U.S. patent application number 15/917293 was filed with the patent office on 2018-10-25 for engineered integrin binding peptide compositions.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Jennifer R. Cochran, Richard H. Kimura, Aron M. Levin.
Application Number | 20180303902 15/917293 |
Document ID | / |
Family ID | 39283352 |
Filed Date | 2018-10-25 |
United States Patent
Application |
20180303902 |
Kind Code |
A1 |
Cochran; Jennifer R. ; et
al. |
October 25, 2018 |
ENGINEERED INTEGRIN BINDING PEPTIDE COMPOSITIONS
Abstract
Engineered peptides that bind with high affinity (low
equilibrium dissociation constant (Kd)) to the cell surface
receptors of fibronectin (.alpha..sub.5.beta..sub.1) or vitronectin
(.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 integrins)
are disclosed as useful as imaging tissue. These peptides are based
on a molecular scaffold into which a subsequence containing the RGD
integrin-binding motif has been inserted. The subsequence (RGD
mimic) comprises about 9-13 amino acids, and the RGD contained
within the subsequence can be flanked by a variety of amino acids,
the sequence of which was determined by sequential rounds of
selection (in vitro evolution). The molecular scaffold is
preferably based on a knottin, e.g., EETI (Trypsin inhibitor 2
(Trypsin inhibitor II) (EETI-II) [Ecballium elaterium (Jumping
cucumber)], AgRP (Agouti-related protein), and Agatoxin IVB, which
peptides have a rigidly defined three-dimensional conformation. It
is demonstrated that EETI tolerates mutations in other loops and
that the present peptides may be used as imaging agents.
Inventors: |
Cochran; Jennifer R.;
(Stanford, CA) ; Kimura; Richard H.; (Menlo Park,
CA) ; Levin; Aron M.; (Menlo Park, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Stanford |
CA |
US |
|
|
Family ID: |
39283352 |
Appl. No.: |
15/917293 |
Filed: |
March 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15007091 |
Jan 26, 2016 |
9913878 |
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15917293 |
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14028348 |
Sep 16, 2013 |
9265845 |
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15007091 |
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12418376 |
Apr 3, 2009 |
8536301 |
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14028348 |
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PCT/US2007/021218 |
Oct 3, 2007 |
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12418376 |
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60849259 |
Oct 4, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/06 20130101;
A61K 45/06 20130101; G01N 33/57492 20130101; A61K 51/082 20130101;
C07K 14/47 20130101; A61K 49/0056 20130101; A61K 38/08 20130101;
A61K 38/1709 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; G01N 33/574 20060101 G01N033/574; A61K 49/00 20060101
A61K049/00; C07K 14/47 20060101 C07K014/47; A61K 51/08 20060101
A61K051/08; A61K 38/08 20060101 A61K038/08; A61K 38/06 20060101
A61K038/06; A61K 45/06 20060101 A61K045/06 |
Goverment Interests
STATEMENT OF GOVERNMENTAL SUPPORT
[0002] This invention was made with Government support under
contract 5K01CA104706 awarded by the National Institutes of Health.
The Government has certain rights in the invention.
Claims
1.-20. (canceled)
21. A method comprising administering a therapeutically effective
amount of an integrin binding peptide to an individual in need
thereof, wherein the integrin binding peptide comprises a knottin
protein scaffold comprising an engineered integrin binding loop
that binds to at least one of .alpha.v.beta.5 integrin,
.alpha.v.beta.3 integrin and .alpha.5.beta.1 integrin, and wherein
the integrin binding peptide comprises an amino acid sequence at
least 90% identical to the amino acid sequence of a peptide of any
one of SEQ ID NO:23 through SEQ ID NO:52.
22. The method according to claim 21, wherein the integrin binding
peptide is administered parenterally to the individual.
23. The method according to claim 21, wherein the integrin binding
peptide is administered orally to the individual.
24. The method according to claim 21, wherein the individual has a
proliferative disease.
25. The method according to claim 24, wherein the proliferative
disease is cancer.
26. The method according to claim 21, wherein the integrin binding
peptide is administered to block angiogenesis in the
individual.
27. The method according to claim 21, wherein the integrin binding
peptide is administered to promote cell adhesion in the
individual.
28. The method according to claim 21, wherein the integrin binding
peptide comprises an amino acid sequence at least 95% identical to
the amino acid sequence of a peptide of any one of SEQ ID NO:23
through SEQ ID NO:52.
29. The method according to claim 21, wherein the integrin binding
peptide comprises the amino acid sequence of a peptide of any one
of SEQ ID NO:23 through SEQ ID NO:52.
30. The method according to claim 21, wherein the integrin binding
peptide comprises the amino acid sequence of the peptide of SEQ ID
NO:49.
31. The method according to claim 21, wherein the integrin binding
peptide comprises the amino acid sequence of the peptide of SEQ ID
NO:50.
32. The method according to claim 21, wherein the integrin binding
peptide is conjugated to an agent.
33. The method according to claim 32, wherein the agent is a
chemotherapeutic agent.
34. The method according to claim 32, wherein the agent is a
half-life extending moiety.
35. The method according to claim 34, wherein the half-life
extending moiety is polyethylene glycol (PEG).
36. The method according to claim 32, wherein the agent is an
imaging agent.
37. The method according to claim 36, wherein the imaging agent is
a positron emission tomography (PET)-based imaging agent.
38. The method according to claim 37, wherein the (PET)-based
imaging agent comprises .sup.18F or .sup.64Cu.
39. The method according to claim 36, wherein the imaging agent is
a single photon emission computed tomography (SPECT) imaging
agent.
40. The method according to claim 39, wherein the SPECT imaging
agent is Indium-111, technetium-99m, or lodine-131.
41. A method of imaging a tissue highly expressing an endothelial
integrin that is at least one of .alpha.v.beta.5 integrin,
.alpha.v.beta.3 integrin and .alpha.5.beta.1 integrin, comprising
contacting said tissue with an integrin binding peptide, wherein
said integrin binding peptide comprises a knottin protein scaffold
comprising an engineered integrin binding loop that binds to at
least one of .alpha.v.beta.5 integrin, .alpha.v.beta.3 integrin and
.alpha.5.beta.1 integrin, wherein said integrin binding peptide
comprises an amino acid sequence at least 90% identical to the
amino acid sequence of a peptide of any one of SEQ ID NO:23 through
SEQ ID NO:52, and wherein said integrin binding peptide further
comprises a label that can be detected when the integrin binding
peptide is bound to the tissue, thereby imaging the tissue.
42. An integrin binding peptide or a pharmaceutical composition
comprising said integrin binding peptide, wherein said integrin
binding peptide comprises a knottin protein scaffold comprising an
engineered integrin binding loop that binds to at least one of
.alpha.v.beta.5 integrin, .alpha.v.beta.3 integrin and
.alpha.5.beta.1 integrin, and wherein said integrin binding peptide
comprises an amino acid sequence at least 90% identical to the
amino acid sequence of a peptide of any one of SEQ ID NO:23 through
SEQ ID NO:52.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/007,091 filed on Jan. 26, 2016, which is a
continuation of U.S. patent application Ser. No. 14/028,348 filed
on Sep. 16, 2013, now U.S. Pat. No. 9,265,845 issued on Feb. 23,
2016, which is a divisional of U.S. patent application Ser. No.
12/418,376 filed on Apr. 3, 2009, now U.S. Pat. No. 8,536,301
issued on Sep. 17, 2013, which is a continuation in part of
PCT/US2007/021218, filed on Oct. 3, 2007, which claims priority to
U.S. Provisional Patent Application No. 60/849,259 filed on Oct. 4,
2006, all of which are hereby incorporated by reference in their
entireties.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The present invention relates to the field of engineered
peptides, and to the field of peptides which bind to integrins,
and, particularly to integrin binding as it relates to cell growth
and development.
Related Art
[0004] Integrins are a family of extracellular matrix adhesion
proteins that noncovalently associate into .alpha. and .beta.
heterodimers with distinct cellular and adhesive specificities
(Hynes, 1992; Luscinskas and Lawler, 1994). Cell adhesion, mediated
though integrin-protein interactions, is responsible for cell
motility, survival, and differentiation. Each .alpha. and .beta.
subunit of the integrin receptor contributes to ligand binding and
specificity.
[0005] Protein binding to many different cell surface integrins can
be mediated through the short peptide motif Arg-Gly-Asp (RGD)
(Pierschbacher and Ruoslahti, 1984). These peptides have dual
functions: They promote cell adhesion when immobilized onto a
surface, and they inhibit cell adhesion when presented to cells in
solution. Adhesion proteins that contain the RGD sequence include:
fibronectin, vitronectin, osteopontin, fibrinogen, von Willebrand
factor, thrombospondin, laminin, entactin, tenascin, and bone
sialoprotein (Ruoslahti, 1996). The RGD sequence displays
specificity to about half of the 20 known integrins including the
.alpha..sub.5.beta..sub.1, .alpha..sub.8.beta..sub.1,
.alpha..sub.v.beta..sub.1, .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, .alpha..sub.v.beta..sub.6,
.alpha..sub.v.beta..sub.8, and .alpha..sub.iiib.beta.3 integrins,
and, to a lesser extent, the .alpha..sub.2.beta..sub.1,
.alpha..sub.3.beta..sub.1, .alpha..sub.4.beta..sub.1, and
.alpha..sub.7.beta..sub.1 integrins (Ruoslahti, 1996). In
particular, the .alpha..sub.v.beta..sub.3 integrin is capable of
binding to a large variety of RGD containing proteins including
fibronectin, fibrinogen, vitronectin, osteopontin, von Willebrand
factor, and thrombospondin (Ruoslahti, 1996; Haubner et al., 1997),
while the .alpha..sub.5.beta..sub.1 integrin is more specific and
has only been shown to bind to fibronectin (D'Souza et al.,
1991).
[0006] The linear peptide sequence RGD has a much lower affinity
for integrins than the proteins from which it is derived (Hautanen
et al., 1989). This due to conformational specificity afforded by
folded protein domains not present in linear peptides. Increased
functional integrin activity has resulted from preparation of
cyclic RGD motifs, alteration of the residues flanking the RGD
sequence, and synthesis of small molecule mimetics (reviewed in
(Ruoslahti, 1996; Haubner et al., 1997)).
[0007] The X-ray crystal structure of the 10th type III domain of
fibronectin (Dickinson et al., 1994), and the NMR solution
structures of the murine 9th and 10th type III fibronection domains
(Copie et al., 1998) containing the RGD sequence have been solved.
In these structures, the GRGDSP (SEQ ID NO: 105) amino acid
sequence makes a type II .beta.-hairpin turn that protrudes from
the rest of the fibronectin structure for interaction with integrin
receptors.
[0008] Short RGD peptides also have been shown to assume a type II
.beta.-turn in aqueous solution, as determined by NMR (Johnson et
al., 1993). Conformation and stereochemistry about the RGD motif in
the form of cyclic penta- and hexa-peptides, and
disulfide-constrained peptides have been studied extensively
(reviewed in (Haubner et al., 1997)). Previous approaches have
shown that combinations of natural and unnatural amino acids,
peptidomimetics, or disulfide bonds flanking the RGD motif have
been necessary to create high affinity, biologically active
.beta.-turn structures. The recent structure of an RGD 3-loop mimic
bound to .alpha..sub.v.beta..sub.3 (Xiong et al., 2002) has shed
some interesting light on the nature of the ligand-receptor
interaction and has validated the body of work encompassing the
ligand-based design strategy.
[0009] Previously, phage display technology has been used to
isolate cyclic peptides specific to different integrin receptors.
When a random linear hexapeptide library displayed on phage was
panned with immobilized integrin, the amino acid sequence CRGDCL
(SEQ ID NO: 1) was isolated (Koivunen et al., 1993). It was
determined that this peptide was 10-fold more potent than linear
RGD hexapeptides in inhibiting the binding of attachment of
.alpha..sub.5.beta..sub.1 expressing cells to fibronectin (Koivunen
et al., 1993). This cyclic peptide also inhibited cell adhesion
mediated by .alpha..sub.v.beta..sub.1, .alpha..sub.v.beta.3, and
.alpha..sub.v.beta..sub.5 integrins. In another study, phage
display was used to isolate selective ligands to the
.alpha..sub.5.beta..sub.1 .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, and .mu..sub.I1b .beta..sub.3 integrins
from phage libraries expressing cyclic peptides (Koivunen et al.,
1995). It was determined that each of the four integrins studied
primarily selected RGD-containing sequences, but preferred
different ring sizes and flanking residues around the RGD motif. A
cyclic peptide, ACRGDGWCG (SEQ ID NO: 2), was isolated that bound
with high affinity to the .alpha..sub.5.beta..sub.1 integrin. In
addition, the cyclic peptide ACDCRGDCFCG (SEQ ID NO: 3), which
contains two disulfide bonds, was shown to be 20-fold more
effective in inhibiting cell adhesion mediated by the
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 integrins
than comparable peptides with one disulfide bond, and 200-fold more
potent than linear RGD peptides.
[0010] Phage display has also been used to isolate novel integrin
binding motifs from peptide libraries. The cyclic peptide CRRETAWAC
(SEQ ID NO: 4) was identified from a random heptapeptide phage
library with flanking cystine residues (Koivunen et al., 1994).
This peptide was specific for binding to the
.alpha..sub.5.beta..sub.1 integrin, and not the
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 integrins,
and was determined to have an overlapping binding site with the RGD
sequence. The peptide NGRAHA (SEQ ID NO: 5) was identified by phage
display libraries as well (Koivunen et al., 1993), but it was later
determined that the receptor for this peptide was aminopeptidase N,
and not integrins as originally thought (Pasqualini et al., 2000).
A synergistic binding site on the 10th domain of fibronectin
(encompassing the sequence RNS) also enhances RGD binding to the
.alpha..sub.5.beta..sub.1 integrin (Koivunen et al., 1994; Obara
and Yoshizato, 1995). In addition, the sequence PHSRN (SEQ ID NO:
6) (from the 9th domain of fibronectin), increases
.alpha..sub.5.beta..sub.1 integrin binding to the RGD peptide in
fibronectin (Aota et al., 1994). The sequence ACGSAGTCSPHLRRP (SEQ
ID NO: 7) was identified from a 15-mer phage library panned with
.alpha..sub.v.beta..sub.3 integrin. The SAGT (SEQ ID NO: 139)
tetrapeptide is found in the sequence of vitronectin, suggesting
that this may be an accessory site for integrin recognition and
binding (Healy et al., 1995). It has been hypothesized that other
synergy sites may exist (reviewed in Ruoslahti, 1996), suggesting
that random peptide library screening for integrin ligands other
than RGD would be useful.
[0011] The presentation of multiple RGD motifs within one molecule
has been shown to increase integrin binding affinity and activity.
Numerous studies have demonstrated that multivalent clustering of
RGD ligands within a polymer coated surface or bead results in
enhanced cell adhesion, due to increased local concentration of
ligand, or increased ligand/receptor avidity. (Miyamoto et al.,
1995; Maheshwari et al., 2000; Pierschbacher et al., 1994;
Shakesheff et al., 1998). Soluble RGD repeats incorporated into
polypeptides (Saiki, 1997), or linked through a
poly(carboxyethylmethacrylamide) backbone (Komazawa et al., 1993)
have demonstrated an increased potential for inhibition of cancer
metastasis compared to free peptide. More recently, soluble
multivalent polymers of GRGD (SEQ ID NO: 8), and copolymers of GRGD
(SEQ ID NO: 8) and the .alpha..sub.5.beta..sub.1 synergy peptide
SRN have been prepared synthetically through ring-opening
metathesis (Maynard et al., 2001). Homopolymers containing GRGD
(SEQ ID NO: 8) peptides were more potent inhibitors of fibronectin
cell adhesion (IC.sub.50=0.18 mM) than peptide alone
(IC.sub.5o=1.08 mM). Heteropolymers containing both GRGD (SEQ ID
NO: 8) and SRN peptides exhibited an enhanced ability to block
fibronectin adhesion with an IC.sub.50 of 0.03 mM (Maynard et al.,
2001). Although multivalent homo- and hetero-oligomers of integrin
peptides demonstrated increased inhibition of cell adhesion,
improvements in affinity and efficacy are contemplated through the
use of multivalent frameworks.
[0012] The growth of new blood vessels, termed angiogenesis, plays
an important role in development, wound healing, and inflammation
(Folkman and Shing, 1992). Angiogenesis has been implicated in
proliferative disease states such as rheumatoid arthritis, cancer,
and diabetic retinopathy, and therefore is a relevant and
attractive target for therapeutic intervention. In cancer, the
growth and survival of solid tumors is dependent on their ability
to trigger new blood vessel formation to supply nutrients to the
tumor cells (Folkman, 1992). With this new tumor vascularization
comes the ability to release tumor cells into the circulation
leading to metastases. One specific approach to anti-angiogenic
therapy is to inhibit cell adhesion events in endothelial cells.
The .alpha..sub.v.beta..sub.3 (Brooks et al., 1994) and
.alpha..sub.v.beta..sub.5 integrins (Friedlander et al., 1995), and
more recently the .alpha..sub.5.beta..sub.1 integrin (Kim et al.,
2000), have been shown to be required for angiogenesis in vascular
cells. Brooks and colleagues demonstrated that the
.alpha..sub.v.beta..sub.3 integrin was abundantly expressed on
blood vessels, but not on dermis or epithelial cells, and
expression was upregulated on vascular tissue during angiogenesis
(Brooks et al., 1994). In addition, the .alpha..sub.v.beta..sub.1
integrin has been shown to be expressed on the tumor vasculature of
breast, ovarian, prostate, and colon carcinomas, but not on normal
adult tissues or blood vessels (Kim et al., 2000). The
.alpha..sub.v.beta..sub.3 (and .alpha..sub.v.beta..sub.5) integrins
are highly expressed on many tumor cells such as osteosarcomas,
neuroblastomas, carcinomas of the lung, breast, prostate, and
bladder, as well as glioblastomas, and invasive melanomas (reviewed
in (Haubner et al., 1997). It has also been demonstrated that the
expression levels of .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5 by the vascular endothelium of
neuroblastoma was associated with the aggressiveness of the tumor
(Erdreich-Epstein et al., 2000).
[0013] A monoclonal anti-.alpha..sub.v.beta..sub.3 antibody (LM609)
was shown to inhibit angiogenesis by fibroblast growth factor
(FGF), tumor necrosis factor-a, and human melanoma fragments
(Brooks et al., 1994). The humanized version of LM609, termed
Vitaxin, has been shown to suppress tumor growth in animal models
(Brooks et al., 1995), and target angiogenic blood vessels (Sipkins
et al., 1998). Vitaxin has undergone Phase I clinical trials in
humans and appears to be safe and potentially active in disease
stabilization (Gutheil et al., 2000). In another study,
function-blocking anti-.alpha..sub.5.beta..sub.1 monoclonal
antibodies were shown to inhibit cell adhesion to fibronectin, and
inhibit FGF-induced angiogenesis in vivo (Kim et al., 2000). In
addition, RGD peptides selective to .alpha..sub.v (Pasqualini et
al., 1997) and .alpha..sub.5.beta..sub.1 integrins (Kim et al.,
2000) are relevant targets for imaging and therapeutic purposes.
Bacteriophage displaying an RGD peptide (CDCRGDCFC) (SEQ ID NO: 9)
with high affinity to a integrins was shown to localize to tumor
blood vessels when injected into tumor-bearing mice (Ruoslahti,
2000). In other approaches, RGD containing peptides and
peptidomimetics have demonstrated promise in cancer therapy by
binding to overexpressed cell surface integrins and interfering
with angiogenesis and tumor blood supply. Inhibition of
.alpha..sub.v.beta..sub.3 and .mu..sub.v.beta..sub.5 integrins by
cyclic RGD peptides resulted in significant reduction of functional
blood vessel density, and was shown to impair tumor growth and
metastasis in vivo (Brooks et al., 1994; Buerkle et al., 2002). In
addition, the cyclic peptide c(RGDfV) (SEQ ID NO: 10) was shown to
cause .alpha..sub.v.beta..sub.3-mediated apoptosis in human
malignant glioma cells (Chatterjee et al., 2000) and prostate
cancer cells (Chatterjee et al., 2001). The cyclic peptide
antagonist CRRETAWAC (SEQ ID NO: 11), and the nonpeptide antagonist
SJ749, were shown to selectively inhibit
.alpha..sub.5.beta..sub.1-mediated cell adhesion to fibronectin, as
well as block FGF-induced angiogenesis in vivo (Kim et al., 2000).
Of particular interest, the integrin inhibitors seem to have no
effect on normal vessels, and appear to function by specifically
inducing apoptosis in newly budding endothelial cells during
angiogenesis (Brooks et al., 1994), and interfering with the
function of metalloproteinase enzymes required for cellular
invasion (Brooks et al., 1996).
[0014] Radiolabeled integrin antagonists as described below are
useful in tumor targeting and imaging applications. Noninvasive
methods to visualize and quantify integrin expression in vivo are
crucial for clinical applications of integrin antagonists (Brower,
1999). The first generation of radioiodinated cyclic RGD peptides
exhibited high affinity and specificity in vitro and in vivo for
.alpha..sub.v.beta..sub.3 integrins however, exhibited rapid
excretion and accumulation in the liver and intestines, limiting
their application (Haubner et al., 1999). Modifications of these
peptides with a sugar moiety reduced their uptake in the liver, and
increased their accumulation in
.alpha..sub.v.beta..sub.3-expressing tumors in vivo (Haubner et
al., 2001). Noninvasive imaging with an .sup.18F-labeled version of
this glycoRGD peptide by positron emission tomography demonstrated
receptor-specific binding and high tumor to background ratios in
vivo, suggesting suitability for .alpha..sub.v.beta..sub.3
quantification and therapy (Haubner et al., 2001). In addition, RGD
peptides coupled to chelating agents could be radiolabeled with
.sup.111In, .sup.125I, .sup.90Y, and .sup.177Lu, enlarging their
potential for both tumor imaging and radionuclide therapy (van
Hagen et al., 2000). Integrin-specific antibodies can also be
useful for imaging applications. Paramagnetic liposomes coated with
the anti .alpha..sub.v.beta..sub.3 integrin antibody LM609 were
used for detailed imaging of rabbit carcinomas for a noninvasive
means to asses growth and malignancy of tumors (Sipkins et al.,
1998). The small integrin binding proteins described below would
therefore be very amenable to coupling to a variety of
radionuclides and chemotherapeutic agents.
PATENTS AND PUBLICATIONS
[0015] Ruoslahti et al., have obtained a series of patents relating
to RGD peptides. For example, U.S. Pat. No. 5,695,997, entitled
"Tetrapeptide," relates to a method of altering cell attachment
activity of cells, comprising: contacting the cells with a
substantially pure soluble peptide including RGDX where X is any
amino acid and the peptide has cell attachment activity. The patent
further includes an embodiment where X is any amino acid and the
peptide has cell attachment activity and the peptide has less than
about 31 amino acids.
[0016] Similarly, U.S. Pat. No. 4,792,525 relates to a
substantially pure peptide including as the
cell-attachment-promoting constituent the amino acid sequence
Arg-Gly-Asp-R wherein R is Ser, Cys, Thr or other amino acid, said
peptide having cell-attachment promoting activity, and said peptide
not being a naturally occurring peptide.
[0017] U.S. Pat. No. 5,169,930, to Ruoslahti, et al., relates to a
substantially pure integrin receptor characterized in that it
consists of an .alpha..sub.v.beta..sub.1 subunit.
[0018] U.S. Pat. No. 5,536,814, to Ruoslahti, et al., entitled
"Integrin-binding peptides," issued Jul. 16, 1996, discloses a
purified synthetic peptide consisting of certain specified amino
acid sequences.
[0019] U.S. Pat. No. 5,519,005, to Ofer et al., relates to certain
non-peptidic compounds comprising a guanidino and a carboxyl
terminal groups with a spacer sequence of 11 atoms between them,
which are effective inhibitors of cellular or molecular
interactions which depend on RXD or DGR recognition, wherein X is G
(gly), E (glu), Y (tyr), A (ala) or F (phe). These RXD and DGR
analogues are referred to as "RXD surrogates."
[0020] US 2005/0164300 to Artis, et al., published Jul. 28, 2005,
entitled "Molecular scaffolds for kinase ligand development,"
discloses molecular scaffolds that can be used to identify and
develop ligands active on one or more kinases, for example, the PIM
kinases, (e.g., PIM-1, PIM-2, and PIM-3).
[0021] U.S. Pat. No. 6,451,976, to Lu et al., discloses a process
in which dendroaspin, a polypeptide neurotoxin analogue, is
modified by recombinant DNA techniques, particularly "loop
grafting," to provide a modified polypeptide.
[0022] U.S. Pat. No. 6,962,974, to Kalluri et al., issued Nov. 8,
2005, discloses recombinantly-produced Tumstatin, comprising the
NCl domain of the .alpha.3 chain of Type IV collagen, having
anti-angiogenic activity, anti-angiogenic fragments of the isolated
Tumstatin, multimers of the isolated Tumstatin and anti-angiogenic
fragments, and polynucleotides encoding those anti-angiogenic
proteins.
[0023] U.S. Pat. No. 5,766,591, to Brooks et al., relates to a
method of inducing solid tumor regression comprising administering
an RGD-containing integrin .alpha.v.beta.3 antagonist.
[0024] U.S. Pat. No. 5,880,092 to Pierschbacher et al., relates to
a substantially pure compound comprising an Arg-Gly-Asp sequence
stereochemically stabilized through a bridge and having a molecular
weight less than about 5.4 kilodaltons.
[0025] U.S. Pat. No. 5,981,468 to Pierschbacher et al., relates to
a compound having a stabilized stereochemical conformation of a
cyclic RGD peptide.
[0026] Koivunen et al., "Phage Libraries Displaying Cyclic Peptides
with Different Ring Sizes: Ligand Specificities of the RGD-Directed
Integrins," Bio/Technology 13:265-270 (1995) discloses selective
ligands to the cell surface receptors of fibronectin
(.alpha..sub.5.beta..sub.1 integrin), vitronectin
((.alpha..sub.v.beta..sub.3 integrin and .alpha..sub.5.beta..sub.5
integrin and fibrinogen ((.alpha..sub.m.beta..sub.3 integrin from
phage libraries expressing cyclic peptides. A mixture of libraries
was used that express a series of peptides flanked by a cystine
residue on each side (CX5C, CX6C, CX7C) or only on one side (CX9)
of the insert.
[0027] Reiss et al., "Inhibition of platelet aggregation by
grafting RGD and KGD sequences on the structural scaffold of small
disulfide-rich proteins," Platelets 17(3):153-7 (May 2006)
discloses RGD and KGD containing peptide sequences with seven and
11 amino acids, respectively, which were grafted into two cystine
knot microproteins, the trypsin inhibitor EETI-II and the
melanocortin receptor binding domain of the human agouti-related
protein AGRP, as well as into the small disintegrin obtustatin.
[0028] Wu et al., "Stepwise in vitro affinity maturation of
Vitaxin, an .alpha..sub.v.beta..sub.3-specific humanized mAb,"
Proc. Nat. Acad. Sci. Vol. 95, Issue 11, 6037-6042, May 26, 1998,
discloses a focused mutagenesis implemented by codon-based
mutagenesis applied to Vitaxin, a humanized version of the
antiangiogenic antibody LM609 directed against a conformational
epitope of the .alpha..sub.v.beta..sub.3 integrin complex. Wu et
al., "Stepwise in vitro affinity maturation of Vitaxin, an
v3-specific humanized mAb," Proc. Nat. Acad. Sci., Vol. 95, Issue
11, 6037-6042, May 26, 1998, discloses a focused mutagenesis
implemented by codon-based mutagenesis applied to Vitaxin, a
humanized version of the antiangiogenic antibody LM609 directed
against a conformational epitope of the .alpha..sub.v.beta..sub.3
integrin complex.
BRIEF SUMMARY OF THE INVENTION
[0029] The following brief summary is not intended to include all
features and aspects of the present invention, nor does it imply
that the invention must include all features and aspects discussed
in this summary.
[0030] In certain aspects, the present invention comprises an
artificial integrin binding peptide, based on a combination of a
knottin peptide and an engineered loop, where the engineered loop
provides a binding sequence specific to bind to at least one of
.alpha..sub.v.beta..sub.5 integrin, .alpha..sub.v.beta..sub.3
integrin and .alpha..sub.5.beta..sub.1 integrin, said binding
sequence being comprised in a knottin protein scaffold. The knottin
protein provides a "scaffold" due to its relatively rigid
three-dimensional structure. The binding sequence will be an
engineered integrin binding loop between 9 and 13 amino acids long,
said loop comprising the sequence RGD. Said scaffold, except for
the engineered integrin binding loop, is identical or at least
substantially identical to one of: EETI-II, AgRP, mini-AGRP,
agatoxin or miniagatoxin. It is shown here that certain scaffolds
are tolerant to mutations in their loop regions.
[0031] In certain aspects, the present invention comprises an
integrin binding peptide, comprising a binding sequence, which
specifically binds to one or both of .alpha..sub.v.beta.5 and
.alpha..sub.v.beta.3 integrins. Certain sequences also bind only to
.alpha..sub.5.beta..sub.1 integrin. It has been shown that some of
the present peptides will bind to only .alpha..sub.v.beta..sub.5
and .alpha.v.beta.3 integrins and not .alpha..sub.5.beta..sub.1.
The present engineered peptides further comprise a molecular
scaffold which is a knottin protein. As described, the knottin
proteins are characterized by intramolecular bonds which stabilize
them and form a rigid scaffold. A portion of the scaffold, e.g., a
loop beginning at residue 3 of EETI-II, is replaced by a sequence
that has been discovered, though in vitro molecular evolution, to
have superior binding properties. The peptide thus has a scaffold
comprising replacement of a portion of the knottin with an integrin
binding loop between 9 and 13 amino acids long, said peptide
substantially identical to one of: EETI sequences as set forth in
Table 2, AgRP sequences as set forth in Table 3 or mini-RGD-AgRP
sequences as set forth in Table 4.
[0032] The present invention may further be characterized in that
it comprises an integrin binding peptide comprising a molecular
scaffold, wherein the molecular scaffold is covalently linked to
either end of an RGD mimic sequence, which is a loop consisting of
about 8-12 amino acids, which comprise the sequence RGD, and
preferably are selected from the group consisting of XXXRGDXXXXX
(sequence (a)), 11 amino acids and XXRGDXXXX (sequence (b)), 9
amino acids, where X is any amino acid and said mimic sequence is
linked at either end in the vicinity of, preferably immediately
adjacent to, cross-linked residues, e.g., cysteines. The molecular
scaffold is preferably taken from a knottin peptide, and the mimic
sequence is inserted in the scaffold between the two cysteine
residues. The identity of the residues "X" can be varied in that,
together, the X residues flanking the binding motif (RGD, RYD,
etc.), provided a certain structure that will selectively recognize
the ligand, in this case an endothelial integrin. Directed
evolution techniques were used and peptides with surprising
selectivity and binding affinity were obtained. It has been found
that the number of residues on either side of the RGD sequence is
critical, particularly in relation to the three dimensional
structure of the flanking Cys residues. That is, the location of
RGD as after 3 residues and before 5 residues (sequence (a)) is
important with regard to the EETI scaffold, while the location in
sequence (b) is similarly important in the AgRP or agatoxin
scaffold. The present EETI peptides will have 2-5 disulfide
linkages between cysteine residues, where the linkages are not
directly between Cys residues immediately flanking the RGD loop, as
shown in FIG. 3. In other knottins, there may be a disulfide
linkage immediately flanking the loop, but in each case, there are
at least two disulfide linkages, forming a molecular scaffold.
[0033] The present integrin binding peptides will have a specific
affinity for an integrin selected from the group consisting of
.alpha..sub.5.beta..sub.1, .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5, particularly .alpha..sub.v.beta..sub.3.
The present peptides preferably have a Kd less than 100 nM, or,
more preferably less than 70 nM. Also they preferably do not bind
to integrin .alpha.IIb.beta.3, which is found on platelets. The
term Kd means a dissociation constant, as is known in the art;
lower Kd indicates tighter binding between the peptide and the
integrin.
[0034] The molecular scaffold is preferably selected from the group
consisting of EETI, AgRP, and agatoxin.
[0035] The sequences may be taken from a peptide having a sequence
substantially identical to a peptide listed in Table 1 (EETI
scaffold containing native fibronectin loop), Table 2 (EETI mutant,
RGD in loop 4-6), Table 3 (AgRP peptides, RGD in loop) or Table 4
(mini-RGD-AgRP peptides, RGD in loop). Substantial identity may be
regarded as least 70% identical, or at least 90-95% identical.
Substantial identity may be different in the RGD loop and in the
knottin scaffold.
[0036] The peptides of the present invention can be made by
recombinant DNA production techniques, including a vector encoding
a peptide sequence according to the present invention. The DNA
sequences are chosen according to the genetic code, with codon
preferences given according to the host cell, e.g., mammalian,
insect, yeast, etc. The present peptides may also be made by
peptide synthetic methods.
[0037] Thus there is provided a method of inhibiting binding of an
integrin to vitronectin and, in some cases, fibronectin, comprising
contacting said integrin with an integrin binding peptide
comprising a molecular scaffold, wherein the molecular scaffold is
covalently linked to either end of an RGD mimic sequence selected
from the group consisting of XXXRGDXXXXX and XXRGDXXXX, where X is
any amino acid. The present invention has been demonstrated with
comparison to the 10.sup.th domain of fibronectin ("10FN" in the
figures, e.g., FIGS. 4(f) and 4(i)).
[0038] Also provided is a method of treating a proliferative
disease comprising the step of administering to a subject in need
thereof a composition comprising an integrin binding peptide
comprising a molecular scaffold, wherein the molecular scaffold is
covalently linked to either end of an RGD mimic sequence selected
from the group consisting of XXXRGDXXXXX and XXRGDXXXX, where X is
any amino acid. A wide variety of proliferative disorders will
respond to the integrin inhibiting effects of the present peptides,
which have been demonstrated with integrin
.alpha..sub.v.beta..sub.3. .alpha..sub.v.beta..sub.5, and in some
cases .alpha..sub.5.beta..sub.1. For example, adhesive interaction
of vascular cells through this integrin is known to be necessary
for angiogenesis, and an antibody to this integrin has been shown
to block angiogenesis. The present peptides may also be used in
vitro or in vivo, e.g., in bone or tissue grafts, to promote cell
adhesion by binding to cells expressing a selected integrin. The
present peptides may also be used as imaging agents, in recognition
of their affinity for integrins, which are more highly expressed in
certain types of cells. For example, tumor cells express higher
levels of these integrins.
[0039] Also provided is a method for imaging tumors, in which
engineered integrin binding peptides specific for certain integrins
are administered to a living organism, and the binding of the
peptides to sites where endothelial integrins are highly expressed
serves to image tumors. The peptides disclosed here may be
conjugated to a dye or radiolabel for such imaging.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1A shows an example of flow cytometry data depicted as
a dot plot of individual cells. Yeast cells are double-labeled with
a labeled antibody against the c-myc epitope tag (x-axis), and
ligand labeled with another dye (y-axis). Since protein expression
levels on the yeast cell surface are variable, a `diagonal` cell
population results, in which cells that express more protein bind
more ligand; flow cytometry data is depicted as a dot plot of
individual cells.
[0041] FIG. 1B shows a schematic of the present yeast display
system; yeast fusion proteins are expressed on the cell surface
(Boder and Wittrup, 1997). The yeast display construct shown in
FIG. 1A has the general orientation: Aga2-HA-engineered
knottin--c-myc epitope, with the c-myc epitope at the carboxy
terminus of the peptide. The displayed knottin is labeled with a
chicken anti-cmyc antibody, which is then detected with an Alexa
555-labeled anti-chicken secondary antibody. The displayed knottin
is allowed to bind to a test integrin. Bound integrin is detected
with an anti-integrin antibody, labeled with FITC.
[0042] FIG. 2 (left panel) is a schematic representation of an
integrin antagonist having high specificity for one integrin
(.alpha..sub.v.beta..sub.3 here) engineered using yeast display and
flow cytometry enrichment as referred to in FIG. 1. Integrin
antagonists with ultra high specificity will allow for detection
and inhibition of only certain integrins. FIG. 2 (right panel)
shows a high avidity integrin-binding protein in which the integrin
binding proteins described below are presented in a tetravalent
manner through linkage to a GCN 4-zipper, which spontaneously
self-assembles to form a tetramer. Tetravalent presentation of the
integrin antagonists will enhance integrin binding by increasing
the local concentration of antagonist, upon binding of the first
antagonist.
[0043] FIGS. 3A, 3B and 3C show the positions of the Cys-Cys
disulfide linkages in the sequences of knottin proteins EETI-II
(SEQ ID NO: 13), AgRP (SEQ ID NO: 107) and omega agatoxin 4B (SEQ
ID NO: 16). Cysteine residues can be seen to be immediately
flanking the RGD mimic loops, which, in the present engineered
peptides, are between the brackets. For example, in AgRP, it can be
seen that the cysteines flanking the RGD mimic sequence will be
linked to each other, whereas in EETI they are not. The size of the
grafted sequence will depend on the molecular framework structure,
such that shorter loops will be preferred in cases where they are
in the framework adjacent linked cysteines. Other loops between Cys
residues may be engineered according to the present methods.
Disulfide linkages for other knottin proteins are set forth in the
knottin database. FIG. 3A-C is adapted from Biochemistry, 40,
15520-15527 (2001) and J. Biol. Chem., 2003, 278:6314-6322.
[0044] FIG. 4A-FIG. 4I is a series of nine panels, (a) through (i)
from top left to bottom right, showing flow cytometry data obtained
for yeast-displayed RGD-EETI#3 (also called FN-RGD). Panels (a)
through (c) are controls; FL1-H represents the signal generated
from the FITC-labeled integrin antibody, and FL2-H represents the
signal generated from the chicken anti-cmyc antibody+Alexa-555
labeled anti-chicken secondary antibody. Panels (d) through (f) are
histograms of the data presented below in panels (g) (h) and (i).
Panels (g), (h) and (i) (Bottom row) are dot plots of RDG-EETI#3
induced at 30.degree. C., with 100 nM integrin
.alpha..sub.5.beta..sub.3 (e), at 20.degree. C., with 100 nM
integrin .alpha..sub.v.beta..sub.3 (f) and 10FN (10.sup.th domain
of fibronectin) induced at 30.degree. C., with 100 nM integrin
.alpha..sub.v.beta..sub.3. The plots show that the RGD-EETI#3
(FN-RGD) peptide binds better than the 10.sup.th domain of
fibronectin, a natural .alpha..sub.v.beta..sub.3 integrin binder,
and, further, that the peptide folds correctly at both 30.degree.
C. and 20.degree. C. expression.
[0045] FIG. 5A through FIG. 5G is a series of seven panels, (a)
through (g) from top left to bottom right, showing flow cytometry
data obtained for yeast-displayed RGD-AgRP#3 (panel d), Agatoxin #2
(panel e), mini AgRP (panel f) and mini-RGD-Agatoxin (panel g).
Panels (a) through (c) are controls; parameters are FL1-H and FL2-H
are as in FIG. 4; Second row panels (d) and (e) are, respectively,
dot plots of RGD-AgRP#3 with 100 nM integrin
.alpha..sub.v.beta..sub.3 and RGD-agatoxin #2 with 100 nM integrin
.alpha..sub.v.beta..sub.3. Third row panels (f) and (g) are,
respectively, dot plots of mini-RGD-AgRP with 100 nM integrin
.alpha..sub.v.beta..sub.3 and mini-RGD-agatoxin with 100 nM
integrin .alpha..sub.v.beta..sub.3. The plots show that the "mini"
versions of RGD-AgRP #3 and RGD-agatoxin#2 bind to integrin
.alpha..sub.v.beta..sub.3 just as well as the full-length
versions.
[0046] FIG. 6A through FIG. 6M is a series of 13 panels (a) through
(m) showing dot plots of EETI-based RGD mutants obtained by
directed evolution, labeled with 100 nM of
.alpha..sub.v.beta..sub.3 integrin. The first row consists of
controls. FL1H and FL2H are labeled as before. The samples are
labeled from left to right for each row. Samples 1.5B (d), 1.4B
(e), 1.5F (f), 2.4F (g), 2.5A (h), 2.5C (i), 2.5D (j), 2.5F (k),
2.5H (1) and 2.5J (m) represent EETI-based variants as set forth in
Table 2.
[0047] FIG. 7A through FIG. 7M is a series of 13 panels showing dot
plots and histograms (panels (c), (f), (i), (k), and (m)) showing a
control (a), and the samples as labeled in the center column of the
figure, i.e., 1.5B, 2.4F, 2.5A, 2.5D and 2.5J. These peptides are
labeled with 50 nM integrin .alpha..sub.v.beta..sub.3 and further
defined in the table below. The flow cytometry parameters are as
given above. FIG. 7 shows that the best mutants appear to be 1.5B,
2.5A, and 2.5D. This data suggests K.sub.d values of about 50 nM.
When displayed on the yeast cell surface, these mutants bind to
.alpha..sub.v.beta..sub.3 integrin about 2-3.times. better than the
starting mutant RGD-EETI#3 (FN-RGD), although this is a gross
estimate since we did not have enough soluble
.alpha..sub.v.beta..sub.3 integrin to perform full titration
curves.
[0048] FIG. 8A is a series of 4 pictures showing in vivo imaging of
Cy5.5-labeled polypeptides in mice. 1.5 nmol of Cy5.5-labeled
EETI-RGD peptide 2.5D or other indicated peptide was injected by
tail vein into U87MG glioblastoma xenograft mouse models and imaged
at various time points post injection. Arrows indicate the position
of tumors. FIG. 8B is a graph showing quantified tumor/normal
tissue ratio for Cy5.5-labeled 2.5D (top line, triangles) compared
to Cy5.5-labeled FN-RGD (middle line, squares) and Cy5.5-labeled
c(RGDyK) (SEQ ID NO: 140) (middle line, circles). The
tumor/background ratio shows .about.60% greater contrast for the
high affinity evolved 2.5D peptide over the weaker binding FN-RGD
and c(RGDyK) (SEQ ID NO: 140) peptides. Cy5.5-labeled FN-RDG
negative control (bottom line, open squares) indicates background
levels. FIG. 8C is a series of images of different organs showing
uptake of Cy5.5-labeled 2.5D and a comparison peptide, c(RGDyK)
(SEQ ID NO: 140). It can be seen that the tumor took up
significantly more 2.5D than c(RGDyK) (SEQ ID NO: 140), and that
other organs were not significantly showing fluorescence, except
for the kidney, where the peptide would accumulate prior to
excretion.
[0049] FIG. 9 is a graph showing normalized competition plotted
against peptide concentration in an integrin-binding assay on U87MG
glioblastoma cells. Relative polypeptide binding affinity was
measured by competition of .sup.125I-labeled echistatin with
unlabeled echistatin (line 1), 2.5D (line 2), FN-RGD (line 5),
c(RGDyK) (SEQ ID NO: 140) (line 4), and scrambled FN-RDG (line
3).
[0050] FIG. 10A through FIG. 10D is a series of histograms showing
binding specificities to integrins .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, .alpha..sub.5.beta..sub.1, and
.alpha..sub.iib.beta..sub.3 for engineered EETI-RGD peptides
compared to controls. Error bars represent experiments performed in
triplicate. Competition binding of 0.06 nM .sup.125I echistatin
with 5 nM (black bars) and 50 nM (grey bars) unlabeled peptide to
plate-coated integrins was measured. 1=echistatin; 2=c(RGDyK) (SEQ
ID NO: 140); 3=FN-RGD; 4=1.5B; 4=2.5D; 5=2.5F; 6=FN-RDG. The
engineered peptides have very little binding to
.alpha..sub.iib.beta..sub.3 integrin.
[0051] FIG. 11 is a series of histograms showing residue
distribution of mutants isolated from EETI XXXRGDXXXXX library #2.
The distribution of residues in different positions is shown for
each position.
[0052] FIGS. 12A and 12B are a pair of bar graphs showing binding
results of mutagenesis of AgRP loops 1-3 using degenerate codons.
Binding of 50 nM integrin to yeast-displayed AgRP peptide clones
from sort round 4 of degenerate codon libraries is shown. Top graph
shows binding to .alpha.v.beta.3 integrin; bottom graph shows
binding to .alpha.iib.beta.3 integrin. Background, marked "bg"
indicates cells stained with fluorescein-conjugated anti-integrin
antibodies only. Numbers are arbitrary fluorescence units.
[0053] FIG. 13 is a chart showing radioactivity accumulation
quantification in selected organs of U87-MG tumor bearing mice at
30 min, 1 hour, 2 hour, 4 hour and 24 hour after injection of
.sup.64Cu-DOTA-7C AgRP engineered peptide.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Overview
[0054] The present invention involves the selection of a knottin
protein as a peptide framework (scaffold) and replacing a portion
of the sequence that appears on the surface with a specific binding
sequence, e.g., containing an integrin binding sequence (RGD). The
resulting engineered peptides have high affinity and specificity
for selected integrins present on surfaces of tumor cells,
epithelial cells, and the like.
[0055] Directed evolution is a useful technology for creating novel
biomolecules that enhance or mimic protein function. Small
polypeptides with applications as therapeutics and research tools
were developed using directed evolution. These peptides are
amenable to chemical synthesis and offer facile incorporation into
biomaterials. Using molecular cloning, biologically active amino
acid sequences derived from cell adhesion proteins (fibronectin)
were grafted into several stable, constrained knottin peptide
frameworks (EETI, AgRP and Agatoxin IVB) and were shown to bind to
integrin receptors (.alpha..sub.v.beta..sub.3) with modest
affinity. Since polypeptide conformation is critical for high
affinity receptor binding and specificity, prototype molecules were
subjected to affinity maturation using molecular evolution.
Combinatorial libraries of mutants displayed on the yeast cell
surface were screened by flow cytometric sorting to isolate
polypeptides with enhanced integrin binding affinity. These
proteins specifically modulate integrin-mediated cell adhesion and
can serve as molecular imaging agents. These results demonstrate
that naturally occurring constrained peptide scaffolds 1) can be
redirected to function as adhesion molecule mimics and 2) can be
engineered for enhanced integrin binding affinity through directed
evolution.
[0056] The present methods have led to the development of specific
binding peptides against the .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, and, in some cases, the
.alpha.5.beta..sub.1 integrin receptors, which have been implicated
in cell adhesion and angiogenesis of vascular tissue in cancer.
Integrin-specific binders comprised of the cyclic peptide
Arg-Gly-Asp (RGD, discussed in Background) have shown much
therapeutic promise, but can benefit from improvements in affinity
and stability. A novel selection approach based on yeast surface
display was utilized for affinity maturation and stabilization of
molecular scaffolds containing the RGD motif. In addition,
frameworks for multivalent RGD ligand presentation through chemical
crosslinking and protein engineering are presented.
[0057] Knottin proteins containing RGD motifs were assayed for
binding against integrins. It was found that the scaffolds offer an
extremely stable platform for conformationally constrained ligand
presentation and are a useful framework for protein engineering
studies. In addition, multivalent protein scaffolds can be
engineered by replacing multiple binding faces of knottin proteins
with RGD motifs for enhanced integrin binding.
[0058] Multivalent presentation of integrin-specific motifs through
chemical crosslinking is also contemplated here. Receptor
clustering has been shown to be important for high avidity integrin
binding and function. A series of crosslinking agents can be
developed for multivalent integrin ligand presentation using novel
coupling methodology. Synergistic effects have been shown to exist
between RGD and other integrin-specific peptide motifs. Therefore,
cross linkers could also be designed that incorporate
heterofunctional groups to couple different integrin-specific
molecules. These multivalent integrin binding proteins and peptides
can be tested for their ability to enhance integrin binding and
antagonism of cell adhesion.
[0059] Combinatorial mutant libraries of RGD-based knottin
scaffolds expressed on yeast were screened for specific, high
affinity binding against soluble .alpha..sub.v.beta..sub.3 integrin
using flow cytometry.
Definitions
[0060] The term "molecular scaffold" means a polymer having a
predefined three-dimensional structure, into which can be
incorporated a binding loop, which will contain an RGD mimic as
described herein. The term "molecular scaffold" has an
art-recognized meaning (in other contexts), which is also intended
here. For example, a review by Skerra, "Engineered protein
scaffolds for molecular recognition," J. Mol. Recognit. 2000;
13:167-187 describes the following scaffolds: single domains of
antibodies of the immunoglobulin superfamily, protease inhibitors,
helix-bundle proteins, disulfide-knotted peptides and lipocalins.
Guidance is given for the selection of an appropriate molecular
scaffold.
[0061] Incorporation of integrin binding motifs into a molecular
(e.g., protein) scaffold offers a framework for ligand presentation
that is more rigid and stable than linear or cyclic peptide loops.
In addition, the conformational flexibility of small peptides in
solution is high, and results in large entropic penalties upon
binding. Incorporation of an RGD motif into a protein scaffold
provides conformational constraints that are required for high
affinity integrin binding, (as evidenced by the CDCRGDCFC (SEQ ID
NO: 12) peptide described above (Koivunen et al., 1995)).
Furthermore, the scaffold provides a platform to carry out protein
engineering studies such as affinity or stability maturation.
[0062] Characteristics of a desirable scaffold for protein design
and engineering include 1) high stability in vitro and in vivo, 2)
the ability to replace amino acid regions of the scaffold with
other sequences without disrupting the overall fold, 3) the ability
to create multifunctional or bispecific targeting by engineering
separate regions of the molecule, and 4) a small size to allow for
chemical synthesis and incorporation of non-natural amino acids if
desired. Scaffolds derived from human proteins are favored for
therapeutic applications to reduce toxicity or immunogenicity
concerns, but are not always a strict requirement. Other scaffolds
that have been used for protein design include fibronectin (Koide
et al., 1998), lipocalin (Beste et al., 1999), cytotoxic T
lymphocyte-associated antigen 4 (CTLA-4) (Hufton et al., 2000), and
tendamistat (McConnell and Hoess, 1995; Li et al., 2003). While
these scaffolds have proved to be useful frameworks for protein
engineering, molecular scaffolds such as knottins have a distinct
advantage: their small size.
[0063] The term "proliferative diseases" refers to diseases in
which some tissue in a patient proliferates at a greater than
normal rate. Proliferative diseases may be cancerous or
non-cancerous. Non-cancerous proliferative diseases include
epidermic and dermoid cysts, lipomas, adenomas, capillary and
cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas,
nephromas, myofibromatosis, osteoplastic tumors, other dysplastic
masses and the like.
[0064] The types of proliferative diseases which may be treated or
imaged with compounds and compositions of the present invention
include epidermic and dermoid cysts, lipomas, adenomas, capillary
and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas,
nephromas, myofibromatosis, osteoplastic tumors, other dysplastic
masses and the like.
[0065] The types of cancers which may be treated or imaged with
compounds and compositions of the present invention include: breast
carcinoma, bladder carcinoma, brain cancer, colorectal carcinoma,
esophageal carcinoma, gastric carcinoma, germ cell carcinoma e.g.,
testicular cancer, gynecologic carcinoma, hepatocellular carcinoma,
small cell lung carcinoma, non-small cell lung carcinoma,
lymphomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma, malignant
melanoma, multiple myeloma, neurologic carcinoma, ovarian
carcinoma, pancreatic carcinoma, prostate carcinoma, renal cell
carcinoma, Ewings sarcoma, osteosarcoma, soft tissue sarcoma,
pediatric malignancies and the like.
[0066] The term "effective amount" means an amount of a compound of
the present invention that is capable of modulating binding of an
integrin to a cognate ligand.
[0067] The term "knottin protein" means a structural family of
small proteins, typically 25-40 amino acids, that bind to a range
of molecular targets like proteins, sugars and lipids. Their
three-dimensional structure is essentially defined by a peculiar
arrangement of three to five disulfide bonds. A characteristic
knotted topology with one disulfide bridge crossing the macro-cycle
limited by the two other intrachain disulfide bonds, which was
found in several different microproteins with the same cysteine
network, lent its name to this class of biomolecules. Although
their secondary structure content is generally low, the knottins
share a small triple-stranded antiparallel .beta.-sheet, which is
stabilized by the disulfide bond framework. Biochemically
well-defined members of the knottin family, also called cysteine
knot proteins, include the trypsin inhibitor EETI-II from Ecballium
elaterium seeds, the neuronal N-type Ca.sup.2+ channel blocker
.omega.-conotoxin from the venom of the predatory cone snail Conus
geographus, agouti-related protein (See Millhauser et al., "Loops
and Links: Structural Insights into the Remarkable Function of the
Agouti-Related Protein," Ann. N.Y. Acad. Sci., Jun. 1, 2003;
994(1): 27-35), the omega agatoxin family, etc.
[0068] Knottin proteins are shown in FIG. 3 as having a
characteristic disulfide linking structure. This structure is also
illustrated in Gelly et al., "The KNOTTIN website and database: a
new information system dedicated to the knottin scaffold," Nucleic
Acids Research, 2004, Vol. 32, Database issue D156-D159. A
triple-stranded -sheet is present in many knottins. The cysteines
involved in the knot are shown as connected by lines in FIG. 3
indicating which Cys residues are linked to each other. The spacing
between Cys residues is important in the present invention, as is
the molecular topology and conformation of the RGD-containing
integrin binding loop. These attributes are critical for high
affinity integrin binding. The RGD mimic loop is inserted between
knottin Cys residues, but the length of the loop must be adjusted
for optimal integrin binding depending on the three-dimensional
spacing between those Cys residues. For example, if the flanking
Cys residues are linked to each other, the optimal loop may be
shorter than if the flanking Cys residues are linked to Cys
residues separated in primary sequence. Otherwise, particular amino
acid substitutions can be introduced that constrain a longer
RGD-containing loop into an optimal conformation for high affinity
integrin binding.
[0069] The term "amino acid" includes both naturally occurring and
synthetic amino acids and includes both the D and L form of the
acids as well as the racemic form. More specifically, amino acids
contain up to ten carbon atoms. They may contain an additional
carboxyl group, and heteroatoms such as nitrogen and sulfur.
Preferably the amino acids are .alpha. and .beta.-amino acids. The
term .alpha.-amino acid refers to amino acids in which the amino
group is attached to the carbon directly attached to the carboxyl
group, which is the .alpha.-carbon. The term .beta.-amino acid
refers to amino acids in which the amino group is attached to a
carbon one removed from the carboxyl group, which is the
.beta.-carbon. The amino acids described here are referred to in
standard IUPAC single letter nomenclature, with "X" meaning any
amino acid.
[0070] The term "EETI" means Protein Data Bank Entry (PDB) 2ETI.
Its entry in the Knottin database is EETI-II. It has the
sequence
TABLE-US-00001 (SEQ ID NO: 13) GC PRILMR
[CKQDSDC]LAGCV[CGPNGFC]G,
[0071] The bold and underlined portion is replaced above and in the
examples below by the present RGD mimic sequence(s). Loops 2 and 3,
including the end defining cysteines, are show in brackets. These
loops can also be varied without affecting binding efficiency, as
is demonstrated below.
[0072] The term "AgRP" means PDB entry 1HYK. Its entry in the
Knottin database is SwissProt AGRP_HUMAN, where the full-length
sequence of 129 amino acids may be found. It comprises the sequence
beginning at amino acid 87. An additional G is added to this
construct. It also includes a C105A mutation described in Jackson,
et al. 2002 Biochemistry, 41, 7565.
TABLE-US-00002 (SEQ ID NO: 14)
GCVRLHESCLGQQVPCCDPCATCYCRFFNAFCYCR-KLGTAMNPCSRT
[0073] The dashed portion shows a fragment omitted in the "mini"
version, below. The bold and underlined portion, from loop 4, is
replaced by the RGD sequences described below.
[0074] The term "mini" in reference to AgRP means PDB entry 1MRO.
It is also SwissProt AGRP_HUMAN. It has the sequence, similar to
that given above,
TABLE-US-00003 (SEQ ID NO: 15)
GCVRLHESCLGQQVPCCDPAATCYCRFFNAFCYCR
where the italicized "A" represents an amino acid substitution
which eliminates a possible dimer forming cystine. (Cystine herein
refers to the single amino acid; cysteine to the dimer.). The bold
and underlined portion, from loop 4, is replaced by the below
described he RGD sequences.
[0075] The term "agatoxin" means omega agatoxin PDB 1OMB and the
SwissProt entry in the knottin database TOG4B_AGEAP. It has the
sequence
TABLE-US-00004 (SEQ ID NO: 16)
EDN-CIAEDYGKCTWGGTKCCRGRPCRCSMIGTNCECT-PRLIMEGLSFA
[0076] The dashes indicate portions of the peptide omitted for the
"mini" agatoxin. As shown in Table 3, an additional glycine is
added to the N-terminus of the mini-construct. The bold and
underlined portion is replaced by the below described he RGD
sequences.
[0077] The term "substantial identity" in the context of a peptide
indicates that a peptide comprises a sequence with at least 70%
sequence identity to a reference sequence, preferably 80%, more
preferably 85%, most preferably at least 90% or at least 95%
sequence identity to the reference sequence over a specified
comparison window, which in this case is either the entire peptide,
a molecular scaffold portion, or a binding loop portion
(.about.9-11 residues). Preferably, optimal alignment is conducted
using the homology alignment algorithm of Needleman and Wunsch
(1970) J. Mol. Biol., 48:443 453. An indication that two peptide
sequences are substantially identical is that one peptide is
immunologically reactive with antibodies raised against the second
peptide. Another indication for present purposes, that a sequence
is substantially identical to a specific sequence explicitly
exemplified is that the sequence in question will have an integrin
binding affinity at least as high as the reference sequence. Thus,
a peptide is substantially identical to a second peptide, for
example, where the two peptides differ only by a conservative
substitution. "Conservative substitutions" are well known, and
exemplified, e.g., by the PAM 250 scoring matrix. Peptides that are
"substantially similar" share sequences as noted above except that
residue positions that are not identical may differ by conservative
amino acid changes. As used herein, "sequence identity" or
"identity" in the context of two nucleic acid or polypeptide
sequences makes reference to the residues in the two sequences that
are the same when aligned for maximum correspondence over a
specified comparison window. When percentage of sequence identity
is used in reference to proteins it is recognized that residue
positions which are not identical often differ by conservative
amino acid substitutions, where amino acid residues are substituted
for other amino acid residues with similar chemical properties
(e.g., charge or hydrophobicity) and therefore do not change the
functional properties of the molecule. When sequences differ in
conservative substitutions, the percent sequence identity may be
adjusted upwards to correct for the conservative nature of the
substitution. Sequences that differ by such conservative
substitutions are said to have "sequence similarity" or
"similarity." Means for making this adjustment are well known to
those of skill in the art. Typically this involves scoring a
conservative substitution as a partial rather than a full mismatch,
thereby increasing the percentage sequence identity. Thus, for
example, where an identical amino acid is given a score of 1 and a
non-conservative substitution is given a score of zero, a
conservative substitution is given a score between zero and 1. The
scoring of conservative substitutions is calculated, e.g., as
implemented in the NIH Multiple alignment workshop
(http://helixweb.nih.gov/multi-align/). Three-dimensional tools may
also be used for sequence comparison.
[0078] As used herein, "percentage of sequence identity" means the
value determined by comparing two optimally aligned sequences over
a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0079] The term "endothelial integrin" is used in its conventional
sense and means integrins expressed on the outer apical pole of the
surface epithelium, and are involved in angiogenesis. More specific
details are found at J. Clin. Invest., 110:913-914 (2002).
[0080] The term "optical label" is used in its conventional sense
to mean, e.g., Cy-5.5 and other dyes useful as near infrared
imaging agents. A variety of optical labels can be used in the
practice of the invention and include, for example,
4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine
and derivatives: acridine, acridine isothiocyanate;
5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);
4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;
N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY;
Brilliant Yellow; coumarin and derivatives; coumarin,
7-amino-4-methylcoumarin (AMC, Coumarin 120),
7-amino-4-trifluoromethylcouluarin (Coumarin 151); cyanine dyes;
cyanosine; 4',6-diaminidino-2-phenylindole (DAPI);
5'5''-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;
diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS,
dansylchloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate
(DABITC); eosin and derivatives; eosin, eosin isothiocyanate,
erythrosin and derivatives; erythrosin B, erythrosin,
isothiocyanate; ethidium; fluorescein and derivatives;
5-carboxyfluorescein (FAM),
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),
2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein,
fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;
IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho
cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;
B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives:
pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum
dots; Reactive Red 4 (Cibacron.TM. Brilliant Red 3B-A) rhodamine
and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine
(R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod),
rhodamine B, rhodamine 123, rhodamine X isothiocyanate,
sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative
of sulforhodamine 101 (Texas Red);
N,N,N',N'tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl
rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);
riboflavin; rosolic acid; terbium chelate derivatives; Cyanine-3
(Cy3); Cyanine-5 (Cy5); Cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD
700; IRD 800; La Jolta Blue; phthalo cyanine; and naphthalo
cyanine. Other useful labels include the Alexa Fluor.RTM. dyes from
Invitrogen, which are sulfonated dyes, based on aminocoumarin,
rhodamine, etc.
[0081] The term "positron-emitting label" is used in its
conventional sense and means a label for detection by a positron
emission camera, as in positron emission tomography, in which the
label is attached, e.g., via a chelator, to a peptide according to
the present invention. The most common labels used positron
emitting nuclei in PET are .sup.11C, .sup.13N, .sup.15O and
.sup.18F. Positron emitters zirconium-89 (.sup.89Zr) and iodine-124
(.sup.124I) are also contemplated for their long half life. Other
labels include in particular .sup.94mTc, .sup.68Ga and .sup.18F,
.sup.64Cu, .sup.86Y, and .sup.76Br.
[0082] The term "engineered integrin binding loop" means a primary
sequence of about 9-13 amino acids which have been created ab
initio through experimental methods such as directed molecular
evolution to bind to endothelial integrins. That is, the sequence
contains an RGD sequence or the like, placed between amino acids
which are particular to the scaffold and the binding specificity
desired. The RGD (RYD, etc) binding sequence is not simply taken
from a natural binding sequence of a known protein.
EXPERIMENTAL
Library Creation
[0083] In order to generate a randomized library of RGD mimic
sequences, oligonucleotides were prepared which coded for various
RGD mimic sequences as they were to be contained within a selected
knottin scaffold. Since the knottin/RGD engineered sequence was
relatively short, the DNA used to express the engineered protein in
yeast could be prepared synthetically. The DNA sequences to be
ligated into the yeast display vector were obtained from
MWG-BIOTECH Inc., High Point, North Carolina. Where an amino acid
was to be varied, twenty different codons, each coding for a
different amino acid, were synthesized for a given position.
Randomized oligonucleotide synthesis has been used to create a
coding cassette in which about 5 to about 15 amino acids are
randomized (see, e.g., Burritt et al., (1996) Anal. Biochem. 238:1
13; Lowman (1997) Annu. Rev. Biophys. Biomol. Struct. 26:410 24;
Wilson (1998) Can. J. Microbiol. 44:313 329).
[0084] The yeast display vector used for evolution of improved
mutants is called "pCT". The vector is further described in US
2004/0146976 to Wittrup, et al., published Jul. 29, 2004, entitled
"Yeast cell surface display of proteins and uses thereof." As
described there, the vector provides a genetic fusion of the N
terminus of a polypeptide of interest to the C-terminus of the
yeast Aga2p cell wall protein. The outer wall of each yeast cell
can display approximately 10.sup.4-10.sup.5 protein agglutinins.
The vector contains the specific restriction sites and illustrates
the transcriptional regulation by galactose, the N-terminal HA and
C-terminal c-myc epitope tags and the Factor Xa protease cleavage
site.
[0085] The vector used in the present work contained NheI (GCTAGC)
(SEQ ID NO: 17) and BamHI (GGATCC) (SEQ ID NO: 18) restriction
sites for specific insertion of the RGD mimic coding sequence.
Labeling Yeast-Displayed Polypeptides
[0086] Below is a typical protocol to label a yeast library samples
for sorting by flow cytometry (FACS): [0087] 1. Want
2.times.10.sup.6 cells, OD.sub.600 of 1.0.apprxeq.10.sup.7 cells/mL
[0088] 2. Add 1 mL PBS/BSA (phosphate buffered saline containing 1
mg/mL bovine serum albumin) to wash cells [0089] 3. Spin down cells
3 min at 8000 RPM [0090] 4. Remove supernatant using vacuum [0091]
5. Re-suspend in 40 .mu.L PBS/BSA containing proper amount of
integrin (100 nM [2.5 .mu.L of stock .alpha..sub.v.beta..sub.3]; no
anti-cmyc at this point) [0092] 6. Incubate for 1.5 h at r.t
(w/tumbling) [0093] 7. Add 1:250 dilution of (chick anti-cmyc) to
labeling solution [0094] 8. DO NOT wash cells at this point. [0095]
9. Incubate 1 h at 4.degree. C. (w/tumbling) [0096] 10. Keep on ice
after this step. [0097] 11. Spin down cells 3 min at 8000 RPM,
4.degree. C. and vacuum supernatant [0098] 12. Repeat wash steps
2-4 [0099] 13. Re-suspend in 40 .mu.L PBS/BSA containing proper
amount of secondary labels (secondary labeling is simultaneous)
[0100] i. Anti-integrin Ab (FITC conj): 1:25 dilution+Anti-chick
(Alexa 555): 1:100 dilution [0101] ii. Positive control: Anti-chick
(Alexa 555): 1:100 dilution (for FACS compensation) [0102] iii.
Positive control: Anti-chick (Alexa 488): 1:100 dilution (for FACS
compensation) [0103] 14. Incubate on ice 30 min and keep in dark
(lid on ice bucket) [0104] 15. Spin down cells at 3 min at 8000
RPM, 4.degree. C. and vacuum supernatant [0105] 16. Repeat steps
2-4: Add 1 mL PBS/BSA, pellet cells, vacuum supernatant [0106] 17.
Leave pelleted cells on ice until use.
Fluorescent Cell Sorting
[0107] Commercially available flow cytometers can measure
fluorescence emissions at the single-cell level at four or more
wavelengths, at a rate of approximately 50,000 cells per second
(Ashcroft and Lopez, 2000). Typical flow cytometry data are shown
in FIG. 4-7, in which yeast have been labeled with two different
color fluorescent probes to measure protein expression levels and
bound soluble ligand (in this case integrin receptor). A `diagonal`
population of cells results due to variation in protein expression
levels on a per cell basis: cells that express more protein will
bind more ligand. The equilibrium binding constant (K.sub.d) can be
determined by titration of soluble ligand, and the dissociation
rate constant (k.sub.off) can be measured through competition
binding of unlabeled ligand. With yeast, a monodispersity of
tethered proteins exists over the cell surface, and soluble ligand
are used for binding and testing, such that avidity effects are not
observed, unlike other display methods using immobilized ligands.
To date, the properties of most proteins expressed on the yeast
cell surface mimic what is seen in solution in terms of stability
and binding affinity (Bader et al., 2000; Feldhaus et al., 2003;
Holler et al., 2000; VanAntwerp and Wittrup, 2000). See, also,
Weaver-Feldhaus et al., "Directed evolution for the development of
conformation-specific affinity reagents using yeast display,"
Protein Engineering Design and Selection Sep. 26, 2005
18(11):527-536.
[0108] Cell sorting was carried out on a FACSVantage (BD
Biosciences) multiparameter laser flow cytometer and cell sorter.
Before sorting, fluorescent staining was carried out as described
above, so that analysis of integrin binding and c-myc expression
levels were detected, as described above. Cells with the highest
levels of integrin binding, normalized for c-myc expression levels,
were gated and sorted into a collection tube containing culture
media. Sorted clones were propagated in culture and flow cytometric
screening was repeated several times to obtain an enriched
population of yeast-displayed peptides with high affinity integrin
binding.
[0109] After obtaining a pool of cells with high integrin binding
affinity, single yeast clones were obtained by plating onto Petri
dishes. Plasmid DNA was obtained from the entire yeast population,
transformed to E. coli and then individual E. coli clones were
selected for plasmid recovery and sequencing to determined the aa
composition of individual mutants. The DNA sequences of
representative peptides are given below.
Sequence Design: EETI-II Scaffold and Mini-AgRP Scaffold
[0110] The sequences listed below were generated from three
different yeast displayed combinatorial libraries--two libraries
based on the EETI-II scaffold and one library based on the
Mini-AgRP scaffold. All libraries were sorted by fluorescence
activated cell sorting (FACS). Mutants from each round were
isolated and sequenced.
[0111] The EETI-II-based library was:
TABLE-US-00005 (SEQ ID NO: 19)
GCXXXRGDXXXXXCKQDSDCLAGCVCGPNGFCG,
where X=any amino acid. This library produced mutants 1.x, listed
below. A follow-up library was made in a similar manner in an
attempt to improve the mutants made from the original library just
mentioned. These resultant mutants are labeled 2.x, listed
below.
[0112] In the above sequence EETI-II loops 2 and 3 (from left to
right) are also underlined, but not bolded. Flanking cysteines are
not underlined. As will be discussed below, these sequences have
been shown to be tolerant to diversity without affecting the
binding capacity of the binding loop.
[0113] The above-described library was also prepared with the
insert XXRGDXXXX and these EETI-II peptides were mixed with the
library shown here before sorting. However, no sequences from this
library were isolated. This indicates the importance of having the
proper number of flanking residues around the RGD sequence in this
scaffold.
EETI-II Based Sequences:
TABLE-US-00006 [0114] TABLE 1 Sequences wherein the RGD motif (in
italics) is found in the insert at positions 3-5. Peptide
identifier Sequence SEQ ID NO: RGD-EETI#2 GCTG
SPASSKCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 20) RGD-EETI#3 GCVT
SPASSCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 21)
[0115] RGD-EETI#3 had binding estimated to be in the range of Kd
100-200 nM. RGD-EETI#2 had approximately half the affinity of
RGD-EETI#3. The bolded sequences were chosen for initial loop
design from native fibronectin RGD loop sequences.
[0116] Variants based on RGD-EETI#3 above were prepared, with the
RGD motif at amino acid positions 6-8, where the sequence RGD is
italicized in 1.4A. In other words, the starting library was
GCXXXRGDXXXXXCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 22)
[0117] The integrin-binding loop was inserted after the second
residue and the first Cys.
TABLE-US-00007 TABLE 2 EETI sequences wherein the RGD motif (in
italics in 1.4A) is found in the insert at positions 4-6. Peptide
identifier Sequence SEQ ID NO: 1.4A GCAEP MPWTWCKQDSDCLAGCVCGPNGFCG
(SEQ ID NO: 23) 1.4B GCVGGRGDWSPKWCKQDSDCPAGCVCGPNGFCG (SEQ ID NO:
24) 1.4C GCAELRGDRSYPECKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 25) 1.4E
GCRLPRGDVPRPHCKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 26) 1.4H
GCYPLRGDNPYAACKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 27) 1.5B
GCTIGRGDWAPSECKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 28) 1.5F
GCHPPRGDNPPVTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 29) 2.3A
GCPEPRGDNPPPSCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 30) 2.3B
GCLPPRGDNPPPSCKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 31) 2.3C
GCHLGRGDWAPVGCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 32) 2.3D
GCNVGRGDWAPSECKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 33) 2.3E
GCFPGRGDWAPSSCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 34) 2.3F
GCPLPRGDNPPTECKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 35) 2.3G
GCSEARGDNPRLSCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 36) 2.3H
GCLLGRGDWAPEACKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 37) 2.31
GCHVGRGDWAPLKCKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 38) 2.3J
GCVRGRGDWAPPSCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 39) 2.4A
GCLGGRGDWAPPACKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 40) 2.4C
GCFVGRGDWAPLTCKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 41) 2.4D
GCPVGRGDWSPASCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 42) 2.4E
GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 43) 2.4F
GCYQGRGDWSPSSCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 44) 2.4G
GCAPGRGDWAPSECKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 45) 2.4J
GCVQGRGDWSPPSCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 46) 2.5A
GCHVGRGDWAPEECKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 47) 2.5C
GCDGGRGDWAPPACKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 48) 2.5D
GCPQGRGDWAPTSCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 49) 2.5F
GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 50) 2.5H
GCPCIGRGDWAPEWCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 51) 2.5J
GCPRGRGDWSPPACKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 52)
[0118] Thus there has been described an engineered integrin binding
peptide comprising a scaffold sequence and an RGD insert, both of
which may be modified as described.
[0119] The EETI-II scaffold as described above reveals a number of
specific sequences, which form the EETI-II knottin scaffold as
illustrated in FIG. 3, having three disulfide linkages. The native
sequence, which is replaced by the insert is shown in brackets in
FIG. 3 and in bold underline above; the insert is shown in bold
underline in Tables 1 and 2. The scaffold sequence may be varied,
as the primary function of the scaffold is to maintain the
orientation of the RGD insert. The scaffold should have the GPNG
(SEQ ID NO: 109) sequence, which is known to be needed for folding
(Wentzel, et al., "Sequence Requirements of the GPNG (SEQ ID NO:
109) .beta.-Turn of the Ecballum elaterium Trypsin Inhibitor
Explored by Combinatorial Library Screening," J. Biol. Chem.
274(30):21037-21043 (1999)). Lysine 15 may be removed for ease of
synthesis and labeling, and replaced with a less reactive residue.
It can also be seen that the sequence CLAG (SEQ ID NO: 110) has
been varied, e.g., CPAG (SEQ ID NO: 111), CQAG (SEQ ID NO: 112),
CRAG (SEQ ID NO: 113). These mutations were isolated from the
library; however, are thought to have arisen from primer errors,
since mutagenesis was not performed at this amino acid
position.
[0120] The RGD insert, on either side of the linked Cys residues,
comprises the sequence RGD within an 11 amino acid sequence of 11
amino acids replaces the native sequence, with R at the 4.sup.th
position. Putting R in the 3.sup.rd position (EETI #2) was found to
decrease binding in the peptides tested. That is, the inserts,
which had the sequence
X.sub.1X.sub.2X.sub.3R.sub.4G.sub.5D.sub.6X.sub.7X.sub.8X.sub.9X-
.sub.10X.sub.11 (sequence (a)) were superior to
X.sub.1X.sub.2R.sub.3G.sub.4D.sub.5X.sub.6X.sub.7X.sub.8X.sub.9
X.sub.10X.sub.11 (sequence (b)), where the subscript indicates
position in the insert). The length of the loop is based on the
distance between the adjacent Cys residues, but may be varied
between about 9 and 13 residues. As shown in FIG. 3, for EETI-II
the adjacent Cys residues are not linked to each other; rather they
are linked to other Cys residues in a peptide "scaffold," which is
a knottin peptide such as listed above in Tables 1-4.
EETI Sequence Variations
[0121] The RGD-containing loop may be varied from the specific
sequences disclosed. For example the loop sequence of 2.5F,
PLPRGDNPPTE (SEQ ID NO: 106) (See below) may be varied in the 8
non-underlined residues to a certain degree without affecting
binding specificity and potency. For example, if three of the
eleven residues were varied, one would have about 70% identity to
2.5D. For guidance in selecting which residues to vary, histograms
in FIG. 11 present information on likely residues for each
position. For example, in position -3 (the first X, one would most
likely use a proline residue, based on isolated mutants that had
positive integrin binding. However, His or Leu are also possible
choices, as shown by their higher incidence in mutants with good
integrin binding properties.
[0122] The sequences from Table 2 were aligned using NPS @: Network
Protein Sequence Analysis, TIBS 2000 March Vol. 25, No. 3
[291]:147-150, Combet C., Blanchet C., Geourjon C. and Deleage G.
The alignment was performed at
http://npsa-pbil.ibcp.fr/cgi-bin/align_multalin.pl, using default
parameters. Residues conserved for 90% or more (upper-case
letters): 24 is 72.73%. The sequences in Table 2 are considered
substantially identical to the consensus sequence.
GCPXGRGDWAPPSCKQDSDCRAGCVCGPNGFCG, where X=any amino acid. (SEQ ID
NO: 53)
Agouti-Related Protein (AgRP) and Agatoxin Sequences:
[0123] The two wild-type proteins AgRP and Agatoxin are quite
different in sequence, but they have the same three-dimensional
fold. As a result, any RGD sequence that works in AgRP will work in
Agatoxin, and vice versa.
[0124] The following sequences illustrate various RGD mimics,
showing improvements in integrin binding properties obtained by the
yeast display molecular evolution process described above. The
integrin binding properties of the peptides were RGD-AgRP #1<#2,
<#3:
TABLE-US-00008 TABLE 3 AgRP peptides ID no. Sequence SEQ ID NO:
RGD- GCVRLHESCLGQQVPCCDPCATCYCRGDCYCRKLGTAMNPCSRT (SEQ ID NO: 54)
AgRP#1 RGD- GCVRLHESCLGQQVPCCDPCATCYCTGRGDSCYCRKLGTAMNPCSRT (SEQ ID
NO: 55) AgRP#2 RGD- CVRLHESCLGQQVPCCDPCATCYCTGRGDSPASCYCRKLGTAMNPCS
(SEQ ID NO: 56) AgRP#3 RT Mini-
GCVRLHESCLGQQVPCCDPAATCYCTGRGDSPASCYCR (SEQ ID NO: 57) RGD-AgRP
Mini- GCIAEDYGKCTWGGTKCCRGRPCRCTGRGDSPASCECT (SEQ ID NO: 58) RGD-
Agatoxin
[0125] A shortened version of AgRP was also prepared. The
Mini-AgRP-based starting library was:
TABLE-US-00009 (SEQ ID NO: 59)
GCVRLHESCLGQQVPCCDPAATCYCXXRGDXXXXCYCR.
[0126] Variants based on Mini-RGD-AgRP isolated by the techniques
described above are shown below.
TABLE-US-00010 TABLE 4 Mini-RGD-AgRP peptides Number Sequence SEQ
ID NO: 3A GCVRLHESCLGQQVPCCDPAATCYCVVRGDWRKRCYCR (SEQ ID NO: 60) 3B
GCVRLHESCLGQQVPCCDPAATCYCEERGDMLEKCYCR (SEQ ID NO: 61) 3C
GCVRLHESCLGQQVPCCDPAATCYCETRGDGKEKCYCR (SEQ ID NO: 62) 3D
GCVRLHESCLGQQVPCCDPAATCYCQWRGDGDVKCYCR (SEQ ID NO: 63) 3E
GCVRLHESCLGQQVPCCDPAATCYCSRRGDMRERCYCR (SEQ ID NO: 64) 3F
GCVRLHESCLGQQVPCCDPAATCYCQYRGDGMKHCYCR (SEQ ID NO: 65) 3G
GCVRLHESCLGQQVPCCDPAATCYCTGRGDTKVLCYCR (SEQ ID NO: 66) 3H
GCVRLHESCLGQQVPCCDPAATCYCVERGDMKRRCYCR (SEQ ID NO: 67) 3I
GCVRLHESCLGQQVPCCDPAATCYCTGRGDVRMNCYCR (SEQ ID NO: 68) 3J
GCVRLHESCLGQQVPCCDPAATCYCVERGDGMSKCYCR (SEQ ID NO: 69) 4A
GCVRLHESCLGQQVPCCDPAATCYCRGRGDMRRECYCR (SEQ ID NO: 70) 4B
GCVRLHESCLGQQVPCCDPAATCYCEGRGDVKVNCYCR (SEQ ID NO: 71) 4C
GCVRLHESCLGQQVPCCDPAATCYCVGRGDEKMSCYCR (SEQ ID NO: 72) 4D
GCVRLHESCLGQQVPCCDPAATCYCVSRGDMRKRCYCR (SEQ ID NO: 73) 4E
GCVRLHESCLGQQVPCCDPAATCYCERRGDSVKKCYCR (SEQ ID NO: 74) 4F
GCVRLHESCLGQQVPCCDPAATCYCEGRGDTRRRCYCR (SEQ ID NO: 75) 4G
GCVRLHESCLGQQVPCCDPAATCYCEGRGDVVRRCYCR (SEQ ID NO: 76) 4H
GCVRLHESCLGQQVPCCDPAATCYCKGRGDNKRKCYCR (SEQ ID NO: 77) 4I
GCVRLHESCLGQQVPCCDPAXTCYCKGRGDVRRVCYCR (SEQ ID NO: 78) 4J
GCVRLHESCLGQQVPCCDPAATCYCVGRGDNKVKCYCR (SEQ ID NO: 79) 5A
GCVRLHESCLGQQVPCCDPAATCYCVGRGDNRLKCYCR (SEQ ID NO: 80) 5B
GCVRLHESCLGQQVPCCDPAATCYCVERGDGMKKCYCR (SEQ ID NO: 81) 5C
GCVRLHESCLGQQVPCCDPAATCYCEGRGDMRRRCYCR (SEQ ID NO: 82) 5D
GCVRLHESCLGQQVPCCDPAATCYCQGRGDGDVKCYCR (SEQ ID NO: 83) 5E
GCVRLHESCLGQQVPCCDPAATCYCSGRGDNDLVCYCR (SEQ ID NO: 84) 5F
GCVRLHESCLGQQVPCCDPAATCYCVERGDGMIRCYCR (SEQ ID NO: 85) 5G
GCVRLHESCLGQQVPCCDPAATCYCSGRGDNDLVCYCR (SEQ ID NO: 86) 5H
GCVRLHESCLGQQVPCCDPAATCYCEGRGDMKMKCYCR (SEQ ID NO: 87) 5I
GCVRLHESCLGQQVPCCDPAATCYCIGRGDVRRRCYCR (SEQ ID NO: 88) 5J
GCVRLHESCLGQQVPCCDPAATCYCEERGDGRKKCYCR (SEQ ID NO: 89) 6B
GCVRLHESCLGQQVPCCDPAATCYCEGRGDRDMKCYCR (SEQ ID NO: 90) 6C
GCVRLHESCLGQQVPCCDPAATCYCTGRGDEKLRCYCR (SEQ ID NO: 91) 6E
GCVRLHESCLGQQVPCCDPAATCYCVERGDGNRRCYCR (SEQ ID NO: 92) 6F
GCVRLHESCLGQQVPCCDPAATCYCESRGDVVRKCYCR (SEQ ID NO: 93) 7C
GCVRLHESCLGQQVPCCDPAATCYCYGRGDNDLRCYCR (SEQ ID NO: 94)
Anti-angiogenic activity
[0127] The present peptides have been shown to bind to integrins
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5, by in
vitro experiments in which the peptides were incubated with soluble
integrins as described above.
[0128] Based on present knowledge of cell adhesion and
tumorigenesis, it may be expected that the present peptides will
function in vivo as well as in vitro, and that they will exhibit
anti-angiogenic and anti-proliferative activity. It is known that
the integrin .alpha..sub.v.beta..sub.3 is required for
angiogenesis, see Brooks et al., Science 264:569-571. In order to
demonstrate and evaluate such activity, a number of assays, known
in the art are included within the present concepts.
[0129] Such assays include, but are not limited to, assays of
endothelial cell proliferation, endothelial cell migration, cell
cycle analysis, and endothelial cell tube formation, detection of
apoptosis, e.g., by apoptotic cell morphology or Annexin V-FITC
assay, chorioallantoic membrane (CAM) assay, and inhibition of
renal cancer tumor growth in nude mice. Examples of such assays are
given in U.S. Pat. No. 6,962,974 to Kalluri, issued Nov. 8, 2005,
entitled "Anti-angiogenic proteins and fragments and methods of use
thereof." For example, C-PAE cells are grown to confluence in DMEM
with 10% fetal calf serum (FCS) and kept contact inhibited for 48
hours. Control cells are 786-O (renal carcinoma) cells, PC-3 cells,
HPEC cells, and A-498 (renal carcinoma) cells. Cells are harvested
with typsinization (Life Technologies/Gibco BRL, Gaithersburg, Md.,
USA). A suspension of 12,500 cells in DMEM with 1% FCS is added to
each well of a 24-well plate coated with 10 .mu.g/ml fibronectin.
The cells are incubated for 24 hours at 37.degree. C. with 5%
CO.sub.2 and 95% humidity. Medium is removed and replaced with DMEM
containing 0.5% FCS and 3 ng/ml bFGF (R&D Systems, Minneapolis,
Minn., USA). Cells are treated with concentrations of the present
engineered peptides ranging from 0.01 to 50 gig/ml. All wells
receive 1 pt Curie of .sup.3H-thymidine at the time of treatment.
After 24 hours, medium is removed and the wells are washed with
PBS. Cells are extracted with IN NaOH and added to a scintillation
vial containing 4 ml of ScintiVerse II (Fisher Scientific,
Pittsburgh, Pa., USA) solution. Thymidine incorporation is measured
using a scintillation counter. The showing incorporation of
.sup.3H-thymidine into C-PAE cells treated with varying amounts of
the peptides will show inhibition of cell division.
[0130] For animal testing, about two million 786-0 cells are
injected subcutaneously into 7- to 9-week-old male athymic nude
mice. In the first group of mice, the tumors are allowed to grow to
about 700 mm.sup.3. In a second group of mice, the tumors are
allowed to group to 100 mm.sup.3. The engineered peptide (e.g.,
EETI-1.5B, 2.5A, and 2.5D), in sterile PBS is injected I.P. daily
for 10 days, at a concentration of 20 mg/kg for the mice with
tumors of 700 mm.sup.3, and 10 mg/kg for the mice with tumors of
100 mm3. Control mice receive either BSA or the PBS vehicle. The
results will show a change in tumor volume from 700 mm.sup.3 for 10
mg/kg peptide treated, BSA-treated (+), and control mice. Tumors in
the peptide-treated mice will shrink, while tumors in BSA-treated
and control mice will grow.
[0131] In another known protocol (See again U.S. Pat. No.
6,962,974), about 5 million PC-3 cells (human prostate
adenocarcinoma cells) are harvested and injected subcutaneously
into 7- to 9-week-old male athymic nude mice. The tumors grow for
10 days, and are then measured with Vernier calipers. The tumor
volume is calculated using the standard formula, and animals are
divided into groups of 5-6 mice. Experimental groups are injected
I.P. daily with a test engineered peptide (10 mg/kg/day) or a
control drug (e.g., an anti-integrin antibody) (10 mg/kg/day). The
control group receives PBS each day. The results will show that an
engineered peptide inhibits the growth of tumors as well, or
slightly better, than did the control drug. The experiment may be
repeated at different dosages and times.
EETI, AgRP and Agatoxin Peptide Constructs
[0132] The peptides specifically set forth above may be modified in
a number of ways. For example, the peptides may be further
cross-linked internally, or may be cross linked to each other, or
the RGD mimic loops may be grafted onto other cross linked
molecular scaffolds. There are a number of commercially available
crosslinking reagents for preparing protein or peptide
bioconjugates. Many of these crosslinkers allow dimeric homo- or
heteroconjugation of biological molecules through free amine or
sulfhydryl groups in protein side chains. More recently, other
crosslinking methods involving coupling through carbohydrate groups
with hydrazide moieties have been developed. These reagents have
offered convenient, facile, crosslinking strategies for researchers
with little or no chemistry experience in preparing
bioconjugates.
[0133] The present peptides may be produced by recombinant DNA or
may be synthesized in solid phase using a peptide synthesizer,
which has been done for the peptides of all three scaffolds
described here. They may further be capped at their N-termini by
reaction with fluorescein isothiocyanate (FITC) or other labels,
and, still further, may be synthesized with amino acid residues
selected for additional crosslinking reactions. TentaGel S RAM Fmoc
resin (Advanced ChemTech) may be used to give a C-terminal amide
upon cleavage. B-alanine is used as the N-terminal amino acid to
prevent thiazolidone formation and release of fluorescein during
peptide deprotection (Hermanson, 1996). Peptides are cleaved from
the resin and side-chains are deprotected with 8% trifluoroacetic
acid, 2% triisopropylsilane, 5% dithiothreitol, and the final
product is recovered by ether precipitation. Peptides are purified
by reverse phase HPLC using an acetonitrile gradient in 0.1%
trifluoroacetic acid and a C4 or C18 column (Vydac) and verified
using matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF) or electrospray ionization-mass
spectrosometry (ESI-MS).
[0134] When the present peptides are produced by recombinant DNA,
expression vectors encoding the selected peptide are transformed
into a suitable host. The host should be selected to ensure proper
peptide folding and disulfide bond formation as described above.
Certain peptides, such as EETI-II can fold properly when expressed
in prokaryotic hosts such as bacteria.
[0135] Exemplary DNA sequences used for the present peptides are
given below:
TABLE-US-00011 RGD-EETI#3-based hits (DNA sequences) 1.4B (SEQ ID
NO: 95) GGGTGCGTGGGGGGGAGAGGGGATTGGAGCCCGAAGTGGTGCAAACAGGAC
TCCGACTGCCCGGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 1.5B (SEQ ID NO:
96) GGGTGCACGATCGGGAGAGGGGATTGGGCCCCCTCGGAGTGCAAACAGGAC
TCCGACTGCCTGGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 1.5F (SEQ ID NO:
97) GGGTGCCACCCGCCGAGAGGGGATAACCCCCCCGTGACTTGCAAACAGGAC
TCCGACTGCCTGGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 2.4F (SEQ ID NO:
98) GGGTGCTATCAAGGAAGAGGGGATTGGTCTCCTTCATCGTGCAAACAGGAC
TCCGACTGCCCAGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 2.5A (SEQ ID NO:
99) GGGTGCCATGTAGGAAGAGGGGATTGGGCTCCTGAAGAGTGCAAACAGGAC
TCCGACTGCCAAGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 2.5C (SEQ ID NO:
100) GGGTGCGATGGAGGAAGAGGGGATTGGGCTCCTCCAGCGTGCAAACAGGAC
TCCGACTGCCGAGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 2.5D (SEQ ID NO:
101) GGGTGCCCTCAAGGAAGAGGGGATTGGGCTCCTACATCGTGCAAACAGGAC
TCCGACTGCCGAGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 2.5F (SEQ ID NO:
102) GGGTGCCCTCGACCAAGAGGGGATAACCCTCCTCTAACGTGCAAACAGGAC
TCCGACTGCCTAGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 2.5H (SEQ ID NO:
103) GGGTGCCCTCAAGGAAGAGGGGATTGGGCTCCTGAATGGTGCAAACAGGAC
TCCGACTGCCCAGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 2.5J (SEQ ID NO:
104) GGGTGCCCTCGAGGAAGAGGGGATTGGTCTCCTCCAGCGTGCAAACAGGAC
TCCGACTGCCAAGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA
[0136] Dimeric, trimeric, and tetrameric complexes of the present
peptides can be formed through genetic engineering of the above
sequences or by reaction of the synthetic cross-linkers with
engineered peptides carrying an introduced cysteine residue, for
example on the C-terminus of the peptide. These oligomeric peptide
complexes can be purified by gel filtration. Oligomers of the
present peptides can be prepared by preparing vectors encoding
multiple peptide sequences end-to-end. Also, multimers may be
prepared by complexing the peptides, such as, e.g., described in
U.S. Pat. No. 6,265,539. There, an active HIV peptide is prepared
in multimer form by altering the amino-terminal residue of the
peptide so that it is peptide-bonded to a spacer peptide that
contains an amino-terminal lysyl residue and one to about five
amino acid residues such as glycyl residues to form a composite
polypeptide. Alternatively, each peptide is synthesized to contain
a cystine (Cys) residue at each of its amino- and carboxy-termini.
The resulting di-cystine-terminated (di-Cys) peptide is then
oxidized to polymerize the di-Cys peptide monomers into a polymer
or cyclic peptide multimer. Multimers may also be prepared by solid
phase peptide synthesis utilizing a lysine core matrix. The present
peptides may also be prepared as nanoparticles. See, "Multivalent
Effects of RGD Peptides Obtained by Nanoparticle Display," Montet,
et al., J. Med. Chem.; 2006; 49(20) pp 6087-6093. EETI dimerization
may be carried out with the present EETI-II peptides according to
EETI-II dimerization paper that just came out: "Grafting of
thrombopoietin-mimetic peptides into cystine knot miniproteins
yields high-affinity thrombopoietin antagonist and agonists,"
Krause, et al., FEBS Journal; 2006; 274 pp 86-95.
[0137] One may also prepare chemically synthesized peptide-based
crosslinking reagents for use in cross-linking the present
peptides. The peptide may further contain a fluorescent label
(fluorescein) and two or more thiol-reactive maleimide groups
introduced at lysine residues spaced along a flexible backbone
composed of glycine, serine, and glutamic acid (Cochran and Stern,
2000; Cochran et al., 2000). The non-repeating backbone amino acid
sequences are designed to be water-soluble with little propensity
to form an ordered structure, and to provide sufficient length and
flexibility to allow integrin binding side chains to bind
simultaneously to a cell surface. Maleimide-to-maleimide distances
for the cross-linkers, in extended conformations for allowing
pendant groups to present peptides in the same plane, are
approximately 45 Angstroms for the dimeric cross-linkers, and 50
Angstroms for the trimeric, and tetrameric cross linkers, as
estimated from molecular models.
[0138] Other reagents would allow multivalent presentation of
integrin binding peptides or small protein scaffolds.
Ruthenium-based metathesis catalysts would allow site-specific
crosslinking of alkene functional groups incorporated into amino
acid side chains. The ability to specifically couple biomolecules
using a chemical strategy that does not rely on natural amino acids
would be extremely useful in creating small oligomeric peptide and
protein motifs. An example is illustrated in FIG. 2. An amphipathic
helix is derived from the coiled coil helix of the transcription
factor GCN4, in which hydrophobic positions of heptad repeat have
been exchanged to insert RGD mimics. Further details are given in
Pack et al., "Tetravalent miniantibodies with high avidity
assembling in Escherichia coli.," J Mol Biol. 1995 Feb. 10;
246(1):28-34.
[0139] Synergistic sites on fibronectin and other adhesion proteins
have been identified for enhanced integrin binding (Ruoslahti,
1996; Koivunen et al., 1994; Aota et al., 1994; Healy et al.,
1995). The ability to incorporate different integrin-specific
motifs into one soluble molecule would have an important impact on
therapeutic development. Crosslinkers with heterofunctional
specificity may be used for creating integrin-binding proteins with
synergistic binding effects. In addition, these same crosslinkers
could easily be used to create bispecific targeting molecules, or
as vehicles for delivery of radionuclides or toxic agents for
imaging and therapeutic applications.
Methods of Use
[0140] The present engineered peptides may be used in a variety of
ways. If the peptides are attached to a surface, they may be used
to attract/recruit cells to grow on the surface. For example, the
present peptides may be applied to prosthetic devices, implants,
bone grafts, and the like to promote tissue growth and healing at
the site. They may be attached to culture dishes and promote
attachment and differentiation of cells in culture. In addition,
the present engineered peptides may be used to modulate cell
binding to selected integrins such as .alpha..sub.5.beta..sub.1,
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5,
particularly .alpha..sub.v.beta..sub.3, by adding the peptides to a
cell culture to prevent cells expressing these integrins from
adhering to a substrate. In a series of experiments using the
present RGD-containing peptides to block adhesion of U87MG
glioblastoma cells to vitronectin-coated plates, it was found that
the present peptides 2.5F, 2.5D, and 1.5D all blocked adhesion
better than controls and comparative compounds FN-RGD and c(RGDyK)
(SEQ ID NO: 140) (data not shown). Also, the present 2.5F, 2.5D and
1.5B peptides were tested for blocking adhesion of U87MG
glioblastoma cells to fibronectin-coated plates. In this case, only
echistatin and polypeptide 2.5F blocked adhesion of U87MG
glioblastoma cells to the fibronectin-coated plates. This confirms
that the RGD-miniprotein 2.5F binds with strong affinity to the
.alpha..sub.5.beta..sub.1 integrin subtype.
[0141] These binding studies show that the present peptides can be
used in soluble form to modulate binding of cells to known cell
culture substrates (extracellular matrix). Cell binding to
integrins can be used to modulate stem cell self-renewal or
differentiation. For example, it is known that stem cells express
higher levels of the beta 1-integrin family of extracellular matrix
receptors than transit amplifying cells and this can be used to
isolate each subpopulation of keratinocyte and to determine its
location within the epidermis. See, Watt, "Epidermal stem cells:
markers, patterning and the control of stem cell fate," Philos
Trans R Soc Lond B Biol Sci., 1998 Jun. 29; 353(1370): 831-837.
Alternatively, the engineered peptides may be prepared and coated
on plates or incorporated into polymers or other biomaterials and
used as a cell culture substrate to promote adhesion by the
selected integrin.
[0142] The present peptides may also be used to treat proliferative
diseases, when administered in soluble form. By attaching to
cellular integrins, they block attachment of the cells and inhibit
their growth and development. The present peptides will therefore
find use in cancer therapy. The instant peptides are also useful in
combination with known anti-cancer agents. Such known anti-cancer
agents include the following: estrogen receptor modulators,
androgen receptor modulators, retinoid receptor modulators,
cytotoxic agents, antiproliferative agents, prenyl-protein
transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease
inhibitors, reverse transcriptase inhibitors, and other
angiogenesis inhibitors. The instant compounds are also useful when
coadminsitered with radiation therapy. The present peptides may
also be chemically linked to cytotoxic agents. They may also be
used with other angiogenesis inhibitors. "Angiogenesis inhibitors"
refers to compounds that inhibit the formation of new blood
vessels, regardless of mechanism. Examples of angiogenesis
inhibitors include, but are not limited to, tyrosine kinase
inhibitors, such as inhibitors of the tyrosine kinase receptors
Fit-1 (VEGFR1) and Flk-1/KDR (VEGFR20), inhibitors of
epidermal-derived, fibroblast-derived, or platelet derived growth
factors, MMP (matrix metalloprotease) inhibitors,
interferon-..alpha.., interleukin-12, pentosan polysulfate,
cyclooxygenase inhibitors, including nonsteroidal
anti-inflammatories (NSAIDs) like aspirin and ibuprofen as well as
selective cyclooxygenase-2 inhibitors like celecoxib and rofecoxib
(PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch.
Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec., Vol. 238, p. 68
(1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol.
313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p. 107. (1996);
Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol. 57,
p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol., Med.,
Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)),
carboxyamidotriazole, combretastatin A-4, squalamine,
6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,
troponin-1, angiotensin II antagonists (see Fernandez et al., J.
Lab. Clin. Med. 105:141 145 (1985)), and antibodies to VEGF (see;
Nature Biotechnology, Vol. 17, pp. 963 968 (October 1999); Kim et
al., Nature, 362, 841 844 (1993).
[0143] The present peptides may also be used in vitro as cell
labeling reagents, and in vivo as imaging or diagnostic agents,
binding to cells, such as tumor cells, which express high levels of
a specific integrin.
Synthesis of Soluble Peptides
Folding Conditions for EETI-II Polypeptides
[0144] In preparing the present peptides, it is essential that the
correct disulfide linkages be formed, and that the peptide be
correctly folded. Glutathione-assisted oxidative folding of the
cystine-knot was used. An exemplary protocol for EETI-II is given
below. Large scale folding reactions were performed with 20% DMSO
(v/v) in 0.1 M ammonium bicarbonate, pH 9 and 2.5 mM reduced
glutathione while gently rocking overnight. The final oxidized
product was purified by semi-preparative HPLC using various linear
gradients of solvent A and solvent B. Following purification, the
peptide was lyophilized and stored until used. Working
concentrations of pure peptide dissolved in purified water were
determined by amino acid analysis. The purified peptide was
analyzed by HPLC and ESI-MS.
[0145] Solvent (A) is 99.9% water 0.1% TFA, (B) is 10% water 90%
MeCN and 0.1% TFA.
Folding Conditions for Mini-AGRP Polypeptides
Tris pH 8.0
[0146] 10 mM reduced glutathione 2 mM oxidized glutathione
0.5 M DMSO
[0147] with or without 2-4M guanidine (depending on the peptide)
1-3 days at room temperature.
[0148] Data on synthesized Agouti peptides: The peptides were
tested for activity; the results are as follows, with the peptide
designation corresponding to the sequence given in Table 4: IC50's
(obtained by competing off binding of .sup.125I-echistatin as
described above)
WT--1.4.+-.0.7 .mu.M
3F--880.+-.340 nM
6E--130.+-.20 nM
6F--410.+-.80 nM
7C--23.+-.4 nM
Imaging Probes
[0149] The present polypeptides target .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, and in some cases
.alpha..sub.5.beta..sub.1 integrin receptors. They do not bind to
other integrins tested. Thus, these engineered integrin-binding
polypeptides have broad diagnostic and therapeutic applications in
a variety of human cancers that specifically overexpress the above
named integrins. As described below, these polypeptides bind with
high affinity to both detergent-solubilized and tumor cell surface
integrin receptors. Furthermore, when used as optical imaging
agents in mouse xenograft models, the tumor/background signal ratio
generated by these engineered polypeptides is approximately 60%
greater than that elicited by an alternative pentapeptide currently
under pre-clinical development. Also, the present engineered
high-affinity integrin-binding polypeptides can also be labeled
with positron emitting isotopes and changed from optical imaging
probes to robust positron emission tomography (PET)-based imaging
agents. In a clinical setting, these polypeptides will be used to
visualize integrin expression in the human body for diagnostic and
management applications in cancer. They can be coupled to
radionuclides for therapeutic purposes. They offer advantages of
stability, which reduces toxic uptake of free radionuclides by the
kidneys.
[0150] As described above, it is known that the integrin
.alpha..sub.v.beta..sub.3 is expressed during angiogenesis. The
.alpha..sub.v.beta..sub.3 (and .alpha..sub.v.beta..sub.5) integrins
are also highly expressed on many tumor cells including
osteosarcomas, neuroblastomas, carcinomas of the lung, breast,
prostate, and bladder, glioblastomas, and invasive melanomas The
.alpha..sub.v.beta..sub.3 integrin has been shown to be expressed
on tumor cells and/or the vasculature of breast, ovarian, prostate,
and colon carcinomas, but not on normal adult tissues or blood
vessels. Therefore, noninvasive methods to visualize and quantify
integrin expression in vivo are crucial for patient-specific
treatment of cancer with integrin antagonists. Also, the
.alpha..sub.5.beta..sub.1 has been shown to be expressed on tumor
cells and/or the vasculature of breast, ovarian, prostate, and
colon carcinomas, but not on normal adult tissue or blood vessels.
The present, small, conformationally-constrained polypeptides
(about 33 amino acids) are so constrained by intramolecular bonds,
such as shown in FIG. 3. For example, EETI-II has three disulfide
linkages. This will make it more stable in vivo. These peptides
target .alpha..sub.v integrins alone, or both .alpha..sub.v and
.alpha..sub.5.beta..sub.1 integrins. Until now, it is believed that
the development of a single agent that can bind
.alpha..sub.v.beta..sub.3, .alpha..sub.v.beta..sub.5, and
.alpha..sub.5.beta..sub.1 integrins with high affinity and
specificity has not been achieved. Since all three of these
integrins are expressed on tumors and are involved in mediating
angiogenesis and metastasis, a broad spectrum targeting agent
(i.e., .alpha..sub.v.beta..sub.3, .alpha..sub.v.beta..sub.5, and
.alpha..sub.5.beta..sub.1) will likely be more effective for
diagnostic and therapeutic applications.
[0151] The present engineered polypeptides (termed
RGD-miniproteins) have several advantages over previously
identified integrin-targeting compounds. They possess a compact,
disulfide-bonded core that confers proteolytic resistance and
exceptional in vivo stability.
[0152] Our studies indicate their half-life in mouse serum to be
approximately 90 hours (data not shown). Their larger size
(.about.3-4 kDa) and enhanced affinity compared to RGD-based cyclic
peptides confer enhanced pharmacokinetics and biodistribution for
molecular imaging and therapeutic applications. This is described
in connection with FIG. 8. These RGD-miniproteins are small enough
to allow for chemical synthesis and site-specific conjugation of
imaging probes, radioisotopes, or chemotherapeutic agents.
Furthermore, they can easily be chemically modified to further
improve in vivo properties if necessary. The imaging study shown in
FIG. 8 shows tumor localization by peptide 2.5D. The tumor is
indicated by an arrow. The near infrared fluorescent Cy5.5 label
study provides guidance for the preparation of these polypeptides
as .sup.18F and .sup.64Cu-labeled PET imaging probes. In the
clinical setting, these imaging agents will play a critical role in
identifying patients whose cancer would benefit most from specific
integrin-targeted therapy, and will provide a molecular rationale
for why treatments may later fail if tumors cease to express these
integrins. They may also serve to stage cancer when coupled with
existing PET tracers such as 2-fluoro-2-deoxy-glucose (FDG). In
addition, as described above, these RGD-miniproteins may be used
for treatment of a variety of human cancers, as RGD-based targeting
agents have been shown to have therapeutic efficacy through
caspase-mediated apoptosis and cell death (see, Brooks, P. C.,
Montgomery, A M., Rosenfeld, M., Reisfeld, R A, Hu, T., Klier, G.
& Cheresh, D. A. (1994). Integrin alpha v beta 3 antagonists
promote tumor regression by inducing apoptosis of angiogenic blood
vessels. Cell 79, 1157-64.; Chatterjee, S., Brite, K. H. &
Matsumura, A (2001). Induction of apoptosis of integrin-expressing
human prostate cancer cells by cyclic Arg-Gly-Asp peptides. Clin
Cancer Res 7, 3006-11.
[0153] Polypeptide Synthesis and Folding:
[0154] RGD-miniproteins described below were synthesized using
standard Fmoc-based solid phase peptide synthesis with a CS Bio
CS336S automated synthesizer (Menlo Park, Calif.). The polypeptides
originally contained a lysine at position 15 that was mutated to a
serine to facilitate chemical coupling of imaging probes
specifically to the N-terminus. Crude polypeptide was purified by
reversed phase HPLC using a C.sub.18 column (Vydac). The correct
molecular mass was verified using electrospray mass spectrometry.
Polypeptides were folded with the assistance of dimethyl sulfoxide
and glutathione. Folded polypeptides exhibit a distinct
chromatographic profile that allows them to be purified from
unfolded or misfolded species by reversed-phase HPLC.
[0155] Binding to Tumor Cells Overexpressing
.alpha..sub.v.beta..sub.3 Integrins:
[0156] Referring now to FIG. 9, RGD-miniproteins were tested for
their ability to compete for cell surface integrin binding with
.sup.125I-labeled echistatin, a protein which binds the
.alpha..sub.v.beta..sub.3 integrin with a K.sub.D of 0.3 nM. U87MG
glioblastoma cells, which express .about.10.sup.5
.alpha..sub.v.beta..sub.3 integrin receptors per cell, were used
for these studies. We compared the receptor binding affinity of
loop-grafted FN-RGD (designated FN-RGD), and three of our
affinity-matured mutants, designated Miniprotein 1.5B, 2.5D, or
2.5F (see Table 2), to that of c(RGDyK) (SEQ ID NO: 140), a
pentapeptide currently under pre-clinical development for molecular
imaging applications. An EETI-based polypeptide with a scrambled
RDG amino acid sequence, designated FN-RDG, served as a negative
control. All of the RGD-containing peptides inhibited the binding
of .sup.125I-labeled echistatin to U87MG cells in a dose dependent
manner. Their IC.sub.50 values (corresponding to data in FIG. 9)
are shown in the Table 5 below.
TABLE-US-00012 TABLE 5 IC50 values of 1.5B, 2.5D and 2.5F c(RGDyK)
(SEQ ID NO: Loop-grafted Miniprotein Miniprotein Miniprotein Cy5.5
Echistatin 140) FN-RGD 1.5B 2.5D 2.5F IC.sub.50 No 4.9 .+-. 1.0 nM
860 .+-. 400 nM 370 .+-. 150 nM 13 .+-. 3.3 nM 16 .+-. 6.1 nM 26
.+-. 5.4 nM IC.sub.50 Yes 2.6 .+-. 0.2 nM 62.9 .+-. 4.1 nM 33.9
.+-. 13 nM 6.4 .+-. 3.3 nM 4.2 .+-. 0.9 nM 3.4 .+-. 0.8 nM
[0157] The above table shows competition binding of engineered
peptides with .sup.125I-echistatin to U87MG tumor cells.
Half-maximal inhibitory concentrations (IC.sub.50) represent the
standard deviation of data measured on at least three separate
days. Data for unlabeled and Cy5.5-labeled peptides are shown.
Site-specific labeling of RGD miniproteins with a near-infrared
optical imaging probe: The free N-terminal amine of our
polypeptides was used for site-specific attachment of Cy5.5, a near
infrared imaging probe.
[0158] Our evolved mutants were shown to bind to U87MG cells with a
50 to 80-fold higher affinity than both of the parental
loop-grafted FN-RGD and c(RGDyK) (SEQ ID NO: 140).
[0159] Unique Integrin Binding Specificities:
[0160] Since U87MG cells have been shown to express
.alpha..sub.v.beta..sub.3, .alpha..sub.v.beta..sub.5, and
.alpha..sub.5.beta..sub.1 integrins, it was necessary to use
another means to measure integrin-binding specificity. This was
done by competition of .sup.125I-echistatin to
detergent-solubilized integrin receptors coated onto microtiter
plates (see FIG. 10). As expected, echistatin binds strongly to all
of the tested integrins. The scrambled FN-RDG miniprotein, the
negative control, did not bind to any of the integrins used in this
study. All peptides bound to .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5 integrins to some degree, with the
engineered RGD-miniproteins 1.5B, 2.5D, and 2.5F showing the
strongest levels of binding. This is consistent with previous
studies which have shown .alpha..sub.v integrin receptors can
accommodate a wide range of RGD-containing cyclic structures.
Interestingly, the RGD-miniprotein 2.5F binds with strong affinity
to the .alpha..sub.5.beta..sub.1 integrin subtype, while
RGD-miniproteins 1.5B and 2.5D exhibit only minimal binding to this
receptor. However, since .alpha..sub.5.beta..sub.1 integrins are
expressed on many tumors and are all involved in mediating
angiogenesis and metastasis, a broad spectrum agent that targets
all three integrins will be useful for diagnostic and therapeutic
applications. With the exception of echistatin, all of the
RGD-containing peptides bound weakly to the
.alpha..sub.iib.beta..sub.3 receptor, showing the specificity of
the present peptides for the .alpha.v and .alpha.5-containing
integrin heterodimers. This characteristic is valuable for
molecular imaging and therapeutic applications, since binding to a
.alpha..sub.iib.beta..sub.3 on platelet cells prevents blood
clotting and would lead to non-specific in vivo effects.
TABLE-US-00013 1.5B: (SEQ ID NO: 28)
GCTIGRGDWAPSECKQDSDCLAGCVCGPNGFCG 2.5 D (SEQ ID NO: 49)
GCPQGRGDWAPTSCKQDSDCRAGCVCGPNGFCG 2.5F (SEQ ID NO: 50)
GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG
[0161] Viewing 1.5B and 2.5D together, one observes the identical
"GRGDWAP" (SEQ ID NO: 108) motif that these peptides have in
common, and would be expected to confer the integrin specificity
observed. On the other hand, the unique specificity of 2.5F (strong
affinity to the .alpha..sub.5.beta..sub.1 integrin subtype), is
reflected in the additional proline residues.
[0162] The proline rich sequence of 2.5F may constrain the binding
epitope into a favorable conformation for these high affinity
interactions. We are now pursuing molecular modeling and structural
studies to provide insight into the unique binding integrin
specificity of this peptide.
[0163] In another experiment, the ability of engineered knottin
peptides to block U87MG cell adhesion to vitronectin- and
fibronectin-coated microtiter plates. Vitronectin is a natural
ligand for several integrins, including .alpha..sub.v.beta..sub.3
and .alpha..sub.v.beta..sub.5. The RGD-containing peptides were all
able to inhibit U87MG cell adhesion to vitronectin-coated plates in
a dose-responsive manner. The IC.sub.50 values of cell adhesion
correlated with the .sup.125I-echistatin competition binding data
(above), indicating that inhibition of cell adhesion is directly
related to integrin binding events. Fibronectin also binds to
several integrins, (including .alpha..sub.4.beta..sub.3 and
.alpha..sub.5.beta..sub.1), but the .alpha..sub.5.beta..sub.1
integrin receptor is generally selective for fibronectin. We found
that only echistatin and knottin peptide 2.5F were able to block
U87MG cell adhesion to fibronectin-coated plates, consistent with
their ability to bind both .alpha..sub.v and
.alpha..sub.5.beta..sub.1 integrins with high affinity. The FN-RDG2
negative control was not able to inhibit U87MG cell adhesion to
vitronectin or fibronectin, as expected.
Engineered Knottin Peptides Exhibit High Stability in Serum
[0164] The stability of the knottin peptide 2.5D upon exposure to
human or mouse serum at 37.degree. C. was measured. Reversed-phase
HPLC was used to quantify the amount of intact knottin peptide
remaining at various times post incubation. We found that
approximately 90% of the peptide remained after incubation for 24 h
in human and mouse serum, with approximately 70% remaining after 96
h.
Soluble Expression of Engineered AgRP Peptides
[0165] P. pastoris was chosen for recombinant expression of the
engineered AgRP peptides, as it has been successfully used to
express proteins with disulfide bonds and significant secondary
structure. The eukaryotic quality control machinery in the
secretory pathway of yeast should help ensure proper folding and
high levels of soluble expression of the AgRP peptides, which have
four disulfide bonds and complex folds. Using conditions and
procedures described in the P. pastoris expression kit (Invitrogen
K1750-01), AgRP clones 6C, 7A (named above as 5E), 7C, 7E, and 7J
(named above as 6B) were produced in yields of 3-10 mg/L
culture.
TABLE-US-00014 TABLE 5 Sequences of Additional AgRP mutants
isolated from flow cytometry sort rounds 6 and 7. Clone Loop 4
sequence 7A (5E) (SEQ ID NO:114)
GCVRLHESCLGQQVPCCDPAATCYCSGRGDNDLVCYCR 7B (SEQ ID NO: 115)
GCVRLHESCLGQQVPCCDPAATCYCKGRGDARLQCYCR 7E (SEQ ID NO: 116)
GCVRLHESCLGQQVPCCDPAATCYCVGRGDDNLKCYCR 7J (6B) (SEQ ID NO: 90)
GCVRLHESCLGQQVPCCDPAATCYCEGRGDRDMKCYCR
[0166] The engineered AgRP peptides were expressed with N-terminal
FLAG epitope tags (DYKDDDDK) SEQ ID NO: 138 and C-terminal
hexahistidine tags for use as handles in purification and cell
binding assays through antibody detection. The expressed peptides
were purified by Ni-affinity chromatography and determined to be
>90% pure by reversed-phase HPLC and gel-filtration
chromatography. SDS-PAGE analysis was performed on reduced and
non-reduced. Peptide composition was confirmed and exact
concentrations were determined by amino acid analysis (data not
shown), and masses were obtained by MALDI-TOF mass
spectrometry.
[0167] To determine whether the FLAG and hexahistidine tags would
interfere with .alpha.v.beta.3 integrin binding, one of the AgRP
clones, 7C, was prepared without epitope tags by solid-phase
peptide synthesis using standard Fmoc chemistry. The crude, reduced
peptide was purified by reversed-phase HPLC, then oxidized with
glutathione and DMSO, as previously described. The fully oxidized
peptide was purified from unfolded and misfolded states by
reversed-phase HPLC, and the mass was confirmed by mass
spectrometry.
[0168] To compare the .alpha.v.beta.3 integrin binding affinities
of synthetic and recombinant AgRP peptides, a competition binding
assay using U87MG glioblastoma cells, which express approximately
10.sup.5 .alpha.v.beta.3 receptors per cell, as well as
.alpha.v.beta.5 and .alpha.5.beta.1 integrins. Recombinant AgRP
peptide 7C (20 nM) was pre-incubated with 10.sup.5 cells and
binding was then competed off using synthetic AgRP peptide 7C at
concentrations ranging from 1 nM to 500 nM. After washing, the
cells were stained with a fluorescein-conjugated anti-FLAG antibody
and then analyzed by flow cytometry. Competition of synthetic
peptide by recombinant peptide was performed analogously. These
experiments gave essentially identical half-maximal inhibitory
concentration (IC.sub.50) values (22.+-.3 nM and 23.+-.6 nM,
respectively; suggesting that both recombinant and synthetic AgRP
peptides are correctly folded and that the FLAG and His epitope
tags on the recombinant peptide do not interfere with integrin
binding.
Integrin Binding Affinity and Specificity of Engineered AgRP
Peptides.
[0169] Direct equilibrium binding titrations of the recombinant
engineered AgRP peptides were performed on U87MG glioblastoma
cells. Peptides were incubated with cells for 3 h at 4.degree. C.,
followed by staining with a fluorescein-conjugated anti-His
antibody and analysis by flow cytometry. Equilibrium binding
constant (K.sub.D) values were obtained by fitting plots of
concentration versus mean fluorescence intensity to a sigmoidal
curve using KaleidaGraph software.
TABLE-US-00015 TABLE 6 Binding and cell adhesion data summary for
engineered AgRP peptides. binding (K.sub.D; nM) adhesion
(IC.sub.50; nM) Clone U87MG K562-.alpha.v.beta.3
K562-.alpha.v.beta.3 6C 13 .+-. 2 15 .+-. 4 650 .+-. 250 7A 0.78
.+-. 0.37 0.89 .+-. 0.36 61 .+-. 19 7C 1.8 .+-. 0.6 2.4 .+-. 0.8 12
.+-. 6 7E 1.4 .+-. 0.5 1.6 .+-. 1.1 9.9 .+-. 5.8 7J 8.3 .+-. 1.8 16
.+-. 8 200 .+-. 150 Echistatin nd nd 3.2 .+-. 2.7 nd = not
determined
[0170] As shown above, all five engineered AgRP peptides tested
bound with low nanomolar to high picomolar affinity, and the
tightest binder, 7A, isolated from sort round 7, showed 17-fold
improvement over the worst binder, 6C, isolated from sort round 6.
The saturation levels for the different clones vary roughly with
affinity. This suggests that the mutants have different binding
off-rates that dictate their K.sub.D values, with weaker binding
clones having faster off-rates. Alternatively, the differences in
saturation levels could be due to integrin receptor clustering that
is differentially elicited or stabilized by clones with varying
affinities.
[0171] To determine the binding specificities of the AgRP peptides
for .alpha.v.beta.3 integrin versus the .alpha.v.beta.5 and
.alpha.5.beta.1 integrins also expressed on U87MG cells, K562
leukemia cells that had been stably transfected with individual
.alpha. and .beta. integrin subunits were used. The engineered AgRP
peptides were tested for binding to untransfected K562 cells, which
intrinsically express .alpha.5.beta.1 integrin. Equilibrium binding
assays were performed on the untransfected K562 cells as described
above for the U87MG cells. Three peptide concentrations were
tested. Negligible signal over background levels (cells stained
with fluorescein-conjugated anti-His antibody alone) even at 500
nM, the highest concentration tested, was observed. This
demonstrated that the engineered AgRP peptides do not appreciably
bind to .alpha.5.beta.1 integrin, and that the K562 cells
transfected to express other integrins would be useful in
determining integrin-binding specificity.
[0172] The engineered AgRP peptides were tested for binding to K562
cells expressing .alpha.v.beta.5 .alpha.iii.beta.3 or
.alpha.v.beta.3 integrins. The peptides were tested at three
concentrations with very little signal over background for cells
expressing .alpha.v.beta.5 integrin, even at 500 nM, the highest
concentration. The peptides bound weakly to the K562 cells
expressing .alpha.ii.beta.3 integrins and, as expected, strongly to
the K562 cells expressing .alpha.v.beta.3 integrins. In order to
determine K.sub.D values for the engineered AgRP peptides against
K562-.alpha.ii.beta.3 and K562-.alpha.v.beta.3 cells, binding
titrations were done over a larger range of concentrations. K.sub.D
values for AgRP peptide binding to K562-.alpha.v.beta.3 cells were
essentially identical to the K.sub.D values obtained for the U87MG
cells, indicating that binding of engineered AgRP peptides to U87MG
cells is mediated by .alpha.v.beta.3 integrin (Table 7). K.sub.D
values for peptide binding to K562-.alpha.iib.beta.3 cells could
not be determined because binding was still increasing at the
highest concentration (5 .mu.M) of peptides tested. However, from
the data, it may be estimated that the K.sub.D values for AgRP
peptide binding to the K562-.alpha.iib.beta.3 cells are much
greater than 100 nM.
Inhibition of Vitronectin-Mediated Cell Adhesion by Engineered AgRP
Peptides.
[0173] To determine whether the engineered AgRP peptides could
inhibit cell adhesion mediated by vitronectin, the primary ligand
for .alpha.v.beta.3 integrin, K562-.alpha.v.beta.3 cells were
incubated with varying concentrations of peptides in microtiter
wells coated with vitronectin to determine the ability of the
peptides to inhibit cell adhesion. The engineered AgRP peptides
were able to block vitronectin-mediated adhesion of the
K562-.alpha.v.beta.3 cells with IC.sub.50 values ranging from 9.9
to 650 nM (Table 7). The IC.sub.50 values for inhibition of cell
adhesion were 6- to 67-fold greater than the K.sub.D values against
the K562-.alpha.v.beta.3 cells. This difference may be a result of
multivalent interactions between the cell surface .alpha.v.beta.3
integrins and the immobilized vitronectin, thereby making it more
difficult for the peptides to compete.
[0174] Whether the engineered AgRP peptides could block
vitronectin-mediated adhesion to the U87MG cells was tested using
an analogous assay. However, adhesion of the U87MG cells was only
partially blocked by the peptides, even at concentrations up to 1
.mu.M. In contrast, the RGD-containing disintegrin echistatin,
which binds strongly to .alpha.v.beta.3, .alpha.v.beta.5,
.alpha.5.beta.1, and .alpha.iib.beta.3 integrins, blocked U87MG
cell adhesion to vitronectin with an IC.sub.50 of 5.8 nM. The AgRP
peptides may not effectively block U87MG adhesion compared to the
K562-.alpha.v.beta.3 cells because the .alpha.v.beta.5 integrins
co-expressed on the surface of the U87MG cells could also
contribute to vitronectin-mediated adhesion and compensate for the
loss of .alpha.v.beta.3 integrin function. These data provide
further evidence that the engineered AgRP peptides bind to
.alpha.v.beta.3 but not to .alpha.v.beta.5 integrins.
Mutagenesis of AgRP Loops 1, 2 and 3.
[0175] Modification of the remaining AgRP loops (loops 1, 2, or 3)
were studied for their effect on integrin binding affinity or
specificity, and/or on the tolerance of the other AgRP loops to
mutagenesis for further protein engineering studies. This is
illustrated as follows:
TABLE-US-00016 (SEQ ID NO: 117)
GCXXXXXXXCLGQQVPCCDPAATCYCYGRGDNDLRCYCR
[0176] In this sequence loops one and four are underlined.
TABLE-US-00017 (SEQ ID NO: 118)
GCVRLHESCXXXXXXXCCDPAATCYCYGRGDNDLRCYCR
[0177] In this sequence loops two and four are underlined.
TABLE-US-00018 (SEQ ID NO: 119)
GCVRLHESCLGQQVPCCXXXXXXCYCYGRGDNDLRCYCR
[0178] In this sequence loops three and four are underlined. Loop 4
contains the engineered regions previously described, and loops 1-3
are being modified in this example.
[0179] Yeast-displayed libraries using clone 7C as a starting
point, with loops 1, 2, or 3 as shown above individually
substituted with randomized sequences using degenerate codons were
prepared. These randomized loop libraries were subjected to four
rounds of screening by FACS to ascertain whether it would be
possible to select mutants that retained binding to .alpha.v.beta.3
integrin. In each screening round, the yeast were labeled for
peptide expression using the cMyc epitope and incubated with 50 nM
.alpha.v.beta.3 integrin, followed by staining with fluorescently
labeled secondary antibodies. Although the initial libraries showed
significantly diminished binding to .alpha.v.beta.3 integrin
compared to the parent clone 7C, mutants that retained affinity for
.alpha.v.beta.3 integrin were enriched after four rounds of
screening (data not shown).
[0180] After sort round four, six yeast-displayed clones were
chosen at random from each loop-mutagenized library (Table 8), and
were tested for their ability to bind integrins.
TABLE-US-00019 TABLE 7 Modified Loop Sequences of AgRP Loops 1, 2
and 3 Clone.sup.a Modified Loop Sequence 1-1 ASGSGDP SEQ ID NO: 120
1-2 RPLGDAG SEQ ID NO: 121 1-3 LAGLSGP SEQ ID NO: 122 1-4 RSASVGG
SEQ ID NO: 123 1-5 IASGLFG SEQ ID NO: 124 1-6 DLYGSHD SEQ ID NO:
125 2-1 GGSVGVE SEQ ID NO: 126 2-2 DPRVGVR SEQ ID NO: 127 2-3
ADTLMAA SEQ ID NO: 128 2-4 EWGRGGD SEQ ID NO: 129 2-5 GSWGTLA SEQ
ID NO: 130 2-6 WGSILGH SEQ ID NO: 131 3-1 GTPKPE SEQ ID NO: 132 3-2
SRSDAH SEQ ID NO: 133 3-3 SGLGNR SEQ ID NO: 134 3-4 QGREQS SEQ ID
NO: 135 3-5 TVTNSR SEQ ID NO: 136 3-6 TSKQHH SEQ ID NO: 137 aFirst
number indicates mutated loop, second number indicates clone
number.
[0181] Yeast displaying each mutant were treated with 50 nM
.alpha.v.beta.3, 50 nM .alpha.iib .beta.3, or 50 nM .alpha.v
.beta.5 integrin, then stained with an appropriate
fluorescein-conjugated anti-integrin antibody and analyzed by flow
cytometry. None of the clones bound to .alpha.v .beta.5 integrin
(data not shown). In contrast, all of the clones bound
.alpha.v.beta.3 integrin at levels close to that of the parent
clone 7C. The clones also weakly bound to .alpha.iib .beta.3
integrin, albeit at lower levels compared to the parent clone 7C.
The ratio of .alpha.v .beta.3 binding to .alpha.iib .beta.3 binding
was increased for all of the clones over the parent clone 7C. These
data suggest that AgRP loops 1, 2, and 3 are in principle tolerant
to mutagenesis and they may contribute to binding specificity
through either direct integrin contacts or through structural
changes in the engineered AgRP peptides.
[0182] The above binding studies demonstrate that the engineered
AgRP peptides are selective for .alpha.v .beta.3 over several other
integrins that also recognize RGD sequences, namely .alpha.v
.beta.5, .alpha..beta.1, and .alpha.iib .beta.3. The engineered
peptides did not bind at all to .alpha.v .beta.5 or
.alpha.5.beta.1, whereas weak binding was observed against
.alpha.iib .beta.3 integrin. It has been a challenge in the past to
select peptides that are selective for .alpha.v .beta.3 over
.alpha.iib .beta.3 and vice versa. Without wishing to be bound by
any theory, it is believed that the examples here provide guidance
for design of such selective peptides. RGD ligands bind to .alpha.v
.beta.3 and .alpha.iib .beta.3 near .beta.-propeller loops of the a
subunit that form a cap subdomain and a so-called
specificity-determining loop in the .beta.3 subunit. Structural
differences between .alpha.v and .alpha.iib are responsible for
variations in the cap subdomain and the 33 specificity-determining
loop, while the remainder of the .alpha.v and .alpha.iib
1-propeller structures are conserved. Consequently, specificity of
RGD-containing peptides for .alpha.v .beta.3 versus .alpha.iib
.beta.3 is controlled by the deeper .beta.-propeller pocket of
.alpha.iib. The Arg residue in RGD must be in an extended
conformation to reach into the .alpha.iib pocket to hydrogen bond
with .alpha.iib-Asp224, while .alpha.v-Asp150 and .alpha.v-Asp218
residues are found in a shallower pocket. Furthermore, .alpha.iib
.beta.3 shows a preference for aliphatic residues flanking RGD, as
.alpha.v-Asp218 is replaced by a hydrophobic Phe231 in .alpha.iib.
It is postulated that the engineered AgRP peptides tested here have
predominantly hydrophilic or charged residues flanking RGD, which
may clash with .alpha.iib-Phe231.
Mutagenesis and Screening of AgRP Loop 1, 2, and 3 Libraries
[0183] Libraries of AgRP clone 7C (Table 8) with loop 1, 2, or 3
substituted with 7, 7, or 6 randomized amino acid residues,
respectively, were prepared as described above. For screening, each
library was incubated with 50 nM .alpha.v .beta.3 integrin and
2.times.10.sup.7 cells were sorted by FACS as described above.
Three additional rounds of FACS were performed to enrich for a pool
of clones that retained binding to .alpha.v .beta.3 integrin. In
these subsequent sort rounds the libraries were over sampled at
least 10-fold to ensure diversity was maintained, but the integrin
concentration was kept at 50 nM. Clones from the fourth round of
sorting were isolated and sequenced as described above.
Recombinant and Synthetic Production of Engineered AgRP Mutants
[0184] Peptides were expressed recombinantly using the Multi-Copy
Pichia Expression Kit (Invitrogen K1750-01). The open reading frame
encoding the clone of interest was inserted into pPIC9K plasmid
between the AvrII and MluI restriction sites. In addition, DNA
encoding for a FLAG tag was inserted between SnaBI and AvrII sites,
while DNA encoding for a hexahistidine tag was inserted between
MluI and NotI restriction sites. .about.10 .mu.g of plasmid was
linearized by cutting with SacI then electroporated into the P.
pastoris strain GS115. Yeast were allowed to recover on RDB plates
and were then transferred to YPD plates containing 4 mg/mL
geneticin. Geneticin-resistant colonies were grown in BMGY and then
induced in BMMY. Cultures were grown for 3 days with methanol
concentration maintained at .about.0.5%.
[0185] AgRP Clone 7C was also prepared without FLAG or His tags
using solid-phase peptide synthesis on a CS Bio peptide synthesizer
(Menlo Park, Calif.) using standard Fmoc chemistry. The peptide was
purified by reversed-phase HPLC and then folded using 4 M
guanidine, 10 mM reduced glutathione, 2 mM oxidized glutathione,
and 0.5 M DMSO at pH 7.5. The correctly folded peptide was
separated from unfolded and partly folded peptides by
reversed-phase HPLC, where it appeared as a single peak with a
shorter retention time than unfolded or misfolded precursors. All
peptides, recombinant and synthetic, were characterized by amino
acid analysis (AAA Service Laboratory, Damascus, Oreg.) and
MALDI-TOF mass spectrometry (Stanford Protein and Nucleic Acid
Facility), which gave a single peak corresponding to the fully
folded protein containing four disulfide bonds.
Cell Binding Assays
[0186] All cell lines were cultured at 37.degree. C. with 5%
CO.sub.2. Adherent U87MG cells were obtained from ATCC and cultured
in DMEM media (Gibco 11995) supplemented with 10% fetal bovine
serum. Untransfected K562 cells (.alpha.5.beta.1-positive) were
obtained from ATCC and cultured in suspension in IMDM media (Gibco
12440) supplemented with 10% fetal bovine serum. K562 cells stably
transfected with .alpha.v .beta.3, .alpha.v .beta.5, or .alpha.iib
.beta.3 integrins were obtained from S. Blystone (Blystone, S. D.,
Graham, I. L., Lindberg, F. P. & Brown, E. J. (1994). Integrin
avb3 differentially regulates adhesive and phagocytic functions of
the fibronectin receptor .alpha.5b1. J. Cell Biol. 127, 1129-1137)
and were grown in media supplemented with 10 .mu.g/mL geneticin.
Equilibrium binding assays were performed with 10.sup.5 cells per
reaction. Cells were suspended in IBB with varying amounts of
engineered AgRP peptide at 4.degree. C. for 3 h with gentle
rocking. The cells were washed and resuspended in BPBS with a 1:40
dilution of fluorescein-conjugated anti-6.times.-His antibody
(Bethyl A190-113F) and incubated on ice for 20 min. After washing,
the cells were analyzed by flow cytometry using a BD FACSCalibur
instrument and CellQuest software (Becton Dickinson, Franklin
Lakes, N.J.). Mean fluorescence intensity values for each cell
population was plotted against concentration on a log scale. Data
was fit to sigmoidal curves to obtain equilibrium dissociation
constants using KaleidaGraph (Synergy Software), and is presented
as average values with standard deviations. Each assay was
performed a minimum of three times.
Modifications of EETI-II Loops 2 and 3
[0187] This example involves the creation of EETI loop-substituted
libraries in which a single cysteine-flanked loop of EETI (loop 2
or loop 3) was substituted with randomized amino acid sequences of
variegated lengths in order to explore the tolerance of the EETI
scaffold for different loop sizes and amino acid compositions.
Libraries were generated by overlap extension PCR using degenerate
NNS oligonucleotides (N=any nucleotide, S=G or C). Six libraries in
total were generated: two libraries of EETI loop 2 variants with
substitution lengths of 7 amino acids (EL2-7) and 9 amino acids
(EL2-9), and four libraries of EETI loop 3 variants with
substitution lengths of 6 amino acids (EL3-6), 7 amino acids
(EL3-7), 8 amino acids (EL3-8), and 9 amino acids (EL3-9). EETI
loop 1, which is responsible for binding to trypsin, was used as a
handle to probe the structural integrity of the EETI
loop-substituted clones. Library DNA was electroporated into the S.
cerevisiae EBY100 strain with linearized yeast-display plasmid as
previously described. By performing dilution plating, it was
estimated that the sizes of the loop-substituted libraries ranged
from 5.times.10.sup.6-1.times.10.sup.7 transformants. At least 50
clones from each of the six original libraries were sequenced to
confirm that the substituted loops were of the correct lengths and
had diverse amino acid compositions. The amino acid frequencies of
the loop-substituted regions were similar to those expected for a
degenerate NNS codon library.
Isolation of EETI Loop-Substituted Trypsin-Binding Clones
[0188] It has been previously demonstrated that retention of the
correct pairings of disulfide-bonded cystines in EETI can be
examined by testing for trypsin binding. Each of the EETI
loop-substituted libraries was screened for clones that were both
displayed on the yeast cell surface (as detected by indirect
immunofluorescence against the C-terminal cMyc epitope tag) and
properly folded (as determined by their ability to bind
fluorescently-labeled trypsin) using dual-color
fluorescence-activated cell sorting (FACS). By performing repeated
rounds of FACS on each yeast-displayed library, each time
collecting the top 1-2% of trypsin-binding clones, EETI
loop-substituted clones that retained the highest levels of
trypsin-binding ability were enriched. After four rounds of
sorting, a pool of clones that showed moderate to wild-type levels
of trypsin binding had been isolated from each library.
[0189] The amino acid frequencies of the enriched library
populations also differed from their original, unsorted
counterparts. Notably, glycine was enriched in all EETI
loop-substituted libraries compared to the starting libraries, and
cysteine virtually disappeared from all trypsin-binding clones,
except in the EL3-7 library. Apart from glycine, hydrophilic
residues predominated in the loop-substituted positions of enriched
clones, which was expected given their solvent-accessibility. EETI
loop 2-substituted clones were relatively tolerant of diversity
across all loop positions. Glycine comprised approximately 25-30%
of all amino acids in EETI loop 2-substituted trypsin-binding
clones at all positions in the loop except the second. On average,
EETI loop 2-substituted sequences of both 7- and 9-amino acids
contained approximately 2 glycine residues per clone. Proline and
serine, residues that commonly populate turn segments, predominated
in the second position of EETI loop 2-substituted variants (FIG.
5).
[0190] The overall diversities of EETI loop 3-substituted clones
were slightly higher than those of loop 2-substituted clones. The
greatest levels of diversity occurred in the middle positions of
the substituted loops of loop 3 variants while the first,
penultimate, and final positions had the lowest diversities.
Approximately 75% of all EL3-9 clones began with one of four
preferred residues: asparagine, arginine, valine, and histidine.
The most common amino acids for the penultimate and final positions
of EETI loop 3 were glycine and tyrosine, respectively; nearly a
quarter of all loop 3 substituted sequences from enriched clones
ended in a glycine-tyrosine doublet. We observed the aforementioned
trends in tolerated EETI loop 3 substituted clones across all loop
lengths.
[0191] EETI loop 3 was very tolerant of substitution with 6, 8, and
9 amino acid sequences, but surprisingly did not appear to be
tolerant of a 7-amino acid loop.
[0192] The Table 9 below show frequencies of amino acid
substitutions in different positions, taken from the enriched
library:
TABLE-US-00020 TABLE 8 Amino Acid Substitutions EETI Loop 3
Substituted with 9 Amino Acids Loop 1 N (36%) R (15%) V (15%)
Position 2 R (19%) K (15%) T (13%) N (10%) P (10%) 3 N (21%) T
(21%) R (11%) 4 N (17%) T (17%) R (15%) 5 R (19%) G (13%) N (12%) H
(10%) 6 G (17%) N (13%) T (13%) R (12%) D (10%) 7 P (12%) T (12%) L
(10%) M (10%) R (10%) 8 G (60%) S (17%) 9 Y (85%) EETI Loop 3
Substituted with 8 Amino Acids Loop 1 R (27%) I (20%) V (12%) N
(11%) L (9%) Position 2 H (16%) R (12%) N (11%) G (9%) K (9%) S
(9%) 3 S (18%) N (16%) R (12%) T (12%) 4 G (20%) R (18%) K (12%) T
(9%) 5 R (14%) H (11%) G (11%) Q (11%) 6 R (16%) G (14%) H (9%) 7 G
(38%) S (14%) A (11%) R (11%) K (9%) 8 Y (50%) F (14%) T (11%) EETI
Loop 3 Substituted with 7 Amino Acids Loop 1 R (18%) N (17%) D
(11%) L (11%) I (9%) S (9%) Position 2 R (14%) S (12%) G (11%) L
(11%) P (11%) T (9%) 3 G (18%) S (16%) N (11%) P (11%) R (9%) T
(9%) 4 P (12%) T (12%) S (9%) Y (9%) 5 C (14%) G (14%) P (12%) R
(9%) T (9%) 6 G (36%) R (20%) A (9%) 7 Y (38%) F (20%) C (18%) EETI
Loop 3 Substituted with 6 Amino Acids Loop 1 D (27%) N (27%) H
(18%) Position 2 T (21%) K (10%) P (10%) Q (10%) R (10%) S (10%) 3
R (24%) D (10%) L (10%) 4 S (34%) T (14%) D (10%) 5 G (34%) R (16%)
N (14%) 6 Y (31%) T (23%) EETI Loop 2 Substituted with 7 Amino
Acids Loop 1 G (32%) S (19%) V (9%) Position 2 P (21%) S (19%) V
(9%) 3 G (26%) S (13%) V (9%) 4 G (24%) L (11%) A (9%) 5 G (43%) S
(13%) 6 G (21%) S (21%) L (13%) V (9%) 7 G (19%) S (15%) R (13%) V
(11%) L (9%) 8 S (23%) G (17%) R (11%) 9 G (38%) P (13%) A (9%) Q
(9%) S (9%) EETI Loop 2 Substituted with 9 Amino Acids Loop 1 G
(26%) D (18%) V (11%) N (10%) Position 2 P (44%) G (21%) 3 G (41%)
A (10%) 4 G (21%) L (13%) V (11%) A (10%) 5 G (33%) S (15%) R (10%)
6 G (38%) S (13%) 7 G (44%) T (11%) E (10%)
Imaging
[0193] Site-Specific Labeling with Imaging Probe:
[0194] The free N-terminal amine of the engineered peptide was used
for site-specific attachment of Cy5.5, a near-infrared imaging
probe. Cy5.5 monofunctional N-hydroxysuccinimide ester (Amersham
Biosciences) was covalently-coupled to all of the polypeptides
described above, and the complexes were purified by reversed-phase
HPLC. The molecular masses of the conjugated polypeptides were
confirmed by mass spectrometry (data not shown). Interestingly,
Cy5.5 conjugation slightly increased the affinity of the
polypeptides to U87MG cells; however, the Cy5.5-labeled FN-RDG
negative control exhibited no binding.
In Vivo Optical Imaging of Tumors in Mouse U87MG Xenograft
Models
[0195] Whole-body imaging of subcutaneous mouse xenografts were
imaged with the IVIS 200 system (Xenogen) and quantified with
Living Image 2.50.1 software. FIG. 8A shows typical NIR fluorescent
images of athymic nude mice bearing subcutaneous U87MG glioblastoma
tumors after intravenous (iv) injection of 1.5 nmol of
Cy5.5-labeled RGD-miniprotein 2.5D, or the Cy5.5-labeled FN-RDG
negative control. The fluorescence intensity of the tumor to normal
tissue (T/N) ratio as a function of time is depicted in FIG. 8B,
which also includes the corresponding values for iv injection of
Cy5.5-labeled loop grafted FN-RGD and c(RGDyK) (SEQ ID NO: 140).
The Cy5.5-labeled RGD-miniprotein 2.5D shows approximately a 60%
greater T/N ratio at both early and late time points compared to
both the FN-RGD and the c(RGDyK) (SEQ ID NO: 140) pentapeptide.
These results indicate that integrin binding affinity plays a role
in tumor targeting, and provides a strong foundation for clinical
translation of RGD-miniproteins as .sup.18F and .sup.64Cu-labeled
PET imaging probes.
Preparation of Radiolabeled Integrin-Binding Polypeptides for
microPET Imaging.
[0196] Integrin binding polypeptides are conjugated to .sup.18F and
.sup.64Cu radioprobes for microPET imaging, which is PET based
imaging in small animals. Both radioprobes are studied due to
potential differences in metabolism, pharmacokinetics, and
biodistribution.
[0197] Polypeptide Synthesis:
[0198] Polypeptides are synthesized, folded, purified, and
characterized as described above.
Preparation of 4-[.sup.18F] fluorobenzoyl-labeled polypeptides
([.sup.18F] FB)
[0199] [.sup.18F] FB-labeled polypeptides are prepared by
conjugation of the N-terminal amine with N-succinimidyl
4-[.sup.18F]fluorbenzoate ([.sup.18F]SFB) under slightly basic
conditions as previously described. (See, Chen, X., Liu, S., Hou,
Y., Tohme, M., Park, R, Bading, J. R & Conti, P. S. (2004).
MicroPET imaging of breast cancer alpha v-integrin expression with
.sup.64Cu-labeled dimeric RGD peptides. Mol Imaging Bioi 6, 350-9.;
and Chen, X., Park, R, Tohme, M., Shahinian, A H., Bading, J. R
& Conti, P. S. (2004). MicroPET and autoradiographic imaging of
breast cancer alpha v-integrin expression using .sup.18F- and
.sup.64Cu labeled RGD peptide. Bioconjug Chem 15, 41-9).
[0200] Briefly, [.sup.18F]SFB are purified by semipreparative HPLC,
and the appropriate fraction are collected, diluted with water and
trapped by a C-18 cartridge. The cartridge will then be washed with
water and blown dried with Argon. [.sup.18F]SFB is eluted with
acetonitrile and rotovapped to dryness. The dried [.sup.18F]SFB is
dissolved in dimethyl sulfoxide and allowed to react with
polypeptides in sodium phosphate buffer. Final purification is done
by semipreparative HPLC. (See, Zhang, X., Xiong, Z., Wu, Y., Cai,
W., Tseng, J. R, Gambhir, S. S. & Chen, X. (2006). Quantitative
PET imaging of tumor integrin alphavbeta3 expression with
18F-FRGD2. J Nucl Med 47, 113-21.)
[0201] Before .sup.18F is used, synthesis and purification
conditions should be first validated with nonradioactive
.sup.19F-labeled polypeptides, and confirmed using mass
spectrometry.
[0202] Preparation of DOTA-Conjugated Polypeptides:
[0203] 1,4,7,10-tetradodecane-N, N', N'', N'''-tetraacetic acid
(DOTA) are conjugated to polypeptides in a manner similar to that
described before. (See, Cheng, Z., Xiong, Z., Subbarayan, M., Chen,
X. & Gambhir, S. S. (2007).) Briefly, DOTA is activated with
1-ethyl-3-[3-(dimethylamino)propyl]carboiimide at pH 5.5 for 30
minutes (4.degree. C.) with a molar ratio of
DOTA:EDC:N-hydroxysulfonosuccinimide=1:1:0.8. Polypeptides are then
be added to the prepared sulfosuccinimidyl ester of DOTA in a
stoichiometry of 5:1. The reaction is mixed at pH 8.5-9.0 overnight
(4.degree. C.). The resulting DOTA-conjugated polypeptides are then
purified by reversed phase HPLC on a semipreparative C-18 column,
and stored as a lyophilized solid. The mass is verified by
electrospray mass spectrometry.
Preparation of [.sup.64Cu]-DOTA-polypeptide radiotracers
[0204] The DOTA-conjugated polypeptides are radiolabeled with
.sup.64Cu by incubation of 5 mCi .sup.64CuCl.sub.2 in 0.1 N NaOAc,
pH 5.5 for 1 h at 50.degree. C., and terminated with
trifluoroacetic acid. The radiolabeled complex is then be purified
by HPLC, dried by rotovap, reconstituted in phosphate buffered
saline and passed through a 0.22 11 m filter for animal
experiments. Before .sup.64Cu is used, synthesis and purification
conditions should be first validated with nonradioactive "mock"
Cu-DOTA-conjugated polypeptides, and confirmed using mass
spectrometry.
In-Vitro Characterization of Radiolabeled Integrin-Binding
Polypeptides for microPET Imaging
[0205] Nonradioactive versions of all polypeptides (mock-PET
tracers) are tested first to determine if conjugation alters their
stability or integrin binding affinity. This is not expected, since
the conjugation chemistry will occur at a site in the polypeptide
that is distant from the RGD-based integrin binding loop, where
prior Cy5.5 conjugation has shown little effect (FIG. 8).
[0206] .alpha..sub.v.beta..sub.3 integrin binding assay: An
.alpha..sub.v.beta..sub.3 integrin receptor binding assay is
performed to determine the relative affinities of the mock-PET
tracers compared to their unlabeled polypeptides. Briefly,
2.times.10.sup.5 U87MG glioblastoma cells are incubated with 0.06
nM .sup.125I-echistatin in integrin binding buffer (25 mM Tris-HCl,
pH 7.4, 150 mM NaCl.sub.2 1 mM CaCl.sub.2 1 mM MgCl.sub.2, 1 mM
MnCl.sub.2, 0.1% BSA), in the presence of increasing concentrations
of mock-PET tracers at room temperature. After incubation for 3 h,
cells are pelleted by centrifugation at 1500 RPM and washed three
times in binding buffer to remove unbound ligands. The
radioactivity remaining in the cell pellet is measured by .gamma.
counting. IC.sub.50 values are determined by plotting the %
competition and using a four-point binding equation (Kaleidagraph)
to fit the data.
[0207] In-Vitro Serum Stability Studies:
[0208] Nonradioactive mock-PET tracers are incubated with mouse and
human serum for various time points at 37.degree. C., and aliquots
are acidified and flash frozen. The aliquots are then thawed and
microcentrifuged at high speeds to remove aggregates. The soluble
fraction is passed through a Sep-Pak C.sub.18 cartridge (Waters
Corp), and rinsed several times with water containing 0.1% TFA
(solvent A). The cartridge-bound PET-tracers are eluted with 90%
acetonitrile containing 0.1% TFA, lyophilized, and resuspended in
solvent A. The solution is passed through a NANOSEP (Pall Corp) 10
kDa cutoff filter and analyzed by reversed-phase HPLC to determine
the amount of polypeptide-conjugate remaining.
[0209] In-Vivo Metabolic Stability Study:
[0210] The metabolic stability of radiolabeled PET tracers is
evaluated in normal athymic nude mice. These animals are sacrificed
and dissected at various time points (30 min, 60 min, 120 min)
after injection of radiotracer via the tail vein. Blood is
immediately be centrifuged at 15,000 g for 5 min. Liver and kidneys
are homogenized and extracted with phosphate buffered saline (PBS)
and centrifuged at 15,000 g for 5 min. The extracted organ
fractions and a urine sample are separately passed through Sep-Pak
C18 cartridges (Waters Corp) to collect the radioactive polypeptide
tracers. The PET tracers are eluted with 90% acetonitrile
containing 0.1% TFA, lyophilized, and resuspended in solvent A. The
solution are analyzed by reversed-phase HPLC to determine how much
of the tracer is intact post injection and the clearance half-life
from different organs.
MicroPET Imaging in Mouse Tumor Models Using Radiolabeled
Integrin-Binding Polypeptides
[0211] To assess the potential of integrin-binding polypeptides as
clinical imaging agents, six polypeptides (c(RGDyK) (SEQ ID NO:
140), FN-RDG, FN-RGD, Miniprotein 1.5B, Miniprotein 2.5D, and
Miniprotein 2.5F are conjugated to .sup.18F or .sup.64Cu. Three
polypeptide concentrations are tested, ranging from pmol to nmol.
Each imaging study is performed in replicates with three mice.
[0212] U87MG Glioblastoma Xenograft Mouse Model:
[0213] All animal procedures are performed in the Stanford Small
Animal Imaging Facility, according to protocols approved by the
Stanford University Administrative Panels on Laboratory Animal
Care. The U87MG glioblastoma cell line (ATCC, Manassas, Va.) is
maintained at 37.degree. C. in a humidified atmosphere containing
5% CO.sub.2 in Isocove's modified Dulbecco's medium supplemented
with 5% heat-inactivated fetal bovine serum (Invitrogen Carlsbad
Calif.) and penicillin/streptomycin as an antibiotic. Female
athymic nude mice (nu/nu) obtained from Charles River Laboratories,
Inc (Cambridge, Mass.) 4 to 6 weeks of age, are subcutaneously
injected on the shoulder with 2.times.10.sup.7 U87MG glioblastoma
cells suspended in 100-uL of phosphate buffered saline. Tumors are
allowed to grow to approximately 1 cm for the microPET imaging
experiments.
[0214] MicroPET Imaging:
[0215] MicroPET imaging of tumor-bearing mice is performed on a
microPET R4 rodent model scanner (Concorde Microsystems Inc,
Knoxville, Tenn.). U87MG tumor bearing mice are injected with PET
imaging agent via the tail vein. At various times post injection,
the mice are anesthetized with 2% isoflurane, and 10 min static
scans are obtained. Images are reconstructed by a two-dimensional
ordered expectation maximum subset algorithm as previously
described. Regions of interest (ROI) are drawn over the tumor on
decay corrected whole body images and ROI derived % injected dose
per gram of tissue is determined. Statistical analysis is performed
using the student's t-test for unpaired data. A 95% confidence
level is used to determine statistical significance.
[0216] Female athymic mice bearing U87MG tumors were injected with
80-150 .mu.Ci of .sup.64Cu-DOTA-knottin 2.5D or 7-9 .mu.Ci of
[.sup.18F]-FB-E[knottin 2.5D]. Static images were taken at various
timepoints post injection using a microPET R4 rodent model scanner
(Concorde Microsystems Inc, Knoxville, Tenn.). Both the .sup.64Cu-
and .sup.18F-labeled knottin 2.5D clearly targeted the U87MG tumor,
with high contrast relative to the contralateral background. Uptake
was also observed in the kidney as both probes cleared through the
bladder. Probe uptake in the tumor was essentially blocked by
preinjection of the unlabeled peptide demonstrating specific
targeting of the tumor.
[0217] In Vivo Biodistribution Studies:
[0218] Mice are sacrificed by exanguinations at various time points
postinjection. Blood, tumor and the major organs and tissues are
collected, wet-weighed and measured in a .gamma.-counter. The %
injected dose per gram is determined for each sample. For each
mouse, radioactivity of the tissue samples is calibrated against a
known aliquot of the injectate. Values are reported as the
mean.+-.standard deviation.
[0219] The following Table 10 quantifies the .sup.64Cu-DOTA knottin
uptake by direct tissue sampling of the mice up to 24 hours post
injection and is reported in % ID (percent injected dose)/gram.
Similar results were obtained with [.sup.18F]-FB-E[knottin
2.5D].
TABLE-US-00021 TABLE 9 Organ Uptake Mean 0.5 h 1 h 2 h 4 h 25 h
Tumor 4.94 4.47 2.89 2.24 1.31 Liver 1.39 1.29 0.98 0.92 0.53
Kidney 7.26 4.06 3.45 3.26 1.75 Muscle 0.45 0.28 0.07 0.06 0.03
[0220] The above results compare favorably with imaging done with
cyclic RGD.
[0221] In previous work, glycosylation or polyethylene glycol
modification of the c(RGDyK) (SEQ ID NO: 140) pentapeptide was
shown to enhance its pharmacokinetic profiles compared to the
unmodified c(RGDyK) (SEQ ID NO: 140) PET tracer. (See, Chen, X.,
Hou, Y., Tohme, M., Park, R, Khankaldyyan, V., Gonzales-Gomez, I.,
Bading, J. R, Laug, W. E. & Conti, P. S. (2004). Pegylated
Arg-Gly-Asp peptide: .sup.64 Cu labeling and PET imaging of brain
tumor alphavbeta3-integrin expression. J Nucl Med 45, 1776-83., and
Haubner, R, Wester, H. J., Burkhart, F., Senekowitsch-Schmidtke, R,
Weber, W., Goodman, S. L., Kessler, H. & Schwaiger, M. (2001).
Glycosylated RGD-containing peptides: tracer for tumor targeting
and angiogenesis imaging with improved biokinetics. J Nucl Med 42,
326-36.)
[0222] Moreover, [.sup.18F] Galacto-c(RGDfK) has recently been used
in humans for PET-based clinical trials, and its uptake was shown
to correlate well with expression .alpha.v.beta.3 in several
different human tumors. (See, Beer, A J., Haubner, R, Wolf, I.,
Goebel, M., Luderschmidt, S., Niemeyer, M., Grosu, A L., Martinez,
M. J., Wester, H. J., Weber, W. A & Schwaiger, M. (2006).
PET-based human dosimetry of 18F-galacto-RGD, a new radiotracer for
imaging alpha v beta3 expression. J Nucl Med 47, 7639., and
Haubner, R, Weber, W. A, Beer, A J., Vabuliene, E., Reim, D.,
Sarbia, M., Becker, K. F., Goebel, M., Hein, R, Wester, H. J.,
Kessler, H. & Schwaiger, M. (2005). Noninvasive visualization
of the activated alpha v beta 3 integrin in cancer patients by
positron emission tomography and [18F]Galacto-RGD. PLoS Med 2,
e70.) Similar polypeptide modifications can be applied here if PET
imaging data indicates poor pharmacokinetics or biodistribution in
vivo.
Other Labeling Strategies
[0223] Peptides with paramagnetic ions as labels for use in
magnetic resonance imaging have also been described (Lauffer, R. B.
Magnetic Resonance in Medicine 1991 22:339-342). The label used
will be selected in accordance with the imaging modality to be
used. For example, radioactive labels such as Indium-111,
Technetium-99m or Iodine-131 can be used for planar scans or single
photon emission computed tomography (SPECT). Positron emitting
labels such as Fluorine-19 can be used in positron emission
tomography. Paramagnetic ions such as Gadlinium (III) or Manganese
(II) can be used in magnetic resonance imaging (MRI). Presence of
the label, as compared to imaging of normal tissue, permits
determination of the spread of the cancer. The amount of label
within an organ or tissue also allows determination of the presence
or absence of cancer in that organ or tissue. .sup.90Y and
.sup.177Lu may be used for therapy.
DOTA Chemical Conjugation and .sup.64Cu Radiolabeling of Peptides
2.5D and 2.5F
[0224] Peptides 2.5D and 2.5F, containing the engineered sequences
flanking the RGD integrin binding motif, were radiolabeled, as were
controls containing the native fibronectin RGD sequence and a
scrambled RGD sequence (RDG instead of RGD).
[0225] The radiolabel was coupled at the amino terminus through
1,4,7,10-tetradodecane-N, N', N'', N'''-tetraacetic acid (DOTA;
Sigma Aldrich) which was activated with
1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC; Pierce) and
N-hydroxysulfonosuccinimide (SNHS; Pierce) in water (pH 5.5) for 40
min at room temperature using a 1:1:1 molar ratio of DOTA:EDC:SNHS.
Peptides were dissolved in 300 .mu.L of sodium phosphate buffer (30
mM, pH 8.5), and added to the above in-situ prepared
sulfosuccinimidyl ester of DOTA (DOTA-OSSu). A molar excess of
DOTA-OSSu was used to drive the conjugation reaction to completion.
The reaction was allowed to proceed at room temperature for 1 h and
mixed at 4.degree. C. overnight. The resulting DOTA-peptide
conjugates were purified by reversed-phase HPLC and stored as a
lyophilized solid. The product masses were verified by ESI-MS and
MALDI-TOF-MS and peptide concentrations were determined by amino
acid analysis.
[0226] The DOTA-conjugated peptides (25 .mu.g) were radiolabeled
with .sup.64Cu by incubating with 2-3 mCi .sup.64CuCl.sub.2
(University of Wisconsin-Madison, Madison, Wis.) in 0.1 N sodium
acetate (pH 6.3) for 1 h at 45.degree. C. The reaction was
terminated with the addition of EDTA. The radiolabeled complexes
were purified using a PD-10 column (Amersham) or by radio-HPLC
using a gamma detector, dried by rotary evaporation, reconstituted
in PBS, and passed through a 0.22 .mu.m filter for animal
experiments. The radiochemical purity, determined as the ratio of
the main product peak to other peaks, was determined by HPLC to be
>95%. The radiochemical yield, determined as the ratio of final
activity of the product over the starting activity used for the
reaction, was usually over 80%. At least 7 radiolabeling reactions
were performed for experiments run on different days.
[0227] A U87MG glioblastoma xenograft mouse model was used. U87MG
cells were maintained at 37.degree. C. in a humidified atmosphere
containing 5% CO.sub.2 in Dulbecco's modified eagle medium, 10%
heat-inactivated fetal bovine serum, and penicillin-streptomycin
(all from Invitrogen). Animal procedures were carried out according
to a protocol by Stanford University Administrative Panels on
Laboratory Animal Care. Female athymic nude mice (nu/nu), obtained
at 4-6 weeks of age (Charles River Laboratories, Inc.), were
injected subcutaneously in the right or left shoulder with
2.times.10.sup.7 U87MG glioblastoma cells suspended in 100 .mu.L of
PBS. Mice were used for in vivo imaging studies when their tumors
reached approximately 8 to 10 millimeters in diameter.
[0228] U87MG tumor-bearing mice (n=3 or more for each probe) were
injected with .about.100 .mu.Ci of .sup.64Cu-DOTA-conjugated
peptides via the tail vein and imaged with a microPET R4 rodent
model scanner (Siemens Medical) using 3 or 5 min static scans. For
blocking experiments, mice were co-injected with 330 .mu.g
(.about.0.5 .mu.mol) of unlabeled c(RGDyK) (SEQ ID NO: 140). Images
were reconstructed by a two dimensional ordered expectation maximum
subset algorithm and calibrated as previously described (Wu, Y,
Zhang, X, Xiong, Z, et al. microPET imaging of glioma integrin
{alpha}v{beta}3 expression using (64)Cu-labeled tetrameric RGD
peptide. J Nucl Med 2005; 46:1707-18). ROIs were drawn over the
tumor on decay-corrected whole body images using ASIPro VM software
(Siemens Medical). The mean counts per pixel per minute were
obtained from the ROI and converted to counts per milliliter per
minute with a calibration constant. ROIs were converted to
counts/g/min, and % ID/g values were determined assuming a tissue
density of 1 g/mL. No attenuation correction was performed.
[0229] Knottin peptides 2.5D and 2.5F were shown to bind to U87MG
cells with a significantly stronger affinity (IC.sub.50=19.+-.6 nM
and 26.+-.5 nM, respectively) than both the loop-grafted FN-RGD2
(IC.sub.50=370.+-.150 nM) and c(RGDyK) (SEQ ID NO: 140)
(IC.sub.50=860.+-.400 nM) peptides. FN-RDG2 was not able to compete
for .sup.125I-echistatin binding to U87MG cells, as expected. Next,
DOTA-conjugated peptides were shown to bind to U87MG cells in a
dose-dependent manner with affinities that were comparable to the
unmodified peptides. Since U87MG cells have been shown to express
.alpha..sub.v.beta..sub.5, and .alpha..sub.5.beta..sub.1 integrins
in addition to .alpha..sub.v.beta..sub.3 integrin, we measured
integrin binding specificity by competition of .sup.125I-echistatin
to detergent-solubilized .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, .alpha..sub.5.beta..sub.1, and
.alpha..sub.iib.beta..sub.3 integrin receptors coated onto
microtiter plates. Unlabeled echistatin, our positive control,
bound strongly to all of the tested integrins, in agreement with
previous reports. All RGD-containing peptides bound to
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 integrins
to some degree, with the knottin peptides 2.5D, and 2.5F showing
the strongest levels of binding compared to FN-RGD2 and c(RGDyK)
(SEQ ID NO: 140). DOTA-conjugated FN-RDG2, our negative control
(having the RGD sequence switched to RDG), did not bind to any of
the integrins used in this study.
[0230] Tumor uptake at 1 h post injection for two high affinity
(IC.sub.50.about.20 nM).sup.64Cu-DOTA-conjugated knottin peptides
was 4.47.+-.1.21 and 4.56.+-.0.64% injected dose/gram (% ID/g),
compared to a low affinity knottin peptide (IC.sub.50.about.0.4
.mu.M; 1.48.+-.0.53% ID/g) and c(RGDyK) (SEQ ID NO: 140)
(IC.sub.50.about.1 .mu.M; 2.32.+-.0.55% ID/g), a low affinity
cyclic pentapeptide under clinical development. Furthermore,
.sup.64Cu-DOTA-conjugated knottin peptides generated lower levels
of non-specific liver uptake (.about.2% ID/g) compared to c(RGDyK)
(SEQ ID NO: 140) (.about.4% ID/g) 1 h post injection. MicroPET
imaging results were confirmed by in vivo biodistribution studies.
.sup.64Cu-DOTA-conjugated knottin peptides were stable in mouse
serum, and in vivo metabolite analysis showed minimal degradation
in the blood or tumor upon injection. Thus, engineered
integrin-binding knottin peptides show great potential as clinical
diagnostics for a variety of cancers.
[0231] The above results showed that Cy5.5- (optical label
described above) and .sup.64Cu-DOTA-conjugated FN-RGD2 knottin
peptides, which bind to integrins with affinities in the low
micromolar range, generated significantly weaker imaging signals
compared to knottin peptides 2.5D and 2.5F. These results strongly
suggest that integrin binding affinity influences tumor uptake of
knottin peptides, although other factors such as hydrophobicity can
also affect tissue biodistribution. Interestingly, in PET studies
knottin peptide 2.5F exhibited slower tumor washout compared to
2.5D, resulting in much higher tumor/blood ratios 4 h post
injection. This could be due to the ability of knottin 2.5F to bind
more tightly to .alpha..sub.5.beta..sub.1 integrins compared to
knottin 2.5D or potential differences in peptide hydrophobicity,
charge, or off-rates of integrin receptor binding. Finally, we
demonstrated that knottin peptides were stable in vitro upon
prolonged serum incubation, and in vivo in the tumor and blood
during the timeframe in which imaging experiments were performed.
In addition to increased tumor uptake, high affinity
.sup.64Cu-DOTA-labeled knottin peptides 2.5D and 2.5F demonstrated
more favorable tissue distribution as shown by lower liver uptake
compared to .sup.64Cu-DOTA-c(RGDyK). PEGylated versions of the
knottin peptides, as well as oligomeric knottin proteins that
present multiple integrin-binding RGD motifs may be synthesized
according to known methods and used in the imaging application
described here. Based on the teachings of the present disclosure,
it may be expected that these peptides will elicit enhanced tissue
distribution and/or tumor uptake compared to unmodified knottin
peptides, much like that observed with PEGylated and multivalent
c(RGDyK) (SEQ ID NO: 140) peptides, respectively.
Imaging with Agouti Peptide 7C Labeled with DOTA-.sup.64Cu
[0232] This example was carried out in similar manner to that
above. A .sup.64Cu Labeled AgRP loop 4 RDG mutant (peptide 7C,
described above). It was shown by HPLC radiochromatogram to be
essentially pure. Its binding activity was demonstrated with a
U87MG cell .sup.125I-echistatin competition binding assay, as
described above. 7C showed better binding than 3F, 6E or 6F. In
vitro cell uptake of the .sup.64Cu labeled 7C on U87-MG cells could
be blocked with cRGDyK (SEQ ID NO: 140). Biodistribution studies
showed preferential uptake of the labeled 7C by a U87-MG tumor
implanted in nude mice. Of non-tumor tissue, the kidneys were shown
to have the highest uptake. These data are shown in FIG. 13.
Peptide Formulations
[0233] The present invention also encompasses a pharmaceutical
composition useful in the treatment of cancer, comprising the
administration of a therapeutically effective amount of the
compounds of this invention, with or without pharmaceutically
acceptable carriers or diluents. Suitable compositions of this
invention include aqueous solutions comprising compounds of this
invention and pharmacologically acceptable carriers, e.g., saline,
at a pH level, e.g., 7.4. The solutions may be introduced into a
patient's bloodstream by local bolus injection.
[0234] When a compound according to this invention is administered
into a human subject, the daily dosage will normally be determined
by the prescribing physician with the dosage generally varying
according to the age, weight, and response of the individual
patient, as well as the severity of the patient's symptoms.
[0235] In one exemplary application, a suitable amount of compound
is administered to a mammal undergoing treatment for cancer.
Administration occurs in an amount between about 0.1 mg/kg of body
weight to about 60 mg/kg of body weight per day, preferably of
between 0.5 mg/kg of body weight to about 40 mg/kg of body weight
per day.
[0236] The pharmaceutical composition may be administered
parenterally, topically, orally or locally. It is preferably given
by parenteral, e.g., subcutaneous, intradermal or intramuscular
route, preferably by subcutaneous or intradermal route, in order to
reach proliferating cells in particular (e.g., potential metastases
and tumor cells). Within the scope of tumor therapy the peptide may
also be administered directly into a tumor.
[0237] The composition according to the invention for parenteral
administration is generally in the form of a solution or suspension
of the peptide in a pharmaceutically acceptable carrier, preferably
an aqueous carrier. Examples of aqueous carriers that may be used
include water, buffered water, saline solution (0.4%), glycine
solution (0.3%), hyaluronic acid and similar known carriers. Apart
from aqueous carriers it is also possible to use solvents such as
dimethylsulphoxide, propyleneglycol, dimethylformamide and mixtures
thereof. The composition may also contain pharmaceutically
acceptable excipients such as buffer substances and inorganic salts
in order to achieve normal osmotic pressure and/or effective
lyophilization. Examples of such additives are sodium and potassium
salts, e.g., chlorides and phosphates, sucrose, glucose, protein
hydrolysates, dextran, polyvinylpyrrolidone or polyethylene glycol.
The compositions may be sterilized by conventional methods, e.g.,
by sterile filtration. The composition may be decanted directly in
this form or lyophilized and mixed with a sterile solution before
use.
[0238] In one embodiment, the pharmaceutical composition according
to the invention is in the form of a topical formulation, e.g., for
dermal or transdermal application. The pharmaceutical composition
may, for example, take the form of hydrogel based on polyacrylic
acid or polyacrylamide (such as Dolobene.RTM., Merckle), as an
ointment, e.g., with polyethyleneglycol (PEG) as the carrier, like
the standard ointment DAB 8 (50% PEG 300, 50% PEG 1500), or as an
emulsion, especially a microemulsion based on water-in-oil or
oil-in-water, optionally with added liposomes. Suitable permeation
accelerators (entraining agents) include sulphoxide derivatives
such as dimethylsulphoxide (DMSO) or decylmethylsulphoxide
(decyl-MSO) and transcutol (diethyleneglycolmonoethylether) or
cyclodextrin, as well as pyrrolidones, e.g., 2-pyrrolidone,
N-methyl-2-pyrrolidone, 2-pyrrolidone-5-carboxylic acid or the
biodegradable N-(2-hydroxyethyl)-2-pyrrolidone and the fatty acid
esters thereof, urea derivatives such as dodecylurea,
1,3-didodecylurea and 1,3-diphenylurea, terpenes, e.g., D-limonene,
menthone, a-terpinol, carvol, limonene oxide or 1,8-cineol.
[0239] Other formulations are aerosols, e.g., for administering as
a nasal spray or for inhalation.
[0240] The composition according to the invention may also be
administered by means of liposomes which may take the form of
emulsions, foams, micelles, insoluble monolayers, phospholipid
dispersions, lamella layers and the like. These act as carriers for
conveying the peptides to their target of a certain tissue, e.g.,
lymphoid tissue or tumor tissue or to increase the half-life of the
peptides. The present peptides may also be formulated for oral
peptide delivery, e.g., with organic acids to inactivate digestive
enzymes and a detergent, or bile acid for temporarily opening up
the tight junctions within the intestine to facilitate transport
into the bloodstream. The present peptides may also be conjugated
to carriers such as polyethylene glycol, or modified by
glycosylation, or acylation for improvement of circulatory
half-life.
[0241] If the composition according to the invention is in the form
of a topical formulation it may also contain UV-absorbers in order
to act, for example, as a sun protection cream, for example, when
the formulation is used prophylactically against melanoma.
[0242] The person skilled in the art will find suitable
formulations and adjuvants in standard works such as "Remington's
Pharmaceutical Sciences," 1990.
CONCLUSION
[0243] The above specific description is meant to exemplify and
illustrate the invention and should not be seen as limiting the
scope of the invention, which is defined by the literal and
equivalent scope of the appended claims. Any patents or
publications mentioned in this specification, including the below
cited references are indicative of levels of those skilled in the
art to which the patent pertains and are intended to convey details
of the invention which may not be explicitly set out but which
would be understood by workers in the field Such patents or
publications are hereby incorporated by reference to the same
extent as if each was specifically and individually incorporated by
reference, as needed for the purpose of describing and enabling the
methods and materials.
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Sequence CWU 1
1
14016PRTArtificial sequenceSynthetic polypeptide 1Cys Arg Gly Asp
Cys Leu 1 5 29PRTArtificial sequenceSynthetic polypeptide 2Ala Cys
Arg Gly Asp Gly Trp Cys Gly 1 5 311PRTArtificial sequenceSynthetic
polypeptide 3Ala Cys Asp Cys Arg Gly Asp Cys Phe Cys Gly 1 5 10
49PRTArtificial sequenceSynthetic polypeptide 4Cys Arg Arg Glu Thr
Ala Trp Ala Cys 1 5 56PRTArtificial sequenceSynthetic polypeptide
5Asn Gly Arg Ala His Ala 1 5 65PRTArtificial sequenceSynthetic
polypeptide 6Pro His Ser Arg Asn 1 5 715PRTArtificial
sequenceSynthetic polypeptide 7Ala Cys Gly Ser Ala Gly Thr Cys Ser
Pro His Leu Arg Arg Pro 1 5 10 15 84PRTArtificial sequenceSynthetic
polypeptide 8Gly Arg Gly Asp 1 99PRTArtificial sequenceSynthetic
polypeptide 9Cys Asp Cys Arg Gly Asp Cys Phe Cys 1 5
105PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(4)..(4)This amino acid may be D-Phe. 10Arg
Gly Asp Phe Val 1 5 119PRTArtificial sequenceSynthetic polypeptide
11Cys Arg Arg Glu Thr Ala Trp Ala Cys 1 5 129PRTArtificial
sequenceSynthetic polypeptide 12Cys Asp Cys Arg Gly Asp Cys Phe Cys
1 5 1328PRTEcballium elaterium 13Gly Cys Pro Arg Ile Leu Met Arg
Cys Lys Gln Asp Ser Asp Cys Leu 1 5 10 15 Ala Gly Cys Val Cys Gly
Pro Asn Gly Phe Cys Gly 20 25 1447PRTHomo sapiens 14Gly Cys Val Arg
Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp
Pro Cys Ala Thr Cys Tyr Cys Arg Phe Phe Asn Ala Phe Cys 20 25 30
Tyr Cys Arg Lys Leu Gly Thr Ala Met Asn Pro Cys Ser Arg Thr 35 40
45 1535PRTArtificial sequenceSynthetic polypeptide 15Gly Cys Val
Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys
Asp Pro Ala Ala Thr Cys Tyr Cys Arg Phe Phe Asn Ala Phe Cys 20 25
30 Tyr Cys Arg 35 1648PRTAgelenopsis aperta 16Glu Asp Asn Cys Ile
Ala Glu Asp Tyr Gly Lys Cys Thr Trp Gly Gly 1 5 10 15 Thr Lys Cys
Cys Arg Gly Arg Pro Cys Arg Cys Ser Met Ile Gly Thr 20 25 30 Asn
Cys Glu Cys Thr Pro Arg Leu Ile Met Glu Gly Leu Ser Phe Ala 35 40
45 176DNAArtificial sequenceSynthetic nucleic acid 17gctagc
6186DNAArtificial sequenceSynthetic nucleic acid 18ggatcc
61933PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(3)..(5)Xaa may be any amino
acid.MISC_FEATURE(9)..(13)Xaa may be any amino acid. 19Gly Cys Xaa
Xaa Xaa Arg Gly Asp Xaa Xaa Xaa Xaa Xaa Cys Lys Gln 1 5 10 15 Asp
Ser Asp Cys Leu Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20 25
30 Gly 2033PRTArtificial sequenceSynthetic polypeptide 20Gly Cys
Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Leu Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 2133PRTArtificial sequenceSynthetic polypeptide 21Gly Cys
Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Leu Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 2233PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(3)..(5)Xaa may be any amino
acid.MISC_FEATURE(9)..(13)Xaa may be any amino acid. 22Gly Cys Xaa
Xaa Xaa Arg Gly Asp Xaa Xaa Xaa Xaa Xaa Cys Lys Gln 1 5 10 15 Asp
Ser Asp Cys Leu Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20 25
30 Gly 2333PRTArtificial sequenceSynthetic polypeptide 23Gly Cys
Ala Glu Pro Arg Gly Asp Met Pro Trp Thr Trp Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Leu Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 2433PRTArtificial sequenceSynthetic polypeptide 24Gly Cys
Val Gly Gly Arg Gly Asp Trp Ser Pro Lys Trp Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Pro Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 2533PRTArtificial sequenceSynthetic polypeptide 25Gly Cys
Ala Glu Leu Arg Gly Asp Arg Ser Tyr Pro Glu Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Leu Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 2633PRTArtificial sequenceSynthetic polypeptide 26Gly Cys
Arg Leu Pro Arg Gly Asp Val Pro Arg Pro His Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Gln Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 2733PRTArtificial sequenceSynthetic polypeptide 27Gly Cys
Tyr Pro Leu Arg Gly Asp Asn Pro Tyr Ala Ala Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Arg Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 2833PRTArtificial sequenceSynthetic polypeptide 28Gly Cys
Thr Ile Gly Arg Gly Asp Trp Ala Pro Ser Glu Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Leu Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 2933PRTArtificial sequenceSynthetic polypeptide 29Gly Cys
His Pro Pro Arg Gly Asp Asn Pro Pro Val Thr Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Leu Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 3033PRTArtificial sequenceSynthetic polypeptide 30Gly Cys
Pro Glu Pro Arg Gly Asp Asn Pro Pro Pro Ser Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Arg Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 3133PRTArtificial sequenceSynthetic polypeptide 31Gly Cys
Leu Pro Pro Arg Gly Asp Asn Pro Pro Pro Ser Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Gln Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 3233PRTArtificial sequenceSynthetic polypeptide 32Gly Cys
His Leu Gly Arg Gly Asp Trp Ala Pro Val Gly Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Pro Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 3333PRTArtificial sequenceSynthetic polypeptide 33Gly Cys
Asn Val Gly Arg Gly Asp Trp Ala Pro Ser Glu Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Pro Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 3433PRTArtificial sequenceSynthetic polypeptide 34Gly Cys
Phe Pro Gly Arg Gly Asp Trp Ala Pro Ser Ser Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Arg Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 3533PRTArtificial sequenceSynthetic polypeptide 35Gly Cys
Pro Leu Pro Arg Gly Asp Asn Pro Pro Thr Glu Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Gln Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 3633PRTArtificial sequenceSynthetic polypeptide 36Gly Cys
Ser Glu Ala Arg Gly Asp Asn Pro Arg Leu Ser Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Arg Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 3733PRTArtificial sequenceSynthetic polypeptide 37Gly Cys
Leu Leu Gly Arg Gly Asp Trp Ala Pro Glu Ala Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Arg Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 3833PRTArtificial sequenceSynthetic polypeptide 38Gly Cys
His Val Gly Arg Gly Asp Trp Ala Pro Leu Lys Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Gln Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 3933PRTArtificial sequenceSynthetic polypeptide 39Gly Cys
Val Arg Gly Arg Gly Asp Trp Ala Pro Pro Ser Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Pro Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 4033PRTArtificial sequenceSynthetic polypeptide 40Gly Cys
Leu Gly Gly Arg Gly Asp Trp Ala Pro Pro Ala Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Arg Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 4133PRTArtificial sequenceSynthetic polypeptide 41Gly Cys
Phe Val Gly Arg Gly Asp Trp Ala Pro Leu Thr Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Gln Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 4233PRTArtificial sequenceSynthetic polypeptide 42Gly Cys
Pro Val Gly Arg Gly Asp Trp Ser Pro Ala Ser Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Arg Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 4333PRTArtificial sequenceSynthetic polypeptide 43Gly Cys
Pro Arg Pro Arg Gly Asp Asn Pro Pro Leu Thr Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Leu Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 4433PRTArtificial sequenceSynthetic polypeptide 44Gly Cys
Tyr Gln Gly Arg Gly Asp Trp Ser Pro Ser Ser Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Pro Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 4533PRTArtificial sequenceSynthetic polypeptide 45Gly Cys
Ala Pro Gly Arg Gly Asp Trp Ala Pro Ser Glu Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Gln Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 4633PRTArtificial sequenceSynthetic polypeptide 46Gly Cys
Val Gln Gly Arg Gly Asp Trp Ser Pro Pro Ser Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Pro Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 4733PRTArtificial sequenceSynthetic polypeptide 47Gly Cys
His Val Gly Arg Gly Asp Trp Ala Pro Glu Glu Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Gln Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 4833PRTArtificial sequenceSynthetic polypeptide 48Gly Cys
Asp Gly Gly Arg Gly Asp Trp Ala Pro Pro Ala Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Arg Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 4933PRTArtificial sequenceSynthetic polypeptide 49Gly Cys
Pro Gln Gly Arg Gly Asp Trp Ala Pro Thr Ser Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Arg Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 5033PRTArtificial sequenceSynthetic polypeptide 50Gly Cys
Pro Arg Pro Arg Gly Asp Asn Pro Pro Leu Thr Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Leu Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 5133PRTArtificial sequenceSynthetic polypeptide 51Gly Cys
Pro Gln Gly Arg Gly Asp Trp Ala Pro Glu Trp Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Pro Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 5233PRTArtificial sequenceSynthetic polypeptide 52Gly Cys
Pro Arg Gly Arg Gly Asp Trp Ser Pro Pro Ala Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Gln Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 5333PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(4)..(4)Xaa may be any amino acid. 53Gly Cys
Pro Xaa Gly Arg Gly Asp Trp Ala Pro Pro Ser Cys Lys Gln 1 5 10 15
Asp Ser Asp Cys Arg Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20
25 30 Gly 5444PRTArtificial sequenceSynthetic polypeptide 54Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Cys Ala Thr Cys Tyr Cys Arg Gly Asp Cys Tyr Cys Arg 20
25 30 Lys Leu Gly Thr Ala Met Asn Pro Cys Ser Arg Thr 35 40
5547PRTArtificial sequenceSynthetic polypeptide 55Gly Cys Val Arg
Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp
Pro Cys Ala Thr Cys Tyr Cys Thr Gly Arg Gly Asp Ser Cys 20 25 30
Tyr Cys Arg Lys Leu Gly Thr Ala Met Asn Pro Cys Ser Arg Thr 35 40
45 5649PRTArtificial sequenceSynthetic polypeptide 56Cys Val Arg
Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys Cys 1 5 10 15 Asp
Pro Cys Ala Thr Cys Tyr Cys Thr Gly Arg Gly Asp Ser Pro Ala 20 25
30 Ser Cys Tyr Cys Arg Lys Leu Gly Thr Ala Met Asn Pro Cys Ser Arg
35 40 45 Thr 5738PRTArtificial sequenceSynthetic polypeptide 57Gly
Cys Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10
15 Cys Asp Pro Ala Ala Thr Cys Tyr Cys Thr Gly Arg Gly Asp Ser Pro
20 25 30 Ala Ser Cys Tyr Cys Arg 35 5838PRTArtificial
sequenceSynthetic polypeptide 58Gly Cys Ile Ala Glu Asp Tyr Gly Lys
Cys Thr Trp Gly Gly Thr Lys 1 5 10 15 Cys Cys Arg Gly Arg Pro Cys
Arg Cys Thr Gly Arg Gly Asp Ser Pro 20 25 30 Ala Ser Cys Glu Cys
Thr 35 5938PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(26)..(27)Xaa may be any amino
acid.MISC_FEATURE(31)..(34)Xaa may be any amino acid. 59Gly Cys Val
Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys
Asp Pro Ala Ala Thr Cys Tyr Cys Xaa Xaa Arg Gly Asp Xaa Xaa 20 25
30 Xaa Xaa Cys Tyr Cys Arg 35 6038PRTArtificial sequenceSynthetic
polypeptide 60Gly Cys Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln
Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys Tyr Cys Val Val
Arg Gly Asp Trp Arg 20 25 30 Lys Arg Cys Tyr Cys Arg 35
6138PRTArtificial sequenceSynthetic polypeptide 61Gly Cys Val Arg
Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp
Pro Ala Ala Thr Cys Tyr Cys Glu Glu Arg Gly Asp Met Leu 20 25 30
Glu Lys Cys Tyr Cys Arg 35 6238PRTArtificial sequenceSynthetic
polypeptide 62Gly Cys Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln
Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys Tyr Cys Glu Thr
Arg Gly Asp Gly Lys 20 25 30 Glu Lys Cys Tyr Cys Arg 35
6338PRTArtificial sequenceSynthetic polypeptide 63Gly Cys Val Arg
Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp
Pro Ala Ala Thr Cys Tyr Cys Gln Trp Arg Gly Asp Gly Asp 20 25 30
Val Lys Cys Tyr Cys Arg 35 6438PRTArtificial sequenceSynthetic
polypeptide 64Gly Cys Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln
Val Pro Cys 1 5
10 15 Cys Asp Pro Ala Ala Thr Cys Tyr Cys Ser Arg Arg Gly Asp Met
Arg 20 25 30 Glu Arg Cys Tyr Cys Arg 35 6538PRTArtificial
sequenceSynthetic polypeptide 65Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Gln Tyr Arg Gly Asp Gly Met 20 25 30 Lys His Cys Tyr Cys
Arg 35 6638PRTArtificial sequenceSynthetic polypeptide 66Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Ala Ala Thr Cys Tyr Cys Thr Gly Arg Gly Asp Thr Lys 20
25 30 Val Leu Cys Tyr Cys Arg 35 6738PRTArtificial
sequenceSynthetic polypeptide 67Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Val Glu Arg Gly Asp Met Lys 20 25 30 Arg Arg Cys Tyr Cys
Arg 35 6838PRTArtificial sequenceSynthetic polypeptide 68Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Ala Ala Thr Cys Tyr Cys Thr Gly Arg Gly Asp Val Arg 20
25 30 Met Asn Cys Tyr Cys Arg 35 6938PRTArtificial
sequenceSynthetic polypeptide 69Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Val Glu Arg Gly Asp Gly Met 20 25 30 Ser Lys Cys Tyr Cys
Arg 35 7038PRTArtificial sequenceSynthetic polypeptide 70Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Ala Ala Thr Cys Tyr Cys Arg Gly Arg Gly Asp Met Arg 20
25 30 Arg Glu Cys Tyr Cys Arg 35 7138PRTArtificial
sequenceSynthetic polypeptide 71Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Glu Gly Arg Gly Asp Val Lys 20 25 30 Val Asn Cys Tyr Cys
Arg 35 7238PRTArtificial sequenceSynthetic polypeptide 72Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Ala Ala Thr Cys Tyr Cys Val Gly Arg Gly Asp Glu Lys 20
25 30 Met Ser Cys Tyr Cys Arg 35 7338PRTArtificial
sequenceSynthetic polypeptide 73Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Val Ser Arg Gly Asp Met Arg 20 25 30 Lys Arg Cys Tyr Cys
Arg 35 7438PRTArtificial sequenceSynthetic polypeptide 74Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Ala Ala Thr Cys Tyr Cys Glu Arg Arg Gly Asp Ser Val 20
25 30 Lys Lys Cys Tyr Cys Arg 35 7538PRTArtificial
sequenceSynthetic polypeptide 75Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Glu Gly Arg Gly Asp Thr Arg 20 25 30 Arg Arg Cys Tyr Cys
Arg 35 7638PRTArtificial sequenceSynthetic polypeptide 76Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Ala Ala Thr Cys Tyr Cys Glu Gly Arg Gly Asp Val Val 20
25 30 Arg Arg Cys Tyr Cys Arg 35 7738PRTArtificial
sequenceSynthetic polypeptide 77Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Lys Gly Arg Gly Asp Asn Lys 20 25 30 Arg Lys Cys Tyr Cys
Arg 35 7838PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(21)..(21)Xaa may be any amino acid. 78Gly
Cys Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10
15 Cys Asp Pro Ala Xaa Thr Cys Tyr Cys Lys Gly Arg Gly Asp Val Arg
20 25 30 Arg Val Cys Tyr Cys Arg 35 7938PRTArtificial
sequenceSynthetic polypeptide 79Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Val Gly Arg Gly Asp Asn Lys 20 25 30 Val Lys Cys Tyr Cys
Arg 35 8038PRTArtificial sequenceSynthetic polypeptide 80Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Ala Ala Thr Cys Tyr Cys Val Gly Arg Gly Asp Asn Arg 20
25 30 Leu Lys Cys Tyr Cys Arg 35 8138PRTArtificial
sequenceSynthetic polypeptide 81Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Val Glu Arg Gly Asp Gly Met 20 25 30 Lys Lys Cys Tyr Cys
Arg 35 8238PRTArtificial sequenceSynthetic polypeptide 82Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Ala Ala Thr Cys Tyr Cys Glu Gly Arg Gly Asp Met Arg 20
25 30 Arg Arg Cys Tyr Cys Arg 35 8338PRTArtificial
sequenceSynthetic polypeptide 83Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Gln Gly Arg Gly Asp Gly Asp 20 25 30 Val Lys Cys Tyr Cys
Arg 35 8438PRTArtificial sequenceSynthetic polypeptide 84Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Ala Ala Thr Cys Tyr Cys Ser Gly Arg Gly Asp Asn Asp 20
25 30 Leu Val Cys Tyr Cys Arg 35 8538PRTArtificial
sequenceSynthetic polypeptide 85Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Val Glu Arg Gly Asp Gly Met 20 25 30 Ile Arg Cys Tyr Cys
Arg 35 8638PRTArtificial sequenceSynthetic polypeptide 86Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Ala Ala Thr Cys Tyr Cys Ser Gly Arg Gly Asp Asn Asp 20
25 30 Leu Val Cys Tyr Cys Arg 35 8738PRTArtificial
sequenceSynthetic polypeptide 87Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Glu Gly Arg Gly Asp Met Lys 20 25 30 Met Lys Cys Tyr Cys
Arg 35 8838PRTArtificial sequenceSynthetic polypeptide 88Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Ala Ala Thr Cys Tyr Cys Ile Gly Arg Gly Asp Val Arg 20
25 30 Arg Arg Cys Tyr Cys Arg 35 8938PRTArtificial
sequenceSynthetic polypeptide 89Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Glu Glu Arg Gly Asp Gly Arg 20 25 30 Lys Lys Cys Tyr Cys
Arg 35 9038PRTArtificial sequenceSynthetic polypeptide 90Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Ala Ala Thr Cys Tyr Cys Glu Gly Arg Gly Asp Arg Asp 20
25 30 Met Lys Cys Tyr Cys Arg 35 9138PRTArtificial
sequenceSynthetic polypeptide 91Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Thr Gly Arg Gly Asp Glu Lys 20 25 30 Leu Arg Cys Tyr Cys
Arg 35 9238PRTArtificial sequenceSynthetic polypeptide 92Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Ala Ala Thr Cys Tyr Cys Val Glu Arg Gly Asp Gly Asn 20
25 30 Arg Arg Cys Tyr Cys Arg 35 9338PRTArtificial
sequenceSynthetic polypeptide 93Gly Cys Val Arg Leu His Glu Ser Cys
Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys
Tyr Cys Glu Ser Arg Gly Asp Val Val 20 25 30 Arg Lys Cys Tyr Cys
Arg 35 9438PRTArtificial sequenceSynthetic polypeptide 94Gly Cys
Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15
Cys Asp Pro Ala Ala Thr Cys Tyr Cys Tyr Gly Arg Gly Asp Asn Asp 20
25 30 Leu Arg Cys Tyr Cys Arg 35 9599DNAArtificial
sequenceSynthetic polypeptide 95gggtgcgtgg gggggagagg ggattggagc
ccgaagtggt gcaaacagga ctccgactgc 60ccggctggct gcgtttgcgg gcccaacggt
ttctgcgga 999699DNAArtificial sequenceSynthetic polypeptide
96gggtgcacga tcgggagagg ggattgggcc ccctcggagt gcaaacagga ctccgactgc
60ctggctggct gcgtttgcgg gcccaacggt ttctgcgga 999799DNAArtificial
sequenceSynthetic polypeptide 97gggtgccacc cgccgagagg ggataacccc
cccgtgactt gcaaacagga ctccgactgc 60ctggctggct gcgtttgcgg gcccaacggt
ttctgcgga 999899DNAArtificial sequenceSynthetic polypeptide
98gggtgctatc aaggaagagg ggattggtct ccttcatcgt gcaaacagga ctccgactgc
60ccagctggct gcgtttgcgg gcccaacggt ttctgcgga 999999DNAArtificial
sequenceSynthetic polypeptide 99gggtgccatg taggaagagg ggattgggct
cctgaagagt gcaaacagga ctccgactgc 60caagctggct gcgtttgcgg gcccaacggt
ttctgcgga 9910099DNAArtificial sequenceSynthetic polypeptide
100gggtgcgatg gaggaagagg ggattgggct cctccagcgt gcaaacagga
ctccgactgc 60cgagctggct gcgtttgcgg gcccaacggt ttctgcgga
9910199DNAArtificial sequenceSynthetic polypeptide 101gggtgccctc
aaggaagagg ggattgggct cctacatcgt gcaaacagga ctccgactgc 60cgagctggct
gcgtttgcgg gcccaacggt ttctgcgga 9910299DNAArtificial
sequenceSynthetic polypeptide 102gggtgccctc gaccaagagg ggataaccct
cctctaacgt gcaaacagga ctccgactgc 60ctagctggct gcgtttgcgg gcccaacggt
ttctgcgga 9910399DNAArtificial sequenceSynthetic polypeptide
103gggtgccctc aaggaagagg ggattgggct cctgaatggt gcaaacagga
ctccgactgc 60ccagctggct gcgtttgcgg gcccaacggt ttctgcgga
9910499DNAArtificial sequenceSynthetic polypeptide 104gggtgccctc
gaggaagagg ggattggtct cctccagcgt gcaaacagga ctccgactgc 60caagctggct
gcgtttgcgg gcccaacggt ttctgcgga 991056PRTArtificial
sequenceSynthetic polypeptide 105Gly Arg Gly Asp Ser Pro 1 5
10611PRTArtificial sequenceSynthetic polypeptide 106Pro Leu Pro Arg
Gly Asp Asn Pro Pro Thr Glu 1 5 10 10746PRTArtificial
sequenceSynthetic polypeptide 107Cys Val Arg Leu His Glu Ser Leu
Cys Gly Gln Gln Val Pro Cys Cys 1 5 10 15 Asp Pro Cys Ala Thr Cys
Tyr Cys Arg Phe Phe Asn Ala Phe Cys Tyr 20 25 30 Cys Arg Lys Leu
Gly Thr Ala Met Asn Pro Cys Ser Arg Thr 35 40 45 1087PRTArtificial
sequenceSynthetic polypeptide 108Gly Arg Gly Asp Trp Ala Pro 1 5
1094PRTArtificial sequenceSynthetic polypeptide 109Gly Pro Asn Gly
1 1104PRTArtificial sequenceSynthetic polypeptide 110Cys Leu Ala
Gly 1 1114PRTArtificial sequenceSynthetic polypeptide 111Cys Pro
Ala Gly 1 1124PRTArtificial sequenceSynthetic polypeptide 112Cys
Gln Ala Gly 1 1134PRTArtificial sequenceSynthetic polypeptide
113Cys Arg Ala Gly 1 11438PRTArtificial sequenceSynthetic
polypeptide 114Gly Cys Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln
Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys Tyr Cys Ser Gly
Arg Gly Asp Asn Asp 20 25 30 Leu Val Cys Tyr Cys Arg 35
11538PRTArtificial sequenceSynthetic polypeptide 115Gly Cys Val Arg
Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10 15 Cys Asp
Pro Ala Ala Thr Cys Tyr Cys Lys Gly Arg Gly Asp Ala Arg 20 25 30
Leu Gln Cys Tyr Cys Arg 35 11638PRTArtificial sequenceSynthetic
polypeptide 116Gly Cys Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln
Val Pro Cys 1 5 10 15 Cys Asp Pro Ala Ala Thr Cys Tyr Cys Val Gly
Arg Gly Asp Asp Asn 20 25 30 Leu Lys Cys Tyr Cys Arg 35
11739PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(3)..(9)Xaa may be any amino acid. 117Gly
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Leu Gly Gln Gln Val Pro 1 5 10
15 Cys Cys Asp Pro Ala Ala Thr Cys Tyr Cys Tyr Gly Arg Gly Asp Asn
20 25 30 Asp Leu Arg Cys Tyr Cys Arg 35 11839PRTArtificial
sequenceSynthetic polypeptideMISC_FEATURE(10)..(16)Xaa may be any
amino acid. 118Gly Cys Val Arg Leu His Glu Ser Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Cys Cys Asp Pro Ala Ala Thr Cys Tyr Cys Tyr
Gly Arg Gly Asp Asn 20 25 30 Asp Leu Arg Cys Tyr Cys Arg 35
11939PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(18)..(23)Xaa may be any amino acid. 119Gly
Cys Val Arg Leu His Glu Ser Cys Leu Gly Gln Gln Val Pro Cys 1 5 10
15 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Tyr Cys Tyr Gly Arg Gly Asp Asn
20 25 30 Asp Leu Arg Cys Tyr Cys Arg 35 1207PRTArtificial
sequenceSynthetic polypeptide 120Ala Ser Gly Ser Gly Asp Pro 1 5
1217PRTArtificial sequenceSynthetic polypeptide 121Arg Pro Leu Gly
Asp Ala Gly 1 5 1227PRTArtificial sequenceSynthetic polypeptide
122Leu Ala Gly Leu Ser Gly Pro 1 5 1237PRTArtificial
sequenceSynthetic polypeptide 123Arg Ser Ala Ser Val Gly Gly 1 5
1247PRTArtificial sequenceSynthetic polypeptide 124Ile Ala Ser Gly
Leu Phe Gly 1 5 1257PRTArtificial sequenceSynthetic polypeptide
125Asp Leu Tyr Gly Ser His Asp 1 5 1267PRTArtificial
sequenceSynthetic polypeptide 126Gly Gly Ser Val Gly Val Glu 1 5
1277PRTArtificial sequenceSynthetic polypeptide 127Asp Pro Arg Val
Gly Val Arg 1 5 1287PRTArtificial sequenceSynthetic polypeptide
128Ala Asp Thr Leu Met Ala Ala 1 5 1297PRTArtificial
sequenceSynthetic polypeptide 129Glu Trp Gly Arg Gly Gly Asp 1 5
1307PRTArtificial sequenceSynthetic polypeptide 130Gly Ser Trp Gly
Thr Leu Ala 1 5 1317PRTArtificial sequenceSynthetic polypeptide
131Trp Gly Ser Ile Leu Gly His 1 5 1326PRTArtificial
sequenceSynthetic polypeptide 132Gly Thr Pro Lys Pro Glu 1 5
1336PRTArtificial
sequenceSynthetic polypeptide 133Ser Arg Ser Asp Ala His 1 5
1346PRTArtificial sequenceSynthetic polypeptide 134Ser Gly Leu Gly
Asn Arg 1 5 1356PRTArtificial sequenceSynthetic polypeptide 135Gln
Gly Arg Glu Gln Ser 1 5 1366PRTArtificial sequenceSynthetic
polypeptide 136Thr Val Thr Asn Ser Arg 1 5 1376PRTArtificial
sequenceSynthetic polypeptide 137Thr Ser Lys Gln His His 1 5
1388PRTArtificial sequenceSynthetic polypeptide 138Asp Tyr Lys Asp
Asp Asp Asp Lys 1 5 1394PRTArtificial sequenceSynthetic polypeptide
139Ser Ala Gly Thr 1 1405PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(4)..(4)This amino acid may be D-Tyr. 140Arg
Gly Asp Tyr Lys 1 5
* * * * *
References