID
NO:5) and a thrombin-cleavable linker EFGGLVPRGS (SEQ ID NO:6).
[0091] 1. Preparation
[0092] The fusion 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. [0093] a. Recombinant
Preparation
[0094] The fusion peptide 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.
[0095] Somewhat more particularly, the DNA sequence encoding an
IGFBP-3 fusion protein 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 Laboratorv
Manual (2d ed.), Cold Spring Harbor Laboratory, N.Y., 1989).
[0096] The parent DNA is then inserted into an appropriate plasmid
or vector that is used to transform a host cell. In general,
plasmid vectors containing replication and control sequences that
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 that encode proteins or
peptides that are capable of providing phenotypic selection in
transformed cells.
[0097] 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).
[0098] A preferred vector is pET21a. This vector is driven by the
T7 promoter and is available, for example, from Novagene, Inc. and
described in Studier et al., Methods Enzymol., 185: 60-89 (1990).
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. Another suitable
vector is pB0475, which contains origins of replication for phage
and E. coli that allow it to be shared between such hosts, thereby
facilitating both mutagenesis and expression (Cunningham et al.,
Science 243: 1330-1336 (1989); U.S. Pat. No. 5,580,723).
[0099] 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.
[0100] The host cell may be prokaryotic or eukaryotic. Prokaryotes
are preferred for cloning and expressing DNA sequences to produce
the fusion protein. 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 c600hfl, 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 BL21 (Stratagene), which is deficient in the
OmpT and Lon proteases, which may interfere with isolation of
intact recombinant proteins, and useful with T7 promoter-driven
vectors, such as the pET vectors. Another suitable prokaryote is E.
coli W3110 (ATCC No. 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.
[0101] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for fusion-protein-encoding vectors. Saccharomyces cerevisiae is a
commonly used lower eukaryotic host microorganism. Others include
Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140
(1981); EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S.
Pat. No. 4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991))
such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et
al., J. Bacteriol., 154(2):737-742 (1983)), K. fragilis (ATCC
12,424), K. bulgaricus (ATCC No. 16,045), K. wickeramii (ATCC No.
24,178), K. waltii (ATCC No. 56,500), K. drosophilarum (ATCC No.
36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K.
thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia
pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol.,
28:265-278 (1988)); Candida; Trichoderna reesia (EP 244,234);
Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA,
76:5259-5263 (1979)); Schwanniomyces such as Schwanniomyces
occidentalis (EP 394,538 published 31 Oct. 1990); and filamentous
fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO
91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A.
nidulans (Ballance et al., Biochem. Biophys. Res. Commun.,
112:284-289 (1983); Tilburn et al., Gene, 26:205-221 (1983); Yelton
et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 (1984)) and A.
niger (Kelly and Hynes, EMBO J, 4:475-479 (1985)). Methylotropic
yeasts are suitable herein and include, but are not limited to,
yeast capable of growth on methanol selected from the genera
consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces,
Torulopsis, and Rhodotorula. A list of specific species that are
exemplary of this class of yeasts may be found in C. Anthony, The
Biochemistry of Methylotrophs, 269 (1982).
[0102] Other suitable host cells for the expression of fusion
proteins are derived from multicellular organisms. Examples of
invertebrate cells include insect cells such as Drosophila S2 and
Spodoptera Sf9, as well as plant cells. Examples of useful
mammalian host cell lines include Chinese hamster ovary (CHO) and
COS cells. More specific examples include monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC No. CRL 1651); human embryonic
kidney line (293 or 293 cells subcloned for growth in suspension
culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinese
hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl.
Acad. Sci. USA, 77:4216(1980)); mouse sertoli cells (TM4, Mather,
Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC No.
CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary
tumor (MMT 060562, ATCC No. CCL51). The selection of the
appropriate host cell is deemed to be within the skill in the
art.
[0103] Preferably, the Z-domain portion of the fusion protein can
be secreted by the cell (has a signal sequence, for example),
making it possible to isolate and purify the fusion protein from
the culture medium and eliminating the necessity of destroying the
host cells that 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.
[0104] 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.
[0105] 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. [0106] b. Synthetic Preparation
[0107] 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 a-amino acid to a suitable resin. Such a starting
material can be prepared by attaching an a-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.
[0108] 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 fusion
protein. 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.
[0109] Another coupling method involves use of a suitable coupling
agent such as N,N'-dicyclohexylcarbodiimide or
N,N'-diisopropylcarbodiimide. Other appropriate coupling agents,
apparent to those skilled in the art, are disclosed in E. Gross
& J. Meienhofer, The Peptides: Analysis. Structure, Biology,
Vol. 1: Major Methods of Peptide Bond Formation (Academic Press,
New York, 1979).
[0110] 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).
[0111] In the selection of a particular side-chain protecting group
to be used in synthesizing the peptides, the following general
rules are followed. An a-amino protecting group (a) must render the
a-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 fusion
protein, 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
a-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.
[0112] 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)isopropyloxycarbonyl 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:
[0113] (1) for an a-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)-isopropyloxycarbonyl, allyloxycarbonyl and the
like; (c) cycloalkyl urethane-type protecting groups, such as
cyclopentyloxycarbonyl, adamantyloxycarbonyl, and
cyclohexyloxycarbonyl; and d) allyloxycarbonyl. The preferred
a-amino protecting groups are BOC or FMOC.
[0114] (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.
[0115] (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.
[0116] (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.
[0117] (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.
[0118] (6) for the imidazole nitrogen of His, the tosyl moiety is
suitably employed.
[0119] (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.
[0120] (8) for the side-chain amino group of Asn or Gln, xanthyl
(Xan) is preferably employed.
[0121] (9) for Met, the amino acid is preferably left
unprotected.
[0122] (10) for the thio group of Cys, p-methoxybenzyl is typically
employed.
[0123] An appropriately selected protecting group, in the case of
Lys, BOC, protects the C-terminal amino acid, e.g., Lys, at the
N-amnino position. 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 25EC 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 OEC 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.
[0124] 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 carbodimide or
diisopropylcarbodiimide.
[0125] 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
a., Anal. Biochem, 34: 595 (1970). The coupling reactions can be
performed automatically using well-known methods, for example, a
BIOSEARCH 9500.TM. peptide synthesizer.
[0126] 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
chloromethylated 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
reagents that are known to be capable of breaking an ester linkage
and of penetrating the resin matrix can cleave the anchoring
bond.
[0127] 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 chioromethylated rcsin 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.
[0128] 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. Pep. 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.
[0129] 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.
[0130] 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.
[0131] 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.
[0132] 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 group 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.
[0133] 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, sulflhydryl 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.
[0134] The peptides of this invention also may be conformationally
stabilized by cyclization. The peptides ordinarily are cyclized by
covalently bonding the NB 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 NB
and C-terminal domains. The linking sites thus generally will be
between the side chains of the residues.
[0135] Many suitable methods per se are known for preparing mono-
or poly-cyclized peptides as contemplated herein. Lys/Asp
cyclization has been accomplished using
sodium-tert-butyloxycarbonyl (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.
[0136] 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).
[0137] 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.
[0138] 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.
[0139] If in the peptides being created carbon atoms bonded to four
nonidentical substituents are asymmetric, then the peptides may
exist as diastereoisomers, 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 R) or S) and both are within the scope of the
present invention.
[0140] In another embodiment, the fragment of the fusion protein
herein may be affinity matured so that it has better affinity
toward the IGF-I and/or IGF-II than the parent fragment. Such
affinity maturation can be done, for example, through phage
display, rational mutagenesis, random mutagenesis, or DNA shuffling
and phage display, or by any such other means known in the art for
effecting this change. See the references noted above in the
definition section.
[0141] 2. Uses There are many advantages to using the fusion
proteins for the applications disclosed herein. For example,
mammalian systems can be expensive for the industrial production of
wild-type IGFBP-3. On the other hand, peptide fusions are expected
to be cheaper and easier to produce than wild-type IGFBP-3 using
either synthetic chemical methods or highly efficient biological
production systems well known to those skilled in the art.
[0142] The peptides herein may be useful in diagnostic assays,
e.g., for detecting expression of IGF-1 in specific cells, tissues,
or serum.
[0143] For diagnostic applications, the peptide typically will be
labeled with a detectable moiety. Numerous labels are available
that can be generally grouped into the following categories:
[0144] (a) Radioisotopes, such as 35S, 14C, I211, 3H, and I311, are
available. The peptide can be labeled with the radioisotope using
the techniques described in Current Protocols in Immunology,
Volumes 1 and 2, Coligen et al., ed. (Wiley-Interscience: New York,
1991), for example, and radioactivity can be measured using
scintillation counting.
[0145] (b) Fluorescent labels such as rare-earth chelates (europium
chelates) or fluorescein and its derivatives, rhodainine and its
derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are
available. The fluorescent labels can be conjugated to the peptide
using the techniques disclosed in Current Protocols in Immunology,
supra, for example. Fluorescence can be quantified using a
fluorimeter.
[0146] (c) Various enzyme-substrate labels are available and U.S.
Pat. No. 4,275,149 provides a review of some of these. The enzyme
generally catalyzes a chemical alteration of the chromogenic
substrate that can be measured using various techniques. For
example, the enzyme may catalyze a color change in a substrate,
which can be measured spectrophotometrically. Alternatively, the
enzyme may alter the fluorescence or chemiluminescence of the
substrate. Techniques for quantifying a change in fluorescence are
described above. The chemiluminescent substrate becomes
electronically excited by a chemical reaction and may then emit
light that can be measured (using a chemiluminometer, for example)
or donates energy to a fluorescent acceptor. Examples of enzymatic
labels include luciferases (e.g., firefly luciferase and bacterial
luciferase; U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as horseradish peroxidase (HRPO), alkaline
phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide
oxidases (e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as
uricase and xanthine oxidase), lactoperoxidase, microperoxidase,
and the like. Techniques for conjugating enzymes to antibodies are
described in O'Sullivan et al., "Methods for the Preparation of
Enzyme-Antibody Conjugates for use in Enzyme Immunoassay," in
Methods in Enzym. (ed J. Langone & H. Van Vunakis), 73:147-166
(Academic Press, New York, 1981).
[0147] Examples of enzyme-substrate combinations include, for
example:
[0148] (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase
as a substrate, wherein the hydrogen peroxidase oxidizes a dye
precursor (e.g., orthophenylene diamine (OPD) or
3,3',5,5'-tetramethyl benzidine hydrochloride (TMB));
[0149] (ii) alkaline phosphatase (AP) with para-nitrophenyl
phosphate as chromogenic substrate; and
[0150] (iii) beta-D-galactosidase (beta-D-Gal) with a chromogenic
substrate (e.g., p-nitrophenyl-beta-D-galactosidase) or fluorogenic
substrate (4-methylumbellifuryl-beta-D-galactosidase).
[0151] Numerous other enzyme-substrate combinations are available
to those skilled in the art. For a general review of these, see
U.S. Pat. Nos. 4,275,149 and 4,318,980.
[0152] Sometimes, the label is indirectly conjugated with the
peptide. The skilled artisan will be aware of various techniques
for achieving this. For example, the peptide can be conjugated with
biotin and any of the three broad categories of labels mentioned
above can be conjugated with avidin, or vice versa. Biotin binds
selectively to avidin and thus, the label can he conjugated with
the peptide in this indirect manner. Alternatively, to achieve
indirect conjugation of the label with the peptide, the peptide is
conjugated with a small hapten (e.g., digoxin) and one of the
different types of labels mentioned above is conjugated with an
anti-hapten peptide (e.g., anti-digoxin antibody). Thus, indirect
conjugation of the label with the peptide can be achieved.
[0153] In another embodiment of the invention, the peptide need not
be labeled, and the presence thereof can be detected using a
labeled antibody that binds to the peptide.
[0154] The peptide of the present invention may be employed in any
known assay method, such as competitive binding assays, direct and
indirect sandwich assays, and immunoprecipitation assays. Zola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC
Press, Inc. 1987).
[0155] The peptide may also be used for in vivo diagnostic assays.
Generally, the peptide is labeled with a radionuclide (such as
111In, 99Tc, 14C, 131I, 125I, 3H, 32P or 35S) so that the
antigenorcells expressing it can be localized using
immunoscintiography.
[0156] Other uses include mapping binding epitopes of bplS and
IGFBP-3 and other IGF peptide agonists. In addition, the IGFBP-3
fragments that bind to IGF-I or IGF-II or fusion proteins
comprising them can be used in cell-based assays. Certain such
assays comprise contacting the cell with the fusion protein rather
than with native-sequence human IGFBP-3 and determining if a
biological activity attributable to native-sequence human IGFBP-3,
native-sequence human IGF-I or native-sequence IGF-II, or an
agonist of said IGF-I or said IGF-II is observed. In one
embodiment, the biological activity is apoptosis of native-sequence
human IGFBP-3 that is independent of IGF-I. In another example, the
assay is an IGF-dependent KIRA phosphorylation assay. This assay is
an IGF-I kinase receptor activation assay, which is a direct
activity assay for the human Type 1 receptor. When a receptor in
the tyrosine kinase family, such as the Type 1 IGF receptor, is
activated, it is phosphorylated on tyrosine residues. In this assay
cells containing the Type 1 IGF receptor are activated in vitro,
then disrupted, and antibodies against the receptor are used to
precipitate the IGF receptor. Next, an anti-phosphotyrosine
antibody is used to assay the amount of Type 1 IGF receptor that is
phosphorylated. If a fixed number of cells are used, then the
amount of receptor that is phosphorylated is a direct measure of
the activity of a molecule on the Type 1 IGF receptor. In this KIRA
assay, cells such as a breast cancer cell line are treated with
IGF-I or IGF-II plus the fusion protein and a biological activity
of the fusion protein is determined by the amount of receptor that
is phosphorylated. KIRA assays are described further in U.S. Pat.
No. 6,251,865 using MCF-7 breast cancer cells as one embodiment,
and in Chen et al., Am. J. Physiol. Endocrinol. Metab., 284:
E1149-E1155 (2003).
[0157] In yet another embodiment, the biological activity is
inhibition of binding of radiolabeled IGF-I or IGF-II to the
cells.
[0158] A further example is a method comprising pre-treating breast
cancer cells with an IGFBP-3 fragment or fusion protein thereof
that binds to IGF-I or IGF-II for at least about 24 hours prior to
treating the cells with an apoptotic factor, such as, for example,
a chemotherapeutic agent, e.g., doxorubicin or paclitaxel, and
native-sequence human IGFBP-3 or said IGFBP-3 fragment or fusion
protein, and determining if the pre-treatment or treatment enhances
the apoptosis induced by the treatment with apoptotic factor, or if
the amounts of pre-treatment or treatment are effective for that
purpose.
[0159] Kits are also contemplated for this invention. The kit
generally comprises a container containing a composition comprising
the fusion protein and instructions for its use, e.g., in an assay.
A typical kit would comprise a container, preferably a vial, for
the fusion protein formulation comprising the fusion protein in a
buffer and instructions, such as a product insert or label,
directing the user to utilize the formulation, e.g., for mapping
epitopes or cell-based assays. The kit optionally includes a
container, preferably a vial, for an agent to be used with the
fusion protein.
[0160] 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
Production of IGFBP-3 and miniBP-3 Fusion Protein
Introduction:
[0161] Native-sequence human IGFBP-3 and miniBP-3 fusion protein
were prepared and assessed for direct binding to native-sequence
human IGF-I and IGF-II in BIAcore assays.
[0162] Based upon the results of experiments using the miniBP-3
fusion protein described below, it is predicted that molecules of
the type claimed herein should decrease active IGF levels.
Materials and Methods:
[0163] Expression and Purification of Wild-Type IGFBP-3 and
miniBP-3 Fusion and Cleaved miniBP-3
[0164] Wild-type IGFBP-3 and miniBP-3 were produced in E. coli
using the vector pET21a (Novagene) as follows:
[0165] All routine chemical reagents were purchased from Sigma
Chemical Co. (St Louis, Mo.) or Fisher Scientific (Fair Lawn,
N.J.). Restriction enzymes and the T4 DNA ligase were obtained from
New England Biolabs (Beverley, Mass.). Oligonucleotide synthesis
reagents, DNA sequencing kits, and PCR kits were obtained from PE
Biosystems (Foster City, Calif.). dNTPs, IPTG and ATP were
purchased from Boehringer-Mannheim (Indianapolis, Ind.). DNA
polymerase and E. coli strain BL21 were purchased from Stratagene
(La Jolla, Calif.).
[0166] Plasmid pET21a was purchased from Novagen Inc. (Madison,
Wis.). The affinity column was from Pharmacia (Piscataway, N.J.).
LB medium was prepared according to the standard formula (Sambrook
et al., supra). HEPES and CHAPS buffers and DTT are obtainable from
the sources indicated below. Periplasmic extraction buffer
consisted of 10 mM TRIS-HCl, pH 8.5 and 1 mM EDTA. TE buffer
consisted of 10 mM TRIS-HCl, pH 8.0 and 1 mM EDTA.
[0167] Oligodeoxyribonucleotides were synthesized using a 394
automated DNA synthesizer from PE Biosystems. PCR and sequencing
primers were purified by ethanol precipitation and dissolved in TE
buffer. The Z domain was amplified by PCR of vector pA-100-Z
(derived from the construct as described in Dennis et al.,
Biochemistry, 40: 9513 (2001)) using the primers: 5'-ACT AAA TAT
GCT AGC GCC GTA GAC AAC AAA TTC AAC AAA G-3' (SEQ ID NO:13) and
5'-ATA TTT AGT GAA TTC CTT AGG CGC CTG AGC ATC ATT TAG-3' (SEQ ID
NO:14). The amplified Z domain was digested with NheI and EcoRI and
ligated into the cloning vector pET21a (Novagen) treated with the
same pair of restriction enzymes to generate pET21a-Z domain
fusion. The insertion of the Z domain was confirmed by
sequencing.
[0168] The coding region of miniBP-3 fusion was synthesized by PCR
using the gene assembly method of Stemmer et al., Gene, 164: 49-53
(1995). The oligonucleotides used for gene assembly had the
sequences: 5'-ACT AAA TAG AAT TCG GCG GTG ATG ATG ACG ACA AAG CCC
TGA GCG AAG GTC AGC CGT GCG GTA TTT AT-3' (SEQ ID NO:15), 5'-GCT
CGG CTG GCA ACG CAG ACC GCT ACC GCA ACG TTC GGT ATA AAT ACC GCA
CGG-3' (SEQ ID NO:16), 5'-CGT TGC CAG CCG AGC CCG GAT GAA GCC CGT
CCG CTG CAG GCC CTG CTG GAT GGT CGT GGT CTG TGC-3' (SEQ ID NO:17),
and 5'-TAT TTA GTA AGC TTC TAA TAG GCA CGC AGA CGG CTA ACG GCG CTG
GCG TTA ACG CAC AGA CCA CGA CC-3' (SEQ ID NO:18). The assembled
gene was amplified using the following oligonucleotides: 5'-ACT AAA
TAG AAT TCG GCG GTG ATG-3' (SEQ ID NO:19) and 5'-TAT TTA GTA AGC
TTC TAA TAG GCA CG-3' (SEQ ID NO:20).
[0169] The gene encoding full-length fusion miniBP-3 protein was
amplified with the primers using a PCR System 9700.TM. thermocycler
(PE Biosystems) as described in Cao et al., Gene 197:
205-214(1997). The amplified product was digested with Eco RI and
Hind III and ligated into the pET21a-Z-domain-containing vector
treated with the same pair of restriction enzymes to generate
pET21a-miniBP-3 fusion. The insertion of the miniBP-3 fragment was
confirmed by sequencing. The enterokinase cleavage site EFGGDDDDK
(SEQ ID NO:4) was later replaced with the caspase-3 cleavage site
EFGGDLVD (SEQ ID NO:7) using the QUIKCHANGE.TM. Site-Directed
Mutagenesis Kit. These two mutagenic primers had the sequences:
5'-GGA ATT CGG CGG TGA TCT GGT GGA TGC CCT GAG CGA AGG-3' (SEQ ID
NO:21) and 5'-CCT TCG CTC AGG GCA TCC ACC AGA TCA CCG CCG AAT
TCC-3' (SEQ ID NO:22). The mutation was confirmed by
sequencing.
[0170] This vector was transformed into E. coli strain BL21
(Stratagene). Inserts in pET expression vectors were sequenced in
both orientations to ensure that the plasmid constructs were free
of PCR or ligation errors.
[0171] The cells were propagated overnight at 37.degree. C. in 2YT
medium (Sambrook et al, supra) containing 50 .mu.g/ml of
ampicillin. Overnight cultures were diluted 100-fold into the same
medium, grown until the optical density of the culture reached 0.5
at 600 nm, then induced by the addition of
isopropyl-beta-D-thiogalactopyranoside (IPTG) to a final
concentration of 50 .mu.M and grown for an additional 4 hours under
the same conditions. Cells were collected by centrifugation,
frozen/thawed at -20/4.degree. C. in periplasmic extraction buffer,
and clarified by centrifugation as described by Cao et al.,
supra.
[0172] The periplasmic extracts from the centrifugation were
subjected to chromatography using an IgG SEPHAROSE.TM. ion-exchange
column (Amersham Biosciences) as described in Nilssen et al.,
supra. Briefly, the column was washed with 5 volumes 50 mM TRIS
buffer, pH 7.6, 150 mM NaCl and 0.05% TWEEN-20.TM. buffer (TST),
then alternately with 0.5 M acetic acid (HAc), pH 3.4 and TST for
two cycles. The column was equilibrated with TST and the fusion
protein was captured on the column, as described in Nilsson et al.,
supra. The column was washed with 10 bed volumes TST and 2 bed
volumes of 5 mM ammonium acetate, pH 5.0 before elution.
[0173] After the fusion protein was eluted with 4 bed volumes of
0.5M HAc it was incubated overnight at 4.degree. C. with 125 nM
caspase-3 (kindly provided by Dr. Guy Salvesen, The Burnham
Institute, La Jolla, Calif.) in 100 mM Hepes buffer (ProSciTech),
0.1% CHAPS lysis buffer (Chemicon International), 0.5 mM
dithiothreitol (DTT) (JT Baker), pH 7.5. Cleaved miniBP-3 was
recovered from the supernatant of the reaction after
centrifugation. The purity of the fusion protein was verified by
SDS-PAGE analysis followed by visualizing the overloaded gel with
routine Coomassie brilliant blue R staining. See FIG. 2, which
shows the SDS-PAGE analysis for the various stages.
[0174] BL21 cells expressing wild-type IGFBP-3 were cultured and
expression was induced as with miniBP-3. The wild-type IGFBP-3 was
extracted from inclusion bodies and refolded in vitro using
standard conditions. Purification was achieved using ion-exchange
chromatography on both Q and S SEPHAROSE.TM. (Amersham Biosciences)
columns and hydrophobic-interaction chromatography on a phenyl
SUPEROSE.TM. (Amersham Biosciences) colurn using standard
techniques.
Biosensor Kinetic Measurements
[0175] The binding affinities of wild-type IGFBP-3 and miniBP-3
fusion protein for IGF-I and IGF-II were determined using a
BIAcore.TM. -2000 real time kinetic interaction analysis system
(BlAcore, Inc., Piscataway, N.J.) to measure association (ka) and
dissociation (kd) rates. Two types of chips were prepared for this
purpose.
[0176] CM5 Chip Preparation:
[0177] Carboxymethylated dextran biosensor chips (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, wild-type IGFBP-3 and mutants in
10 mM sodium acetate, pH 4, were injected onto the biosensor chip
at a concentration of 0.2 mg/ml to yield approximately 450-700 RU's
(resonance-response units) of covalently coupled protein. Unreacted
groups were blocked with an injection of 1 M ethanolamine.
[0178] Kinetic measurements were carried out by injecting two-fold
serial dilutions of IGF-I or IGF-II in PBST running buffer (PBS,
0.05% TWEEN-20.TM. buffer, 0.01% sodium azide) at 25.degree. C.
using a flow rate of 20 or 50:l/min. IGF concentrations ranged
between 0.4 nM and 50 nM for IGFBP-3 and 100 nM to 25:M for
miniBP-3 fusion. For both proteins, association and dissociation
rates were calculated using a 1:1 Langmuir association model in the
BIAcore.TM. evaluation software. For wild-type IGFBP-3, the
equilibrium dissociation constant was calculated as kd/ka. For
miniBP-3 fusion, the equilibrium dissociation constant was
calculated by plotting equilibrium binding data in GraphPad
Prism.TM. software (GraphPad Software Inc., San Diego, Calif.) and
fitting to a one-site binding model.
[0179] SA Chip Preparation:
[0180] Streptavidin-coated chips were conditioned according to the
manufacturer's instructions (BIAcore, Inc.) prior to injection of
0.02 mg/ml of biotinylated IGF-I or IGF-II in 10 mM sodium acetate
pH 4. Biotinylated IGF-I and IGF-II were prepared using EZ-Link
biotinylation reagents according to the manufacturer's instructions
(Pierce). Between 100 and 1000 RUs were immobilized on the
chip.
[0181] Kinetic measurements were carried out by injecting two-fold
serial dilutions of miniBP-3 fusion protein in PBST running buffer
at 50:l/min. Concentrations were between 250 nM and 32:M.
[0182] Competition binding experiments were carried out as follows.
15 nM to 100:M bp15 peptide and 20 nM IGFBP-3 were incubated for 1
hour at room temperature before injection at 20:l/min in PBST over
immobilized biotinylated IGF-I and -II. Equilibrium binding data
was plotted in GraphPad Prism.TM. software and fit to a one-site
competition-binding model.
Results:
[0183] The wild-type GFBP-3 and miniBP-3 fusion protein were
submitted for kinetic analysis using a BIAcore.TM. instrument and
tested for binding affinity to IGF-I and IGF-II. The results are
shown in Table I for wild-type IGFBP-3 and Table II for miniBP-3
fusion protein.
TABLE-US-00003 TABLE I Kinetic Parameters for the Interaction of
Purified Wild-type IGFBP-3 with IGF-I and -II Determined by BIAcore
.TM. Analysis k.sub.a (.times.10.sup.4 M.sup.-1 s.sup.-1) k.sub.d
(.times.10.sup.-4 s.sup.-1) K.sub.D (.times.10.sup.-9 M) IGF-I 715
.+-. 3.66 7.58 .+-. 0.07 0.11 .+-. 0.001 IGF-II 348 .+-. 1.02 4.07
.+-. 0.05 0.12 .+-. 0.001
TABLE-US-00004 TABLE II Kinetic Parameters for the Interaction of
Purified MiniBP-3 Fusion Protein with IGF-I and -II Determined by
BIAcore .TM. Analysis k.sub.a (.times.10.sup.4 M.sup.-1 s.sup.-1)
k.sub.d (s.sup.-1) K.sub.D (.times.10.sup.-6 M) IGF-I 9.13 .+-.
0.87 0.59 .+-. 0.04 4.64 .+-. 0.17 IGF-II 7.59 .+-. 1.35 0.08 .+-.
<0.01 4.66 .+-. 0.06
[0184] The results in Table I (see also FIGS. 3 and 4), showing
that IGFBP-3 has high affinity for IGF-I and IGF-II, compare
favorably with other measurements in the literature. The results in
Table II (see also FIGS. 5 and 6) as compared to Table I show that
the miniBP-3 fusion has lower affinity for IGF-I and IGF-II
compared to wild-type IGFBP-3. Without being limited to any one
theory, it is believed that this is primarily due to increases in
off-rate. Studies with other fragments of IGFBP-3 display similar
findings, as shown in Table III.
TABLE-US-00005 TABLE III Kinetic Parameters for the Interaction of
Purified N-Terminal Fragments of IGFBP-3 with IGF-I and -II
Determined by BIAcore .TM. Analysis k.sub.a K.sub.A Fragment
(.times.10.sup.4 M.sup.-1 s.sup.-1) k.sub.d (s.sup.-1)
(.times.10.sup.5 M.sup.-1) Reference N-88 14.7 .+-. 1.6 1.26 .+-.
0.28 .times. 10.sup.-2 103 .+-. 8 Galanis et al. (2001), supra
IGFBP-3.sup.1-97 3.2 .+-. 1.1 5.55 .+-. 1.40 .times. 10.sup.-3 61.4
.+-. 19.8 Vorwerk et al. (2002), supra
EXAMPLE 2
Binding of Peptide bp15 to MiniBP-3 Fusion
[0185] Peptide bp15 (SEEVCWPVAEWYLCN) (SEQ ID NO:11) was identified
by phage display (Lowman et al., Biochemistry, 1998, supra). It
competes with IGF-I and -II for binding to IGFBP-3 as determined by
BIAcore.TM. analysis as described in Example I. Therefore, bp15 was
tested to see if it would bind to the miniBP-3 fusion. Compare
FIGS. 7 and 8 for competitive binding data for bp15 to IGFBP-3 and
to the miniBP-3 fusion, respectively.
[0186] It can be seen that peptide bp15 does bind to the miniBP-3
fusion, but with reduced affinity compared to wild-type IGFBP-3.
Exact affinity was unable to be determined due to lack of
saturation.
Discussion:
[0187] The miniBP-3 fusion has affinity for IGF-I and IGF-II. This
affinity is reduced compared to that of other N-terminal fragments
of IGFBP-3. Nevertheless, the miniBP-3 fusion contains at least
part of the bp15 peptide-binding site. N-terminal fragments of
IGFBP-3 have also been associated with IGF-independent effects of
IGFBP-3. See, for example, Angelloz-Nicoud et al., Growth Hormone
& IGF Research, 8: 71-75 (1990); Lalou et al., Endocrinology,
137 (8): 3206-3212 (1996); Yamanaka et al., Endocrinology, 140 (3):
1319-1328 (1999); Maile et al., Endocrinology, 140 (9): 4040-4045
(1999); Salahifar et al., Growth Hormone & IGF Research,
10:367-377(2000); and Bernard et al., Biochem. Biophys. Res. Comm.,
293: 55-60 (2002).
[0188] Cell-based assays using a fusion protein containing miniBP-3
or an IGFBP-3 fragment can be developed to detect these
IGF-independent events. Thus, in experiments performed by Gill et
al., J. Biol. Chem., 272: 25602-25607 (1997) and Fowler et al.,
Int. J. Cancer, 88: 448-453 (2000), breast cancer cells are treated
with IGFBP-3 for 24 hours prior to the addition of paclitaxel (and
IGFBP-3). Although treatment of the cells with IGFBP-3 alone is not
sufficient to induce apoptosis, pre-treatment with IGFBP-3 enhances
the apoptosis induced by paclitaxel treatment. The miniBP-3 fusion
protein, or fragments of IGFBP-3 that bind to IGF-I or IGF-II and
fusion proteins thereof, are expected to be useful in this assay
instead of wild-type IGFBP-3 if they contain the activity
responsible for potentiation of cell death. An advantage of using
the fusion protein or fragments is that they are easier to make in
large quantities than intact IGFBP-3, as noted above.
[0189] Additionally to these apoptosis assays, cell-based assays
can be used for IGF-dependent KIRA assays and radiolabeled IGF
binding inhibition, because wild-type IGFBP-3 does not inhibit
binding on some cells, perhaps, without being limited to any one
theory, due to binding to its own receptor. In an exemplary
cell-based IGF-1 KIRA assay, a KIRA for measuring the activation of
the human type 1 IGF-1 receptor is performed using human MCF-7
cells. Cells are grown overnight in 96-well plates with medium
(50:50 F12/DMEM, Gibco). Supernatants are decanted, and stimulation
media (50:50 F12/DMEM with 25 mM HEPES and 2.0% BSA) containing
either controls (2 nM IGF-1 preincubated with wild-type IGFBP-1 or
IGFBP-3) or experimental samples (fusion protein containing
miniBP-3 preincubated for 30 min. with 2 nM IGF-1) are added. After
15-minute stimulation the cells are lysed, and added to apolyclonal
anti-IGF-1R (3B7; SantaCruz Biotech) coated overnight on
immunosorbant plates. Detection ELISA is performed, and the KIRA
results would be expected to show about the same inhibition for the
fusion protein containing miniBP-3 as, or possibly more inhibition
of IGF-I receptor binding than, that seen for wild-type IGFBP-3,
and the inhibition is likely to be improved if the miniBP-3
fragment is affinity matured by phage display or other means as
would be known to those skilled in the art.
[0190] 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
221264PRTHomo sapiens 1Gly Ala Ser Ser Gly Gly Leu Gly Pro Val Val
Arg Cys Glu Pro1 5 10 15Cys Asp Ala Arg Ala Leu Ala Gln Cys Ala Pro
Pro Pro Ala Val 20 25 30Cys Ala Glu Leu Val Arg Glu Pro Gly Cys Gly
Cys Cys Leu Thr 35 40 45Cys Ala Leu Ser Glu Gly Gln Pro Cys Gly Ile
Tyr Thr Glu Arg 50 55 60Cys Gly Ser Gly Leu Arg Cys Gln Pro Ser Pro
Asp Glu Ala Arg 65 70 75Pro Leu Gln Ala Leu Leu Asp Gly Arg Gly Leu
Cys Val Asn Ala 80 85 90Ser Ala Val Ser Arg Leu Arg Ala Tyr Leu Leu
Pro Ala Pro Pro 95 100 105Ala Pro Gly Asn Ala Ser Glu Ser Glu Glu
Asp Arg Ser Ala Gly 110 115 120Ser Val Glu Ser Pro Ser Val Ser Ser
Thr His Arg Val Ser Asp 125 130 135Pro Lys Phe His Pro Leu His Ser
Lys Ile Ile Ile Ile Lys Lys 140 145 150Gly His Ala Lys Asp Ser Gln
Arg Tyr Lys Val Asp Tyr Glu Ser 155 160 165Gln Ser Thr Asp Thr Gln
Asn Phe Ser Ser Glu Ser Lys Arg Glu 170 175 180Thr Glu Tyr Gly Pro
Cys Arg Arg Glu Met Glu Asp Thr Leu Asn 185 190 195His Leu Lys Phe
Leu Asn Val Leu Ser Pro Arg Gly Val His Ile 200 205 210Pro Asn Cys
Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg 215 220 225Pro Ser
Lys Gly Arg Lys Arg Gly Phe Cys Trp Cys Val Asp Lys 230 235 240Tyr
Gly Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp 245 250
255Val His Cys Tyr Ser Met Gln Ser Lys 26024PRTArtificial
sequenceSequence is synthesized 2Asp Leu Val Asp134PRTArtificial
sequenceSequence is synthesized 3Asp Glu Met Asp144PRTArtificial
sequenceSequence is synthesized 4Asp Glu Met Asp158PRTArtificial
sequenceSequence is synthesized 5Glu Phe Gly Gly Asp Asp Asp Lys1
5610PRTArtificial sequenceSequence is synthesized 6Glu Phe Gly Gly
Leu Val Pro Arg Gly Ser1 5 1078PRTArtificial sequenceSequence is
synthesized 7Glu Phe Gly Gly Asp Leu Val Asp1 588PRTArtificial
sequenceSequence is synthesized 8Glu Phe Gly Gly Asp Glu Met Asp1
598PRTArtificial sequenceSequence is synthesized 9Glu Phe Gly Gly
Asp Ala Val Asp1 51058PRTArtificial sequenceSequence is synthesized
10Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu1 5 10
15Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe 20 25
30Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu 35 40
45Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50
551115PRTArtificial sequenceSequence is synthesized 11Ser Glu Glu
Val Cys Trp Pro Val Ala Glu Trp Tyr Leu Cys Asn1 5 10
1512264PRTHomo sapiens 12Gly Ala Ser Ser Ala Gly Leu Gly Pro Val
Val Arg Cys Glu Pro1 5 10 15Cys Asp Ala Arg Ala Leu Ala Gln Cys Ala
Pro Pro Pro Ala Val 20 25 30Cys Ala Glu Leu Val Arg Glu Pro Gly Cys
Gly Cys Cys Leu Thr 35 40 45Cys Ala Leu Ser Glu Gly Gln Pro Cys Gly
Ile Tyr Thr Glu Arg 50 55 60Cys Gly Ser Gly Leu Arg Cys Gln Pro Ser
Pro Asp Glu Ala Arg 65 70 75Pro Leu Gln Ala Leu Leu Asp Gly Arg Gly
Leu Cys Val Asn Ala 80 85 90Ser Ala Val Ser Arg Leu Arg Ala Tyr Leu
Leu Pro Ala Pro Pro 95 100 105Ala Pro Gly Asn Ala Ser Glu Ser Glu
Glu Asp Arg Ser Ala Gly 110 115 120Ser Val Glu Ser Pro Ser Val Ser
Ser Thr His Arg Val Ser Asp 125 130 135Pro Lys Phe His Pro Leu His
Ser Lys Ile Ile Ile Ile Lys Lys 140 145 150Gly His Ala Lys Asp Ser
Gln Arg Tyr Lys Val Asp Tyr Glu Ser 155 160 165Gln Ser Thr Asp Thr
Gln Asn Phe Ser Ser Glu Ser Lys Arg Glu 170 175 180Thr Glu Tyr Gly
Pro Cys Arg Arg Glu Met Glu Asp Thr Leu Asn 185 190 195His Leu Lys
Phe Leu Asn Val Leu Ser Pro Arg Gly Val His Ile 200 205 210Pro Asn
Cys Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys Arg 215 220 225Pro
Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp Cys Val Asp Lys 230 235
240Tyr Gly Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp 245
250 255Val His Cys Tyr Ser Met Gln Ser Lys 2601340DNAArtificial
sequenceSequence is synthesized 13actaaatatg ctagcgccgt agacaacaaa
ttcaacaaag 401439DNAArtificial sequenceSequence is synthesized
14atatttagtg aattccttag gcgcctgagc atcatttag 391568DNAArtificial
sequenceSequence is synthesized 15actaaataga attcggcggt gatgatgacg
acaaagccct gagcgaaggt 50cagccgtgcg gtatttat 681654DNAArtificial
sequenceSequence is synthesized 16gctcggctgg caacgcagac cgctaccgca
acgttcggta taaataccgc 50acgg 541766DNAArtificial sequenceSequence
is synthesized 17cgttgccagc cgagcccgga tgaagcccgt ccgctgcagg
ccctgctgga 50tggtcgtggt ctgtgc 661868DNAArtificial sequenceSequence
is synthesized 18tatttagtaa gcttctaata ggcacgcaga cggctaacgg
cgctggcgtt 50aacgcacaga ccacgacc 681924DNAArtificial
sequenceSequence is synthesized 19actaaataga attcggcggt gatg
242026DNAArtificial sequenceSequence is synthesized 20tatttagtaa
gcttctaata ggcacg 262139DNAArtificial sequenceSequence is
synthesized 21ggaattcggc ggtgatctgg tggatgccct gagcgaagg
392239DNAArtificial sequenceSequence is synthesized 22ccttcgctca
gggcatccac cagatcaccg ccgaattcc 39
* * * * *