U.S. patent application number 13/227213 was filed with the patent office on 2012-02-16 for peptides that specifically bind hgf receptor (cmet) and uses thereof.
Invention is credited to Daniel T. DRANSFIELD, Robert Charles Ladner, Palaniappa Nanjappan, Aaron K. Sato, Regi Thomas.
Application Number | 20120039802 13/227213 |
Document ID | / |
Family ID | 32962606 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120039802 |
Kind Code |
A1 |
DRANSFIELD; Daniel T. ; et
al. |
February 16, 2012 |
PEPTIDES THAT SPECIFICALLY BIND HGF RECEPTOR (CMET) AND USES
THEREOF
Abstract
A polypeptide or multimeric polypeptide construct having the
ability to bind to cMet or a complex comprising cMet and HGF, and
methods for use are disclosed.
Inventors: |
DRANSFIELD; Daniel T.;
(Hanson, MA) ; Sato; Aaron K.; (Richmond, CA)
; Ladner; Robert Charles; (Ijamsville, MD) ;
Thomas; Regi; (Princeton, NJ) ; Nanjappan;
Palaniappa; (Princeton, NJ) |
Family ID: |
32962606 |
Appl. No.: |
13/227213 |
Filed: |
September 7, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12635193 |
Dec 10, 2009 |
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13227213 |
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12012011 |
Jan 30, 2008 |
8044175 |
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12635193 |
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10792582 |
Mar 3, 2004 |
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12012011 |
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60451588 |
Mar 3, 2003 |
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Current U.S.
Class: |
424/1.69 ;
424/9.1; 424/9.5; 424/9.6 |
Current CPC
Class: |
C07K 14/4753 20130101;
B82Y 5/00 20130101; A61K 47/60 20170801; A61K 49/0056 20130101;
C07K 7/08 20130101; A61K 47/543 20170801; A61K 51/08 20130101; A61K
47/665 20170801; A61K 49/0043 20130101; C07K 14/00 20130101; A61P
35/00 20180101; A61P 43/00 20180101 |
Class at
Publication: |
424/1.69 ;
424/9.1; 424/9.5; 424/9.6 |
International
Class: |
A61K 51/08 20060101
A61K051/08; A61K 49/22 20060101 A61K049/22; A61P 35/00 20060101
A61P035/00; A61K 49/00 20060101 A61K049/00 |
Claims
1.-32. (canceled)
33. A diagnostic imaging agent comprising: a) a polypeptide which
binds cMet or which binds a complex comprising cMet and HGF,
wherein the polypeptide comprises the amino acid sequence:
C-X2-C-X3-G-P-P-X4-F-X5-C-X6-C; wherein X2=H, Y, N, R, S, D, Q, L
or E; X3=S, G, N, I, K or Y; X4=T, W, R, S, A or Q; X5=E or Q;
X6=W, Y, F, T or A; and, b) a detectable label for diagnostic
imaging.
34. The diagnostic imaging agent of claim 33, wherein the
polypeptide comprises the amino acid sequence:
G-X1-C-X2-C-X3-G-P-P-X4-F-X5-C-X6-C-X7-X8-X9-X10-P; wherein X1=S,
N, A, K, F, D, I, R, G or T; X2=H, Y, N, R, S, D, Q, L or E; X3=S,
G, N, I or Y; X4=T, W, R, S, A or Q; X5=E or Q; X6=W, Y, F, T or A;
X7=Y or F; X8=G, D, A, E, S, R, H, T or V; X9=T, S, V, M, A, L, Q,
E or P; and, X10=E, D, A, N, Y or G.
35. The diagnostic imaging agent of claim 33, wherein the
polypeptide comprises the amino acid sequence:
S-C-X2-C-X3-G-P-P-X4-F-X5-C-X6-C-X7-X8-X9-X10; wherein X2=N, H or
Y; X3=G or S; X4=T; X5=E; X6=W; X7=Y; X8=A, D, E, G or S; X9=S or
T; and, X10=D or E.
36. The diagnostic imaging agent of claim 33, wherein the
polypeptide comprises the amino acid sequence:
E-X12-G-S-C-H-C-S-G-P-.alpha.-T-F-E-C-X6-C-X7; wherein X12=A, E, G
or S; X6=F, W or Y; and, X7=F or Y.
37. The diagnostic imaging agent of claim 33, wherein the
polypeptide comprises the amino acid sequence: TABLE-US-00014
C-Y-C-S-G-P-P-R-F-E-C-W-C or
S-E-T-R-P-T-E-A-G-S-C-Y-C-S-G-P-P-R-F-E-C-W-C-Y-E- T-E-P-T-E.
38. The diagnostic imaging agent of claim 33, wherein the
detectable label is an enzyme, a fluorescent compound, a liposome,
an optical dye, a paramagnetic metal ion, an ultrasound contrast
agent, or a diagnostic radionuclide.
39. The diagnostic imaging agent of claim 38, wherein the
paramagnetic metal ion or the diagnostic radionuclide is complexed
with a chelator.
40. The diagnostic imaging agent of claim 33, wherein the
detectable label is an ultrasound contrast agent or a diagnostic
radionuclide, wherein the diagnostic radionuclide is complexed with
a chelator.
41. The diagnostic imaging agent of claim 33, wherein the
detectable label is detectable by optical imaging, acousto-optical
imaging or sonoluminescent imaging.
42. The diagnostic imaging agent of claim 41, wherein the
detectable label is a photolabel.
43. The diagnostic imaging agent of claim 42, wherein the
photolabel is an optical dye or bioluminescent molecule.
44. The diagnostic imaging agent of claim 33, further comprising a
linker.
45. A method of detecting cMet or a complex comprising cMet and HGF
in a subject, the method comprising: (a) administering to the
subject the diagnostic imaging agent of claim 33; and, (b)
detecting the diagnostic imaging agent in the subject.
46. The method of claim 45, further comprising imaging a portion of
the subject.
47. The method of claim 46, further comprising constructing an
image.
48. The method of claim 47, wherein the image is constructed using
ultrasound energy, radioimaging or light imaging.
49. The diagnostic imaging agent of claim 37, wherein the
detectable label is an enzyme, a fluorescent compound, a liposome,
an optical dye, a paramagnetic metal ion, an ultrasound contrast
agent, or a diagnostic radionuclide.
50. The diagnostic imaging agent of claim 49, wherein the
paramagnetic metal ion, or the diagnostic radionuclide is complexed
with a chelator.
51. The diagnostic imaging agent of claim 37, wherein the
detectable label is an ultrasound contrast agent or a diagnostic
radionuclide, wherein the diagnostic radionuclide is complexed with
a chelator.
52. The diagnostic imaging agent of claim 37, wherein the
detectable label is detectable by optical imaging, acousto-optical
imaging or sonoluminescent imaging.
53. The diagnostic imaging agent of claim 52, wherein the
detectable label is a photolabel.
54. The diagnostic imaging agent of claim 53, wherein the
photolabel is an optical dye or bioluminescent molecule.
55. The diagnostic imaging agent of claim 37, further comprising a
linker.
56. A method of detecting cMet or a complex comprising cMet and HGF
in a subject, the method comprising: (a) administering to the
subject the diagnostic imaging agent of claim 37; and, (b)
detecting the diagnostic imaging agent in the subject.
57. The method of claim 56, further comprising imaging a portion of
the subject.
58. The method of claim 56, further comprising constructing an
image.
59. The method of claim 58, wherein the image is constructed using
ultrasound energy, radioimaging or light imaging.
Description
[0001] This application claims priority under 35 USC .sctn.119(e)
to U.S. Provisional Patent Application Ser. No. 60/451,588, filed
on Mar. 3, 2003, the entire contents of which is hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Hepatocyte growth factor (also known as scatter factor) is a
multi-functional growth factor involved in various physiological
processes such as embryogenesis, wound healing and angiogenesis. It
has become apparent that HGF, through interactions with its high
affinity receptor (cMet), is involved in tumor growth, invasion and
metastasis. In fact, dysregulated cMet expression (for example, the
overexpression of cMet in neoplastic epithelium of colorectal
adenomas and in other carcinomas as compared to normal mucosa)
and/or activity, as well as hyperactivity of the cMet receptor
through an autocrine stimulatory loop with HGF, has been
demonstrated in a variety of tumor tissues and induces oncogenic
transformation of specific cell lines.
[0003] In general, HGF is produced by the stromal cells, which form
part of many epithelial tumors; however, it is believed that the
production of HGF by tumor cells themselves comprises the main
pathway leading to the hyperproliferation of specific tumors.
HGF/cMet autocrine stimulatory loops have been detected in,
gliomas, osteosarcomas, and mammary, prostate, breast, lung and
other carcinomas.
[0004] Interrupting the HGF interaction with the cMet receptor
slows tumor progression in animal models. In addition to
stimulating proliferation of certain cancer cells through
activation of cMet, HGF also protects against DNA-damaging
agent-induced cytotoxicity in a variety of cell lines susceptible
to hyperproliferative phenotypes (e.g., breast cancer). Therefore,
preventing HGF from binding to cMet could predispose certain cancer
cells to the cytotoxicity of certain drugs.
[0005] In addition to hyperproliferative disorders, cMet also has
been linked to angiogenesis. For example, stimulation of cMet leads
to the production of vascular endothelial growth factor (VEGF),
which, in turn, stimulates angiogenesis. Additionally, stimulation
of cMet also has been implicated in promoting wound healing.
[0006] In addition to identifying the cMet receptor as a
therapeutic target for hyperproliferative disorders, angiogenesis
and wound healing, the large discrepancy between expression levels
of neoplastic and corresponding normal tissues indicates that cMet
is an attractive target for imaging applications directed to
hyperproliferative disorders.
SUMMARY OF THE INVENTION
[0007] The present invention relates to peptides, peptide complexes
and compositions having the ability to bind to cMet and antagonize
hepatocyte growth factor (HGF) activity by preventing HGF from
binding to cMet. In addition, this invention relates to such
peptides, peptide complexes and compositions having the ability to
bind to cMet for the purpose of detecting and targeting this
receptor, inhibiting cMet activity independent of HGF antagonistic
properties, and for the purpose of diagnostic imaging. The
involvement of the HGF/cMet axis in a variety of cellular functions
including cellular proliferation, wound healing and angiogenesis,
leading to hyperproliferative diseases such as cancer, make the
present invention particularly useful for interrupting HGF-mediated
physiological events, for targeting substances, e.g., therapeutics,
including radiotherapeutics, to such sites, and for imaging
important sites of cellular hyperproliferation.
[0008] In answer to the need for improved materials and methods for
detecting, localizing, imaging, measuring and possibly inhibiting
or affecting, e.g., hyperproliferation and/or angiogenesis, it has
been surprisingly discovered that twelve classes of non-naturally
occurring polypeptides bind specifically to cMet. Appropriate
labeling of such polypeptides provides detectable imaging agents
that can bind, e.g., at high concentration, to cMet-expressing
cells or cells exhibiting HGF/cMet complexes, providing specific
imaging agents for sites of cellular proliferation and/or
angiogenesis. The cMet binding polypeptides of the instant
invention can thus be used in the detection and diagnosis of such
hyperproliferative-related and/or angiogenesis-related disorders.
Conjugation or fusion of such polypeptides with effective agents
such as cMet inhibitors or tumoricidal agents also can be used to
treat pathogenic tumors, e.g., by causing the conjugate or fusion
to "home" to the site of active proliferation and/or angiogenesis,
thereby providing an effective means for treating pathogenic
conditions associated with hyperproliferation and/or
angiogenesis.
[0009] This invention pertains to cMet binding polypeptides, and
includes use of a single binding polypeptide as a monomer or in a
multimeric or polymeric construct as well as use of more than one
binding polypeptide of the invention in multimeric or polymeric
constructs. Binding polypeptides according to this invention are
useful in any application where binding, inhibiting, detecting or
isolating cMet, or fragments thereof retaining the polypeptide
binding site, is advantageous. A particularly important aspect of
such binding polypeptides is the inhibition of cMet activity,
either through competition with HGF for cMet binding, or by
directly inhibiting cMet activity irrespective of whether HGF is
bound or not. For example, in some cases, cMet signaling can occur
in the absence of HGF binding, in such situations, a binding
polypeptide that inhibits cMet signaling activity irrespective of
whether HGF is bound, would be useful in inhibiting cMet
signaling.
[0010] Another particularly advantageous use of the binding
polypeptides disclosed herein is in a method of imaging cellular
proliferation and/or angiogenesis in vivo. The method entails the
use of specific binding polypeptides according to the invention for
detecting a site of cellular proliferation and/or angiogenesis,
where the binding polypeptides have been detectably labeled for use
as imaging agents, including magnetic resonance imaging (MRI)
contrast agents, x-ray imaging agents, radiopharmaceutical imaging
agents, ultrasound imaging agents, and optical imaging agents.
[0011] Yet another advantageous use of the cMet binding
polypeptides disclosed herein is to target therapeutic agents,
(including compounds capable of providing a therapeutic,
radiotherapeutic or cytotoxic effect) or delivery vehicles for
therapeutics (including drugs, genetic material, etc.) to sites of
hyperproliferation and/or angiogenesis or other tissue expressing
cMet.
[0012] The cMet receptor is part of the receptor tyrosine kinase
family of signaling molecules. For the purposes of the present
invention, receptor tyrosine kinase function can include any one
of: oligomerization of the receptor, receptor phosphorylation,
kinase activity of the receptor, recruitment of downstream
signaling molecules, induction of genes, induction of cell
proliferation, induction of cell migration, or combination thereof.
"Heteromeric" molecules, used herein to refer to molecules
containing more than one cMet binding peptide as described herein,
such that each binding peptide of the heteromeric molecule binds to
a different site, e.g., "epitope", of cMet, also are encompassed by
the present invention. For example, heteromeric constructs of
binding polypeptides provided herein could, for example, bind, via
one binding peptide, to, for example, the HGF binding site of cMet,
while another binding peptide of the heteromeric molecule binds to
a different high affinity binding site of cMet. Targeting two or
more distinct epitopes on cMet with a single binding construct can
greatly improve the ability of the construct to inhibit HGF binding
and/or receptor function (such inhibition can occur by direct
inhibition of cMet irrespective of HGF binding). Even binding
peptides with weak ability to block receptor activity can be used
to generate heteromeric constructs having improved ability to block
HGF-dependent and HGF-independent receptor function.
[0013] Therefore, the present invention is drawn to constructs
comprising means for producing multimeric molecules comprising two
or more binding polypeptides, at least one of which binds cMet. In
one embodiment, the multimeric constructs comprise two or more
copies of a single binding polypeptide or nucleotide sequence that
encode two or more copies of a single binding polypeptide. In
another embodiment, the multimeric constructs of the present
invention comprise two or more binding polypeptides or nucleotide
sequence that encode two or more binding polypeptides, such that at
least two of the binding polypeptides in the construct are specific
for different epitopes of cMet. These constructs also are referred
to herein as "heteromeric constructs", "heteromultimers", etc. The
constructs of the present invention also can include unrelated, or
control peptide. The constructs can include two or more, three or
more, or four or more binding polypeptides or the nucleotide
sequences that encode such polypeptides. Based on the teachings
provided herein, one of ordinary skill in the art is able to
assemble the binding polypeptides provided herein into multimeric
constructs and to select multimeric constructs having improved
properties, such as improved ability to bind the target molecule,
or improved ability to inhibit receptor tyrosine kinase function.
Such multimeric constructs having improved properties are included
in the present invention.
[0014] Consensus sequences from the screen of the cyclic/linear
peptide libraries have been determined based on the twelve classes
of specific cMet binding polypeptides shown in Table 6. In specific
embodiments, cMet binding polypeptides of the invention comprise
one or more of these sequences. Such preferred cMet binding
polypeptides include polypeptides with the potential to form a
cyclic or loop structure between invariant cysteine residues
comprising.
[0015] The polypeptides described herein can have additional amino
acids attached at either or both of the - and C-terminal ends. In
preferred embodiments, binding polypeptides according to the
invention can be prepared having N-terminal and/or C-terminal
flanking peptides of one or more, preferably two, amino acids
corresponding to the flanking peptides of the display construct of
the phage selectant from which the binding polypeptides were
isolated. Preferred N-terminal flanking peptides include Gly-Ser-
(most preferably for TN6 sequences), Ala-Gly- (most preferably for
TN8 and TN9 sequences), Gly-Ser- (most preferably for TN10 and TN11
sequences), Gly-Asp-(most preferably for TN12 sequences), Ala-Gln-
(most preferably for linear sequences). Preferred C-terminal
flanking peptides include -Ala-Pro (most preferably for TN6
sequences), -Gly-Thr (most preferably for TN8 and TN9 sequences),
-Ala-Pro (most preferably for TN10 and TN11 sequences), -Asp-Pro
(most preferably for TN12 sequences), -Asp-Phe (most preferably for
linear sequences). Single terminal amino acids also can be added to
the binding polypeptides of the invention, and preferred terminal
amino acids will correspond to the parental phage display
construct, e.g., most preferably, N-terminal amino acids will be
selected from Gly- (most preferably for TN6, TN8 and TN9
sequences), Ser- (most preferably for TN10 and TN11 sequences),
Asp- (most preferably for TN12 sequences), and Gln- (most
preferably for linear sequences), and most preferably C-terminal
amino acids will be selected from -Gly (most preferably for TN6,
TN8 and TN9, and linear sequences), -Ala (most preferably for TN10
and TN11 sequences), and -Asp (most preferably for TN12 sequences).
Conservative substitutions (i.e., substitute amino acids selected
within the following groups: {Arg, His, Lys}, {Glu, Asp}, {Asn,
Cys, Glu, Gly, Ser, Thr, Tyr}, {Ala, Ile, Leu, Met, Phe, Pro, Trp,
Val}) for such flanking amino acids also are contemplated.
[0016] Examination of the sequence information and binding data
from the isolates of libraries containing polypeptides with the
potential to form loop structures (e.g., libraries designated TN6,
TN8, TN9, TN10, TN11 and TN12; the number refers to the number of
amino acids in the sequence from cysteine to cysteine;
additionally, the linear display library, LN20, also was screened)
identifies an additional series of cMet binding polypeptides. A
consensus motif was obtained from this initial screen of a TN9
library (CxGpPxFxC; SEQ ID NO:512). The consensus sequence was
derived from the sequences listed in Table 6. This consensus
sequence along with sequence trends in the cMet binding peptides
identified from the linear peptide library was used to design a
second generation library that was used in a secondary screen.
Sequences from both screens were used to identify twelve classes of
cMet binding motifs listed in Table 6.
[0017] Another aspect of the present invention relates to
modifications of the polypeptides of the invention to provide
specific cellular proliferation and/or angiogenesis imaging agents
by detectably labeling a polypeptide or multimeric polypeptide
construct according to the present invention. Such detectable
labeling can involve radiolabeling, enzymatic labeling, or labeling
with MR paramagnetic chelates or microparticles; incorporation into
ultrasound bubbles, microparticles, microspheres, emulsions, or
liposomes; or conjugation with optical dyes.
[0018] In another aspect of the present invention, methods for
isolating cMet-expressing cells using the present binding
polypeptides or multimeric polypeptide construct are provided.
[0019] Additionally, the cMet binding polypeptides or multimeric
polypeptide construct of the invention can be used as therapeutic
agents, either alone in a pharmaceutically acceptable composition
or conjugated to (or in combination with) other therapeutic agents.
The compositions can be used to treat diseases or conditions
involving cellular proliferation, angiogenesis and/or wound
healing.
[0020] When used as therapeutic agents, it may be advantageous to
enhance the serum residence time of the peptides. This can be
accomplished by: a) conjugating to the peptide a moiety, such as
maleimide, that reacts with free sulfhydryl groups on serum
proteins, such as serum albumin, b) conjugating to the peptide a
moiety, such as a fatty acid, that binds non-covalently to serum
proteins, especially serum albumin, c) conjugating to the peptide a
polymer, such as polyethylene glycol (PEG), that is known to
enhance serum residence time, and d) fusing DNA that encodes the
cMet-binding peptide to DNA that encodes a serum protein such as
human serum albumin or an antibody and expressing the encoded
fusion protein.
[0021] In another aspect of the invention, methods of screening
polypeptides identified by phage display for their ability to bind
to cells expressing the target are provided. These methods permit
rapid screening of the binding ability of polypeptides, including
polypeptides with monomeric affinities that are too low for
evaluation in standard cell-binding assays. Additionally, these
methods can be used to rapidly assess the stability of the peptides
in the presence of serum.
[0022] In one embodiment, the present invention is directed to a
polypeptide or multimeric polypeptide construct having the ability
to bind to cMet or a complex comprising cMet and HGF comprising an
amino acid sequence comprising
Cys-X.sub.1-Gly-X.sub.2-Pro-X.sub.3-Phe-X.sub.4-Cys, wherein
X.sub.1, X.sub.2, X.sub.3 and X.sub.4 can be any amino acid. In a
particular embodiment, X.sub.2 is Pro.
[0023] In another embodiment, the polypeptides of the invention
further comprises N-terminal and/or C-terminal flanking peptides of
one or more amino acids. For example, the polypeptide can comprise
a modification selected from the group consisting of an amino acid
substitution, and amide bond substitution, a D-amino acid
substitution, a glycosylated amino acid, a disulfide mimetic
substitution, an amino acid translocation, a retro-inverso peptide,
a peptoid, a retro-inverso peptoid and a synthetic peptide. In
another embodiment, any of the polypeptides described herein can be
conjugated to a detectable label or a therapeutic agent, optionally
further comprising a linker or spacer between the polypeptide and
the detectable label or the therapeutic agent. In a particular
embodiment, the detectable label or the therapeutic agent is
selected from the group consisting of: an enzyme, a fluorescent
compound, a liposome, an optical dye, a paramagnetic metal ion, an
ultrasound contrast agent and a radionuclide. In a particular
embodiment, the therapeutic agent or detectable label comprises a
radionuclide. For example, the radionuclide can be one or more
selected from the group consisting of .sup.18F, .sup.124I,
.sup.125I, .sup.131I, .sup.123I, .sup.77Br, .sup.76Br, .sup.99mTc,
.sup.51Cr, .sup.67Ga, .sup.68Ga, .sup.47Sc, .sup.51Cr, .sup.167Tm,
.sup.141Ce, .sup.111In, .sup.168Yb, .sup.175Yb, .sup.140La,
.sup.90Y, .sup.88Y, .sup.153Sm, .sup.166Ho, .sup.165Dy, .sup.166Dy,
.sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.97Ru, .sup.103Ru, .sup.186Re,
.sup.188Re, .sup.203Pb, .sup.211Bi, .sup.212Bi, .sup.213Bi,
.sup.214Bi, .sup.105Rh, .sup.109Pd, .sup.117mSn, .sup.149Pm,
.sup.161Tb, .sup.177Lu, .sup.198Au and .sup.199Au. In another
embodiment, the therapeutic agent or detectable label further
comprises a chelator. For example, the chelator can comprise a
compound selected from the group consisting of formula 20, 21, 22,
23a, 23b, 24a, 24b and 25. In a particular embodiment, the
radionuclide is .sup.99mTc or .sup.111In. In another embodiment,
the radionuclide is selected from the group consisting of
.sup.177Lu, .sup.90Y, .sup.153Sm and .sup.166Ho. In another
embodiment, the detectable label comprises an ultrasound contrast
agent. For example, the ultrasound contrast agent can comprise a
phospholipid stabilized microbubble or a microballoon comprising a
gas, e.g., a fluorinated gas. In another embodiment, the detectable
label comprises a paramagnetic metal ion and a chelator. Another
aspect of the invention is directed to any of the polypeptides of
the invention, wherein the therapeutic agent is selected from the
group consisting of: a bioactive agent, a cytotoxic agent, a drug,
a chemotherapeutic agent or a radiotherapeutic agent. In other
embodiments, the polypeptide has an apparent K.sub.D for cMet of
cMet/HGF complex of less than about 10 .mu.M, less than about 1.0
.mu.M, less than about 0.1 .mu.M or less than about 1 nM.
[0024] In one embodiment, the present invention is directed to a
polypeptide or multimeric polypeptide construct having the ability
to bind to cMet or a complex comprising cMet and HGF comprising an
amino acid sequence of one of the following classes: Class 1:
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.4-X.sub.5-X.sub.6-X.sub.7-Cys-X.sub.8-X-
.sub.9-X.sub.10 (TN46), wherein X.sub.1 is Phe, Leu, Ser, Trp, Tyr
or Met; X.sub.2 is Ile, Tyr, His, Thr or Asn; X.sub.3 is Ile, Leu,
Asp, Met, Phe or Ser; X.sub.4 is Arg, Asn, Glu, Pro or Trp; X.sub.5
is Glu, Gly, Leu, Pro, Thr, Trp or Tyr; X.sub.6 is Asp, Gln, Glu
Gly, Phe, Ser, Thr or Trp; X.sub.7 is Ala, Arg, Asn, Gln, Glu, Gly,
Phe, or Trp; X.sub.8 is Gly, Asn, His, Arg, Met, Ile, Asp, Val or
Thr; X.sub.9 is Ser, Lys, Phe, Met, Thr, Asp or Leu; and X.sub.10
is Ser, Pro, Thr, Leu, Tyr, Asn, His, Glu or Trp; or Class II:
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub-
.9-Cys-X.sub.10-X.sub.11-X.sub.12 (TN8), wherein X.sub.1 is Gly,
Val, Trp, Thr, Lys or Gln; X.sub.2 is Trp, Tyr, Leu, Phe or Thr;
X.sub.3 is Trp, Glu, Phe, Ile, Leu and Ser; X.sub.4 is Asn, Gln or
Glu; X.sub.5 is Leu, Glu or Trp; X.sub.6 is Glu, Ser or Tyr;
X.sub.7 is Glu, Met or Pro; X.sub.8 is Met, Ser or Trp; X.sub.9 is
Leu, Phe or Val; X.sub.10 is Asp, Glu or Trp; X.sub.11 is Met, Phe
or Trp; and X.sub.12 is Gln, Leu or Trp; or Class III:
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.4-Gly-X.sub.5-Pro-X.sub.6-Phe-X.sub.7-C-
ys-X.sub.8-X.sub.9 (TN9), wherein X.sub.1 is Glu, Ser, Trp or Tyr;
X.sub.2 is Phe, Thr or Trp; X.sub.3 is His, Phe or Trp; X.sub.1 is
Ala, Lys, Ser or Thr; X.sub.5 is Pro or Trp; X.sub.6 is Ser or Thr;
X.sub.7 is Glu or Ser; X.sub.8 is Ile, Tip or Tyr; and X.sub.9 is
Glu, Met, Tip or Tyr; or Class IV-1:
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.4-Gly-Pro-Pro-X.sub.5-Phe-X.sub.6-Cys-T-
rp-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11 (TN9), wherein X.sub.1
is Arg, Asp, Asn, Ile or Ser; X.sub.2 is Leu, Ile, Phe, Tip or Val;
X.sub.3 is Asn, Gln, His, Leu, Tyr or Val; X.sub.4 is Leu, Lys or
Ser; X.sub.5 is Ala, Ser, Thr or Trp; X.sub.6 is Leu, Ser or Tip;
X.sub.7 is Leu, Ser or Trp; X.sub.8 is Phe or Tyr; X.sub.9 is Asp,
Glu, Gly or Val; X.sub.10 is Met, Pro, Thr or Ser; and X.sub.11 is
Glu or Gly; or Class IV-2:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-Trp-X.sub.5-Cys-X.sub.6-Gly-Pro-Pro-Thr-P-
he-Glu-Cys-Trp-X.sub.7-X.sub.8 (TN9), wherein X.sub.1 is Asp, Glu
or Val; X.sub.2 is Ala, Asp, Gly, Ser or Val; X.sub.3 is Asp, Gly,
Ser or Val; X.sub.4 is Arg, Asn, Gly, Ser or Thr; X.sub.5 is Gln or
His; X.sub.6 is Asn, Lys or Ser; X.sub.7 is Ser or Tip; and X.sub.8
is Phe or Tyr; or Class V:
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.su-
b.8-X.sub.9-X.sub.10-X.sub.11-Cys-X.sub.12-X.sub.13-X.sub.14
(TN10), wherein X.sub.1 is His, Phe, Pro, Thr or Trp; X.sub.2 is
Ala, Arg, Glu, His, Lys or Phe; X.sub.3 is Met, Phe, Pro, Thr or
Val; X.sub.4 is His, Leu, Met, Phe or Trp; X.sub.5 is Arg, Asp,
Glu, Gly, Met or Trp; X.sub.6 is Glu, Gly, Ile, Lys, Phe or Pro;
X.sub.7 is Asp, Phe, Pro, Ser, Trp or Tyr; Xg is Ala, Arg, Asn, Phe
or Ser; X.sub.9 is Ala, Gln, Gly, Leu or Phe; X.sub.10 is Gln, Gly,
Ile, Leu, Tip or Tyr; X.sub.11 is Arg, Asp, Phe, Pro, Tyr or Val;
X.sub.12 is Asn, Gln, His, Ile or Thr; X.sub.13 is Ala, Asn, Asp,
Glu or His; and X.sub.14 is Asn, Gln, Glu, His or Val; or Class VI:
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.s-
ub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-Cys-X.sub.13-X.sub.14-X.sub.15,
wherein X.sub.1 is Gln, Gly, Met, Phe or Ser; X.sub.2 is Asn, Gln,
Leu or Met; X.sub.3 is Arg, Asn, Gly, His or Ile; X.sub.4 is Asn,
Asp, Leu, Thr or Tip; X.sub.5 is Arg, Gln, Thr, Tyr or Val; X.sub.6
is Glu, Gly, Leu, Met or Thr; X.sub.7 is Ala, Asn, Asp, His, Ile,
Leu or Ser; X.sub.8 is Arg, Gln, Ser, Thr or Tyr; X.sub.9 is Asp,
Gly, Ile or Phe; X.sub.10 is Gln, Phe or Thr; X.sub.11 is Gln, His,
Phe, Pro, Ser or Tyr; X.sub.12 is Asn, Asp, Phe, Pro or Ser;
X.sub.13 is Ala, Asn, Gly, Leu or Ser; X.sub.14 is Arg, Pro, Ser or
Val; and X.sub.15 is Asp, Glu, Leu or Met; or Class VIII:
X.sub.1-X.sub.2-X.sub.3-Cys-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub-
.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-Cys-X.sub.14-X.sub.15-X.sub.16,
wherein X.sub.1 is Ala, His, Leu, Phe or Tyr; X.sub.2 is Arg, Asp,
Leu, Ser or Tyr; X.sub.3 is Glu, Met or Tip; X.sub.4 is Asp, Gln,
Glu, Phe or Ser; X.sub.5 is Glu, Ile, Phe or Tip; X.sub.6 is Asn,
Asp or Ser; X.sub.7 is Asn, Asp or Leu; X.sub.8 is Asp, Glue or
Lys; X.sub.9 is Gly, Phe or Thr; X.sub.10 is Gly, Phe, Tip or Tyr;
X.sub.11 is Glu, Ser or Tip; X.sub.12 is Glu, Phe, Tyr or Val;
X.sub.13 is Glu, Lys, Thr or Val; X.sub.14 is Glu or Tip; X.sub.15
is Asp, Phe, Pro, Ser or Trp; and X.sub.16 is Ala, Asn or Ile; or
Class IX-1:
Ser-Cys-X.sub.1-Cys-X.sub.2-Gly-Pro-Pro-Thr-Phe-Glu-Cys-Trp-Cys-Tyr-X.sub-
.3-X.sub.4-X.sub.5, wherein X.sub.1 is Asn, His or Tyr; X.sub.2 is
Gly or Ser; X.sub.3 is Ala, Asp, Glu, Gly or Ser; X.sub.4 is Ser or
Thr; and X.sub.5 is Asp or Glu; or Class IX-2:
Glu-X.sub.1-Gly-Ser-Cys-His-Cys-Ser-Gly-Pro-Pro-Thr-Phe-Glu-Cys-X.sub.2-C-
ys-X.sub.3, wherein X.sub.1 is Ala, Glu, Gly or Ser; X.sub.2 is
Phe, Trp or Tyr; and X.sub.3 is Phe or Tyr.
[0025] In another embodiment, the invention is directed to a
polypeptide or multimeric polypeptide construct having the ability
to bind to cMet or a complex comprising cMet and HGF comprising an
amino acid sequence, wherein the amino acid sequence comprises at
least six amino acids out of a contiguous stretch of nine amino
acids from a sequence selected from the group consisting of SEQ ID
NOS:1-511. In a particular embodiment, the polypeptide, used as
either a monomer or in a multimeric construct, can be selected from
the group consisting of SEQ ID NOS:1-511, SEQ ID NOS:1-10, SEQ ID
NOS:11-47, SEQ ID NOS:48-101, SEQ ID NOS:102-364, SEQ ID
NOS:365-370, SEQ ID NOS:371-387, SEQ ID NO:388 or SEQ ID NO:399,
SEQ ID NOS:390-404, SEQ ID NOS:405-447, SEQ ID NO:448, SEQ ID
NOS:449-496 and SEQ ID NOS:497-511.
[0026] In another embodiment, the invention is directed to a method
for isolating phage that bind cMet or a complex comprising cMet and
HGF, comprising the steps of: immobilizing cMet or a complex
comprising cMet and HGF on a solid support; contacting a library of
potential cMet or cMet/HGF complex binding phage with the solid
support to bind cMet or cMet/HGF binding phage in the library; and
removing the unbound portion of the phage library from the solid
support, thereby isolating phage that bind cMet or a complex
comprising cMet and HGF.
[0027] In another embodiment, the invention is directed to a method
of detecting cMet or a complex comprising cMet and HGF in an animal
or human subject and optionally imaging at least a portion of the
animal or human subject comprising the steps of: detectably
labeling a polypeptide or multimeric polypeptide construct having
the ability to bind to cMet or a complex comprising cMet and HGF
comprising an amino acid sequence comprising
Cys-X.sub.1-Gly-X.sub.2-Pro-X.sub.3-Phe-X.sub.4-Cys, wherein
X.sub.1, X.sub.2, X.sub.3 and X.sub.4 can be any amino acid;
administering to the subject the labeled polypeptide or multimeric
polypeptide construct; and, detecting the labeled polypeptide or
construct in the subject, and, optionally, constructing an image,
thereby detecting cMet or a complex comprising cMet and HGF.
[0028] In a particular embodiments, the methods of the invention
encompass methods wherein the label is selected from the group
consisting of: an enzyme, a fluorescent compound, an ultrasound
contrast agent, a liposome and an optical dye, wherein the label
optionally further comprises a linker and/or a spacer. In
particular embodiment, the ultrasound contrast agent is a
phospholipid stabilized microbubble or a microballoon comprising a
gas, e.g., a fluorinated gas. In other embodiments, the label is a
radioactive label or a paramagnetic metal atom, and optionally
further comprises a linker or a spacer. In another embodiment, the
radioactive label comprises a radionuclide selected from the group
consisting of: .sup.18F, .sup.124I, .sup.125I, .sup.131I,
.sup.123I, .sup.77Br, .sup.76Br, .sup.99mTc, .sup.51Cr, .sup.67Ga,
.sup.68Ga, .sup.47Sc, .sup.51Cr, .sup.167Tm, .sup.141Ce,
.sup.111In, .sup.168Yb, .sup.175Yb, .sup.140La, .sup.90Y, .sup.88Y,
.sup.153Sm, .sup.166Ho, .sup.165Dy, .sup.166Dy, .sup.62Cu,
.sup.64Cu, .sup.67Cu, .sup.97Ru, .sup.103Ru, .sup.186Re,
.sup.188Re, .sup.203Pb, .sup.211Bi, .sup.212Bi, .sup.213Bi,
.sup.214Bi, .sup.105Rh, .sup.109Pd, .sup.117mSn, .sup.149Pm,
.sup.161Tb, .sup.177Lu, .sup.198Au and .sup.199Au. In another
embodiment, the radioactive label further comprises a chelator,
e.g., chelators selected from the group consisting of: formula 20,
21, 22, 23a, 23b, 24a, 24b and 25. In another embodiment, the
radionuclide is .sup.99mTc or .sup.111In. In a particular
embodiment, the paramagnetic label comprises a paramagnetic metal
atom selected from the group consisting of: Mn.sup.2+, Cu.sup.2+,
Fe.sup.2+, Co.sup.2+, Ni.sup.2+, Gd.sup.3+, Eu.sup.3+, Dy.sup.3+,
Pr.sup.3+, Cr.sup.3+, Co.sup.3+, Fe.sup.3+, Ti.sup.3+, Tb.sup.3+,
Nd.sup.3+, sm.sup.3+, Ho.sup.3+, Er.sup.3+, Pa.sup.4+ and
Eu.sup.2+. In another embodiment, the paramagnetic label further
comprises a chelator, e.g., a chelator is selected from the group
consisting of: DTPA, DO3A, DOTA, EDTA, TETA, EHPG, HEED, NOTA,
DOTMA, TETMA, PDTA, TTHA, LICAM, and MECAM. In particular
embodiments, detection of the labeled polypeptide or multimeric
polypeptide construct is indicative of a hyperproliferative
disorder. In other embodiments, detection of the labeled
polypeptide or multimeric polypeptide construct is indicative of
angiogenesis or neovascularization. In particular embodiments, the
label is an ultrasound contrast agent that comprises a fluorinated
gas selected from the group of SF.sub.6 freons, CF.sub.4,
C.sub.2F.sub.6, C.sub.3F.sub.8, C.sub.4F.sub.10i CBrF.sub.3,
CCl.sub.2F.sub.2, C.sub.2CIF.sub.5, CBrCIF.sub.2 and
perfluorocarbons. In particular embodiments, the ultrasound
contrast agent comprises a perfluorocarbon gas having the formula
C.sub.nF.sub.n+2 wherein n is from 1 to 12.
[0029] In another embodiment, the invention is directed to a method
of detecting cMet or a complex comprising cMet and HGF in an animal
or human subject and optionally imaging at least a portion of the
animal or human subject comprising the steps of: detectably
labeling a polypeptide or multimeric polypeptide construct having
the ability to bind to cMet or a complex comprising cMet and HGF
comprising an amino acid sequence, wherein the amino acid sequence
comprises at least six amino acids out of a contiguous stretch of
nine amino acids from a sequence selected from the group consisting
of SEQ ID NOS:1-511; administering to the subject the labeled
polypeptide or construct; and, detecting the labeled polypeptide or
construct in the subject, and, optionally, constructing an image,
thereby detecting cMet or a complex comprising cMet and HGF.
[0030] In another embodiment, the invention is directed to a method
of treating a condition involving activation of cMet, comprising
administering to an animal or human subject in need of treatment
for such a condition a composition comprising a polypeptide or
multimeric polypeptide construct having the ability to bind to cMet
or a complex comprising cMet and HGF comprising an amino acid
sequence comprising
Cys-X.sub.1-Gly-X.sub.2-Pro-X.sub.3-Phe-X.sub.4-Cys, wherein
X.sub.1, X.sub.2, X.sub.3 and X.sub.4 can be any amino acid. In
another embodiment, the invention is directed to a method of
treating a condition involving activation of cMet, comprising
administering to an animal or human subject in need of treatment
for such a condition a composition comprising a polypeptide or
multimeric polypeptide construct having the ability to bind to cMet
or a complex comprising cMet and HGF comprising an amino acid
sequence, wherein the amino acid sequence comprises at least six
amino acids out of a contiguous stretch of nine amino acids from a
sequence selected from the group consisting of SEQ ID NOS:1-511. In
a particular embodiment, the condition is solid tumor growth, e.g.,
wherein the tumor is selected from the group consisting of breast,
thyroid, glioblastoma, prostate, malignant mesothelioma,
colorectal, hepatocellular, hepatobiliary, renal, osteosarcoma and
cervical. In a particular embodiment, the polypeptide or multimeric
polypeptide construct can be conjugated to a tumoricidal agent.
[0031] In another embodiment, the invention is directed to a
recombinant bacteriophage displaying any one or more of the
polypeptides or multimeric polypeptide construct described herein
or having any one or more of the consensus sequences described
herein, such that the phage has the ability to bind to cMet or a
complex comprising cMet and HGF, and wherein the polypeptide is
displayed on the surface of the recombinant bacteriophage.
[0032] In another embodiment, the invention is directed to a
magnetic resonance imaging contrast agent comprising a composition
comprising a polypeptide having the ability to bind to cMet or a
complex comprising cMet and HGF comprising an amino acid sequence
comprising Cys-X.sub.1-Gly-X.sub.2-Pro-X.sub.3-Phe-X.sub.4-Cys,
wherein X.sub.1, X.sub.2, X.sub.3 and X.sub.4 can be any amino
acid, or wherein the amino acid sequence comprises at least six
amino acids out of a contiguous stretch of nine amino acids from a
sequence selected from the group consisting of SEQ ID NOS:1-511. In
a particular embodiment, the magnetic resonance imaging contrast
agent further comprises at least one paramagnetic metal atom, e.g.,
at least one chelator selected from the group consisting of: DTPA,
DOTA, EDTA, TETA, EHPG, HBED, NOTA, DOTMA, TETMA, PDTA, TTHA,
LICAM, and MECAM. In particular embodiments, the chelator is
selected from the group consisting of: diethylenetriamine,
tetraazacyclododecane and a carboxymethyl-substituted derivative
thereof. In other embodiments, the paramagnetic metal atom is
selected from the group consisting of Mn.sup.2+, Cu.sup.2+,
Fe.sup.2+, Co.sup.2+, Ni.sup.2+, Gd.sup.3+, Eu.sup.3+, Dy.sup.3+,
Pr.sup.3+, Cr.sup.3+, Co.sup.3+, Fe.sup.3+, Ti.sup.3+, Tb.sup.3+,
Nd.sup.3+, Sm.sup.3+, Ho.sup.3+, Er.sup.3+, Pa.sup.4+ and
Eu.sup.2+. In a particular embodiment, the multivalent cation is
Gd.sup.3+.
[0033] In another embodiment, the invention is directed to a method
for identifying cMet or cMet/HGF complex binding compounds
comprising the steps of: utilizing a cMet or cMet/HGF complex
binding polypeptide or multimeric polypeptide construct having the
ability to bind to cMet or a complex comprising cMet and HGF
comprising an amino acid sequence comprising
Cys-X.sub.1-Gly-X.sub.2-Pro-X.sub.3-Phe-X.sub.4-Cys, wherein
X.sub.1, X.sub.2, X.sub.3 and X.sub.4 can be any amino acid, to
form a complex with a cMet or cMet/HGF complex target; contacting
the complex with one or more potential cMet or cMet/HGF complex
binding compounds; and determining whether the potential cMet or
cMet/HGF complex binding compound competes with the cMet or
cMet/HGF complex binding polypeptide to form a complex with the
cMet or cMet/HGF complex target.
[0034] In one embodiment, the invention is directed to a diagnostic
imaging contrast agent comprising a polypeptide or multimeric
polypeptide construct having the ability to bind to cMet or a
complex comprising cMet and HGF comprising an amino acid sequence
comprising Cys-X.sub.1-Gly-X.sub.2-Pro-X.sub.3-Phe-X.sub.4-Cys,
wherein X.sub.1, X.sub.2, X.sub.3 and X.sub.4 can be any amino
acid, or wherein the amino acid sequence comprises at least six
amino acids out of a contiguous stretch of nine amino acids from a
sequence selected from the group consisting of SEQ ID
NOS:1-511.
[0035] In another embodiment, the invention is directed to a method
of medical imaging comprising the steps of administering to an
animal or human subject a pharmaceutical preparation of a contrast
agent comprising at least one polypeptide or multimeric polypeptide
construct having the ability to bind to cMet or a complex
comprising cMet and HGF comprising an, amino acid sequence
comprising Cys-X.sub.1-Gly-X.sub.2-Pro-X.sub.3-Phe-X.sub.4-Cys,
wherein X.sub.1, X.sub.2, X.sub.3 and X.sub.4 can be any amino
acid, and imaging the contrast agent by a method selected from the
group consisting of: magnetic resonance imaging, ultrasound
imaging, optical imaging, sonoluminescence imaging, photoacoustic
imaging, and nuclear imaging. In another embodiment, the amino acid
sequence comprises at least six amino acids out of a contiguous
stretch of nine amino acids from a sequence selected from the group
consisting of SEQ ID NOS:1-511, and imaging the contrast agent by a
method selected from the group consisting of: magnetic resonance
imaging, ultrasound imaging, optical imaging, sonoluminescence
imaging, photoacoustic imaging, and nuclear imaging.
[0036] In another embodiment, the invention is directed to a method
of radiotherapy comprising administering to an animal or human
subject in need of such therapy a compound comprising at least one
polypeptide or multimeric polypeptide construct having the ability
to bind to cMet or a complex comprising cMet and HGF comprising an
amino acid sequence comprising
Cys-X.sub.1-Gly-X.sub.2-Pro-X.sub.3-Phe-X.sub.4-Cys, wherein
X.sub.1, X.sub.2, X.sub.3 and X.sub.4 can be any amino acid, or
wherein the amino acid sequence comprises at least six amino acids
out of a contiguous stretch of nine amino acids from a sequence
selected from the group consisting of SEQ ID NOS:1-511, conjugated
to a radionuclide useful for radiotherapy. In a particular
embodiment, the compound further comprises a chelator, e.g., a
compound selected from the group consisting of: formula 20, 21, 22,
23a, 23b, 24a, 24b and 25. In another embodiment, the compound
further comprises a spacer or linker. In a particular embodiment,
the radionuclide can be .sup.185Re, .sup.188Re, .sup.177Lu,
.sup.90Y, .sup.153Sm or .sup.166Ho.
[0037] In another embodiment, the invention is directed to a kit
for preparation of a radiopharmaceutical comprising a polypeptide
or multimeric polypeptide construct having the ability to bind to
cMet or a complex comprising cMet and HGF comprising an amino acid
sequence comprising
Cys-X.sub.1-Gly-X.sub.2-Pro-X.sub.3-Phe-X.sub.4-Cys, wherein
X.sub.1, X.sub.2, X.sub.3 and X.sub.4 can be any amino acid, or
wherein the amino acid sequence comprises at least six amino acids
out of a contiguous stretch of nine amino acids from a sequence
selected from the group consisting of SEQ ID NOS:1-511, a chelator
for a radionuclide, and a reducing agent.
[0038] In another embodiment, the invention is directed to a method
of targeting genetic material to cMet-expressing cells comprising
administering to an animal or a human in need of such genetic
material a polypeptide or multimeric polypeptide construct having
the ability to bind to cMet or a complex comprising cMet and HGF
comprising an amino acid sequence comprising
Cys-X.sub.1-Gly-X.sub.2-Pro-X.sub.3-Phe-X.sub.4-Cys, wherein
X.sub.1, X.sub.2, X.sub.3 and X.sub.4 can be any amino acid, or
wherein the amino acid sequence comprises at least six amino acids
out of a contiguous stretch of nine amino acids from a sequence
selected from the group consisting of SEQ ID NOS:1-511, conjugated
to or associated with the genetic material or a delivery vehicle
containing such genetic material.
[0039] In another embodiment, the invention is directed to a method
of screening binding polypeptides identified by phage display for
their ability to bind to cells expressing the cMet or cMet/HGF
target comprising the steps of preparing multimeric constructs
including one or more binding polypeptides; contacting the
multimeric constructs with cells expressing the target and
assessing the ability of the multimeric constructs to bind to the
target. In a particular embodiment, the cells can be engineered by
recombinant DNA technology to express the target. In another
embodiment, the multimeric constructs can be detectably labeled. In
another embodiment, the ability of the multimeric constructs to
bind to the target is assessed in the presence of serum. In another
embodiment, the multimeric construct can comprise biotinylated
binding polypeptides complexed with avidin, streptavidin or
neutravidin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIGS. 1A-1C are representations of mimics, which can be
employed to mimic structural motifs and turn features in a peptide
and simultaneously provide stability to proteolysis and enhance
other properties (structure 1A: Hart, S, and Etzkorn, F., 1999. J.
Org. Chem., 64:2998-2999; structure 1B: Hanessian, S, and
McNaughton-Smith, G., "Synthesis of a Versatile Peptidomimetic
Scaffold" in Methods in Molecular Medicine, Vol. 23:
Peptidomimetics Protocols, W. Kazmierski, Ed. (Humana Press Inc.,
Totowa, N.J., 1999), Chapter 10, pp. 161-174; structure 1C: WO
01/16135.
[0041] FIG. 2 is a representation of the amino acids (4),
containing an aminoalcohol function, and (5) containing an
alkoxyamino function.
[0042] FIG. 3 is a representation depicting the cyclization of
Cysteine with a pendant bromoacetamide function (this process is
referred to herein as "scheme 1").
[0043] FIG. 4 is a representation showing intramolecular
cyclization of suitably located vicinal amino mercaptan functions
and aldehyde functions to provide thiazolidines that result in the
formation of a bicyclic peptide, one ring of which is that formed
by the residues in the main chain, and the second ring being the
thiazolidine ring (this process is referred to herein as "scheme
2").
[0044] FIG. 5 is a representation showing how a lactam function,
available by intramolecular coupling via standard peptide coupling
reagents (such as HATU, PyBOP etc) can act as a surrogate for the
disulfide bond. The Dde/Dmab approach is shown (and is referred to
herein as "scheme 3").
[0045] FIG. 6 is a representation showing the Grubbs reaction
(referred to herein as "scheme 4").
[0046] FIGS. 7A and 7B are chemical structures of phospholipid
moieties.
[0047] FIGS. 8A-F depict structures of preferred metal
chelators.
[0048] FIG. 9 is a schematic representation of the selection
strategy that was employed to identify cMet binding polypeptides.
TEA=triethylamine, Bead Infection=capture of non-eluted phage that
remained bound to the cMet-Fc/protein-A beads.
[0049] FIG. 10 illustrates the growth inhibitory properties of
cMet-binding peptide SEQ ID NO:365.
[0050] FIG. 11 shows a schematic diagram for the preparation of SEQ
ID NO:514 conjugated to a 6-PnAO-Glut moiety, (referred to herein
as "scheme 5").
[0051] FIG. 12 shows a schematic diagram for the preparation of a
heterodimer containing SEQ ID NOS: 514 and 515 joined by a
K(PnAO6-Glut) linker (referred to herein as "scheme 5").
[0052] FIGS. 13A-13C show the chemical structures of three
heterodimers as follows: FIG. 13A shows SEQ ID NO:514 linked to SEQ
ID NO:515 (Ac-GSPEMCMMFPFLYPCNHHAPGGGK
{PnAO6-Glut-K[Ac-GSFFPCWRIDRFGYCHANAPGGGKJJ-Glut]-NH2}-NH2); FIG.
13B shows SEQ ID NO:515 linked to SEQ ID NO:516
(Ac-GSFFPCWRIDRFGYCHANAPGGGK
{PnAO6-Glut-K[Ac-AQEWEREYFVDGFWGSWFGIPHGGGK(JJ-Glut)-NH2]}-NH2);
and FIG. 13C shows SEQ ID NO:514 linked to SEQ ID NO:517
(Ac-GSPEMCMMFPFLYPCNIIHAPGGGK
{PnAO6-Glut-K[Ac-GDYSECFFEPDSFEVKCYDRDPGGGK(JJ-Glut)-NH2]}-NH2).
[0053] FIG. 14 is a graphical representation of data showing
binding of derivatives of SEQ ID NO:514 with different spacer
length and biotin. Derivatives have none, one J and two J spacers
respectively in between the targeting sequence and biotin.
DETAILED DESCRIPTION OF THE INVENTION
[0054] A description of preferred embodiments of the invention
follows.
[0055] The present invention provides novel binding moieties that
bind to the hepatocyte growth factor receptor ("HGFr" or "cMet").
Such binding moieties make possible the efficient detection,
imaging and localization of activated cells exhibiting upregulated
cMet expression and binding of HGF to cMet. Such activated cells
are initiators of cellular proliferation, and therefore the
polypeptides described herein provide a means of detecting,
monitoring and localizing sites of proliferation. In particular,
the binding moieties of this invention, which include polypeptides
and multimeric polypeptide constructs, when appropriately labeled,
are useful for detecting, imaging and localizing tumors or other
proliferative disorders that result from dysregulated cellular
proliferation (e.g., cancer). Thus, the binding polypeptides and
multimeric polypeptide constructs of the invention can be used to
form a variety of diagnostic and therapeutic agents for diagnosing
and treating neoplastic tumor growth or other proliferative
disorders. In addition, the binding polypeptides and multimeric
polypeptide constructs can themselves be used as therapeutic
agents.
[0056] Specific cMet binding polypeptides according to the present
invention were isolated initially by screening of phage display
libraries, that is, populations of recombinant bacteriophage
transformed to express an exogenous peptide on their surface. In
order to isolate new polypeptide binding moieties for a particular
target, such as cMet, screening of large peptide libraries, for
example using phage display techniques, is especially advantageous,
in that very large numbers (e.g., 5.times.10.sup.9) of potential
binders can be tested and successful binders isolated in a short
period of time.
[0057] In order to prepare a phage library of displaying
polypeptides to screen for binding polypeptides such as cMet
binding polypeptides and/or polypeptides that bind to a complex
comprising HGF bound to cMet, a candidate binding domain is
selected to serve as a structural template for the peptides to be
displayed in the library. The phage library is made up of a
multiplicity of analogues of the parental domain or template. The
binding domain template can be a naturally occurring or synthetic
protein, or a region or domain of a protein. The binding domain
template can be selected based on knowledge of a known interaction
between the binding domain template and the binding target, but
this is not critical. In fact, it is not essential for the selected
domain to act as a template for the library or have any affinity
for the target at all; its purpose is to provide a structure from
which a multiplicity (library) of similarly structured polypeptides
(analogues) can be generated, which multiplicity of analogs will
include one or more analogs that exhibit the desired binding
properties (and any other properties screened for).
[0058] In selecting the parental binding domain or template on
which to base the variegated amino acid sequences of the library,
an important consideration is how the variegated peptide domains
will be presented to the target, i.e., in what conformation the
peptide analogues will come into contact with the target. In phage
display methodologies, for example, the analogs are generated by
insertion of synthetic DNA encoding the analogs into phage,
resulting in display of the analog on the surfaces of the phage.
Such libraries of phage, such as M13 phage, displaying a wide
variety of different polypeptides, can be prepared using techniques
as described, e.g., in Kay et al., Phage Display of Peptides and
Proteins: A Laboratory Manual (Academic Press, Inc., San Diego,
1996) and U.S. Pat. No. 5,223,409 (Ladner et al.), incorporated
herein by reference.
[0059] In isolating the specific polypeptides according to this
invention, seven cyclic peptide (or "loop") libraries, designated
TN6, TN7, TN8, TN9, TN10, TN11, TN12, and a linear library,
designated LN20, were initially screened. Each library was
constructed for expression of diversified polypeptides on M13
phage. The seven libraries having a "TN" designation were designed
to display a short, variegated exogenous peptide loop of 6, 7, 8,
9, 10, 11 or 12 amino acids, respectively, on the surface of M13
phage, at the amino terminus of protein III. The libraries are
designated TN6 (having a potential 3.3.times.10.sup.12 amino acid
sequence diversity), TN7 (having a potential 1.2.times.10.sup.14
amino acid sequence diversity), TN8 (having a potential
2.2.times.10.sup.15 amino acid sequence diversity), TN9 (having a
potential 4.2.times.10.sup.16 amino acid sequence diversity, TN10
(having a potential 3.0.times.10.sup.16 amino acid sequence
diversity), TN11 (having a potential 1.5.times.10.sup.19 amino acid
sequence diversity), TN12 (having a sequence diversity of
4.6.times.10.sup.19), and LN20 (having a potential
3.8.times.10.sup.25 amino acid sequence diversity).
[0060] The TN6 library was constructed to display a single
microprotein binding loop contained in a 12-amino acid template.
The TN6 library utilized a template sequence of
Xaa1-Xaa2-Xaa3-Cys-Xaa5-Xaa6 Xaa7-Xaa8-Cys-Xaa10-Xaa11-Xaa12. The
amino acids at positions 2, 3, 5, 6, 7, 8, 10, and 11 of the
template were varied to permit any amino acid except cysteine
(Cys). The amino acids at positions 1 and 12 of the template were
varied to permit any amino acid except cysteine (Cys), glutamic
acid (Glu), isoleucine (Ile), Lysine (Lys), methionine (Met), and
threonine (Thr).
[0061] The TN7 library was constructed to display a single
microprotein binding loop contained in a 13-amino acid template.
The TN7 library utilized a template sequence of
Xaa1-Xaa2-Xaa3-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Cys-Xaa11-Xaa12-Xaa13.
The amino acids at amino acid positions 1, 2, 3, 5, 6, 7, 8, 9, 11,
12, and 13 of the template were varied to permit any amino acid
except cysteine (Cys).
[0062] The TN8 library was constructed to display a single
microprotein binding loop contained in a 14-amino acid template,
The TN8 library utilized a template sequence of
Xaa1-Xaa2-Xaa3-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Cys-Xaa12-Xaa13-Xaa14.
The amino acids at position 1, 2, 3, 5, 6, 7, 8, 9, 10, 12, 13, and
14 in the template were varied to permit any amino acid except
cysteine (Cys).
[0063] The TN9 library was constructed to display a single
microprotein binding loop contained in a 15-amino acid template.
The TN9 library utilized a template sequence
Xaa1-Xaa2-Xaa3-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Cys-Xaa13-Xaa14-X-
aa15. The amino acids at position 1, 2, 3, 5, 6, 7, 8, 9, 10, 11,
13, 14 and 15 in the template were varied to permit any amino acid
except cysteine (Cys).
[0064] The TN10 library was constructed to display a single
microprotein binding loop contained in a 16-amino acid template.
The TN10 library utilized a template sequence Xaa1Xaa2-Xaa3
Cys-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Cys-Xaa14-Xaa15-Xaa16.
The amino acids at positions 1, 2, 15, and 16 in the template were
varied to permit any amino acid selected from a group of 10 amino
acids: D, F, H, L, N, P, R, S, W, or Y). The amino acids at
positions 3 and 14 in the template were varied to permit any amino
acid selected from a group of 14 amino acids: A, D, F, G, H, L, N,
P, Q, R, S, V, W, or Y). The amino acids at positions 5, 6, 7, 8,
9, 10, 11, and 12 in the template were varied to permit any amino
acid except cysteine (Cys).
[0065] The TN11 library was constructed to display a single
microprotein binding loop contained in a 17-amino acid template.
The TN11 library utilized a template sequence
Xaa1-Xaa2-Xaa3-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12
Xaa13-Cys-Xaa15-Xaa16-Xaa17. The amino acids at positions 1 through
3, 5 through 13, and 15 through 17 in the template were varied to
permit any amino acid except cysteine (Cys).
[0066] The TN12 library was constructed to display a single
microprotein binding loop contained in an 18-amino acid template.
The TN12 library utilized a template sequence
Xaa1-Xaa2-Xaa3-Cys-Xaa5-Xaa6-Xaa7-Xaa8
Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Cys-Xaa16-Xaa17-Xaa18. The amino
acids at position 1, 2, 17, and 18 in the template were varied to
permit any amino acid selected from a group of 12 amino acids: A,
D, F, G, H, L, N, P, R, S, W, or Y). The amino acids at positions
3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 16 were varied to permit
any amino acid except cysteine (Cys).
[0067] The LN20 library was constructed to display multiple linear
peptides on the surface of a phage. Each phage, however, displays
multiple copies of the same sequence. Therefore, a single phage
will display, for example, five copies of a particular sequence, a
different phage will display, for example, five copies of a
different sequence, etc. The linear peptides are provided in a
20-amino acid template. The amino acids at each position in the
template were varied to permit any amino acid except cysteine
(Cys).
[0068] The binding polypeptides provided herein can include
additions or truncations in the - and/or C-termini. Such modified
binding polypeptides are expected to bind cMet. For example, a
-GGGK linker (SEQ ID NO:513) can be present at the N-terminus of
the binding polypeptides provided herein. Other linkers, such as
-GSGK (SEQ ID NO:651), or -GSGSK (SEQ ID NO:652) could be used.
Binding polypeptides comprising the loop portion of the templates
and sequences provided herein are expected to bind cMet and also
are encompassed by the present invention. The loop portion of the
templates and sequences includes the sequences between and
including the two cysteine residues that are expected to form a
disulfide bond, thereby generating a peptide loop structure.
Furthermore, the binding polypeptides of the present invention can
include additional amino acid residues at the - and/or
C-termini.
[0069] The phage display libraries were created by making a
designed series of mutations or variations within a coding sequence
for the polypeptide template, each mutant sequence encoding a
peptide analog corresponding in overall structure to the template
except having one or more amino acid variations in the sequence of
the template. The novel variegated (mutated) DNA provides sequence
diversity, and each transformant phage displays one variant of the
initial template amino acid sequence encoded by the DNA, leading to
a phage population (library) displaying a vast number of different
but structurally related amino acid sequences. The amino acid
variations are expected to alter the binding properties of the
binding peptide or domain without significantly altering its
structure, at least for most substitutions. It is preferred that
the amino acid positions that are selected for variation (variable
amino acid positions) will be surface amino acid positions, that
is, positions in the amino acid sequence of the domains that, when
the domain is in its most stable conformation, appear on the outer
surface of the domain (i.e., the surface exposed to solution). Most
preferably the amino acid positions to be varied will be adjacent
or close together, so as to maximize the effect of
substitutions.
[0070] As indicated previously, the techniques discussed in Kay et
al., Phage Display of Peptides and Proteins: A Laboratory Manual
(Academic Press, Inc., San Diego, 1996) and U.S. Pat. No. 5,223,409
are particularly useful in preparing a library of potential binders
corresponding to the selected parental template. Libraries as
discussed above were prepared according to such techniques, and
they were screened for cMet binding polypeptides against an
immobilized target, as explained in the examples to follow.
[0071] In a typical screen, a phage library is contacted with and
allowed to bind the target, or a particular subcomponent thereof.
To facilitate separation of binders and non-binders, it is
convenient to immobilize the target on a solid support. Phage
bearing a target-binding moiety form a complex with the target on
the solid support whereas non-binding phage remain in solution and
can be washed away with excess buffer. Bound phage are then
liberated from the target by changing the buffer to an extreme pH
(pH 2 or pH 10), changing the ionic strength of the buffer, adding
denaturants, or other known means. To isolate the binding phage
exhibiting the polypeptides of the present invention, a protein
elution is performed, i.e., some phage are eluted from the target
using HGF in solution (competitive elution). Additionally, for
example, very high affinity binding phage that could not be
competed off during the overnight HGF incubation were captured by
using the phage still bound to substrate for infection of E. coli
cells.
[0072] The recovered phage can then be amplified through infection
of bacterial cells and the screening process can be repeated with
the new pool that is now depleted in non-binders and enriched for
binders. The recovery of even a few binding phage is sufficient to
carry the process to completion. After a few rounds of selection,
the gene sequences encoding the binding moieties derived from
selected phage clones in the binding pool are determined by
conventional methods, described below, revealing the peptide
sequence that imparts binding affinity of the phage to the target.
When the selection process works, the sequence diversity of the
population falls with each round of selection until desirable
binders remain. The sequences converge on a small number of related
binders, typically 10-50 out of about 10.sup.9 to 10.sup.10
original candidates from each library. An increase in the number of
phage recovered at each round of selection, and of course, the
recovery of closely related sequences are good indications that
convergence of the library has occurred in a screen. After a set of
binding polypeptides is identified, the sequence information can be
used to design other secondary phage libraries, biased for members
having additional desired properties.
[0073] Formation of the disulfide binding loop is advantageous
because it leads to increased affinity and specificity for such
peptides. However, in serum, the disulfide bond can be opened by
free cysteines or other thiol-containing molecules. Thus, it could
be useful to modify the cysteine residues to replace the disulfide
cross-link with another less reactive linkage. The
--CH.sub.2--S--S--CH.sub.2-- cross-link has a preferred geometry in
which the dihedral bond between sulfurs is close to 90 degrees, but
the exact geometry is determined by the context of other side
groups and the binding state of the molecule. Preferred
modifications of the closing cross-link of the binding loop will
preserve the overall bond lengths and angles as much as possible.
Suitable such alternative cross--links include thioether linkages
such as --CH.sub.2--S--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--S--CH.sub.2--,
--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--; lactam or amide
linkages such as --CH.sub.2--NH--CO--CH.sub.2-- and
--CH.sub.2--CO--NH--CH.sub.2--; ether linkages such as
--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--; alkylene bridges
such as --(CH.sub.2).sub.n-- (where n=4, 5, or 6); the linkage
--CH.sub.2--NH--CO--NH--CH.sub.2--, and similar groups known in the
art.
[0074] Although polypeptides containing a stable disulfide-linked
binding loop are most preferred, linear polypeptides derived from
the foregoing sequences can be readily prepared, e.g., by
substitution of one or both cysteine residues, which may retain at
least some of the cMet binding activity of the original polypeptide
containing the disulfide linkage. In making such substitutions for
Cys, the amino acids Gly, Ser, and Ala are preferred, and it also
is preferred to substitute both Cys residues, so as not to leave a
single Cys that could cause the polypeptide to dimerize or react
with other free thiol groups in a solution. All such linearized
derivatives that retain cMet binding properties are within the
scope of this invention.
[0075] Direct synthesis of the polypeptides of the invention can be
accomplished using conventional techniques, including solid-phase
peptide synthesis, solution-phase synthesis, etc. Solid-phase
synthesis is preferred (see, for example, Stewart et al.,
Solid-Phase Peptide Synthesis (W.H. Freeman Co., San Francisco,
1989); Merrifield, J., 1963, Am. Chem. Soc., 85:2149-2154;
Bodanszky and Bodanszky, The Practice of Peptide Synthesis
(Springer-Verlag, New York, 1984)), incorporated herein by
reference.
[0076] Polypeptides according to the invention can also be prepared
commercially by companies providing peptide synthesis as a service
(e.g., BACHEM Bioscience, Inc., King of Prussia, Pa.; Quality
Controlled Biochemicals, Inc., Hopkinton, Mass.).
[0077] Automated peptide synthesis machines, such as manufactured
by Perkin-Elmer Applied Biosystems, also are available.
[0078] The polypeptide compound is preferably purified after it has
been isolated or synthesized by either chemical or recombinant
techniques. For purification purposes, there are many standard
methods that may be employed, including reversed-phase high
pressure liquid chromatography (RP-HPLC) using an alkylated silica
column such as C.sub.4-, C.sub.8- or C.sub.18-silica. A gradient
mobile phase of increasing organic content is generally used to
achieve purification, for example, acetonitrile in an aqueous
buffer, usually containing a small amount of trifluoroacetic acid.
Ion-exchange chromatography can also be used to separate peptides
based on their charge. The degree of purity of the polypeptide can
be determined by various methods, including identification of a
major large peak on HPLC. A polypeptide that produces a single peak
that is at least 95% of the input material on an HPLC column is
preferred. Even more preferable is a polypeptide that produces a
single peak that is at least 97%, at least 98%, at least 99% or
even 99.5% or more of the input material on an HPLC column.
[0079] To ensure that the peptide obtained using any of the
techniques described above is the desired peptide for use in
compositions of the present invention, analysis of the peptide
composition can be carried out. Such composition analysis can be
conducted using high resolution mass spectrometry to determine the
molecular weight of the peptide. Alternatively, the amino acid
content of the peptide can be confirmed by hydrolyzing the peptide
in aqueous acid, and separating, identifying and quantifying the
components of the mixture using HPLC, or an amino acid analyzer.
Protein sequenators, which sequentially degrade the peptide and
identify the amino acids in order, can also be used to determine
the sequence of the peptide.
[0080] cMet binding polypeptides according to the present invention
also can be produced using recombinant DNA techniques, utilizing
nucleic acids (polynucleotides) encoding the polypeptides according
to this invention and then expressing them recombinantly, i.e., by
manipulating host cells by introduction of exogenous nucleic acid
molecules in known ways to cause such host cells to produce the
desired cMet binding polypeptides. Such procedures are within the
capability of those skilled in the art (see, for example, Davis et
al., Basic Methods in Molecular Biology (1986)), incorporated by
reference. Recombinant production of short peptides, such as those
described herein, might not be practical in comparison to direct
synthesis, however recombinant means of production can be very
advantageous where a cMet binding moiety of this invention is
incorporated in a hybrid polypeptide or fusion protein.
[0081] In the practice of the present invention, a determination of
the affinity of the cMet binding moiety for cMet relative to
another protein or target is a useful measure, and is referred to
as specificity for cMet. Standard assays for quantitating binding
and determining affinity include equilibrium dialysis, equilibrium
binding, gel filtration, or the monitoring of numerous
spectroscopic changes (such as a change in fluorescence
polarization) that result from the interaction of the binding
moiety and its target. These techniques measure the concentration
of bound and free ligand as a function of ligand (or protein)
concentration. The concentration of bound polypeptide ([Bound]) is
related to the concentration of free polypeptide ([Free]) and the
concentration of binding sites for the polypeptide, i.e., on cMet,
(N), as described in the following equation:
[Bound]=N.times.[Free]/((1/K.sub.a)+[Free]).
A solution of the data to this equation yields the association
constant, K.sub.a, a quantitative measure of the binding affinity.
The association constant, Ic is the reciprocal of the dissociation
constant, K.sub.D. The K.sub.D is more frequently reported in
measurements of affinity. Preferred cMet binding polypeptides have
a K.sub.D for cMet in the range of, for example, less than 1
nanomolar (nM), 1 nM to 100 micromolar (.mu.M), which includes
K.sub.D values of less than 10 nM, less than 20 nM, less than 40
nM, less than 60 nM, less than 80 nM, less than 1 .mu.M, less than
5 .mu.M, less than 10 .mu.M, less than 20 .mu.M, less than 40
.mu.M, less than 60 .mu.M, and less than 80 .mu.M.
[0082] Where cMet binding moieties are employed as imaging agents,
other aspects of binding specificity become important; imaging
agents operate in a dynamic system in that binding of the imaging
agent to the target (cMet, e.g., on activated cells) might not be
in a stable equilibrium state throughout the imaging procedure. For
example, when the imaging agent is initially injected, the
concentration of imaging agent and of agent-target complex rapidly
increases. Shortly after injection, however, the circulating (free)
imaging agent starts to clear through the kidneys or liver, and the
plasma concentration of imaging agent begins to drop. This drop in
the concentration of free imaging agent in the plasma eventually
causes the agent-target complex to dissociate. The usefulness of an
imaging agent depends on the difference in rate of agent-target
dissociation relative to the clearing rate of the agent. Ideally,
the dissociation rate will be slow compared to the clearing rate,
resulting in a long imaging time during which there is a high
concentration of agent-target complex and a low concentration of
free imaging agent (background signal) in the plasma.
[0083] Quantitative measurement of dissociation rates can be
performed using several methods known in the art, such as fiber
optic fluorimetry (see, for example, Anderson and Miller, 1988,
Clin. Chem., 34:1417-21), surface plasmon resonance (see, for
example, Malmborg et al., 1996, J. Immunol. Methods, 198:51-7; and
Schuck, 1997, Curr. Op. Biotechnol., 8:498-502), resonant mirror,
and grating coupled planar waveguiding (see, for example,
Hutchinson, 1995, Molec. Biotechnol., 3:47-54). Automated
biosensors are commercially available for measuring binding
kinetics: BIAcore surface plasmon resonance sensor (Biacore AB,
Uppsala SE), IAsys resonant mirror sensor (Fisons Applied Sensor
Technology, Cambridge GB), BIOS-1 grated coupled planar waveguiding
sensor (Artificial Sensor Instruments, Zurich CH).
Methods of Screening Polypeptides Identified by Phage Display For
Their Ability To Bind To Cells Expressing The Target
[0084] In another aspect of the invention, methods of screening
binding polypeptides identified by phage display for their ability
to bind to cells expressing the target (and not to cells that do
not express the target) are provided. These methods address a
significant problem associated with screening peptides identified
by phage display: frequently the peptides so identified do not have
sufficient affinity for the target to be screened against
target-expressing cells in conventional assays. However,
ascertaining that a particular phage-identified peptide binds to
cells that express the target (and does not bind to cells that do
not) is a critical piece of information in identifying binding
peptides that are potential in vivo targeting moieties, whether
they are used as monomers or as part of a multimeric construct. The
method takes advantage of the increase in affinity and avidity
associated with multivalent binding and permit screening of
polypeptides with low affinities against target-expressing
cells.
[0085] The method generally consists of preparation and screening
of multimeric constructs including one or more binding
polypeptides. For example, polypeptides identified by phage display
as binding to a target are biotinylated and complexed with avidin,
streptavidin or neutravidin to form tetrameric constructs. These
tetrameric constructs are then incubated with cells that express
the desired target and cells that do not, and binding of the
tetrameric construct is detected. Binding can be detected using any
method of detection known in the art. For example, to detect
binding the avidin, streptavidin, or neutravidin may be conjugated
to a detectable marker (e.g., a radioactive label, a fluorescent
label, or an enzymatic label which undergoes a color change, such
as HRP (horse radish peroxidase), TMB (tetramethyl benzidine) or
alkaline phosphatase).
[0086] The biotinylated peptides are preferably complexed with
neutravidin-HRP. Neutravidin exhibits lower non-specific binding to
molecules than the other alternatives due to the absence of lectin
binding carbohydrate moieties and cell adhesion receptor-binding
RYD domain in neutravidin (Hiller, Y. et al., 1987. Biochem. J.,
248:167-171; Alon, R. et al., 1990. Biochem. Biophys. Res. Commun.,
170:1236-41).
[0087] The tetrameric constructs can be screened against cells that
naturally express the target or cells that have been engineered via
recombinant DNA technologies to express the target (e.g.,
transfectants, transformants, etc.). If cells that have been
transfected to express the target are used, mock transfected cells
(i.e., cells transfected without the genetic material encoding the
target) can be used as a control.
[0088] The tetrameric complexes can optionally be screened in the
presence of serum. Thus, the assay also can be used to rapidly
evaluate the effect of serum on the binding of peptides to the
target.
[0089] The methods disclosed herein are particularly useful in
preparing and evaluating combinations of distinct binding
polypeptides for use in dimeric or multimeric targeting constructs
that contain two or more binding polypeptides. Use of biotin/avidin
complexes allows for relatively easy preparation of tetrameric
constructs containing one to four different binding peptides.
Furthermore, it has now been found that affinity and avidity of a
targeting construct can be increased by inclusion of two or more
targeting moieties that bind to different epitopes on the same
target. The screening methods described herein are useful in
identifying combinations of binding polypeptides that could have
increased affinity when included in such multimeric constructs.
[0090] In a preferred embodiment, the screening methods described
herein can be used to screen cMet binding polypeptides identified
by phage display, such as those described herein. These methods can
be used to assess the specific binding of cMet binding polypeptides
to cells that express cMet or have been engineered to express cMet.
Tetrameric complexes of biotinylated cMet binding polypeptides of
the invention and, for example, neutravidin-HRP can be prepared and
screened against cells transfected to express cMet as well as mock
transfected cells, which do not express cMet.
[0091] The assay can be used to identify cMet binding polypeptides
that bind specifically to cMet-expressing cells (and do not bind to
cells that do not express cMet) even when the monodentate K.sub.D
of the polypeptide is on the order of 200 nM-300 nM. The assay can
be used to screen homotetrameric constructs containing four copies
of a single cMet binding polypeptide of the invention as well as
heterotetrameric (constructs containing two or more different cMet
binding polypeptides). The methods described herein are
particularly useful for assessing combinations of cMet binding
polypeptides for use in multimeric constructs, particularly
constructs containing two or more cMet binding polypeptides that
bind to different epitopes of cMet.
[0092] The assay also can be used to assess the effect of serum on
the cMet binding polypeptides.
Modification or Optimization of cMet Binding Polypeptides
[0093] As discussed, modification or optimization of cMet binding
polypeptides is within the scope of the invention and the modified
or optimized polypeptides are included within the definition of
"cMet binding polypeptides". Specifically, a polypeptide sequence
identified by phage display can be modified to optimize its
potency, pharmacokinetic behavior, stability and/or other
biological, physical and chemical properties.
Substitution of Amino Acid Residues
[0094] For example, one can make the following isosteric and/or
conservative amino acid changes in the parent polypeptide sequence
with the expectation that the resulting polypeptides would have a
similar or improved profile of the properties described above:
[0095] Substitution of alkyl-substituted hydrophobic amino acids:
including alanine, leucine, isoleucine, valine, norleucine,
S-2-aminobutyric acid, S-cyclohexylalanine or other simple
alpha-amino acids substituted by an aliphatic side chain from C1-10
carbons including branched, cyclic and straight chain alkyl,
alkenyl or alkynyl substitutions.
[0096] Substitution of aromatic-substituted hydrophobic amino
acids: including phenylalanine, tryptophan, tyrosine,
biphenylalanine, 1-naphthylalanine, 2-naphthylalanine,
2-benzothienylalanine, 3-benzothienylalanine, histidine, amino,
alkylamino, dialkylamino, aza, halogenated (fluoro, chloro, bromo,
or iodo) or alkoxy (from C.sub.1-C.sub.4)-substituted forms of the
previous listed aromatic amino acids, illustrative examples of
which are: 2-,3- or 4-aminophenylalanine, 2-,3- or
4-chlorophenylalanine, 2-,3- or 4-methylphenylalanine, 2-,3- or
4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or
5-methoxytryptophan, 2'-, 3'-, or 4'-amino-, 2'-, 3'-, or
4'-chloro-, 2,3, or 4-biphenylalanine, 2', -3', - or 4'-methyl-2, 3
or 4-biphenylalanine, and 2- or 3-pyridylalanine.
[0097] Substitution of amino acids containing basic functions:
including arginine, lysine, histidine, ornithine,
2,3-diaminopropionic acid, homoarginine, alkyl, alkenyl, or
aryl-substituted (from C.sub.1-C.sub.10 branched, linear, or
cyclic) derivatives of the previous amino acids, whether the
substituent is on the heteroatoms (such as the alpha nitrogen, or
the distal nitrogen or nitrogens, or on the alpha carbon, in the
pro-R position for example. Compounds that serve as illustrative
examples include: N-epsilon-isopropyl-lysine,
3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine,
N,N-gamma, gamma'-diethyl-homoarginine. Included also are compounds
such as alpha methyl arginine, alpha methyl 2,3-diaminopropionic
acid, alpha methyl histidine, alpha methyl ornithine where alkyl
group occupies the pro-R position of the alpha carbon. Also
included are the amides formed from alkyl, aromatic, heteroaromatic
(where the heteroaromatic group has one or more nitrogens, oxygens
or sulfur atoms singly or in combination) carboxylic acids or any
of the many well-known activated derivatives such as acid
chlorides, active esters, active azolides and related derivatives)
and lysine, ornithine, or 2,3-diaminopropionic acid.
[0098] Substitution of acidic amino acids: including aspartic acid,
glutamic acid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl,
and heteroaryl sulfonamides of 2,4-diaminopriopionic acid,
ornithine or lysine and tetrazole-substituted alkyl amino
acids.
[0099] Substitution of side chain amide residues: including
asparagine, glutamine, and alkyl or aromatic substituted
derivatives of asparagine or glutamine.
[0100] Substitution of hydroxyl containing amino acids: including
serine, threonine, homoserine, 2,3-diaminopropionic acid, and alkyl
or aromatic substituted derivatives of serine or threonine. It is
also understood that the amino acids within each of the categories
listed above can be substituted for another of the same group.
Substitution of Amide Bonds
[0101] Another type of modification within the scope of the
invention is to substitute the amide bonds within the backbone of
the polypeptide. For example, to reduce or eliminate undesired
proteolysis, or other degradation pathways that diminish serum
stability, resulting in reduced or abolished bioactivity, or to
restrict or increase conformational flexibility, one can substitute
amide bonds within the backbone of the peptides with functionality
that mimics the existing conformation or alters the conformation in
the manner desired. Such modifications can produce increased
binding affinity or improved pharmacokinetic behavior. It is
understood that those knowledgeable in the art of peptide synthesis
can make the following amide bond changes for any amide bond
connecting two amino acids with the expectation that the resulting
peptides could have the same or improved activity: insertion of
alpha-N-methylamides or peptide amide backbone thioamides, removal
of the carbonyl to produce the cognate secondary amines,
replacement of one amino acid with an aza-amino acid to produce
semicarbazone derivatives, and use of E-olefins and substituted
E-olefins as amide bond surrogates.
Introduction of D-Amino Acids
[0102] Another approach within the scope of the invention is the
introduction of D-alanine, or another D-amino acid, distal or
proximal to the labile peptide bond. In this case it is also
understood to those skilled in the art that such D-amino acid
substitutions can, and at times, must be made, with D-amino acids
whose side chains are not conservative replacements for those of
the L-amino acid being replaced. This is because of the difference
in chirality and hence side-chain orientation, which could result
in the accessing of a previously unexplored region of the binding
site of the target that has moieties of different charge,
hydrophobicity, steric requirements etc. than that serviced by the
side chain of the replaced L-amino acid.
[0103] Modifications To Improve Pharmacokinetic or Pharmacodynamic
Properties
[0104] It also is understood that use of one or more cMet binding
polypeptides in a particular application could be benefitted by
modifications of the peptide or formulations of the peptide to
improve pharmacokinetic and pharmacodynamic behavior. It is
expected that the properties of the peptide can be changed by
attachment of moieties anticipated to bring about the desired
physical or chemical properties. Such moieties can be appended to
the peptide using acids or amines, via amide bonds or urea bonds,
respectively, to the - or C-terminus of the peptide, or to the
pendant amino group of a suitably located lysine or lysine
derivative, 2,3-diaminopropionic acid, ornithine, or other amino
acid in the peptide that possesses a pendant amine group or a
pendant alkoxyamine or hydrazine group. Conversely acidic amino
acid side-chains such as those of Asp or Glu can be selectively
unmasked and amidated with amines bearing the desired modifying
functionality, or they can be modified in this manner before
incorporation into the peptide chain. The moieties introduced can
be groups that are hydrophilic, basic, or nonpolar alkyl or
aromatic groups depending on the peptide of interest and the extant
requirements for modification of its properties.
Glycosylation of Amino Acid Residues
[0105] Yet another modification within the scope of the invention
is glycosylation of one or more amino acid residues (e.g., serine
or threonine residues) in the cMet binding polypeptide.
Glycosylation, which can be carried out using standard conditions,
can be used to enhance solubility, alter pharmacokinetics and
pharmacodynamics or to enhance binding via a specific or
non-specific interaction involving the glycosidic moiety.
Formation of Salts
[0106] It also is within the scope of the invention to form
different salts that could increase or decrease the water
solubility or the ease of formulation of these peptides. These may
include, but are not restricted to, N-methylglucamine (meglumine),
acetate, oxalates, ascorbates, etc.
Structural Modifications which Retain Structural Features
[0107] Yet another modification within the scope of the invention
is truncation of cyclic polypeptides. The cyclic nature of many
polypeptides of the invention limits the conformational space
available to the peptide sequence, particularly within the cycle.
Therefore truncation of the peptide by one or more residues distal
or even proximal to the cycle, at either the N-terminal or
C-terminal region could provide truncated peptides with similar or
improved biological activity. A unique sequence of amino acids,
even as small as three amino acids, which is responsible for the
binding activity, can be identified, as noted for RGD peptides
(Plow, E. et al., 1987. Blood, 70:110-5; Oldberg, A. et al., 1988.
J. Biol. Chem., 263:19433-19436; Taub, R. et al., 1989. J. Biol.
Chem., 264:259-65; Andrieux, A. et al., 1989. J. Biol. Chem.,
264:9258-65; and U.S. Pat. Nos. 5,773,412 and 5,759,996, each of
which is incorporated herein by reference).
[0108] It also has been shown in the literature that large peptide
cycles can be substantially shortened, eliminating extraneous amino
acids, but substantially including the critical binding residues.
See, U.S. Pat. No. 5,556,939, incorporated by reference herein.
[0109] The shortened cyclic peptides can be formed using disulfide
bonds or amide bonds of suitably located carboxylic acid groups and
amino groups.
[0110] Furthermore, D-amino acids can be added to the peptide
sequence to stabilize turn features (especially in the case of
glycine). In another approach alpha, beta, gamma or delta dipeptide
or turn mimics (such as .alpha., .beta., .gamma., or .delta. turn
mimics), some of which are shown in FIGS. 1A-1C, can be employed to
mimic structural motifs and turn features in a peptide and
simultaneously provide stability from proteolysis and enhance other
properties such as, for example, conformational stability and
solubility (structure 1A: Hart et al., J. Org. Chem., 64, 2998-2999
(1999); structure 1B: Hanessian et al., "Synthesis of a Versatile
Peptidomimetic Scaffold" in Methods in Molecular Medicine, Vol. 23:
Peptidomimetics Protocols, W. Kazmierski, Ed. (Humana Press Inc.,
Totowa, N.J., 1999), Chapter 10, pp. 161-174; structure 1C: WO
01/16135.
Substitution of Disulfide Mimetics
[0111] Also within the scope of the invention is the substitution
of disulfide mimetics for disulfide bonds within the cMet binding
peptides of the invention.
[0112] Where disulfide-containing peptides are employed in
generating .sup.99mTc-based radiopharmaceuticals, or other useful
radiopharmaceuticals based on other isotopes, a significant problem
is the presence of the disulfide bond. For example, the integrity
of the disulfide bond is difficult to maintain during procedures
designed to incorporate .sup.99mTc via routes that are reliant upon
the reduction of pertechnetate ion and subsequent incorporation of
the reduced Tc species into substances bearing Tc-specific
chelating groups. This is because the disulfide bond is rather
easily reduced by the reducing agents commonly used in kits devised
for one-step preparation of radiopharmaceuticals. Therefore, the
ease with which the disulfide bond can be reduced during Tc
chelation may require substitution with mimetics of the disulfide
bonds. Accordingly, another modification within the scope of the
invention is to substitute the disulfide moiety with mimetics
utilizing the methods disclosed herein or known to those skilled in
the art, while retaining the activity and other desired properties
of the cMet-binding polypeptides of the invention.
1.) Oxime Linker
[0113] The oxime moiety has been employed as a linker by
investigators in a number of contexts (Wahl, F. and Mutter, M.,
1996. Tetrahedron Lett., 37:6861-6864). As shown in FIG. 2, the
amino acids 4, containing an aminoalcohol function, and 5
containing an alkoxyamino function, can be incorporated into the
peptide chain, not necessarily at the end of the peptide chain.
After formation of the peptide the side-chain protecting groups can
be removed. The aldehyde group is then unmasked and an oxime
linkage is formed.
2.) Lanthionine Linker
[0114] Lanthionines are cyclic sulfides, wherein the disulfide
linkage (S--S) is replaced by a carbon-sulfur (C--S) linkage. Thus,
the lability to reduction is far lower. Lanthionines can be
prepared by a number of methods including those discussed
below.
1) Preparation of Lanthionines using Bromoacetylated Peptides
[0115] Lanthionines can be readily prepared using known methods
(Robey, F. and Fields, R., 1989. Anal. Biochem., 177:373-377;
Inman, J. et al., 1991. Bioconjug. Chem., 2:458-463; Ploinsky, A.
et al., 1992. J. Med. Chem., 35:4185-4194; Mayer et al., "Peptides,
Frontiers of Peptide Science", in Proceedings of the 15th American
Peptide Symposium, Tam and Kaumaya (Eds.), Jun. 14-19, 1995,
Nashville, Tenn. (Klumer Academic Pub., Boston), pp. 291-292; Wakao
et al., Jpn. Kokai Tokyo Koho, JP 07300452 A2 (1995)). Preparation
of peptides using Boc automated peptide synthesis followed by
coupling the peptide terminus with bromoacetic acid gives
bromoacetylated peptides in good yield. Cleavage and deprotection
of the peptides can be accomplished using HF/anisole. If the
peptide contains a cysteine group its reactivity can be controlled
with low pH. If the pH of the medium is raised to 6-7 then either
polymerization or cyclization of the peptide takes place.
Polymerization is favored at high (100 mg/mL) concentration whereas
cyclization is favored at lower concentrations (1 mg/mL), e.g., 6
cyclizes to 7 (referred to herein as "scheme 1" as shown in FIG.
3). Inman et al. demonstrated the use of
Na-(Boc)-Ne-[N-(bromoacetyl)-.beta.-alanyl]R-lysine as a carrier of
the bromoacetyl group that could be employed in Boc peptide
synthesis thus allowing placement of a bromoacetyl bearing moiety
anywhere in a sequence. In preliminary experiments they found that
peptides with 4-6 amino acids separating the bromoacetyl-lysine
derivative from a cysteine tend to cyclize, indicating the
potential utility of this strategy.
2) Preparation of Lanthionines Via Cysteine Thiol Addition to
Acrylamides
[0116] Several variants of this strategy can be implemented.
Resin-bound serine can be employed to prepare the lanthionine ring
on resin either using a bromination-dehydrobromination-thiol
addition sequence or by dehydration with disuccinimidyl carbonate
followed by thiol addition. Conjugate addition of thiols to
acrylamides has also been amply demonstrated and a reference to the
addition of 2-mercaptoethanol to acrylamide is provided (Wakao et
al., Jpn. Kokai Tokyo Koho, JP 07300452 A2, 1995).
3) Diaryl Ether or Diarylamine Linkage From Intramolecular
Cyclization of Aryl Boronic Acids and Tyrosine
[0117] The reaction of arylboronic acids with phenols, amines and
heterocyclic amines in the presence of cupric acetate, in air, at
ambient temperature, in dichloromethane using either pyridine or
triethylamine as a base to provide unsymmetrical diaryl ethers and
the related amines in good yields (as high as 98%) has been
reported (Evans, D. et al., 1998. Tetrahedron Lett., 39:2937-2940;
Chan, D. et al., 1998. Tetrahedron Lett., 39:2933-2936; Lam, P. et
al., 1998. Tetrahedron Lett., 39:2941-2944). In the case of
N-protected tyrosine derivatives as the phenol component the yields
were also as high as 98%. This demonstrates that amino acid amides
(peptides) are expected to be stable to the transformation and that
yields are high. Precedent for an intramolecular reaction exists in
view of the facile intramolecular cyclizations of peptides to
lactams, intramolecular biaryl ether formation based on the SNAr
reaction and the generality of intramolecular cyclization reactions
under high dilution conditions or on resin, wherein the
pseudo-dilution effect mimics high dilution conditions.
4) Formation of Cyclic Peptides with a Thiazolidine Linkage via
Intramolecular Reaction of Peptide Aldehydes with Cysteine
Moieties
[0118] Another approach that may be employed involves
intramolecular cyclization of suitably located vicinal amino
mercaptan functions (usually derived from placement of a cysteine
at a terminus of the linear sequence or tethered to the sequence
via a side-chain nitrogen of a lysine, for example) and aldehyde
functions to provide thiazolidines that result in the formation of
a bicyclic peptide, one ring of which is that formed by the
residues in the main chain, and the second ring being the
thiazolidine ring. Scheme 2 (FIG. 4) provides an example. The
required aldehyde function can be generated by sodium metaperiodate
cleavage of a suitably located vicinal aminoalcohol function, which
can be present as an unprotected serine tethered to the chain by
appendage to a side chain amino group of a lysine moiety. In some
cases the required aldehyde function is generated by unmasking of a
protected aldehyde derivative at the C-terminus or the N-terminus
of the chain (Botti, P. et al., 1996. J. Am. Chem. Soc.,
118:10018-10034).
5) Lactams Based on Intramolecular Cyclization of Pendant Amino
Groups with Carboxyl Groups on Resin.
[0119] Macrocyclic peptides can be prepared by lactam formation by
either head-to-tail or by pendant group cyclization. The basic
strategy is to prepare a fully protected peptide wherein it is
possible to remove selectively an amine protecting group and a
carboxy protecting group. Orthogonal protecting schemes have been
developed. Of those that have been developed the allyl, trityl and
Dde methods have been employed most (Mellor et al., "Synthesis of
Modified Peptides", in Fmoc Solid Phase Synthesis: A Practical
Approach, White and Chan (eds) (Oxford University Press, New York,
2000), Ch. 6, pp. 169-178). The Dde approach is of interest because
it utilizes similar protecting groups for both the carboxylic acid
function (Dmab ester) and the amino group (Dde group). Both are
removed with 2-10% hydrazine in DMF at ambient temperature.
Alternately the Dde can be used for the amino group and the allyl
group can be used for the carboxyl.
[0120] A lactam function, available by intramolecular coupling via
standard peptide coupling reagents (such as HATU, PyBOP etc) can
act as a surrogate for the disulfide bond. The Dde/Dmab approach is
shown in Scheme 3 (FIG. 5).
[0121] Thus, a linear sequence containing, for example, the
Dde-protected lysine and Dmab ester can be prepared on a
Tentagel-based Rink amide resin at low load (.about.0.1-0.2
mmol/g). Deprotection of both functions with hydrazine is then
followed by on-resin cyclization to give the desired products.
Subsequently cleavage from resin and purification may also be
carried out. For functionalization of the N-terminus of the peptide
it is understood that diamino acids such as
trans-4-(iv-Dde)methylaminocyclohexane carboxylic acid or
4-(iv-Dde)methylamino benzoic acid would be required. An
alternative scenario is to employ the safety catch method to
intramolecular lactam formation during cleavage from the resin.
6) Cyclic Peptides Based on Olefin Metathesis
[0122] The Grubbs reaction (Scheme 4, FIG. 6) involves the
metathesis/cyclization of olefin bonds (Schuster et al., 1997.
Angew. Chem. Int. Edn Engl., 36:2036-2056; Miller et al., 1996. J.
Am. Chem. Soc., 118:9606-9614). It is readily seen that if the
starting material is a diolefin 16 that the resulting product will
be cyclic compound 17. The reaction has been applied to creation of
cycles from olefin-functionalized peptides (Pernerstorfer et al.,
1997. Chem. Commun., 20:1949-50; Clark et al., 1999. Chem. Eur. J.,
5:782-792; Blackwell et al., 1998 Angew. Chem. Int. Ed.,
37:3281-3284; Ripka, A. et al., 1998. Bioorg. Med. Chem. Lett.,
8:357-360; Miller et al., 1996. J. Am. Chem. Soc., 118:9606-9614;
Clark et al., 1995. J. Am. Chem. Soc., 117:12364-12365; Miller et
al., 1995. J. Am. Chem. Soc., 117:5855-5856). One can prepare
either C-allylated amino acids or possibly N-allylated amino acids
and employ them in this reaction in order to prepare carba-bridged
cyclic peptides as surrogates for disulfide bond containing
peptides.
[0123] One also can prepare novel compounds with olefinic groups.
Functionalization of the tyrosine hydroxyl with an
olefin-containing tether is one option. The lysine E-amino group is
another option with appendage of the olefin-containing unit as part
of an acylating moiety, for example. If instead the lysine side
chain amino group is alkylated with an olefin containing tether, it
can still function as a point of attachment for a reporter as well.
The use of 5-pentenoic acid as an acylating agent for the lysine,
ornithine, or diaminopropionic side chain amino groups is another
possibility. The length of the olefin-containing tether can also be
varied in order to explore structure activity relationships.
Manipulation of Peptide Sequences
[0124] Other modifications within the scope of the invention
include manipulations of peptide sequences, which can be expected
to yield peptides with similar or improved biological properties.
These include amino acid translocations (swapping amino acids in
the sequence), use of retro-inverso peptides in place of the
original sequence or a modified original sequence, peptoids and
retro-inverso peptoid sequences. Structures wherein specific
residues are peptoid instead of peptidic, which result in hybrid
molecules, neither completely peptidic nor completely peptoid, are
anticipated as well.
Linkers
[0125] Additionally, modifications within the invention include
introduction of linkers or spacers between the targeting sequence
of the binding moiety or binding polypeptide and the detectable
label or therapeutic agent. For example, use of such
linkers/spacers can improve the relevant properties of the binding
peptides (e.g., increase serum stability, etc.). These linkers can
include, but are not restricted to, substituted or unsubstituted
alkyl chains, polyethylene glycol derivatives, amino acid spacers,
sugars, or aliphatic or aromatic spacers common in the art.
[0126] For example, suitable linkers include homobifunctional and
heterobifunctional cross-linking molecules. The homobifunctional
molecules have at least two reactive functional groups, which are
the same. The reactive functional groups on a homobifunctional
molecule include, for example, aldehyde groups and active ester
groups. Homobifunctional molecules having aldehyde groups include,
for example, glutaraldehyde and subaraldthyde.
[0127] Homobifunctional linker molecules having at least two active
ester units include esters of dicarboxylic acids and
N-hydroxysuccinimide. Some examples of such N-succinimidyl esters
include disuccinimidyl suberate and dithio-bis-(succinimidyl
propionate), and their soluble bis-sulfonic acid and bis-sulfonate
salts such as their sodium and potassium salts.
[0128] Heterobifunctional linker molecules have at least two
different reactive groups. Some examples of heterobifunctional
reagents containing reactive disulfide bonds include N-succinimidyl
3-(2-pyridyl-dithio)propionate (Carlsson of al., 1978. Biochem. J.,
173:723-737), sodium
S-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and
4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene.
N-succinimidyl 3-(2-pyridyldithio)propionate is preferred. Some
examples of heterobifunctional reagents comprising reactive groups
having a double bond that reacts with a thiol group include
succinimidyl 4-(N-maleimidomethyl)cyclohexahe-1-carboxylate and
succinimidyl m-maleimidobenzoate. Other heterobifunctional
molecules include succinimidyl 3-(maleimido)propionate,
sulfosuccinimidyl 4-(p-maleimido-phenyl)butyrate, sulfosuccinimidyl
4-(N-maleimidomethyl-cyclohexane)-1-carboxylate,
maleimidobenzoyl-5N-hydroxy-succinimide ester.
[0129] Furthermore, linkers that are combinations of the molecules
and/or moieties described above, can also be employed to confer
special advantage to the properties of the peptide. Lipid molecules
with linkers may be attached to allow formulation of ultrasound
bubbles, liposomes or other aggregation based constructs. Such
constructs could be employed as agents for targeting and delivery
of a diagnostic reporter, a therapeutic agent (e.g., a chemical
"warhead" for therapy), or a combination of these.
Multimeric Constructs of cMet Binding Polypeptides
[0130] Constructs employing dimers, multimers or polymers of one or
more cMet binding polypeptides of the invention are also
contemplated. Indeed, there is ample literature evidence that the
binding of low potency peptides or small molecules can be
substantially increased by the formation of dimers and multimers.
Thus, dimeric and multimeric constructs (both homogeneous and
heterogeneous) are within the scope of the instant invention. The
polypeptide sequences in the dimeric constructs can be attached at
their N- or C-terminus or the N-epsilon nitrogen of a suitably
placed lysine moiety (or another function bearing a selectively
derivatizable group such as a pendant oxyamino or other
nucleophilic group), or can be joined together via one or more
linkers (e.g., those discussed herein) employing the appropriate
attachment chemistry. This coupling chemistry can include amide,
urea, thiourea, oxime, or aminoacetylamide (from chloro- or
bromoacetamide derivatives, but is not so limited). For example,
methods to prepare dimeric or multimeric constructs of cMet binding
polypeptides of the invention include at least those discussed
below.
Method A
[0131] Fully protected cMet-binding peptides can be built up on
Ellman-type safety catch resin using automated or manual Fmoc
peptide synthesis protocols (Backes et al., 1996. J. Am. Chem.
Soc., 118:3055-56). Separately, using standard methods known in the
art of peptide synthesis, a di-lysine derivative can be constructed
on 2-chlorotrityl resin (Fields et al., "Principles and Practice of
Solid Phase Synthesis" in Synthetic Peptides, A Users Guide, Grant,
Ed. (W.H. Freeman Co., New York, 1992), Ch. 3, pp. 77-183; Barlos
et al., "Convergent Peptide Synthesis" in Fmoc Solid Phase Peptide
Synthesis, Chan, W. C. and White, P. D., Eds. (Oxford University
Press, New York, 2000), Ch. 9, pp. 215-228). Liberation of this
from the 2-chlorotrityl resin without removal of the side-chain
protecting groups, activation of the carboxyl group and coupling to
any amine-functionalized labeling group provides a di-lysine
derivative whose protected pendant nitrogen atoms can be unmasked
to give two free amino groups. The prior-mentioned safety-catch
resin is activated and the desired N-deprotected labeling
group-functionalized di-lysine derivative is added to the activated
safety-catch resin. The pendant amino groups are acylated by the
carboxy-terminus of the safety-catch resin-bound peptide, which is
now detached from the resin and represents an integral part of the
di-lysine structure. An excess of the safety-catch resin-bound
peptide can be employed to insure complete reaction of the amino
groups of the di-lysine construct. Optimization of the ratio of the
reacting partners in this scheme optimizes the yield. The
protecting groups on the cMet-binding peptides are removed
employing trifluoroacetic acid based cleavage protocols.
[0132] The synthesis of dimeric and multimeric constructs wherein
two or more cMet-binding peptides are present in one construct is
easily accomplished. Orthogonal protection schemes (such as an
allyloxycarbonyl group on one nitrogen and an Fmoc group on the
other, or employing the Fmoc group in conjunction with the iV-Dde
protecting group on the other, for example) can be employed to
distinguish the pendant nitrogen atoms of the di-lysine derivatives
described above. Unmasking of one of the amino groups, followed by
reaction of the resulting product with an activated safety-catch
resin-bound cMet-binding peptide as described above, provides a
di-lysine construct having a single cMet-binding peptide attached.
Removal of the second protecting group unmasks the remaining
nitrogen (Mellor et al., "Synthesis of Modified Peptides" in Fmoc
Solid Phase Peptide Synthesis, Chan, W. C. and White, P. D., Eds.
(Oxford University Press, New York, 2000), Chapt. 6, pp. 169-176).
The resulting product can be reacted with a second safety-catch
resin bearing another cMet-binding peptide to provide a
fully-protected homodimeric construct, which after removal of
protecting groups with trifluoroacetic acid, provides the desired
material.
Method B
[0133] A cMet-binding peptide is assembled on a Rink-amide resin by
automated or manual peptide coupling methods, usually employing
Fmoc peptide synthesis protocols. The peptide can possess a
C-terminus or N-terminus functionalized with a linker or a
linker-labeling group construct that may possess an additional
nucleophilic group such as the .epsilon.-amino group of a lysine
moiety, for example. Cleavage of the protecting groups is
accomplished employing trifluoroacetic acid with appropriate
modifiers depending on the nature of the peptide. The fully
deprotected peptide is then reacted with a large excess of a
bifunctional electrophile such as the commercially available
glutaric acid bis-N-hydroxysuccinimide ester (Tyger Scientific,
Inc., Princeton, N.J.). The resulting monoamidated,
mono-N-hydroxysuccinimidyl ester of glutaric acid is then treated
with an additional equivalent of the same peptide, or an equivalent
of a different cMet-binding peptide. Purification of the resulting
material by HPLC affords the desired homo-dimeric construct bearing
a suitable labeling group.
Method C
[0134] A modular scheme can be employed to prepare dimeric or
higher multimeric constructs bearing suitable labeling groups as
defined above. In a simple illustration, fmoc-lysine(iV-Dde) Rink
amide resin is treated with piperidine to remove the fmoc moiety.
Then a labeling function, such as biotin, 5-carboxyfluorescein or
N,N-dimethyl-Gly-Ser(O-t-Bu)-Cys(Acm)-Gly-OH is coupled to the
nitrogen atom. The resin is next treated with hydrazine to remove
the iV-Dde group. After thorough washing, the resin is treated with
cyanuric chloride and a hindered base such as diisopropylethylamine
in a suitable solvent such as DMF, NMP or dichloromethane to
provide a monofunctionalized dichlorotriazine bound to the resin.
Subsequent successive displacement of the remaining chlorine atoms
by two equivalents of a cMet-binding peptide provides a resin-bound
homo-dimeric labeling group-functionalized construct (Falorni, M.
et al., 1998. Tetrahedron Lett., 39:7607-7610; Johnson, C. et al.,
1998. Tetrahedron, 54:4097-4106; Stankova, M. and Lebl, M., 1996.
Mol. Divers., 2:75-80). The incoming peptides can be protected or
unprotected as the situation warrants. Cleavage of protecting
groups is accomplished employing trifluoroacetic acid-based
deprotection reagents as described above, and the desired materials
are purified by high performance liquid chromatography.
[0135] It is understood that in each of these methods lysine
derivatives can be serially employed to increase the multiplicity
of the multimers. The use of related, more rigid molecules bearing
the requisite number of masked, or orthogonally protected nitrogen
atoms to act as scaffolds to vary the distance between the
cMet-binding peptides, to increase the rigidity of the construct
(by constraining the motion and relative positions of the
cMet-binding peptides relative to each other and the reporter) is
entirely within the scope of methods A-C and all other methods
described herein. The references cited above are incorporated by
reference herein in their entirety.
[0136] Uses for cMet Binding Polypeptides and Multimeric Peptide
Constructs
[0137] The cMet binding moieties of the invention also have utility
in the treatment of a variety of disease states, including those
associated with cellular proliferation (e.g., hyperproliferation,
e.g., cancer). The cMet binding moieties of the invention (e.g.,
polypeptides and multimeric polypeptide constructs) can themselves
be used as therapeutics or could be used to localize one or more
therapeutic agents (e.g., a chemotherapeutic, a radiotherapeutic,
genetic material, etc.) to cMet-expressing cells, including sites
of cellular proliferation. Any suitable method of assaying or
imaging cMet also can be employed. The cMet binding moieties
according to this invention are useful for detection and/or imaging
of cMet in vitro or in vivo, and particularly for detection and/or
imaging of sites of angiogenesis, in which HGF and cMet are
intimately involved, as explained herein.
In Vitro
[0138] For detection of HGF or cMet in solution, a binding
polypeptide or multimeric polypeptide construct according to the
invention can be detectably labeled, e.g., fluorescently labeled,
enzymatically labeled, or labeled with a radioactive or
paramagnetic metal, then contacted with the solution, and
thereafter formation of a complex between the binding polypeptide
and the cMet target can be detected. As an example, a fluorescently
labeled cMet binding peptide can be used for in vitro cMet or
HGF/cMet complex detection assays, wherein the peptide is added to
a solution to be tested for cMet or HGF/cMet complex under
conditions allowing binding to occur. The complex between the
fluorescently labeled cMet binding peptide and cMet or HGF/cMet
complex target can be detected and quantified by, for example,
measuring the increased fluorescence polarization arising from the
cMet or HGF/cMet complex-bound peptide relative to that of the free
peptide.
[0139] Alternatively, a sandwich-type "ELISA" assay can be used,
wherein a cMet binding polypeptide is immobilized on a solid
support such as a plastic tube or well, then the solution suspected
of containing cMet or HGF/cMet complex target is contacted with the
immobilized binding moiety, non-binding materials are washed away,
and complexed polypeptide is detected using a suitable detection
reagent, such as a monoclonal antibody recognizing cMet or HGF/cMet
complex. The monoclonal antibody is detectable by conventional
means known in the art, including being detectably labeled, e.g.,
radiolabeled, conjugated with an enzyme such as horseradish
peroxidase and the like, or fluorescently labeled, etc.
[0140] For detection or purification of soluble cMet or HGF/cMet
complex in or from a solution, binding polypeptides or multimeric
polypeptide construct of the invention can be immobilized on a
solid substrate such as a chromatographic support or other matrix
material, then the immobilized binder can be loaded or contacted
with the solution under conditions suitable for formation of a
binding polypeptide/cMet complex. The non-binding portion of the
solution can be removed and the complex can be detected, for
example, using an anti-HGF or anti-HGF/cMet complex antibody, or an
anti-binding polypeptide antibody, or the cMet or HGF/cMet complex
target can be released from the binding moiety at appropriate
elution conditions.
[0141] The biology of cellular proliferation and the roles of HGF
and cMet in initiating and maintaining it have been investigated by
many researchers and continues to be an active field for research
and development. In furtherance of such research and development, a
method of purifying bulk amounts of cMet or HGF/cMet complex in
pure form is desirable, and the binding polypeptides and multimeric
polypeptide constructs according to this invention are especially
useful for that purpose, using the general purification methodology
described above.
In Vivo
Diagnostic Imaging
[0142] A particularly preferred use for the polypeptides and
multimeric polypeptide constructs according to the present
invention is for creating visually readable images of cMet
expressing tissue, such as, for example, neoplastic tumors, which
exhibit hyperproliferation. The cMet binding polypeptides and
multimeric polypeptide constructs disclosed herein can be converted
to imaging reagents by conjugating the polypeptides with a label
appropriate for diagnostic detection, optionally via a linker.
Preferably, a peptide or multimeric polypeptide construct
exhibiting much greater specificity for cMet or HGF/cMet than for
other serum proteins is conjugated or linked to a label appropriate
for the detection methodology to be employed. For example, the cMet
or HGF/cMet complex binding polypeptide can be conjugated with or
without a linker to a paramagnetic chelate suitable for Magnetic
Resonance Imaging (MRI), with a radiolabel suitable for x-ray,
Positron Emission Tomography (PET) or scintigraphic imaging
(including a chelator for a radioactive metal), with an ultrasound
contrast agent (e.g., a stabilized microbubble, a microballoon, a
microsphere or what has been referred to as a gas filled
"liposome") suitable for ultrasound detection, or with an optical
imaging dye.
[0143] Suitable linkers can include those discussed herein,
including substituted or unsubstituted alkyl chains, amino acid
chains (e.g., polyglycine), polyethylene glycols, polyamides, and
other linkers known in the art.
[0144] In general, the technique of using a detectably labeled cMet
binding moiety is based on the premise that the label generates a
signal that is detectable outside a patient's body. For example,
when the detectably labeled cMet binding moiety is administered to
the patient in which it is desirable to detect, e.g.,
hyperproliferation, the high affinity of the cMet binding moiety
for cMet causes the binding moiety to bind to the site of
hyperproliferation and accumulate label at the site. Sufficient
time is allowed for the labeled binding moiety to localize at the
site of proliferation. The signal generated by the labeled peptide
is detected by a scanning device that will vary according to the
type of label used, and the signal is then converted to an image of
the site of proliferation.
[0145] In another embodiment, rather than directly labeling a cMet
binding polypeptide or multimeric polypeptide construct with a
detectable label or radiotherapeutic construct, one or more
peptides or constructs of the invention can be conjugated with for
example, avidin, biotin, or an antibody or antibody fragment that
will bind the detectable label or radiotherapeutic. For example,
one or more cMet-binding peptides can be conjugated to streptavidin
(potentially generating multivalent binding) for in vivo binding to
cMet-expressing cells. After the unbound targeting construct is
cleared from the body, a biotinylated detectable label or
radiotherapeutic construct (e.g., a chelate molecule complexed with
a radioactive metal) can be infused and will rapidly concentrate at
the site where the targeting construct is bound. This approach in
some situations can reduce the time required after administering
the detectable label until imaging can take place. It also can
increase signal to noise ratio in the target site, and decrease the
dose of the detectable label or radiotherapeutic construct
required. This is particularly useful when a radioactive label or
radiotherapeutic is used as the dose of radiation that is delivered
to normal but radiation-sensitive sites in the body, such as
bone-marrow, kidneys, and liver is decreased. This approach,
sometimes referred to as pre-targeting or two-step, or three-step
approaches was reviewed by S. F. Rosebrough in Q. J. Nucl. Med.,
40:234-251 (1996), which is incorporated by reference herein.
A. Magnetic Resonance Imaging
[0146] The cMet binding moieties of the present invention can
advantageously be conjugated with a paramagnetic metal chelate in
order to form a contrast agent for use in MRI. Preferred
paramagnetic metal ions have atomic numbers 21-29, 42, 44, or
57-83. This includes ions of the transition metal or lanthanide
series which have one, and more preferably five or more, unpaired
electrons and a magnetic moment of at least 1.7 Bohr magneton.
Preferred paramagnetic metals include, but are not limited to,
chromium (III), manganese (II), manganese (III), iron (II), iron
(III), cobalt (II), nickel (II), copper (II), praseodymium (III),
neodymium (III), samarium (III), gadolinium (III), terbium (III),
dysprosium (III), holmium erbium (III), europium (III) and
ytterbium (III), chromium (III), iron (III), and gadolinium (III).
The trivalent cation, Gd.sup.3+, is particularly preferred for MRI
contrast agents, due to its high relaxivity and low toxicity, with
the further advantage that it exists in only one biologically
accessible oxidation state, which minimizes undesired metabolysis
of the metal by a patient. Another useful metal is Cr.sup.3+, which
is relatively inexpensive. Gd(III) chelates have been used for
clinical and radiologic MR applications since 1988, and
approximately 30% of MR exams currently employ a gadolinium-based
contrast agent.
[0147] The practitioner will select a metal according to dose
required to detect cellular proliferation and considering other
factors such as toxicity of the metal to the subject. See, Tweedle
et al., Magnetic Resonance Imaging (2nd ed.), vol. 1, Partain et
al., Eds. (W.B. Saunders Co. 1988), pp. 796-797. Generally, the
desired dose for an individual metal will be proportional to its
relaxivity, modified by the biodistribution, pharmacokinetics and
metabolism of the metal.
[0148] The paramagnetic metal chelator is a molecule having one or
more polar groups that act as a ligand for, and complex with, a
paramagnetic metal. Suitable chelators are known in the art and
include acids with methylene phosphonic acid groups, methylene
carbohydroxarnine acid groups, carboxyethylidene groups, or
carboxymethylene groups. Examples of chelators include, but are not
limited to, diethylenetriaminepentaacetic acid (DTPA),
1,4,7,10-tetraazacyclo-tetradecane-1,4,7,10-tetraacetic acid
(DOTA), 1-substituted
1,4,7,-tricarboxymethyl-1,4,7,10-teraazacyclododecane (DO3A),
ethylenediaminetetraacetic acid (EDTA), and
1,4,8,11-tetra-azacyclotetradecane-1,4,8,11-tetraacetic acid
(TETA). Additional chelating ligands are ethylene
bis-(2-hydroxy-phenylglycine) (EHPG), and derivatives thereof,
including 5-C1-EHPG, 5-Br-EHPG, 5-Me-EHPG, 5-t-Bu-EHPG, and
5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid
(benzo-DTPA) and derivatives thereof, including dibenzo-DTPA,
phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzyl DTPA; bis-2
(hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and
derivatives thereof; the class of macrocyclic compounds which
contain at least 3 carbon atoms, more preferably at least 6, and at
least two heteroatoms (0 and/or N), which macrocyclic compounds can
consist of one ring, or two or three rings joined together at the
hetero ring elements, e.g., benzo-DOTA, dibenzo-DOTA, and
benzo-NOTA, where NOTA is 1,4,7-triazacyclononane
N,N',N''-triacetic acid, benzo-TETA, benzo-DOTMA, where DOTMA is
1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyl tetraacetic
acid), and benzo-TETMA, where TETMA is
1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic
acid); derivatives of 1,3-propylene-diaminetetraacetic acid (PDTA)
and triethylenetetraaminehexaacetic acid (TTNA); derivatives of
1,5,10?N,N,N''-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM);
and 1,3,5-N,N',N''-tris(2,3-dihydroxybenzoyl)aminomethylbenzene
(MECAM). A preferred chelator for use in the present invention is
DTPA, and the use of DO3A is particularly preferred. Examples of
representative chelators and chelating groups contemplated by the
present invention are described in WO 98/18496, WO 86/06605, WO
91/03200, WO 95/28179, WO 96/23526, WO 97/36619, PCT/US98/01473,
PCT/US98/20182, and U.S. Pat. No. 4,899,755, U.S. Pat. No.
5,474,756, U.S. Pat. No. 5,846,519 and U.S. Pat. No. 6,143,274, all
of which are hereby incorporated by reference.
[0149] In accordance with the present invention, the chelator of
the MRI contrast agent is coupled to the cMet binding polypeptide.
The positioning of the chelate should be selected so as not to
interfere with the binding affinity or specificity of the cMet
binding polypeptide. Preferably, the chelate will be appended
either to the N-terminus or the C-terminus, however the chelate
also can be attached anywhere within the sequence. In preferred
embodiments, a chelator having a free central carboxylic acid group
(e.g., DTPA-Asp(.beta.-COOH)-)OtBu) makes it easy to attach at the
N-terminus of the peptide by formation of an amide bond. The
chelate also can be attached at the C-terminus with the aid of a
linker. Alternatively, isothiocyanate conjugation chemistry can be
employed as a way of linking the appropriate isothiocyanate group
bearing DTPA to a free amino group anywhere within the peptide
sequence.
[0150] In general, the cMet binding moiety can be bound directly or
covalently to the metal chelator (or other detectable label), or it
can be coupled or conjugated to the metal chelator using a linker,
which can be, without limitation, amide, urea, acetal, ketal,
double ester, carbonyl, carbamate, thiourea, sulfone, thioester,
ester, ether, disulfide, lactone, imine, phosphoryl, or
phosphodiester linkages; substituted or unsubstituted saturated or
unsaturated alkyl chains; linear, branched, or cyclic amino acid
chains of a single amino acid or different amino acids (e.g.,
extensions of the N- or C-terminus of the cMet binding moiety);
derivatized or underivatized polyethylene glycols (PEGS),
polyoxyethylene, or polyvinylpyridine chains; substituted or
unsubstituted polyamide chains; derivatized or underivatized
polyamine, polyester, polyethylenimine, polyacrylate, poly(vinyl
alcohol), polyglycerol, or oligosaccharide (e.g., dextran) chains;
alternating block copolymers; malonic, succinic, glutaric, adipic
and pimelic acids; caproic acid; simple diamines and dialcohols;
any of the other linkers disclosed herein; or any other simple
polymeric linkers known in the art (see, for example, WO 98/18497
and WO 98/18496). Preferably the molecular weight of the linker can
be tightly controlled. The molecular weights can range in size from
less than 100 to greater than 1000. Preferably the molecular weight
of the linker is less than 100. In addition, it can be desirable to
utilize a linker that is biodegradable in vivo to provide efficient
routes of excretion for the imaging reagents of the present
invention. Depending on their location within the linker, such
biodegradable functionalities can include ester, double ester,
amide, phosphoester, ether, acetal, and ketal functionalities.
[0151] In general, known methods can be used to couple the metal
chelate and the cMet binding moiety using such linkers (WO
95/28967, WO 98/18496, WO 98/18497 and discussion therein). The
cMet binding moiety can be linked through an N- or C-terminus via
an amide bond, for example, to a metal coordinating backbone
nitrogen of a metal chelate or to an acetate arm of the metal
chelate itself. The present invention contemplates linking of the
chelate on any position, provided the metal chelate retains the
ability to bind the metal tightly in order to minimize toxicity.
Similarly, the cMet binding moiety can be modified or elongated in
order to generate a locus for attachment to a metal chelate,
provided such modification or elongation does not eliminate its
ability to bind cMet.
[0152] MRI contrast reagents prepared according to the disclosures
herein can be used in the same manner as conventional MRI contrast
reagents. When imaging a site of hyperproliferation, for example,
certain MR techniques and pulse sequences can be preferred to
enhance the contrast of the site to the background blood and
tissues. These techniques include (but are not limited to), for
example, black blood angiography sequences that seek to make blood
dark, such as fast spin echo sequences (Alexander, A. et al., 1998.
Magn. Reson. Med., 40: 298-310) and flow-spoiled gradient echo
sequences (Edelman, R. et al., 1990. Radiology, 177: 45-50). These
methods also include flow independent techniques that enhance the
difference in contrast, such as inversion-recovery prepared or
saturation-recovery prepared sequences that will increase the
contrast between angiogenic tumor and background tissues. Finally,
magnetization transfer preparations also can improve contrast with
these agents (Goodrich, K. et al., 1996. Invest. Radial., 31:
323-32).
[0153] The labeled reagent is administered to the patient in the
form of an injectable composition. The method of administering the
MRI contrast agent is preferably parenterally, meaning
intravenously, intraarterially, intrathecally, interstitially, or
intracavitarilly. For imaging active angiogenesis, intravenous or
intraarterial administration is preferred. For MRI, it is
contemplated that the subject will receive a dosage of contrast
agent sufficient to enhance the MR signal at the site of
angiogenesis at least 10%. After injection with the cMet binding
moiety-containing MRI reagent, the patient is scanned in the MRI
machine to determine the location of any sites of
hyperproliferation. In therapeutic settings, upon identification of
a site of hyperproliferation (e.g., tumor), a tumoricidal agent or
anti-hyperproliferative agent (e.g., inhibitors of HGF) can be
immediately administered, if necessary, and the patient can be
subsequently scanned to visualize tumor regression or arrest of
angiogenesis.
B. Ultrasound Imaging
[0154] When ultrasound is transmitted through a substance, the
acoustic properties of the substance will depend upon the velocity
of the transmissions and the density of the substance. Changes in
the acoustic properties will be most prominent at the interface of
different substances (solids, liquids, gases). Ultrasound contrast
agents are intense sound wave reflectors because of the acoustic
differences between the agent and the surrounding tissue. Gas
containing or gas generating ultrasound contrast agents are
particularly useful because of the acoustic difference between
liquid (e.g., blood) and the gas-containing or gas generating
ultrasound contrast agent. Because of their size, ultrasound
contrast agents comprising microbubbles, microballoons, and the
like can remain for a longer time in the blood stream after
injection than other detectable moieties; a targeted cMet-specific
ultrasound agent therefore could demonstrate superior imaging of
sites of hyperproliferation (e.g., cancer) and angiogenesis.
[0155] In this aspect of the invention, the cMet binding moiety can
be linked to a material that is useful for ultrasound imaging. For
example, one or more cMet binding polypeptide or multimeric
polypeptide constructs can be linked to materials employed to form
vesicles (e.g., microbubbles, microballoons, microspheres, etc.),
or emulsions containing a liquid or gas, which functions as the
detectable label (e.g., an echogenic gas or material capable of
generating an echogenic gas). Materials for the preparation of such
vesicles include surfactants, lipids, sphingolipids, oligolipids,
phospholipids, proteins, polypeptides, carbohydrates, and synthetic
or natural polymeric materials (WO 98/53857, WO 98/18498, WO
98/18495, WO 98/18497, WO 98/18496, and WO 98/18501, incorporated
herein by reference in their entirety).
[0156] For contrast agents comprising suspensions of stabilized
microbubbles (a preferred embodiment), phospholipids, and
particularly saturated phospholipids are preferred. Examples of
suitable phospholipids include esters of glycerol with one or two
(the same or different) fatty acids molecules and with phosphoric
acid, wherein the phosphoric acid residue is in turn bonded to a
hydrophilic group, such as choline, serine, inositol, glycerol,
ethanolamine, and the like groups. Fatty acids present in the
phospholipids are in general long chain aliphatic acids, typically
containing from 12 to 24 carbon atoms, preferably from 14 to 22,
that can be saturated or can contain one or more unsaturations.
Examples of suitable fatty acids are lauric acid, myristic acid,
palmitic acid, stearic acid, arachidonic acid, behenic acid, oleic
acid, linoleic acid, and linolenic acid. Mono esters of
phospholipid are also known in the art as the "lyso" forms of the
phospholipids. Further examples of phospholipid are phosphatidic
acids, i.e., the diesters of glycerol-phosphoric acid with fatty
acids, sphingomyelins, i.e., those phosphatidylcholine analogs
where the residue of glycerol diester with fatty acids is replaced
by a ceramide chain, cardiolipins, i.e., the esters of
1,3-diphosphatidylglycerol with a fatty acid, gangliosides,
cerebrosides, etc.
[0157] As used herein, the term "phospholipids" includes naturally
occurring, semisynthetic or synthetically prepared products that
can be employed either singularly or as mixtures.
[0158] Examples of naturally occurring phospholipids are natural
lecithins (phosphatidylcholine (PC) derivatives) such as,
typically, soya bean or egg yolk lecithins. Examples of
semisynthetic phospholipids are the partially or fully hydrogenated
derivatives of the naturally occurring lecithins.
[0159] Examples of synthetic phospholipids are, e.g.,
dilauryloyl-phosphatidylcholine ("DLPC"),
dimyristoylphosphatidylcholine ("DMPC"),
dipalmitoyl-phosphatidylcholine ("DPPC"),
diarachidoylphosphatidylcholine ("DAPC"),
distearoyl-phosphatidylcholine ("DSPC"),
1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"),
1-pahnitoyl-2-myristoylphosphatidylcholine ("PMPC"),
1-palmitoyl-2-stearoylphosphatid-ylcholine ("PSPC"),
1-stearoyl-2-palmitoyl-phosphatidylcholine ("SPPC"),
dioleoylphosphatidylycholine ("DOPC"), 1,2
Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),
dilauryloyl-phosphatidylglycerol ("DLPG") and its alkali metal
salts, diarachidoylphosphatidylglycerol ("DAPG") and its alkali
metal salts, dimyristoylphosphatidylglycerol ("DMPG") and its
alkali metal salts, dipalmitoyl phosphatidylglycerol ("DPPG") and
its alkali metal salts, distearolyphosphatidylglycerol ("DSPG") and
its alkali metal salts, dioleoylphosphatidylglycerol ("DOPG") and
its alkali metal salts, dimyristoyl phosphatidic acid ("DMPA") and
its alkali metal salts, dipalmitoyl phosphatidic acid ("DPPA") and
its alkali metal salts, distearoyl phosphatidic acid ("DSPA"),
diarachidoyl phosphatidic acid ("DAPA") and its alkali metal salts,
dimyristoyl phosphatidyl-ethanolamine ("DMPE"), dipalmitoyl
phosphatidylethanolamine ("DPPE"), distearoyl
phosphatidyl-ethanolamine ("DSPE"), dimyristoyl phosphatidylserine
("DMPS"), diarachidoyl phosphatidylserine ("DAPS"), dipalmitoyl
phosphatidylserine ("DPPS"), distearoylphosphatidylserine ("DSPS"),
dioleoylphosphatidylserine ("DOPS"), dipalmitoyl sphingomyelin
("DPSP"), and distearoyl sphingomyelin ("DSSP"). In a preferred
embodiment, at least one of the phospholipid moieties has the
structure shown in FIG. 7A or 7B, and described in U.S. Pat. No.
5,686,060, which is herein incorporated by reference.
[0160] Other preferred phospholipids include
dipalmitoylphosphatidylcholine, dipalmitoylphosphatidic acid and
dipalmitoylphosphatidylserine. The compositions also can contain
PEG-4000 and/or palmitic acid. Any of the gases disclosed herein or
known to the skilled artisan can be employed; however, inert gases,
such as SF6 or fluorocarbons like CF4, C3F8 and C4F10, are
preferred.
[0161] The preferred gas-filled microbubbles of the invention can
be prepared by means known in the art, such as, for example, by a
method described in any one of the following patents: EP 554213,
U.S. Pat. No. 5,413,774, U.S. Pat. No. 5,578,292, EP 744962, EP
682530, U.S. Pat. No. 5,556,610, U.S. Pat. No. 5,846,518, U.S. Pat.
No. 6,183,725, EP 474833, U.S. Pat. No. 5,271,928, U.S. Pat. No.
5,380,519, U.S. Pat. No. 5,531,980, U.S. Pat. No. 5,567,414, U.S.
Pat. No. 5,658,551, U.S. Pat. No. 5,643,553, U.S. Pat. No.
5,911,972, U.S. Pat. No. 6,110,443, U.S. Pat. No. 6,136,293, EP
619743, U.S. Pat. No. 5,445,813, U.S. Pat. No. 5,597,549, U.S. Pat.
No. 5,686,060, U.S. Pat. No. 6,187,288, and U.S. Pat. No.
5,908,610, which are incorporated by reference herein in their
entirety.
[0162] The preferred microbubble suspensions of the present
invention can be prepared from phospholipids using known processes
such as a freeze-drying or spray-drying solutions of the crude
phospholipids in a suitable solvent or using the processes set
forth in EP 554213; U.S. Pat. No. 5,413,774; U.S. Pat. No.
5,578,292; EP 744962; EP 682530; U.S. Pat. No. 5,556,610; U.S. Pat.
No. 5,846,518; U.S. Pat. No. 6,183,725; EP 474833; U.S. Pat. No.
5,271,928; U.S. Pat. No. 5,380,519; U.S. Pat. No. 5,531,980; U.S.
Pat. No. 5,567,414; U.S. Pat. No. 5,658,551; U.S. Pat. No.
5,643,553; U.S. Pat. No. 5,911,972; U.S. Pat. No. 6,110,443; U.S.
Pat. No. 6,136,293; EP 619743; U.S. Pat. No. 5,445,813; U.S. Pat.
No. 5,597,549; U.S. Pat. No. 5,686,060; U.S. Pat. No. 6,187,288;
and U.S. Pat. No. 5,908,610, which are incorporated by reference
herein in their entirety. Most preferably, the phospholipids are
dissolved in an organic solvent and the solution is dried without
going through a liposome formation stage. This can be done by
dissolving the phospholipids in a suitable organic solvent together
with a hydrophilic stabilizer substance or a compound soluble both
in the organic solvent and water and freeze-drying or spray-drying
the solution. In this embodiment the criteria used for selection of
the hydrophilic stabilizer is its solubility in the organic solvent
of choice. Examples of hydrophilic stabilizer compounds soluble in
water and the organic solvent are, e.g., a polymer, like polyvinyl
pyrrolidone (PVP), polyvinyl alcohol (PVA), polyethylene glycol
(PEG), etc., malic acid, glycolic acid, maltol, and the like. Such
hydrophilic compounds also aid in homogenizing the microbubbles
size distribution and enhance stability under storage. Any suitable
organic solvent can be used as long as its boiling point is
sufficiently low and its melting point is sufficiently high to
facilitate subsequent drying. Typical organic solvents include, for
example, dioxane, cyclohexanol, tertiary butanol,
tetrachlorodifluoro ethylene (C.sub.2Cl.sub.4F.sub.2) or
2-methyl-2-butanol. 2-methyl-2-butanol and C.sub.2Cl.sub.4F.sub.2
are preferred.
[0163] Prior to formation of the suspension of microbubbles by
dispersion in an aqueous carrier, the freeze dried or spray dried
phospholipid powders are contacted with air or another gas. When
contacted with the aqueous carrier the powdered phospholipids whose
structure has been disrupted will form lamellarized or laminarized
segments that will stabilize the microbubbles of the gas dispersed
therein. This method permits production of suspensions of
microbubbles, which are stable even when stored for prolonged
periods, and are obtained by simple dissolution of the dried
laminarized phospholipids, which have been stored under a desired
gas, without shaking or any violent agitation.
[0164] Unless it contains a hyperpolarized gas, known to require
special storage conditions, the lyophilized or freeze-dried residue
can be stored and transported without need of temperature control
of its environment and in particular it can be supplied to
hospitals and physicians for on site formulation into a
ready-to-use administrable suspension without requiring such users
to have special storage facilities.
[0165] Preferably in such a case it can be supplied in the form of
a two component kit. The two component kit can include two separate
containers or a dual-chamber container. In the former case
preferably the container is a conventional septum-sealed vial,
wherein the vial containing the lyophilized residue of step b) is
sealed with a septum through which the carrier liquid can be
injected using an optionally pre-filled syringe. In such a case the
syringe used as the container of the second component is also used
then for injecting the contrast agent. In the latter case,
preferably the dual-chamber container is a dual-chamber syringe and
once the lyophilizate/freeze-dried residue has been reconstituted
and then suitably mixed or gently shaken, the container can be used
directly for injecting the contrast agent. In both cases means for
directing or permitting application of sufficient bubble forming
energy into the contents of the container are provided. However, as
noted above, in the stabilized contrast agents the size of the gas
microbubbles is substantially independent of the amount of
agitation energy applied to the reconstituted dried product.
Accordingly no more than gentle hand shaking is generally required
to give reproducible products with consistent microbubble size.
[0166] It can be appreciated by one ordinary skilled in the art
that other two-chamber reconstitution systems capable of combining
the dried powder with the aqueous solution in a sterile manner are
also within the scope of the present invention. In such systems, it
is particularly advantageous if the aqueous phase can be interposed
between the water-insoluble gas and the environment, to increase
shelf life of the product. Where a material necessary for forming
the contrast agent is not already present in the container (e.g., a
cMet binding moiety of the invention to be linked to the
phospholipid during reconstitution), it can be packaged with the
other components of the kit, preferably in a form or container
adapted to facilitate ready combination with the other components
of the kit.
[0167] No specific containers, vial or connection systems are
required; the present invention can use conventional containers,
vials and adapters. The only requirement is a good seal between the
stopper and the container. The quality of the seal, therefore,
becomes a matter of primary concern; any degradation of seal
integrity could allow undesirables substances to enter the vial. In
addition to assuring sterility, vacuum retention is essential for
products stoppered at ambient or reduced pressures to assure safe
and proper reconstitution. As to the stopper, it may be a compound
or multicomponent formulation based on an elastomer, such as
poly(isobutylene) or butyl rubber.
[0168] Alternatively, microbubbles can be prepared by suspending a
gas in an aqueous solution at high agitation speed, as disclosed,
e.g., in WO 97/29783. A further process for preparing microbubbles
is disclosed in co-pending European patent application no.
03002373, herein incorporated by reference, which comprises
preparing an emulsion of an organic solvent in an aqueous medium in
the presence of a phospholipid and subsequently lyophilizing said
emulsion, after optional washing and/or filtration steps.
[0169] Additives known to those of ordinary skill in the art can be
included in the suspensions of stabilized microbubbles. For
instance, non-film forming surfactants, including polyoxypropylene
glycol and polyoxyethylene glycol and similar compounds, as well as
various copolymers thereof; fatty acids such as myristic acid,
palmitic acid, stearic acid, arachidonic acid or their derivatives,
ergosterol, phytosterol, sitosterol, lanosterol, tocopherol, propyl
gallate, ascorbyl palmitate and butylated hydroxytoluene may be
added. The amount of these non-film forming surfactants is usually
up to 50% by weight of the total amount of surfactants but
preferably between 0 and 30%.
[0170] In ultrasound applications the contrast agents formed by
phospholipid stabilized microbubbles can, for example, be
administered in doses such that the amount of phospholipid injected
is in the range 0.1 to 200 .mu.g/kg body weight, preferably from
about 0.1 to 30 .mu.g/kg.
[0171] Other gas containing suspensions include those disclosed in,
for example, U.S. Pat. No. 5,798,091, WO 97/29783, also EP 881 915,
incorporated herein by reference in their entirety. These agents
can be prepared as described in U.S. Pat. No. 5,798,091 or
WO97/29783.
[0172] Another preferred ultrasound contrast agent comprises
microballoons. The term "microballoon" refers to gas filled bodies
with a material boundary or envelope. More on microballoon
formulations and methods of preparation can be found in EP 324 938
(U.S. Pat. No. 4,844,882); U.S. Pat. No. 5,711,933; U.S. Pat. No.
5,840,275; U.S. Pat. No. 5,863,520; U.S. Pat. No. 6,123,922; U.S.
Pat. No. 6,200,548; U.S. Pat. No. 4,900,540; U.S. Pat. No.
5,123,414; U.S. Pat. No. 5,230,882; U.S. Pat. No. 5,469,854; U.S.
Pat. No. 5,585,112; U.S. Pat. No. 4,718,433; U.S. Pat. No.
4,774,958; WO 95/01187; U.S. Pat. No. 5,529,766; U.S. Pat. No.
5,536,490; and U.S. Pat. No. 5,990,263, the contents of which are
incorporated herein by reference.
[0173] The preferred microballoons have an envelope including a
biodegradable physiologically compatible polymer or, a
biodegradable solid lipid. The polymers useful for the preparation
of the microballoons of the present invention can be selected from
the biodegradable physiologically compatible polymers, such as any
of those described in any of the following patents: EP 458745; U.S.
Pat. No. 5,711,933; U.S. Pat. No. 5,840,275; EP 554213; U.S. Pat.
No. 5,413,774; and U.S. Pat. No. 5,578,292, the entire contents of
which are incorporated herein by reference. In particular, the
polymer can be selected from biodegradable physiologically
compatible polymers, such as polysaccharides of low water
solubility, polylactides and polyglycolides and their copolymers,
copolymers of lactides and lactones such as .epsilon.-caprolactone,
.gamma.-valerolactone and polypeptides. Other suitable polymers
include poly(ortho)esters (see for instance U.S. Pat. No.
4,093,709; U.S. Pat. No. 4,131,648; U.S. Pat. No. 4,138,344; U.S.
Pat. No. 4,180,646); polylactic and polyglycolic acid and their
copolymers, for instance DEXON (Heller, J., 1980. Biomaterials,
1:51-57); poly(DL-lactide-co-e-caprolactone),
poly(DL-lactide-co-.gamma.-valerolactone),
poly(DL-lactide-co-.gamma.-butyrolactone), polyalkylcyanoacrylates;
polyamides, polyhydroxybutyrate; polydioxanone;
poly-.beta.-aminoketones (Polymer, 23:1693 (1982));
polyphosphazenes (Allcock, H., 1976. Science, 193:1214-1219); and
polyanhydrides. The microballoons of the present invention can also
be prepared according to the methods of WO 96/15815, incorporated
herein by reference, where the microballoons are made from a
biodegradable membrane comprising biodegradable lipids, preferably
selected from mono- di-, tri-glycerides, fatty acids, sterols,
waxes and mixtures thereof. Preferred lipids are di- or
tri-glycerides, e.g. di- or tri-myristin, -palmityn or -stearin, in
particular tripalmitin or tristearin.
[0174] The microballoons can employ any of the gases disclosed
herein of known to the skilled artisan; however, inert gases such
as fluorinated gases are preferred. The microballoons can be
suspended in a pharmaceutically acceptable liquid carrier with
optional additives known to those of ordinary skill in the art and
stabilizers.
[0175] Microballoons-containing contrast agents are typically
administered in doses such that the amount of wall-forming polymer
or lipid is from about 10 .mu.g/kg to about 20 .mu.g/kg of body
weight.
[0176] Other gas-containing contrast agent formulations include
microparticles (especially aggregates of microparticles) having gas
contained therein or otherwise associated therewith (for example
being adsorbed on the surface thereof and/or contained within
voids, cavities or pores therein). Methods for the preparation of
these agents are as described in EP 0122624; EP 0123235; EP
0365467; U.S. Pat. No. 5,558,857; U.S. Pat. No. 5,607,661; U.S.
Pat. No. 5,637,289; U.S. Pat. No. 5,558,856; U.S. Pat. No.
5,137,928; WO 95/21631 or WO 93/13809, incorporated herein by
reference in their entirety.
[0177] Any of these ultrasound compositions also should be, as far
as possible, isotonic with blood. Hence, before injection, small
amounts of isotonic agents can be added to any of above ultrasound
contrast agent suspensions. The isotonic agents are physiological
solutions commonly used in medicine and they comprise aqueous
saline solution (0.9% NaCl), 2.6% glycerol solution, 5% dextrose
solution, etc. Additionally, the ultrasound compositions can
include standard pharmaceutically acceptable additives, including,
for example, emulsifying agents, viscosity modifiers,
cryoprotectants, lyoprotectants, bulking agents etc.
[0178] Any biocompatible gas can be used in the ultrasound contrast
agents useful in the invention. The term "gas" as used herein
includes any substances (including mixtures) substantially in
gaseous form at the normal human body temperature. The gas may thus
include, for example, air, nitrogen, oxygen, CO.sub.2, argon, xenon
or krypton, fluorinated gases (including for example,
perfluorocarbons, SF.sub.6, SeF.sub.6) a low molecular weight
hydrocarbon (e.g., containing from 1 to 7 carbon atoms), for
example, an alkane such as methane, ethane, a propane, a butane or
a pentane, a cycloalkane such as cyclopropane, cyclobutane or
cyclopentene, an alkene such as ethylene, propene, propadiene or a
butene, or an alkyne such as acetylene or propyne and/or mixtures
thereof. However, fluorinated gases are preferred. Fluorinated
gases include materials which contain at least one fluorine atom
such as SF.sub.6, freons (organic compounds containing one or more
carbon atoms and fluorine, i.e., CF.sub.4, C.sub.2F.sub.6,
C.sub.3F.sub.8, C.sub.4F.sub.8, C.sub.4F.sub.10, CBrF.sub.3,
CCl.sub.2F.sub.2, C.sub.2ClF.sub.5, and CBrClF.sub.2) and
perfluorocarbons. The term perfluorocarbon refers to compounds
containing only carbon and fluorine atoms and includes, in
particular, saturated, unsaturated, and cyclic perfluorocarbons.
The saturated perfluorocarbons, which are usually preferred, have
the formula CF.sub.n+2, where n is from 1 to 12, preferably from 2
to 10, most preferably from 3 to 8 and even more preferably from 3
to 6. Suitable perfluorocarbons include, for example, CF.sub.4,
C.sub.2F.sub.6, C.sub.3F.sub.8 C.sub.4F.sub.8, C.sub.4F.sub.10,
C.sub.5F.sub.12, C.sub.6F.sub.12, C.sub.7F.sub.14, C.sub.8F.sub.18,
and C.sub.9F.sub.20. Most preferably the gas or gas mixture
comprises SF6 or a perfluorocarbon selected from the group
consisting of C.sub.3F.sub.8 C.sub.4F.sub.8, C.sub.4F.sub.10,
C.sub.6F.sub.12, C.sub.6F.sub.12, C.sub.7F.sub.14, C.sub.8F.sub.18,
with C.sub.4F.sub.10 being particularly preferred. See also WO
97/29783, WO 98/53857, WO 98/18498, WO 98/18495, WO 98/18496, WO
98/18497, WO 98/18501, WO 98/05364, WO 98/17324.
[0179] In certain circumstances it can be desirable to include a
precursor to a gaseous substance (e.g., a material that is capable
of being converted to a gas in vivo, often referred to as a "gas
precursor"). Preferably the gas precursor and the gas it produces
are physiologically acceptable. The gas precursor can be
pH-activated, photo-activated, temperature activated, etc. For
example, certain perfluorocarbons can be used as temperature
activated gas precursors. These perfluorocarbons, such as
perfluoropentane, have a liquid/gas phase transition temperature
above room temperature (or the temperature at which the agents are
produced and/or stored) but below body temperature; thus they
undergo a phase shift and are converted to a gas within the human
body.
[0180] The gas can comprise a mixture of gases. The following
combinations are particularly preferred gas mixtures: a mixture of
gases (A) and (B) in which, at least one of the gases (B), present
in an amount of between 0.5-41% by vol., has a molecular weight
greater than 80 daltons and is a fluorinated gas and (A) is
selected from the group consisting of air, oxygen, nitrogen, carbon
dioxide and mixtures thereof, the balance of the mixture being gas
A.
[0181] Since ultrasound vesicles can be larger than the other
detectable labels described herein, they can be linked or
conjugated to a plurality of cMet binding polypeptides or
multimeric polypeptide constructs in order to increase the
targeting efficiency of the agent. Attachment to the ultrasound
contrast agents described above (or known to those skilled in the
art) can be via direct covalent bond between the cMet binding
polypeptide and the material used to make the vesicle or via a
linker, as described previously. For example, see WO 98/53857
generally for a description of the attachment of a peptide to a
bifunctional PEG linker, which is then reacted with a liposome
composition (Lanza, G. et al., 1997. Ultrasound Med. Biol.,
23:863-870).). The structure of these compounds typically
comprises: [0182] a) A hydrophobic portion, compatible with the
material forming the envelope of the microbubble or of the
microballoon, in order to allow an effective incorporation of the
compound in the envelope of the vesicle; said portion is typically
a lipid moiety (e.g., dipalmitin, distearoil); [0183] b) A spacer
(typically PEGs of different molecular weights), which can be
optional in some cases (microbubbles may, for instance, prove
difficult to freeze dry if the spacer is too long) or preferred in
some others (e.g., peptides can be less active when conjugated to a
microballoon with a short spacer); [0184] c) A reactive group
capable of reacting with a corresponding reactive moiety on the
peptide to be conjugated (e.g., maleimido with the --SH group of
cysteine).
[0185] A number of methods can be used to prepare suspensions of
microbubbles conjugated to cMet binding polypeptides. For example,
one can prepare maleimide-derivatized microbubbles by incorporating
5% (w/w) of N-MPB-PE
(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-4-(p-maleimido-phenyl
butyramide), (Avanti Polar-Lipids, Inc., Alabaster, Ala.) in the
phospholipid formulation. Then, solutions of mercaptoacetylated
cMet-binding peptides (10 mg/mL in DMF), which have been incubated
in deacetylation solution (50 mM sodium phosphate, 25 mM EDTA, 0.5
M hydroxylamine.HCl, pH 7.5) are added to the maleimide-activated
microbubble suspension. After incubation in the dark, under gentle
agitation, the peptide conjugated microbubbles can be purified by
centrifugation.
[0186] Alternatively, cMet-binding polypeptide conjugated
microbubbles can be prepared using biotin/avidin. For example,
avidin-conjugated microbubbles can be prepared using a
maleimide-activated phospholipid microbubble suspension, prepared
as described above, which is added to mercaptoacetylated-avidin
(which has been incubated with deacetylation solution).
Biotinylated cMet-binding peptides (prepared as described herein)
are then added to the suspension of avidin-conjugated microbubbles,
yielding a suspension of microbubbles conjugated to cMet-binding
peptides.
[0187] Ultrasound imaging techniques, which can be used in
accordance with the present invention, include known techniques,
such as color Doppler, power Doppler, Doppler amplitude, stimulated
acoustic imaging, and two- or three-dimensional imaging techniques.
Imaging may be done in harmonic (resonant frequency) or fundamental
modes, with the second harmonic preferred.
C. Optical Imaging, Sonoluminescence or Photoacoustic Imaging
[0188] In accordance with the present invention, a number of
optical parameters can be employed to determine the location of
cMet or HGF/cMet complex with in vivo light imaging after injection
of the subject with an optically-labeled cMet binding polypeptides.
Optical parameters to be detected in the preparation of an image
may include transmitted radiation, absorption, fluorescent or
phosphorescent emission, light reflection, changes in absorbance
amplitude or maxima, and elastically scattered radiation. For
example, biological tissue is relatively translucent to light in
the near infrared (NIR) wavelength range of 650-1000 nm. NIR
radiation can penetrate tissue up to several centimeters,
permitting the use of the cMet binding polypeptides or multimeric
polypeptide constructs of the present invention for optical imaging
of cMet or HGF/cMet complex in vivo.
[0189] The cMet binding polypeptides or multimeric polypeptide
constructs can be conjugated with photolabels, such as, for
example, optical dyes, including organic chromophores or
fluorophores, having extensive delocalized ring systems and having
absorption or emission maxima in the range of 400-1500 nm. The cMet
binding polypeptide or multimeric polypeptide construct can
alternatively be derivatized with a bioluminescent molecule. The
preferred range of absorption maxima for photolabels is between 600
and 1000 nm to minimize interference with the signal from
hemoglobin. Preferably, photoabsorption labels have large molar
absorptivities, e.g., greater than 10.sup.5 cm.sup.-1M.sup.-1,
while fluorescent optical dyes will have high quantum yields.
Examples of optical dyes include, but are not limited to those
described in WO 98/18497, WO 98/18496, WO 98/18495, WO 98/18498, WO
98/53857, WO 96/17628, WO 97/18841, WO 96/23524, WO 98/47538, and
references cited therein. The photolabels can be covalently linked
directly to the cMet binding peptide or linked to the cMet binding
peptide or multimeric polypeptide construct via a linker, as
described previously.
[0190] After injection of the optically-labeled cMet binding
moiety, the patient is scanned with one or more light sources
(e.g., a laser) in the wavelength range appropriate for the
photolabel employed in the agent. The light used can be
monochromatic or polychromatic and continuous or pulsed.
Transmitted, scattered, or reflected light is detected via a
photodetector tuned to one or multiple wavelengths to determine the
location of cMet or HGF/cMet complex in the subject. Changes in the
optical parameter can be monitored over time to detect accumulation
of the optically-labeled reagent at the site of hyperproliferation.
Standard image processing and detecting devices can be used in
conjunction with the optical imaging reagents of the present
invention.
[0191] The optical imaging reagents described above also can be
used for acousto-optical or sonoluminescent imaging performed with
optically-labeled imaging agents (see, U.S. Pat. No. 5,171,298, WO
98/57666, and references cited therein). In acousto-optical
imaging, ultrasound radiation is applied to the subject and affects
the optical parameters of the transmitted, emitted, or reflected
light. In sonoluminescent imaging, the applied ultrasound actually
generates the light detected. Suitable imaging methods using such
techniques are described in WO 98/57666.
D. Nuclear Imaging (Radionuclide Imaging) and Radiotherapy
[0192] The cMet binding moieties can be conjugated with a
radionuclide reporter appropriate for scintigraphy, SPECT, or PET
imaging and/or with a radionuclide appropriate for radiotherapy.
Constructs in which the cMet binding moieties are conjugated with
both a chelator for a radionuclide useful for diagnostic imaging
and a chelator useful for radiotherapy are within the scope of the
invention.
[0193] For use as a PET agent a peptide or multimeric polypeptide
construct is complexed with one of the various positron emitting
metal ions, such as .sup.51Mn, .sup.52Fe, .sup.60Cu, .sup.68Ga,
.sup.72As, .sup.94 mTc, or .sup.110In. The binding moieties of the
invention can also be labeled by halogenation using radionuclides
such as .sup.18F, .sup.124I, .sup.125I, .sup.131I, .sup.123I,
.sup.77Br, and .sup.76Br. Preferred metal radionuclides for
scintigraphy or radiotherapy include .sup.99mTc, .sup.51 Cr,
.sup.67Ga, .sup.68Ga .sup.47Sc, .sup.51Cr, .sup.167Tm, .sup.141Ce,
.sup.111In, .sup.168Yb, .sup.175Yb, .sup.140La, .sup.90Y, .sup.88Y,
.sup.153Sm, .sup.166Ho, .sup.165Dy, .sup.166Dy, .sup.62Cu,
.sup.64Cu, .sup.67Cu, .sup.97Ru, .sup.186Re, .sup.188Re,
.sup.203Pb, .sup.211Bi, .sup.212Bi, .sup.213Bi, .sup.214Bi,
.sup.165Dy, .sup.166Dy, .sup.62Cu, .sup.97Ru, .sup.103Ru,
.sup.186Re, .sup.188Re, .sup.203Pb, .sup.211Bi, .sup.212Bi,
.sup.213Bi, .sup.214Bi, .sup.105Rh, .sup.109Pd, .sup.117mSn,
.sup.149Pm, .sup.161Tb, .sup.177Lu, .sup.198Au and .sup.199Au. The
choice of metal will be determined based on the desired therapeutic
or diagnostic application. For example, for diagnostic purposes the
preferred radionuclides include .sup.64Cu, .sup.67Ga, .sup.68Ga,
.sup.99mTc, and .sup.111In. For therapeutic purposes, the preferred
radionuclides include .sup.64Cu, .sup.90Y, .sup.105Rh, .sup.111In,
.sup.117mSn, .sup.149Pm, .sup.153Sm, .sup.161Tb, .sup.166Dy,
.sup.166Ho, .sup.175Yb, .sup.177Lu, .sup.186/188Re, and .sup.199Au.
.sup.99mTc is particularly preferred for diagnostic applications
because of its low cost, availability, imaging properties, and high
specific activity. The nuclear and radioactive properties of
.sup.99mTc make this isotope an ideal scintigraphic imaging agent.
This isotope has a single photon energy of 140 keV and a
radioactive half-life of about 6 hours, and is readily available
from a .sup.99Mo-.sup.99mTc generator.
[0194] The metal radionuclides may be chelated by, for example,
linear, macrocyclic, terpyridine, and N.sub.3S, N.sub.2S.sub.2, or
N.sub.4 chelants (see also, U.S. Pat. No. 5,367,080, U.S. Pat. No.
5,364,613, U.S. Pat. No. 5,021,556, U.S. Pat. No. 5,075,099, U.S.
Pat. No. 5,886,142), and other chelators known in the art
including, but not limited to, HYNIC, DTPA, EDTA, DOTA, DO3A, TETA,
and bisamino bisthiol (BAT) chelators (see also U.S. Pat. No.
5,720,934). For example, N.sub.4 chelators are described in U.S.
Pat. No. 6,143,274; U.S. Pat. No. 6,093,382; U.S. Pat. No.
5,608,110; U.S. Pat. No. 5,665,329; U.S. Pat. No. 5,656,254; and
U.S. Pat. No. 5,688,487. Certain N.sub.3S chelators are described
in PCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in U.S. Pat.
No. 5,662,885; U.S. Pat. No. 5,976,495; and U.S. Pat. No.
5,780,006. The chelator also can include derivatives of the
chelating ligand mercapto-acetyl-acetyl-glycyl-glycine (MAG3),
which contains an N.sub.3S, and N.sub.2S.sub.2 systems such as MAMA
(monoamidemonoaminedithiols), DADS (N.sub.2S diaminedithiols),
CODADS and the like. These ligand systems and a variety of others
are described in, for example, Liu, S, and Edwards, D., 1999. Chem.
Rev., 99:2235-2268, and references therein.
[0195] The chelator also can include complexes containing ligand
atoms that are not donated to the metal in a tetradentate array.
These include the boronic acid adducts of technetium and rhenium
dioximes, such as are described in U.S. Pat. No. 5,183,653; U.S.
Pat. No. 5,387,409; and U.S. Pat. No. 5,118,797, the disclosures of
which are incorporated by reference herein, in their entirety.
[0196] In another embodiment, disulfide bonds of a cMet binding
polypeptide of the invention are used as two ligands for chelation
of a radionuclide such as .sup.99mTc. In this way the peptide loop
is expanded by the introduction of Tc (peptide-S--S-peptide changed
to peptide-S--Tc--S-peptide). This also has been used in other
disulfide containing peptides in the literature (Chen, J. et al.,
2001. J. Nucl. Med., 42:1847-1855) while maintaining biological
activity. The other chelating groups for Tc can be supplied by
amide nitrogens of the backbone, another cystine amino acid or
other modifications of amino acids.
[0197] Particularly preferred metal chelators include those of
Formula 20, 21, 22, 23a, 23b, 24a, 24b and 25, set forth in FIGS.
8A-8F. Formulae 20-22 are particularly useful for lanthanides such
as paramagnetic Gd.sup.3+ and radioactive lanthanides such as
.sup.177Lu, .sup.90Y, .sup.153Sm, .sup.111In, or .sup.166Ho.
Formulae 23a-24b are particularly useful for radionuclides
.sup.99mTc, .sup.186Re, or .sup.188Re. Formula 25 is particularly
useful for .sup.99mTc. These and other metal chelating groups are
described in U.S. Pat. No. 6,093,382 and U.S. Pat. No. 5,608,110,
which are incorporated by reference herein in their entirety.
Additionally, the chelating group of formula 22 is described in,
for example, U.S. Pat. No. 6,143,274; the chelating group of
formula 24 is described in, for example, U.S. Pat. No. 5,627,286
and U.S. Pat. No. 6,093,382, and the chelating group of formula 25
is described in, for example, U.S. Pat. No. 5,662,885; U.S. Pat.
No. 5,780,006; and U.S. Pat. No. 5,976,495.
[0198] For formulae 24a and 24b of FIG. 8E, X is either CH.sub.2 or
O; Y is C.sub.1-C.sub.10 branched or unbranched alky, aryl,
aryloxy, arylamino, arylaminoacyl, or arylalkyl comprising
C.sub.1-C.sub.10 branched or unbranched alkyl groups, hydroxy or
C.sub.1-C.sub.10 branched or unbranched polyhydroxyalkyl groups,
C.sub.1-C.sub.10 branched or unbranched hydroxy or polyalkoxyalkyl
or polyhydroxy-polyalkoxyalkyl groups; J is C(.dbd.O)-,
OC(.dbd.O)-, SO.sub.2-, NC(.dbd.O)-, NC(.dbd.S)-, N(Y),
NC(.dbd.NCH.sub.3)-, NC(.dbd.NH)-, N.dbd.N-, homopolyamides or
heteropolyamines derived from synthetic or naturally occurring
amino acids; and n is 1-100. Other variants of these structures are
described, for example, in U.S. Pat. No. 6,093,382. The disclosures
of each of the foregoing patents, applications and references are
incorporated by reference herein, in their entirety.
[0199] The chelators can be covalently linked directly to the cMet
binding moiety or multimeric polypeptide construct or linked to the
cMet binding polypeptide via a linker, as described previously, and
then directly labeled with the radioactive metal of choice (see, WO
98/52618, U.S. Pat. No. 5,879,658, and U.S. Pat. No.
5,849,261).
[0200] Complexes of radioactive technetium are particularly useful
for diagnostic imaging and complexes of radioactive rhenium are
particularly useful for radiotherapy. In forming a complex of
radioactive technetium with the reagents of this invention, the
technetium complex, preferably a salt of .sup.99mTc pertechnetate,
is reacted with the reagent in the presence of a reducing agent.
Preferred reducing agents are dithionite, stannous and ferrous
ions; the most preferred reducing agent is stannous chloride. Means
for preparing such complexes are conveniently provided in a kit
form comprising a sealed vial containing a predetermined quantity
of a reagent of the invention to be labeled and a sufficient amount
of reducing agent to label the reagent with .sup.99mTc.
Alternatively, the complex can be formed by reacting a peptide of
this invention conjugated with an appropriate chelator with a
pre-formed labile complex of technetium and another compound known
as a transfer ligand. This process is known as ligand exchange and
is well known to those skilled in the art. The labile complex can
be formed using such transfer ligands as tartrate, citrate,
gluconate or mannitol, for example. Among the .sup.99mTc
pertechnetate salts useful with the present invention are included
the alkali metal salts such as the sodium salt, or ammonium salts
or lower alkyl ammonium salts.
[0201] Preparation of the Complexes of the Present Invention where
the Metal is radioactive rhenium can be accomplished using rhenium
starting materials in the +5 or +7 oxidation state. Examples of
compounds in which rhenium is in the Re(V11) state are
NH.sub.4ReO.sub.4 or KReO.sub.4. Re(V) is available as, for
example, [ReOCl.sub.4](NBu.sub.4), [ReOCl.sub.4](AsPh.sub.4),
ReOCl.sub.3(PPh.sub.3).sub.2 and as ReO.sub.2(pyridine).sup.4+,
where Ph is phenyl and Bu is n-butyl. Other rhenium reagents
capable of forming a rhenium complex also can be used.
[0202] Radioactively labeled scintigraphic imaging agents provided
by the present invention are encompassed having a suitable amount
of radioactivity. Generally, the unit dose to be administered has a
radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi
to 20 mCi. The solution to be injected at unit dosage is from about
0.01 mL to about 10 mL. In forming .sup.99mTc radioactive
complexes, it is generally preferred to form radioactive complexes
in solutions containing radioactivity at concentrations of from
about 0.01 mCi to 100 mCi per mL.
[0203] Typical doses of a radionuclide-labeled cMet binding imaging
agents according to the invention provide 10-20 mCi. After
injection of the cMet-specific radionuclide imaging agent into the
patient, a gamma camera calibrated for the gamma ray energy of the
nuclide incorporated in the imaging agent is used to image areas of
uptake of the agent and quantify the amount of radioactivity
present in the site. Imaging of the site in vivo can take place in
a matter of a few minutes. However, imaging can take place, if
desired, in hours or even longer, after the radiolabeled peptide is
injected into a patient. In most instances, a sufficient amount of
the administered dose will accumulate in the area to be imaged
within about 0.1 of an hour to permit the taking of
scintiphotos.
[0204] Proper dose schedules for the radiotherapeutic compounds of
the present invention are known to those skilled in the art. The
compounds can be administered using many methods including, but not
limited to, a single or multiple IV or IP injections, using a
quantity of radioactivity that is sufficient to cause damage or
ablation of the targeted cMet-expressing tissue, but not so much
that substantive damage is caused to non-target (normal tissue).
The quantity and dose required is different for different
constructs, depending on the energy and half-life of the isotope
used, the degree of uptake and clearance of the agent from the body
and the mass of the tumor. In general, doses can range from a
single dose of about 30-50 mCi to a cumulative dose of up to about
3 Ci.
[0205] The radiotherapeutic compositions of the invention can
include physiologically acceptable buffers, and can require
radiation stabilizers to prevent radiolytic damage to the compound
prior to injection. Radiation stabilizers are known to those
skilled in the art, and can include, for example, para-aminobenzoic
acid, ascorbic acid, gentistic acid and the like.
[0206] A single, or multi-vial kit that contains all of the
components needed to prepare the complexes of this invention, other
than the radionuclide, is an integral part of this invention.
[0207] A single-vial kit preferably contains a chelating ligand, a
source of stannous salt, or other pharmaceutically acceptable
reducing agent, and is appropriately buffered with pharmaceutically
acceptable acid or base to adjust the pH to a value of about 3 to
about 9. The quantity and type of reducing agent used would depend
on the nature of the exchange complex to be formed. The proper
conditions are well known to those that are skilled in the art. It
is preferred that the kit contents be in lyophilized form. Such a
single vial kit can optionally contain labile or exchange ligands
such as glucoheptonate, gluconate, mannitol, malate, citric or
tartaric acid and can also contain reaction modifiers such as
diethylenetriamine-pentaacetic acid (DPTA), ethylenediamine
tetraacetic acid (EDTA), or .alpha., .beta., or .gamma.
cyclodextrin that serve to improve the radiochemical purity and
stability of the final product. The kit also can contain
stabilizers, bullring agents such as mannitol, that are designed to
aid in the freeze-drying process, and other additives known to
those skilled in the art.
[0208] A multi-vial kit preferably contains the same general
components but employs more than one vial in reconstituting the
radiopharmaceutical. For example, one vial can contain all of the
ingredients that are required to form a labile Tc(V) complex on
addition of pertechnetate (e.g., the stannous source or other
reducing agent). Pertechnetate is added to this vial, and after
waiting an appropriate period of time, the contents of this vial
are added to a second vial that contains the ligand, as well as
buffers appropriate to adjust the pH to its optimal value. After a
reaction time of about 5 to 60 minutes, the complexes of the
present invention are formed. It is advantageous that the contents
of both vials of this multi-vial kit be lyophilized. As above,
reaction modifiers, exchange ligands, stabilizers, bulking agents,
etc. can be present in either or both vials.
Therapeutic Applications
[0209] The cMet binding polypeptides and multimeric polypeptide
constructs of the present invention can be used to present, treat
or improve the activity of therapeutic agents such as
anti-proliferative or tumoricidal agents against undesired cellular
proliferation (such as occurs in neoplastic tumors, e.g., cancer,
by providing or improving their affinity for cMet and their
residence time at a HGF/cMet complex on proliferating cells, such
as, for example, epithelial cells) for diseases associated with
cMet, including, but not limited to, diseases related to cMet
activity. In this aspect of the invention, hybrid agents are
provided by conjugating a cMet binding polypeptide or multimeric
polypeptide construct according to the invention with a therapeutic
agent. The therapeutic agent can be a radiotherapeutic, discussed
above, a drug, chemotherapeutic or tumoricidal agent, genetic
material or a gene delivery vehicle, etc. The cMet binding
polypeptide moiety portion of the conjugate causes the therapeutic
to "home" to the sites of cMet or HGF/cMet complex (i.e., activated
epithelial cells), and to improve the affinity of the conjugate for
the endothelium, so that the therapeutic activity of the conjugate
is more localized and concentrated at the sites of cellular
proliferation. In addition, these cMet binding moieties can inhibit
HGF-mediated signaling events by preventing HGF from binding to
cMet. Such conjugates will be useful in treating hyperproliferative
disorders, especially neoplastic tumor growth and metastasis, in
mammals, including humans. The method comprises administering to a
mammal in need thereof an effective amount of a cMet binding
polypeptide or multimeric polypeptide construct according to the
invention conjugated with a therapeutic agent. The invention also
provides the use of such conjugates in the manufacture of a
medicament for the treatment of angiogenesis associated diseases in
mammals, including humans.
[0210] Suitable therapeutic agents for use in this aspect of the
invention include, but are not limited to: antineoplastic agents,
such as platinum compounds (e.g., spiroplatin, cisplatin, and
carboplatin), methotrexate, adriamycin, mitomycin, ansamitocin,
bleomycin, cytosine, arabinoside, arabinosyl adenine,
mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan
(e.g., PAM, L-PAM, or phenylalanine mustard), mercaptopurine,
mitotane, procarbazine hydrochloride, dactinomycin (actinomycin D),
daunorubcin hydrochloride, doxorubicin hydrochloride, taxol,
mitomycin, plicamycin (mithramycin), aminoglutethimide,
estramustine phosphate sodium, flutamide, leuprolide acetate,
megestrol acetate, tamoxifen citrate, testoiactone, trilostane,
amsacrine (m-AMSA), aparaginase (L-aparaginase), Erwina
aparaginase, etoposide (VP-16), interferon CX-2a, Interferon CX-2b,
teniposide (VM-26, vinblastine sulfate (VLB), vincristine sulfate,
bleomycin sulfate, adriamycin, and arabinosyl; anti-angiogenic
agents such as tyrosine kinase inhibitors with activity toward
signaling molecules important in angiogenesis and/or tumor growth
such as SU5416 and SU6668 (Sugen/Pharmacia and Upjohn), endostatin
(EntreMed), angiostatin (EntreMed), Combrestatin (Oxigene),
cyclosporine, 5-fluorouracil, vinblastine, doxorubicin, paclitaxel,
daunorubcin, immunotoxins; coagulation factors; antivirals such as
acyclovir, amantadine azidothymidine (AZT or Zidovudine), ribavirin
and vidarabine monohydrate (adenine arahinoside, ara-A);
antibiotics, antimalarials, antiprotozoans such as chloroquine,
hydroxychloroquine, metroidazole, quinine and meglumine antimonate;
anti-inflammatories such as diflunisal, ibuprofen, indomethacin,
meclofenamate, mefenamic acid, naproxen, oxyphenbutazone,
phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and
salicylates.
[0211] In one embodiment of the invention, the therapeutic agent
can be associated with an ultrasound contrast agent composition in
which cMet binding moieties of the invention are linked to the
material employed to form the vesicles as described herein. After
administration of the ultrasound contrast agent and the optional
imaging of the contrast agent bound to the tissue expressing cMet
or HGF/cMet complex, the tissue can be irradiated with an energy
beam (preferably ultrasonic, e.g., with a frequency of from 0.3 to
3 MHz), to rupture or burst the microvesicles. The therapeutic
effect of the therapeutic agent can thus be enhanced by the energy
released by the rupture of the microvesicles, in particular causing
an effective delivery of the therapeutic agent to the targeted
tissue. For instance, the therapeutic agent can be associated with
the targeted ultrasound contrast agent and delivered as described
in U.S. Pat. No. 6,258,378, herein incorporated by reference.
[0212] The cMet binding polypeptides and multimeric polypeptide
constructs of the present invention also can be used to target
genetic material to cMet-expressing cells. Thus, they can be useful
in gene therapy, particularly for treatment of hyperproliferative
disorders. In this embodiment, genetic material or one or more
delivery vehicles containing genetic material useful in treating a
hyperproliferative disorder can be conjugated to one or more cMet
binding moieties of the invention and administered to a patient.
The genetic material can include nucleic acids, such as RNA or DNA,
of either natural or synthetic origin, including recombinant RNA
and DNA and antisense RNA and DNA. Types of genetic material that
can be used include, for example, genes carried on expression
vectors such as plasmids, phagemids, cosmids, yeast artificial
chromosomes (YACs) and defective or "helper" viruses, antigene
nucleic acids, both single and double stranded RNA and DNA and
analogs thereof, such as phosphorothioate and phosphorodithioate
oligodeoxynucleotides. Additionally, the genetic material can be
combined, for example, with lipids, proteins or other polymers.
Delivery vehicles for genetic material can include, for example, a
virus particle, a retroviral or other gene therapy vector, a
liposome, a complex of lipids (especially cationic lipids) and
genetic material, a complex of dextran derivatives and genetic
material, etc.
[0213] In a preferred embodiment the constructs of the invention
are utilized in gene therapy for treatment of hyperproliferative
disorders. In this embodiment, genetic material, or one or more
delivery vehicles containing genetic material, e.g., useful in
treating a hyperproliferative disorder, can be conjugated to one or
more cMet binding polypeptides or multimeric polypeptide constructs
of the invention and administered to a patient.
[0214] Constructs including genetic material and the cMet-binding
moieties of the invention can be used, in particular, to
selectively introduce genes into proliferating cancer cells (e.g.,
epithelial cells), which can be useful to treat cancer.
[0215] Therapeutic agents and the cMet binding moieties of the
invention can be linked or fused in known ways, optionally using
the same type of linkers discussed elsewhere in this application.
Preferred linkers will be substituted or unsubstituted alkyl
chains, amino acid chains, polyethylene glycol chains, and other
simple polymeric linkers known in the art. More preferably, if the
therapeutic agent is itself a protein, for which the encoding DNA
sequence is known, the therapeutic protein and cMet binding
polypeptide can be coexpressed from the same synthetic gene,
created using recombinant DNA techniques, as described above. The
coding sequence for the cMet binding polypeptide can be fused in
frame with that of the therapeutic protein, such that the peptide
is expressed at the amino- or carboxy-terminus of the therapeutic
protein, or at a place between the termini, if it is determined
that such placement would not destroy the required biological
function of either the therapeutic protein or the cMet binding
polypeptide. A particular advantage of this general approach is
that concatamerization of multiple, tandemly arranged cMet binding
polypeptides is possible, thereby increasing the number and
concentration of cMet binding sites associated with each
therapeutic protein. In this manner cMet binding avidity is
increased, which would be expected to improve the efficacy of the
recombinant therapeutic fusion protein.
[0216] Additionally, constructs including cMet binding polypeptides
of the present invention can themselves be used as therapeutics to
treat a number of diseases associated with cMet activity. For
example, where binding of a protein or other molecule (e.g., a
growth factor, hormone etc.) is necessary for or contributes to a
disease process and a binding moiety inhibits such binding,
constructs including such binding moieties could be useful as
therapeutics. Similarly, where binding of a binding moiety itself
inhibits a disease process, constructs containing such binding
moieties also could be useful as therapeutics.
[0217] The binding of HGF to cMet results in the activation of
numerous intracellular signal transduction pathways leading to
hyperproliferation of various cells. As such, in one embodiment,
constructs including cMet binding polypeptides that inhibit the
binding of HGF to cMet (or otherwise inhibit activation of cMet)
can be used as anti-neoplastic agents. In addition, as binding of
HGF and activation of cMet is implicated in angiogenic activity, in
another embodiment, constructs including cMet binding polypeptides
that inhibit the binding of HGF to cMet, or otherwise inhibit
activation of cMet, can be used as anti-angiogenic agents. Certain
constructs of the invention including monomers, multimers and
heteromultimers that inhibit activation of cMet are also discussed
in the Examples, and include, for example, SEQ ID NO:365 (FIG. 10).
The binding polypeptides and constructs thereof of the present
invention are useful as therapeutic agents for treating conditions
that involve endothelial and/or epithelial cells expressing cMet.
Because an important function of endothelium is angiogenesis, or
the formation of blood vessels, the polypeptides and constructs
thereof are particularly useful for treating conditions that
involve angiogenesis and/or hyperproliferation. Conditions that
involve angiogenesis include, for example, solid tumors, tumor
metastases and benign tumors. Tumors caused by cMet activation or
through angiogenesis are well known in the art and include, for
example, breast, thyroid, glioblastoma, prostate, malignant
mesothelioma, colorectal, hepatocellular, hepatobiliary, renal,
osteosarcoma and cervical. Additional tumors and related disorders
are listed in Table I of U.S. Pat. No. 6,025,331, issued Feb. 15,
2000 to Moses, et al., the teachings of which are incorporated
herein by reference. Benign tumors include, for example,
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic granulomas. Other relevant diseases that involve
angiogenesis and/or hyperproliferation include for example,
rheumatoid arthritis, psoriasis, and ocular diseases, such as
diabetic retinopathy, retinopathy of prematurity, macular
degeneration, corneal graft rejection, neovascular glaucoma,
retrolental fibroplasia, rebeosis, Osler-Webber Syndrome,
myocardial angiogenesis, plaque neovascularization, telangiectasia,
hemophiliac joints, angiofibroma and wound granulation. Other
relevant diseases or conditions that involve blood vessel growth
include intestinal adhesions, atherosclerosis, scleroderma, and
hypertropic scars, and ulcers. Furthermore, the binding
polypeptides and constructs thereof of the present invention can be
used to reduce or prevent uterine neovascularization required for
embryo implantation, for example, as a birth control agent.
[0218] The binding polypeptides, multimeric polypeptide constructs
and constructs conjugates thereof can be administered to an
individual over a suitable time course depending on the nature of
the condition and the desired outcome. They binding, polypeptides
and constructs thereof can be administered prophylactically, e.g.,
before the condition is diagnosed or to an individual predisposed
to a condition. The binding polypeptides multimeric polypeptide
constructs and conjugates and constructs thereof can be
administered while the individual exhibits symptoms of the
condition or after the symptoms have passed or otherwise been
relieved (such as after removal of a tumor). In addition, they
binding polypeptides and constructs thereof of the present
invention can be administered a part of a maintenance regimen, for
example to prevent or lessen the recurrence or the symptoms or
condition. As described below, the binding polypeptides multimeric
polypeptide constructs and conjugates and constructs thereof of the
present invention can be administered systemically or locally.
[0219] The quantity of material administered will depend on the
seriousness of the condition. For example, for treatment of a
hyperproliferative disorder, e.g., in the case of neoplastic tumor
growth, the position and size of the tumor will affect the quantity
of material to be administered. The precise dose to be employed and
mode of administration must per force, in view of the nature of the
complaint, be decided according to the circumstances by the
physician supervising treatment. In general, dosages of the agent
conjugate polypeptides, multimeric polypeptide constructs and
conjugates of the present invention will follow the dosages that
are routine for the therapeutic agent alone, although the improved
affinity of a binding polypeptide or multimeric polypeptide
construct of the invention for its target can allow for a decrease
in the standard dosage.
[0220] Such conjugate pharmaceutical compositions are preferably
formulated for parenteral administration, and most preferably for
intravenous or intra-arterial administration. Generally, and
particularly when administration is intravenous or intra-arterial,
pharmaceutical compositions can be given as a bolus, as two or more
doses separated in time, or as a constant or non-linear flow
infusion.
[0221] As used herein the term "therapeutic" includes at least
partial alleviation of symptoms of a given condition. The binding
polypeptides, multimeric constructs and constructs conjugates
thereof of the present invention do not have to produce a complete
alleviation of symptoms to be useful. For example, treatment of an
individual can result in a decrease in the size of a tumor or
diseased area, or prevention of an increase in size of the tumor or
diseased area. Treatment also can prevent or lessen the number or
size of metastatic outgrowths of the main tumor(s).
[0222] Symptoms that can be alleviated include physiological
characteristics such as cMet activity. The binding polypeptides
multimeric polypeptide constructs and conjugates and constructs
thereof of the present invention can inhibit activity of cMet and
its homologs by binding to cMet and inhibiting its activity or by
binding to cMet and inhibiting HGF from activating this receptor.
Such inhibition can be detected, for example, by measuring the
phosphorylation state of the receptor in the presence of or after
treatment with the binding polypeptides or constructs thereof.
Based on the teachings provided herein, one of ordinary skill in
the art would know how and be able to administer a suitable dose of
binding polypeptide, multimeric polypeptide constructs and
conjugates or construct thereof as provided herein, and measure the
effect of treatment on the parameter of interest. For example, the
size of the area of interest (e.g., the tumor or lesion) can be
measured before and after treatment. Cells or cMet itself can be
isolated from the sample and used in assays described herein.
[0223] The dosage of the polypeptides multimeric polypeptide
constructs and conjugates and constructs thereof can depend on the
age, sex, health, and weight of the individual, as well as the
nature of the condition and overall treatment regimen. The
biological effects of the polypeptides multimeric polypeptide
constructs and conjugates and constructs thereof are described
herein. Therefore, based on the biological effects of the binding
polypeptides multimeric polypeptide constructs and conjugates and
constructs provided herein, and the desired outcome of treatment,
the preferred dosage is determinable by one of ordinary skill in
the art through routine optimization procedures. Typically, the
daily regimen is in the range of about 0.1 mg/kg to about 1
mg/kg.
[0224] The binding polypeptides moieties and constructs conjugates
thereof provided herein can be administered as the sole active
ingredient, optionally together with a pharmaceutically acceptable
excipient, or can be administered together (e.g., simultaneously or
sequentially) with other binding polypeptides and constructs
thereof, other therapeutic agents, or combination thereof. In
addition, the binding polypeptides moieties and conjugate
constructs thereof can be conjugated to therapeutic agents, for
example, to improve specificity, residence time in the body, or
therapeutic effect. Such othertherapeutic agents include, for
example, other anti-proliferative compounds, and tumoricidal
compounds. The therapeutic agent also can include antibodies.
Furthermore, the binding polypeptide multimeric polypeptide
constructs and constructs thereof of the present invention can be
used as a cancer cell homing device. Therefore, they binding
polypeptide or constructs thereof can may be conjugated to nucleic
acid encoding, for example, a therapeutic polypeptide, in order to
target the nucleic acid to stromal cells. Once exposed to the
nucleic acid conjugated binding polypeptide moiety or conjugate
thereof, the stromal cells can internalize and express the
conjugated nucleic acid, thereby delivering the therapeutic peptide
to the target cells.
[0225] The binding polypeptides, multimeric polypeptide constructs
and conjugates and constructs thereof can be administered locally
or systemically by any suitable route. Suitable routes of
administration include, but are not limited to, topical
application, transdermal, parenteral, gastrointestinal,
intravaginal, and transalveolar. Compositions for the desired route
of administration can be prepared by any of the methods well known
in the pharmaceutical arts, for example, as described in Remington:
The Science and Practice of Pharmacy, 20th ed., Lippincott,
Williams and Wilkins, 2000.
[0226] For topical application, the binding polypeptides,
multimeric polypeptide constructs and conjugates thereof can be
suspended, for example, in a cream, gel or rinse that allows the
polypeptides or constructs to penetrate the skin and enter the
blood stream, for systemic delivery, or contact the area of
interest, for localized delivery. Compositions suitable for topical
application include any pharmaceutically acceptable base in which
the polypeptides or constructs are at least minimally soluble.
[0227] For transdermal administration, the polypeptides, multimeric
polypeptide constructs and conjugates thereof can be applied in
pharmaceutically acceptable suspension together with a suitable
transdermal device or "patch". Examples of suitable transdermal
devices for administration of the polypeptides or constructs of the
present invention are described, for example, in U.S. Pat. No.
6,165,458, issued Dec. 26, 2000 to Foldvari et al., and U.S. Pat.
No. 6,274,166B1, issued Aug. 4, 2001 to Sintov et al., the
teachings of which are incorporated herein by reference.
[0228] For parenteral administration, the polypeptides, multimeric
polypeptide constructs and conjugates thereof can be injected
intravenously, intramuscularly, intraperitoneally, or
subcutaneously. Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous buffer.
Other pharmaceutically acceptable carriers include, but are not
limited to, sterile water, saline solution, and buffered saline
(including buffers like phosphate or acetate), alcohol, vegetable
oils, polyethylene glycols, gelatin, lactose, amylose, magnesium
stearate, talc, silicic acid, paraffin, etc. Where necessary, the
composition also can include a solubilizing agent and a local
anaesthetic such as lidocaine to ease pain at the site of the
injection, preservatives, stabilizers, wetting agents, emulsifiers,
salts, lubricants, etc. as long as they do not react deleteriously
with the active compounds. Similarly, the composition may comprise
conventional excipients, i.e. pharmaceutically acceptable organic
or inorganic carrier substances suitable for parenteral, enteral or
intranasal application which do not deleteriously react with the
active compounds. Generally, the ingredients will be supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent in activity units. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade "water for injection" or saline. Where the composition is to
be administered by injection, an ampoule of sterile water for
injection or saline can be provided so that the ingredients can be
mixed prior to administration.
[0229] For gastrointestinal and intravaginal administration, the
polypeptides, multimeric polypeptide constructs and conjugates
thereof can be incorporated into pharmaceutically acceptable
powders, pills or liquids, and suppositories for rectal or vaginal
administration.
[0230] For transalveolar, buccal or pulmonary administration, the
polypeptides, multimeric polypeptide constructs and conjugates
thereof can be suspended in a pharmaceutically acceptable excipient
suitable for aerosolization and inhalation or as a mouthwash.
Devices suitable for transalveolar administration such as atomizers
and vaporizers also are included within the scope of the invention.
Suitable formulations for aerosol delivery of polypeptides, etc.
using buccal or pulmonary routes can be found, for example in U.S.
Pat. No. 6,312,665B1, issued Nov. 6, 2001 to Pankaj Modi, the
teachings of which are incorporated herein by reference.
[0231] In addition, the polypeptides, multimeric polypeptide
constructs and conjugates thereof of the present invention can be
administered nasally or ocularly, where the polypeptide or
construct is suspended in a liquid pharmaceutically acceptable
agent suitable for drop-wise dosing.
[0232] The polypeptides, multimeric polypeptide constructs and
conjugates thereof of the present invention can be administered
such that the polypeptide, etc. is released in the individual over
an extended period of time (sustained or controlled release). For
example, the polypeptide, multimeric polypeptide constructs and
conjugates thereof can be formulated into a composition such that a
single administration provides delivery of the polypeptide, etc.
for at least one week, or over the period of a year or more.
Controlled release systems include monolithic or reservoir-type
microcapsules, depot implants, osmotic pumps, vesicles, micelles,
liposomes, transdermal patches and iontophoretic devices. In one
embodiment, the polypeptides, multimeric polypeptide constructs and
conjugates thereof of the present invention are encapsulated or
admixed in a slowly degrading, non-toxic polymer. Additional
formulations suitable for controlled release of the polypeptides,
multimeric polypeptide constructs and conjugates thereof provided
herein are described in U.S. Pat. No. 4,391,797, issued Jul. 5,
1983, to Folkman et al., the teachings of which are incorporated
herein by reference.
[0233] Another suitable method for delivering the polypeptides of
the present to an individual is via in viva production of the
polypeptide. A gene encoding the polypeptide can be administered to
the individual such that the encoded polypeptide is expressed. The
gene can be transiently expressed. In a particular embodiment, the
gene encoding the polypeptide is transfected into cells that have
been obtained from the patient, a method referred to as ex vivo
gene therapy. Cells expressing the polypeptide are then returned to
the patient's body. Methods of ex vivo gene therapy are well known
in the art and are described, for example, in U.S. Pat. No.
4,391,797, issued Mar. 21, 1998 to Anderson et al., the teachings
of which are incorporated herein by reference.
[0234] Isolation of cMet binding moieties polypeptides and
preparation and use of cMet binding moieties and conjugates thereof
in accordance with this invention will be further illustrated in
the following examples. The specific parameters included in the
following examples are intended to illustrate the practice of the
invention, and they are not presented to in any way limit the scope
of the invention.
EXAMPLES
Example 1
Method for Identification of cMet-Binding Polypeptides
[0235] A four-pronged selection strategy using a variety of
peptide-displaying phage libraries was utilized to screen for
cMet-binding polypeptides. Both the extracellular domain of the
cMet receptor (expressed as an Fc-fusion protein) and the
colorectal cancer cell line, DLD-1, which express high levels of
cMet on their cell surface, were used as tools for the
selections.
[0236] Briefly, the selections involved either using the soluble
cMet-Fc-fusion protein or DLD-1 cells as the target. Specific
elutions with HGF (first for 1 hour and then overnight to identify
both low and high affinity cMet binders) were performed.
Additionally, while using the soluble cMet receptor, all
peptide-displaying phage that remained bound to the receptor were
harvested to identify peptides that did not bind to the ligand
binding site, but could nevertheless be potentially developed into
imaging agents. FIG. 9 illustrates the selection strategy that was
employed. Briefly, 21 different selection campaign/elution
combinations were performed with each library pool. An additional
10 selection campaigns representing rounds 3 and 4 using the
soluble Met-Fc fusion protein were also performed. HGF elutions
were at a concentration of 100 ng/mL.
Example 2
Determination of Peptide-Display Phage Binding to Soluble cMet-Fc
Fusion Protein "Protein Phage Elisas"
[0237] Protein phage ELISAs using peptide-displaying phage isolates
from the various selection campaigns were performed to determine
specificity of the peptides for cMet versus an unrelated Fc-fusion
protein (TRAIL-Fe). Briefly, 384-well plates were coated overnight
at 4 C with 0.5 .mu.g/mL of cMet-Fc fusion protein or TRAIL-Fc
fusion protein (background). The plates were blocked for 2 hours 37
C with 3% (w/v) BSA in PBS containing 0.05% (v/v) Tween-20 (PBST).
The plates were washed with PBST and 100 .mu.L of
peptide-displaying phage were added to each well. The plates were
incubated for 2 hours at room temperature and washed with PBST.
cMet-binding peptide-displaying phage were detected using an
HRP-conjugated anti-M13 antibody.
[0238] The peptide-displaying phage that demonstrated a>3-fold
binding to cMet-Fc fusion protein versus TRAIL-Fe fusion protein
are herein referred to as "positive hits". The positive hits
identified in the above screen were subjected to DNA sequencing.
From subsequent sequence analysis, 187 unique peptide sequences
were identified. The corresponding amino acid sequences of the
cMet-binding phage-displayed peptides are listed in Table 1 (SEQ ID
NO: 001-101, 365-387, 390-404, 449-496).
Example 3
Determination of cMet Binding in a Cellular Model
[0239] Whole cell ELISAs were performed to assess whether the
positive hits demonstrated specific binding to cell
surface-expressed human cMet.
[0240] Whole cell ELISAs were performed using 3T3 cells that
over-express human cMet. 3T3 cells that do not express cMet
("non-expressing cells") were used as a control cell line. Briefly,
96-well plates were seeded with 10.sup.5 cells per well. The plates
were centrifuged for 5 minutes at 1600 rpm to pellet the cells. The
resulting cell layer was fixed with 0.1% (v/v) glutaraldehyde for
12 minutes at 37 C. The cells were washed with PBS and subsequently
blocked with 3% BSA in PBST for 1 hour at 37 C. Peptide-displaying
phage also were blocked in the above solution for 1 hour at 37 C.
100 .mu.L of blocked phage was then added to each well and the
plates were incubated for 1 hour at room temperature. The plates
were washed with PBST. cMet-binding peptide-displaying phage were
detected using an HRP-conjugated anti-M13 antibody.
Example 4
HGF Competition Protein ELISAs
[0241] HGF competition protein ELISAs were performed in an attempt
to determine whether any of the cMet-binding peptides compete with
HGF for a similar binding site on cMet. This competition ELISA
identifies peptides that serve as "HGF antagonistic peptides",
peptides that block HGF-mediated signaling events (e.g.,
proliferation). These assays were conducted using the
peptide-displaying phage discovered from the initial selection and
screening campaigns using the first generation peptide libraries.
Briefly, 96-well plates were coated overnight at 4 C with 0.5
.mu.g/mL of cMet-Fc fusion protein or TRAIL-Fc fusion protein
(background). The plates were blocked for 2 hours at 37 C with 3%
BSA in PBST. The plates were washed with PBST, and 100 .mu.L of HGF
(either at 100 ng/mL or 500 ng/mL in PBST) was added to each well.
The plates were incubated for 30 minutes at room temperature after
which the plates were washed with PBST and 70 .mu.L of HGF (143
ng/mL or 714 ng/mL) or 70 mL of PBST was added to the respective
wells. This was followed by an addition of 30 .mu.L of
peptide-displaying phage overnight culture to each well. The plates
were incubated for 2 hours at room temperature, washed with PBST
and cMet-binding peptide-displaying phage was detected using an
HRP-conjugated anti-M13 antibody.
[0242] Data for the protein ELISAs, whole cell ELISAs and the HGF
competition experiments is presented in Table 7.
Example 5
Peptide Synthesis and Fluorescein Labeling
[0243] A select number of cMet-binding peptides corresponding to
positive phage isolates were synthesized on a solid phase matrix
using 9-fluorenylmethoxycarbonyl protocols. These peptides were
purified with reverse phase chromatography. Peptide masses were
confirmed by electrospray mass spectrometry, and peptides were
quantified by measuring absorbance at 280 nm. For synthesis, two
N-terminal and two C-terminal amino acids from the phage vector
sequence from which the peptide was excised were retained, and a
linker, e.g., -Gly-Gly-Gly-Lys-NH.sub.2 (SEQ ID NO:513) was added
to the C-terminus of each peptide. Each peptide was N-terminally
acetylated. Selected lysine residues were protected with
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde)
where appropriate. The protecting group allows for selective
coupling to the C-terminal lysine, is not removed during peptide
cleavage, but can be removed after coupling with 2% hydrazine in
DMF or 0.5 M hydroxylamine, pH 8, in water.
[0244] Each peptide was labeled with fluorescein on the C-terminal
lysine using fluorescein (N-hydroxysuccinimide ester derivative) or
fluorescein isothiocyanate (FITC) in DMF with 2%
diisopropylethylamine (DIPEA). In the case where the peptide
contained an ivDde protected lysine, the reaction was quenched by
the addition of 2% hydrazine, which reacts with all free
NHS-fluorescein and removes the internal protecting group. For all
other peptides, the reaction was quenched by the addition of an
equal volume of 0.5M hydroxylamine, pH 8. The quenched reactions
were then diluted with water to less than 10% DMF and then purified
using C18 reverse phase chromatography. The peptides were verified
by analyzing them for expected mass using an LC-MS system (HP1100
HPLC with in-line SCIEX AP150 single quadrapole mass spectrometer),
and the purity of the peptides was determined.
Example 6
Fluorescence Anisotropy Measurements
[0245] Fluorescence anisotropy measurements were performed in
384-well microplates in a volume of 10 .mu.L in binding buffer
(PBS, 0.01% Tween-20, pH 7.5) using a Tecan Polarion fluorescence
polarization plate reader (Caracas, Venezuela). The concentration
of fluorescein-labeled peptide was held constant (20 nM) and the
concentration of cMet-Fc fusion protein (or similar target) was
varied. Binding mixtures were equilibrated for 10 minutes in the
microplate at 30 C before measurement. The observed change in
anisotropy was fit to the equation below via nonlinear regression
to obtain the apparent K.sub.D. This equation (1) assumes that the
synthetic peptide and cMet form a reversible complex in solution
with 1:1 stoichiometry.
r obs = r free + ( r bound - r free ) ( K D + cMet + P ) - ( K D +
cMet + P ) 2 - 4 cMet P 2 P ##EQU00001##
where r.sub.obs is the observed anisotropy, r.sub.free is the
anisotropy of the free peptide, r.sub.bound is the anisotropy of
the bound peptide, K.sub.D is the apparent dissociation constant,
cMet is the total cMet concentration, and P is the total
fluorescein-labeled peptide concentration. K.sub.D was calculated
in a direct binding assay (K.sub.D,B) and therefore these values
represent cMet binding to the fluorescein labeled peptide.
Example 7
Peptide Competition Fluorescence Polarization Assays
[0246] Peptide competition fluorescence polarization assays were
performed to determine which peptides compete with each other for
binding to cMet. This would identify potential heteromeric peptide
complexes that exhibit higher affinity for the cMet receptor than
an individual peptide alone.
[0247] Briefly, cross competition of cMet-binding peptides was
performed on a Cartesian liquid handler (Irvine, Calif.) in a 3
.mu.L total reaction volume. Flourescein-labeled peptides were
diluted to a final concentration of 20 nM and unlabeled competitor
peptides were diluted to a final concentration of 10 .mu.M. cMet-Fc
fusion protein was diluted to the K.sub.D for each
fluorescein-labeled peptide in the reaction. Binding mixtures were
equilibrated for 10 minutes on the microplate at 30 C before
measuring any changes in anisotropy. From these studies, three
pairs of cMet-binding peptides were identified as being
non-competitors and represent ideal candidates for heteromeric
cMet-binding peptide complexes (see Table 9).
Example 8
General Procedure for Preparation of Heteromeric cMet-Binding
Peptide Complexes
[0248] Each of the dimers consists of a Tc-chelating 6-PnAO ligand
bearing sequence (generally referred to as A) and a spacer
functionalized (spacer=JJ; J=8-Amino-3,6-dioxaoctanoic acid)
portion (generically referred to as B). Compound B was treated with
a 10-fold excess of glutaric acid his NHS ester (Tyger Scientific,
Princeton, N.J.) and .about.20-fold excess of diisopropylethylamine
at ambient temperature in DMF for 30 minutes. The reaction mixture
was diluted with ether (15-fold by volume) which led to the
precipitation of the mono-NHS ester of the glutarylated peptide.
The ether was decanted and the solid washed thrice more with ether,
which removed any traces of unreacted glutaric acid bis NHS ester.
The resulting solid was resuspended in dry DMF and the compound A
(1 equiv) was added followed by diisopropylethylamine (20 equiv)
and the mixture was stirred for 24 hours at ambient temperature.
The mixture was diluted with water (50-fold) and the mixture was
directly loaded onto a reverse-phase HPLC column, which was eluted
with a gradient of acetonitrile (0.1% TFA) into water (0.1% TFA).
Fractions containing the desired product were combined and
lyophilized to provide the desired materials.
Specific Example
Preparation of Heterodimeric cMet-Binding Peptides Complexes
1) Preparation of a PnAOG-Glut Modified SEQ ID NO:514 Peptide (A
Type A Compound)
[0249] To a solution of 6-Glutaryl-PnAO (40 mg, 0.1 mmol) in dry
DMF (0.2 mL) was added N-hydroxysuccinimide (NHS, 14 mg, 0.12 mmol)
and diisopropylcarbodiimide (DIC, 15 mg, 0.12 mmol) and stirred for
4 h at room temperature. Ether:hexane (5 mL, 1:1) was added to the
reaction mixture. The mixture was stirred and the supernatant
solution was removed by decantation, leaving behind the paste in
the flask. The paste was washed with ether:hexane (1:1) (3.times.5
mL) and dissolved in dry DMF (0.2 mL). To this solution were added
the K-(ivDde)-modified SEQ ID NO:518 (50 mg, 0.017 mmol) and
diisopropylethylamine (DIEA, 10 mg, 0.08 mmol) and the resultant
mixture was stirred for 18 hours. Hydrazine (10 .mu.L) was added
and the solution was stirred for 30 min. The reaction mixture was
diluted with water (20 mL), loaded onto a reversed-phase (C18) HPLC
column, and eluted with water (0.1% TFA)-acetonitrile (0.1% TFA)
system. Fractions containing the required product (>95% purity)
were collected and freeze-dried to provide SEQ ID
NO:518-(6-PnAO-Glut)) (see Scheme 5 as shown in FIG. 11) as a
colorless fluffy solid. The yield was 25.1 mg (47.4%).
2) Preparation of Dimer Containing SEQ ID NO:514 Linked to SEQ ID
NO:515
[0250] To a solution of the peptide containing SEQ ID NO:515 (a
type B compound) (10 mg, 0.0034 mmol) and diisopropylethylamine (10
mg, 0.08 mmol) in dry DMF (0.2 mL) was added disuccinimidyl
glutarate (10 mg, 0.031 mmol) and stirred at room temperature for
30 min. The reaction mixture was diluted with ether (3 mL) and
stirred. The supernatant was decanted, leaving behind the
semi-solid in the flask. This process of washing the reaction
product was repeated with ether (3.times.5 mL). The semi-solid thus
obtained was dissolved in dry DMF (0.2 mL) and the peptide SEQ ID
NO:514-(6-PnAO-Glut)) (10 mg, 0.0032 mmol) and
diisopropylethylamine (10 mg, 0.08 mmol) were added and stirred the
reaction mixture for 24 h at room temperature. The reaction mixture
was diluted with water (10 mL), loaded onto a reversed-phase (C18)
HPLC column, and eluted with water (0.1% TFA)-acetonitrile (0.1%
TFA) system. Fractions containing the required product (>95%
purity) were collected and freeze-dried to provide the heterodimer
having SEQ ID NO:514 linked to SEQ ID NO:515 via a 6-PnAO-Glut
linkage (see Scheme 6 as shown in FIG. 12) as a colorless fluffy
solid. Yield: 6.7 mg (33%). The structures for this and other
heterodimers are shown in FIGS. 13A-13C.
Example 9
Cellular Proliferation Assay
[0251] Cellular proliferation assays were performed to identify
cMet-binding peptides that antagonize HGF-stimulated proliferation.
These in vitro studies utilized a leomyosarcoma cell line,
SK-LMS-1, in which cells proliferate in response to HGF. SK-LMS-1
cells were seeded on 96-well plates at a density of 2000
cells/well. After a 24 hour incubation at 37 C, the cells were
starved in culture media containing 0.1% BSA instead of 10% fetal
bovine serum for 36 hours at 37 C. Fresh starvation media with or
without a cMet-binding peptide (10 .mu.M) was added to the
respective wells and the cells were incubated for 2 hours at 37 C.
DMF was used as the control vehicle and did not receive a
cMet-binding peptide. HGF was then added at a concentration of
either 50 ng/mL or 100 ng/mL and the cells were incubated for an
additional 12 hours at 37 C. Proliferation was assessed by
measuring the incorporation of BrdU (Calbiochem, San Diego, Calif.)
as described by the manufacturer. Results are shown for SEQ ID
NO:365 (FIG. 10).
Example 10
Design of a Second Generation cMet-Binding Peptide Library
[0252] Initial selection from linear and cyclic peptide libraries
identified a number of positive hits for cMet. The TN9 hits
contained a highly conserved motif (CxGpPxFxC, SEQ ID NO:512, the
`p` is less strongly selected than are the uppercase amino acids).
A library was constructed having both cyclic and linear members and
was built in phage having a gene III stump display.
TABLE-US-00001 TABLE 1 TN9 and linear components in the second
generation library: Libraries of TN9s for cMet (cMet TN9 2nd lib
#1) Component 1: TN9 consensus with 3 AA left extension S M G S E T
R P T ctcagcagtcactgtct tCC ATG Ggt tct gaa act cgc cct aca
NcoI..... e a g s w h C s G P P t F e C w w y jej jqz jjz ejz zjj
qez tgt ejz ggt cct cct eqj ttc jej tgc zjj zjj zez G T E P T E A S
gga acg gag ccg act gaa GCT AGC Gtga ctctgacagtctctgt NheI... (SEQ
ID NO: 518) cMet TN9 2nd lib #2: TN9 consensus with 3 AA right
extension. S M G S E T R P T ctcagcagtcactgtct tcc atg ggt tct gAa
act cgc cct AcA NcoI..... E A G s w h C s G P P t F e C w w y GAG
GCT GGT ejz zjj qez tgt ejz ggt cct cct eqj ttc jej tgc zjj zjj zez
g t e P T E R P S S S jjz eqj jej ccg AcT gAA cgt cct agt GCT AGC
Gtga ctctgacagtctctgt NheI... (SEQ ID NO: 519) cMet TN9 2nd lib #3
SIQCKGPPWFSCAMY (SEQ ID NO: 537) with 3 AA extension on left S M G
S E T R P T ctcagcagtcactgtct tcc atg ggt tct gaa act cgc cct AcA
NcoI..... e a g s i q C k G P P w F s C a m y jej jqz jjz ejz ezz
qej tgc eej ggt cct cct zjj ttc ejz tgt jqj ezj zez G T E P T E A S
A ggA Acg gAg ccg AcT gAA GCT AGC Gtga ctctgacagtctctgt NheI...
(SEQ ID NO: 520) cMet TN9 2nd lib #4 SIQCKGPPWFSCAMY (SEQ ID NO:
537) with 3 AA extension on right S M G S E T R P T
ctcagcagtcactgtct tcc atg ggt tct gaa act cgc cct AcA NcoI..... E A
G s i q C k G P P w F s C a m y gag gcc ggt ejz ezz qej tgc eej ggt
cct cct zjj ttc ejz tgt jqj ezj zez g t e P T E R P S S A jjz eqj
jej ccg AcT gAA cgt cct agt GCT AGC Gtga ctctgacagtctctgt NheI...
(SEQ ID NO: 521) cMet TN9 5th lib 330-F05 YYGCKGPPTFECQWM (SEQ ID
NO: 531)with 3 AA extension on right three peptides have the core
sequence CKGPPTFEC (SEQ ID NO: 653) S M G S E T R P T
ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cct AcA NcoI..... E A
G y y g C k G P P t F e C q w m GAG GCT GGT zez zez jjz tgc eej ggt
cct cct eqz ttc jej tgt qee zjj ezj g t e P T E R P S S S jjz eqj
jej ccg AcT gAA cgt cct agt GCT AGC Gtga ctctgacagtctctgt NheI...
(SEQ ID NO: 522) cMet TN9 6th lib: 550-G12 AFFCSGPPTFMCSLY (SEQ ID
NO: 536) with 3 AA extension on right two peptides have the core
sequence CSGPPTFMC (SEQ ID NO: 654) S M G S E T R P T
ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cct AcA NcoI..... E A
G a f f C s G p P t F m C s l y GAG GCT GGT jqz zzq zzq tgt zqz ggt
qqj cct eqz ttc ezj tgc ejq qzz zez g t e P T E R P S S S jjz eqj
jej ccg AcT gAA cgt cct agt GCT AGC Gtga ctctgacagtctctgt NheI...
(SEQ ID NO: 523) cMet TN9 7th lib, three AA to left and let first P
of gPP vary. S M G S E T R P T ctcagcagtcactgtct tCC ATG Ggt tct
gaa act cgc cct aca NcoI..... e a g q f k C a G p P s F a C w m t
jej jqz jjz qej zzq eej tgt jqz ggt qqj ccg ejz ttc jqq tgt zjj ezj
eqq G T E P T E A S gga acg gag ccg act gaa GCT AGC Gtga
ctctgacagtctctgt NheI... (SEQ ID NO: 524) E = 0.64A + 0.12C + 0.12G
+ 0.12T Q = 0.12A + 0.64C + 0.12G + 0.12T J = 0.12A + 0.12C + 0.64G
+ 0.12T Z = 0.12A + 0.12C + 0.12G + 0.64T Note: (0.64).sup.36 =
1.E-7 (0.64).sup.39 = 2.5E-8
Example 11
Analysis of 94-E08 and Other Linear Peptides Selected for Binding
cMet
[0253] The linear isolate 94-E08 (SEQ ID NO:454) has high affinity
for cMet yet there were few other peptides isolated that had any
homology to 94-E08 and those that did have very limited similarity
over very short regions. Thus, three variable oligonucleotides
based on 94-E08 were made: (1) vary the first 13 codons, keeping
the last 7 constant; (2) vary 13 of the first 18, keeping 5 that
showed some similarity to other isolates fixed; and (3) vary the
last 13 codons, keeping the first 5 fixed, see table 4 below.
TABLE-US-00002 TABLE 4 Component #8 with variation in the first 13
positions (SEQ ID NO: 594). S M G S E 5'-tcactgtct tCC ATG Ggt tct
gaa- Scab.......| NcoI | y d t w v f q f i h zez jez eqz zjj jzj
zzz qej zzz ezz qez- e v p G E L V A M Q jej jzj qqj ggt gag ctg
gtt gct atg cag- G G S G T E A S ggt ggt agt ggt act gaa GCT AGC
Gtga ctctgac-3' | NheI |Scab........ Component#9 Fix five AAs and
extend variegation to position 18 (SEQ ID NO: 595). S M G S E
5'-tcactgtct tCC ATG Ggt tct gaa- Scab.......| NcoI | y D T w v F q
f i h zez gat act zjj jzj ttt qej zzz ezz qez- E V p g e l v a M Q
gag gtt qqj jjz jej qzj jzj jqj atg caa! G G S G T E A S ggt ggt
agt ggt act gaa GCT AGC Gtga ctctgac-3' | NheI |Scab........
Component#10 Fix first seven AAs and vary last 13 (SEQ ID NO: 596).
S M G S E 5'-tcactgtct tCC ATG Ggt tct gaa- Scab.......| NcoI | Y D
T W V F Q F i h tat gat act tgg gtt ttt caa ttt ezz qez- e v p g e
l v a m q jej jzz qqj jjz jej qzj jzj jqj ezj qzz! G G S G T E A S
ggt ggt agt ggt act gaa GCT AGC Gtga ctctgac-3' | NheI
|Scab........
Oligonucleotide design for construction of the second generation
peptide library (SEQ ID NOS:597-646; N.B. oligonucleotides marked
"[RC]" consist of the reverse complement of the sequence
shown):
TABLE-US-00003 vg#1 NcoI.... (CM2_ZTPSAlt) 5'-tcactgtct tcc atg ggt
tct gAa- 3' (CM2_TPLalt) 5'-tcactgtct tcc atg ggt tct gAa act cgc
cct AcA-3' (CM2_ZTPS) 5'-ctcagcagtcactgtct tcc at-3' (CM2_TPLong)
5'-ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cct AcA-3'
(CM2_V1) 5'-tct gAa act cgc cct AcA- jej jqz jjz ejz zjj qez tgt
ejz ggt cct cct eqj ttc jej tgc zjj zjj zez- gga acg gag ccg act
gaa gct-3' (CM2_BPL1) [RC] 5'-gga acg gag ccg act gaa GCT AGC Gtga
ctctgacagtctctgt-3' (CM2_XBPS) [RC] 5'-CA Gtga ctctgacagtctctgt-3'
(BPL1_CM2) [RC] 5'-gga acg gag ccg act gaa GCT AGC Gtga ctctgac-3'
(XBPS_CM2) [RC] 5'-act gaa GCT AGC Gtga ctctgac-3' vg#2 NheI...
(CM2_ZTPS) 5'-ctcagcagtcactgtct tcc at-3' (CM2_TPLong)
5'-ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cct AcA-3'
(CM2_V2) 5'-tct gAa act cgc cct AcA- GAG GCT GGT ejz zjj qez tgt
ejz ggt cct cct eqj ttc jej tgc zjj zjj zez- jjz eqj jej ccg AcT
gAA cgt cct agt g-3' (CM2_2BPL) [RC] 5'-ccg AcT gAA cgt cct agt GCT
AGC Gtga ctctgacagtctctgt-3' (CM2_XBPS) [RC] 5'-CA Gtga
ctctgacagtctctgt-3' (BPL2_CM2) [RC] 5'-ccg AcT gAA cgt cct agt GCT
AGC Gtga ctctgac-3' (XPL2_CM2) [RC] 5'-ct agt GCT AGC Gtga
ctctgac-3' vg#3 (CM2_ZTPS) 5'-ctcagcagtcactgtct tcc at-3'
(CM2_TPLong) 5'-ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cct
AcA-3' (CM2_V3) 5'-tct gaa act cgc cct AcA- jej jqz jjz ejz ezz qej
tgc eej ggt cct cct zjj ttc ejz tgt jqj ezj zez- ggA Acg gAg ccg
AcT gAA GC-3' (CM2_BPL1) [RC] 5'-gga acg gag ccg act gaa GCT AGC
Gtga ctctgacagtctctgt-3' (CM2_XBPS) [RC] 5'-CA Gtga
ctctgacagtctctgt-3' vg#4 (CM2_ZTPS) 5'-ctcagcagtcactgtct tcc at-3'
(CM2_TPLong) 5'-ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cct
AcA-3' (CM2_V4) 5'-tct gaa act cgc cct AcA- gag gcc ggt ejz ezz qej
tgc eej ggt cct cct zjj ttc ejz tgt jqj ezj zez- jjz eqj jej ccg
AcT gAA cgt cct agt GC-3' (CM2_2BPL) [RC] 5'-ccg AcT gAA cgt cct
agt GCT AGC Gtga ctctgacagtctctgt-3' (CM2_XBPS) [RC] 5'-CA Gtga
ctctgacagtctctgt-3' vg#5 (CM2_ZTPS) 5'-ctcagcagtcactgtct tcc at-3'
(CM2_TPLong) 5'-ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cct
AcA-3' (CM2_V5) 5'-tct gAa act cgc cct AcA-' GAG GCT GGT zez zez
jjz tgc eej ggt cct cct eqz ttc jej tgt qee zjj ezj- jjz eqj jej
ccg AcT gAA cgt cct agt GC-3' (CM2_2BPL) [RC] 5'-ccg AcT gAA cgt
cct agt GCT AGC Gtga ctctgacagtctctgt-3' (CM2_XBPS) [RC] 5'-CA Gtga
ctctgacagtctctgt-3' vg#6 (CM2_ZTPS) 5'-ctcagcagtcactgtct tcc at-3'
(CM2_TPLong) 5'-ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cct
AcA-3' (CM2_V6) 5'-tct gAa act cgc cct AcA- GAG GCT GGT jqz zzq zzq
tgt zqz ggt qqj cct eqz ttc ezj tgc ejq qzz zez jjz eqj jej ccg AcT
gAA cgt cct agt GC-3' (CM2_2BPL) [RC] 5'-ccg AcT gAA cgt cct agt
GCT AGC Gtga ctctgacagtctctgt-3' (CM2_XBPS) [RC] 5'-CA Gtga
ctctgacagtctctgt-3' vg#7 (CM2_ZTPS) 5'-ctcagcagtcactgtct tcc at-3'
(CM2_TPLong) 5'-ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cct
AcA-3' (CM2_V7) 5'-tct gaa act cgc cct aca- jej jqz jjz qej zzq eej
tgt jqz ggt qqj ccg ejz zzq jqq tgt zjj ezj eqq- gga acg gag ccg
act gaa GC-3' (CM2_BPL1) [RC] 5'-gga acg gag ccg act gaa GCT AGC
Gtga ctctgacagtctctgt-3' (CM2_XBPS) [RC] 5'-CA Gtga
ctctgacagtctctgt-3'
Component #8 Vary the First 13 Positions.
TABLE-US-00004 [0254] (CM2_ZTPSAlt) 5'-tcactgtct tcc atg ggt tct
gAa-3' (CM2C8vg) 5'-tcactgtct tCC ATG Ggt tct gaa - zez jez eqz zjj
jzj zzz qej zzz ezz qez - jej jzj qqj ggt gag ctg gtt gct atg cag -
ggt ggt agt ggt act gaa GCT (L20botamp) 5'-ggt ggt agt ggt act gaa
GCT AGC [RC] Gtga ctct-3'
Component #9 Fix Five AAs and Extend Variegation to Position
18.
TABLE-US-00005 [0255] (CM2_ZTPSAlt) 5'-tcactgtct tcc atg ggt tct
gAa-3' (CM2C9vg) 5'-tcactgtct tCC ATG Ggt tct gaa - zez gat act zjj
jzj ttt qej zzz ezz qez - gag gtt qqj jjz jej qzj jzj jqj atg caa -
ggt ggt agt ggt act gaa GCT-3' (L20botamp) 5'-ggt ggt agt ggt act
gaa GCT AGC [RC] Gtga ctct-3'
Component #10 Fix First Seven AAs and Vary Last 13.
TABLE-US-00006 [0256] (CM2_ZTPSAlt) 5'-tcactgtct tcc atg ggt tct
gAa-3' (CM2C10vg) 5'-tcactgtct tCC ATG Ggt tct gaa - tat gat act
tgg gtt ttt caa ttt ezz qez - jej jzz qqj jjz jej qzj jzj jqj ezj
qzz - ggt ggt agt ggt act gaa GCT-3' (L20botamp) 5'-ggt ggt agt ggt
act gaa GCT AGC [RC] Gtga ctct-3'
Example 12
Construction of a Second Generation cMet-Binding Peptide
Library
[0257] The phage vector DY3P82 was digested with NheI and NcoI,
cleaned and treated with alkaline phosphatase. The 10 templates,
CM2-V1 through CM2-V7, plus CM2-V8vg, CM2-V9vg and CM2-V10vg, were
amplified separately, using the primer pairs listed in Table 5
below.
TABLE-US-00007 TABLE 5 Template Sense Antisense CM2_V1 CM2_TPLONG
CM2_BPL1 CM2_V2 CM2_TPLONG CM2_BPL1 CM2_V3 CM2_TPLONG CM2_BPL1
CM2_V4 CM2_TPLONG CM2_BPL1 CM2_V5 CM2_TPLONG CM2_BPL1 CM2_V6
CM2_TPLONG CM2_BPL1 CM2_V7 CM2_TPLONG CM2_BPL1 CM2_V8vg CM2_ZTPSALT
L20BOTAMP CM2_V9vg CM2_ZTPSALT L20BOTAMP CM2_V10vg CM2_ZTPSALT
L20BOTAMP
[0258] Each sample was digested separately with NheI and NcoI,
extracted with phenol/chloroform, and mixed in an equimolar ratio
prior to performing the ligation. A vector:insert ratio of 1:5 was
used. Ligated DNA constructs were electroporated into DH5.alpha.
cells. The resulting library size was 1.12.times.10.sup.8 different
transformants.
Example 13
Measurement of Binding of Peptide Dimers to cMet
[0259] Using a BIAcore machine, the binding constants were
determined for the peptide dimers (shown in FIGS. 13A-13C) binding
to immobilized cMet-Fc.
[0260] Three densities of cMet-Fc (R&D Systems) were
cross-linked to the dextran surface of a CM5 sensor chip by the
standard amine coupling procedure (3 .mu.M solution diluted 1:100,
1:50, or 1:20 with 50 mM acetate, pH 5.5). Flow cell 1 was
activated and then blocked to serve as a reference subtraction.
Final immobilization levels achieved: R.sub.L Fc 2 cMet-Fc=2582
R.sub.L Fc 3 cMet-Fc=5048 R.sub.L Fe 4 cMet-Fc=9721
[0261] Experiments were performed in PBST buffer (5.5 mM phosphate,
pH 7.65, 0.15 M NaCl)+0.05% (v/v) Tween-20). Peptide dimers were
dissolved in deionized H.sub.2O to 1 mg/mL solutions. Dimers were
diluted to 50 nM in PBS. Serial dilutions were performed to produce
25, 12.5, 6.25, and 3.125 nM solutions. All samples were injected
in duplicate. For association, dimers were injected at 30
.mu.L/minute for 3 minutes using the kinject program. Following a
10-minute dissociation, any remaining peptide was stripped from the
cMet surface with two quick injects of 4M MgCl.sub.2 for 2 minutes
at 50 .mu.L/minute. Sensorgrams were analyzed using BIAevaluation
software 3.1. The heterodimer, Ac-GSPEMCMMFPFLYPCNHHAPGGGK
{PnAO6-Glut-K[Ac-GSFFPCWRIDRFGYCHANAPGGGKJJ-Glut]-NH.sub.2}-NH.sub.2
(SEQ ID NO 514 linked to SEQ ID NO:515), exhibits a K.sub.D of 0.79
nM.
Example 14
Enhancing the Serum Residence of cMet-Binding Peptides: Conjugation
to Maleimide
[0262] It is known in the art that compounds that contain maleimide
and other groups that can react with thiols react with thiols on
serum proteins, especially serum albumin, when the compounds are
injected. The adducts have serum life times similar to serum
albumin, more than 14 days in humans for example.
[0263] Methods are available that allow for the direct synthesis of
maleimide-labeled linear peptides encompassed by the present
invention (Holmes, D. et al., 2000. Bioconjug. Chem.,
11:439-444.).
[0264] Peptides that include disulfides can be derivatized with
maleimide in one of several ways. For example, a third cysteine can
be added at the carboxy terminus. The added cysteine is protected
with protecting group that is orthogonal to the type of groups used
for the cysteines that are to form the disulfide. The disulfide is
formed by selectively deprotecting the intended cysteines and
oxidizing the peptide. The final cysteine is then deprotected and
the peptide reacted with a large molar excess of a bismaleimide.
The resulting compound has one of the maleimides free to react with
serum albumin or other thiol-containing serum proteins.
[0265] Alternatively, a cyclic peptide of the present invention is
synthesized with a lysine-containing C-terminal extention, such as
-GGGK (SEQ ID NO:513). Lysines of the cMet-binding motif are
protected with ivDde and the C-terminal lysine is deprotected. This
lysine is reacted with a maleimide-containing compound, such as
N-[e-maleimidocaproyloxy]succinimide ester (Pierce Biotechnology,
Rockford, Ill.) or N-[a-Maleimidoacetoxy]succinimide ester (Pierce
Biotechnology).
Example 15
Enhancing the Serum Residence of cMet-Binding Peptides: Conjugation
to a Moiety that Binds Serum Albumin Non-Covalently
[0266] Polypeptides having a molecular weight less than 50-60 kDa
are rapidly excreted. Many small molecules, such as fatty acids,
bind to serum albumin. Attaching a fatty acid or other serum
albumin binding moiety to a peptide causes it to bind non
covalently to serum albumin and can greatly prolong serum
residence. Fatty acids attached to peptides of the present
invention should contain at least 12 carbons, preferably at least
14 carbons and, more preferably at least 16 carbons. The fatty acid
could be straight-chain or branched. The fatty acid could be
saturated or unsaturated. Palmate
(CH.sub.3--(CH.sub.2).sub.14--CO-- is a preferred fatty acid. This
binding in serum can reduce the rate of excretion (Knudsen, L. et
al., 2000. J. Med. Chem., 43:1664-1669). Using methods known in the
art, serum-albumin-binding moieties can be conjugated to any one of
the peptides or multimeric polypeptide binding constructs herein
disclosed. The serum-albumin-binding moiety can be joined to the
cMet-binding peptide through a linker. The linker can be peptidic
or otherwise, such as PEG. Linkers of zero to about thirty atoms
are preferred. It is preferred that the linker be hydrophilic. The
serum-albumin-binding moiety can be conjugated to the cMet-binding
peptide or construct at either end or though a side group of an
appended amino acid. Suitable side groups include lysine and
cysteine. Such compounds also can comprise, for example, chelators
for radionuclides, or other detectable labels or therapeutic
constructs, as discussed herein. A cMet peptide or construct joined
to a serum-albumin-binding moiety will bind cMet.
Example 16
Enhancing the Serum Residence of cMet-Binding Peptides: Conjugation
to PEG
[0267] Attachment of PEG to proteins and peptides enhances the
serum residence of these molecules. Attachment of PEG (linear or
branched) to a cMet-binding peptide or multimeric polypeptide
construct is expected give substantial enhancement of serum
residence time. The molecular weight of the PEG be at least 10 kDa,
more preferably at least 20 kDa, and most preferably 30 kDa or
more. The PEG can be attached at the N- or C-terminus. Methods of
attaching PEG to peptides are well known in the art. PEG can be
attached to reactive side groups such as lysine or cysteine.
Example 17
Enhancing the Serum Residence of cMet-Binding Peptides: Fusion to
Serum Protein
[0268] Proteins comprising serum albumin (SA) and other proteins
have enhanced serum residence times. The amino-acid sequence of
human SA (hSA) is shown in Table 10. Table 11 shows a fusion
protein comprising of (SEQ ID NO:657), mature hSA, and SEQ ID
NO:658. The cMet-binding peptides are separated from mature hSA by
linkers that are rich in glycine to allow flexible spacing. One
need not use all of hSA to obtain an injectable protein that will
have an enhanced serum residence time. Chemical groups, such as
maleimide and alpha bromo carboxylates, react with the unpaired
cysteine (residue 34) to form stable adducts. Thus, one can attach
a single chelator to hSA fusion proteins so that the adduct will
bind a radionuclide. One can prepare a chelator with a maleimide
group and couple that to hSA or an hSA derivative. Alternatively,
hSA or an hSA derivative can be reacted with a bismaleimide and a
chelator carrying a reactive thiol could be reacted with the
bismaleimide-derivatized hSA.
[0269] Construction of genes that encode a given amino-acid
sequence are known in the art. Expression of HSA fusions in
Saccharomyces cerevisiae is known in the art.
Example 18
Pretargeting Radioactivity or Toxins to cMet Expressing Tumors
[0270] Conventional radioimmune cancer therapy is plagued by two
problems. The generally attainable targeting ratio (ratio of
administered dose localizing to tumor versus administered dose
circulating in blood or ratio of administered dose localizing to
tumor versus administered dose migrating to bone marrow) is low.
Also, the absolute dose of radiation or therapeutic agent delivered
to the tumor is insufficient in many cases to elicit a significant
tumor response. Improvement in targeting ratio or absolute dose to
tumor would be of great importance for cancer therapy.
[0271] The present invention provides methods of increasing active
agent localization at a target cell site of a mammalian recipient.
The methods include, for example, a) administering to a recipient a
fusion protein comprising a targeting moiety and a member of a
ligand-anti-ligand binding pair; b) thereafter administering to the
recipient a clearing agent capable of directing the clearance of
circulating fusion protein via hepatocyte receptors of the
recipient, wherein the clearing agent incorporates a member of the
ligand-anti-ligand binding pair; and c) subsequently administering
to the recipient an active agent comprising a ligand/anti-ligand
binding pair member.
[0272] It is known in the art that hexoses, particularly the
hexoses galactose, glucose, mannose, mannose-6-phosphate,
N-acetylglucosamine, pentamannosyl phosphate,
N-acetylgalactosamine, thioglycosides of galactose, and mixtures
thereof are effective in causing hepatic clearance. Binding of
sugars to hepatic receptors is not, however, the only means of
directing a molecule to the liver.
[0273] Clearance of carcinoembryonic antigen (CEA) from the
circulation is by binding to Kupffer cells in the liver. We have
shown that CEA binding to Kupffer cells occurs via a peptide
sequence YPELPK representing amino acids 107-112 of the CEA
sequence. This peptide sequence is located in the region between
the N-terminal and the first immunoglobulin like loop domain. Using
native CEA and peptides containing this sequence complexed with a
heterobifunctional crosslinking agent and ligand blotting with
biotinylated CEA and NCA we have shown binding to an 80 kD protein
on the Kupffer cell surface. This binding protein may be important
in the development of hepatic metastases. (Thomas, P. et al., 1992.
Biochem. Biophys. Res. Commun., 188:671-677)
[0274] To use YPELPK (SEQ ID NO:655) as a clearance agent, one
fuses this sequence via a linker to a moiety that binds the fusion
protein (Ab). For example, if the Ab has affinity for DOTA/Re, one
would make a derivative having YPELPK attached to DOTA/Re; for
example, rvYPELPKpsGGG-DOTA. `rvYPELPKps` is a fragment of CEA
which includes the YPELPK sequence identified by Thomas et al.
(supra). Any convenient point on DOTA can be use for attachment.
RVYPELPKPSGGG-DOTA/cold Re (SEQ ID NO:656) would then be used as a
clearing agent. The Fab corresponding to the fusion Ab would have
affinity for the clearing agent of Kd<100 nM, preferably
Kd<10 nM, and most preferably Kd<1 nM.
[0275] The therapeutic agent would contain DOTA/.sup.185Re. In a
preferred embodiment, the therapeutic agent would contain two or
more DOTA moieties so that the Ab immobilized on the tumor would
bind the bis-DOTA compound with high avidity. The two DOTA moieties
would preferably be connected with a hydrophilic linker of ten to
thirty units of PEG. PEG is a preferred linker because it is not
degraded, promotes solubility. Ten to thirty units of PEG is not
sufficient to give the bis DOTA compound a very long serum
residence time. A half life of 30 minutes to 10 hours is
acceptable. The serum half life should be longer than the
radioactive half life of the radionuclide used so that most of the
radiation is delivered to the tumor or to the external
environment.
[0276] In one embodiment, a "fusion protein" of the present
invention comprises at least one Wet-binding peptide fused to the
amino terminus or the carboxy terminus of either the light chain
(LC) or the heavy chain (HC) of a human antibody. Optionally and
preferably, two or more cMet-binding peptides are fused to the
antibody. The antibody is picked to have high affinity for a small
molecule that can be made radioactive or have a toxin attached.
Preferably, the affinity of the Fab corresponding to the Ab has
affinity for the small molecule with K.sub.d less than 100 nM, more
preferably less than 10 nM, and most preferably less than 1 nM. The
small molecule could be a chelator capable of binding a useful
radioactive atom, many of which are listed herein. The small
molecule could be a peptide having one or more tyrosines to which
radioactive iodine can be attached without greatly affecting the
binding property of the peptide.
[0277] Any cMet-binding peptide (CMBP) of the present invention can
be fused to either end of either chain of an antibody that is
capable of binding a small radioactive compound. Useful embodiments
include:
1) CMBP#1::link::LC/HC, 2) LC::link::CMBP#1/HC, 3)
LC/CMBP#1::link::HC, 4) LC/HC::link::CMBP#1, 5)
CMEBP#1::link1::LC::link2::CMBP#2/HC, 6)
LC/CMBP#1::link1::HC::link2::CMBP#2, 7)
CMBP#1::link1::LC/CMBP#2::link2::HC, 8)
CMBP#1::link1::LC/HC::link2:: CMBP#2, 9)
LC::link1::CMBP#1/CMBP#2::link2::HC, 10)
LC::link1::CMBP#1/HC::link2:: CMBP#2, 11)
CMBP#1::link1::LC::link2::CMBP#2/CMBP#3::link3::HC, 12)
CMBP#1::link1::LC::link2::CMBP#2/HC::link3::CMBP#3, 13)
CMBP#3::link3::LC/CMBP#1::link1 14)
LC::link3::CMBP#3/CMBP#1::link1::HC::link2::CMBP#2, and 15)
CMBP#1::link1::LC::link2::CMBP#2/CMBP#3::link3::HC: link4:: CMBP#4.
In cases (5)-(15), the linkers (shown as "link1", "link2", "link3",
and "link4") can be the same or different or be absent. These
linkers, if present, are preferably hydrophilic, protease
resistant, non-toxic, non-immunogenic, and flexible. Preferably,
the linkers do not contain glycosylation sites or sequences known
to cause hepatic clearance. A length of zero to fifteen amino acids
is preferred. The cMet-binding peptides (CMBP#1, #2, #3, and #4)
could be the same or different. If the encoded amino-acid sequences
are the same, it is preferred that the DNA encoding these sequences
is different.
[0278] Since antibodies are dimeric, each fusion protein will
present two copies of each of the fused peptides. In case (15),
there will be eight CMBPs present and binding to cMet-displaying
cells should be highly avid. It is possible that tumor penetration
will be aided by moderate cMet affinity in each of the CMBPs rather
than maximal affinity.
[0279] The fusion protein is produced in eukaryotic cells so that
the constant parts of the HC will be glycosylated. Preferably, the
cells are mammalian cells, such as CHO cells.
[0280] The fusion proteins are injected into a patient and time is
allowed for the fusion protein to accumulate at the tumor. A
clearing agent is injected so that fusion protein that has not
become immobilized at the tumor will be cleared. In previous
pretargeting methods, the antibody combining site has been used to
target to the tumor and biotin/avidin or biotin/streptavidin has
been used to attach the radioactive or toxic agent to the
immobilized antibody. The biotin/avidin or streptavidin binding is
essentially irreversible. Here we fuse a target-binding peptide to
the antibody which is picked to bind a radioactive or toxic agent.
Because the fusion protein contains 2, 4, 6, or 8 CMBPs, binding of
the fusion protein to the tumor is very avid. A clearing agent that
will cause fusion protein not immobilized at the tumor to clear can
be administered between 2 and 48 hours of the injection of the
fusion protein. Because the clearance agent is monomeric in the
moiety that binds the antibody, complexes of clearance agent and
immobilized fusion protein will not have very long life times.
Within 4 to 48 hours of injecting clearance agent, the immobilized
antibody will have lost any clearance agent that binds there. The
active agent is, preferably, dimeric in the moiety that binds the
fusion protein. The active agent is injected between 2 and
.about.48 hours of injection of clearance agent.
Example 19
Binding of cMet Binding Peptides/Avidin HRP Complex to MDA-MB-231
Cells
[0281] The spacer length requirements for the binding of a
biotinylated derivative of a cMet binding peptide, SEQ ID NO:514,
to cMet expressing MDA-MB-231 cells were determined. In order to
decide the spacer length to be placed in between peptide and
biotin, derivatives were synthesized with no spacer, a single
spacer, J, and two spacers, JJ. These three different derivatives
of cMet-binding peptide SEQ ID NO:514 and a control peptide that
does not bind to cMet, were tested as tetrameric complexes with
neutravidin HRP for their ability to bind cMet expressing MB-231
cells. All three tetrameric complexes of cMet-binding peptides
bound to the MB231 cells as compared to control peptide; however,
the peptide with the JJ spacer exhibited the best K.sub.D (12.62
nM). This suggests that inclusion of two spacers (JJ) between the
cMet-binding peptide and the biotin is better than one or no
spacer.
Cell Culture: MDA-MB231 cells were obtained from ATCC and grown as
monolayer culture in their recommended media plus 1 mL/L pen/strep
(InVitrogen, Carlsbad, Calif.). Cells were split the day before the
assay, 35000 cells were added to each well of a 96-well plate.
Binding of Peptide/Neutravidin HRP to MDA-MB-231 Cells
[0282] Complexes of control peptide, and the SEQ ID NO:514
derivatives described above, with neutravidin-HRP, were prepared as
described above and tested for their ability to bind MDA-MB-231
cells. During the peptide/neutravidin-HRP complex preparation, a
7.5-fold excess of biotinylated peptides over neutravidin-HRP was
used to make sure that all four biotin binding sites on neutravidin
were occupied. After complex formation, the excess of free
biotinylated peptides was removed using soft release
avidin-sepharose to avoid any competition between free biotinylated
peptides and neutravidin HRP-complexed biotinylated peptides. The
experiment was performed at several different concentrations of
peptide/neutravidin-HRP, from 0.28 nM to 33.33 nM, to generate
saturation binding curves for derivatives without a J spacer and
with a single J spacer (FIG. 14), and 0.28 nM to 16.65 nM to
generate a saturation binding curve for the derivative with the JJ
spacer (FIG. 14). In order to draw the saturation binding curve,
the background binding of the control peptide/neutravidin HRP
complex was subtracted from the binding of the SEQ ID NO:514
derivatives in complex with neutravidin-HRP for each concentration
tested. Therefore, absorbance on the Y-axis of FIG. 14 is
differential absorbance (cMet-binding peptide minus control
peptide) and not the absolute absorbance. Analysis of the
saturation binding data in FIG. using Graph Pad Prism software
(version 3.0) yielded a K.sub.D of 12.62 nM (+/-3.16) for the
tetrameric derivative with the JJ spacer, 155.4 nM (+/-86.56) for
the tetrameric derivative with the J spacer and 123.8 nM (+/-37.71)
for the tetrameric derivative without a spacer peptide complexes.
These binding constants are, as expected, lower than that measured
by FP for the related monodentate peptide SEQ ID NO:514 (880
nM).
Results: It is evident from FIG. 14 that the derivative with the JJ
spacer showed much better binding to cMet on MDA-MB-231 cells than
either of the other two derivatives, with a K.sub.D of 12.62 nM
after subtracting binding of control peptide as background binding
(n=1). This suggests that a certain minimum spacer length may be
required to be able to reach multiple different binding sites on
cells and thus achieve multimeric binding. This minimum spacer
length could depend on the spacing between different target
molecules on cells. As was the case where the binding target was
KDR, the neutravidin-HRP assay with biotinylated peptides
identified with phage display was useful for identifying peptides
capable of binding to an immobilized target even when the affinity
of the monomeric binding sequence is too low for an ELISA-type
assay (with washing steps after binding) to work well.
TABLE-US-00008 TABLE 6 cMet-binding peptide sequences SEQ ID NO:
Isolate Sequence CLASS I TN6: SEQ ID NO: 001 571-C05,
GSWIICWWDNCGSSAP SEQ ID NO: 002 465-A06, GSYYDCREFQCNKPAP SEQ ID
NO: 003 465-D09, GSSHLCNPEFCHFTAP SEQ ID NO: 004 569-H10,
GSMLMCELWWCRFLAP SEQ ID NO: 005 470-E11, GSLIFCPYGECMMYAP SEQ ID
NO: 006 452-F01, GSEYSCRTSRCIFSAP SEQ ID NO: 007 569-C03,
GSFILCWWTFCDTNAP SEQ ID NO: 008 574-H03, GSSTICPGTACVDHAP SEQ ID
NO: 009 567-C08, GSLIICWWSWCDKQAP SEQ ID NO: 010 561-C08,
GSFNICPYQWCTLWAP Consensus Motif:
G-S-X1-X2-X3-C-X4-X5-X6-X7-C-X8-X9-X10- A-P-G-G-K; where X1 is F,
L, S, W, Y, or M; X2 is I, Y, H, T, or N; X3 is I, L, D, M, F, or
S, preferably I; X4 is P, R, W, N, or E, preferably W or P; X5 is
W, Y, E, P, L, T, or G; X6 is S, T, D, F, E, W, G, or Q; X7 is F,
W, N, Q, E, R, or A; X8 is G, N, H, R, M, I, D, V, or T; X9 is S,
K, F, M, T, D, or L; and X10 is S, P, T, L, Y, N, H, Q, or W. CLASS
II TN8: SEQ ID NO: 011 573-F04, AGGFACGPPWDICWMFGT SEQ ID NO: 012
570-E07, AGAWNCEYPTFICEWQGA SEQ ID NO: 013 456-E04,
AGNWICNLSEMRCYPKGT SEQ ID NO: 014 434-E12, AGDGWCMAWPEICEWLGT SEQ
ID NO: 015 489-A04, AGLYLCDLSIMYCFFQGT SEQ ID NO: 016 484-D08,
AGWWSCQWELNVCIWQGT SEQ ID NO: 017 482-D02, AGYYHCIDDFPQCKWMGT SEQ
ID NO: 018 437-A09, AGWFECEFGFWGCNWLGT SEQ ID NO: 019 352-E04,
AGTVYCSWESSECWWVGT SEQ ID NO: 020 376-E05, AGVWICRVWDDECFFQGT SEQ
ID NO: 021 482-A12, AGDHYCWEEWWFCWDSGT SEQ ID NO: 022 423-C11,
AGVLQCIGFEWFCDIWGT SEQ ID NO: 023 499-C09, AGVIVCNLSMMYCLYPGT SEQ
ID NO: 024 457-A09, AGYPECKDNYHWCEWKGT SEQ ID NO: 025 573-E07,
AGWTWCDLSMMSCIFHGT SEQ ID NO: 026 465-F08, AGVTNCNLSTMFCFLHGT SEQ
ID NO: 027 465-E09, AGTLSCSEEYKSCQLQGT SEQ ID NO: 028 444-B08,
AGTIRCNLAMMVCMFEGT SEQ ID NO: 029 465-E11, AGQYLCTQAALGCPEWGT SEQ
ID NO: 030 465-D12, AGQMWCAEKNSKCYQWGT SEQ ID NO: 031 470-A02,
AGQAVCEWGPFWCQMQGT SEQ ID NO: 032 465-C01, AGPYSCHSESHDCKLMGT SEQ
ID NO: 033 448-H02, AGPLFCFEWPSLCHWGGT SEQ ID NO: 034 465-D01,
AGNLPCHWNMSVCDHQGT SEQ ID NO: 035 571-C11, AGMDFCEGFWFLCIGNAT SEQ
ID NO: 036 465-B11, AGLLGCWDMPMECTGEGT SEQ ID NO: 037 442-E08,
AGKYMCEGFEWFCEMWGT SEQ ID NO: 038 465-C11, AGKTVCQKWESVCSGMGT SEQ
ID NO: 039 465-F10, AGKQWCVVWEETCDQLGT SEQ ID NO: 040 471-A11,
AGIWFCNNEEKSCWAYGT SEQ ID NO: 041 465-C07, AGHTICQHKALGCPANGT SEQ
ID NO: 042 465-D04, AGHFECPKHQYMCDMPGT SEQ ID NO: 043 445-E04,
AGGNWCSFYEELCEWLGT SEQ ID NO: 044 465-B06, AGGHWCLELKHLCPPYGT SEQ
ID NO: 045 470-C02, AGFWDCGWMMQDCHMHGT SEQ ID NO: 046 458-B05,
ADAWMCEYFQWNCGDKGT SEQ ID NO: 047 545-E08, GDGFLCRWENGWCEFWDP
Consensus Motif: A-G-X1-X2-X3-C-X4-X5-X6-X7-X8-X9-C-X10-
X11-X12-G-T-G-G-G-K; where X1 is any amino acid other than C,
preferably G, V, W, T, K, Q; X2 is any amino acid other than C,
preferably W, Y, L, F, T; X3 is any amino acid other than C,
preferably W, E, F, I, L, S X4 is any amino acid other than C,
preferably E, N, Q; X5 is any amino acid other than C, preferably
W, L, E; X6 is any amino acid other than C, preferably E, S, Y; X7
is any amino acid other than C, preferably E, M, P; X8 is any amino
acid other than C, preferably M, S, W; X9 is any amino acid other
than C, preferably F, L, V; X10 is any amino acid other than C,
preferably E, D, W; X11 is any amino acid other than C, preferably
W, F, M; and X12 is any amino acid other than C, preferably Q, W,
L. CLASS III TN9 #1: SEQ ID NO: 048 325-H05, AGSIQCKGPPWFSCAMYGT
SEQ ID NO: 049 330-F05, AGYYGCKGPPTFECQWMGT SEQ ID NO: 050 333-F09,
AGQFKCAGPPSFACWMTGT SEQ ID NO: 051 336-G04, AGWFQCKGPPSFECERHGT SEQ
ID NO: 052 334-G06, AGWTHCIGPPTFECIPMGT SEQ ID NO: 053 330-B07,
AGSFACKGPPTFACVEFGT SEQ ID NO: 054 330-C10, AGNYFCAGSPSFSCYFMGT SEQ
ID NO: 055 331-G04, AGSWHCAGPPSFECWEFGT SEQ ID NO: 056 548-F06,
AGWISCAGPPTFACWPGGT SEQ ID NO: 057 538-F08, AGFVNCKGPPTFECILTGT SEQ
ID NO: 058 547-H07, AGDWICHGPPMFECEWVGT SEQ ID NO: 059 323-A11,
AGYTSCVGPPSFECTPYGT SEQ ID NO: 060 333-H03, AGYFECKGPPTFECWLSGT SEQ
ID NO: 061 329-D02, AGHAWCSGPPRFECWPPGT SEQ ID NO: 062 550-C09,
AGHYWCAGPPTFICMGPGT SEQ ID NO: 063 548-E08, AGETTCLGWPTFVCVDYGT SEQ
ID NO: 064 332-A05, AGHGTCRGWPTFECIYFGT SEQ ID NO: 065 330-C01,
AGDWHCQGPPAFMCWMIGT SEQ ID NO: 066 545-A09, AGLPKCSGPPWFSCYYGGT SEQ
ID NO: 067 334-C08, AGGWECTGPPWFQCGYYGT SEQ ID NO: 068 333-C05,
AGDIVCTGHPYFECWSWGT SEQ ID NO: 069 551-B02, AGTWHCAGPPWFTCYMDGT SEQ
ID NO: 070 551-G12, AGSWECTGPPSFHCQWYGT SEQ ID NO: 071 330-G09,
AGHWICVGPPTFSCQWHGT SEQ ID NO: 072 331-F01, AGEWWCHGPPEFLCYWTGT SEQ
ID NO: 073 274-B07, AGETVCYWLNGWFCVDDGT SEQ ID NO: 074 335-D11,
AGSIQCVGPPSFECTPYGT SEQ ID NO: 075 336-D07, AGYSVCKGYPSFECAFFGT SEQ
ID NO: 076 332-C03, AGVNSCLGPPTFECYQMGT SEQ ID NO: 077 331-D03,
AGYWHCKGPPHFACEFHGT SEQ ID NO: 078 331-G06, AGNWICTGPPSFGCWYHGT SEQ
ID NO: 079 552-G03, AGYWSCAGPPMFMCTWQGT SEQ ID NO: 080 552-G11,
AGYWDCKGPPHFFCEWHGT SEQ ID NO: 081 550-G08, AGYFHCSGSPWFQCDYYGT SEQ
ID NO: 082 550-G12, AGWYNCSGENFWNCKWIGT SEQ ID NO: 083 552-A01,
AGWSDCLGPPQFTCVHWGT SEQ ID NO: 084 548-C06, AGTMYCLGPPTFICQQYGT SEQ
ID NO: 085 545-B12, AGSYWCSGPPTFMCRYEGT SEQ ID NO: 086 549-F06,
AGSTDCRGHPTFECWGWGT SEQ ID NO: 087 552-F01, AGSSPCKGWPTFECYFYGT SEQ
ID NO: 088 547-H12, AGSIACTGWPYFSCIDLGT SEQ ID NO: 089 550-F11,
AGQFYCSGPPTFQCIMIGT SEQ ID NO: 090 548-D08, AGPWKCTGPPTFSCIQFGT SEQ
ID NO: 091 549-D02, AGNYWCSGPPSFICHAVGT SEQ ID NO: 092 552-F02,
AGMTLCAGPPTFECYEVGT SEQ ID NO: 093 545-E04, AGETKCSGPPYFYCWMEGT SEQ
ID NO: 094 545-E05, AGETFCVGNPSFECWSWGT SEQ ID NO: 095 547-H03,
AGETFCSGWPTFECMQWGT SEQ ID NO: 096 552-G09, AGEIFCVGPPTFTCMWTGT SEQ
ID NO: 097 550-A08, AGDFICQGPPSFVCTNIGT SEQ ID NO: 098 550-G07,
AGAFFCSGPPTFMCSLYGT SEQ ID NO: 099 551-A05, AGWGWCSGPPMFMCTEYGT SEQ
ID NO: 100 548-C10, GSEFECTGWPEFRCYEYAP SEQ ID NO: 101 465-C10,
GSILYCINRNDPQCPYTAP Consensus Motif:
G-X1-X2-X3-C-X4-G-X5-P-X6-F-X7-C-X8-X9- X10-G-T; where: X1 is any
amino acid other than C, preferably E, S, Y, or W; X2 is any amino
acid other than C, preferably W, T, or F; X3 is any amino acid
other than C, preferably W, H, or F; X4 is any amino acid other
than C, preferably A, K, S, or T; X5 is any amino acid other than
C, preferably P or W; X6 is any amino acid other than C, preferably
T or S; X7 is any amino acid other than C, preferably E or S; X8 is
any amino acid other than C, preferably W, Y, or I; X9 is any amino
acid other than C, preferably W, Y, M, or E; and X10 is any amino
acid other than C, preferably Y.
CLASS IV TN9 #2: SEQ ID NO: 102 605-G10,
SETRPTEAGDLICSGPPTFICTLYHTEPTE SEQ ID NO: 103 593-C01,
SETRPTQAVRSQCSGPPTFECWYFGTEPTE SEQ ID NO: 104 592-C01,
SETRPTEGGSWYCSGPPAFECWWYGTEPTE SEQ ID NO: 105 591-E01,
SETRPTVASRWHCNGPPTFECWRYGTEPTE SEQ ID NO: 106 590-E01,
SETRPTEAGTFHCSGPPTFECWSYGPKPTE SEQ ID NO: 107 589-B01,
SETRPTEAGSLWCMGPPWFCCVIYGTQPTE SEQ ID NO: 108 607-A02,
SETRPTEAGILHCSGPPTFECWWNYTEPTE SEQ ID NO: 109 590-F01,
SETRPTESGRVHCPGPPWFRCARNGTEPTE SEQ ID NO: 110 589-C01,
SETRPTAAGRILCTGPPWFSCAMYGTEPTE SEQ ID NO: 111 606-B11,
SETRPTEAADWLCSGPPTFECWWFGTEPTE SEQ ID NO: 112 593-E01,
SETRPTQVGRWQCDGPPTFACRSYGTEPTE SEQ ID NO: 113 592-F12,
SETRPTEAGSTKCSGPPTFECWWFDTEPTE SEQ ID NO: 114 590-F07,
SETRPTVAGSWHCSGPPTFECWWYGTEPTE SEQ ID NO: 115 588-D02,
SETRPTEAGRNHCKGPPGFRCAMTDTEPTE SEQ ID NO: 116 607-H09,
SETRPTETDFVYCRGPPTFECWWYGTEPTE SEQ ID NO: 117 590-H01,
SETRPTSSGSRHCKGPPTFECWGYGTEPTE SEQ ID NO: 118 589-F01,
SETRPTEAGSWRCSGPPTFECWWYETSPTE SEQ ID NO: 119 608-F11,
SETRPTDAIRSYCSGPPTFECWWFGTEPTE SEQ ID NO: 120 606-D11,
SETRPTEAGSWNCSGPPAFECWWYGSEPTE SEQ ID NO: 121 604-D04,
SETRPTEAGSWQCSGPPTFECWSFGTEPTE SEQ ID NO: 122 602-A11,
SETRPTEAGSWHCNGPPTFECWWYDMEPTE SEQ ID NO: 123 593-F02,
SETRPTEAGRVSCLGPPTFECWWFVPEPTE SEQ ID NO: 124 591-H05,
SETRPTDAGSWRCAGPPTFECWWFGTEPTE SEQ ID NO: 125 590-H06,
SETRPTEPVTWQCTGPPTFECWWLGTEPTE SEQ ID NO: 126 588-F10,
SETRPTDAVSTHCNGPPTFECYIYGTEPTE SEQ ID NO: 127 608-G03,
SETRPTVAESWYCVGPPSFECWWYGTEPTE SEQ ID NO: 128 604-D09,
SETRPTEAGSWNCSGPPTFECWSYQTEPTE SEQ ID NO: 129 602-A12,
SETRPTEAGSGHCNGPPTFKCWWYDMEPTE SEQ ID NO: 130 592-G11,
SETRPTDQDSWQCSGPPTFECWWYGTEPTE SEQ ID NO: 131 588-G01,
SETRPTESTQVQCAGPPSFACWMTGTEPTE SEQ ID NO: 132 606-E05,
SETRPTEVESWHCSGPPTFECWWYGTEPTE SEQ ID NO: 133 594-C07,
SETRPTEAGSFHCSGPPTFECWLYWTDPTE SEQ ID NO: 134 592-H01,
SETRPTEAGQFGCKGPPPFECKLMGRVPTE SEQ ID NO: 135 605-C05,
SETRPTDTVTWHCNGPPTFECWWYGTEPTE SEQ ID NO: 136 594-E08,
SETRPTEADRWHCDGPPTFECWWYGTEPTE SEQ ID NO: 137 593-B11,
SETRPTEAGSIQCVGPPWFSCRMYVTEPTE SEQ ID NO: 138 590-C01,
SETRPTVSGSWQCVGPPTFECWSYGTEPTE SEQ ID NO: 139 612-G11,
SETRPTENGSWHCNGPPTFECWWYGTEPTE SEQ ID NO: 140 612-E08,
SETRPTEAGSWHCSGPPIFECWWYDMEPTE SEQ ID NO: 141 612-A02,
SETRPTVDGGWHCNGPPTFECWMYGTEPTE SEQ ID NO: 142 611-G01,
SETRPTDAGTWNCTGPPSFECWWFGTEPTE SEQ ID NO: 143 610-G04,
SETRPTWDGKWHCSGPPTFECWWYGTEPTE SEQ ID NO: 144 610-E06,
SETRPTEAGSWRCSGPPTFECWWYYTEPTE SEQ ID NO: 145 610-C06,
SETRPTEAGNWLCSGPPTFECWWYVTGPTE SEQ ID NO: 146 610-A04,
SETRPTEGGNWHCSGPPTFECWLYGTEPTE SEQ ID NO: 147 612-D02,
SETRPTEAGGWHCSGPPTFECWWFNNEPTE SEQ ID NO: 148 612-A12,
SETRPTEVISWHCSGPPTFECYRYGTEPTE SEQ ID NO: 149 611-D03,
SETRPTEVGSWHCNGPPTFECWWYGTEPTE SEQ ID NO: 150 610-G10,
SETRPTLASTWYCSGPPTFECWWYGTEPTE SEQ ID NO: 151 610-A11,
SETRPTEAGGWYCKGPPTFECWWDGTEPTE SEQ ID NO: 152 612-H02,
SETRPTEAGGWFCSGPPTFECWWYDTVPTE SEQ ID NO: 153 612-B01,
SETRPTEAATWQCSGPPTFECWGYGTEPTE SEQ ID NO: 154 610-C12,
SETRPTEAGDYVCVGPPTFECYLMDAEPTE SEQ ID NO: 155 610-B01,
SETRPTEAGGWYCSGPPSFECWSYGTEPTE SEQ ID NO: 156 612-H04,
SETRPTESSSWHCSGPPTFECWRFGTEPTE SEQ ID NO: 157 612-B09,
SETRPTEAGSWYCSGPPTFECWWYYAEPTE SEQ ID NO: 158 611-G07,
SETRPTLAGNWQCSGPPTFECWWYGTEPTE SEQ ID NO: 159 611-E10,
SETRPTEAGSWHCNGPPTFECWQYGTEPTE SEQ ID NO: 160 610-H02,
SETRPTEAGSWECHGPPSFECWWYGTEPTE SEQ ID NO: 161 610-D03,
SETRPTEAGSWRCSGPPTFECWWYDAEPTE SEQ ID NO: 162 610-B03,
SETRPTEAGSWNCAGPPTFECWWYGTEPTE SEQ ID NO: 163 612-H05,
SETRPTEAGSFYCSGPPTFECWQYVPEPTE SEQ ID NO: 164 612-F05,
SETRPTEAGSWMCSGPPTFECWQYFTEPTE SEQ ID NO: 165 612-B10,
SETRPTEAGSLHCSGPPTFECWWWETEPTE SEQ ID NO: 166 611-E11,
SETRPTEEGVWHCNGPPTFECWWYGTEPTE SEQ ID NO: 167 610-F08,
SETRPTEAGRWNCSGPPTFECWWYSTEPTE SEQ ID NO: 168 610-D05,
SETRPTEAGSWRCSGPPTFECWWFGTEPTE SEQ ID NO: 169 610-B04,
SETRPTQAVSSYCSGPPTFECWSFGTEPTE SEQ ID NO: 170 612-B12,
SETRPTEAGRSYCSGPPTFECWWYATEPTE SEQ ID NO: 171 611-H01,
SETRPTVVAKVHCAGPPTFECWTYGTEPTE SEQ ID NO: 172 610-H05,
SETRPTEPGSWHCSGPPTFVCWWWGTEPTE SEQ ID NO: 173 610-F10,
SETRPTEAGRWHCSGPPTFECWWHDTEPTE SEQ ID NO: 174 612-H07,
SETRPTEAGSWQCTGPPTFECWGYVEEPTE SEQ ID NO: 175 612-G09,
SETRPTEAGSWQCGGPPTFECWWYYTGPTE SEQ ID NO: 176 612-F08,
SETRPTEAGSWYCTGPPTFECWLYETYPTE SEQ ID NO: 177 611-H08,
SETRPTAAWSGSCSGPPSFECWNYGTEPTE SEQ ID NO: 178 610-E01,
SETRPTEAGSWQCSGPPTFACWWYGTEPTE SEQ ID NO: 179 610-B09,
SETRPTEAGILHCSGPPTFECWWEVMEPTE SEQ ID NO: 180 612-E07,
SETRPTEAGRVACSGPPTFECWSYDEEPTE SEQ ID NO: 181 612-C11,
SETRPTEAGNWECQGPPTFECWWFGTEPTE SEQ ID NO: 182 610-E04,
SETRPTLASNGYCNGPPTFECWHYGTEPTE SEQ ID NO: 183 610-B12,
SETRPTEAGSFHCSGPPTFECIWYGSEPTE SEQ ID NO: 184 616-B11,
SETRPTEAGSWYCSGPPTFACWWDGTEPTE SEQ ID NO: 185 615-H08,
SETRPTQGDNWNCSGPPTFECWWYGTEPTE SEQ ID NO: 186 615-B11,
SETRPTEAGRWHCNGPPTFECWRYDYDPTE SEQ ID NO: 187 614-C07,
SETRPTEAYSWECTGPPMFECWWYGTEPTE SEQ ID NO: 188 613-H12,
SETRPTEVVDWHCSGPPQFECWWYGTEPTE SEQ ID NO: 189 613-F02,
SETRPTEAGSWNCSGPPTFECWWYGSEPTE SEQ ID NO: 190 613-D05,
SETRPTASGSWHCSGPPTFECWIFGTEPTE SEQ ID NO: 191 612-H12,
SETRPTEAGAWYCMGPPTFECWWYDRGPTE SEQ ID NO: 192 616-D05,
SETRPTEAGGLHCSGPPTFECWWYDTEPTE SEQ ID NO: 193 615-C01,
SETRPTVGGSWDCKGPPTFECWSYGTEPTE SEQ ID NO: 194 614-E09,
SETRPTEAGAWSCLGPPTFECWWYGTEPTE SEQ ID NO: 195 614-A03,
SETRPTEAGSLHCSGPPTFECWWFDTEPTE SEQ ID NO: 196 616-C02,
SETRPTAGRSWECSGPPTFECWVFGTEPTE SEQ ID NO: 197 615-C04,
SETRPTDNGSWHCNGPPTFECWWYGTEPTE SEQ ID NO: 198 614-C12,
SETRPTEAGSWQCKGPPTFECWWYGTEPTE SEQ ID NO: 199 615-C11,
SETRPTEVGNYKCSGPPTFECWWYGTEPTE SEQ ID NO: 200 614-H08,
SETRPTEAGSWHCVGPPTFECWGYVTEPTE SEQ ID NO: 201 614-E11,
SETRPTEAGSFVCKGPPTFECYWFGQDPTE SEQ ID NO: 202 616-E10,
SETRPTEAGSWHCSGPPTFECWWYGPDPTE SEQ ID NO: 203 615-D02,
SETRPTEAERWHCSGPPTFECWWYGTEPTE SEQ ID NO: 204 614-F04,
SETRPTEAGSWHCSGPPTFECWFYVKEPTE SEQ ID NO: 205 614-D06,
SETRPTEAGSWDCSGPPTFECWWFGTEPTE SEQ ID NO: 206 614-B08,
SETRPTEPAGWECRGPPSFECLWYGTEPTE SEQ ID NO: 207 613-H01,
SETRPTDAGPWNCTGPPSFECWWYGTEPTE SEQ ID NO: 208 613-E04,
SETRPTEARGWHCSGPPTFECWLWGTEPTE SEQ ID NO: 209 613-B08,
SETRPTEAGRWNCSGPPTFECWQYEMDPTE SEQ ID NO: 210 615-D04,
SETRPTEAGSWYCSGPPTFECFWYDTEPTE SEQ ID NO: 211 615-A05,
SETRPTESGSWHCSGPPTFECWWFGTEPTE SEQ ID NO: 212 614-E04,
SETRPTEAGSWLCTGPPTFECWWFDTDPTE SEQ ID NO: 213 613-E06,
SETRPTEPSHWHCVGPPTFACWWYVTDPTE SEQ ID NO: 214 613-C05,
SETRPTEAGSWYCSGPPMFECYLFVTEPTE SEQ ID NO: 215 616-C07,
SETRPTEAVNWHCLGPPSFECWQFGTEPTE SEQ ID NO: 216 615-G02,
SETRPTEAGSWHCSGPPTFECWWYGTDPTE SEQ ID NO: 217 615-E06,
SETRPTEAGSWHCSGPPTFECWSFVSLPTE SEQ ID NO: 218 615-A08,
SETRPTEGSEWSCIGPPSFECWWYGTEPTE SEQ ID NO: 219 614-G01,
SETRPTEDGYWNCSGPPTFECWWHGTEPTE SEQ ID NO: 220 613-D01,
SETRPTEAGSWSCSGPPTFECWPYYTEPTE SEQ ID NO: 221 614-G02,
SETRPTEAGSWYCSGPPTFECWWYWPEPTE SEQ ID NO: 222 614-E06,
SETRPTDDGRWSCAGPPTFECWRYGTEPTE SEQ ID NO: 223 620-E11,
SETRPTEGGSWSCGGPPTFECWWFGTEPTE SEQ ID NO: 224 620-A11,
SETRPTVTGSWYCSGPPTFECWWYGTEPTE SEQ ID NO: 225 618-F04,
SETRPTEASSWYCTGPPAFECWWYGTEPTE
SEQ ID NO: 226 617-G06, SETRPTEAGSWLCSGPPTFECWWYGTEPTE SEQ ID NO:
227 616-G06, SETRPTESVRWYCSGPPTFECWWYGTEPTE SEQ ID NO: 228 620-F10,
SETRPTEAGRLVCSGPPTFMCRTYATDPTE SEQ ID NO: 229 619-G04,
SETRPTEAGSWECTGPPWFVCRQYAIEPTE SEQ ID NO: 230 618-F12,
SETRPTEAGYLYCSGPPTFECWWYDTMPTE SEQ ID NO: 231 618-B06,
SETRPTEAGSWHCSGPPTFECWWFGTEPTE SEQ ID NO: 232 617-E09,
SETRPTEAGNWHCLGPPTFECWWYGTEPTE SEQ ID NO: 233 616-F10,
SETRPTEAGSWHCSGPPTFECWWYDTEPTE SEQ ID NO: 234 620-B11,
SETRPTESGGWYCSGPPAFECWWYGTEPTE SEQ ID NO: 235 619-G07,
SETRPTVAGAVSCSGPPTFECWWYGTEPTE SEQ ID NO: 236 619-E11,
SETRPTEAGRWYCSGPPTFECWWFLPDPTE SEQ ID NO: 237 619-B12,
SETRPTEAGGWHCSGPPSFECWWFDTVPTE SEQ ID NO: 238 618-G11,
SETRPTGVGGWYCSGPPSFECWLYGTEPTE SEQ ID NO: 239 618-B11,
SETRPTQADYLHCSGPPTFECFWYGTEPTE SEQ ID NO: 240 617-F01,
SETRPTGDGNWHCNGPPTFECWRFGTEPTE SEQ ID NO: 241 617-B01,
SETRPTEASNYHCIGPPTFECFWYGTEPTE SEQ ID NO: 242 616-G12,
SETRPTEAGDWLCKGPPTFECWWQVTDPTE SEQ ID NO: 243 620-G01,
SETRPTEAGSWHCNGPPTFECWWYSSDPTE SEQ ID NO: 244 620-C10,
SETRPTEDGGWRCSGPPTFECWWYGTEPTE SEQ ID NO: 245 619-G09,
SETRPTEAGRIECKGPPWFSCVIYGTEPTE SEQ ID NO: 246 619-F06,
SETRPTGGGSWNCSGPPTFECWWYGTEPTE SEQ ID NO: 247 618-C03,
SETRPTEAGSLYCSGPPTFECWWYITHPTE SEQ ID NO: 248 617-F02,
SETRPTEAGRWHCSGPPRFECWWYDTEPTE SEQ ID NO: 249 616-H01,
SETRPTEYGSWHCSGPPTFECWYHGTEPTE SEQ ID NO: 250 618-D01,
SETRPTEAGNWHCSGPPSFECWWYATEPTE SEQ ID NO: 251 617-F03,
SETRPTEQGSWHCKGPPTFECWSYGTEPTE SEQ ID NO: 252 616-H03,
SETRPTDAANYHCSGPPTFECWWYGTEPTE SEQ ID NO: 253 616-G02,
SETRPTEAGSWYCSGPPMFECWWLAEEPTE SEQ ID NO: 254 620-G09,
SETRPTEAGGWYCSGPPAFECWWYATEPTE SEQ ID NO: 255 620-D12,
SETRPTEAGIWSCSGPPTFECWWYESSPTE SEQ ID NO: 256 619-A09,
SETRPTEEGLRVCSGPPTFECWWYGTEPTE SEQ ID NO: 257 618-D06,
SETRPTEAGSWLCFGPPTFECWSFGTEPTE SEQ ID NO: 258 617-H12,
SETRPTVAGSWDCSGPPTFECWWYGTEPTE SEQ ID NO: 259 616-H05,
SETRPTKADNWHCSGPPTFECWWYGTEPTE SEQ ID NO: 260 619-H10,
SETRPTEAGIVYCSGPPTFECWWFGTEPTE SEQ ID NO: 261 619-D03,
SETRPTEAGYWHCLGPPTFECWWYVKEPTE SEQ ID NO: 262 618-D12,
SETRPTEPGLLHCSGPPTFECWWYGTEPTE SEQ ID NO: 263 620-E04,
SETRPTEASSWYCSGPPSFECWWYGTEPTE SEQ ID NO: 264 620-A05,
SETRPTEAGSWHCLGPPTFECWWYVKEPTE SEQ ID NO: 265 619-D04,
SETRPTEAGIILCKGPPWFSCDIYDTGPTE SEQ ID NO: 266 618-A11,
SETRPTAAGNWHCSGPPTFECWAYGTEPTE SEQ ID NO: 267 617-D07,
SETRPTVGGSWYCSGPPTFECWSYGTEPTE SEQ ID NO: 268 627-A10,
SETRPTEDGWLDCKGPPTFECWWYGTEPTE SEQ ID NO: 269 626-H02,
SETRPTEDGNWHCSGPPTFECWSYGTEPTE SEQ ID NO: 270 626-F06,
SETRPTEAGSWHCSGPPTFECWYYWPEPTE SEQ ID NO: 271 624-D02,
SETRPTEAGSLYCSGPPMFECWWYDWYPTE SEQ ID NO: 272 622-D09,
SETRPTEAGGWYCMGPPAFECWWYASEPTE SEQ ID NO: 273 621-F11,
SETRPTNAGSWYCSGPPTFECWWYGTEPTE SEQ ID NO: 274 621-B11,
SETRPTEASRWHCNGPPTFECWWYGTEPTE SEQ ID NO: 275 627-B03,
SETRPTEAGSFVCSGPPTFECWWYNTGPTE SEQ ID NO: 276 626-H03,
SETRPTEAGSWHCSGPPTFECWSYGTEPTE SEQ ID NO: 277 626-F07,
SETRPTESDIWLCSGPPTFECWWYGTEPTE SEQ ID NO: 278 626-D02,
SETRPTDADPWHCSGPPTFECWWFGTEPTE SEQ ID NO: 279 625-B03,
SETRPTEAGVVLCSGPPTFECWWYDTEPTE SEQ ID NO: 280 622-D10,
SETRPTEVGSVHCSGPPTFECWWFGTEPTE SEQ ID NO: 281 621-G02,
SETRPTEAGRWLCSGPPTFECWEYDTEPTE SEQ ID NO: 282 621-E04,
SETRPTDAGWLQCSGPPTFECWWYGTEPTE SEQ ID NO: 283 621-B12,
SETRPTEASRRHCNGPPTFECWRYGTEPTE SEQ ID NO: 284 626-H04,
SETRPTEAGRWYCSGPPTFECWLFVEEPTE SEQ ID NO: 285 626-F11,
SETRPTAADSWQCSGPPTFECWSFGTEPTE SEQ ID NO: 286 626-D03,
SETRPTEAGSWHCGGPPTFECWMYVTEPTE SEQ ID NO: 287 626-A02,
SETRPTDDGSWYCSGPPTFECWWYGTEPTE SEQ ID NO: 288 623-E07,
SETRPTEAGYWHCLGPPTFECWWYDMEPTE SEQ ID NO: 289 622-G09,
SETRPTEAGILRCSGPPTFECWYYETEPTE SEQ ID NO: 290 622-E05,
SETRPTEDVSVHCAGPPTFECWLYGTEPTE SEQ ID NO: 291 622-B12,
SETRPTEEGVFQCVGPPTFECWWYGTEPTE SEQ ID NO: 292 621-G07,
SETRPTEDGGFFCSGPPTFECWWYGTEPTE SEQ ID NO: 293 621-E07,
SETRPTEPGSWHCSGPPTFECWWYGTEPTE SEQ ID NO: 294 621-C01,
SETRPTEAGSWHCSGPPTFECWWYDRAPTE SEQ ID NO: 295 626-A05,
SETRPTEAGTWYCSGPPTFECWYYATEPTE SEQ ID NO: 296 623-G02,
SETRPTEAGSLYCSGPPAFECYWYGTVPTE SEQ ID NO: 297 622-H11,
SETRPTDPGVLHCSGPPTFECWWFGTEPTE SEQ ID NO: 298 622-C04,
SETRPTEAGTWYCLGPPTFECWSFWQDPTE SEQ ID NO: 299 621-G11,
SETRPTEAGRWGCSGPPTFECWWYVAEPTE SEQ ID NO: 300 621-C07,
SETRPTEAGIWHCAGPPTFICWLYETEPTE SEQ ID NO: 301 627-C03,
SETRPTEAGSWHCSGPPSFECWQYSTEPTE SEQ ID NO: 302 626-D12,
SETRPTEAGSWQCSGPPTFECWVYETEPTE SEQ ID NO: 303 626-A06,
SETRPTEAGSWYCSGPPTFECWWYDVGPTE SEQ ID NO: 304 623-H02,
SETRPTDEVSWECRGPPTFECWWYGTEPTE SEQ ID NO: 305 623-B05,
SETRPTEGGSWVCSGPPTFECWWYGTEPTE SEQ ID NO: 306 622-E10,
SETRPTEYGSWYCSGPPTFECWWLGTEPTE SEQ ID NO: 307 622-C06,
SETRPTEAGVWLCSGPPTFECWWYDTDPTE SEQ ID NO: 308 621-H03,
SETRPTMAGSYYCSGPPTFECWVYGTEPTE SEQ ID NO: 309 621-E11,
SETRPTEAGYVQCYGPPSFVCHPMVPDPTE SEQ ID NO: 310 621-C08,
SETRPTEDGFVLCKGPPWFSCEMYGTEPTE SEQ ID NO: 311 627-C04,
SETRPTEAGGWNCSGPPTFECWWYVTEPTE SEQ ID NO: 312 626-A07,
SETRPTEDGSWECFGPPTFECWSYGTEPTE SEQ ID NO: 313 623-H08,
SETRPTDAVSYVCKGPPTFECWWYGTEPTE SEQ ID NO: 314 622-F05,
SETRPTEARSWHCSGPPTFECWWYGTEPTE SEQ ID NO: 315 627-A04,
SETRPTASVSWHCSGPPTFECWSYGTEPTE SEQ ID NO: 316 626-G05,
SETRPTEAGSWYCSGPPTFECWYYDMDPTE SEQ ID NO: 317 623-H11,
SETRPTEAGSWLCSGPPTFECWWFGTEPTE SEQ ID NO: 318 622-F11,
SETRPTGDGSWYCSGPPTFECWWLGTEPTE SEQ ID NO: 319 621-F03,
SETRPTEAGSWYCSGPPTFECWWYFLDPTE SEQ ID NO: 320 626-F01,
SETRPTEAGGWYCSGPPTFECWWFATEPTE SEQ ID NO: 321 621-F04,
SETRPTEAGDLDCLGPPTFICRIYGTEPTE SEQ ID NO: 322 630-F06,
SETRPTEAGSWQCVGPPTFECWSFGTEPTE SEQ ID NO: 323 630-A03,
SETRPTEADSWYCSGPPTFECWLFGTEPTE SEQ ID NO: 324 629-F10,
SETRPTQADSWYCSGPPTFECWWWGTEPTE SEQ ID NO: 325 629-D11,
SETRPTEAFSWDCSGPPTFECWWFGTEPTE SEQ ID NO: 326 629-B06,
SETRPTEAGSWQCSGPPVFECWWYDTEPTE SEQ ID NO: 327 628-H01,
SETRPTEAGNVQCSGPPTFECWWFDTEPTE SEQ ID NO: 328 628-F03,
SETRPTEAGSVVCSGPPRFECWAFVTEPTE SEQ ID NO: 329 627-G02,
SETRPTEDGTLHCSGPPTFACWWYGTEPTE SEQ ID NO: 330 629-E01,
SETRPTDAEVWVCNGPPTFECWWYGTEPTE SEQ ID NO: 331 628-H09,
SETRPTEDVTFHCSGPPTFECWLYGTEPTE SEQ ID NO: 332 628-A05,
SETRPTSDFDWHCKGPPTFECWSYGTEPTE SEQ ID NO: 333 627-G04,
SETRPTEADSWYCSGPPTFECWWYVPEPTE SEQ ID NO: 334 630-A05,
SETRPTDDGNWYCSGPPTFECWWYGTEPTE SEQ ID NO: 335 629-E03,
SETRPTEAGSWYCSGPPTFECWRYDTDPTE SEQ ID NO: 336 629-C02,
SETRPTEAGPWSCSGPPTFECWWFDTEPTE SEQ ID NO: 337 628-H10,
SETRPTEAGMFLCSGPPAFECWWYDTEPTE SEQ ID NO: 338 628-F12,
SETRPTEAGSLYCSGPPTFECWLYDVEPTE SEQ ID NO: 339 627-D12,
SETRPTEAGQWNCSGPPTFECWWYDIEPTE SEQ ID NO: 340 630-G02,
SETRPTEAGSWYCSGPPTFECWWFETEPTE SEQ ID NO: 341 629-E06,
SETRPTEAGSFVCSGPPTFECWGYVTEPTE SEQ ID NO: 342 628-D07,
SETRPTQDGTWFCSGPPTFECWWYGTEPTE SEQ ID NO: 343 627-E06,
SETRPTEGDSWHCAGPPTFECWWYGTEPTE SEQ ID NO: 344 629-E07,
SETRPTEAGSWSCSGPPTFECWSYGTEPTE SEQ ID NO: 345 629-C11,
SETRPTEAGRIQCSGPPTFECWWYDEEPTE SEQ ID NO: 346 629-A03,
SETRPTEAGTIVCKGPPWFSCEIYETEPTE SEQ ID NO: 347 628-A12,
SETRPTEAGDWYCSGPPAFECWEYLGEPTE SEQ ID NO: 348 627-E08,
SETRPTEAGSWFCSGPPSFECWSYVTEPTE SEQ ID NO: 349 629-E08,
SETRPTEAGSWHCSGPPAFECWWYDNEPTE SEQ ID NO: 350 629-B02,
SETRPTEAGRWTCSGPPTFECWWYVSDPTE SEQ ID NO: 351 628-E06,
SETRPTEAGEWYCGGPPTFECWWFDTAPTE
SEQ ID NO: 352 627-G09, SETRPTEAGSWHCSGPPSFECWWFDTGPTE SEQ ID NO:
353 631-A11, SETRPTEAGSFICSGPPTFECWWYGTEPTE SEQ ID NO: 354 630-C10,
SETRPTEDVRWYCSGPPTFECWWFGTEPTE SEQ ID NO: 355 628-B08,
SETRPTEAGSWYCSGPPTFECWWYVPEPTE SEQ ID NO: 356 629-F03,
SETRPTEAGNWLCSGPPAFECWWFVAEPTE SEQ ID NO: 357 632-A09,
SETRPTEAGSWYCSGPPTFECWWYGTEPTE SEQ ID NO: 358 632-G07,
SETRPTEAGDWLCAGPPTFECWWWGTDPTE SEQ ID NO: 359 631-F12,
SETRPTEAGSWHCVGPPTFECWWFDTEPTE SEQ ID NO: 360 633-A02,
SETRPTEAGEWSCSGPPTFECWWWDMEPTE SEQ ID NO: 361 633-B06,
SETRPTYYVSWYCSGPPTFECWSYGTEPTE SEQ ID NO: 362 632-D11,
SETRPTEDGSWYCSGPPTFECWWYGTEPTE SEQ ID NO: 363 631-D10,
SETRPTEDGTWYCSGPPTFECWWYGTEPTE SEQ ID NO: 364 633-F09,
SETRPTETDSWVCSGPPTFECWWYGTEPTE Consensus Motif#1:
G-X1-X2-X3-C-X4-G-P-P-X5-F-X6-C-X7-X8- X9-X10-X11-X12-P-T-E, where:
X1 is any amino acid other than C, preferably S, R, I, D, or N; X2
is any amino acid other than C, preferably W, L, F, V, or I; X3 is
any amino acid other than C, preferably H, Y, L, Q, N, or V; X4 is
any amino acid other than C, preferably S, K, or L; X5 is any amino
acid other than C, preferably T, S, A, or W; X6 is any amino acid
other than C, preferably E or S; X7 is any amino acid other than C,
preferably W; X8 is any amino acid other than C, preferably W, S,
or L; X9 is any amino acid other than C, preferably Y or F; X10 is
any amino acid other than C, preferably D, G, V, or E; X11 is any
amino acid other than C, preferably T, P, M, or S; and X12 is any
amino acid other than C, preferably E or G. Motif#2:
T-X1-X2-X3-X4-X5-X6-C-X7-G-P-P-X8-F-X9-C-X10-X11- X12-G, where: X1
is any amino acid other than C, preferably E, D, or V; X2 is any
amino acid other than C, preferably A, D, G, S, or V; X3 is any
amino acid other than C, preferably G, V, D, or S; X4 is any amino
acid other than C, preferably S, N, R, T, or G; X5 is any amino
acid other than C, preferably W; X6 is any amino acid other than C,
preferably H or Q; X7 is any amino acid other than C, preferably S,
N, or K; X8 is any amino acid other than C, preferably T; X9 is any
amino acid other than C, preferably E; X10 is any amino acid other
than C, preferably W; X11 is any amino acid other than C,
preferably W or S; and X12 is any amino acid other than C,
preferably Y or F. CLASS V TN10: SEQ ID NO: 365 545-C02,
GSWRFCGGEYSFQVCQDVAP SEQ ID NO: 366 546-E02, GSHHTCLDGFAGWRCTEVAP
SEQ ID NO: 367 545-C11, GSFAPCGWPSFAIDCIAEAP SEQ ID NO: 368
549-G01, GSTKVCHEKWNQLFCHNQAP SEQ ID NO: 369 548-F07,
GSPEMCMMFPFLYPCNHHAP SEQ ID NO: 370 551-H10, GSFFPCWRIDRFGYCHANAP
Consensus Motif: S-X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X10-X11-
C-X12-X13-X14-A-P, where X1 is one of W, H, F, T, or P; X2 is one
of R, H, A, K, E, or F; X3 is one of F, T, P, V, or M; X4 is one of
F, L, H, M, or W; X5 is one of G, D, W, E, M, or R; X6 is one of E,
G, P, K, F, or I; X7 is one of Y, F, S, W, P, or D; X8 is one of S,
A, F, N, or R; X9 is one of F, G, A, Q, or L; X10 is one of Q, W,
I, L, Y, or G; X11 is one of V, R, D, F, P, or Y; X12 is one of Q,
T, I, H, or N; X13 is one of D, E, A, N, or H; and X14 is one of V,
E, Q, H, or N CLASS VI TN11 #1: SEQ ID NO: 371 443-H10,
GSQQICDRKEYRFQACLSDAP SEQ ID NO: 372 557-A12, GSTMSCWRWGRDAYSCNQMAP
SEQ ID NO: 373 465-A03, GSSQICAVYLDDTHNCERHAP SEQ ID NO: 374
446-E12, GSSHCNQMITPWQNCGMRAP SEQ ID NO: 375 445-E06,
GSSARCDELINDFHSCLVMAP SEQ ID NO: 376 452-A03, GSRFHCWQGDLMQTYCMPMAP
SEQ ID NO: 377 465-C06, GSQNNCEYGSRGSSFCLAMAP SEQ ID NO: 378
441-H01, GSMNMCDTTDEISPTCHPSAP SEQ ID NO: 379 443-D04,
GSMLGCLFEHQNKYDCYVLAP SEQ ID NO: 380 445-G12, GSLYRCLGEASPTPPCAYEAP
SEQ ID NO: 381 442-E03, GSGMGCHQVNISTGDCAEDAP SEQ ID NO: 382
453-A05, GSGDPCSPGPSINGHCSVMAP SEQ ID NO: 383 445-E07,
GSFWNCTTDLGAMSDCGFFAP SEQ ID NO: 384 451-B12, GSFTACNKTSTTRQPCNPYAP
SEQ ID NO: 385 465-B07, GSELFCFYHHQGYEGCDVLAP SEQ ID NO: 386
451-C06, GSDMNCTVLAQDQIFCFREAP SEQ ID NO: 387 445-E11,
GSAGWCYTMNYVDQLCTYMAP Consensus Motif:
S-X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X10-X11- X12-C-X13-X14-X15-A-P,
where X1 is any amino acid other than C, preferably S, F, G, M, or
Q; X2 is any amino acid other than C, preferably M, L, N, or Q; X3
is any amino acid other than C, preferably N, G, H, I, or R; X4 is
any amino acid other than C, preferably D, L, N, T, or W; X5 is any
amino acid other than C, preferably Q, T, R, V, or Y; X6 is any
amino acid other than C, preferably G, E, L, M, or T; X7 is any
amino acid other than C, preferably A, D, H, I, L, N, or S; X8 is
any amino acid other than C, preferably Q, R, S, T, or Y; X9 is any
amino acid other than C, preferably D, G, I, or P; X10 is any amino
acid other than C, preferably T, F, or Q; X11 is any amino acid
other than C, preferably Q, F, H, P, S, or Y; X12 is any amino acid
other than C, preferably D, F, N, P, or S; X13 is any amino acid
other than C, preferably L, A, G, N, or S; X14 is any amino acid
other than C, preferably V, P, R, or Y; and X15 is any amino acid
other than C, preferably M, D, E, or L. CLASS VII TN11 #2 SEQ ID
NO: 388 593-G11, SETRPTEAGMCACRGPPAFVCQWYGSEPTE SEQ ID NO: 389
631-E12, SETRPTEAGSCHCSGPPTFECWSYVTEPTE CLASS VIII TN12: SEQ ID NO:
390 546-G02, GDYDYCDFDLETYIPECHSYDP SEQ ID NO: 391 333-C03,
GDDFHCEFIDDYQSEICYFNDP SEQ ID NO: 392 549-G05,
GDLLVCKFDDKFWTETCEWADP SEQ ID NO: 393 546-B01,
GDSYNCSWDSKTFEVTCLYADP SEQ ID NO: 394 551-D02,
GDASWCDENSPAAWFYCELWDP SEQ ID NO: 395 334-F05,
GDLLGCGYQEKGGEYKCRFNDP SEQ ID NO: 396 330-G02,
GDPWWCFEKDSFIPFACWHHDP SEQ ID NO: 397 316-F08,
GDYYQCQFSKDMYSERCWPYDP SEQ ID NO: 398 332-H09,
GDNRFCSWVYNVDDWWCVDNDP SEQ ID NO: 399 545-H12,
GDYSECFFEPDSFEVKCYDRDP SEQ ID NO: 400 548-G05,
GDYRMCQISDMWGNYECSSDDP SEQ ID NO: 401 547-C09,
GDPDECQLNRETFEVWCPWHDP SEQ ID NO: 402 545-F04,
GDHRKCEISAKTHEVTCYDNDP SEQ ID NO: 403 552-F06,
GDHLTCEFRDDGWKEHCWWSDP SEQ ID NO: 404 531-E11,
GDASMCYDGLALRWDQCWPHDP Consensus Motif:
D-X1-X2-X3-C-X4-X5-X-6-X7-X8-X9-X10- X11-X12-X13-C-X14-X15-X16-D-P,
where X1 is any amino acid other than C, preferably Y, A, H, L, or
P; X2 is any amino acid other than C, preferably L, R, S, D, or Y;
X3 is any amino acid other than C, preferably E, M, or W; X4 is any
amino acid other than C, preferably E, Q, D, F, or S; X5 is any
amino acid other than C, preferably F, I, W, or E; X6 is any amino
acid other than C, preferably D, S, or N; X7 is any amino acid
other than C, preferably D, S, or L; X8 is any amino acid other
than C, preferably D, K, or E; X9 is any amino acid other than C,
preferably T, F, or G; X10 is any amino acid other than C,
preferably F, W, Y, or G; X11 is any amino acid other than C,
preferably E, S, or W; X12 is any amino acid other than C,
preferably E, V, F, or Y; X13 is any amino acid other than C,
preferably T, E, K, or V; X14 is any amino acid other than C,
preferably W or E; X15 is any amino acid other than C, preferably
D, W, F, P, or S; and X16 is any amino acid other than C,
preferably N, I, or A. CLASS IX TN9 #3: SEQ ID NO: 405 606-B08,
SETRPTEAGSCHCSGPPTFQCWCYEVEPTE SEQ ID NO: 406 602-G12,
SETRPTEAGSCHCSGPPTFECWCYGTEPTE SEQ ID NO: 407 603-E09,
SETRPTGESDCHCSGPPTFECYCYGTEPTE SEQ ID NO: 408 606-C12,
SETRPTESGNCYCSGPPWFECWCYGTEPTE SEQ ID NO: 409 603-H03,
SETRPTEAGACRCSGPPTFECYCYDMAPTE SEQ ID NO: 410 604-G01,
SETRPTEAGSCYCSGPPRFECWCYETEPTE SEQ ID NO: 411 602-G04,
SETRPTEAGSCHCSGPPSFECWCFGTEPTE SEQ ID NO: 412 611-G11,
SETRPTVSVSCSCGGPPTFECWCFGTEPTE SEQ ID NO: 413 611-F02,
SETRPTEAGSCHCNGPPTFECFCFGTEPTE SEQ ID NO: 414 610-G02,
SETRPTEAGSCYCGGPPSFECWCYGTEPTE SEQ ID NO: 415 614-E08,
SETRPTEAGSCHCSGPPTFECWCYGSNPTE SEQ ID NO: 416 615-A01,
SETRPTEAGSCHCSGPPAFECWCYRAEPTE SEQ ID NO: 417 617-H02,
SETRPTEAGSCDCSGPPTFECWCFGTEPTE
SEQ ID NO: 418 616-F12, SETRPTEAGKCHCGGPPSFECWCYATEPTE SEQ ID NO:
419 620-G06, SETRPTEAGKCHCSGPPTFECTCYHTDPTE SEQ ID NO: 420 627-B04,
SETRPTEAGFCQCSGPPAFECWCYDTEPTE SEQ ID NO: 421 627-B06,
SETRPTEAVSCECKGPPTFECWCFGTEPTE SEQ ID NO: 422 626-H05,
SETRPTEAGDCHCSGPPTFECWCYGTEPTE SEQ ID NO: 423 626-D11,
SETRPTEAGACDCIGPPTFECWCYDTYPTE SEQ ID NO: 424 626-E05,
SETRPTEAGNCLCSGPPTFECACYHSEPTE SEQ ID NO: 425 621-D01,
SETRPTEAGSCHCSGPPTFQCWCYSTEPTE SEQ ID NO: 426 622-A10,
SETRPTEAGICHCSGPPTFECWCYATEPTE SEQ ID NO: 427 630-D09,
SETRPTEEGSCHCSGPPTFECWCFGTEPTE SEQ ID NO: 428 628-D01,
SETRPTEAGICNCSGPPTFECWCYSMGPTE SEQ ID NO: 429 628-F11,
SETRPTQGGNCHCSGPPTFECWCYGTEPTE SEQ ID NO: 430 628-D04,
SETRPTEAGSCNCSGPPTFECYCYTLDPTE SEQ ID NO: 431 630-G01,
SETRPTDNGSCQCSGPPTFECWCFGTEPTE SEQ ID NO: 432 627-G06,
SETRPTESGSCHCSGPPTFECWCYGTEPTE SEQ ID NO: 433 630-G05,
SETRPTEAGSCNCSGPPSFECWCYVTEPTE SEQ ID NO: 434 630-C03,
SETRPTEGGSCYCGGPPTFECWCYGTEPTE SEQ ID NO: 435 627-G07,
SETRPTEAGRCHCSGPPTFECWCYVQEPTE SEQ ID NO: 436 630-H10,
SETRPTESGSCLCSGPPQFECWCYGTEPTE SEQ ID NO: 437 628-B01,
SETRPTETDSCHCIGPPTFECWCYGTEPTE SEQ ID NO: 438 630-F01,
SETRPTEAGFCRCSGPPTFECWCYDTEPTE SEQ ID NO: 439 629-D01,
SETRPTEHGSCNCYGPPTFECWCYGTEPTE SEQ ID NO: 440 633-G02,
SETRPTALGGCLCSGPPTFECWCYGTEPTE SEQ ID NO: 441 631-F07,
SETRPTEGGSCECSGPPTFECWCYGTEPTE SEQ ID NO: 442 633-G08,
SETRPTEEGSCHCSGPPAFECWCYGTEPTE SEQ ID NO: 443 632-H07,
SETRPTEAGTCYCSGPPTFECWCYGTEPTE SEQ ID NO: 444 631-D03,
SETRPTEDGSCHCSGPPRFECWCYGTEPTE SEQ ID NO: 445 633-G12,
SETRPTEAGSCHCSGPPTFECWCYSTEPTE SEQ ID NO: 446 633-H03,
SETRPTEAGSCYCSGPPTFECWCYAEEPTE SEQ ID NO: 447 632-F05,
SETRPTEAGSCHCSGPPTFECWCFEPEPTE Motif13-1
G-X1-C-X2-C-X3-G-P-P-X4-F-X5-C-X6-C-X7-X8-X9-X10- P, where X1 is
any amino acid other than C, preferably S; X2 is any amino acid
other than C, preferably H, Y, or N; X3 is any amino acid other
than C, preferably S or G; X4 is any amino acid other than C,
preferably T; X5 is any amino acid other than C, preferably E; X6
is any amino acid other than C, preferably W; X7 is any amino acid
other than C, preferably Y; X8 is any amino acid other than C,
preferably G, D, A, E, or S; X9 is any amino acid other than C,
preferably T or S; and X10 is any amino acid other than C,
preferably E or D. Motif13-2 is
T-X1-X2-X3-X4-C-X5-C-X6-G-P-P-X7-F-E-C-X8-C- X9-G where: X1 is any
amino acid other than C, preferably E; X2 is any amino acid other
than C, preferably A, S, E, or G; X3 is any amino acid other than
C, preferably G; X4 is any amino acid other than C, preferably S;
X5 is any amino acid other than C, preferably H; X6 is any amino
acid other than C, preferably S; X7 is any amino acid other than C,
preferably T; X8 is any amino acid other than C, preferably W, Y,
or F; and X9 is any amino acid other than C, preferably Y or F.
CLASS X SEQ ID NO: 448 606-E11, SEYPTWVSKEFHECAGELVAMQGGSGTE CLASS
XI Linear #1: SEQ ID NO: 449 525-A07, AQQASRFTFTDGDSYWWFEDF SEQ ID
NO: 450 528-F05, AQIQGIQKTEQGEFYWFNWFPA SEQ ID NO: 451 524-E09,
AQREVEEPYWYLDFLSSWRMHE SEQ ID NO: 452 96-H12,
AQRPEAHYKLAMSYPIIPRTKT SEQ ID NO: 453 118-A08,
AQRWSSPGMSQSFVLEWKWNDN SEQ ID NO: 454 94-E08,
AQYDTWVFQFIHEVPGELVAMQ SEQ ID NO: 455 119-F06, AQMYQTPDGVIGKFVDWMFN
SEQ ID NO: 456 95-A11, AQVGSPMLPSWFSFEANWSS SEQ ID NO: 457 94-H04,
AQNAVVPPPMLWSIYWDYGREG SEQ ID NO: 458 94-F07,
AQPYYELQDADMLLVVALLSTG SEQ ID NO: 459 103-G08,
AQVGTAEAIMFSDVEDTGVHKF SEQ ID NO: 460 118-C07,
AQFPLEFDVPNFSYHWLVSFNP SEQ ID NO: 461 104-C09,
AQDLKPWTAGWEPPWLWTDRGP SEQ ID NO: 462 117-F08,
AQHQYGQMMVLHIQYDMGEFIP SEQ ID NO: 463 76-D09,
AQSPYIFPIDDSGRQIFVIQWG SEQ ID NO: 464 93-C08,
AQVPDWLSAVVIEKLIEYGMMV SEQ ID NO: 465 92-B05,
AQFDRYWHFAWMDVSFSSGQSG SEQ ID NO: 466 116-H02,
AQKETWEFFDIVYGSGWKFNSP SEQ ID NO: 467 02-B08,
AQHSVQRQMDVWMPVQFMAGFT SEQ ID NO: 468 117-F03,
AQEWQTWTWNMIEVISENKTP SEQ ID NO: 469 127-A07, AQGFELWVDHTRNFFIAISP
SEQ ID NO: 470 94-B08, AQAYEWWADESIFNHGYYWGHQ SEQ ID NO: 471
115-G02, AQDPGFSKHSMGHGYPSKMNWG SEQ ID NO: 472 130-E10,
AQEWEREYFVDGFWGSWFGIPH SEQ ID NO: 473 136-D01,
AQMGHHWDVQWDYKLFHVARGD SEQ ID NO: 474 15-D02,
AQELFQILEKQMWSDFMEWATP SEQ ID NO: 475 79-B02,
AQHWDYDSGSDFWFPVFFLEHH SEQ ID NO: 476 94-A06,
AQHGYLSPLKQYQMSHVEFWTY SEQ ID NO: 477 94-G02,
AQFSGLVMYGRTHEVQWTFGSM SEQ ID NO: 478 75-B12,
AQAEWVITSEEFYWKMADFGPP SEQ ID NO: 479 117-F04, AQWPHDGLVHWGEVIMLRF
SEQ ID NO: 480 151-B08, AQWNQWDEFMWFLNPPPIGLMW SEQ ID NO: 481
117-E09, AQDNTADQMFNGFHVLAMYMV SEQ ID NO: 482 93-B10,
AQSDHDHAHWGVKHWPFRRYQ SEQ ID NO: 483 98-F05, AQLFQYLWHDDPQGAFFQLSMW
SEQ ID NO: 484 118-B12, AQHVVTLTLIQMPFAFNFEPRM SEQ ID NO: 485
27-D10, AQVGESLDDGWTFFSDKWFDFF SEQ ID NO: 486 122-D07,
AQFMYEKEHYVMSISLPGLWFY SEQ ID NO: 487 149-E06,
AQHMDPAEWDWFIRIYSPVVNP SEQ ID NO: 488 166-H04,
AQMWHRVHDPGYTFEVTWLWDN SEQ ID NO: 489 96-D06,
AQWNWDMGFMWTTDSAQVQPSM SEQ ID NO: 490 103-C04,
AQKTWFLEADLFQMFQEVTWQF SEQ ID NO: 491 527-E08,
AQWGAVDNDWYDWEMEQIWMFE SEQ ID NO: 492 524-H02,
AQVEDMATVHFKFNPATHEVIW SEQ ID NO: 493 523-A04,
AQRDYLFYWNDGSYQPWQVFVG SEQ ID NO: 494 524-D07,
AQQWMFQIHQSMAWPYEWIDSY SEQ ID NO: 495 522-H03,
AQGIAWQLEWSYMPQSPPSFDR SEQ ID NO: 496 527-A10,
AQGGRYPFYDTDWFKWEMYVL CLASS XII Linear #2 SEQ ID NO: 497 594-F01,
SEEDTWLFWQIIEVPVGQVLMQGGSGTE SEQ ID NO: 498 592-E11,
SEYDTLLFQRTGEVVGKLGSMQGGSGTE SEQ ID NO: 499 591-G09,
SEYDTWVFQFMLEVPGSWMARLGGSGTE SEQ ID NO: 500 601-G11,
SEYDTWIFQFYREVPGVPGAMQGGSGTE SEQ ID NO: 501 592-G01,
SEVDTGVQLLTHEGPGELVAMQGGSGTE SEQ ID NO: 502 591-H01,
SESDTWVFQLIHEVPASVVAMQGGSGTE SEQ ID NO: 503 592-G05,
SEYDTWVFQFRHGVKAQLVAMRGGSGTE SEQ ID NO: 504 606-D12,
SEYDSRVFQYAPEVAGQVEAMQGGSGTE SEQ ID NO: 505 592-B01,
SEDESRVVQFQHEVSGELVAMQGGSGTE SEQ ID NO: 506 591-A06,
SEQDTFVFMYNGEVSGDMVAMQGGSGTE SEQ ID NO: 507 588-H01,
SEYDTWVFQFRRQVPGVLETMLGGSGTE SEQ ID NO: 508 589-A01,
SEQETLVFAVIDGDPGELVAMQGGSGTE SEQ ID NO: 509 619-F10,
SEYDTWVFQFIHVARGEMEGTLGGSGTE SEQ ID NO: 510 592-B01,
SEDESRVVQFQHEVSGELVAMQGGSGTE SEQ ID NO: 511 591-A06,
SEQDTFVFMYNGEVSGDMVAMQGGSGTE
TABLE-US-00009 TABLE 7 Protein WC HGF 100 HGF 500 SEQ ID NO:
Isolate ELISA ELISA ng/mL ng/mL CLASS I SEQ ID 571-C05 4.9 1.30
102% 74% NO: 001 SEQ ID 465-A06 4.4 1.33 56% 32% NO: 002 SEQ ID
465-D09 3.2 1.30 90% 70% NO: 003 SEQ ID 569-H10 3.4 1.27 98% 83%
NO: 004 SEQ ID 470-E11 3.5 1.33 55% 127% NO: 005 SEQ ID 452-F01 3.2
1.33 117% 110% NO: 006 SEQ ID 569-C03 3.4 1.30 95% 89% NO: 007 SEQ
ID 574-H03 3.2 1.27 88% 18% NO: 008 SEQ ID 567-C08 3.8 1.27 85% 94%
NO: 009 SEQ ID 561-C08 3.0 1.37 92% 96% NO: 010 CLASS II SEQ ID
573-F04 5.6 1.30 76% 71% NO: 011 SEQ ID 570-E07 4.5 1.27 81% 71%
NO: 012 SEQ ID 456-E04 3.9 1.40 82% 81% NO: 013 SEQ ID 434-E12 4.8
1.33 117% 41% NO: 014 SEQ ID 489-A04 4.3 1.33 30% 13% NO: 015 SEQ
ID 484-D08 4.1 1.33 105% 90% NO: 016 SEQ ID 482-D02 3.9 1.37 66%
44% NO: 017 SEQ ID 437-A09 3.9 1.13 89% 78% NO: 018 SEQ ID 352-E04
3.9 1.37 88% 74% NO: 019 SEQ ID 376-E05 3.7 1.37 122% 121% NO: 020
SEQ ID 482-A12 3.5 1.37 98% 79% NO: 021 SEQ ID 423-C11 3.4 1.40
132% 75% NO: 022 SEQ ID 499-C09 3.2 1.33 91% 70% NO: 023 SEQ ID
457-A09 14.5 1.30 27% 67% NO: 024 SEQ ID 573-E07 3.2 1.37 77% 82%
NO: 025 SEQ ID 465-F08 3.8 1.30 68% 116% NO: 026 SEQ ID 465-E09 3.6
1.30 60% 77% NO: 027 SEQ ID 444-B08 3.6 1.43 111% 93% NO: 028 SEQ
ID 465-E11 4.3 1.23 33% 124% NO: 029 SEQ ID 465-D12 3.2 1.27 34% 0%
NO: 030 SEQ ID 470-A02 3.2 1.30 78% 62% NO: 031 SEQ ID 465-C01 3.2
1.27 267% 23% NO: 032 SEQ ID 448-H02 3.8 1.43 113% 92% NO: 033 SEQ
ID 465-D01 3.3 1.30 235% 134% NO: 034 SEQ ID 571-C11 3.5 1.23 107%
72% NO: 035 SEQ ID 465-B11 3.6 1.27 97% 89% NO: 036 SEQ ID 442-E08
4.1 1.43 81% 75% NO: 037 SEQ ID 465-C11 3.1 1.30 41% 4% NO: 038 SEQ
ID 465-F10 3.7 1.33 61% 42% NO: 039 SEQ ID 471-A11 3.0 1.37 85% 80%
NO: 040 SEQ ID 465-C07 3.1 1.27 102% 138% NO: 041 SEQ ID 465-D04
3.1 1.23 77% 31% NO: 042 SEQ ID 445-E04 4.2 1.37 127% 102% NO: 043
SEQ ID 465-B06 4.1 1.23 89% 57% NO: 044 SEQ ID 470-C02 3.9 1.33
340% 227% NO: 045 SEQ ID 458-B05 4.5 1.33 201% 247% NO: 046 SEQ ID
545-E08 4.7 1.30 81% 57% NO: 047 CLASS III SEQ ID 325-H05 15.9 1.47
41% 32% NO: 048 SEQ ID 330-F05 13.8 1.33 51% 27% NO: 049 SEQ ID
333-F09 14.8 1.43 52% 32% NO: 050 SEQ ID 336-G04 5.4 1.33 46% 23%
NO: 051 SEQ ID 334-G06 8.0 1.30 56% 43% NO: 052 SEQ ID 330-B07 18.1
1.27 58% 40% NO: 053 SEQ ID 330-C10 13.4 1.33 48% 25% NO: 054 SEQ
ID 331-G04 18.3 1.47 56% 36% NO: 055 SEQ ID 548-F06 14.3 1.23 76%
18% NO: 056 SEQ ID 538-F08 12.3 1.23 55% 43% NO: 057 SEQ ID 547-H07
15.9 1.17 60% 45% NO: 058 SEQ ID 323-A11 21.2 1.43 41% 18% NO: 059
SEQ ID 333-H03 8.1 1.43 55% 37% NO: 060 SEQ ID 329-D02 3.2 1.27 53%
31% NO: 061 SEQ ID 550-C09 10.2 1.40 25% 25% NO: 062 SEQ ID 548-E08
5.3 1.27 102% 50% NO: 063 SEQ ID 332-A05 6.0 1.40 40% 21% NO: 064
SEQ ID 330-C01 4.7 1.30 58% 43% NO: 065 SEQ ID 545-A09 13.5 1.30
44% 22% NO: 066 SEQ ID 334-C08 8.0 1.47 70% 57% NO: 067 SEQ ID
333-C05 6.3 1.33 83% 66% NO: 068 SEQ ID 551-B02 9.0 1.30 69% 43%
NO: 069 SEQ ID 551-G12 3.9 1.37 88% 46% NO: 070 SEQ ID 330-G09 13.5
1.40 42% 26% NO: 071 SEQ ID 331-F01 12.6 1.47 77% 73% NO: 072 SEQ
ID 274-B07 7.8 1.10 342% 296% NO: 073 SEQ ID 335-D11 6.7 1.37 56%
37% NO: 074 SEQ ID 336-D07 5.8 1.33 44% 37% NO: 075 SEQ ID 332-C03
5.7 1.20 37% 95% NO: 076 SEQ ID 331-D03 5.5 1.40 64% 55% NO: 077
SEQ ID 331-G06 4.7 1.40 59% 51% NO: 078 SEQ ID 552-G03 10.7 1.27
101% 83% NO: 079 SEQ ID 552-G11 7.4 1.23 55% 41% NO: 080 SEQ ID
550-G08 9.1 1.40 79% 58% NO: 081 SEQ ID 550-G12 14.3 1.43 61% 79%
NO: 082 SEQ ID 552-A01 3.9 1.33 76% 81% NO: 083 SEQ ID 548-C06 13.0
1.23 94% 77% NO: 084 SEQ ID 545-B12 17.1 1.27 51% 42% NO: 085 SEQ
ID 549-F06 5.2 1.30 96% 40% NO: 086 SEQ ID 552-F01 4.8 1.30 56% 37%
NO: 087 SEQ ID 547-H12 5.6 1.10 92% 81% NO: 088 SEQ ID 550-F11 12.4
1.23 58% 23% NO: 089 SEQ ID 548-D08 19.5 1.23 97% 62% NO: 090 SEQ
ID 549-D02 8.9 1.27 47% 36% NO: 091 SEQ ID 552-F02 12.3 1.23 60%
40% NO: 092 SEQ ID 545-E04 16.3 1.23 48% 17% NO: 093 SEQ ID 545-E05
10.3 1.27 70% 32% NO: 094 SEQ ID 547-H03 16.2 1.23 109% 53% NO: 095
SEQ ID 552-G09 9.7 1.27 98% 68% NO: 096 SEQ ID 550-A08 8.4 1.27 52%
51% NO: 097 SEQ ID 550-G07 6.2 1.27 63% 36% NO: 098 SEQ ID 551-A05
4.0 1.30 68% 42% NO: 099 SEQ ID 548-C10 8.4 1.20 69% 57% NO: 100
SEQ ID 465-C10 3.0 1.27 95% 71% NO: 101 CLASS V SEQ ID 545-C02 26.3
1.33 54% 31% NO: 365 SEQ ID 546-E02 10.4 1.33 74% 54% NO: 366 SEQ
ID 545-C11 7.7 1.30 77% 50% NO: 367 SEQ ID 549-G01 7.0 1.27 62% 18%
NO: 368 SEQ ID 548-F07 27.5 2.43 54% 37% NO: 369 SEQ ID 551-H10
13.3 1.87 88% 49% NO: 370 CLASS VI SEQ ID 443-H10 3.4 1.40 124%
143% NO: 371 SEQ ID 557-A12 4.6 1.37 87% 62% NO: 372 SEQ ID 465-A03
4.0 1.30 33% 17% NO: 373 SEQ ID 446-E12 3.3 1.37 73% 83% NO: 374
SEQ ID 445-E06 4.3 1.33 83% 73% NO: 375 SEQ ID 452-A03 3.0 1.30
140% 112% NO: 376 SEQ ID 465-C06 6.4 1.23 184% 104% NO: 377 SEQ ID
441-H01 3.6 1.40 91% 69% NO: 378 SEQ ID 443-D04 3.2 1.43 69% 73%
NO: 379 SEQ ID 445-G12 4.0 1.37 85% 52% NO: 380 SEQ ID 442-E03 3.9
1.43 130% 81% NO: 381 SEQ ID 453-A05 4.5 1.33 51% 28% NO: 382 SEQ
ID 445-E07 3.1 1.37 82% 64%
NO: 383 SEQ ID 451-B12 3.1 1.37 61% 27% NO: 384 SEQ ID 465-B07 4.8
1.27 111% 79% NO: 385 SEQ ID 451-C06 3.0 1.37 108% 86% NO: 386 SEQ
ID 445-E11 3.7 1.43 69% 79% NO: 387 CLASS VIII SEQ ID 546-G02 16.1
1.27 32% 19% NO: 390 SEQ ID 333-C03 12.4 1.37 52% 43% NO: 391 SEQ
ID 549-G05 23.7 1.47 28% 21% NO: 392 SEQ ID 546-B01 8.4 1.20 95%
77% NO: 393 SEQ ID 551-D02 13.4 1.37 91% 70% NO: 394 SEQ ID 334-F05
13.5 1.40 58% 29% NO: 395 SEQ ID 330-G02 7.4 1.30 37% 31% NO: 396
SEQ ID 316-F08 7.0 1.30 .sup. 72% - 38% NO: 397 SEQ ID 332-H09 6.2
1.30 50% 43% NO: 398 SEQ ID 545-H12 11.3 1.30 74% 60% NO: 399 SEQ
ID 548-G05 6.1 1.30 110% 47% NO: 400 SEQ ID 547-C09 4.3 1.23 50%
32% NO: 401 SEQ ID 545-F04 5.2 1.17 143% 114% NO: 402 SEQ ID
552-F06 11.1 1.23 82% 32% NO: 403 SEQ ID 531-E11 3.4 1.30 61% 33%
NO: 404 CLASS XI SEQ ID 525-A07 7.0 1.17 93% 88% NO: 449 SEQ ID
528-F05 4.3 1.10 84% 81% NO: 450 SEQ ID 524-E09 8.2 1.33 100% 93%
NO: 451 SEQ ID 96-H12 35.3 1.37 88% 64% NO: 452 SEQ ID 118-A08 11.3
1.30 85% 74% NO: 453 SEQ ID 94-E08 8.9 1.23 102% 74% NO: 454 SEQ ID
119-F06 8.0 1.33 4% 27% NO: 455 SEQ ID 95-A11 7.0 1.30 109% 108%
NO: 456 SEQ ID 94-H04 7.0 1.37 150% 101% NO: 457 SEQ ID 94-F07 6.1
1.20 106% 104% NO: 458 SEQ ID 103-G08 5.7 1.33 140% 95% NO: 459 SEQ
ID 118-C07 5.6 1.27 100% 84% NO: 460 SEQ ID 104-C09 5.0 1.30 64%
50% NO: 461 SEQ ID 117-F08 4.5 1.27 102% 270% NO: 462 SEQ ID 76-D09
4.4 1.23 79% 87% NO: 463 SEQ ID 93-C08 4.4 1.37 101% 96% NO: 464
SEQ ID 92-B05 4.3 1.20 94% 94% NO: 465 SEQ ID 116-H02 4.0 1.23 84%
72% NO: 466 SEQ ID 02-B08 3.9 1.30 84% 96% NO: 467 SEQ ID 117-F03
3.8 1.40 104% 93% NO: 468 SEQ ID 127-A07 3.8 1.20 101% 107% NO: 469
SEQ ID 94-B08 3.8 1.20 111% 121% NO: 470 SEQ ID 115-G02 3.7 1.27
59% 0% NO: 471 SEQ ID 130-E10 3.7 1.80 100% 92% NO: 472 SEQ ID
136-D01 3.7 1.23 85% 149% NO: 473 SEQ ID 15-D02 3.6 1.23 97% 118%
NO: 474 SEQ ID 79-B02 3.5 1.30 102% 86% NO: 475 SEQ ID 94-A06 3.5
1.17 84% 96% NO: 476 SEQ ID 94-G02 3.5 1.30 108% 76% NO: 477 SEQ ID
75-B12 3.4 1.23 95% 108% NO: 478 SEQ ID 117-F04 3.3 1.37 93% 91%
NO: 479 SEQ ID 151-B08 3.3 1.23 102% 368% NO: 480 SEQ ID 117-E09
3.3 1.37 109% 102% NO: 481 SEQ ID 93-B10 3.1 1.20 0% 0% NO: 482 SEQ
ID 98-F05 3.1 1.23 88% 57% NO: 483 SEQ ID 118-B12 3.1 1.30 98% 112%
NO: 484 SEQ ID 27-D10 3.0 1.17 111% 131% NO: 485 SEQ ID 122-D07 3.0
1.63 102% 92% NO: 486 SEQ ID 149-E06 3.0 1.80 80% 86% NO: 487 SEQ
ID 166-H04 3.0 1.27 77% 85% NO: 488 SEQ ID 96-D06 3.0 1.37 154%
151% NO: 489 SEQ ID 103-C04 3.0 1.40 73% 86% NO: 490 SEQ ID 527-E08
3.2 1.23 98% 95% NO: 491 SEQ ID 524-H02 3.2 1.53 26% 25% NO: 492
SEQ ID 523-A04 5.5 1.30 133% 143% NO: 493 SEQ ID 524-D07 3.9 1.23
105% 104% NO: 494 SEQ ID 522-H03 4.5 1.17 107% 94% NO: 495 SEQ ID
527-A10 3.8 1.30 84% 78% NO: 496 Note: Protein ELISAs were measured
as fold over background (cMet-Fc vs. TRAIL-Fc) Whole Cell ELISAs
were measured as fold over background (3T3 cells expressing human
cMet vs. non-expressing 3T3 cells) HGF competition ELISA measured
as a % of binding in the absence of HGF.
TABLE-US-00010 TABLE 8 Fluorescence polarization analysis of select
peptides from first generation peptide library positive hits SEQ ID
NO: Isolate Kd (human) Kd (mouse) CLASS I SEQ ID NO: 001 571-C05
0.20 3.50 CLASS III SEQ ID NO: 048 325-H05 3.50 NT SEQ ID NO: 051
336-G04 3.20 NT SEQ ID NO: 052 334-G06 2.70 NT SEQ ID NO: 053
330-B07 2.90 NT SEQ ID NO: 055 331-G04 0.90 1.10 SEQ ID NO: 056
548-F06 2.70 NT SEQ ID NO: 059 323-A11 4.30 NT SEQ ID NO: 061
329-D02 5.20 NT SEQ ID NO: 067 334-C08 1.65 NT SEQ ID NO: 068
333-C05 2.80 NT SEQ ID NO: 071 330-G09 1.85 NT SEQ ID NO: 072
331-F01 0.98 NT SEQ ID NO: 074 335-D11 3.30 NT SEQ ID NO: 078
331-G06 2.90 NT CLASS V SEQ ID NO: 369 548-F07 0.88 NB SEQ ID NO:
370 551-H10 0.22 NB CLASS VIII SEQ ID NO: 390 546-G02 1.50 NT SEQ
ID NO: 391 333-C03 1.80 NT SEQ ID NO: 399 545-H12 1.15 NB CLASS XI
SEQ ID NO: 449 525-A07 6.90 NT SEQ ID NO: 450 528-F05 2.70 NT SEQ
ID NO: 451 524-E09 2.00 NT SEQ ID NO: 452 96-H12 >2.00 NT SEQ ID
NO: 453 118-A08 >2.00 NT SEQ ID NO: 454 94-E08 0.93 NT SEQ ID
NO: 456 95-A11 2.30 NT SEQ ID NO: 458 94-F07 3.75 NT SEQ ID NO: 459
103-G08 >2.00 NT SEQ ID NO: 461 104-C09 >2.00 NT SEQ ID NO:
462 117-F08 >2.00 NT SEQ ID NO: 463 76-D09 >2.00 NT SEQ ID
NO: 464 93-C08 >2.00 NT SEQ ID NO: 466 116-H02 >2.00 NT SEQ
ID NO: 467 02-B08 >2.00 NT SEQ ID NO: 469 127-A07 2.40 NT SEQ ID
NO: 472 130-E10 2.60 7.65 SEQ ID NO: 475 79-B02 1.90 NT SEQ ID NO:
479 117-F04 1.70 NT SEQ ID NO: 492 524-H02 0.80 NT Kd values are in
.mu.M. NB = no binding, NT = not tested
TABLE-US-00011 TABLE 9 cMet-binding heteromeric peptide complexes
SEQ ID NO: Isolate CLASS PAIR I SEQ ID NO: 472 130-E10 XI SEQ ID
NO: 370 551-H10 V PAIR II SEQ ID NO: 369 548-F07 V SEQ ID NO: 370
551-H10 V PAIR III SEQ ID NO: 370 551-H10 V SEQ ID NO: 399 545-H12
VIII
TABLE-US-00012 TABLE 10 Amino-acid sequence of Mature HSA from
GenBank entry AAN17825 DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV
KLVNEVTEFA KTCVADESAE NCDKSLHTLF GDKLCTVATL RETYGEMADC CAKQEPERNE
CFLQHKDDNP NLPRLVRPEV DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR
YKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLS
QRFPKAEFAE VSKLVTDLTK VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE
KPLLEKSHCI AEVENDEMPA DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPD
YSVVLLLRLA KTYKTTLEKC CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE
YKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKH PEAKRMPCAE DYLSVVLNQL
CVLHEKTPVS DRVTKCCTES LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE
RQIKKQTALV ELVKHKPKAT KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASR
AALGL (SEQ ID NO: 647)
TABLE-US-00013 TABLE 11 Amino-acid Sequence of SEQ ID NO:
648::HSA::SEQ ID NO: 649 GSFFPCWRIDRFGYCHANAP GSGGSGG DAHKSEVAHR
FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA KTCVADESAE NCDKSLHTLF
GDKLCTVATL RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV DVMCTAFHDN
EETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQ AADKAACLLP KLDELRDEGK
ASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK VHTECCHGDL
LECADDRADL AKYICENQDS ISSKLKECCE KPLLEKSHCI AEVENDEMPA DLPSLAADFV
ESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYKTTLEKC CAAADPHECY
AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVR YTKKVPQVST PTLVEVSRNL
GKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES LVNRRPCFSA
LEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKAT KEQLKAVMDD
FAAFVEKCCK ADDKETCFAE EGKKLVAASR AALGL GSGGEGGSG GSWIICWWDNCGSSAP
(SEQ ID NO: 650)
[0283] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
619116PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 1Gly Ser Trp Ile Ile Cys Trp Trp Asp Asn Cys Gly
Ser Ser Ala Pro1 5 10 15216PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 2Gly Ser Tyr Tyr Asp Cys
Arg Glu Phe Gln Cys Asn Lys Pro Ala Pro1 5 10 15316PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 3Gly
Ser Ser His Leu Cys Asn Pro Glu Phe Cys His Phe Thr Ala Pro1 5 10
15416PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 4Gly Ser Met Leu Met Cys Glu Leu Trp Trp Cys Arg
Phe Leu Ala Pro1 5 10 15516PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 5Gly Ser Leu Ile Phe Cys
Pro Tyr Gly Glu Cys Met Met Tyr Ala Pro1 5 10 15616PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 6Gly
Ser Glu Tyr Ser Cys Arg Thr Ser Arg Cys Ile Phe Ser Ala Pro1 5 10
15716PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 7Gly Ser Phe Ile Leu Cys Trp Trp Thr Phe Cys Asp
Thr Asn Ala Pro1 5 10 15816PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 8Gly Ser Ser Thr Ile Cys
Pro Gly Thr Ala Cys Val Asp His Ala Pro1 5 10 15916PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 9Gly
Ser Leu Ile Ile Cys Trp Trp Ser Trp Cys Asp Lys Gln Ala Pro1 5 10
151016PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 10Gly Ser Phe Asn Ile Cys Pro Tyr Gln Trp Cys Thr
Leu Trp Ala Pro1 5 10 151118PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 11Ala Gly Gly Phe Ala Cys
Gly Pro Pro Trp Asp Ile Cys Trp Met Phe1 5 10 15Gly
Thr1218PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 12Ala Gly Ala Trp Asn Cys Glu Tyr Pro Thr Phe Ile
Cys Glu Trp Gln1 5 10 15Gly Ala1318PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 13Ala
Gly Asn Trp Ile Cys Asn Leu Ser Glu Met Arg Cys Tyr Pro Lys1 5 10
15Gly Thr1418PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 14Ala Gly Asp Gly Trp Cys Met Ala Trp
Pro Glu Ile Cys Glu Trp Leu1 5 10 15Gly Thr1518PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 15Ala
Gly Leu Tyr Leu Cys Asp Leu Ser Ile Met Tyr Cys Phe Phe Gln1 5 10
15Gly Thr1618PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 16Ala Gly Trp Trp Ser Cys Gln Trp Glu
Leu Asn Val Cys Ile Trp Gln1 5 10 15Gly Thr1718PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 17Ala
Gly Tyr Tyr His Cys Ile Asp Asp Phe Pro Gln Cys Lys Trp Met1 5 10
15Gly Thr1818PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 18Ala Gly Trp Phe Glu Cys Glu Phe Gly
Phe Trp Gly Cys Asn Trp Leu1 5 10 15Gly Thr1918PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 19Ala
Gly Thr Val Tyr Cys Ser Trp Glu Ser Ser Glu Cys Trp Trp Val1 5 10
15Gly Thr2018PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 20Ala Gly Val Trp Ile Cys Arg Val Trp
Asp Asp Glu Cys Phe Phe Gln1 5 10 15Gly Thr2118PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 21Ala
Gly Asp His Tyr Cys Trp Glu Glu Trp Trp Phe Cys Trp Asp Ser1 5 10
15Gly Thr2218PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 22Ala Gly Val Leu Gln Cys Ile Gly Phe
Glu Trp Phe Cys Asp Ile Trp1 5 10 15Gly Thr2318PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 23Ala
Gly Val Ile Val Cys Asn Leu Ser Met Met Tyr Cys Leu Tyr Pro1 5 10
15Gly Thr2418PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 24Ala Gly Tyr Pro Glu Cys Lys Asp Asn
Tyr His Trp Cys Glu Trp Lys1 5 10 15Gly Thr2518PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 25Ala
Gly Trp Thr Trp Cys Asp Leu Ser Met Met Ser Cys Ile Phe His1 5 10
15Gly Thr2618PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 26Ala Gly Val Thr Asn Cys Asn Leu Ser
Thr Met Phe Cys Phe Leu His1 5 10 15Gly Thr2718PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 27Ala
Gly Thr Leu Ser Cys Ser Glu Glu Tyr Lys Ser Cys Gln Leu Gln1 5 10
15Gly Thr2818PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 28Ala Gly Thr Ile Arg Cys Asn Leu Ala
Met Met Val Cys Met Phe Glu1 5 10 15Gly Thr2918PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 29Ala
Gly Gln Tyr Leu Cys Thr Gln Ala Ala Leu Gly Cys Pro Glu Trp1 5 10
15Gly Thr3018PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 30Ala Gly Gln Met Trp Cys Ala Glu Lys
Asn Ser Lys Cys Tyr Gln Trp1 5 10 15Gly Thr3118PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 31Ala
Gly Gln Ala Val Cys Glu Trp Gly Pro Phe Trp Cys Gln Met Gln1 5 10
15Gly Thr3218PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 32Ala Gly Pro Tyr Ser Cys His Ser Glu
Ser His Asp Cys Lys Leu Met1 5 10 15Gly Thr3318PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 33Ala
Gly Pro Leu Phe Cys Phe Glu Trp Pro Ser Leu Cys His Trp Gly1 5 10
15Gly Thr3418PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 34Ala Gly Asn Leu Pro Cys His Trp Asn
Met Ser Val Cys Asp His Gln1 5 10 15Gly Thr3518PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 35Ala
Gly Met Asp Phe Cys Glu Gly Phe Trp Phe Leu Cys Ile Gly Asn1 5 10
15Ala Thr3618PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 36Ala Gly Leu Leu Gly Cys Trp Asp Met
Pro Met Glu Cys Thr Gly Glu1 5 10 15Gly Thr3718PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 37Ala
Gly Lys Tyr Met Cys Glu Gly Phe Glu Trp Phe Cys Glu Met Trp1 5 10
15Gly Thr3818PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 38Ala Gly Lys Thr Val Cys Gln Lys Trp
Glu Ser Val Cys Ser Gly Met1 5 10 15Gly Thr3918PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 39Ala
Gly Lys Gln Trp Cys Val Val Trp Glu Glu Thr Cys Asp Gln Leu1 5 10
15Gly Thr4018PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 40Ala Gly Ile Trp Phe Cys Asn Asn Glu
Glu Lys Ser Cys Trp Ala Tyr1 5 10 15Gly Thr4118PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 41Ala
Gly His Thr Ile Cys Gln His Lys Ala Leu Gly Cys Pro Ala Asn1 5 10
15Gly Thr4218PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 42Ala Gly His Phe Glu Cys Pro Lys His
Gln Tyr Met Cys Asp Met Pro1 5 10 15Gly Thr4318PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 43Ala
Gly Gly Asn Trp Cys Ser Phe Tyr Glu Glu Leu Cys Glu Trp Leu1 5 10
15Gly Thr4418PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 44Ala Gly Gly His Trp Cys Leu Glu Leu
Lys His Leu Cys Pro Pro Tyr1 5 10 15Gly Thr4518PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 45Ala
Gly Phe Trp Asp Cys Gly Trp Met Met Gln Asp Cys His Met His1 5 10
15Gly Thr4618PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 46Ala Asp Ala Trp Met Cys Glu Tyr Phe
Gln Trp Asn Cys Gly Asp Lys1 5 10 15Gly Thr4718PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 47Gly
Asp Gly Phe Leu Cys Arg Trp Glu Asn Gly Trp Cys Glu Phe Trp1 5 10
15Asp Pro4819PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 48Ala Gly Ser Ile Gln Cys Lys Gly Pro
Pro Trp Phe Ser Cys Ala Met1 5 10 15Tyr Gly Thr4919PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 49Ala
Gly Tyr Tyr Gly Cys Lys Gly Pro Pro Thr Phe Glu Cys Gln Trp1 5 10
15Met Gly Thr5019PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 50Ala Gly Gln Phe Lys Cys Ala Gly Pro
Pro Ser Phe Ala Cys Trp Met1 5 10 15Thr Gly Thr5119PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 51Ala
Gly Trp Phe Gln Cys Lys Gly Pro Pro Ser Phe Glu Cys Glu Arg1 5 10
15His Gly Thr5219PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 52Ala Gly Trp Thr His Cys Ile Gly Pro
Pro Thr Phe Glu Cys Ile Pro1 5 10 15Met Gly Thr5319PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 53Ala
Gly Ser Phe Ala Cys Lys Gly Pro Pro Thr Phe Ala Cys Val Glu1 5 10
15Phe Gly Thr5419PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 54Ala Gly Asn Tyr Phe Cys Ala Gly Ser
Pro Ser Phe Ser Cys Tyr Phe1 5 10 15Met Gly Thr5519PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 55Ala
Gly Ser Trp His Cys Ala Gly Pro Pro Ser Phe Glu Cys Trp Glu1 5 10
15Phe Gly Thr5619PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 56Ala Gly Trp Ile Ser Cys Ala Gly Pro
Pro Thr Phe Ala Cys Trp Pro1 5 10 15Gly Gly Thr5719PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 57Ala
Gly Phe Val Asn Cys Lys Gly Pro Pro Thr Phe Glu Cys Ile Leu1 5 10
15Thr Gly Thr5819PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 58Ala Gly Asp Trp Ile Cys His Gly Pro
Pro Met Phe Glu Cys Glu Trp1 5 10 15Val Gly Thr5919PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 59Ala
Gly Tyr Thr Ser Cys Val Gly Pro Pro Ser Phe Glu Cys Thr Pro1 5 10
15Tyr Gly Thr6019PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 60Ala Gly Tyr Phe Glu Cys Lys Gly Pro
Pro Thr Phe Glu Cys Trp Leu1 5 10 15Ser Gly Thr6119PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 61Ala
Gly His Ala Trp Cys Ser Gly Pro Pro Arg Phe Glu Cys Trp Pro1 5 10
15Pro Gly Thr6219PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 62Ala Gly His Tyr Trp Cys Ala Gly Pro
Pro Thr Phe Ile Cys Met Gly1 5 10 15Pro Gly Thr6319PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 63Ala
Gly Glu Thr Thr Cys Leu Gly Trp Pro Thr Phe Val Cys Val Asp1 5 10
15Tyr Gly Thr6419PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 64Ala Gly His Gly Thr Cys Arg Gly Trp
Pro Thr Phe Glu Cys Ile Tyr1 5 10 15Phe Gly Thr6519PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 65Ala
Gly Asp Trp His Cys Gln Gly Pro Pro Ala Phe Met Cys Trp Met1 5 10
15Ile Gly Thr6619PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 66Ala Gly Leu Pro Lys Cys Ser Gly Pro
Pro Trp Phe Ser Cys Tyr Tyr1 5 10 15Gly Gly Thr6719PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 67Ala
Gly Gly Trp Glu Cys Thr Gly Pro Pro Trp Phe Gln Cys Gly Tyr1 5 10
15Tyr Gly Thr6819PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 68Ala Gly Asp Ile Val Cys Thr Gly His
Pro Tyr Phe Glu Cys Trp Ser1 5 10 15Trp Gly Thr6919PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 69Ala
Gly Thr Trp His Cys Ala Gly Pro Pro Trp Phe Thr Cys Tyr Met1 5 10
15Asp Gly Thr7019PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 70Ala Gly Ser Trp Glu Cys Thr Gly Pro
Pro Ser Phe His Cys Gln Trp1 5 10 15Tyr Gly Thr7119PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 71Ala
Gly His Trp Ile Cys Val Gly Pro Pro Thr Phe Ser Cys Gln Trp1 5 10
15His Gly Thr7219PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 72Ala Gly Glu Trp Trp Cys His Gly Pro
Pro Glu Phe Leu Cys Tyr Trp1 5 10 15Thr Gly Thr7319PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 73Ala
Gly Glu Thr Val Cys Tyr Trp Leu Asn Gly Trp Phe Cys Val Asp1 5 10
15Asp Gly Thr7419PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 74Ala Gly Ser Ile Gln Cys Val Gly Pro
Pro Ser Phe Glu Cys Thr Pro1 5 10 15Tyr Gly Thr7519PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 75Ala
Gly Tyr Ser Val Cys Lys Gly Tyr Pro Ser Phe Glu Cys Ala Phe1 5 10
15Phe Gly Thr7619PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 76Ala Gly Val Asn Ser Cys Leu Gly Pro
Pro Thr Phe Glu Cys Tyr Gln1 5 10 15Met Gly Thr7719PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 77Ala
Gly Tyr Trp His Cys Lys Gly Pro Pro His Phe Ala Cys Glu Phe1 5 10
15His Gly Thr7819PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 78Ala Gly Asn Trp Ile Cys Thr Gly Pro
Pro Ser Phe Gly Cys Trp Tyr1 5 10 15His Gly Thr7919PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 79Ala
Gly Tyr Trp Ser Cys Ala Gly Pro Pro Met Phe Met Cys Thr Trp1 5 10
15Gln Gly Thr8019PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 80Ala Gly Tyr Trp Asp Cys Lys Gly Pro
Pro His Phe Phe Cys Glu Trp1 5 10 15His Gly Thr8119PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 81Ala
Gly Tyr Phe His Cys Ser Gly Ser Pro Trp Phe Gln Cys Asp Tyr1 5 10
15Tyr Gly Thr8219PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 82Ala Gly Trp Tyr Asn Cys Ser Gly Glu
Asn Phe Trp Asn Cys Lys Trp1 5 10 15Ile Gly Thr8319PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 83Ala
Gly Trp Ser Asp Cys Leu Gly Pro Pro Gln Phe Thr Cys Val His1 5 10
15Trp Gly Thr8419PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 84Ala Gly Thr Met Tyr Cys Leu Gly Pro
Pro Thr Phe Ile Cys
Gln Gln1 5 10 15Tyr Gly Thr8519PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 85Ala Gly Ser Tyr Trp Cys
Ser Gly Pro Pro Thr Phe Met Cys Arg Tyr1 5 10 15Glu Gly
Thr8619PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 86Ala Gly Ser Thr Asp Cys Arg Gly His Pro Thr Phe
Glu Cys Trp Gly1 5 10 15Trp Gly Thr8719PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 87Ala
Gly Ser Ser Pro Cys Lys Gly Trp Pro Thr Phe Glu Cys Tyr Phe1 5 10
15Tyr Gly Thr8819PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 88Ala Gly Ser Ile Ala Cys Thr Gly Trp
Pro Tyr Phe Ser Cys Ile Asp1 5 10 15Leu Gly Thr8919PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 89Ala
Gly Gln Phe Tyr Cys Ser Gly Pro Pro Thr Phe Gln Cys Ile Met1 5 10
15Ile Gly Thr9019PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 90Ala Gly Pro Trp Lys Cys Thr Gly Pro
Pro Thr Phe Ser Cys Ile Gln1 5 10 15Phe Gly Thr9119PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 91Ala
Gly Asn Tyr Trp Cys Ser Gly Pro Pro Ser Phe Ile Cys His Ala1 5 10
15Val Gly Thr9219PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 92Ala Gly Met Thr Leu Cys Ala Gly Pro
Pro Thr Phe Glu Cys Tyr Glu1 5 10 15Val Gly Thr9319PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 93Ala
Gly Glu Thr Lys Cys Ser Gly Pro Pro Tyr Phe Tyr Cys Trp Met1 5 10
15Glu Gly Thr9419PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 94Ala Gly Glu Thr Phe Cys Val Gly Asn
Pro Ser Phe Glu Cys Trp Ser1 5 10 15Trp Gly Thr9519PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 95Ala
Gly Glu Thr Phe Cys Ser Gly Trp Pro Thr Phe Glu Cys Met Gln1 5 10
15Trp Gly Thr9619PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 96Ala Gly Glu Ile Phe Cys Val Gly Pro
Pro Thr Phe Thr Cys Met Trp1 5 10 15Thr Gly Thr9719PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 97Ala
Gly Asp Phe Ile Cys Gln Gly Pro Pro Ser Phe Val Cys Thr Asn1 5 10
15Ile Gly Thr9819PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 98Ala Gly Ala Phe Phe Cys Ser Gly Pro
Pro Thr Phe Met Cys Ser Leu1 5 10 15Tyr Gly Thr9919PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence 99Ala
Gly Trp Gly Trp Cys Ser Gly Pro Pro Met Phe Met Cys Thr Glu1 5 10
15Tyr Gly Thr10019PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 100Gly Ser Glu Phe Glu Cys Thr Gly
Trp Pro Glu Phe Arg Cys Tyr Glu1 5 10 15Tyr Ala
Pro10119PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 101Gly Ser Ile Leu Tyr Cys Ile Asn Arg Asn Asp Pro
Gln Cys Pro Tyr1 5 10 15Thr Ala Pro10230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
102Ser Glu Thr Arg Pro Thr Glu Ala Gly Asp Leu Ile Cys Ser Gly Pro1
5 10 15Pro Thr Phe Ile Cys Thr Leu Tyr His Thr Glu Pro Thr Glu 20
25 3010330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 103Ser Glu Thr Arg Pro Thr Gln Ala
Val Arg Ser Gln Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Tyr
Phe Gly Thr Glu Pro Thr Glu 20 25 3010430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
104Ser Glu Thr Arg Pro Thr Glu Gly Gly Ser Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Ala Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3010530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 105Ser Glu Thr Arg Pro Thr Val Ala
Ser Arg Trp His Cys Asn Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Arg
Tyr Gly Thr Glu Pro Thr Glu 20 25 3010630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
106Ser Glu Thr Arg Pro Thr Glu Ala Gly Thr Phe His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Ser Tyr Gly Pro Lys Pro Thr Glu 20
25 3010730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 107Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Leu Trp Cys Met Gly Pro1 5 10 15Pro Trp Phe Cys Cys Val Ile
Tyr Gly Thr Gln Pro Thr Glu 20 25 3010830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
108Ser Glu Thr Arg Pro Thr Glu Ala Gly Ile Leu His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Asn Tyr Thr Glu Pro Thr Glu 20
25 3010930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 109Ser Glu Thr Arg Pro Thr Glu Ser
Gly Arg Val His Cys Pro Gly Pro1 5 10 15Pro Trp Phe Arg Cys Ala Arg
Asn Gly Thr Glu Pro Thr Glu 20 25 3011030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
110Ser Glu Thr Arg Pro Thr Ala Ala Gly Arg Ile Leu Cys Thr Gly Pro1
5 10 15Pro Trp Phe Ser Cys Ala Met Tyr Gly Thr Glu Pro Thr Glu 20
25 3011130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 111Ser Glu Thr Arg Pro Thr Glu Ala
Ala Asp Trp Leu Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Gly Thr Glu Pro Thr Glu 20 25 3011230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
112Ser Glu Thr Arg Pro Thr Gln Val Gly Arg Trp Gln Cys Asp Gly Pro1
5 10 15Pro Thr Phe Ala Cys Arg Ser Tyr Gly Thr Glu Pro Thr Glu 20
25 3011330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 113Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Thr Lys Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Asp Thr Glu Pro Thr Glu 20 25 3011430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
114Ser Glu Thr Arg Pro Thr Val Ala Gly Ser Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3011530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 115Ser Glu Thr Arg Pro Thr Glu Ala
Gly Arg Asn His Cys Lys Gly Pro1 5 10 15Pro Gly Phe Arg Cys Ala Met
Thr Asp Thr Glu Pro Thr Glu 20 25 3011630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
116Ser Glu Thr Arg Pro Thr Glu Thr Asp Phe Val Tyr Cys Arg Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3011730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 117Ser Glu Thr Arg Pro Thr Ser Ser
Gly Ser Arg His Cys Lys Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Gly
Tyr Gly Thr Glu Pro Thr Glu 20 25 3011830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
118Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Arg Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Glu Thr Ser Pro Thr Glu 20
25 3011930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 119Ser Glu Thr Arg Pro Thr Asp Ala
Ile Arg Ser Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Gly Thr Glu Pro Thr Glu 20 25 3012030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
120Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Asn Cys Ser Gly Pro1
5 10 15Pro Ala Phe Glu Cys Trp Trp Tyr Gly Ser Glu Pro Thr Glu 20
25 3012130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 121Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Gln Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Ser
Phe Gly Thr Glu Pro Thr Glu 20 25 3012230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
122Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp His Cys Asn Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Asp Met Glu Pro Thr Glu 20
25 3012330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 123Ser Glu Thr Arg Pro Thr Glu Ala
Gly Arg Val Ser Cys Leu Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Val Pro Glu Pro Thr Glu 20 25 3012430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
124Ser Glu Thr Arg Pro Thr Asp Ala Gly Ser Trp Arg Cys Ala Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Phe Gly Thr Glu Pro Thr Glu 20
25 3012530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 125Ser Glu Thr Arg Pro Thr Glu Pro
Val Thr Trp Gln Cys Thr Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Leu Gly Thr Glu Pro Thr Glu 20 25 3012630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
126Ser Glu Thr Arg Pro Thr Asp Ala Val Ser Thr His Cys Asn Gly Pro1
5 10 15Pro Thr Phe Glu Cys Tyr Ile Tyr Gly Thr Glu Pro Thr Glu 20
25 3012730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 127Ser Glu Thr Arg Pro Thr Val Ala
Glu Ser Trp Tyr Cys Val Gly Pro1 5 10 15Pro Ser Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3012830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
128Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Asn Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Ser Tyr Gln Thr Glu Pro Thr Glu 20
25 3012930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 129Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Gly His Cys Asn Gly Pro1 5 10 15Pro Thr Phe Lys Cys Trp Trp
Tyr Asp Met Glu Pro Thr Glu 20 25 3013030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
130Ser Glu Thr Arg Pro Thr Asp Gln Asp Ser Trp Gln Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3013130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 131Ser Glu Thr Arg Pro Thr Glu Ser
Thr Gln Val Gln Cys Ala Gly Pro1 5 10 15Pro Ser Phe Ala Cys Trp Met
Thr Gly Thr Glu Pro Thr Glu 20 25 3013230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
132Ser Glu Thr Arg Pro Thr Glu Val Glu Ser Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3013330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 133Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Phe His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Leu
Tyr Trp Thr Asp Pro Thr Glu 20 25 3013430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
134Ser Glu Thr Arg Pro Thr Glu Ala Gly Gln Phe Gly Cys Lys Gly Pro1
5 10 15Pro Pro Phe Glu Cys Lys Leu Met Gly Arg Val Pro Thr Glu 20
25 3013530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 135Ser Glu Thr Arg Pro Thr Asp Thr
Val Thr Trp His Cys Asn Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3013630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
136Ser Glu Thr Arg Pro Thr Glu Ala Asp Arg Trp His Cys Asp Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3013730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 137Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Ile Gln Cys Val Gly Pro1 5 10 15Pro Trp Phe Ser Cys Arg Met
Tyr Val Thr Glu Pro Thr Glu 20 25 3013830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
138Ser Glu Thr Arg Pro Thr Val Ser Gly Ser Trp Gln Cys Val Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Ser Tyr Gly Thr Glu Pro Thr Glu 20
25 3013930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 139Ser Glu Thr Arg Pro Thr Glu Asn
Gly Ser Trp His Cys Asn Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3014030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
140Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp His Cys Ser Gly Pro1
5 10 15Pro Ile Phe Glu Cys Trp Trp Tyr Asp Met Glu Pro Thr Glu 20
25 3014130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 141Ser Glu Thr Arg Pro Thr Val Asp
Gly Gly Trp His Cys Asn Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Met
Tyr Gly Thr Glu Pro Thr Glu 20 25 3014230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
142Ser Glu Thr Arg Pro Thr Asp Ala Gly Thr Trp Asn Cys Thr Gly Pro1
5 10 15Pro Ser Phe Glu Cys Trp Trp Phe Gly Thr Glu Pro Thr Glu 20
25 3014330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 143Ser Glu Thr Arg Pro Thr Trp Asp
Gly Lys Trp His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3014430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
144Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Arg Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Tyr Thr Glu Pro Thr Glu 20
25 3014530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 145Ser Glu Thr Arg Pro Thr Glu Ala
Gly Asn Trp Leu Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Val Thr Gly Pro Thr Glu 20 25 3014630PRTArtificial
SequenceSynthetically generated cMet-binding
peptide sequence 146Ser Glu Thr Arg Pro Thr Glu Gly Gly Asn Trp His
Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Leu Tyr Gly Thr Glu
Pro Thr Glu 20 25 3014730PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 147Ser Glu Thr Arg Pro Thr
Glu Ala Gly Gly Trp His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys
Trp Trp Phe Asn Met Glu Pro Thr Glu 20 25 3014830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
148Ser Glu Thr Arg Pro Thr Glu Val Ile Ser Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Tyr Arg Tyr Gly Thr Glu Pro Thr Glu 20
25 3014930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 149Ser Glu Thr Arg Pro Thr Glu Val
Gly Ser Trp His Cys Asn Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3015030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
150Ser Glu Thr Arg Pro Thr Leu Ala Ser Thr Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3015130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 151Ser Glu Thr Arg Pro Thr Glu Ala
Gly Gly Trp Tyr Cys Lys Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Asp Gly Thr Glu Pro Thr Glu 20 25 3015230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
152Ser Glu Thr Arg Pro Thr Glu Ala Gly Gly Trp Phe Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Asp Thr Val Pro Thr Glu 20
25 3015330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 153Ser Glu Thr Arg Pro Thr Glu Ala
Ala Thr Trp Gln Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Gly
Tyr Gly Thr Glu Pro Thr Glu 20 25 3015430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
154Ser Glu Thr Arg Pro Thr Glu Ala Gly Asp Tyr Val Cys Val Gly Pro1
5 10 15Pro Thr Phe Glu Cys Tyr Leu Met Asp Ala Glu Pro Thr Glu 20
25 3015530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 155Ser Glu Thr Arg Pro Thr Glu Ala
Gly Gly Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Ser Phe Glu Cys Trp Ser
Tyr Gly Thr Glu Pro Thr Glu 20 25 3015630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
156Ser Glu Thr Arg Pro Thr Glu Ser Ser Ser Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Arg Phe Gly Thr Glu Pro Thr Glu 20
25 3015730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 157Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Tyr Ala Glu Pro Thr Glu 20 25 3015830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
158Ser Glu Thr Arg Pro Thr Leu Ala Gly Asn Trp Gln Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3015930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 159Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp His Cys Asn Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Gln
Tyr Gly Thr Glu Pro Thr Glu 20 25 3016030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
160Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Glu Cys His Gly Pro1
5 10 15Pro Ser Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3016130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 161Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Arg Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Asp Ala Glu Pro Thr Glu 20 25 3016230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
162Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Asn Cys Ala Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3016330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 163Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Phe Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Gln
Tyr Val Pro Glu Pro Thr Glu 20 25 3016430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
164Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Met Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Gln Tyr Phe Thr Glu Pro Thr Glu 20
25 3016530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 165Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Leu His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Trp Glu Thr Glu Pro Thr Glu 20 25 3016630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
166Ser Glu Thr Arg Pro Thr Glu Glu Gly Val Trp His Cys Asn Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3016730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 167Ser Glu Thr Arg Pro Thr Glu Ala
Gly Arg Trp Asn Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Ser Thr Glu Pro Thr Glu 20 25 3016830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
168Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Arg Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Phe Gly Thr Glu Pro Thr Glu 20
25 3016930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 169Ser Glu Thr Arg Pro Thr Gln Ala
Val Ser Ser Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Ser
Phe Gly Thr Glu Pro Thr Glu 20 25 3017030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
170Ser Glu Thr Arg Pro Thr Glu Ala Gly Arg Ser Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Ala Thr Glu Pro Thr Glu 20
25 3017130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 171Ser Glu Thr Arg Pro Thr Val Val
Ala Lys Val His Cys Ala Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Thr
Tyr Gly Thr Glu Pro Thr Glu 20 25 3017230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
172Ser Glu Thr Arg Pro Thr Glu Pro Gly Ser Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Val Cys Trp Trp Trp Gly Thr Glu Pro Thr Glu 20
25 3017330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 173Ser Glu Thr Arg Pro Thr Glu Ala
Gly Arg Trp His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
His Asp Thr Glu Pro Thr Glu 20 25 3017430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
174Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Gln Cys Thr Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Gly Tyr Val Glu Glu Pro Thr Glu 20
25 3017530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 175Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Gln Cys Gly Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Tyr Thr Gly Pro Thr Glu 20 25 3017630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
176Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Tyr Cys Thr Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Leu Tyr Glu Thr Tyr Pro Thr Glu 20
25 3017730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 177Ser Glu Thr Arg Pro Thr Ala Ala
Trp Ser Gly Ser Cys Ser Gly Pro1 5 10 15Pro Ser Phe Glu Cys Trp Asn
Tyr Gly Thr Glu Pro Thr Glu 20 25 3017830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
178Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Gln Cys Ser Gly Pro1
5 10 15Pro Thr Phe Ala Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3017930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 179Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ile Leu His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Glu Val Met Glu Pro Thr Glu 20 25 3018030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
180Ser Glu Thr Arg Pro Thr Glu Ala Gly Arg Val Ala Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Ser Tyr Asp Glu Glu Pro Thr Glu 20
25 3018130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 181Ser Glu Thr Arg Pro Thr Glu Ala
Gly Asn Trp Glu Cys Gln Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Gly Thr Glu Pro Thr Glu 20 25 3018230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
182Ser Glu Thr Arg Pro Thr Leu Ala Ser Asn Gly Tyr Cys Asn Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp His Tyr Gly Thr Glu Pro Thr Glu 20
25 3018330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 183Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Phe His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Ile Trp
Tyr Gly Ser Glu Pro Thr Glu 20 25 3018430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
184Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Ala Cys Trp Trp Asp Gly Thr Glu Pro Thr Glu 20
25 3018530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 185Ser Glu Thr Arg Pro Thr Gln Gly
Asp Asn Trp Asn Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3018630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
186Ser Glu Thr Arg Pro Thr Glu Ala Gly Arg Trp His Cys Asn Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Arg Tyr Asp Tyr Asp Pro Thr Glu 20
25 3018730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 187Ser Glu Thr Arg Pro Thr Glu Ala
Tyr Ser Trp Glu Cys Thr Gly Pro1 5 10 15Pro Met Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3018830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
188Ser Glu Thr Arg Pro Thr Glu Val Val Asp Trp His Cys Ser Gly Pro1
5 10 15Pro Gln Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3018930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 189Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Asn Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Ser Glu Pro Thr Glu 20 25 3019030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
190Ser Glu Thr Arg Pro Thr Ala Ser Gly Ser Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Ile Phe Gly Thr Glu Pro Thr Glu 20
25 3019130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 191Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ala Trp Tyr Cys Met Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Asp Arg Gly Pro Thr Glu 20 25 3019230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
192Ser Glu Thr Arg Pro Thr Glu Ala Gly Gly Leu His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Asp Thr Glu Pro Thr Glu 20
25 3019330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 193Ser Glu Thr Arg Pro Thr Val Gly
Gly Ser Trp Asp Cys Lys Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Ser
Tyr Gly Thr Glu Pro Thr Glu 20 25 3019430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
194Ser Glu Thr Arg Pro Thr Glu Ala Gly Ala Trp Ser Cys Leu Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3019530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 195Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Leu His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Asp Thr Glu Pro Thr Glu 20 25 3019630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
196Ser Glu Thr Arg Pro Thr Ala Gly Arg Ser Trp Glu Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Val Phe Gly Thr Glu Pro Thr Glu 20
25 3019730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 197Ser Glu Thr Arg Pro Thr Asp Asn
Gly Ser Trp His Cys Asn Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3019830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
198Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Gln Cys Lys Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3019930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 199Ser Glu Thr Arg Pro Thr Glu Val
Gly Asn Tyr Lys Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3020030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
200Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp His Cys Val Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Gly Tyr Val Thr Glu Pro Thr Glu 20
25 3020130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 201Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Phe Val Cys Lys Gly Pro1 5 10 15Pro Thr Phe Glu Cys Tyr Trp
Phe Gly Gln Asp Pro Thr Glu 20 25
3020230PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 202Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp His
Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Pro Asp
Pro Thr Glu 20 25 3020330PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 203Ser Glu Thr Arg Pro Thr
Glu Ala Glu Arg Trp His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys
Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20 25 3020430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
204Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Phe Tyr Val Lys Glu Pro Thr Glu 20
25 3020530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 205Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Asp Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Gly Thr Glu Pro Thr Glu 20 25 3020630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
206Ser Glu Thr Arg Pro Thr Glu Pro Ala Gly Trp Glu Cys Arg Gly Pro1
5 10 15Pro Ser Phe Glu Cys Leu Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3020730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 207Ser Glu Thr Arg Pro Thr Asp Ala
Gly Pro Trp Asn Cys Thr Gly Pro1 5 10 15Pro Ser Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3020830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
208Ser Glu Thr Arg Pro Thr Glu Ala Arg Gly Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Leu Trp Gly Thr Glu Pro Thr Glu 20
25 3020930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 209Ser Glu Thr Arg Pro Thr Glu Ala
Gly Arg Trp Asn Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Gln
Tyr Glu Met Asp Pro Thr Glu 20 25 3021030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
210Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Phe Trp Tyr Asp Thr Glu Pro Thr Glu 20
25 3021130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 211Ser Glu Thr Arg Pro Thr Glu Ser
Gly Ser Trp His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Gly Thr Glu Pro Thr Glu 20 25 3021230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
212Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Leu Cys Thr Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Phe Asp Thr Asp Pro Thr Glu 20
25 3021330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 213Ser Glu Thr Arg Pro Thr Glu Pro
Ser His Trp His Cys Val Gly Pro1 5 10 15Pro Thr Phe Ala Cys Trp Trp
Tyr Val Thr Asp Pro Thr Glu 20 25 3021430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
214Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Met Phe Glu Cys Tyr Leu Phe Val Thr Glu Pro Thr Glu 20
25 3021530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 215Ser Glu Thr Arg Pro Thr Glu Ala
Val Asn Trp His Cys Leu Gly Pro1 5 10 15Pro Ser Phe Glu Cys Trp Gln
Phe Gly Thr Glu Pro Thr Glu 20 25 3021630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
216Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Asp Pro Thr Glu 20
25 3021730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 217Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Ser
Phe Val Ser Leu Pro Thr Glu 20 25 3021830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
218Ser Glu Thr Arg Pro Thr Glu Gly Ser Glu Trp Ser Cys Ile Gly Pro1
5 10 15Pro Ser Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3021930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 219Ser Glu Thr Arg Pro Thr Glu Asp
Gly Tyr Trp Asn Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
His Gly Thr Glu Pro Thr Glu 20 25 3022030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
220Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Ser Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Pro Tyr Tyr Thr Glu Pro Thr Glu 20
25 3022130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 221Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Trp Pro Glu Pro Thr Glu 20 25 3022230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
222Ser Glu Thr Arg Pro Thr Asp Asp Gly Arg Trp Ser Cys Ala Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Arg Tyr Gly Thr Glu Pro Thr Glu 20
25 3022330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 223Ser Glu Thr Arg Pro Thr Glu Gly
Gly Ser Trp Ser Cys Gly Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Gly Thr Glu Pro Thr Glu 20 25 3022430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
224Ser Glu Thr Arg Pro Thr Val Thr Gly Ser Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3022530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 225Ser Glu Thr Arg Pro Thr Glu Ala
Ser Ser Trp Tyr Cys Thr Gly Pro1 5 10 15Pro Ala Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3022630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
226Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Leu Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3022730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 227Ser Glu Thr Arg Pro Thr Glu Ser
Val Arg Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3022830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
228Ser Glu Thr Arg Pro Thr Glu Ala Gly Arg Leu Val Cys Ser Gly Pro1
5 10 15Pro Thr Phe Met Cys Arg Thr Tyr Ala Thr Asp Pro Thr Glu 20
25 3022930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 229Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Glu Cys Thr Gly Pro1 5 10 15Pro Trp Phe Val Cys Arg Gln
Tyr Ala Ile Glu Pro Thr Glu 20 25 3023030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
230Ser Glu Thr Arg Pro Thr Glu Ala Gly Tyr Leu Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Asp Thr Met Pro Thr Glu 20
25 3023130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 231Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Gly Thr Glu Pro Thr Glu 20 25 3023230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
232Ser Glu Thr Arg Pro Thr Glu Ala Gly Asn Trp His Cys Leu Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3023330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 233Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Asp Thr Glu Pro Thr Glu 20 25 3023430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
234Ser Glu Thr Arg Pro Thr Glu Ser Gly Gly Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Ala Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3023530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 235Ser Glu Thr Arg Pro Thr Val Ala
Gly Ala Val Ser Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3023630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
236Ser Glu Thr Arg Pro Thr Glu Ala Gly Arg Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Phe Leu Pro Asp Pro Thr Glu 20
25 3023730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 237Ser Glu Thr Arg Pro Thr Glu Ala
Gly Gly Trp His Cys Ser Gly Pro1 5 10 15Pro Ser Phe Glu Cys Trp Trp
Phe Asp Thr Val Pro Thr Glu 20 25 3023830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
238Ser Glu Thr Arg Pro Thr Gly Val Gly Gly Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Ser Phe Glu Cys Trp Leu Tyr Gly Thr Glu Pro Thr Glu 20
25 3023930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 239Ser Glu Thr Arg Pro Thr Gln Ala
Asp Tyr Leu His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Phe Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3024030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
240Ser Glu Thr Arg Pro Thr Gly Asp Gly Asn Trp His Cys Asn Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Arg Phe Gly Thr Glu Pro Thr Glu 20
25 3024130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 241Ser Glu Thr Arg Pro Thr Glu Ala
Ser Asn Tyr His Cys Ile Gly Pro1 5 10 15Pro Thr Phe Glu Cys Phe Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3024230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
242Ser Glu Thr Arg Pro Thr Glu Ala Gly Asp Trp Leu Cys Lys Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Gln Val Thr Asp Pro Thr Glu 20
25 3024330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 243Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp His Cys Asn Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Ser Ser Asp Pro Thr Glu 20 25 3024430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
244Ser Glu Thr Arg Pro Thr Glu Asp Gly Gly Trp Arg Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3024530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 245Ser Glu Thr Arg Pro Thr Glu Ala
Gly Arg Ile Glu Cys Lys Gly Pro1 5 10 15Pro Trp Phe Ser Cys Val Ile
Tyr Gly Thr Glu Pro Thr Glu 20 25 3024630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
246Ser Glu Thr Arg Pro Thr Gly Gly Gly Ser Trp Asn Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3024730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 247Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Leu Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Ile Thr His Pro Thr Glu 20 25 3024830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
248Ser Glu Thr Arg Pro Thr Glu Ala Gly Arg Trp His Cys Ser Gly Pro1
5 10 15Pro Arg Phe Glu Cys Trp Trp Tyr Asp Thr Glu Pro Thr Glu 20
25 3024930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 249Ser Glu Thr Arg Pro Thr Glu Tyr
Gly Ser Trp His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Tyr
His Gly Thr Glu Pro Thr Glu 20 25 3025030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
250Ser Glu Thr Arg Pro Thr Glu Ala Gly Asn Trp His Cys Ser Gly Pro1
5 10 15Pro Ser Phe Glu Cys Trp Trp Tyr Ala Thr Glu Pro Thr Glu 20
25 3025130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 251Ser Glu Thr Arg Pro Thr Glu Gln
Gly Ser Trp His Cys Lys Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Ser
Tyr Gly Thr Glu Pro Thr Glu 20 25 3025230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
252Ser Glu Thr Arg Pro Thr Asp Ala Ala Asn Tyr His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3025330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 253Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Met Phe Glu Cys Trp Trp
Leu Ala Glu Glu Pro Thr Glu 20 25 3025430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
254Ser Glu Thr Arg Pro Thr Glu Ala Gly Gly Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Ala Phe Glu Cys Trp Trp Tyr Ala Thr Glu Pro Thr Glu 20
25 3025530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 255Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ile Trp Ser Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Glu Ser Ser Pro Thr Glu 20 25 3025630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
256Ser Glu Thr Arg Pro Thr Glu Glu Gly Leu Arg Val Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3025730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 257Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Leu Cys Phe Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp
Ser
Phe Gly Thr Glu Pro Thr Glu 20 25 3025830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
258Ser Glu Thr Arg Pro Thr Val Ala Gly Ser Trp Asp Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3025930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 259Ser Glu Thr Arg Pro Thr Lys Ala
Asp Asn Trp His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3026030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
260Ser Glu Thr Arg Pro Thr Glu Ala Gly Ile Val Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Phe Gly Thr Glu Pro Thr Glu 20
25 3026130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 261Ser Glu Thr Arg Pro Thr Glu Ala
Gly Tyr Trp His Cys Leu Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Val Lys Glu Pro Thr Glu 20 25 3026230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
262Ser Glu Thr Arg Pro Thr Glu Pro Gly Leu Leu His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3026330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 263Ser Glu Thr Arg Pro Thr Glu Ala
Ser Ser Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Ser Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3026430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
264Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp His Cys Leu Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Val Lys Glu Pro Thr Glu 20
25 3026530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 265Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ile Ile Leu Cys Lys Gly Pro1 5 10 15Pro Trp Phe Ser Cys Asp Ile
Tyr Asp Thr Gly Pro Thr Glu 20 25 3026630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
266Ser Glu Thr Arg Pro Thr Ala Ala Gly Asn Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Ala Tyr Gly Thr Glu Pro Thr Glu 20
25 3026730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 267Ser Glu Thr Arg Pro Thr Val Gly
Gly Ser Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Ser
Tyr Gly Thr Glu Pro Thr Glu 20 25 3026830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
268Ser Glu Thr Arg Pro Thr Glu Asp Gly Trp Leu Asp Cys Lys Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3026930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 269Ser Glu Thr Arg Pro Thr Glu Asp
Gly Asn Trp His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Ser
Tyr Gly Thr Glu Pro Thr Glu 20 25 3027030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
270Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Tyr Tyr Trp Pro Glu Pro Thr Glu 20
25 3027130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 271Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Leu Tyr Cys Ser Gly Pro1 5 10 15Pro Met Phe Glu Cys Trp Trp
Tyr Asp Trp Tyr Pro Thr Glu 20 25 3027230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
272Ser Glu Thr Arg Pro Thr Glu Ala Gly Gly Trp Tyr Cys Met Gly Pro1
5 10 15Pro Ala Phe Glu Cys Trp Trp Tyr Ala Ser Glu Pro Thr Glu 20
25 3027330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 273Ser Glu Thr Arg Pro Thr Asn Ala
Gly Ser Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3027430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
274Ser Glu Thr Arg Pro Thr Glu Ala Ser Arg Trp His Cys Asn Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3027530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 275Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Phe Val Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Asn Thr Gly Pro Thr Glu 20 25 3027630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
276Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Ser Tyr Gly Thr Glu Pro Thr Glu 20
25 3027730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 277Ser Glu Thr Arg Pro Thr Glu Ser
Asp Ile Trp Leu Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3027830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
278Ser Glu Thr Arg Pro Thr Asp Ala Asp Pro Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Phe Gly Thr Glu Pro Thr Glu 20
25 3027930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 279Ser Glu Thr Arg Pro Thr Glu Ala
Gly Val Val Leu Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Asp Thr Glu Pro Thr Glu 20 25 3028030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
280Ser Glu Thr Arg Pro Thr Glu Val Gly Ser Val His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Phe Gly Thr Glu Pro Thr Glu 20
25 3028130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 281Ser Glu Thr Arg Pro Thr Glu Ala
Gly Arg Trp Leu Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Glu
Tyr Asp Thr Glu Pro Thr Glu 20 25 3028230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
282Ser Glu Thr Arg Pro Thr Asp Ala Gly Trp Leu Gln Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3028330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 283Ser Glu Thr Arg Pro Thr Glu Ala
Ser Arg Arg His Cys Asn Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Arg
Tyr Gly Thr Glu Pro Thr Glu 20 25 3028430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
284Ser Glu Thr Arg Pro Thr Glu Ala Gly Arg Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Leu Phe Val Glu Glu Pro Thr Glu 20
25 3028530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 285Ser Glu Thr Arg Pro Thr Ala Ala
Asp Ser Trp Gln Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Ser
Phe Gly Thr Glu Pro Thr Glu 20 25 3028630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
286Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp His Cys Gly Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Met Tyr Val Thr Glu Pro Thr Glu 20
25 3028730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 287Ser Glu Thr Arg Pro Thr Asp Asp
Gly Ser Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3028830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
288Ser Glu Thr Arg Pro Thr Glu Ala Gly Tyr Trp His Cys Leu Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Asp Met Glu Pro Thr Glu 20
25 3028930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 289Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ile Leu Arg Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Tyr
Tyr Glu Thr Glu Pro Thr Glu 20 25 3029030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
290Ser Glu Thr Arg Pro Thr Glu Asp Val Ser Val His Cys Ala Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Leu Tyr Gly Thr Glu Pro Thr Glu 20
25 3029130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 291Ser Glu Thr Arg Pro Thr Glu Glu
Gly Val Phe Gln Cys Val Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3029230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
292Ser Glu Thr Arg Pro Thr Glu Asp Gly Gly Phe Phe Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3029330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 293Ser Glu Thr Arg Pro Thr Glu Pro
Gly Ser Trp His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3029430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
294Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Asp Arg Ala Pro Thr Glu 20
25 3029530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 295Ser Glu Thr Arg Pro Thr Glu Ala
Gly Thr Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Tyr
Tyr Ala Thr Glu Pro Thr Glu 20 25 3029630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
296Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Leu Tyr Cys Ser Gly Pro1
5 10 15Pro Ala Phe Glu Cys Tyr Trp Tyr Gly Thr Val Pro Thr Glu 20
25 3029730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 297Ser Glu Thr Arg Pro Thr Asp Pro
Gly Val Leu His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Gly Thr Glu Pro Thr Glu 20 25 3029830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
298Ser Glu Thr Arg Pro Thr Glu Ala Gly Thr Trp Tyr Cys Leu Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Ser Phe Trp Gln Asp Pro Thr Glu 20
25 3029930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 299Ser Glu Thr Arg Pro Thr Glu Ala
Gly Arg Trp Gly Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Val Ala Glu Pro Thr Glu 20 25 3030030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
300Ser Glu Thr Arg Pro Thr Glu Ala Gly Ile Trp His Cys Ala Gly Pro1
5 10 15Pro Thr Phe Ile Cys Trp Leu Tyr Glu Thr Glu Pro Thr Glu 20
25 3030130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 301Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp His Cys Ser Gly Pro1 5 10 15Pro Ser Phe Glu Cys Trp Gln
Tyr Ser Thr Glu Pro Thr Glu 20 25 3030230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
302Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Gln Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Val Tyr Glu Thr Glu Pro Thr Glu 20
25 3030330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 303Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Asp Val Gly Pro Thr Glu 20 25 3030430PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
304Ser Glu Thr Arg Pro Thr Asp Glu Val Ser Trp Glu Cys Arg Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3030530PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 305Ser Glu Thr Arg Pro Thr Glu Gly
Gly Ser Trp Val Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3030630PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
306Ser Glu Thr Arg Pro Thr Glu Tyr Gly Ser Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Leu Gly Thr Glu Pro Thr Glu 20
25 3030730PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 307Ser Glu Thr Arg Pro Thr Glu Ala
Gly Val Trp Leu Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Asp Thr Asp Pro Thr Glu 20 25 3030830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
308Ser Glu Thr Arg Pro Thr Met Ala Gly Ser Tyr Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Val Tyr Gly Thr Glu Pro Thr Glu 20
25 3030930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 309Ser Glu Thr Arg Pro Thr Glu Ala
Gly Tyr Val Gln Cys Tyr Gly Pro1 5 10 15Pro Ser Phe Val Cys His Pro
Met Val Pro Asp Pro Thr Glu 20 25 3031030PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
310Ser Glu Thr Arg Pro Thr Glu Asp Gly Phe Val Leu Cys Lys Gly Pro1
5 10 15Pro Trp Phe Ser Cys Glu Met Tyr Gly Thr Glu Pro Thr Glu 20
25 3031130PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 311Ser Glu Thr Arg Pro Thr Glu Ala
Gly Gly Trp Asn Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Val Thr Glu Pro Thr Glu 20 25 3031230PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
312Ser Glu Thr Arg Pro Thr Glu Asp Gly Ser Trp Glu Cys Phe Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Ser Tyr Gly Thr Glu Pro Thr Glu 20
25 3031330PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 313Ser Glu Thr Arg Pro Thr Asp Ala
Val Ser Tyr Val Cys Lys Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3031430PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 314Ser Glu Thr Arg Pro Thr Glu Ala
Arg Ser Trp His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3031530PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
315Ser Glu Thr Arg Pro Thr Ala Ser Val Ser Trp His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Ser Tyr Gly Thr Glu Pro Thr Glu 20
25 3031630PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 316Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Tyr
Tyr Asp Met Asp Pro Thr Glu 20 25 3031730PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
317Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Leu Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Phe Gly Thr Glu Pro Thr Glu 20
25 3031830PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 318Ser Glu Thr Arg Pro Thr Gly Asp
Gly Ser Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Leu Gly Thr Glu Pro Thr Glu 20 25 3031930PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
319Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Phe Leu Asp Pro Thr Glu 20
25 3032030PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 320Ser Glu Thr Arg Pro Thr Glu Ala
Gly Gly Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Ala Thr Glu Pro Thr Glu 20 25 3032130PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
321Ser Glu Thr Arg Pro Thr Glu Ala Gly Asp Leu Asp Cys Leu Gly Pro1
5 10 15Pro Thr Phe Ile Cys Arg Ile Tyr Gly Thr Glu Pro Thr Glu 20
25 3032230PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 322Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Gln Cys Val Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Ser
Phe Gly Thr Glu Pro Thr Glu 20 25 3032330PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
323Ser Glu Thr Arg Pro Thr Glu Ala Asp Ser Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Leu Phe Gly Thr Glu Pro Thr Glu 20
25 3032430PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 324Ser Glu Thr Arg Pro Thr Gln Ala
Asp Ser Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Trp Gly Thr Glu Pro Thr Glu 20 25 3032530PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
325Ser Glu Thr Arg Pro Thr Glu Ala Phe Ser Trp Asp Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Phe Gly Thr Glu Pro Thr Glu 20
25 3032630PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 326Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Gln Cys Ser Gly Pro1 5 10 15Pro Val Phe Glu Cys Trp Trp
Tyr Asp Thr Glu Pro Thr Glu 20 25 3032730PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
327Ser Glu Thr Arg Pro Thr Glu Ala Gly Asn Val Gln Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Phe Asp Thr Glu Pro Thr Glu 20
25 3032830PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 328Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Val Val Cys Ser Gly Pro1 5 10 15Pro Arg Phe Glu Cys Trp Ala
Phe Val Thr Glu Pro Thr Glu 20 25 3032930PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
329Ser Glu Thr Arg Pro Thr Glu Asp Gly Thr Leu His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Ala Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3033030PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 330Ser Glu Thr Arg Pro Thr Asp Ala
Glu Val Trp Val Cys Asn Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3033130PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
331Ser Glu Thr Arg Pro Thr Glu Asp Val Thr Phe His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Leu Tyr Gly Thr Glu Pro Thr Glu 20
25 3033230PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 332Ser Glu Thr Arg Pro Thr Ser Asp
Phe Asp Trp His Cys Lys Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Ser
Tyr Gly Thr Glu Pro Thr Glu 20 25 3033330PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
333Ser Glu Thr Arg Pro Thr Glu Ala Asp Ser Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Val Pro Glu Pro Thr Glu 20
25 3033430PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 334Ser Glu Thr Arg Pro Thr Asp Asp
Gly Asn Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3033530PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
335Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Arg Tyr Asp Thr Asp Pro Thr Glu 20
25 3033630PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 336Ser Glu Thr Arg Pro Thr Glu Ala
Gly Pro Trp Ser Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Asp Thr Glu Pro Thr Glu 20 25 3033730PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
337Ser Glu Thr Arg Pro Thr Glu Ala Gly Met Phe Leu Cys Ser Gly Pro1
5 10 15Pro Ala Phe Glu Cys Trp Trp Tyr Asp Thr Glu Pro Thr Glu 20
25 3033830PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 338Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Leu Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Leu
Tyr Asp Val Glu Pro Thr Glu 20 25 3033930PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
339Ser Glu Thr Arg Pro Thr Glu Ala Gly Gln Trp Asn Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Asp Ile Glu Pro Thr Glu 20
25 3034030PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 340Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Glu Thr Glu Pro Thr Glu 20 25 3034130PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
341Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Phe Val Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Gly Tyr Val Thr Glu Pro Thr Glu 20
25 3034230PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 342Ser Glu Thr Arg Pro Thr Gln Asp
Gly Thr Trp Phe Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3034330PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
343Ser Glu Thr Arg Pro Thr Glu Gly Asp Ser Trp His Cys Ala Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3034430PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 344Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Ser Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Ser
Tyr Gly Thr Glu Pro Thr Glu 20 25 3034530PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
345Ser Glu Thr Arg Pro Thr Glu Ala Gly Arg Ile Gln Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Asp Glu Glu Pro Thr Glu 20
25 3034630PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 346Ser Glu Thr Arg Pro Thr Glu Ala
Gly Thr Ile Val Cys Lys Gly Pro1 5 10 15Pro Trp Phe Ser Cys Glu Ile
Tyr Glu Thr Glu Pro Thr Glu 20 25 3034730PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
347Ser Glu Thr Arg Pro Thr Glu Ala Gly Asp Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Ala Phe Glu Cys Trp Glu Tyr Leu Gly Glu Pro Thr Glu 20
25 3034830PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 348Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp Phe Cys Ser Gly Pro1 5 10 15Pro Ser Phe Glu Cys Trp Ser
Tyr Val Thr Glu Pro Thr Glu 20 25 3034930PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
349Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp His Cys Ser Gly Pro1
5 10 15Pro Ala Phe Glu Cys Trp Trp Tyr Asp Asn Glu Pro Thr Glu 20
25 3035030PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 350Ser Glu Thr Arg Pro Thr Glu Ala
Gly Arg Trp Thr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Val Ser Asp Pro Thr Glu 20 25 3035130PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
351Ser Glu Thr Arg Pro Thr Glu Ala Gly Glu Trp Tyr Cys Gly Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Phe Asp Thr Ala Pro Thr Glu 20
25 3035230PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 352Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Trp His Cys Ser Gly Pro1 5 10 15Pro Ser Phe Glu Cys Trp Trp
Phe Asp Thr Gly Pro Thr Glu 20 25 3035330PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
353Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Phe Ile Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3035430PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 354Ser Glu Thr Arg Pro Thr Glu Asp
Val Arg Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Phe Gly Thr Glu Pro Thr Glu 20 25 3035530PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
355Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Val Pro Glu Pro Thr Glu 20
25 3035630PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 356Ser Glu Thr Arg Pro Thr Glu Ala
Gly Asn Trp Leu Cys Ser Gly Pro1 5 10 15Pro Ala Phe Glu Cys Trp Trp
Phe Val Ala Glu Pro Thr Glu 20 25 3035730PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
357Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3035830PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 358Ser Glu Thr Arg Pro Thr Glu Ala
Gly Asp Trp Leu Cys Ala Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Trp Gly Thr Asp Pro Thr Glu 20 25 3035930PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
359Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Trp His Cys Val Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Phe Asp Thr Glu Pro Thr Glu 20
25 3036030PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 360Ser Glu Thr Arg Pro Thr Glu Ala
Gly Glu Trp Ser Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Trp Asp Met Glu Pro Thr Glu 20 25 3036130PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
361Ser Glu Thr Arg Pro Thr Tyr Tyr Val Ser Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Ser Tyr Gly Thr Glu Pro Thr Glu 20
25 3036230PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 362Ser Glu Thr Arg Pro Thr Glu Asp
Gly Ser Trp Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3036330PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
363Ser Glu Thr Arg Pro Thr Glu Asp Gly Thr Trp Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Trp Tyr Gly Thr Glu Pro Thr Glu 20
25 3036430PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 364Ser Glu Thr Arg Pro Thr Glu Thr
Asp Ser Trp Val Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Trp
Tyr Gly Thr Glu Pro Thr Glu 20 25 3036520PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
365Gly Ser Trp Arg Phe Cys Gly Gly Glu Tyr Ser Phe Gln Val Cys Gln1
5 10 15Asp Val Ala Pro 2036620PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 366Gly Ser His His Thr Cys
Leu Asp Gly Phe Ala Gly Trp Arg Cys Thr1 5 10 15Glu Val Ala Pro
2036720PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 367Gly Ser Phe Ala Pro Cys Gly Trp Pro Ser Phe Ala
Ile Asp Cys Ile1 5 10 15Ala Glu Ala Pro 2036820PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
368Gly Ser Thr Lys Val Cys His Glu Lys Trp Asn Gln Leu Phe Cys His1
5 10 15Asn Gln Ala Pro 2036920PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 369Gly Ser Pro Glu Met Cys
Met Met Phe Pro Phe Leu Tyr Pro Cys Asn1 5 10 15His His Ala Pro
2037020PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 370Gly Ser Phe Phe Pro Cys Trp Arg Ile Asp Arg Phe
Gly Tyr Cys His1 5 10
15Ala Asn Ala Pro 2037121PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 371Gly Ser Gln Gln Ile Cys
Asp Arg Lys Glu Tyr Arg Phe Gln Ala Cys1 5 10 15Leu Ser Asp Ala Pro
2037221PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 372Gly Ser Thr Met Ser Cys Trp Arg Trp Gly Arg Asp
Ala Tyr Ser Cys1 5 10 15Asn Gln Met Ala Pro 2037321PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
373Gly Ser Ser Gln Ile Cys Ala Val Tyr Leu Asp Asp Thr His Asn Cys1
5 10 15Glu Arg His Ala Pro 2037420PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
374Gly Ser Ser His Cys Asn Gln Met Ile Thr Pro Trp Gln Asn Cys Gly1
5 10 15Met Arg Ala Pro 2037521PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 375Gly Ser Ser Ala Arg Cys
Asp Glu Leu Ile Asn Asp Phe His Ser Cys1 5 10 15Leu Val Met Ala Pro
2037621PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 376Gly Ser Arg Phe His Cys Trp Gln Gly Asp Leu Met
Gln Thr Tyr Cys1 5 10 15Met Pro Met Ala Pro 2037721PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
377Gly Ser Gln Asn Asn Cys Glu Tyr Gly Ser Arg Gly Ser Ser Phe Cys1
5 10 15Leu Ala Met Ala Pro 2037821PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
378Gly Ser Met Asn Met Cys Asp Thr Thr Asp Glu Ile Ser Pro Thr Cys1
5 10 15His Pro Ser Ala Pro 2037921PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
379Gly Ser Met Leu Gly Cys Leu Phe Glu His Gln Asn Lys Tyr Asp Cys1
5 10 15Tyr Val Leu Ala Pro 2038021PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
380Gly Ser Leu Tyr Arg Cys Leu Gly Glu Ala Ser Pro Thr Pro Pro Cys1
5 10 15Ala Tyr Glu Ala Pro 2038121PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
381Gly Ser Gly Met Gly Cys His Gln Val Asn Ile Ser Thr Gly Asp Cys1
5 10 15Ala Glu Asp Ala Pro 2038221PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
382Gly Ser Gly Asp Pro Cys Ser Pro Gly Pro Ser Ile Asn Gly His Cys1
5 10 15Ser Val Met Ala Pro 2038321PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
383Gly Ser Phe Trp Asn Cys Thr Thr Asp Leu Gly Ala Met Ser Asp Cys1
5 10 15Gly Phe Phe Ala Pro 2038421PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
384Gly Ser Phe Thr Ala Cys Asn Lys Thr Ser Thr Thr Arg Gln Pro Cys1
5 10 15Asn Pro Tyr Ala Pro 2038521PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
385Gly Ser Glu Leu Phe Cys Phe Tyr His His Gln Gly Tyr Glu Gly Cys1
5 10 15Asp Val Leu Ala Pro 2038621PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
386Gly Ser Asp Met Asn Cys Thr Val Leu Ala Gln Asp Gln Ile Phe Cys1
5 10 15Phe Arg Glu Ala Pro 2038721PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
387Gly Ser Ala Gly Trp Cys Tyr Thr Met Asn Tyr Val Asp Gln Leu Cys1
5 10 15Thr Tyr Met Ala Pro 2038830PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
388Ser Glu Thr Arg Pro Thr Glu Ala Gly Met Cys Ala Cys Arg Gly Pro1
5 10 15Pro Ala Phe Val Cys Gln Trp Tyr Gly Ser Glu Pro Thr Glu 20
25 3038930PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 389Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Cys His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Ser
Tyr Val Thr Glu Pro Thr Glu 20 25 3039022PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
390Gly Asp Tyr Asp Tyr Cys Asp Phe Asp Leu Glu Thr Tyr Ile Pro Glu1
5 10 15Cys His Ser Tyr Asp Pro 2039122PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
391Gly Asp Asp Phe His Cys Glu Phe Ile Asp Asp Tyr Gln Ser Glu Ile1
5 10 15Cys Tyr Phe Asn Asp Pro 2039222PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
392Gly Asp Leu Leu Val Cys Lys Phe Asp Asp Lys Phe Trp Thr Glu Thr1
5 10 15Cys Glu Trp Ala Asp Pro 2039322PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
393Gly Asp Ser Tyr Asn Cys Ser Trp Asp Ser Lys Thr Phe Glu Val Thr1
5 10 15Cys Leu Tyr Ala Asp Pro 2039422PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
394Gly Asp Ala Ser Trp Cys Asp Glu Asn Ser Pro Ala Ala Trp Phe Tyr1
5 10 15Cys Glu Leu Trp Asp Pro 2039522PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
395Gly Asp Leu Leu Gly Cys Gly Tyr Gln Glu Lys Gly Gly Glu Tyr Lys1
5 10 15Cys Arg Phe Asn Asp Pro 2039622PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
396Gly Asp Pro Trp Trp Cys Phe Glu Lys Asp Ser Phe Ile Pro Phe Ala1
5 10 15Cys Trp His His Asp Pro 2039722PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
397Gly Asp Tyr Tyr Gln Cys Gln Phe Ser Lys Asp Met Tyr Ser Glu Arg1
5 10 15Cys Trp Pro Tyr Asp Pro 2039822PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
398Gly Asp Asn Arg Phe Cys Ser Trp Val Tyr Asn Val Asp Asp Trp Trp1
5 10 15Cys Val Asp Asn Asp Pro 2039922PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
399Gly Asp Tyr Ser Glu Cys Phe Phe Glu Pro Asp Ser Phe Glu Val Lys1
5 10 15Cys Tyr Asp Arg Asp Pro 2040022PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
400Gly Asp Tyr Arg Met Cys Gln Ile Ser Asp Met Trp Gly Asn Tyr Glu1
5 10 15Cys Ser Ser Asp Asp Pro 2040122PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
401Gly Asp Pro Asp Glu Cys Gln Leu Asn Arg Glu Thr Phe Glu Val Trp1
5 10 15Cys Pro Trp His Asp Pro 2040222PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
402Gly Asp His Arg Lys Cys Glu Ile Ser Ala Lys Thr His Glu Val Thr1
5 10 15Cys Tyr Asp Asn Asp Pro 2040322PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
403Gly Asp His Leu Thr Cys Glu Phe Arg Asp Asp Gly Trp Lys Glu His1
5 10 15Cys Trp Trp Ser Asp Pro 2040422PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
404Gly Asp Ala Ser Met Cys Tyr Asp Gly Leu Ala Leu Arg Trp Asp Gln1
5 10 15Cys Trp Pro His Asp Pro 2040530PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
405Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Cys His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Gln Cys Trp Cys Tyr Glu Val Glu Pro Thr Glu 20
25 3040630PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 406Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Cys His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Cys
Tyr Gly Thr Glu Pro Thr Glu 20 25 3040730PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
407Ser Glu Thr Arg Pro Thr Gly Glu Ser Asp Cys His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Tyr Cys Tyr Gly Thr Glu Pro Thr Glu 20
25 3040830PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 408Ser Glu Thr Arg Pro Thr Glu Ser
Gly Asn Cys Tyr Cys Ser Gly Pro1 5 10 15Pro Trp Phe Glu Cys Trp Cys
Tyr Gly Thr Glu Pro Thr Glu 20 25 3040930PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
409Ser Glu Thr Arg Pro Thr Glu Ala Gly Ala Cys Arg Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Tyr Cys Tyr Asp Met Ala Pro Thr Glu 20
25 3041030PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 410Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Cys Tyr Cys Ser Gly Pro1 5 10 15Pro Arg Phe Glu Cys Trp Cys
Tyr Glu Thr Glu Pro Thr Glu 20 25 3041130PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
411Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Cys His Cys Ser Gly Pro1
5 10 15Pro Ser Phe Glu Cys Trp Cys Phe Gly Thr Glu Pro Thr Glu 20
25 3041230PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 412Ser Glu Thr Arg Pro Thr Val Ser
Val Ser Cys Ser Cys Gly Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Cys
Phe Gly Thr Glu Pro Thr Glu 20 25 3041330PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
413Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Cys His Cys Asn Gly Pro1
5 10 15Pro Thr Phe Glu Cys Phe Cys Phe Gly Thr Glu Pro Thr Glu 20
25 3041430PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 414Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Cys Tyr Cys Gly Gly Pro1 5 10 15Pro Ser Phe Glu Cys Trp Cys
Tyr Gly Thr Glu Pro Thr Glu 20 25 3041530PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
415Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Cys His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Cys Tyr Gly Ser Asn Pro Thr Glu 20
25 3041630PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 416Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Cys His Cys Ser Gly Pro1 5 10 15Pro Ala Phe Glu Cys Trp Cys
Tyr Arg Ala Glu Pro Thr Glu 20 25 3041730PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
417Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Cys Asp Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Cys Phe Gly Thr Glu Pro Thr Glu 20
25 3041830PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 418Ser Glu Thr Arg Pro Thr Glu Ala
Gly Lys Cys His Cys Gly Gly Pro1 5 10 15Pro Ser Phe Glu Cys Trp Cys
Tyr Ala Thr Glu Pro Thr Glu 20 25 3041930PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
419Ser Glu Thr Arg Pro Thr Glu Ala Gly Lys Cys His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Thr Cys Tyr His Thr Asp Pro Thr Glu 20
25 3042030PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 420Ser Glu Thr Arg Pro Thr Glu Ala
Gly Phe Cys Gln Cys Ser Gly Pro1 5 10 15Pro Ala Phe Glu Cys Trp Cys
Tyr Asp Thr Glu Pro Thr Glu 20 25 3042130PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
421Ser Glu Thr Arg Pro Thr Glu Ala Val Ser Cys Glu Cys Lys Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Cys Phe Gly Thr Glu Pro Thr Glu 20
25 3042230PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 422Ser Glu Thr Arg Pro Thr Glu Ala
Gly Asp Cys His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Cys
Tyr Gly Thr Glu Pro Thr Glu 20 25 3042330PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
423Ser Glu Thr Arg Pro Thr Glu Ala Gly Ala Cys Asp Cys Ile Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Cys Tyr Asp Thr Tyr Pro Thr Glu 20
25 3042430PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 424Ser Glu Thr Arg Pro Thr Glu Ala
Gly Asn Cys Leu Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Ala Cys
Tyr His Ser Glu Pro Thr Glu 20 25 3042530PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
425Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Cys His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Gln Cys Trp Cys Tyr Ser Thr Glu Pro Thr Glu 20
25 3042630PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 426Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ile Cys His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Cys
Tyr Ala Thr Glu Pro Thr Glu 20 25 3042730PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
427Ser Glu Thr Arg Pro Thr Glu Glu Gly Ser Cys His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Cys Phe Gly Thr Glu Pro Thr Glu 20
25 3042830PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 428Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ile Cys Asn Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Cys
Tyr Ser Met Gly Pro Thr Glu 20 25 3042930PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
429Ser Glu Thr Arg Pro Thr Gln Gly Gly Asn Cys His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Cys Tyr Gly Thr Glu Pro Thr Glu 20
25 3043030PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 430Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Cys Asn Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Tyr Cys
Tyr Thr Leu Asp Pro Thr Glu 20 25 3043130PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
431Ser Glu Thr Arg Pro Thr Asp Asn Gly Ser Cys Gln Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Cys Phe Gly Thr Glu Pro Thr Glu 20
25 3043230PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 432Ser Glu Thr Arg Pro Thr Glu Ser
Gly Ser Cys His Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Cys
Tyr Gly Thr Glu Pro Thr Glu 20 25 3043330PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
433Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Cys Asn Cys Ser Gly Pro1
5 10 15Pro Ser Phe Glu Cys Trp Cys Tyr Val Thr Glu Pro Thr Glu
20
25 3043430PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 434Ser Glu Thr Arg Pro Thr Glu Gly
Gly Ser Cys Tyr Cys Gly Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Cys
Tyr Gly Thr Glu Pro Thr Glu 20 25 3043530PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
435Ser Glu Thr Arg Pro Thr Glu Ala Gly Arg Cys His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Cys Tyr Val Gln Glu Pro Thr Glu 20
25 3043630PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 436Ser Glu Thr Arg Pro Thr Glu Ser
Gly Ser Cys Leu Cys Ser Gly Pro1 5 10 15Pro Gln Phe Glu Cys Trp Cys
Tyr Gly Thr Glu Pro Thr Glu 20 25 3043730PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
437Ser Glu Thr Arg Pro Thr Glu Thr Asp Ser Cys His Cys Ile Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Cys Tyr Gly Thr Glu Pro Thr Glu 20
25 3043830PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 438Ser Glu Thr Arg Pro Thr Glu Ala
Gly Phe Cys Arg Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Cys
Tyr Asp Thr Glu Pro Thr Glu 20 25 3043930PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
439Ser Glu Thr Arg Pro Thr Glu His Gly Ser Cys Asn Cys Tyr Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Cys Tyr Gly Thr Glu Pro Thr Glu 20
25 3044030PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 440Ser Glu Thr Arg Pro Thr Ala Leu
Gly Gly Cys Leu Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Cys
Tyr Gly Thr Glu Pro Thr Glu 20 25 3044130PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
441Ser Glu Thr Arg Pro Thr Glu Gly Gly Ser Cys Glu Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Cys Tyr Gly Thr Glu Pro Thr Glu 20
25 3044230PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 442Ser Glu Thr Arg Pro Thr Glu Glu
Gly Ser Cys His Cys Ser Gly Pro1 5 10 15Pro Ala Phe Glu Cys Trp Cys
Tyr Gly Thr Glu Pro Thr Glu 20 25 3044330PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
443Ser Glu Thr Arg Pro Thr Glu Ala Gly Thr Cys Tyr Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Cys Tyr Gly Thr Glu Pro Thr Glu 20
25 3044430PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 444Ser Glu Thr Arg Pro Thr Glu Asp
Gly Ser Cys His Cys Ser Gly Pro1 5 10 15Pro Arg Phe Glu Cys Trp Cys
Tyr Gly Thr Glu Pro Thr Glu 20 25 3044530PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
445Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Cys His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Cys Tyr Ser Thr Glu Pro Thr Glu 20
25 3044630PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 446Ser Glu Thr Arg Pro Thr Glu Ala
Gly Ser Cys Tyr Cys Ser Gly Pro1 5 10 15Pro Thr Phe Glu Cys Trp Cys
Tyr Ala Glu Glu Pro Thr Glu 20 25 3044730PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
447Ser Glu Thr Arg Pro Thr Glu Ala Gly Ser Cys His Cys Ser Gly Pro1
5 10 15Pro Thr Phe Glu Cys Trp Cys Phe Glu Pro Glu Pro Thr Glu 20
25 3044828PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 448Ser Glu Tyr Pro Thr Trp Val Ser
Lys Glu Phe His Glu Cys Ala Gly1 5 10 15Glu Leu Val Ala Met Gln Gly
Gly Ser Gly Thr Glu 20 2544921PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 449Ala Gln Gln Ala Ser Arg
Phe Thr Phe Thr Asp Gly Asp Ser Tyr Trp1 5 10 15Trp Phe Glu Asp Phe
2045022PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 450Ala Gln Ile Gln Gly Ile Gln Lys Thr Glu Gln Gly
Glu Phe Tyr Trp1 5 10 15Phe Asn Trp Phe Pro Ala
2045122PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 451Ala Gln Arg Glu Val Glu Glu Pro Tyr Trp Tyr Leu
Asp Phe Leu Ser1 5 10 15Ser Trp Arg Met His Glu
2045222PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 452Ala Gln Arg Pro Glu Ala His Tyr Lys Leu Ala Met
Ser Tyr Pro Ile1 5 10 15Ile Pro Arg Thr Lys Thr
2045322PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 453Ala Gln Arg Trp Ser Ser Pro Gly Met Ser Gln Ser
Phe Val Leu Glu1 5 10 15Trp Lys Trp Asn Asp Asn
2045422PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 454Ala Gln Tyr Asp Thr Trp Val Phe Gln Phe Ile His
Glu Val Pro Gly1 5 10 15Glu Leu Val Ala Met Gln
2045520PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 455Ala Gln Met Tyr Gln Thr Pro Asp Gly Val Ile Gly
Lys Phe Val Asp1 5 10 15Trp Met Phe Asn 2045620PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
456Ala Gln Val Gly Ser Pro Met Leu Pro Ser Trp Phe Ser Phe Glu Ala1
5 10 15Asn Trp Ser Ser 2045722PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 457Ala Gln Asn Ala Val Val
Pro Pro Pro Met Leu Trp Ser Ile Tyr Trp1 5 10 15Asp Tyr Gly Arg Glu
Gly 2045822PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 458Ala Gln Pro Tyr Tyr Glu Leu Gln
Asp Ala Asp Met Leu Leu Val Val1 5 10 15Ala Leu Leu Ser Thr Gly
2045922PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 459Ala Gln Val Gly Thr Ala Glu Ala Ile Met Phe Ser
Asp Val Glu Asp1 5 10 15Thr Gly Val His Lys Phe
2046022PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 460Ala Gln Phe Pro Leu Glu Phe Asp Val Pro Asn Phe
Ser Tyr His Trp1 5 10 15Leu Val Ser Phe Asn Pro
2046122PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 461Ala Gln Asp Leu Lys Pro Trp Thr Ala Gly Trp Glu
Pro Pro Trp Leu1 5 10 15Trp Thr Asp Arg Gly Pro
2046222PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 462Ala Gln His Gln Tyr Gly Gln Met Met Val Leu His
Ile Gln Tyr Asp1 5 10 15Met Gly Glu Phe Ile Pro
2046322PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 463Ala Gln Ser Pro Tyr Ile Phe Pro Ile Asp Asp Ser
Gly Arg Gln Ile1 5 10 15Phe Val Ile Gln Trp Gly
2046422PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 464Ala Gln Val Pro Asp Trp Leu Ser Ala Val Val Ile
Glu Lys Leu Ile1 5 10 15Glu Tyr Gly Met Met Val
2046522PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 465Ala Gln Phe Asp Arg Tyr Trp His Phe Ala Trp Met
Asp Val Ser Phe1 5 10 15Ser Ser Gly Gln Ser Gly
2046622PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 466Ala Gln Lys Glu Thr Trp Glu Phe Phe Asp Ile Val
Tyr Gly Ser Gly1 5 10 15Trp Lys Phe Asn Ser Pro
2046722PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 467Ala Gln His Ser Val Gln Arg Gln Met Asp Val Trp
Met Pro Val Gln1 5 10 15Phe Met Ala Gly Phe Thr
2046821PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 468Ala Gln Glu Trp Gln Thr Trp Thr Trp Asn Met Ile
Glu Val Ile Ser1 5 10 15Glu Asn Lys Thr Pro 2046920PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
469Ala Gln Gly Phe Glu Leu Trp Val Asp His Thr Arg Asn Phe Phe Ile1
5 10 15Ala Ile Ser Pro 2047022PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 470Ala Gln Ala Tyr Glu Trp
Trp Ala Asp Glu Ser Ile Phe Asn His Gly1 5 10 15Tyr Tyr Trp Gly His
Gln 2047122PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 471Ala Gln Asp Pro Gly Phe Ser Lys
His Ser Met Gly His Gly Tyr Pro1 5 10 15Ser Lys Met Asn Trp Gly
2047222PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 472Ala Gln Glu Trp Glu Arg Glu Tyr Phe Val Asp Gly
Phe Trp Gly Ser1 5 10 15Trp Phe Gly Ile Pro His
2047322PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 473Ala Gln Met Gly His His Trp Asp Val Gln Trp Asp
Tyr Lys Leu Phe1 5 10 15His Val Ala Arg Gly Asp
2047422PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 474Ala Gln Glu Leu Phe Gln Ile Leu Glu Lys Gln Met
Trp Ser Asp Phe1 5 10 15Met Glu Trp Ala Thr Pro
2047522PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 475Ala Gln His Trp Asp Tyr Asp Ser Gly Ser Asp Phe
Trp Phe Pro Val1 5 10 15Phe Phe Leu Glu His His
2047622PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 476Ala Gln His Gly Tyr Leu Ser Pro Leu Lys Gln Tyr
Gln Met Ser His1 5 10 15Val Glu Phe Trp Thr Tyr
2047722PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 477Ala Gln Phe Ser Gly Leu Val Met Tyr Gly Arg Thr
His Glu Val Gln1 5 10 15Trp Thr Phe Gly Ser Met
2047822PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 478Ala Gln Ala Glu Trp Val Ile Thr Ser Glu Glu Phe
Tyr Trp Lys Met1 5 10 15Ala Asp Phe Gly Pro Pro
2047919PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 479Ala Gln Trp Pro His Asp Gly Leu Val His Trp Gly
Glu Val Ile Met1 5 10 15Leu Arg Phe 48022PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
480Ala Gln Trp Asn Gln Trp Asp Glu Phe Met Trp Phe Leu Asn Pro Pro1
5 10 15Pro Ile Gly Leu Met Trp 2048121PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
481Ala Gln Asp Asn Thr Ala Asp Gln Met Phe Asn Gly Phe His Val Leu1
5 10 15Ala Met Tyr Met Val 2048221PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
482Ala Gln Ser Asp His Asp His Ala His Trp Gly Val Lys His Trp Pro1
5 10 15Phe Arg Arg Tyr Gln 2048322PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
483Ala Gln Leu Phe Gln Tyr Leu Trp His Asp Asp Pro Gln Gly Ala Phe1
5 10 15Phe Gln Leu Ser Met Trp 2048422PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
484Ala Gln His Val Val Thr Leu Thr Leu Ile Gln Met Pro Phe Ala Phe1
5 10 15Asn Phe Glu Pro Arg Met 2048522PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
485Ala Gln Val Gly Glu Ser Leu Asp Asp Gly Trp Thr Phe Phe Ser Asp1
5 10 15Lys Trp Phe Asp Phe Phe 2048622PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
486Ala Gln Phe Met Tyr Glu Lys Glu His Tyr Val Met Ser Ile Ser Leu1
5 10 15Pro Gly Leu Trp Phe Tyr 2048722PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
487Ala Gln His Met Asp Pro Ala Glu Trp Asp Trp Phe Ile Arg Ile Tyr1
5 10 15Ser Pro Val Val Asn Pro 2048822PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
488Ala Gln Met Trp His Arg Val His Asp Pro Gly Tyr Thr Phe Glu Val1
5 10 15Thr Trp Leu Trp Asp Asn 2048922PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
489Ala Gln Trp Asn Trp Asp Met Gly Phe Met Trp Thr Thr Asp Ser Ala1
5 10 15Gln Val Gln Pro Ser Met 2049022PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
490Ala Gln Lys Thr Trp Phe Leu Glu Ala Asp Leu Phe Gln Met Phe Gln1
5 10 15Glu Val Thr Trp Gln Phe 2049122PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
491Ala Gln Trp Gly Ala Val Asp Asn Asp Trp Tyr Asp Trp Glu Met Glu1
5 10 15Gln Ile Trp Met Phe Glu 2049222PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
492Ala Gln Val Glu Asp Met Ala Thr Val His Phe Lys Phe Asn Pro Ala1
5 10 15Thr His Glu Val Ile Trp 2049322PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
493Ala Gln Arg Asp Tyr Leu Phe Tyr Trp Asn Asp Gly Ser Tyr Gln Pro1
5 10 15Trp Gln Val Phe Val Gly 2049422PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
494Ala Gln Gln Trp Met Phe Gln Ile His Gln Ser Met Ala Trp Pro Tyr1
5 10 15Glu Trp Ile Asp Ser Tyr 2049522PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
495Ala Gln Gly Ile Ala Trp Gln Leu Glu Trp Ser Tyr Met Pro Gln Ser1
5 10 15Pro Pro Ser Phe Asp Arg 2049621PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
496Ala Gln Gly Gly Arg Tyr Pro Phe Tyr Asp Thr Asp Trp Phe Lys Trp1
5 10 15Glu Met Tyr Val Leu 2049728PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
497Ser Glu Glu Asp Thr Trp Leu Phe Trp Gln Ile Ile Glu Val Pro Val1
5 10 15Gly Gln Val Leu Met Gln Gly Gly Ser Gly Thr Glu 20
2549828PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 498Ser Glu Tyr Asp Thr Leu Leu Phe Gln Arg Thr Gly
Glu Val Val Gly1 5 10 15Lys Leu Gly Ser Met Gln Gly Gly Ser Gly Thr
Glu 20 2549928PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 499Ser Glu Tyr Asp Thr Trp Val Phe
Gln Phe Met Leu Glu Val Pro Gly1 5 10 15Ser Trp Met Ala Arg Leu Gly
Gly Ser Gly Thr Glu 20 2550028PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 500Ser Glu Tyr Asp Thr Trp
Ile Phe Gln Phe Tyr Arg Glu Val Pro Gly1 5 10 15Val Pro Gly Ala Met
Gln Gly Gly Ser Gly Thr Glu 20 2550128PRTArtificial
SequenceSynthetically generated cMet-binding peptide
sequence 501Ser Glu Val Asp Thr Gly Val Gln Leu Leu Thr His Glu Gly
Pro Gly1 5 10 15Glu Leu Val Ala Met Gln Gly Gly Ser Gly Thr Glu 20
2550228PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 502Ser Glu Ser Asp Thr Trp Val Phe Gln Leu Ile His
Glu Val Pro Ala1 5 10 15Ser Val Val Ala Met Gln Gly Gly Ser Gly Thr
Glu 20 2550328PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 503Ser Glu Tyr Asp Thr Trp Val Phe
Gln Phe Arg His Gly Val Lys Ala1 5 10 15Gln Leu Val Ala Met Arg Gly
Gly Ser Gly Thr Glu 20 2550428PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 504Ser Glu Tyr Asp Ser Arg
Val Phe Gln Tyr Ala Pro Glu Val Ala Gly1 5 10 15Gln Val Glu Ala Met
Gln Gly Gly Ser Gly Thr Glu 20 2550528PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
505Ser Glu Asp Glu Ser Arg Val Val Gln Phe Gln His Glu Val Ser Gly1
5 10 15Glu Leu Val Ala Met Gln Gly Gly Ser Gly Thr Glu 20
2550628PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 506Ser Glu Gln Asp Thr Phe Val Phe Met Tyr Asn Gly
Glu Val Ser Gly1 5 10 15Asp Met Val Ala Met Gln Gly Gly Ser Gly Thr
Glu 20 2550728PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 507Ser Glu Tyr Asp Thr Trp Val Phe
Gln Phe Arg Arg Gln Val Pro Gly1 5 10 15Val Leu Glu Thr Met Leu Gly
Gly Ser Gly Thr Glu 20 2550828PRTArtificial SequenceSynthetically
generated cMet-binding peptide sequence 508Ser Glu Gln Glu Thr Leu
Val Phe Ala Val Ile Asp Gly Asp Pro Gly1 5 10 15Glu Leu Val Ala Met
Gln Gly Gly Ser Gly Thr Glu 20 2550928PRTArtificial
SequenceSynthetically generated cMet-binding peptide sequence
509Ser Glu Tyr Asp Thr Trp Val Phe Gln Phe Ile His Val Ala Arg Gly1
5 10 15Glu Met Glu Gly Thr Leu Gly Gly Ser Gly Thr Glu 20
2551028PRTArtificial SequenceSynthetically generated cMet-binding
peptide sequence 510Ser Glu Asp Glu Ser Arg Val Val Gln Phe Gln His
Glu Val Ser Gly1 5 10 15Glu Leu Val Ala Met Gln Gly Gly Ser Gly Thr
Glu 20 2551128PRTArtificial SequenceSynthetically generated
cMet-binding peptide sequence 511Ser Glu Gln Asp Thr Phe Val Phe
Met Tyr Asn Gly Glu Val Ser Gly1 5 10 15Asp Met Val Ala Met Gln Gly
Gly Ser Gly Thr Glu 20 255129PRTArtificial SequenceConsensus motif
512Cys Xaa Gly Pro Pro Xaa Phe Xaa Cys1 55134PRTArtificial
SequenceLinker 513Gly Gly Gly Lys151424PRTArtificial
SequenceSynthetically generated peptide 514Gly Ser Pro Glu Met Cys
Met Met Phe Pro Phe Leu Tyr Pro Cys Asn1 5 10 15His His Ala Pro Gly
Gly Gly Lys 2051524PRTArtificial SequenceSynthetically generated
peptide 515Gly Ser Phe Phe Pro Cys Trp Arg Ile Asp Arg Phe Gly Tyr
Cys His1 5 10 15Ala Asn Ala Pro Gly Gly Gly Lys
2051626PRTArtificial SequenceSynthetically generated peptide 516Ala
Gln Glu Trp Glu Arg Glu Tyr Phe Val Asp Gly Phe Trp Gly Ser1 5 10
15Trp Phe Gly Ile Pro His Gly Gly Gly Lys 20 2551726PRTArtificial
SequenceSynthetically generated peptide 517Gly Asp Tyr Ser Glu Cys
Phe Phe Glu Pro Asp Ser Phe Glu Val Lys1 5 10 15Cys Tyr Asp Arg Asp
Pro Gly Gly Gly Lys 20 25518142DNAArtificial SequenceSynthetic
construct 518ctcagcagtc actgtct tcc atg ggt tct gaa act cgc cct aca
nnn nnn 50 Ser Met Gly Ser Glu Thr Arg Pro Thr Glu Ala 1 5 10nnn
nnn nnn nnn tgt nnn ggt cct cct nnn ttc nnn tgc nnn nnn nnn 98Gly
Ser Trp His Cys Ser Gly Pro Pro Thr Phe Glu Cys Trp Trp Tyr 15 20
25gga acg gag ccg act gaa gct agc g tgactctgac agtctctgt 142Gly Thr
Glu Pro Thr Glu Ala Ser 30 35519151DNAArtificial SequenceSynthetic
construct 519ctcagcagtc actgtct tcc atg ggt tct gaa act cgc cct aca
gag gct 50 Ser Met Gly Ser Glu Thr Arg Pro Thr Glu Ala 1 5 10ggt
nnn nnn nnn tgt nnn ggt cct cct nnn ttc nnn tgc nnn nnn nnn 98Gly
Ser Trp His Cys Ser Gly Pro Pro Thr Phe Glu Cys Trp Trp Tyr 15 20
25nnn nnn nnn ccg act gaa cgt cct agt gct agc gtgactctga cagtctctgt
151Xaa Xaa Xaa Pro Thr Glu Arg Pro Ser Ala Ser 30
35520142DNAArtificial SequenceSynthetic construct 520ctcagcagtc
actgtct tcc atg ggt tct gaa act cgc cct aca nnn nnn 50 Ser Met Gly
Ser Glu Thr Arg Pro Thr Glu Ala 1 5 10nnn nnn nnn nnn tgc nnn ggt
cct cct nnn ttc nnn tgt nnn nnn nnn 98Gly Ser Ile Gln Cys Lys Gly
Pro Pro Trp Phe Ser Cys Ala Met Tyr 15 20 25gga acg gag ccg act gaa
gct agc gtgactctga cagtctctgt 142Gly Thr Glu Pro Thr Glu Ala Ser 30
35521151DNAArtificial SequenceSynthetic construct 521ctcagcagtc
actgtct tcc atg ggt tct gaa act cgc cct aca gag gcc 50 Ser Met Gly
Ser Glu Thr Arg Pro Thr Glu Ala 1 5 10ggt nnn nnn nnn tgc nnn ggt
cct cct nnn ttc nnn tgt nnn nnn nnn 98Gly Ser Ile Gln Cys Lys Gly
Pro Pro Trp Phe Ser Cys Ala Met Tyr 15 20 25nnn nnn nnn ccg act gaa
cgt cct agt gct agc gtgactctga cagtctctgt 151Gly Thr Glu Pro Thr
Glu Arg Pro Ser Ala Ser 30 35522151DNAArtificial SequenceSynthetic
construct 522ctcagcagtc actgtct tcc atg ggt tct gaa act cgc cct aca
gag gct 50 Ser Met Gly Ser Glu Thr Arg Pro Thr Glu Ala 1 5 10ggt
nnn nnn nnn tgc nnn ggt cct cct nnn ttc nnn tgt nnn nnn nnn 98Gly
Tyr tyr Gly Cys Lys Gly Pro Pro Thr Phe Glu Cys Gln Trp Met 15 20
25nnn nnn nnn ccg act gaa cgt cct agt gct agc gtgactctga cagtctctgt
151Gly Thr Glu Pro Thr Glu Arg Pro Ser Ala Ser 30
35523151DNAArtificial SequenceSynthetic construct 523ctcagcagtc
actgtct tcc atg ggt tct gaa act cgc cct aca gag gct 50 Ser Met Gly
Ser Glu Thr Arg Pro Thr Glu Ala 1 5 10ggt nnn nnn nnn tgt nnn ggt
nnn cct nnn ttc nnn tgc nnn nnn nnn 98Gly Ala Phe Phe Cys Ser Gly
Pro Pro Thr Phe Met Cys Ser Leu Tyr 15 20 25nnn nnn nnn ccg act gaa
cgt cct agt gct agc gtgactctga cagtctctgt 151Gly Thr Glu Pro Thr
Glu Arg Pro Ser Ala Ser 30 35524142DNAArtificial SequenceSynthetic
construct 524ctcagcagtc actgtct tcc atg ggt tct gaa act cgc cct aca
nnn nnn 50 Ser Met Gly Ser Glu Thr Arg Pro Thr Glu Ala 1 5 10nnn
nnn nnn nnn tgt nnn ggt nnn ccg nnn ttc nnn tgt nnn nnn nnn 98Gly
Gln Phe Lys Cys Ala Gly Pro Pro Ser Phe Ala Cys Trp Met Thr 15 20
25gga acg gag ccg act gaa gct agc gtgactctga cagtctctgt 142Gly Thr
Glu Pro Thr Glu Ala Ser 30 3552519PRTArtificial SequenceConsensus
motif 525Gly Ser Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Ala Pro1 5 10 15Gly Gly Lys52622PRTArtificial SequenceConsensus
motif 526Ala Gly Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa1 5 10 15Gly Thr Gly Gly Gly Lys 2052718PRTArtificial
SequenceConsensus motif 527Gly Xaa Xaa Xaa Cys Xaa Gly Xaa Pro Xaa
Phe Xaa Cys Xaa Xaa Xaa1 5 10 15Gly Thr 52822PRTArtificial
SequenceConsensus motif 528Gly Xaa Xaa Xaa Cys Xaa Gly Pro Pro Xaa
Phe Xaa Cys Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Pro Thr Glu
2052920PRTArtificial SequenceConsensus motif 529Thr Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Gly Pro Pro Xaa Phe Xaa Cys1 5 10 15Xaa Xaa Xaa Gly
2053019PRTArtificial SequenceConsensus motif 530Ser Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa1 5 10 15Xaa Ala Pro
53115PRTArtificial SequenceSynthetically generated peptide 531Tyr
Tyr Gly Cys Lys Gly Pro Pro Thr Phe Glu Cys Gln Trp Met1 5 10
1553220PRTArtificial SequenceConsensus motif 532Ser Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa1 5 10 15Xaa Xaa Ala Pro
2053321PRTArtificial SequenceConsensus motif 533Asp Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys1 5 10 15Xaa Xaa Xaa Asp
Pro 2053420PRTArtificial SequenceExemplary motif 534Gly Xaa Cys Xaa
Cys Xaa Gly Pro Pro Xaa Phe Xaa Cys Xaa Cys Xaa1 5 10 15Xaa Xaa Xaa
Pro 2053520PRTArtificial SequenceExemplary motif 535Thr Xaa Xaa Xaa
Xaa Cys Xaa Cys Xaa Gly Pro Pro Xaa Phe Glu Cys1 5 10 15Xaa Cys Xaa
Gly 2053615PRTArtificial SequenceSynthetically generated peptide
536Ala Phe Phe Cys Ser Gly Pro Pro Thr Phe Met Cys Ser Leu Tyr1 5
10 1553715PRTArtificial SequenceSynthetically generated peptide
537Ser Ile Gln Cys Lys Gly Pro Pro Trp Phe Ser Cys Ala Met Tyr1 5
10 1553812PRTArtificial SequenceExemplary motif 538Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa1 5 1053914PRTArtificial
SequenceExemplary motif 539Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa1 5 1054014PRTArtificial SequenceExemplary motif
540Xaa Xaa Xaa Cys Xaa Gly Xaa Pro Xaa Phe Xaa Cys Xaa Xaa1 5
1054118PRTArtificial SequenceExemplary motif 541Xaa Xaa Xaa Cys Xaa
Gly Pro Pro Xaa Phe Xaa Cys Trp Xaa Xaa Xaa1 5 10 15Xaa Xaa
54218PRTArtificial SequenceExemplary motif 542Xaa Xaa Xaa Xaa Trp
Xaa Cys Xaa Gly Pro Pro Thr Phe Glu Cys Trp1 5 10 15Xaa Xaa
54316PRTArtificial SequenceExemplary motif 543Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa1 5 10
1554417PRTArtificial SequenceExemplary motif 544Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa1 5 10 15Xaa
54518PRTArtificial SequenceExemplary motif 545Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa1 5 10 15Xaa Xaa
54618PRTArtificial SequenceSynthetically generated peptide 546Ser
Cys Xaa Cys Xaa Gly Pro Pro Thr Phe Glu Cys Trp Cys Tyr Xaa1 5 10
15Xaa Xaa54718PRTArtificial SequenceSynthetically generated peptide
547Glu Xaa Gly Ser Cys His Cys Ser Gly Pro Pro Thr Phe Glu Cys Xaa1
5 10 15Cys Xaa 5489PRTArtificial SequenceSynthetically generated
peptide 548Cys Lys Gly Pro Pro Thr Phe Glu Cys1 55499PRTArtificial
SequenceSynthetically generated peptide 549Cys Ser Gly Pro Pro Thr
Phe Met Cys1 5550119DNAArtificial SequenceSynthetic construct
550tcactgtct tcc atg ggt tct gaa nnn nnn nnn nnn nnn nnn nnn nnn
nnn 51 Ser Met Gly Ser Glu Tyr Asp Thr Trp Val Phe Gln Phe Ile 1 5
10nnn nnn nnn nnn ggt gag ctg gtt gct atg cag ggt ggt agt ggt act
99His Glu Val Pro Gly Glu Leu Val Ala Met Gln Gly Gly Ser Gly Thr15
20 25 30gaa gct agc gtgactctga c 119Glu Ala Ser551119DNAArtificial
SequenceSynthetic construct 551tcactgtct tcc atg ggt tct gaa nnn
gat act nnn nnn ttt nnn nnn nnn 51 Ser Met Gly Ser Glu Tyr Asp Thr
Trp Val Phe Gln Phe Ile 1 5 10nnn gag gtt nnn nnn nnn nnn nnn nnn
atg caa ggt ggt agt ggt act 99His Glu Val Pro Gln Glu Leu Val Ala
Met Gln Gly Gly Ser Gly Thr15 20 25 30gaa gct agc gtgactctga c
119Glu Ala Ser552119DNAArtificial SequenceSynthetic construct
552tcactgtct tcc atg ggt tct gaa tat gat act tgg gtt ttt caa ttt
nnn 51 Ser Met Gly Ser Glu Tyr Asp Thr Trp Val Phe Gln Phe Ile 1 5
10nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn ggt ggt agt ggt act
99His Glu Val Pro Gly Glu Leu Val Ala Met Gln Gly Gly Ser Gly Thr15
20 25 30gaa gct agc gtgactctga c 119Glu Ala Ser55324DNAArtificial
SequenceSynthetically generated oligonucleotide 553tcactgtctt
ccatgggttc tgaa 2455436DNAArtificial SequenceSynthetically
generated oligonucleotide 554tcactgtctt ccatgggttc tgaaactcgc
cctaca 3655522DNAArtificial SequenceSynthetically generated
oligonucleotide 555ctcagcagtc actgtcttcc at 2255644DNAArtificial
SequenceSynthetically generated oligonucleotide 556ctcagcagtc
actgtcttcc atgggttctg aaactcgccc taca 4455793DNAArtificial
SequenceSynthetic construct 557tctgaaactc gccctacann nnnnnnnnnn
nnnnnntgtn nnggtcctcc tnnnttcnnn 60tgcnnnnnnn nnggaacgga gccgactgaa
gct 9355844DNAArtificial SequenceSynthetic construct 558ggaacggagc
cgactgaagc tagcgtgact ctgacagtct ctgt 4455922DNAArtificial
SequenceSynthetic construct 559cagtgactct gacagtctct gt
2256035DNAArtificial SequenceSynthetic construct 560ggaacggagc
cgactgaagc tagcgtgact ctgac 3556123DNAArtificial SequenceSynthetic
construct 561actgaagcta gcgtgactct gac 2356222DNAArtificial
SequenceSynthetic construct 562ctcagcagtc actgtcttcc at
2256344DNAArtificial SequenceSynthetic construct 563ctcagcagtc
actgtcttcc atgggttctg aaactcgccc taca 44564100DNAArtificial
SequenceSynthetic construct 564tctgaaactc gccctacaga ggctggtnnn
nnnnnntgtn nnggtcctcc tnnnttcnnn 60tgcnnnnnnn nnnnnnnnnn nccgactgaa
cgtcctagtg 10056544DNAArtificial SequenceSynthetic construct
565ccgactgaac gtcctagtgc tagcgtgact ctgacagtct ctgt
4456622DNAArtificial SequenceSynthetic construct 566cagtgactct
gacagtctct gt 2256735DNAArtificial SequenceSynthetic construct
567ccgactgaac gtcctagtgc tagcgtgact ctgac 3556822DNAArtificial
SequenceSynthetic construct 568ctagtgctag cgtgactctg ac
2256922DNAArtificial SequenceSynthetic construct 569ctcagcagtc
actgtcttcc at 2257044DNAArtificial SequenceSynthetic construct
570ctcagcagtc actgtcttcc atgggttctg aaactcgccc taca
4457192DNAArtificial SequenceSynthetic construct 571tctgaaactc
gccctacann nnnnnnnnnn nnnnnntgcn nnggtcctcc tnnnttcnnn 60tgtnnnnnnn
nnggaacgga gccgactgaa gc 9257244DNAArtificial SequenceSynthetic
construct 572ggaacggagc cgactgaagc tagcgtgact ctgacagtct ctgt
4457322DNAArtificial SequenceSynthetic construct 573cagtgactct
gacagtctct gt 2257422DNAArtificial SequenceSynthetic construct
574ctcagcagtc actgtcttcc at 2257544DNAArtificial SequenceSynthetic
construct 575ctcagcagtc actgtcttcc atgggttctg aaactcgccc taca
44576101DNAArtificial SequenceSynthetic construct 576tctgaaactc
gccctacaga ggccggtnnn nnnnnntgcn nnggtcctcc tnnnttcnnn 60tgtnnnnnnn
nnnnnnnnnn nccgactgaa cgtcctagtg c 10157744DNAArtificial
SequenceSynthetic construct 577ccgactgaac gtcctagtgc tagcgtgact
ctgacagtct ctgt 4457822DNAArtificial SequenceSynthetic construct
578cagtgactct gacagtctct gt 2257922DNAArtificial SequenceSynthetic
construct 579ctcagcagtc actgtcttcc at 2258044DNAArtificial
SequenceSynthetic
construct 580ctcagcagtc actgtcttcc atgggttctg aaactcgccc taca
44581101DNAArtificial SequenceSynthetic construct 581tctgaaactc
gccctacaga ggctggtnnn nnnnnntgcn nnggtcctcc tnnnttcnnn 60tgtnnnnnnn
nnnnnnnnnn nccgactgaa cgtcctagtg c 10158244DNAArtificial
SequenceSynthetic construct 582ccgactgaac gtcctagtgc tagcgtgact
ctgacagtct ctgt 4458322DNAArtificial SequenceSynthetic construct
583cagtgactct gacagtctct gt 2258422DNAArtificial SequenceSynthetic
construct 584ctcagcagtc actgtcttcc at 2258544DNAArtificial
SequenceSynthetic construct 585ctcagcagtc actgtcttcc atgggttctg
aaactcgccc taca 44586101DNAArtificial SequenceSynthetic construct
586tctgaaactc gccctacaga ggctggtnnn nnnnnntgtn nnggtnnncc
tnnnttcnnn 60tgcnnnnnnn nnnnnnnnnn nccgactgaa cgtcctagtg c
10158744DNAArtificial SequenceSynthetic construct 587ccgactgaac
gtcctagtgc tagcgtgact ctgacagtct ctgt 4458822DNAArtificial
SequenceSynthetic construct 588cagtgactct gacagtctct gt
2258922DNAArtificial SequenceSynthetic construct 589ctcagcagtc
actgtcttcc at 2259044DNAArtificial SequenceSynthetic construct
590ctcagcagtc actgtcttcc atgggttctg aaactcgccc taca
4459192DNAArtificial SequenceSynthetic construct 591tctgaaactc
gccctacann nnnnnnnnnn nnnnnntgtn nnggtnnncc gnnnnnnnnn 60tgtnnnnnnn
nnggaacgga gccgactgaa gc 9259244DNAArtificial SequenceSynthetic
construct 592ggaacggagc cgactgaagc tagcgtgact ctgacagtct ctgt
4459322DNAArtificial SequenceSynthetic construct 593cagtgactct
gacagtctct gt 2259424DNAArtificial SequenceSynthetic construct
594tcactgtctt ccatgggttc tgaa 24595105DNAArtificial
SequenceSynthetic construct 595tcactgtctt ccatgggttc tgaannnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnggtgagc tggttgctat gcagggtggt
agtggtactg aagct 10559632DNAArtificial SequenceSynthetic construct
596ggtggtagtg gtactgaagc tagcgtgact ct 3259724DNAArtificial
SequenceSynthetic construct 597tcactgtctt ccatgggttc tgaa
24598105DNAArtificial SequenceSynthetic construct 598tcactgtctt
ccatgggttc tgaannngat actnnnnnnt ttnnnnnnnn nnnngaggtt 60nnnnnnnnnn
nnnnnnnnat gcaaggtggt agtggtactg aagct 10559932DNAArtificial
SequenceSynthetic construct 599ggtggtagtg gtactgaagc tagcgtgact ct
3260024DNAArtificial SequenceSynthetic construct 600tcactgtctt
ccatgggttc tgaa 24601105DNAArtificial SequenceSynthetic construct
601tcactgtctt ccatgggttc tgaatatgat acttgggttt ttcaatttnn
nnnnnnnnnn 60nnnnnnnnnn nnnnnnnnnn nnnnggtggt agtggtactg aagct
10560232DNAArtificial SequenceSynthetic construct 602ggtggtagtg
gtactgaagc tagcgtgact ct 32603585PRTHomo sapiens 603Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe
Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys
Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro
Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu
Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Lys Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Arg Ala Ala Leu Gly Leu 580
58560427PRTArtificial SequenceSynthetically generated peptide
604Gly Ser Phe Phe Pro Cys Trp Arg Ile Asp Arg Phe Gly Tyr Cys His1
5 10 15Ala Asn Ala Pro Gly Ser Gly Gly Ser Gly Gly 20
2560525PRTArtificial SequenceSynthetically generated peptide 605Gly
Ser Gly Gly Glu Gly Gly Ser Gly Gly Ser Trp Ile Ile Cys Trp1 5 10
15Trp Asp Asn Cys Gly Ser Ser Ala Pro 20 25606637PRTArtificial
SequenceSynthetically generated peptide 606Gly Ser Phe Phe Pro Cys
Trp Arg Ile Asp Arg Phe Gly Tyr Cys His1 5 10 15Ala Asn Ala Pro Gly
Ser Gly Gly Ser Gly Gly Asp Ala His Lys Ser 20 25 30Glu Val Ala His
Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala 35 40 45Leu Val Leu
Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu 50 55 60Asp His
Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys65 70 75
80Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu
85 90 95Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr
Gly 100 105 110Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg
Asn Glu Cys 115 120 125Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu
Pro Arg Leu Val Arg 130 135 140Pro Glu Val Asp Val Met Cys Thr Ala
Phe His Asp Asn Glu Glu Thr145 150 155 160Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe 165 170 175Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe 180 185 190Thr Glu
Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys 195 200
205Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg
210 215 220Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe
Lys Ala225 230 235 240Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
Lys Ala Glu Phe Ala 245 250 255Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys Val His Thr Glu Cys 260 265 270Cys His Gly Asp Leu Leu Glu
Cys Ala Asp Asp Arg Ala Asp Leu Ala 275 280 285Lys Tyr Ile Cys Glu
Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu 290 295 300Cys Cys Glu
Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val305 310 315
320Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe
325 330 335Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys
Asp Val 340 345 350Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg
His Pro Asp Tyr 355 360 365Ser Val Val Leu Leu Leu Arg Leu Ala Lys
Thr Tyr Lys Thr Thr Leu 370 375 380Glu Lys Cys Cys Ala Ala Ala Asp
Pro His Glu Cys Tyr Ala Lys Val385 390 395 400Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys 405 410 415Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn 420 425 430Ala
Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro 435 440
445Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys
450 455 460Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp
Tyr Leu465 470 475 480Ser Val Val Leu Asn Gln Leu Cys Val Leu His
Glu Lys Thr Pro Val 485 490 495Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser Leu Val Asn Arg Arg 500 505 510Pro Cys Phe Ser Ala Leu Glu
Val Asp Glu Thr Tyr Val Pro Lys Glu 515 520 525Phe Asn Ala Glu Thr
Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser 530 535 540Glu Lys Glu
Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val545 550 555
560Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp
565 570 575Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp
Lys Glu 580 585 590Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala
Ala Ser Arg Ala 595 600 605Ala Leu Gly Leu Gly Ser Gly Gly Glu Gly
Gly Ser Gly Gly Ser Trp 610 615 620Ile Ile Cys Trp Trp Asp Asn Cys
Gly Ser Ser Ala Pro625 630 6356076PRTArtificial SequenceFragment
from human source 607Tyr Pro Glu Leu Pro Lys1 560813PRTArtificial
SequenceSynthetically generated peptide 608Arg Val Tyr Pro Glu Leu
Pro Lys Pro Ser Gly Gly Gly1 5 1060910PRTArtificial
SequenceSyntheticaly generated peptide 609Arg Val Tyr Pro Glu Leu
Pro Lys Pro Ser1 5 106104PRTArtificial SequenceLinker sequence
610Gly Ser Gly Lys16115PRTArtificial SequenceLinker sequence 611Gly
Ser Gly Ser Lys1 561212PRTArtificial SequenceA template sequence
612Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa1 5
1061313PRTArtificial SequenceA template sequence 613Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa1 5 1061414PRTArtificial
SequenceA template sequence 614Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa1 5 1061515PRTArtificial SequenceA template
sequence 615Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa1 5 10 1561616PRTArtificial SequenceA template sequence 616Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa1 5 10
1561717PRTArtificial SequenceA template sequence 617Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa1 5 10 15Xaa
61818PRTArtificial SequenceA template sequence 618Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa1 5 10 15Xaa Xaa
6199PRTArtificial SequenceSynthetic peptide 619Cys Xaa Gly Xaa Pro
Xaa Phe Xaa Cys1 5
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