U.S. patent application number 13/326870 was filed with the patent office on 2012-04-12 for ligands to radiation-induced molecules.
This patent application is currently assigned to VANDERBILT UNIVERSITY. Invention is credited to Dennis E. Hallahan.
Application Number | 20120089017 13/326870 |
Document ID | / |
Family ID | 46326488 |
Filed Date | 2012-04-12 |
United States Patent
Application |
20120089017 |
Kind Code |
A1 |
Hallahan; Dennis E. |
April 12, 2012 |
LIGANDS TO RADIATION-INDUCED MOLECULES
Abstract
A method for identifying a molecule that binds an irradiated
tumor in a subject and molecules identified thereby. In some
embodiments, the method includes the steps of (a) exposing a tumor
to ionizing radiation; (b) administering to a subject a library of
diverse molecules; and (c) isolating from the tumor one or more
molecules of the library of diverse molecules, whereby a molecule
that binds an irradiated tumor is identified. Also provided are
targeting ligands that bind an irradiated tumor and therapeutic and
diagnostic methods that employ the disclosed targeting ligands.
Inventors: |
Hallahan; Dennis E.;
(Nashville, TN) |
Assignee: |
VANDERBILT UNIVERSITY
Nashville
TN
|
Family ID: |
46326488 |
Appl. No.: |
13/326870 |
Filed: |
December 15, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11592451 |
Nov 3, 2006 |
8101157 |
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13326870 |
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11183325 |
Jul 15, 2005 |
7906102 |
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11592451 |
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10259087 |
Sep 27, 2002 |
7402392 |
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11183325 |
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10689006 |
Oct 20, 2003 |
7306925 |
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11592451 |
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09914605 |
Nov 9, 2001 |
7049140 |
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10689006 |
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10259087 |
Sep 27, 2002 |
7402392 |
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09914605 |
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60328123 |
Oct 3, 2001 |
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Current U.S.
Class: |
600/431 |
Current CPC
Class: |
C07K 16/44 20130101;
C07K 2317/622 20130101; A61K 49/0056 20130101; A61K 51/08 20130101;
A61K 51/082 20130101; A61K 51/1234 20130101; A61K 41/0038 20130101;
A61K 49/0058 20130101; A61K 49/0032 20130101; A61K 51/088 20130101;
A61K 49/0084 20130101; C07K 16/18 20130101; A61B 6/508 20130101;
A61K 51/1045 20130101 |
Class at
Publication: |
600/431 |
International
Class: |
A61B 6/00 20060101
A61B006/00 |
Goverment Interests
GRANT STATEMENT
[0002] This work was supported by grants CA58508, CA70937, CA89888,
CA89674, and CA90949 from the U.S. National Institutes of Health.
Thus, the U.S. government has certain rights in the presently
disclosed subject matter.
Claims
1-57. (canceled)
58. A method for detecting a tumor in a subject, the method
comprising: (a) exposing a target area of the subject where the
presence of a tumor is suspected to ionizing radiation; (b)
administering to the subject a composition to detect the presence
of TIP-1 in the target area, wherein the composition comprises one
or more targeting antibodies, wherein each targeting antibody i. is
capable of binding to a TIP-1 polypeptide expressed by an
irradiated tumor, and ii. is conjugated to a detectable label, and
iii. substantially lacks binding to a control tissue; and (c)
detecting the detectable label to detect the presence of TIP-1,
wherein the presence of TIP-1 indicates the presence of a tumor in
the target area of the subject.
59. The method of claim 58, wherein the antibody that binds to a
TIP-1 polypeptide is selected from the group consisting of a
polyclonal antibody, a monoclonal antibody, a scFv antibody, and an
antibody fragment that binds to a TIP-1 polypeptide.
60. The method of claim 59, wherein the antibody is humanized.
61. The method of claim 58, wherein the exposing comprises exposing
the tumor to less than about 2 Gy ionizing radiation.
62. The method of claim 61, wherein the exposing comprises exposing
the tumor to at least about 2 Gy ionizing radiation.
63. The method of claim 62, wherein the exposing comprises exposing
the tumor to about 10 Gy to about 20 Gy ionizing radiation.
64. The method of claim 58, wherein the administering comprises
administering the targeting antibody by intravascular
provision.
65. The method of claim 58, wherein the administering comprises
administering the targeting antibody subsequent to radiation
exposure.
66. The method of claim 65, wherein the administering comprises
administering the targeting antibody 0 hours to about 24 hours
following radiation exposure.
67. The method of claim 66, wherein the administering comprises
administering the targeting antibody about 4 hours to about 24
hours following radiation exposure.
68. The method of claim 58, wherein the subject is a warm-blooded
vertebrate.
69. The method of claim 58, wherein the detecting comprises
detecting the radionuclide label using positron emission
tomography, single photon emission computed tomography, gamma
camera imaging, or rectilinear scanning.
70. The method of claim 58, wherein the tumor comprises a tumor
selected from the group consisting of bladder carcinoma, breast
carcinoma, cervical carcinoma, cholangiocarcinoma, colorectal
carcinoma, gastric sarcoma, glioma, lung carcinoma, lymphoma,
melanoma, multiple myeloma, osteosarcoma, ovarian carcinoma,
pancreatic carcinoma, prostate carcinoma, stomach carcinoma, a
head, a neck tumor, and a solid tumor.
71. The method of claim 70, wherein the tumor is selected from the
group consisting of a glioma, a melanoma, a lung carcinoma, and a
prostate carcinoma.
72. The method of claim 58, further comprising simultaneously
detecting two or more tumors in the subject.
73. The method of claim 72, wherein the two or more tumors in a
subject comprise two or more tumor types.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 11/183,325, filed Jul. 15, 2005, which itself
is a continuation-in-part of U.S. patent application Ser. No.
10/259,087, filed Sep. 27, 2002, which is based on and claims
priority to U.S. Provisional Patent Application Ser. No.
60/328,123, filed Oct. 3, 2001. This application is also a
continuation-in-part of U.S. patent application Ser. No.
10/689,006, filed Oct. 20, 2003, which itself is a
continuation-in-part of U.S. patent application Ser. No.
09/914,605, filed Aug. 30, 2001, now U.S. Pat. No. 7,049,140, and
U.S. patent application Ser. No. 10/259,087, filed Sep. 27, 2002.
The entire disclosures of this U.S. patent and these patent
applications are herein incorporated by reference in their
entireties.
TECHNICAL FIELD
[0003] The presently disclosed subject matter generally relates to
ligands for radiation guided delivery of an active agent. The
presently disclosed subject matter also provides new
radiation-induced neoantigens that can be targeted by targeting
ligands that specifically bind an irradiated tumor. Also provided
are therapeutic and diagnostic uses for the same.
BACKGROUND
[0004] Tumor-specific drug delivery has the potential to minimize
toxicity to normal tissues and improve the bioavailability of
therapeutic agents to tumor cells (Hallahan at al., 1995b; Arap et
al., 1998). Targeting ligands include antibodies and peptides that
accumulate in tumors by specific binding to target molecules
present on tumor vasculature, endothelial cells associated with
tumor vasculature, and tumor cells. Effective target molecules are
generally cell surface receptors or other molecules present at the
exterior of tumor cells such that they are accessible to targeting
ligands (Hallahan et al., 2001a).
[0005] Existing site-specific drug delivery systems include ligands
that recognize a tumor marker such as Her2/neu (v-erb-b2 avian
erythroblastic leukemia viral oncogene homologue 2), CEA
(carcinoembryonic antigen; Ito et al., 1991), and breast cancer
antigens (Manome et al., 1994; Kirpotin of al., 1997; Becerril et
al., 1999). See also PCT International Publication No. WO 98/10795.
In an effort to identify ligands that are capable of targeting to
multiple tumor types, targeting ligands have been developed that
bind to target molecules present on tumor vasculature (Baillie et
al., 1995; Pasqualini & Ruoslahti, 1996; Arap et al., 1998;
Burg et al., 1999; Ellerby et al., 1999).
[0006] Despite these advances, current methods for targeted drug
delivery are hindered by targeting ligands that also bind normal
tissues and/or a lack of targeting ligands that bind multiple tumor
types. Ideally, a targeting molecule should display specific
targeting in the absence of substantial binding to normal tissues,
and a capacity for targeting to a variety of tumor types and
stages. Thus, there exists a long-felt need in the art for methods
and compositions to achieve site-specific, tumoral delivery of
therapeutic and/or diagnostic agents.
[0007] To meet this need, the presently disclosed subject matter
provides methods for identifying ligands that bind to irradiated
tumors, and ligands that have been bind irradiated tumors and
tissues. Such ligands are useful for guided drug delivery (e.g.,
radiation guided drug delivery), among other applications.
SUMMARY
[0008] This Summary lists several embodiments of the presently
disclosed subject matter, and in many cases lists variations and
permutations of these embodiments. This Summary is merely exemplary
of the numerous and varied embodiments. Mention of one or more
representative features of a given embodiment is likewise
exemplary. Such an embodiment can typically exist with or without
the feature(s) mentioned; likewise, those features can be applied
to other embodiments of the presently disclosed subject matter,
whether listed in this Summary or not. To avoid excessive
repetition, this Summary does not list or suggest all possible
combinations of such features.
[0009] The presently disclosed subject matter provides compositions
for radiation-guided targeting. In some embodiments, the
compositions comprise one or more targeting ligands that bind to a
TIP-1 polypeptide. In some embodiments, the one or more targeting
ligands comprises a peptide or an antibody or derivative thereof
that binds to the TIP-1 polypeptide. In some embodiments, the
peptide that binds to TIP-1 comprises an amino acid sequence as set
forth in SEQ ID NOs: 1, 35, 71-76, and 78-86. In some embodiments,
the antibody or derivative thereof that binds to TIP-1 is selected
from among polyclonal antibodies, monoclonal antibodies, scFv
antibodies, and antibody fragments that bind to TIP-1. In some
embodiments, the antibody or derivative thereof that binds to TIP-1
is humanized. In some embodiments, the one or more targeting
ligands bind to one or more tumor types selected from among an
irradiated glioma, a melanoma, a lung carcinoma, and a prostate
carcinoma. In some embodiments, the composition further comprises a
detectable label, a therapeutic agent, a drug carrier, or
combinations thereof. In some embodiments, the detectable label is
an in vivo detectable label that can be detected using magnetic
resonance imaging, scintigraphic imaging, ultrasound, or
fluorescence. In some embodiments, the in vivo detectable label
comprises a radionuclide label selected from the group consisting
of .sup.131I or .sup.99mTc. In some embodiments, the therapeutic
agent is selected from the group consisting of a radionuclide, a
cytotoxin, a therapeutic gene, and a chemotherapeutic agent. In
some embodiments, the drug carrier is selected from the group
consisting of a viral vector, a liposome, a plasmid, a
microcapsule, and combinations thereof.
[0010] The presently disclosed subject matter also provides methods
for detecting a tumor in a subject. In some embodiments, the
presently disclosed methods comprise (a) exposing a target areas of
the subject where the presence of a tumor is suspected to ionizing
radiation; (b) administering to the subject a composition
comprising one or more targeting ligands conjugated to a detectable
label, wherein the one or more targeting ligands bind to a TIP-1
polypeptide; and (c) detecting the detectable label to thereby
detect the tumor. In some embodiments, the one or more targeting
ligands comprises a peptide or an antibody or derivative thereof
that binds to the TIP-1 polypeptide. In some embodiments, the
peptide that binds to TIP-1 comprises an amino acid sequence as set
forth in SEQ ID NOs: 1, 35, 71-76, and 78-86. In some embodiments,
the antibody or derivative thereof that binds to TIP-1 is selected
from among polyclonal antibodies, monoclonal antibodies, scFv
antibodies, and antibody fragments that bind to TIP-1. In some
embodiments, the antibody or derivative thereof that binds to TIP-1
is humanized. In some embodiments, the exposing comprises exposing
the tumor to less than about 2 Gy ionizing radiation. In some
embodiments, the exposing comprises exposing the tumor to at least
about 2 Gy ionizing radiation. In some embodiments, the exposing
comprises exposing the tumor to about 10 Gy to about 20 Gy ionizing
radiation. In some embodiments, the administering comprises
administering the targeting ligand by intravascular provision. In
some embodiments, the administering comprises administering the
targeting ligand subsequent to radiation exposure. In some
embodiments, the administering comprises administering the
targeting ligand 0 hours to about 24 hours following radiation
exposure. In some embodiments, the administering comprises
administering the targeting ligand about 4 hours to about 24 hours
following radiation exposure. In some embodiments, the subject is a
warm-blooded vertebrate. In some embodiments, the detecting
comprises detecting the radionuclide label using positron emission
tomography, single photon emission computed tomography, gamma
camera imaging, or rectilinear scanning. In some embodiments, the
tumor comprises a tumor selected from the group consisting of
bladder carcinoma, breast carcinoma, cervical carcinoma,
cholangiocarcinoma, colorectal carcinoma, gastric sarcoma, glioma,
lung carcinoma, lymphoma, melanoma, multiple myeloma, osteosarcoma,
ovarian carcinoma, pancreatic carcinoma, prostate carcinoma,
stomach carcinoma, a head, a neck tumor, and a solid tumor. In some
embodiments, the tumor is selected from the group consisting of a
glioma, a melanoma, a lung carcinoma, and a prostate carcinoma.
[0011] In some embodiments, the presently disclosed methods further
comprise simultaneously detecting two or more tumors in the
subject. In some embodiments, the two or more tumors in a subject
comprise two or more tumor types.
[0012] The presently disclosed subject matter also provides methods
for radiation-guided delivery of a composition comprising an active
agent to a target tissue in a subject. In some embodiments, the
presently disclosed methods comprise (a) exposing the target tissue
to ionizing radiation; and (b) administering to the subject a
therapeutic composition, a diagnostic composition, or a combination
thereof, wherein the therapeutic composition, diagnostic
composition, or the combination thereof comprises a composition
comprising one or more targeting ligands that bind to a TIP-1
polypeptide, whereby the composition comprising an active agent is
selectively targeted to the target tissue. In some embodiments, the
active agent comprises a therapeutic agent, a diagnostic agent, or
a combination thereof. In some embodiments, the target tissue
comprises a tumor, tumor vasculature, or a combination thereof. In
some embodiments, the one or more targeting ligands comprises a
peptide or an antibody or derivative thereof that binds to the
TIP-1 polypeptide. In some embodiments, the peptide that binds to
TIP-1 comprises an amino acid sequence as set forth in SEQ ID NOs:
1, 35, 71-76, and 78-86. In some embodiments, the antibody or
derivative thereof that binds to TIP-1 is selected from among
polyclonal antibodies, monoclonal antibodies, scFv antibodies, and
antibody fragments that bind to TIP-1. In some embodiments, the
antibody or derivative thereof that binds to TIP-1 is humanized. In
some embodiments, the tumor is a primary or a metastasized tumor.
In some embodiments, the tumor is selected from a tumor selected
from the group consisting of bladder carcinoma, breast carcinoma,
cervical carcinoma, cholangiocarcinoma, colorectal carcinoma,
gastric sarcoma, glioma, lung carcinoma, lymphoma, melanoma,
multiple myeloma, osteosarcoma, ovarian carcinoma, pancreatic
carcinoma, prostate carcinoma, stomach carcinoma, a head tumor, a
neck tumor, and a solid tumor. In some embodiments, the tumor is
selected from the group consisting of a glioma, a melanoma, a lung
carcinoma, and a prostate carcinoma. In some embodiments, the
exposing comprises exposing the tumor to at least about 2 Gy
ionizing radiation. In some embodiments, the administering
comprises administering the targeting ligand by intravascular
provision. In some embodiments, the administering comprises
administering the targeting ligand subsequent to radiation
exposure. In some embodiments, the administering comprises
administering the targeting ligand 0 hours to about 24 hours
following radiation exposure. In some embodiments, the subject is a
warm-blooded vertebrate.
[0013] The presently disclosed subject matter also provides methods
for screening a plurality of potential ligands for an ability to
bind to a radiation-induced TIP-1 neoantigen present on a cell. In
some embodiments, the presently disclosed methods comprise (a)
contacting the cell with a first solution, the first solution
comprising the plurality of potential ligands; (b) isolating a
second solution, the second solution comprising those potential
ligands that do not bind to the cell; (c) removing any potential
ligands bound to the cell; (d) treating the cell with radiation,
wherein the treating results in a radiation-induced TIP-1
neoantigen being present on the cell; (e) contacting the cell with
the second solution; and (f) detecting binding of a potential
ligand to the radiation-induced TIP-1 neoantigen on the cell. In
some embodiments, the plurality of potential ligands comprises a
plurality of recombinant peptides, a plurality of antibodies or
fragments or derivatives thereof, or combinations thereof. In some
embodiments, the plurality of antibodies or fragments or
derivatives thereof comprises a plurality of phage-displayed
antibodies or fragments or derivatives thereof. In some
embodiments, the plurality of phage-displayed antibodies or
fragments or derivatives thereof comprises a plurality of
phage-displayed single chain variable fragment (scFv) antibodies, a
plurality of phage-displayed Fab fragments, or combinations
thereof. In some embodiments, the phage-displayed antibodies are
humanized. In some embodiments, one or more of the phage-displayed
antibodies further comprises an epitope tag. In some embodiments,
the epitope tag is selected from the group consisting of a c-myc
tag and a histidine tag. In some embodiments, the plurality of
peptides comprises a plurality of peptides of from 4 to 50 amino
acids in length. In some embodiments, the plurality of peptides
comprises a plurality of peptides of from 7 to 50 amino acids in
length. In some embodiments, the plurality of peptides comprises a
plurality of peptides comprising the amino acid sequence HVGGSSV
(SEQ ID NO: 35). In some embodiments, the cell is selected from the
group consisting of a tumor cell and a vascular endothelial cell.
In some embodiments, the vascular endothelial cell is present
within tumor microvasculature. In some embodiments, the detecting
is by a technique selected from ELISA, BIACORE, Western blotting,
immunohistochemistry, fluorometric microvolume assay technology,
mass spectroscopy, MALDI-MS, and MALDI-TOF.
[0014] Accordingly, it is an object of the presently disclosed
subject matter to provide novel targeting ligands that bind
irradiated tumors and therapeutic and/or diagnostic methods using
the same. This and others objects are achieved in whole or in part
by the presently disclosed subject matter.
[0015] An object of the presently disclosed subject matter having
been stated above, other objects and advantages of the presently
disclosed subject matter will become apparent to those skilled in
the art after a study of the following description and non-limiting
Examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic depicting a polyvalent
immunoconjugate. In this Figure, the polyvalent immunoconjugate
comprises nanoparticle to which two antibodies (Antibody 1 and
Antibody 2) and one therapeutic agent (in this case, a gamma
emitter) are complexed.
[0017] FIG. 2 are graphs depicting the results of binding
competition of an scFv antibody that binds to TIP-1 (TIP1-E11) to a
purified TIP-GST fusion protein with a selective synthetic
biotinylated peptide (HGDPNHVGGSSV; SEQ ID NO: 71) derived from
random peptide library as determined by ELISA under different
conditions. The top panel is a graph depicting the results of the
binding competition between the TIP1-E11 scFv antibody (diamonds)
and TIP-1 in the presence of different dilutions of a competitor
peptide having the amino acid sequence HVGGSSV (SEQ ID NO: 35)
(squares). The bottom panel is a graph depicting the results of the
binding competition between the TIP1-E11 scFv antibody (triangles)
and TIP-1 in the presence of different dilutions of a competitor
peptide having the amino acid sequence HVGGSSV (SEQ ID NO: 35)
(crosses) in the presence or absence of guinea pig serum (diamonds)
or mouse (squares) serum.
[0018] FIGS. 3A and 3B depict the results of TIP-1
immunohistochemical staining of tumor sections. Tumors were treated
with 0 Gy (FIG. 3A) or 3 Gy (FIG. 3B), resected and fixed at 6
hours after irradiation. Tumors were sectioned and stained with
polyclonal rabbit anti-mouse antibody to TIP-1. Sections were then
stained by use of Secondary antibody conjugated to HRP. Shown are
depictions of microscopic photographs using 100.times.
objectives.
[0019] FIG. 4 depicts a western blot for TIP-1 protein separated by
PAGE, transferred, and incubated with polyclonal antibody to TIP-1.
Depicted are autoradiographs of TIP-1 protein at 1, 4, and 24 hours
after irradiation with 3 Gy compared to TIP-1 protein in untreated
controls (0 Gy).
[0020] FIGS. 5A-5C depict NIR imaging of labeled HGDPNHVGGSSV (SEQ
ID NO: 71) binding in irradiated orthotopic prostate cancers.
[0021] FIG. 5A presents a transgenic prostate tumor. FIG. 5B
presents the PC3 tumor implanted into the prostate of nude mice.
FIG. 5C presents the negative control scrambled peptide
administered to a mouse bearing irradiated PC3 orthotopic tumor.
All mice were treated with 3 Gy. The arrow in FIG. 5C indicates the
location of the orthotopic prostate tumor.
[0022] FIGS. 6A and 6B depict NIR images demonstrating the
specificity of tumor binding by phage peptides. FIGS. 6A and 6B
depict NIR images of phage displayed RGDGSSV (SEQ ID NO: 75) and
HGDPNHVGGSSV (SEQ ID NO: 71) in mice bearing irradiated tumors in
the left hind limb and untreated control tumor in right leg.
[0023] FIG. 7 is a bar graph showing the percentage of total
emission that is localized to the tumors using different phage
peptides (HSVGGSSV--SEQ ID NO: 35; RGD; and HGSSV--SEQ ID NO: 76).
The gray bars show the percentage of Cy7 labeled phage binding
within irradiated tumor. The black bars show the percentage of Cy 7
labeled phage binding within the untreated (0 Gy) control tumor in
the opposite hind limb. Data were collected when phage were cleared
from the circulation. Bars labeled "other" show the maximal tumor
specific binding of all other phage.
[0024] FIG. 8 depicts NIR images using Xenogen imaging system of Cy
7 labeled HGDPNHVGGSSV (SEQ ID NO: 71) phage. Depictions of
representative photographs of the same mouse from days 1, 3, 7 and
9, following 3 Gy x-irradiation and HGDPNHVGGSSV phage (SEQ ID NO:
71) are shown. The red arrows indicate the jugular catheter
injection site of labeled phage. Red and yellow color intensity
areas are the highest areas of illumination. Color intensity is
overlaid upon photographs of the nude mouse within the Xenogen
imaging system.
[0025] FIG. 9 is two graphs depicting the pharmacokinetics of the
phage binding in tumors. The percentage of phage binding in tumor
and of the rest of the entire body are shown for HGDPNHVGGSSV (SEQ
ID NO: 71) phage (left panel) and RGDGSSV (SEQ ID NO: 75) page
(right panel). Data were acquired from the Xenogen imaging system
using images as shown in FIG. 8. The percentage of labeled phage
binding in tumor is shown in the dotted line and the percentage of
phage in the rest of the body is shown as a solid line over the
course of several days.
[0026] FIGS. 10A-10D are images of immunohistochemistry of phage
antibody staining of tumor sections acquired from mice shown in
FIG. 8. The tumors from mice treated with 3 Gy-irradiation and
phage were fixed at 4 hours (10B) and 7 days (10C & 10D) after
administration of phage.
[0027] Tumors were sectioned and stained with H & E and
antibody that is specific for phage. Brown staining indicates phage
binding.
[0028] FIGS. 11A-11C are images depicting fluorescence-labeled
HGDPNHVGGSSV (SEQ ID NO: 71) peptide within micro-vasculature of
irradiated tumors. The mouse bearing an irradiated PC3 tumor was
irradiated and Cy7-labeled. HGDPNHVGGSSV (SEQ ID NO: 71) was
injected. NIR imaging shows that the peptide binds within tumor
(arrow). Texas red-labeled peptide was injected after treatment
with 3 Gy. Shown is fluorescence microscopy on the right and the
same stained section (FIG. 11B).
[0029] FIG. 12 is a schematic and images showing the HGDPNHVGGSSV
(SEQ ID NO: 71) peptide was bound to strepavidiin by use of the
biotin on a linker polyglycine. The streptavidin is labeled with
Cy7. The Cy7 labeled streptavidin-peptide complexes injected by
jugular vein into tumor bearing mice. The panel on the left shows
the distribution of peptide in untreated mouse. The panel on the
right shows distribution of peptide binding following irradiation
of hind limb tumors.
[0030] FIG. 13 is a set of images showing orthotopic glioblastoma
tumors in the brains of mice are shown on the left. The H460 lung
cancer in the thorax of the mouse and colon cancer is on the right.
Mice on the right are treated with radiation. The mouse on the far
left is the untreated control mouse. The Cy7 labeled
strepavidin-HGDPNHVGGSSV (SEQ ID NO: 71) complex was injected by
tail vein. Mice were imaged using NIR and the Xenogen IVIS system.
Shown is NIR imaging at 48 hours after injection. The arrow
indicates no peptide binding to irradiated liver.
[0031] FIGS. 14A-14C are a set of images showing PC3 tumor
implanted into the prostate of nude mice (FIGS. 14A and 14B) and
the transgenic PTEN prostate tumor (FIG. 14C). All mice were
treated with 3 Gy. FIG. 14B is the negative control peptide
administered to mouse bearing irradiated PC3 orthotopic tumor. The
arrow indicates the location of the orthotopic prostate tumor shown
below. Cy7-labeled HVGGSSV (SEQ ID NO: 35) peptide was imaged by
the Xenogen system.
[0032] FIGS. 15A and 15B are imaging showing orthotopic PC3 tumor
in the nude mouse. In FIG. 15A, the mouse imaged in FIG. 14A was
euthanized and prostate tumor was identified, (Arrow). In FIG. 15B,
clockwise: prostate tumor, heart, liver & lung were then
resected and imaged by NIR using the Xenogen system.
[0033] FIGS. 16A-16C are images showing prostate and seminal
vesicles in the prostate-specific conditional PTEN transgenic. FIG.
16A) The mouse imaged in FIG. 14C was euthanized and prostate tumor
was identified, (Arrow). FIG. 16B) Clockwise: prostate tumor,
heart, liver & lung were then resected and imaged by NIR using
Xenogen imaging system.
[0034] FIGS. 17A-17C are images showing Fluorescent confocal
microscopy of HGDPNHVGGSSV (SEQ ID NO: 71)-strepavidin. FIG. 17A)
HUVEC 0 Gy control cells. FIG. 17B) 4 hours following irradiation 3
Gy. FIG. 17C) HGDPNHVGGSSV (SEQ ID NO: 71) strepavidin incubation
for 24 hours after 3 Gy.
DETAILED DESCRIPTION
I. Definitions
[0035] While the following terms are believed to be well understood
by one of ordinary skill in the art, the following definitions are
set forth to facilitate explanation of the presently disclosed
subject matter.
[0036] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which the presently disclosed subject
matter belongs. Although any methods, devices, and materials
similar or equivalent to those described herein can be used in the
practice or testing of the presently disclosed subject matter,
representative methods, devices, and materials are now
described.
[0037] Following long-standing patent law convention, the terms
"a", "an", and "the" refer to "one or more" when used in this
application, including the claims. Thus, for example, reference to
"a cell" (e.g., "a PEP") includes a plurality of such cells (e.g.,
a plurality of PEPs), and so forth.
[0038] Unless otherwise indicated, all numbers expressing
quantities of ingredients, reaction conditions, and so forth used
in the specification and claims are to be understood as being
modified in all instances by the term "about". Accordingly, unless
indicated to the contrary, the numerical parameters set forth in
this specification and attached claims are approximations that can
vary depending upon the desired properties sought to be obtained by
the presently disclosed subject matter.
[0039] As used herein, the term "about," when referring to a value
or to an amount of mass, weight, time, volume, concentration or
percentage is meant to encompass variations of in some embodiments
.+-.20%, in some embodiments .+-.10%, in some embodiments .+-.5%,
in some embodiments .+-.1%, in some embodiments .+-.0.5%, and in
some embodiments .+-.0.1% from the specified amount, as such
variations are appropriate to perform the disclosed methods or
employ the disclosed compositions.
[0040] As used herein, the term "cell" refers not only to the
particular subject cell (e.g., a living biological cell), but also
to the progeny or potential progeny of such a cell. Because certain
modifications can occur in succeeding generations due to either
mutation or environmental influences, such progeny might not, in
fact, be identical to the parent cell, but are still included
within the scope of the term as used herein.
[0041] The term "ligand" as used herein refers to a molecule or
other chemical entity having a capacity for binding to a target. A
ligand can comprise a peptide, an oligomer, a nucleic acid (e.g.,
an aptamer), a small molecule (e.g., a chemical compound), an
antibody or fragment thereof, a nucleic acid-protein fusion, and/or
any other affinity agent.
[0042] The term "small molecule" as used herein refers to a
compound, for example an organic compound, with a molecular weight
in some embodiments of less than about 1,000 daltons, in some
embodiments less than about 750 daltons, in some embodiments less
than about 600 daltons, and in some embodiments less than about 500
daltons. A small molecule also has a computed log octanol-water
partition coefficient in some embodiments in the range of about -4
to about +14, and in some embodiments in the range of about -2 to
about +7.5.
[0043] The term "target tissue" as used herein refers to an
intended site for accumulation of a ligand following administration
to a subject. For example, the methods disclosed herein can employ
a target tissue comprising an irradiated tumor.
[0044] The term "control tissue" as used herein refers to a site
suspected to substantially lack binding and/or accumulation of an
administered ligand. For example, in accordance with the methods of
the presently disclosed subject matter, a non-irradiated tumor and
a non-cancerous tissue are control tissues.
[0045] The terms "target" or "target molecule" as used herein each
refer to any substance that is specifically bound by a ligand.
Thus, the term "target molecule" encompasses macromolecules
including but not limited to proteins, nucleic acids,
carbohydrates, lipids, and complexes thereof.
[0046] As used herein, the terms "radiation-induced target",
"radiation-induced tumor target", and "radiation-induced
neoantigen" refer to a target molecule in a tumor whose expression,
localization, or ligand-binding capacity is induced by radiation.
Such a target molecule can comprise a molecule at the surface of a
tumor cell, within a tumor cell, or in the extracellular matrix
surrounding a tumor cell. Alternatively, a target molecule can
comprise a molecule present at the surface of or within a vascular
endothelial cell, or at the surface of or within a blood component
such as a platelet or a leukocyte. In some embodiments, a
radiation-induced neoantigen is selected from the group consisting
of P-selectin, E-selectin, Endoglin, .alpha..sub.2b.beta..sub.3
integrin, .alpha..sub.v.beta..sub.3 integrin, and TIP-1.
[0047] In some embodiments, a radiation-induced neoantigen
comprises is a TIP-1 polypeptide. TIP-1 is also known as T-cell
leukemia virus type I binding protein 3 (TAX1BP3), and several
nucleic acid and amino acid sequences for TIP-1 orthologs from
various species are available in the GENBANK.RTM. database. For
example, TIP-1 sequences that are disclosed in the GENBANK.RTM.
database include, but are not limited to TIP-1 nucleic acid and
amino acid sequences from human (NM.sub.--014604 and
NP.sub.--055419), mouse (NM.sub.--029564 and NP.sub.--083840), rat
(NM.sub.--001025419 and NP.sub.--001020590), bovine (BC102510 and
NP.sub.--001029646), and Xenopus (BC063221 and
NP.sub.--989230).
[0048] The term "induce", as used herein to refer to changes
resulting from radiation exposure, encompasses activation of gene
transcription or regulated release of proteins from cellular
storage reservoirs to vascular endothelium. Alternatively,
induction can refer to a process of conformational change, also
called activation, such as that displayed by the glycoprotein
IIb/IIIa integrin receptor upon radiation exposure (Staba et al.,
2000; Hallahan et al., 2001a). See also U.S. Pat. No. 6,159,443.
Additional proteins undergo conformational changes in response to
radiation or other stimuli (e.g., co-culture with tumor cells), and
these conformational change are also intended to be encompassed by
the term "induction". An exemplary protein that undergoes
conformational changes in response to co-culture with tumor cells
and/or exposure to radiation is perlecan (GENBANK.RTM. Accession
Nos. P98160 and NP.sub.--005520).
[0049] The terms "targeting" or "homing", as used herein to
describe the in vivo activity of a ligand following administration
to a subject, each refer to the preferential movement and/or
accumulation of a ligand in a target tissue as compared with a
control tissue.
[0050] The terms "selective targeting" of "selective homing" as
used herein each refer to a preferential localization of a ligand
that results in an amount of ligand in a target tissue that is in
some embodiments about 2-fold greater than an amount of ligand in a
control tissue, in some embodiments about 5-fold or greater than an
amount of ligand in a control tissue, and in some embodiments an
amount that is about 10-fold or greater than an amount of ligand in
a control tissue. The terms "selective targeting" and "selective
homing" also refer to binding or accumulation of a ligand in a
target tissue concomitant with an absence of targeting to a control
tissue, in some embodiments the absence of targeting to all control
tissues.
[0051] The term "absence of targeting" is used herein to describe
substantially no binding or accumulation of a ligand in all control
tissues where an amount of ligand is detectable.
[0052] The terms "targeting ligand", "targeting molecule", "homing
ligand", and "homing molecule" as used herein each refer to a
ligand that displays targeting activity. In some embodiments, a
targeting ligand displays selective targeting.
[0053] The term "binding" refers to an affinity between two
molecules, for example, a ligand and a target molecule. As used
herein, "binding" means a preferential binding of one molecule for
another in a mixture of molecules. The binding of a ligand to a
target molecule can be considered specific if the binding affinity
is in some embodiments about 1.times.10.sup.4 M.sup.-1 to about
1.times.10.sup.6 M.sup.-1 or greater.
[0054] The phrase "specifically (or selectively) binds", when
referring to the binding capacity of a ligand, refers to a binding
reaction which is determinative of the presence of the protein in a
heterogeneous population of proteins and other biological
materials. The phrase "specifically binds" also refers to
selectively targeting, as defined hereinabove.
[0055] The phases "substantially lack binding" or "substantially no
binding", as used herein to describe binding of a ligand in a
control tissue, refers to a level of binding that encompasses
non-specific or background binding, but does not include specific
binding.
[0056] The term "tumor" as used herein refers to both primary and
metastasized solid tumors and carcinomas of any tissue in a
subject, including but not limited to breast; colon; rectum; lung;
oropharynx; hypopharynx; esophagus; stomach; pancreas; liver;
gallbladder; bile ducts; small intestine; urinary tract including
kidney, bladder and urothelium; female genital tract including
cervix, uterus, ovaries (e.g., choriocarcinoma and gestational
trophoblastic disease); male genital tract including prostate,
seminal vesicles, testes and germ cell tumors; endocrine glands
including thyroid, adrenal, and pituitary; skin (e.g., hemangiomas
and melanomas), bone or soft tissues; blood vessels (e.g., Kaposi's
sarcoma); brain, nerves, eyes, and meninges (e.g., astrocytomas,
gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas,
Schwannomas and meningiomas). The term "tumor" also encompasses
solid tumors arising from hematopoietic malignancies such as
leukemias, including chloromas, plasmacytomas, plaques and tumors
of mycosis fungoides and cutaneous T-cell lymphoma/leukemia, and
lymphomas including both Hodgkin's and non-Hodgkin's lymphomas.
[0057] The term "subject" as used herein refers to any invertebrate
or vertebrate species. The methods and compositions disclosed
herein are particularly useful in the treatment and diagnosis of
warm-blooded vertebrates. Thus, the presently disclosed subject
matter concerns mammals and birds. More particularly contemplated
is the treatment and/or diagnosis of mammals such as humans, as
well as those mammals of importance due to being endangered (such
as Siberian tigers), of economic importance (animals raised on
farms for consumption by humans) and/or social importance (animals
kept as pets or in zoos) to humans, for instance, carnivores other
than humans (such as cats and dogs), swine (pigs, hogs, and wild
boars), ruminants (such as cattle, oxen, sheep, giraffes, deer,
goats, bison, and camels), and horses. Also contemplated is the
treatment of birds, including the treatment of those kinds of birds
that are endangered, kept in zoos, as well as fowl, and more
particularly domesticated fowl, e.g., poultry, such as turkeys,
chickens, ducks, geese, guinea fowl, and the like, as they are also
of economic importance to humans. Thus, contemplated is the
treatment of livestock, including, but not limited to, domesticated
swine (pigs and hogs), ruminants, horses, poultry, and the
like.
[0058] The term "about", as used herein when referring to a
measurable value such as an amount of weight, time, dose (e.g.
radiation dose), etc. is meant to encompass variations of in some
embodiments .+-.20%, in some embodiments .+-.10%, in some
embodiments .+-.5%, in some embodiments .+-.1%, and in some
embodiments .+-.0.1% from the specified amount, as such variations
are appropriate to perform the disclosed method.
II. Guided Drug Delivery Using Ligands
[0059] Ionizing radiation induces proteins in tumor vascular
endothelium through transcriptional induction and/or
posttranslational modification of cell adhesion molecules such as
integrins (Hallahan et al., 1995a; Hallahan et al., 1996; Hallahan
et al., 1998; Hallahan & Virudachalam, 1999). For example,
radiation induces activation of the integrin
.alpha..sub.2b.beta..sub.3, also called the fibrinogen receptor, on
platelets. The induced molecules can serve as binding sites for
targeting ligands.
[0060] Although several radiation-induced molecules within tumor
blood vessels have been identified and characterized, the
.alpha..sub.2b.beta..sub.3 target for drug delivery achieves the
greatest site-specific peptide binding within irradiated tumor
blood vessels. .sup.131I-labeled fibrinogen binds specifically to
tumors following exposure to ionizing radiation (U.S. Pat. No.
6,159,443). Peptides within fibrinogen that bind to the
radiation-induced .alpha..sub.2b.beta..sub.3 receptor include
HHLGGAKQAGDV (SEQ ID NO: 16) and the RGD peptide (Hallahan et al.,
2001a).
[0061] The presently disclosed subject matter includes a study of
the targeting activity of .alpha..sub.2b.beta..sub.3 ligands in
tumor-bearing subjects. Example 1 describes guided drug delivery
(e.g., radiation guided drug delivery) in animal models using
ligand-conjugated liposomes and microspheres. Clinical trials using
a radiolabeled .alpha..sub.2b.beta..sub.3 ligand support the
feasibility of guided drug delivery in humans, as described in
Example 2. See also Hallahan et al., 2001a.
[0062] Despite the successes of guided drug delivery using
.alpha..sub.2b.beta..sub.3 ligands in experimental models, the
clinical application of this approach is limited by nonspecific
binding of the targeting ligand at sites other than the tumor
(Hallahan et al., 2001b). In addition, previous observations of
radiation-induced molecules have employed radiation doses that are
sufficient to limit blood flow, as described in Example 3. Thus,
ligands are sought that demonstrate improved tumor specificity and
binding to target molecules induced by reduced radiation doses.
III. Identification of Ligands that Bind Irradiated Tumors
[0063] Approaches for optimizing peptide binding affinity and
specificity have included modification of peptide conformation and
addition of flanking amino acids to extend the minimal binding
motif. For example, amino acids C-terminal to the RGD sequence are
differentially conserved in RGD-containing ligands, and this
variation correlates with differences in binding specificity (Cheng
et al., 1994; Koivunen et al., 1994). Similarly, cyclization of a
prototype RGD peptide to restrict its conformational flexibility
improved interaction of the peptide with the vitronectin receptor,
yet nearly abolished interaction with the fibronectin receptor
(Pierschbacher & Ruoslahti, 1987).
[0064] Despite conservation of binding motifs among ligands that
bind irradiated tumors and recognition of factors that can
influence ligand binding, design of peptide sequences for improved
targeting activity is yet unpredictable. Approaches for identifying
such peptides have therefore relied on high volume screening
methods to select effective motifs from peptide libraries (Koivunen
et al., 1993; Healy et al., 1995). However, the utility of in
vitro-selected peptides is unpredictable in so far as peptide
binding properties are not consistently recapitulated in vivo. To
obviate these challenges, the presently disclosed subject matter
provides a method for in vivo selection of targeting ligands,
described further hereinbelow.
[0065] Using the in vivo selection method disclosed herein, novel
targeting ligands were identified that can be used for guided drug
delivery (e.g., radiation guided drug delivery). Representative
peptide ligands are set forth as SEQ ID NOs: 1-13, 26-69, and
71-86. Representative antibody ligands are set forth as SEQ ID NOs:
18, 20, 22, and 24. The novel ligands display improved specificity
of binding to irradiated tumors and are effective for targeting
using low dose irradiation. The disclosed targeting ligands also
offer benefits including moderate cost of preparation and ease of
handling.
[0066] III.A. Libraries
[0067] As used herein, the term "library" means a collection of
molecules. A library can contain a few or a large number of
different molecules, varying from about ten molecules to several
billion molecules or more. A molecule can comprise a naturally
occurring molecule, or a synthetic molecule (i.e., a molecule that
is not found in nature). Optionally, as described further
hereinbelow, a plurality of different libraries can be employed
simultaneously for in vivo panning.
[0068] Representative libraries include but are not limited to a
peptide library (U.S. Pat. Nos. 6,156,511, 6,107,059, 5,922,545,
and 5,223,409), an oligomer library (U.S. Pat. Nos. 5,650,489 and
5,858,670), an aptamer library (U.S. Pat. Nos. 6,180,348 and
5,756,291), a small molecule library (U.S. Pat. Nos. 6,168,912 and
5,738,996), a library of antibodies or antibody fragments (U.S.
Pat. Nos. 6,174,708, 6,057,098, 5,922,254, 5,840,479, 5,780,225,
5,702,892, and 5,667,988), a library of nucleic acid-protein
fusions (U.S. Pat. No. 6,214,553), and a library of any other
affinity agent that can potentially bind to an irradiated tumor
(e.g., U.S. Pat. Nos. 5,948,635, 5,747,334, and 5,498,538).
[0069] The molecules of a library can be produced in vitro, or they
can be synthesized in vivo, for example by expression of a molecule
in vivo. Also, the molecules of a library can be displayed on any
relevant support, for example, on bacterial pili (Lu et al., 1995)
or on phage (Smith, 1985).
[0070] A library can comprise a random collection of molecules.
Alternatively, a library can comprise a collection of molecules
having a bias for a particular sequence, structure, or
conformation. See e.g., U.S. Pat. Nos. 5,264,563 and 5,824,483.
Methods for preparing libraries containing diverse populations of
various types of molecules are known in the art, for example as
described in U.S. patents cited hereinabove. Numerous libraries are
also commercially available.
[0071] A library useful for in vivo panning as disclosed herein can
comprise in some embodiments a library of ten or more diverse
molecules, in some embodiments a library of one hundred or more
diverse molecules, and in some embodiments a library of one billion
or more diverse molecules. Representative diverse molecules include
peptides, peptide mimetics, proteins, antibodies or fragments
thereof, small molecules, nucleic acids, and combinations thereof.
In some embodiments, a library of peptides, antibodies, or a
combination thereof is used for in vivo panning. A library can
further comprise a library of diverse molecules that is recovered
following in vitro panning.
[0072] In some embodiments of the presently disclosed subject
matter, a peptide library can be used to perform the disclosed in
vivo panning methods. A peptide library comprises peptides
comprising in some embodiments three or more amino acids, in some
embodiments at least five, six, seven, or eight amino acids, in
some embodiments up to 50 amino acids or 100 amino acids, and in
some embodiments up to about 200 to 300 amino acids.
[0073] The peptides can be linear, branched, or cyclic, and can
include nonpeptidyl moieties. The peptides can comprise naturally
occurring amino acids, synthetic amino acids, genetically encoded
amino acids, non-genetically encoded amino acids, and combinations
thereof.
[0074] A biased peptide library can also be used, a biased library
comprising peptides wherein one or more (but not all) residues of
the peptides are constant. For example, an internal residue can be
constant, so that the peptide sequence is represented as: [0075]
(Xaa.sub.1).sub.m-(AA).sub.1-(Xaa.sub.2).sub.n where Xaa.sub.1 and
Xaa.sub.2 are any amino acid, or any amino acid except cysteine,
wherein Xaa.sub.1 and Xaa.sub.2 are the same or different amino
acids, m and n indicate a number Xaa residues, wherein in some
embodiments m and n are independently chosen from the range of 2
residues to 20 residues, in some embodiments m and n are chosen
from the range of 4 residues to 9 residues, and AA is the same
amino acid for all peptides in the library. In some embodiments, AA
is located at or near the center of the peptide. More specifically,
in some embodiments m and n are not different by more than 2
residues; in some embodiments m and n are equal.
[0076] In some embodiments, AA is tryptophan, proline, or tyrosine.
In some embodiments, AA is phenylalanine, histidine, arginine,
aspartate, leucine, or isoleucine. In some embodiments, AA is
asparagine, serine, alanine, or methionine. In some embodiments, AA
is cysteine or glycine.
[0077] A biased library used for in vivo panning also includes a
library comprising molecules previously selected by in vitro
panning methods. See Example 8.
[0078] In some embodiments of the presently disclosed subject
matter, the method for in vivo panning is performed using a phage
peptide library. Phage display is a method to discover peptide
ligands while minimizing and optimizing the structure and function
of proteins. Phage are used as a scaffold to display recombinant
libraries of peptides and provide a means to recover and amplify
the peptides that bind to putative receptor molecules in vivo. In
vivo phage selection simultaneously provides positive and
subtractive screens based on the spatial separation of normal
tissues and tumors. Phage that specifically bind the vasculature of
normal tissues are removed while specific phage that bind target
molecules present in irradiated tumors are enriched through serial
rounds of biopanning.
[0079] The T7 phage has an icosahedral capsid made of 415 proteins
encoded by gene 10 during its lytic phase. The T7 phage display
system has the capacity to display peptides up to 15 amino acids in
size at a high copy number (415 per phage). Unlike filamentous
phage display systems, peptides displayed on the surface of 17
phage are not capable of peptide secretion. T7 phage also replicate
more rapidly and are extremely robust when compared to other phage.
The stability allows for biopanning selection procedures that
require persistent phage infectivity. Accordingly, the use of
T7-based phage display is an aspect of a preferred embodiment of
the presently disclosed subject matter. Example 4 describes a
representative method for preparation of a 17 phage peptide library
that can be used to perform the in vivo panning methods disclosed
herein.
[0080] A phage peptide library to be used in accordance with the
panning methods of the presently disclosed subject matter can also
be constructed in a filamentous phage, for example M13 or
M13-derived phage. In some embodiments, the encoded antibodies are
displayed at the exterior surface of the phage, for example by
fusion to M13 vital protein 8. Methods for preparing M13 libraries
can be found in Sambrook & Russell, 2001, among other
places.
[0081] In some embodiments, the method for in vivo panning is
performed using a phage antibody library, as described in Example
8. Such a library can be constructed, for example, in M13 or
M13-derived phage. See e.g., U.S. Pat. Nos. 6,225,447; 5,580,717;
5,702,892.
[0082] III.B. In Vivo Panning for Ligands that Bind Irradiated
Tumors
[0083] The presently disclosed subject matter provides a method for
in vivo panning for ligands that bind irradiated tumors. As used
herein, the term "in vivo panning" refers to a method of screening
a library for selection of a ligand that homes to an irradiated
tumor.
[0084] The term "in vivo", as used herein to describe methods of
panning or ligand selection, refers to contacting of one or more
ligands to endogenous candidate target molecules, wherein the
candidate target molecules are naturally present in a subject or a
tumor biopsy from a subject, and the contacting occurs in the
subject or in the biopsied tumor. By contrast, "in vitro" panning
refers to contacting a library of candidate ligands with one or
more isolated or recombinantly produced target molecules.
[0085] Thus, in some embodiments a method for in vivo panning as
disclosed herein includes the steps of (a) exposing a tumor to
ionizing radiation; (b) administering to a subject a library of
diverse molecules; (c) procuring the tumor or fraction thereof; and
(d) isolating one or more molecules of the library of diverse
molecules from the tumor, whereby a molecule that binds an
irradiated tumor is identified. When performing the presently
disclosed in vivo panning methods, each of the steps of exposing,
administering, procuring, and isolating can be repeated one or more
times to modify and preferably improve ligand selection.
[0086] The term "administering to a subject", when used to describe
provision of a library of molecules, is used in its broadest sense
to mean that the library is delivered to the irradiated tumor. For
example, a library can be provided to the circulation of the
subject by injection or cannulization such that the molecules can
pass through the tumor.
[0087] The in vivo panning methods of the presently disclosed
subject matter can further comprise administering the library to
isolated tumor cells or to isolated proteins prior to administering
the library to a subject or to a tumor. For example, in vitro
panning methods can be performed to select ligands that bind to
particular tumor neoantigens, followed by performance of the in
vivo panning methods as disclosed herein.
[0088] Thus, in some embodiments a library can be administered to
an isolated tumor or tumor biopsy. Thus, in some embodiments a
method for in vivo panning can also comprise: (a) exposing a tumor
and a control tissue to ionizing radiation; (b) administering to
the tumor and to the control tissue a library of diverse molecules;
(c) detecting one or more molecules of the library that bind to the
tumor and that substantially lack binding to the control tissue,
whereby a molecule that binds an irradiated tumor is identified. In
some embodiments, the methods can further comprise (a) isolating
the tumor and the control tissue, and (b) administering the library
to the tumor and to the control tissue in vitro.
[0089] The in vivo panning methods of the presently disclosed
subject matter can further comprise administering the library to
isolated tumor cells or to isolated proteins prior to administering
the library to a subject or to a tumor. For example, in vitro
panning methods can be performed to select ligands that bind to
particular tumor neoantigens, followed by performance of the in
vivo panning methods as disclosed herein.
[0090] In some embodiments of the presently disclosed subject
matter, the radiation treatment comprises administration of less
than about 2 Gy ionizing radiation. In some embodiments, the
radiation treatment comprises at least about 2 Gy ionizing
radiation, in some embodiments about 2 Gy to about 3 Gy ionizing
radiation, and in some embodiments about 2 Gy to about 6 Gy
ionizing radiation. In some embodiments, radiation treatment
comprises about 10 Gy to about 20 Gy ionizing radiation.
[0091] The methods of the presently disclosed subject matter can be
performed using any tumor-bearing subject or any subject suspected
of having a tumor. In some embodiments, a subject is a warm-blooded
vertebrate, in some embodiments a mammal, and in some embodiments a
human.
[0092] In some embodiments of the presently disclosed subject
matter, a library is administered to a tumor-bearing human subject
following irradiation of the tumor. Methods and appropriate doses
for administration of a library to a human subject are described in
PCT International Publication No. WO 01/09611.
[0093] Example 5 describes a representative procedure for in vivo
panning of phage-displayed peptide ligands that bind to irradiated
tumor vessels in accordance with the presently disclosed subject
matter. Briefly, peptide binding was studied in tumor blood vessels
of 2 distinct tumor models: (1) GL261 glioma, and (2) Lewis lung
carcinoma. Tumors were irradiated with 3 Gy to facilitate
identification of peptide sequences that bind tumors exposed to a
minimal dose of ionizing radiation. Phage were administered by tail
vein injection into tumor bearing mice following irradiation. Phage
were recovered from the tumor thereafter. Following multiple rounds
of sequential in vivo binding to irradiated tumors, phage were
recovered and individual phage were randomly picked and sequenced.
Recovered phage were additionally tested for targeting activity in
an animal model of melanoma, as described in Example 6.
[0094] Example 8 describes a representative procedure for in vivo
panning of phage-displayed ligands comprising single chain
antibodies. The library used for in vivo panning was a biased
library in that a pool of antibody ligands that bind to
radiation-induced antigens were pre-selected in vitro.
[0095] III.C. In Vitro Panning for Nuclear Targeting Ligands
[0096] Example 12 describes a representative procedure for in vitro
panning of phage-displayed peptide ligands that can be used to
target therapeutic and/or diagnostic compositions to the nucleus of
tumor cells. After in vitro panning, the ability of identified
peptides to target tumor cells in vivo are confirmed using the in
vivo panning techniques disclosed herein.
[0097] III.D. Recovery of Targeting Ligands
[0098] Methods for identifying targeting ligands that bind an
irradiated tumor are selected based on one or more characteristics
common to the molecules present in the library. For example, mass
spectrometry and/or gas chromatography can be used to resolve
molecules that home to an irradiated tumor. Thus, where a library
comprises diverse molecules based generally on the structure of an
organic molecule, determining the presence of a parent peak for the
particular molecule can identify a ligand that binds a
radiation-induced target molecule.
[0099] If desired, a molecule can be linked to a tag, which can
facilitate recovery or identification of the molecule. A
representative tag is an oligonucleotide or a small molecule such
as biotin. See e.g., Brenner & Lerner, 1992 and U.S. Pat. No.
6,068,829. In addition, a tag can be a support or surface to which
a molecule can be attached. For example, a support can be a
biological tag such as a virus or virus-like particle such as a
bacteriophage ("phage"); a bacterium; or a eukaryotic cell such as
yeast, an insect cell, or a mammalian cell (e.g., an endothelial
progenitor cell or a leukocyte); or can be a physical tag such as a
liposome or a microbead. In some embodiments, a support has a
diameter less than about 10 .mu.m to about 50 .mu.m in its shortest
dimension, such that the support can pass relatively unhindered
through the capillary beds present in the subject and not occlude
circulation. In addition, a support can be nontoxic and
biodegradable, particularly where the subject used for in vivo
panning is not sacrificed for isolation of library molecules from
the tumor. Where a molecule is linked to a support, the part of the
molecule suspected of being able to interact with a target in a
cell in the subject is preferably positioned so as be able to
participate in the interaction.
[0100] III.E. Peptide Ligands
[0101] A targeting peptide of the presently disclosed subject
matter can be subject to various changes, substitutions,
insertions, and deletions where such changes provide for certain
advantages in its use. Thus, the term "peptide" encompasses any of
a variety of forms of peptide derivatives, that include amides,
conjugates with proteins, cyclized peptides, polymerized peptides,
conservatively substituted variants, analogs, fragments, peptoids,
chemically modified peptides, and peptide mimetics. The terms
"targeting peptide" and "peptide ligand" refer to a peptide as
defined hereinabove that binds to an irradiated tumor. An exemplary
peptide ligand of the presently disclosed subject matter can bind
to an irradiated tumor of in some embodiments at least one tumor
type, in some embodiments two or more tumor types, and in some
embodiments three or more tumor types. In some embodiments, a
targeting ligand can bind to an irradiated glioma, melanoma, and/or
a Lewis Lung carcinoma.
[0102] Peptides of the presently disclosed subject matter can
comprise naturally occurring amino acids, synthetic amino acids,
genetically encoded amino acids, non-genetically encoded amino
acids, and combinations thereof. Peptides can include both L-form
and D-form amino acids.
[0103] Representative non-genetically encoded amino acids include
but are not limited to 2-aminoadipic acid; 3-aminoadipic acid;
.beta.-aminopropionic acid; 2-aminobutyric acid; 4-aminobutyric
acid (piperidinic acid); 6-aminocaproic acid; 2-aminoheptanoic
acid; 2-aminoisobutyric acid; 3-aminoisobutyric acid;
2-aminopimelic acid; 2,4-diaminobutyric acid; desmosine;
2,2'-diaminopimelic acid; 2,3-diaminopropionic acid;
N-ethylglycine; N-ethylasparagine; hydroxylysine;
allo-hydroxylysine; 3-hydroxyproline; 4-hydroxyproline;
isodesmosine; allo-isoleucine; N-methylglycine (sarcosine);
N-methylisoleucine; N-methylvaline; norvaline; norleucine; and
ornithine.
[0104] Representative derivatized amino acids include for example,
those molecules in which free amino groups have been derivatized to
form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy
groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl
groups. Free carboxyl groups can be derivatized to form salts,
methyl and ethyl esters or other types of esters or hydrazides.
Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl
derivatives. The imidazole nitrogen of histidine can be derivatized
to form N-im-benzylhistidine.
[0105] The term "conservatively substituted variant" refers to a
peptide comprising an amino acid residue sequence substantially
identical to a sequence of a reference ligand of radiation-induced
target in which one or more residues have been conservatively
substituted with a functionally similar residue and which displays
the targeting activity as described herein. The phrase
"conservatively substituted variant" also includes peptides wherein
a residue is replaced with a chemically derivatized residue,
provided that the resulting peptide displays targeting activity as
disclosed herein.
[0106] Examples of conservative substitutions include the
substitution of one non-polar (hydrophobic) residue such as
isoleucine, valine, leucine or methionine for another; the
substitution of one polar (hydrophilic) residue for another such as
between arginine and lysine, between glutamine and asparagine,
between glycine and serine; the substitution of one basic residue
such as lysine, arginine or histidine for another; or the
substitution of one acidic residue, such as aspartic acid or
glutamic acid for another.
[0107] Peptides of the presently disclosed subject matter also
include peptides comprising one or more additions and/or deletions
or residues relative to the sequence of a peptide whose sequence is
disclosed herein, so long as the requisite targeting activity of
the peptide is maintained. The term "fragment" refers to a peptide
comprising an amino acid residue sequence shorter than that of a
peptide disclosed herein.
[0108] Additional residues can also be added at either terminus of
a peptide for the purpose of providing a "linker" by which the
peptides of the presently disclosed subject matter can be
conveniently affixed to a label or solid matrix, or carrier. Amino
acid residue linkers are usually at least one residue and can be 40
or more residues, more often 1 to 10 residues, but do alone not
constitute radiation-induced target ligands. Typical amino acid
residues used for linking are tyrosine, cysteine, lysine, glutamic
and aspartic acid, or the like. In addition, a peptide can be
modified by terminal-NH.sub.2 acylation (e.g., acetylation, or
thioglycolic acid amidation) or by terminal-carboxylamidation
(e.g., with ammonia, methylamine, and the like terminal
modifications). Terminal modifications are useful, as is well
known, to reduce susceptibility by proteinase digestion, and
therefore serve to prolong half life of the peptides in solutions,
particularly biological fluids where proteases can be present.
[0109] Peptide cyclization is also a useful terminal modification,
and in some embodiments is particularly preferred also because of
the stable structures formed by cyclization and in view of the
biological activities observed for such cyclic peptides as
described herein. An exemplary method for cyclizing peptides is
described by Schneider & Eberle, 1993. Typically,
tertbutoxycarbonyl protected peptide methyl ester is dissolved in
methanol and sodium hydroxide solution are added and the admixture
is reacted at 20.degree. C. to hydrolytically remove the methyl
ester protecting group. After evaporating the solvent, the
tertbutoxycarbonyl protected peptide is extracted with ethyl
acetate from acidified aqueous solvent. The tertbutoxycarbonyl
protecting group is then removed under mildly acidic conditions in
dioxane cosolvent. The unprotected linear peptide with free amino
and carboxyl termini so obtained is converted to its corresponding
cyclic peptide by reacting a dilute solution of the linear peptide,
in a mixture of dichloromethane and dimethylformamide, with
dicyclohexylcarbodiimide in the presence of 1-hydroxybenzotriazole
and N-methylmorpholine. The resultant cyclic peptide is then
purified by chromatography.
[0110] The term "peptoid" as used herein refers to a peptide
wherein one or more of the peptide bonds are replaced by
pseudopeptide bonds including but not limited to a carba bond
(CH.sub.2--CH.sub.2), a depsi bond (CO--O), a hydroxyethylene bond
(CHOH--CH.sub.2), a ketomethylene bond (CO--CH.sub.2), a
methylene-oxy bond (CH.sub.2--O), a reduced bond (CH.sub.2--NH), a
thiomethylene bond (CH.sub.2--S), a thiopeptide bond (CS--NH), and
an N-modified bond (--NRCO--). See e.g. Corringer et al., 1993;
Garbay-Jaureguiberry et al., 1992; Tung et al., 1992; Urge et al.,
1992; Pavone et al., 1993.
[0111] Peptides of the presently disclosed subject matter,
including peptoids, can be synthesized by any of the techniques
that are known to those skilled in the art of peptide synthesis.
Synthetic chemistry techniques, such as a solid-phase
Merrifield-type synthesis, are preferred for reasons of purity,
antigenic specificity, freedom from undesired side products, ease
of production and the like. A summary of representative techniques
can be found in Stewart & Young, 1969; Merrifield, 1969; Fields
& Noble, 1990; and Bodanszky, 1993. Solid phase synthesis
techniques can be found in Andersson et al., 2000, references cited
therein, and in U.S. Pat. Nos. 6,015,561, 6,015,881, 6,031,071, and
4,244,946. Peptide synthesis in solution is described by Schroder
& Lubke, 1965. Appropriate protective groups usable in such
synthesis are described in the above texts and in McOmie, 1973.
Peptides that include naturally occurring amino acids can also be
produced using recombinant DNA technology. In addition, peptides
comprising a specified amino acid sequence can be purchased from
commercial sources (e.g., Biopeptide Co., LLC of San Diego, Calif.,
United States of America, and PeptidoGenics of Livermore, Calif.,
United States of America).
[0112] The term "peptide mimetic" as used herein refers to a ligand
that mimics the biological activity of a reference peptide, by
substantially duplicating the targeting activity of the reference
peptide, but it is not a peptide or peptoid. In some embodiments, a
peptide mimetic has a molecular weight of less than about 700
daltons.
[0113] In some embodiments, a peptide mimetic can be designed by
(a) identifying the pharmacophoric groups responsible for the
targeting activity of a peptide; (b) determining the spatial
arrangements of the pharmacophoric groups in the active
conformation of the peptide; and (c) selecting a pharmaceutically
acceptable template upon which to mount the pharmacophoric groups
in a manner that allows them to retain their spatial arrangement in
the active conformation of the peptide. For identification of
pharmacophoric groups responsible for targeting activity, mutant
variants of the peptide can be prepared and assayed for targeting
activity.
[0114] Alternatively or in addition, the three-dimensional
structure of a complex of the peptide and its target molecule can
be examined for evidence of interactions, for example the fit of a
peptide side chain into a cleft of the target molecule, potential
sites for hydrogen bonding, etc. The spatial arrangements of the
pharmacophoric groups can be determined by NMR spectroscopy or
X-ray diffraction studies. An initial three-dimensional model can
be refined by energy minimization and molecular dynamics
simulation. A template for modeling can be selected by reference to
a template database and will typically allow the mounting of 2-8
pharmacophores. A peptide mimetic is identified wherein addition of
the pharmacophoric groups to the template maintains their spatial
arrangement as in the peptide.
[0115] A peptide mimetic can also be identified by assigning a
hashed bitmap structural fingerprint to the peptide based on its
chemical structure, and determining the similarity of that
fingerprint to that of each compound in a broad chemical database.
The fingerprints can be determined using fingerprinting software
commercially distributed for that purpose by Daylight Chemical
Information Systems, Inc. (Mission Viejo, Calif., United States of
America) according to the vendor's instructions. Representative
databases include but are not limited to SPREI'95 (InfoChem GmbH of
MUnchen, Germany), Index Chemicus (ISI of Philadelphia, Pa., United
States of America), World Drug Index (Derwent of London, United
Kingdom), TSCA93 (United States Envrionmental Protection Agency),
MedChem (Biobyte of Claremont, Calif., United States of America),
Maybridge Organic Chemical Catalog (Maybridge of Cornwall, United
Kingdom), Available Chemicals Directory (MDL Information Systems of
San Leandro, Calif., United States of America), NCI96 (United
States National Cancer Institute), Asinex Catalog of Organic
Compounds (Asinex Ltd. of Moscow, Russia), and NP (InterBioScreen
Ltd. of Moscow, Russia). A peptide mimetic of a reference peptide
is selected as comprising a fingerprint with a similarity (Tanamoto
coefficient) of at least 0.85 relative to the fingerprint of the
reference peptide. Such peptide mimetics can be tested for bonding
to an irradiated tumor using the methods disclosed herein.
[0116] Additional techniques for the design and preparation of
peptide mimetics can be found in U.S. Pat. Nos. 5,811,392;
5,811,512; 5,578,629; 5,817,879; 5,817,757; and 5,811,515.
[0117] Any peptide or peptide mimetic of the presently disclosed
subject matter can be used in the form of a pharmaceutically
acceptable salt. Suitable acids which are capable of the peptides
with the peptides of the presently disclosed subject matter include
inorganic acids such as trifluoroacetic acid (TFA), hydrochloric
acid (HCl), hydrobromic acid, perchloric acid, nitric acid,
thiocyanic acid, sulfuric acid, phosphoric acetic acid, propionic
acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,
malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic
acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid or
the like. HCl and TFA salts are particularly preferred.
[0118] Suitable bases capable of forming salts with the peptides of
the presently disclosed subject matter include inorganic bases such
as sodium hydroxide, ammonium hydroxide, potassium hydroxide and
the like; and organic bases such as mono-di- and tri-alkyl and aryl
amines (e.g. triethylamine, diisopropyl amine, methyl amine,
dimethyl amine and the like), and optionally substituted
ethanolamines (e.g. ethanolamine, diethanolamine and the like).
[0119] III.F. Antibody Ligands
[0120] A targeting antibody of the presently disclosed subject
matter comprises an antibody identified by the in vivo panning
methods disclosed herein. In some embodiments, an antibody
targeting ligand comprises (a) a polypeptide comprising an amino
acid sequence of SEQ ID NO: 18, 20, 22, or 24; (b) a polypeptide
substantially identical to SEQ ID NO: 18, 20, 22, or 24; (c) a
polypeptide encoded by SEQ ID NO: 17, 19, 21, or 23; or (d) a
polypeptide substantially identical to SEQ ID NO: 17, 19, 21, or
23. Thus, the presently disclosed subject matter also provides in
some embodiments an isolated nucleic acid that encodes an antibody
targeting ligand comprising (a) a nucleic acid molecule comprising
the nucleotide sequence of SEQ ID NO: 17, 19, 21, or 23; or (b) a
nucleic acid molecule substantially identical to SEQ ID NO: 17, 19,
21, or 23.
[0121] When phage-displayed antibodies bind to an antigen, they can
be affinity-purified using the antigen. These affinity-purified
phage can then be used to infect and introduce the antibody gene
back into E. coli. The E. coli can then be grown and induced to
express a soluble, non-phage-displayed, antigen-specific
recombinant antibody. Phage display technology is especially useful
for producing antibodies to antigens that are either poorly
immunogenic or readily degraded and for which monoclonal and/or
polyclonal antibodies are difficult to obtain. P-selectin, like
.alpha..sub.2b.beta..sub.3, is a high priority radiation-induced
neoantigen because it is not accessible to antibodies or
immunoconjugates until after irradiation of tumor vasculature.
Phage scFv antibodies have been developed to these proteins by use
of phage-displayed antibody library containing 2.times.10.sup.9
members. Negative selection of phage can be first performed on a
control tissue, for example untreated vascular endothelium. This
can eliminate antibodies that nonspecifically bind to, for example,
unirradiated endothelial cells. Unbound phage can then be recovered
and incubated with purified radiation-induced neoantigen, for
example, P-selectin or .alpha..sub.2b.beta..sub.3 integrin. High
affinity phage can then be recovered, for example by use of washing
at pH 1.
[0122] The term "isolated", as used in the context of a nucleic
acid or polypeptide, indicates that the nucleic acid or polypeptide
exists apart from its native environment and is not a product of
nature. An isolated nucleic acid or polypeptide can exist in a
purified form or can exist in a non-native environment such as a
transgenic host cell. In one embodiment of the presently disclosed
subject matter, "isolated" refers to the purification of an scFv
antibody from a target tissue to which it has bound.
[0123] Nucleic Acids Encoding Targeting Antibodies. The terms
"nucleic acid molecule" or "nucleic acid" each refer to
deoxyribonucleotides or ribonucleotides and polymers thereof in
single-stranded or double-stranded. Unless specifically limited,
the term encompasses nucleic acids containing known analogues of
natural nucleotides that have similar properties as the reference
natural nucleic acid. The terms "nucleic acid molecule" or "nucleic
acid" can also be used in place of "gene", "cDNA", or "mRNA".
Nucleic acids can be synthesized, or can be derived from any
biological source, including any organism.
[0124] The term "substantially identical", as used herein to
describe a degree of similarity between nucleotide sequences,
refers to two or more sequences that have in some embodiments at
least about least 60%, in some embodiments at least about least
65%, in some embodiments at least about 70%, in some embodiments at
least about least 75%, in some embodiments at least about 80%, in
some embodiments at least about least 85%, in some embodiments at
least about least 90%, in some embodiments at least about least
93%, in some embodiments at least about least 95%, in some
embodiments at least about least 97%, and in some embodiments about
99% nucleotide identity, as measured using one of the following
sequence comparison algorithms (described hereinbelow) or by visual
inspection. The substantial identity exists in nucleotide sequences
of in some embodiments at least about 100 residues, in some
embodiments at least about 150 residues, and in some embodiments in
nucleotide sequences, comprising a full length coding sequence.
[0125] Thus, substantially identical sequences can comprise)
mutagenized sequences, including sequences comprising silent
mutations, or variably synthesized sequences. A mutation or variant
sequence can comprise a single base change.
[0126] Another indication that two nucleotide sequences are
substantially identical is that the two molecules specifically or
substantially hybridize to each other under stringent conditions.
In the context of nucleic acid hybridization, two nucleic acid
sequences being compared can be designated a "probe" and a
"target". A "probe" is a reference nucleic acid molecule, and a
"target" is a test nucleic acid molecule, often found within a
heterogeneous population of nucleic acid molecules. A "target
sequence" is synonymous with a "test sequence".
[0127] An exemplary nucleotide sequence that can be employed for
hybridization studies or assays includes probe sequences that are
complementary to or mimic at least an about 14 to 40 nucleotide
sequence of a nucleic acid molecule of the presently disclosed
subject matter. For this purpose, a probe comprises a region of the
nucleic acid molecule other than a sequence encoding a common
immunoglobulin region. Thus, a probe comprises in some embodiments
a sequence encoding a domain of the antibody that comprises an
antigen-binding site. In some embodiments, probes comprise 14 to 20
nucleotides, or even longer where desired, such as 30, 40, 50, 60,
100, 200, 300 nucleotides or up to the full length of a region of
SEQ ID NO: 17, 19, 21, or 23 that encodes an antigen binding site.
Such fragments can be readily prepared by, for example, chemical
synthesis of the fragment, by application of nucleic acid
amplification technology, or by introducing selected sequences into
recombinant vectors for recombinant production.
[0128] The phrase "hybridizing specifically to" refers to the
binding, duplexing, or hybridizing of a molecule only to a
particular nucleotide sequence under stringent conditions when that
sequence is present in a complex nucleic acid mixture (e.g., total
cellular DNA or RNA).
[0129] The phrase "hybridizing substantially to" refers to
complementary hybridization between a probe nucleic acid molecule
and a target nucleic acid molecule and embraces minor mismatches
that can be accommodated by reducing the stringency of the
hybridization media to achieve the desired hybridization.
[0130] "Stringent hybridization conditions" and "stringent
hybridization wash conditions" in the context of nucleic acid
hybridization experiments such as Southern and Northern blot
analysis are both sequence- and environment-dependent. Longer
sequences hybridize specifically at higher temperatures. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen, 1993. Generally, highly stringent hybridization and wash
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength and pH. Typically, under "stringent
conditions" a probe will hybridize specifically to its target
subsequence, but to no other sequences.
[0131] The T.sub.m is the temperature (under defined ionic strength
and pH) at which 50% of the target sequence hybridizes to a
perfectly matched probe. Very stringent conditions are selected to
be equal to the T.sub.m for a particular probe. An example of
stringent hybridization conditions for Southern or Northern Blot
analysis of complementary nucleic acids having more than about 100
complementary residues is overnight hybridization in 50% formamide
with 1 mg of heparin at 42.degree. C. An example of highly
stringent wash conditions is 15 minutes in 0.1.times.SSC at
65.degree. C. An example of stringent wash conditions is 15 minutes
in 0.2.times.SSC buffer at 65.degree. C. See Sambrook &
Russell, 2001 for a description of SSC buffer.
[0132] Often, a high stringency wash is preceded by a low
stringency wash to remove background probe signal. An example of
medium stringency wash conditions for a duplex of more than about
100 nucleotides, is 15 minutes in 1.times.SSC at 45.degree. C. An
example of low stringency wash for a duplex of more than about 100
nucleotides, is 15 minutes in 4.times. to 6.times.SSC at 40.degree.
C. For short probes (e.g., about 10 to 50 nucleotides), stringent
conditions typically involve salt concentrations of less than about
1M Na.sup.+ ion, typically about 0.01 to 1M Na.sup.+ ion
concentration (or other salts) at pH 7.0-8.3, and the temperature
is typically at least about 30.degree. C. Stringent conditions can
also be achieved with the addition of destabilizing agents such as
formamide. In general, a signal to noise ratio of 2-fold (or
higher) than that observed for an unrelated probe in the particular
hybridization assay indicates detection of a specific
hybridization.
[0133] The following are examples of hybridization and wash
conditions that can be used to identify nucleotide sequences that
are substantially identical to reference nucleotide sequences of
the presently disclosed subject matter: in some embodiments a probe
nucleotide sequence hybridizes to a target nucleotide sequence in
7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at
50.degree. C. followed by washing in 2.times.SSC, 0.1% SDS at
50.degree. C.; in some embodiments a probe and target sequence
hybridize in 7% sodium dodecyl sulfate (SOS), 0.5 M NaPO.sub.4, 1
mM EDTA at 50.degree. C. followed by washing in 1.times.SSC, 0.1%
SDS at 50.degree. C.; in some embodiments a probe and target
sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaPO.sub.4, 1 mM EDTA at 50.degree. C. followed by washing in
0.5.times.SSC, 0.1% SDS at 50.degree. C.; in some embodiments a
probe and target sequence hybridize in 7% sodium dodecyl sulfate
(SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. followed by
washing in 0.1.times.SSC, 0.1% SDS at 50.degree. C.; and in some
embodiments a probe and target sequence hybridize in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
followed by washing in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
[0134] A further indication that two nucleic acid sequences are
substantially identical is that proteins encoded by the nucleic
acids are substantially identical, share an overall
three-dimensional structure, or are biologically functional
equivalents. These terms are defined further hereinbelow. Nucleic
acid molecules that do not hybridize to each other under stringent
conditions are still substantially identical if the corresponding
proteins are substantially identical. This can occur, for example,
when two nucleotide sequences are significantly degenerate as
permitted by the genetic code.
[0135] The term "conservatively substituted variants" refers to
nucleic acid sequences having degenerate codon substitutions
wherein the third position of one or more selected (or all) codons
is substituted with mixed-base and/or deoxyinosine residues. See
Batzer et al., 1991; Ohtsuka et al., 1985; Rossolini et al.,
1994.
[0136] The term "subsequence" refers to a sequence of nucleic acids
that comprises a part of a longer nucleic acid sequence. An
exemplary subsequence is a probe, described hereinabove, or a
primer. The term "primer" as used herein refers to a contiguous
sequence comprising in some embodiments about 8 or more
deoxyribonucleotides or ribonucleotides, in some embodiments about
10-20 nucleotides, and in some embodiments about 20-30 nucleotides
of a selected nucleic acid molecule. The primers of the presently
disclosed subject matter encompass oligonucleotides of sufficient
length and appropriate sequence so as to provide initiation of
polymerization on a nucleic acid molecule of the presently
disclosed subject matter.
[0137] The term "elongated sequence" refers to an addition of
nucleotides (or other analogous molecules) incorporated into the
nucleic acid. For example, a polymerase (e.g., a DNA polymerase)
can add sequences at the 3' terminus of the nucleic acid molecule.
In addition, the nucleotide sequence can be combined with other DNA
sequences, such as promoters, promoter regions, enhancers,
polyadenylation signals, intronic sequences, additional restriction
enzyme sites, multiple cloning sites, and other coding
segments.
[0138] Nucleic acids of the presently disclosed subject matter can
be cloned, synthesized, recombinantly altered, mutagenized, or
combinations thereof. Standard recombinant DNA and molecular
cloning techniques used to isolate nucleic acids are known in the
art. Site-specific mutagenesis to create base pair changes,
deletions, or small insertions are also known in the art. See e.g.,
Sambrook & Russell, 2001; Silhavy et al., 1984; Glover &
Hames, 1995; Ausubel, 1995.
[0139] Single Chain Antibody Polypeptides. The term "substantially
identical", as used herein to describe a level of similarity
between a polypeptide comprising an antibody targeting ligand and a
polypeptide to SCN1A, refers to a sequence having in some
embodiments at least about 45%, in some embodiments at least about
50%, in some embodiments at least about 60%, in some embodiments at
least about 70%, in some embodiments at least about 80%, in some
embodiments at least about 90%, in some embodiments at least about
95%, and in some embodiments at least about 99% sequence identity
to SEQ ID NO: 17, 19, 21, or 23, when compared over the full length
of the single chain polypeptide. The term "full length", as used
herein to describe an antibody targeting ligand, comprises an amino
acid sequence having 254 amino acids. Methods for determining
percent identity are defined hereinbelow.
[0140] Substantially identical polypeptides also encompass two or
more polypeptides sharing a conserved three-dimensional structure.
Computational methods can be used to compare structural
representations, and structural models can be generated and easily
tuned to identify similarities around important active sites or
ligand binding sites. See Saqi et al., 1999; Barton, 1998; Henikoff
et al., 2000; Huang et al., 2000.
[0141] Substantially identical proteins also include proteins
comprising an amino acid sequence comprising amino acids that are
functionally equivalent to amino acids of SEQ ID NOs: 18, 20, 22,
and 24. The term "functionally equivalent" in the context of amino
acid sequences is known in the art and is based on the relative
similarity of the amino acid side-chain substituents. See Henikoff
& Henikoff, 2000. Relevant factors for consideration include
side-chain hydrophobicity, hydrophilicity, charge, and size. For
example, arginine, lysine, and histidine are all positively charged
residues; that alanine, glycine, and serine are all of similar
size; and that phenylalanine, tryptophan, and tyrosine all have a
generally similar shape. By this analysis, described further
hereinbelow, arginine, lysine, and histidine; alanine, glycine, and
serine; and phenylalanine, tryptophan, and tyrosine; are defined
herein as biologically functional equivalents.
[0142] In making biologically functional equivalent amino acid
substitutions, the hydropathic index of amino acids can be
considered. Each amino acid has been assigned a hydropathic index
on the basis of their hydrophobicity and charge characteristics,
these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine
(+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan
(-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);
glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine
(-3.5); lysine (-3.9); and arginine (-4.5).
[0143] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
generally understood in the art (Kyte et al., 1982). It is known
that certain amino acids can be substituted for other amino acids
having a similar hydropathic index or score and still retain a
similar biological activity. In making changes based upon the
hydropathic index, amino acids can be substituted whose hydropathic
indices are in some embodiments within .+-.2 of the original value,
in some embodiments within .+-.1 of the original value, and in some
embodiments within .+-.0.5 of the original value.
[0144] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101 describes that the greatest
local average hydrophilicity of a protein, as governed by the
hydrophilicity of its adjacent amino acids, correlates with its
immunogenicity and antigenicity, e.g., with a biological property
of the protein. It is understood that an amino acid can be
substituted for another having a similar hydrophilicity value and
still obtain a biologically equivalent protein.
[0145] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4).
[0146] In making changes based upon similar hydrophilicity values,
amino acids can be substituted whose hydrophilicity values are in
some embodiments within .+-.2 of the original value, in some
embodiments within .+-.1 of the original value, and in some
embodiments within .+-.0.5 of the original value.
[0147] The term "substantially identical" also encompasses
polypeptides that are biologically functional equivalents. The term
"functional", as used herein to describe polypeptides comprising
antibody targeting ligands, refers two or more antibodies that are
immunoreactive with a same radiation-induced target molecule. In
some embodiments, the two or more antibodies specifically bind a
same target molecule and substantially lack binding to a control
antigen.
[0148] The term "specifically binds", when used to describe binding
of an antibody to a target molecule, refers to binding to a target
molecule in a heterogeneous mixture of other polypeptides.
[0149] The phases "substantially lack binding" or "substantially no
binding", as used herein to describe binding of an antibody to a
control polypeptide or sample, refers to a level of binding that
encompasses non-specific or background binding, but does not
include specific binding.
[0150] Techniques for detecting antibody-target molecule complexes
are known in the art and include but are not limited to
centrifugation, affinity chromatography and other immunochemical
methods. In some embodiments, an antibody-target molecule complex
can be detected following administration of an antibody to a
subject as described in Examples 6 and 7. In some embodiments, an
antibody-target molecule complex can be detected in vivo by
performing radiation-guided drug delivery, wherein the drug
comprises a targeting antibody of SEQ ID NO: 18, 20, 22, or 24 and
a detectable label, as described in Examples 1 and 2. See also,
Manson, 1992; Ishikawa, 1999; Law, 1996.
[0151] The presently disclosed subject matter also provides
functional fragments of an antibody targeting polypeptide. Such
functional portion need not comprise all or substantially all of
the amino acid sequence of SEQ ID NO: 18, 20, 22, or 24.
[0152] The presently disclosed subject matter also includes
functional polypeptide sequences that are longer sequences than
that of SEQ ID NO: 18, 20, 22, or 24. For example, one or more
amino acids can be added to the N-terminus or C-terminus of a
antibody targeting ligand. Methods of preparing such proteins are
known in the art.
[0153] Isolated polypeptides and recombinantly produced
polypeptides can be purified and characterized using a variety of
standard techniques that are known to the skilled artisan. See
e.g., Schroder & Lubke, 1965; Schneider & Eberle, 1993;
Bodanszky, 1993; Ausubel, 1995.
[0154] Nucleotide and Amino Acid Sequence Comparisons. The terms
"identical" or percent "identity" in the context of two or more
nucleotide or polypeptide sequences, refer to two or more sequences
or subsequences that are the same or have a specified percentage of
amino acid residues or nucleotides that are the same, when compared
and aligned for maximum correspondence, as measured using one of
the sequence comparison algorithms disclosed herein or by visual
inspection.
[0155] The term "substantially identical" in regards to a
nucleotide or polypeptide sequence means that a particular sequence
varies from the sequence of a naturally occurring sequence by one
or more deletions, substitutions, or additions, the net effect of
which is to retain biological activity of a gene, gene product, or
sequence of interest.
[0156] For sequence comparison, typically one sequence acts as a
reference sequence to which test sequences are compared. When using
a sequence comparison algorithm, test and reference sequences are
entered into a computer program, subsequence coordinates are
designated if necessary, and sequence algorithm program parameters
are selected. The sequence comparison algorithm then calculates the
percent sequence identity for the designated test sequence(s)
relative to the reference sequence, based on the selected program
parameters.
[0157] Optimal alignment of sequences for comparison can be
conducted, for example, by the local homology algorithm of Smith
& Waterman, 1981, by the homology alignment algorithm of
Needleman & Wunsch, 1970, by the search for similarity method
of Pearson & Lipman, 1988, by computerized implementations of
these algorithms (e.g., programs available in the DISCOVERY
STUDIO.RTM. package from Accelrys, Inc., San Diego, Calif., United
States of America), or by visual inspection. See generally,
Ausubel, 1995.
[0158] An exemplary algorithm for determining percent sequence
identity and sequence similarity is the BLAST algorithm, which is
described in Altschul et al., 1990. Software for performing BLAST
analyses is publicly available through the website of the National
Center for Biotechnology Information. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold. These initial neighborhood
word hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when the cumulative
alignment score falls off by the quantity X from its maximum
achieved value, the cumulative score goes to zero or below due to
the accumulation of one or more negative-scoring residue
alignments, or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength W=11, an expectation E=10,
a cutoff of 100, M=5, N=-4, and a comparison of both strands. For
amino acid sequences, the BLASTP program uses as defaults a
wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62
scoring matrix. See Henikoff & Henikoff, 1992.
[0159] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences. See e.g., Karlin & Altschul,
1993. One measure of similarity provided by the BLAST algorithm is
the smallest sum probability (P(N)), which provides an indication
of the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a test nucleic
acid sequence is considered similar to a reference sequence if the
smallest sum probability in a comparison of the test nucleic acid
sequence to the reference nucleic acid sequence is in some
embodiments less than about 0.1, in some embodiments less than
about 0.01, and in some embodiments less than about 0.001.
IV. Tumor Diagnosis, Treatment, and Imaging
[0160] The presently disclosed subject matter further provides in
some embodiments methods and compositions for guided drug delivery
(e.g., radiation guided drug delivery) to a tumor in a subject. The
term "drug" as used herein refers to any substance having
biological or detectable activity. Thus, the term "drug" includes a
pharmaceutical agent, a diagnostic agent, or a combination thereof.
The term "drug" also includes any substance that is desirably
delivered to a tumor.
[0161] Immunoconjugates compositions of the presently disclosed
subject matter can be monovalent (i.e. they comprise an antibody
that binds to only one epitope present on a radiation-induced
neoantigen) or polyvalent. As used herein, a "polyvalent
immunoconjugate composition" refers to an immunoconjugate
composition that comprises at least two different ligands (for
example, scFv antibodies that bind to radiation-induced
neoantigens) that bind to at least two different targets, at least
one of which is a radiation-induced neoantigen. Thus, in one
embodiment a polyvalent immunoconjugate composition comprises a
plurality of single chain fragment variable (scFv) antibodies,
human Fab antibodies, or combinations thereof, wherein the
plurality of antibodies or antibody fragments bind to a plurality
of different epitopes, and wherein at least one of the epitopes is
present on a radiation-induced neoantigen. In one embodiment, at
least one of the plurality of different epitopes is present on a
vascular endothelial cell.
[0162] An exemplary polyvalent immunoconjugate is depicted in FIG.
1. As shown in FIG. 1, Antibody 1 binds to an epitope present on
endothelium (for example, tumor endothelium), and Antibody 2 binds
to an antigen present on vascular endothelium. One or both of the
epitopes to which Antibody 1 and Antibody 2 bind can be
radiation-induced neoantigens. This Figure depicts the epitopes to
which Antibodies 1 and 2 bind as being different, thus the
immunoconjugate is a polyvalent immunoconjugate.
[0163] However, if Antibody 1 and Antibody 2 bind to the same
epitope present on a radiation-induced neoantigen, the
immunoconjugate would be monovalent.
[0164] In accordance with the presently disclosed subject matter,
immunoconjugate compositions can be used to deliver therapeutic
agents to target tissues. Such therapeutic agents include, but are
not limited to viruses, radionuclides, cytotoxins, therapeutic
genes, and chemotherapeutic agents.
[0165] Also in accordance with the presently disclosed subject
matter, an immunoconjugate composition, the immunoconjugate
composition can further comprise a detectable label. In one
embodiment, the detectable label is detectable in vivo. In this
embodiment, the detectable label comprises a label that can be
detected using magnetic resonance imaging, scintigraphic imaging,
ultrasound, or fluorescence. An exemplary detectable label that can
be used for detection.
[0166] Thus, in some embodiments, a composition is prepared, the
composition comprising a targeting ligand as disclosed herein and a
diagnostic agent. In some embodiments, the composition can be used
for the detection of a tumor in a subject by (a) exposing a
suspected tumor to ionizing radiation; (b) administering to the
subject a targeting ligand of the presently disclosed subject
matter, wherein the ligand comprises a detectable label; and (c)
detecting the detectable label, whereby a tumor is diagnosed. In
some embodiments, a method for detecting a tumor can comprise (a)
exposing a suspected tumor to ionizing radiation; (b) biopsing a
suspected tumor; (c) contacting a targeting ligand of the presently
disclosed subject matter with the suspected tumor in vitro, wherein
the ligand comprises a detectable label; and (d) detecting the
detectable label, whereby a tumor is diagnosed.
[0167] A therapeutic composition of the presently disclosed subject
matter can comprise one or more targeting ligands and a therapeutic
agent, such that the therapeutic agent can be selectively targeted
to an irradiated tumor. Representative therapeutic agents include a
radionuclide, a cytotoxin, a therapeutic gene, and a
chemotherapeutic agent. The one or more targeting ligands can
comprise ligands having diverse molecular features. For example,
one or more targeting ligands can comprise both peptide and
antibody targeting ligands.
[0168] In some embodiments, a therapeutic composition can
additionally comprise a detectable label, in some embodiments a
label that can be detected in vivo. The biodistribution of the
therapeutic composition so prepared can be monitored following
administration to a subject.
[0169] Methods for preparation, labeling, and guided drug delivery
using targeting ligands of the presently disclosed subject matter
are described further hereinbelow. See also Examples 1 and 2.
[0170] IV.A. Therapeutic Agents
[0171] The novel targeting ligands disclosed here are used to
target a therapeutic agent to an irradiated tumor. Representative
therapeutic agents include but are not limited to a nucleic acid
(e.g., a therapeutic gene) and a small molecule. In some
embodiments of the presently disclosed subject matter, an inactive
drug is administered, which is subsequently activated by
irradiation (Hallahan et al., 1995b). For example, therapeutic gene
expression can be regulated by a radiation-induced promoter
(Hallahan et al., 1995b).
[0172] Therapeutic Genes. Angiogenesis and suppressed immune
response play a central role in the pathogenesis of malignant
disease and tumor growth, invasion, and metastasis. Thus, in some
embodiments, a therapeutic gene encodes a polypeptide having an
ability to induce an immune response and/or an anti-angiogenic
response in vivo.
[0173] The term "immune response" is meant to refer to any response
to an antigen or antigenic determinant by the immune system of a
vertebrate subject. Exemplary immune responses include humoral
immune responses (e.g. production of antigen-specific antibodies)
and cell-mediated immune responses (e.g. lymphocyte
proliferation).
[0174] Representative therapeutic proteins with immunostimulatory
effects include but are not limited to cytokines (e.g., IL-2, IL-7,
IL-12, interferons, granulocyte-macrophage colony-stimulating
factor (GM-CSF), tumor necrosis factor alpha (TNF-.alpha.)),
immunomodulatory cell surface proteins (e.g., human leukocyte
antigen (HLA proteins), co-stimulatory molecules, and
tumor-associated antigens. See Kirk & Mule, 2000; Mackensen et
al., 1997; Walther & Stein, 1999; and references cited
therein.
[0175] The term "angiogenesis" refers to the process, by which new
blood vessels are formed. The term "anti-angiogenic response" and
"anti-angiogenic activity" as used herein, each refer to a
biological process wherein the formation of new blood vessels is
inhibited.
[0176] Representative proteins with anti-angiogenic activities that
can be used in accordance with the presently disclosed subject
matter include: thrombospondin I (Kosfeld & Frazier, 1993;
Tolsma et al., 1993; Dameron et al., 1994), metallospondin proteins
(Carpizo & Iruela-Arispe, 2000), class I interferons (Albini et
al., 2000), IL12 (Voest et al., 1995), protamine (Ingber et al.,
1990), angiostatin (O'Reilly et al., 1994), laminin (Sakamoto et
al., 1991), endostatin (O'Reilly et al., 1997), and a prolactin
fragment (Clapp et al., 1993). In addition, several anti-angiogenic
peptides have been isolated from these proteins (Maione et al.,
1990; Eijan et al., 1991; Woltering et al., 1991).
[0177] A gene therapy construct used in accordance with the methods
of the presently disclosed subject matter can also encode a
therapeutic gene that displays both immunostimulatory and
anti-angiogenic activities, for example, IL-12 (see Dias et al.,
1998; and references cited hereinbelow), interferon-a (O'Byrne et
al., 2000), and references cited therein), or a chemokine (Nomura
& Hasegawa, 2000, and references cited therein). In addition, a
gene therapy construct can encode a gene product with
immunostimulatory activity and a gene product having
anti-angiogenic activity. See e.g. Narvaiza et al., 2000.
[0178] Additional compositions useful for cancer therapy include
but are not limited to genes encoding tumor suppressor gene
products/antigens, apoptosis-inducing polypeptides,
antimetabolites, suicide gene products, and combinations thereof.
See Kirk & Mule, 2000; Mackensen et al., 1997; Walther &
Stein, 1999; and references cited therein.
[0179] Therapeutic Compounds. In accordance with the methods of the
presently disclosed subject matter, a therapeutic agent can also
comprise a cytotoxic agent, a chemotherapeutic agent, a
radionuclide, or any other anti-tumor molecule. Studies using
ligand/drug conjugates have demonstrated that a chemotherapeutic
agent can be linked to a ligand to produce a conjugate that
maintains the binding specificity of the ligand and the therapeutic
function of the agent. For example, doxorubicin has been linked to
antibodies or peptides and the ligand/doxorubicin conjugates
display cytotoxic activity (Shih et al., 1994; Lau et al., 1995;
Sivam et al., 1995), PCT International Publication No. WO
98/10795). Similarly, other anthracyclines, including idarubicin
and daunorubocin, have been chemically conjugated to antibodies,
which have facilitated delivery of effective doses of the agents to
tumors (Aboud-Pirak et al., 1989; Rowland et al., 1993). Other
chemotherapeutic agents include cis-platinum (Schechter et al.,
1991), methotrexate (Shawler et al., 1988; Fitzpatrick &
Garnett; 1995) and mitomycin-C (Dillman et al., 1989).
[0180] In some embodiments of the presently disclosed subject
matter, a therapeutic agent comprises a radionuclide. Radionuclides
can be effectively conjugated to antibodies (Hartmann et al., 1994;
Buchsbaum at al., 1995), small molecule ligands (Wilbur, 1992;
Fjalling et al., 1996), and peptides (Boerman et al., 2000;
Krenning & de Jong, 2000; Kwekkeboom et al., 2000; Virgolini et
al., 2001, and references cited therein), such that administration
of the conjugated radionuclide promotes tumor regression.
Representative therapeutic radionuclides and methods for preparing
a radionuclide-labeled agent are described further hereinbelow
under the heading Scinitaraohic Imaging. For therapeutic methods of
the presently disclosed subject matter, a preferred radionuclide
comprises .sup.131I.
[0181] Additional anti-tumor agents that can be conjugated to the
targeting ligands disclosed herein and used in accordance with the
therapeutic methods of the presently disclosed subject matter
include but are not limited to alkylating agents such as melphalan
and chlorambucil (Smyth et al., 1987; Aboud-Pirak et al., 1989;
Rowland at al., 1993), vinca alkaloids such as vindesine and
vinblastine (Aboud-Pirak at al., 1989; Starling et al., 1992),
antimetabolites such as 5-fluorouracil, 5-fluorouridine and
derivatives thereof (Krauer at al., 1992; Henn et al., 1993).
[0182] Nuclear Targeting. In some embodiments, the therapeutic
and/or diagnostic compositions of disclosed herein are targeted to
the nucleus of a cell (e.g., the nucleus of a tumor cell).
Targeting to the cell nucleus can be accomplished using targeting
peptides comprising, in some embodiments, any of SEQ ID NOs: 61-69.
In some embodiments, the targeting is to the nucleus of a tumor
cell. Targeting to the nucleus of a tumor cell can be accomplished
using a targeting ligand that comprises, in some embodiments, a
peptide comprising any of SEQ ID NOs: 61-69. Targeting to the
nucleus of a tumor cell can be accomplished using a targeting
ligand that comprises, in some embodiments, a peptide comprising
any of SEQ ID NOs: 61-69 in addition to a tumor-targeting peptide
as disclosed herein (e.g., SEQ ID NOs: 1-13, 26-60, and 71-86).
[0183] The therapeutic and/or diagnostic compositions that are
targeted to the nucleus can comprise any of the therapeutic and/or
diagnostic entities disclosed herein, including therapeutic agents
and diagnostic agents disclosed herein. In some embodiments the
nuclear targeting composition can be used to deliver additional
therapeutic and diagnostic agents that are therapeutically
effective when delivered to the nucleus. Such agents include, but
are not limited to polypeptides associated with apoptosis
induction, as well as the nucleotide sequences encoding such
polypeptides. Exemplary apoptosis-inducing genes and gene products
include, but are not limited to bax, bak, and DP5.
[0184] IV.B. Preparation of a Therapeutic and/or Diagnostic
Composition
[0185] The presently disclosed subject matter also provides a
method for preparing a composition for guided drug delivery (e.g.,
radiation guided drug delivery). In some embodiments, the method
comprises (a) performing in vivo panning, whereby a ligand that
binds a radiation-induced tumor molecule is identified; and (b)
conjugating the ligand to a drug, whereby a composition for guided
drug delivery is prepared. A drug can further comprise a drug
carrier and can be formulated in any manner suitable for
administration to a subject. In some embodiments, the method
employs a targeting ligand comprising any one of SEQ ID NOs: 1-13,
18, 20, 22, 24-69, and 71-86.
[0186] Drug Carriers. The compositions of the presently disclosed
subject matter can further comprise a drug carrier to facilitate
drug preparation and administration. Any suitable drug delivery
vehicle or carrier can be used, including but not limited to a gene
therapy vector (e.g., a viral vector or a plasmid), a microcapsule,
for example a microsphere or a nanosphere (Manome et al., 1994;
Hallahan, 2001a; Saltzman & Fung, 1997), a peptide (U.S. Pat.
Nos. 6,127,339 and 5,574,172), a glycosaminoglycan (U.S. Pat. No.
6,106,866), a fatty acid (U.S. Pat. No. 5,994,392), a fatty
emulsion (U.S. Pat. No. 5,651,991), a lipid or lipid derivative
(U.S. Pat. No. 5,786,387), collagen (U.S. Pat. No. 5,922,356), a
polysaccharide or derivative thereof (U.S. Pat. No. 5,688,931), a
nanosuspension (U.S. Pat. No. 5,858,410), a polymeric micelle or
conjugate (Goldman et al., 1997 and U.S. Pat. Nos. 4,551,482,
5,714,166, 5,510,103, 5,490,840, and 5,855,900), and a polysome
(U.S. Pat. No. 5,922,545).
[0187] Conjugation of Targeting Ligands. Antibodies, peptides, or
other ligands can be coupled to drugs or drug carriers using
methods known in the art, including but not limited to carbodiimide
conjugation, esterification, sodium periodate oxidation followed by
reductive alkylation, and glutaraldehyde crosslinking. See Goldman
et al., 1997; Cheng, 1996; Neri et al., 1997; Nebel, 1997; Park et
al., 1997; Pasqualini et al., 1997; Bauminger & Wilchek, 1980;
U.S. Pat. No. 6,071,890; and European Patent No. 0 439 095.
[0188] In addition, a targeting peptide or antibody can be
recombinantly expressed. For example, a nucleotide sequence
encoding a targeting peptide or ligand can be cloned into
adenovirus DNA encoding the H1 loop fiber, such that the targeting
peptide or ligand is extracellularly presented. An adenovirus
vector so prepared can be used for guided delivery (e.g., radiation
guided delivery) of a gene therapy construct as disclosed herein. A
modified adenovirus vector encoding the RGD peptide was observed to
transduce the endothelium in tumor blood vessels.
[0189] Formulation. A therapeutic composition, a diagnostic
composition, or a combination thereof, of the presently disclosed
subject matter comprises in some embodiments a pharmaceutical
composition that includes a pharmaceutically acceptable carrier.
Suitable formulations include aqueous and non-aqueous sterile
injection solutions which can contain anti-oxidants, buffers,
bacteriostats, bactericidal antibiotics and solutes which render
the formulation isotonic with the bodily fluids of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
can include suspending agents and thickening agents. The
formulations can be presented in unit-dose or multi-dose
containers, for example sealed ampoules and vials, and can be
stored in a frozen or freeze-dried (lyophilized) condition
requiring only the addition of sterile liquid carrier, for example
water for injections, immediately prior to use. Some exemplary
ingredients are SDS in the range of in some embodiments 0.1 to 10
mg/ml, in some embodiments about 2.0 mg/ml; and/or mannitol or
another sugar in the range of in some embodiments 10 to 100 mg/ml,
in some embodiments about 30 mg/ml; and/or phosphate-buffered
saline (PBS). Any other agents conventional in the art having
regard to the type of formulation in question can be used.
[0190] The therapeutic regimens and pharmaceutical compositions of
the presently disclosed subject matter can be used with additional
adjuvants or biological response modifiers including, but not
limited to, the cytokines IFN-.alpha., IFN-.gamma., IL-2, IL-4,
IL-6, TNF, or other cytokine affecting immune cells.
[0191] IV.C. Administration
[0192] Suitable methods for administration of a therapeutic
composition, a diagnostic composition, or combinations thereof of
the presently disclosed subject matter include but are not limited
to intravascular, subcutaneous, or intratumoral administration. In
some embodiments, intravascular administration is employed. As used
herein, the phrases "intravascular administration" and
"intravascular provision" refer to administration of a composition
directly into the vascular network of a subject. Techniques that
can be employed for intravascular administration of compositions
are known to those of skill in the art, and include, but are not
limited to intravenous administration and intraarterial
administration. An exemplary method of intravascular provision that
is appropriate for rodents is tail vein administration, although it
is understood that any site and method for intravascular
administration can be chosen, depending at least in part on the
species of the subject to which the composition is to be
administered. For delivery of compositions to pulmonary pathways,
compositions can be administered as an aerosol or coarse spray.
[0193] For therapeutic applications, a therapeutically effective
amount of a composition of the presently disclosed subject matter
is administered to a subject. A "therapeutically effective amount"
is an amount of the therapeutic composition sufficient to produce a
measurable biological tumor response (e.g., an immunostimulatory,
an anti-angiogenic response, a cytotoxic response, or tumor
regression). Actual dosage levels of active ingredients in a
therapeutic composition of the presently disclosed subject matter
can be varied so as to administer an amount of the active
compound(s) that is effective to achieve the desired therapeutic
response for a particular subject. The selected dosage level will
depend upon a variety of factors including the activity of the
therapeutic composition, formulation, the route of administration,
combination with other drugs or treatments, tumor size and
longevity, and the physical condition and prior medical history of
the subject being treated. In some embodiments of the presently
disclosed subject matter, a minimal dose is administered, and dose
is escalated in the absence of dose-limiting toxicity.
Determination and adjustment of a therapeutically effective dose,
as well as evaluation of when and how to make such adjustments, are
known to those of ordinary skill in the art of medicine.
[0194] For diagnostic applications, a detectable amount of a
composition of the presently disclosed subject matter is
administered to a subject. A "detectable amount", as used herein to
refer to a diagnostic composition, refers to a dose of such a
composition that the presence of the composition can be determined
in vivo or in vitro. A detectable amount will vary according to a
variety of factors, including but not limited to chemical features
of the drug being labeled, the detectable label, labeling methods,
the method of imaging and parameters related thereto, metabolism of
the labeled drug in the subject, the stability of the label (e.g.
the half-life of a radionuclide label), the time elapsed following
administration of the drug and/or labeled antibody prior to
imaging, the route of drug administration, the physical condition
and prior medical history of the subject, and the size and
longevity of the tumor or suspected tumor. Thus, a detectable
amount can vary and can be tailored to a particular application.
After study of the present disclosure, and in particular the
Examples, it is within the skill of one in the art to determine
such a detectable amount.
[0195] IV.D. Radiation Treatment
[0196] The disclosed targeting ligands are useful for guided drug
delivery (e.g., radiation guided drug delivery). Targeted drug
delivery to a tumor in a subject can be performed by irradiating
the tumor prior to, concurrent with, or subsequent to
administration of a composition of the presently disclosed subject
matter. In accordance with the in vivo panning methods for
discovery of the targeting ligands, the tumor is irradiated in some
embodiments 0 hours to about 24 hours before administration of the
composition, and in some embodiments about 4 hours to about 24
hours before administration of the composition.
[0197] Low doses of radiation can be used for selective targeting
using the peptide ligands disclosed herein. In some embodiments,
the dose of radiation comprises up to about 2 Gy ionizing
radiation. Higher radiation doses can also be used, especially in
the case of local radiation treatment as described hereinbelow.
[0198] Radiation can be localized to a tumor using conformal
irradiation, brachytherapy, or stereotactic irradiation. The
threshold dose for inductive changes can thereby be exceeded in the
target tissue but avoided in surrounding normal tissues. In some
embodiments, doses of at least about 2 Gy ionizing radiation can be
used, and in some embodiments a dose of about 10 Gy to about 20 Gy
ionizing radiation can be used. For treatment of a subject having
two or more tumors, local irradiation enables differential drug
administration and/or dose at each of the two or more tumors.
Alternatively, whole body irradiation can be used, as permitted by
the low doses of radiation required for targeting of ligands
disclosed herein: Radiotherapy methods suitable for use in the
practice of the presently disclosed subject matter can be found in
Leibel & Phillips, 1998, among other sources.
[0199] IV.E. Monitoring Distribution In Vivo
[0200] In some embodiments of the presently disclosed subject
matter, a diagnostic and/or therapeutic composition for guided drug
delivery comprises a label that can be detected in vivo (e.g.,
radiation guided drug delivery). The term "in vivo", as used herein
to describe imaging or detection methods, refer to generally
non-invasive methods such as scintigraphic methods, magnetic
resonance imaging, ultrasound, or fluorescence, each described
briefly hereinbelow. The term "non-invasive methods" does not
exclude methods employing administration of a contrast agent to
facilitate in vivo imaging.
[0201] The label can be conjugated or otherwise associated with a
targeting ligand (e.g., any one of SEQ ID NOs: 1-13, 18, 20, 22,
24-69, and 71-86), a therapeutic, a diagnostic agent, a drug
carrier, or combinations thereof. Following administration of the
labeled composition to a subject, and after a time sufficient for
binding, the biodistribution of the composition can be visualized.
The term "time sufficient for binding" refers to a temporal
duration that permits binding of the labeled agent to a
radiation-induced target molecule.
[0202] Scintigraphic Imaging. Scintigraphic imaging methods include
SPECT (Single Photon Emission Computed Tomography), PET (Positron
Emission Tomography), gamma camera imaging, and rectilinear
scanning. A gamma camera and a rectilinear scanner each represent
instruments that detect radioactivity in a single plane. Most SPECT
systems are based on the use of one or more gamma cameras that are
rotated about the subject of analysis, and thus integrate
radioactivity in more than one dimension. PET systems comprise an
array of detectors in a ring that also detect radioactivity in
multiple dimensions.
[0203] A representative method for SPECT imaging is presented in
Example 2. Other imaging instruments suitable for practicing the
method of the presently disclosed subject matter, and instruction
for using the same, are readily available from commercial sources.
Both PET and SPECT systems are offered by ADAC of Milpitas, Calif.,
United States of America, and Siemens of Hoffman Estates, Ill.,
United States of America. Related devices for scintigraphic imaging
can also be used, such as a radio-imaging device that includes a
plurality of sensors with collimating structures having a common
source focus.
[0204] When scintigraphic imaging is employed, the detectable label
comprises in some embodiments a radionuclide label, in some
embodiments a radionuclide label selected from the group consisting
of .sup.18fluorine, .sup.64copper, .sup.65copper, .sup.67gallium,
.sup.68gallium, .sup.77bromine, .sup.80mbromine, .sup.95ruthenium,
.sup.97ruthenium, .sup.103ruthenium, .sup.105ruthenium,
.sup.99mtechnetium, .sup.107mercury, .sup.203mercury,
.sup.123iodine, .sup.124iodine, .sup.125iodine, .sup.126iodine,
.sup.131iodine, .sup.133iodine, .sup.111indium, .sup.113mindium,
.sup.99mrhenium, .sup.105rhenium, .sup.101rhenium, .sup.186rhenium,
.sup.188rhenium, .sup.121mtellurium, .sup.122mtellurium,
.sup.125mtellurium, .sup.165thulium, .sup.167thulium,
.sup.168thulium, and nitride or oxide forms derived there from. In
some embodiments, the radionuclide label comprises .sup.131iodine
or .sup.99mTc.
[0205] Methods for radionuclide labeling of a molecule so as to be
used in accordance with the disclosed methods are known in the art.
For example, a targeting molecule can be derivatized so that a
radioisotope can be bound directly to it (Yoo et al., 1997).
Alternatively, a linker can be added that to enable conjugation.
Representative linkers include diethylenetriamine pentaacetate
(DTPA)-isothiocyanate, succinimidyl 6-hydrazinium nicotinate
hydrochloride (SHNH), and hexamethylpropylene amine oxime (HMPAO)
(Chattopadhyay et al., 2001; Sagiuchi et al., 2001; Dewanjee et
al., 1994; U.S. Pat. No. 6,024,938). Additional methods can be
found in U.S. Pat. No. 6,080,384; Hnatowich et al., 1996; and
Tavitian et al., 1998.
[0206] When the labeling moiety is a radionuclide, stabilizers to
prevent or minimize radiolytic damage, such as ascorbic acid,
gentisic acid, or other appropriate antioxidants, can be added to
the composition comprising the labeled targeting molecule.
[0207] Magnetic Resonance Imaging (MRI). Magnetic resonance
image-based techniques create images based on the relative
relaxation rates of water protons in unique chemical environments.
As used herein, the term "magnetic resonance imaging". refers to
magnetic source techniques including convention magnetic resonance
imaging, magnetization transfer imaging (MTI), proton magnetic
resonance spectroscopy (MRS), diffusion-weighted imaging (DWI) and
functional MR imaging (fMRI). See Rovaris et al., 2001; Pomper
& Port, 2000; and references cited therein.
[0208] Contrast agents for magnetic source imaging include but are
not limited to paramagnetic or superparamagnetic ions, iron oxide
particles (Weissleder et al., 1992; Shen et al., 1993), and
water-soluble contrast agents. Paramagnetic and superparamagnetic
ions can be selected from the group of metals including iron,
copper, manganese, chromium, erbium, europium, dysprosium, holmium
and gadolinium. Preferred metals are iron, manganese and
gadolinium; most preferred is gadolinium.
[0209] Those skilled in the art of diagnostic labeling recognize
that metal ions can be bound by chelating moieties, which in turn
can be conjugated to a therapeutic agent in accordance with the
methods of the presently disclosed subject matter. For example,
gadolinium ions are chelated by diethylenetriaminepentaacetic acid
(DTPA). Lanthanide ions are chelated by tetraazacyclododocane
compounds. See U.S. Pat. Nos. 5,738,837 and 5,707,605.
Alternatively, a contrast agent can be carried in a liposome
(Schwendener, 1992).
[0210] Images derived used a magnetic source can be acquired using,
for example, a superconducting quantum interference device
magnetometer (SQUID, available with instruction from Quantum Design
of San Diego, Calif., United States of America). See U.S. Pat. No.
5,738,837.
[0211] Ultrasound. Ultrasound imaging can be used to obtain
quantitative and structural information of a target tissue,
including a tumor. Administration of a contrast agent, such as gas
microbubbles, can enhance visualization of the target tissue during
an ultrasound examination. Preferably, the contrast agent can be
selectively targeted to the target tissue of interest, for example
by using a peptide for guided drug delivery (e.g., radiation guided
drug delivery) as disclosed herein. Representative agents for
providing microbubbles in vivo include but are not limited to
gas-filled lipophilic or lipid-based bubbles (e.g., U.S. Pat. Nos.
6,245,318, 6,231,834, 6,221,018, and 5,088,499). In addition, gas
or liquid can be entrapped in porous inorganic particles that
facilitate microbubble release upon delivery to a subject (U.S.
Pat. Nos. 6,254,852 and 5,147,631).
[0212] Gases, liquids, and combinations thereof suitable for use
with the presently disclosed subject matter include air; nitrogen;
oxygen; is carbon dioxide; hydrogen; nitrous oxide; an inert gas
such as helium, argon, xenon or krypton; a sulphur fluoride such as
sulphur hexafluoride, disulphur decafluoride or
trifluoromethylsulphur pentafluoride; selenium hexafluoride; an
optionally halogenated silane such as tetramethylsilane; a low
molecular weight hydrocarbon (e.g. containing up to 7 carbon
atoms), for example an alkane such as methane, ethane, a propane, a
butane or a pentane, a cycloalkane such as cyclobutane or
cyclopentane, an alkene such as propene or a butene, or an alkyne
such as acetylene; an ether; a ketone; an ester; a halogenated low
molecular weight hydrocarbon (e.g. containing up to 7 carbon
atoms); or a mixture of any of the foregoing. Halogenated
hydrocarbon gases can show extended longevity, and thus are
preferred for some applications. Representative gases of this group
include decafluorobutane, octafluorocyclobutane,
decafluoroisobutane, octafluoropropane, octafluorocyclopropane,
dodecafluoropentane, decafluorocyclopentane, decafluoroisopentane,
perfluoropexane, perfluorocyclohexane, perfluoroisohexane, sulfur
hexafluoride, and perfluorooctaines, perfluorononanes;
perfluorodecanes, optionally brominated.
[0213] Attachment of targeting ligands to lipophilic bubbles can be
accomplished via chemical crosslinking agents in accordance with
standard protein-polymer or protein-lipid attachment methods (e.g.,
via carbodiimide (EDC) or thiopropionate (SPDP)). To improve
targeting efficiency, large gas-filled bubbles can be coupled to a
targeting ligand using a flexible spacer arm, such as a branched or
linear synthetic polymer (U.S. Pat. No. 6,245,318). A targeting
ligand can be attached to the porous inorganic particles by
coating, adsorbing, layering, or reacting the outside surface of
the particle with the targeting ligand (U.S. Pat. No.
6,254,852).
[0214] A description of ultrasound equipment and technical methods
for acquiring an ultrasound dataset can be found in Coatney, 2001;
Lees, 2001; and references cited therein.
[0215] Fluorescent Imaging. Non-invasive imaging methods can also
comprise detection of a fluorescent label. A drug comprising a
lipophilic component (therapeutic agent, diagnostic agent, vector,
or drug carrier) can be labeled with any one of a variety of
lipophilic dyes that are suitable for in vivo imaging. See e.g.
Fraser, 1996; Ragnarson et al., 1992; and Heredia et al., 1991.
Representative labels include but are not limited to carbocyanine
and aminostyryl dyes, preferably long chain dialkyl carbocyanines
(e.g., DiI, DiO, and DiD available from Molecular Probes Inc. of
Eugene, Oreg., United States of America) and dialkylaminostyryl
dyes. Lipophilic fluorescent labels can be incorporated using
methods known to one of skill in the art. For example VYBRANT.TM.
cell labeling solutions are effective for labeling of cultured
cells of other lipophilic components (Molecular Probes Inc. of
Eugene, Oreg., United States of America). Preparation of liposomes
comprising a targeting ligand and a DiI detectable label are
described in Example 1.
[0216] A fluorescent label can also comprise sulfonated cyanine
dyes, including Cy5.5 and Cy5 (available from Amersham of Arlington
Heights, Ill., United States of America), IRD41 and IRD700
(available from Li-Cor, Inc. of Lincoln, Nebr.), NIR-1 (available
from Dejindo of Kumamoto, Japan), and LaJolla Blue (available from
Diatron of Miami, Fla., United States of America). See also Licha
et al., 2000; Weissleder et al., 1999; and Vinogradov et al.,
1996.
[0217] In addition, a fluorescent label can comprise an organic
chelate derived from lanthanide ions, for example fluorescent
chelates of terbium and europium (U.S. Pat. No. 5,928,627). Such
labels can be conjugated or covalently linked to a drug as
disclosed therein.
[0218] For in vivo detection of a fluorescent label, an image is
created using emission and absorbance spectra that are appropriate
for the particular label used. The image can be visualized, for
example, by diffuse optical spectroscopy. Additional methods and
imaging systems are described in U.S. Pat. Nos. 5,865,754;
6,083,486; and 6,246,901, among other places.
[0219] IV.F. In Vitro Detection
[0220] The presently disclosed subject matter further provides
methods for diagnosing a tumor, wherein a tumor sample or biopsy is
evaluated in vitro. In some embodiments, a targeting ligand of the
presently disclosed subject matter comprises a detectable label
such as a fluorescent, epitope, or radioactive label, each
described briefly hereinbelow.
[0221] Fluorescence. Any detectable fluorescent dye can be used,
including but not limited to FITC (fluorescein isothiocyanate),
FLUOR X.TM., ALEXA FLUOR.RTM., OREGON GREEN.RTM., TMR
(tetramethylrhodamine), ROX (X-rhodamine), TEXAS RED.RTM.,
BODIPY.RTM. 630/650, and Cy5 (available from Amersham Pharmacia
Biotech of Piscataway, N.J., United States of America, or from
Molecular Probes Inc. of Eugene, Oreg., United States of
America).
[0222] A fluorescent label can be detected directly using emission
and absorbance spectra that are appropriate for the particular
label used. Common research equipment has been developed for in
vitro detection of fluorescence, including instruments available
from GSI Lumonics (Watertown, Mass., United States of America) and
Genetic MicroSystems Inc. (Woburn, Mass., United States of
America). Most of the commercial systems use some form of scanning
technology with photomultiplier tube detection. Criteria for
consideration when analyzing fluorescent samples are summarized by
Alexay et al., 1996.
[0223] Detection of an Epitope. If an epitope label has been used,
a protein or compound that binds the epitope can be used to detect
the epitope. A representative epitope label is biotin, which can be
detected by binding of an avidin-conjugated fluorophore, for
example avidin-FITC, as described in Example 7. Alternatively, the
label can be detected by binding of an avidin-horseradish
peroxidase (HRP) streptavidin conjugate, followed by colorimetric
detection of an HRP enzymatic product. The production of a
colorimetric or luminescent product/conjugate is measurable using a
spectrophotometer or luminometer, respectively.
[0224] Autoradiographic Detection. In the case of a radioactive
label (e.g., .sup.131I or .sup.93mTc) detection can be accomplished
by conventional autoradiography or by using a phosphorimager as is
known to one of skill in the art. A preferred autoradiographic
method employs photostimulable luminescence imaging plates (Fuji
Medical Systems of Stamford, Conn., United States of America).
Briefly, photostimulable luminescence is the quantity of light
emitted from irradiated phosphorous plates following stimulation
with a laser during scanning. The luminescent response of the
plates is linearly proportional to the activity (Amemiya et al.,
1988; Hallahan et al., 2001b).
V. Identification of a Radiation-Induced Target Molecule
[0225] Targeting ligands obtained using the methods disclosed
herein can be used to identify and/or isolate a target molecule
that is recognized by the targeting ligand. Representative methods
include affinity chromatography, biotin trapping, and two-hybrid
analysis, each described briefly hereinbelow.
[0226] Affinity Chromatography. A representative method for
identification of a radiation-induced target molecule is affinity
chromatography. For example, a targeting ligand as disclosed herein
can be linked to a solid support such as a chromatography matrix. A
sample derived from an irradiated tumor is prepared according to
known methods in the art, and such sample is provided to the column
to permit binding of a target molecule. The target molecule, which
forms a complex with the targeting ligand, is eluted from the
column and collected in a substantially isolated form. The
substantially isolated target molecule is then characterized using
standard methods in the art. See Deutscher, 1990.
[0227] Biotin Trapping. A related method employs a biotin-labeled
targeting ligand such that a complex comprising the biotin-labeled
targeting ligand bound to a target molecule can be purified based
on affinity to avidin, which is provided on a support (e.g., beads,
a column). A targeting ligand comprising a biotin label can be
prepared by any one of several methods, including binding of biotin
maleimide [3-(N-maleimidylpropionyl)biocytin] to cysteine residues
of a peptide ligand (Tang & Casey, 1999), binding of biotin to
a biotin acceptor domain, for example that described in K.
pneumoniae oxaloacetate decarboxylase, in the presence of biotin
ligase (Julien et al., 2000), attachment of biotin amine to reduced
sulfhydryl groups (U.S. Pat. No. 5,168,037), and chemical
introduction of a biotin group into a nucleic acid ligand,
(Carninci et al., 1996). In some embodiments, a biotin-labeled
targeting ligand and the unlabeled same target ligand show
substantially similar binding to a target molecule.
[0228] Two-Hybrid Analysis. As another example, targeting ligands
can be used to identify a target molecule using a two-hybrid assay,
for example a yeast two-hybrid or mammalian two-hybrid assay. In
one embodiment of the method, a targeting ligand is fused to a DNA
binding domain from a transcription factor (this fusion protein is
called the "bait"). Representative DNA-binding domains include
those derived from GAL4, LEXA, and mutant forms thereof. One or
more candidate target molecules is fused to a transactivation
domain of a transcription factor (this fusion protein is called the
"prey"). Representative transactivation domains include those
derived from E. coli B42, GAL4 activation domain II, herpes simplex
virus VP16, and mutant forms thereof. The fusion proteins can also
include a nuclear localization signal.
[0229] The transactivation domain should be complementary to the
DNA-binding domain, meaning that it should interact with the
DNA-binding domain so as to activate transcription of a reporter
gene comprising a binding site for the DNA-binding domain.
Representative reporter genes enable genetic selection for
prototrophy (e.g. LEU2, HIS3, or LYS2 reporters) or by screening
with chromogenic substrates (lacZ reporter).
[0230] The fusion proteins can be expressed from a same vector or
different vectors. The reporter gene can be expressed from a same
vector as either fusion protein (or both proteins), or from a
different vector. The bait, prey, and reporter genes are
co-transfected into an assay cell, for example a microbial cell
(e.g., a bacterial or yeast cell), an invertebrate cell (e.g., an
insect cell), or a vertebrate cell (e.g., a mammalian cell,
including a human cell). Cells that display activity of the encoded
reporter are indicative of a binding interaction between the
peptide and the candidate target molecule. The protein encoded by
such a clone is identified using standard protocols known to one of
skill in the art.
[0231] Additional methods for yeast two-hybrid analysis can be
found in Brent & Finley, 1997; Allen et al., 1995; Lecrenier et
al., 1998; Yang et al., 1995; Bendixen et al., 1994; Fuller et al.,
1998; Cohen et al., 1998; Kolonin & Finley, 1998; Vasavada et
al., 1991; Rehrauer et al., 1996; and Fields & Song, 1989.
[0232] Mass Spectroscopy. MALDI-MS can be used to identify
radiation-induced neoantigens that are well suited for
immunoconjugate-mediated drug delivery. These include antigens that
are not expressed in normal vasculature, but are inducible and
tethered within tumor blood vessels and stroma. The host components
of tumors (vasculature and stroma) respond to ionizing radiation
with physiologic responses that occur within most if not all
tumors. These include responses to oxidative stress and tissue
injury such as receptor and enzyme activation. The response in
vasculature of heterotopic tumors implanted into mice is described
herein.
[0233] Novel radiation-induced neoantigens can also be identified
by analyzing the response of human head and neck squamous cell
carcinoma (HNSCC) from biopsies of tumors following irradiation and
characterizing the proteomic response to irradiation within both
microvasculature and stroma. For example, the response of stroma
and endothelium following irradiation of tumors can be analyzed to
detect sites of apoptosis using terminal deoxynucleotidyl
transferase-mediated nick end labeling (TUNEL) staining. Using this
approach, it was observed that irradiated tumor endothelial respond
with apoptosis which provides neoantigenic targets for drug
delivery.
EXAMPLES
[0234] The following Examples have been included to illustrate
modes of the presently disclosed subject matter. These Examples
illustrate standard laboratory practices of the co-inventors. In
light of the present disclosure and the general level of skill in
the art, those of skill will appreciate that the following Examples
are intended to be exemplary only and that numerous changes,
modifications, and alterations can be employed without departing
from the scope of the presently disclosed subject matter.
Example 1
Radiation Guided Delivery of Fibrinogen-Conjugated Liposomes and
Microspheres
[0235] Preparation of Radiolabeled Microspheres. Albumin
microspheres (Martodam et al., 1979) were resuspended using 10 ml
of sterile normal saline (0.9% NaCl). One-half milliliter of the
reconstituted microsphere was added to a 1.5 ml conical
polypropylene tube previously coated with IODO-GEN.RTM. (Pierce
Biotechnology, Inc., Rockford, Ill., United States of America). To
this, 11.3 mCi (418 megabecquerel (MBq)) of .sup.131I (DuPont
Pharmaceuticals, Wilmington, Del., United States of America) was
added in approximately 11 .mu.l of saline and allowed to incubate
at room temperature for 30 minutes. Following incubation, the
microspheres were transferred to a 15 ml sterile centrifuge tube,
diluted to 10 ml with normal saline and centrifuged at 1,500 g for
seven minutes. The supernatant was removed and discarded. The
microspheres were washed one additional time with 10 ml of normal
saline and centrifuged. The microspheres were suspended in 2 ml of
normal saline for injection. Final yield was 4.8 mCi (177.6 MBq) of
radioiodinated microspheres in 2 ml saline. Radiochemical yield was
42.4%.
[0236] Preparation of Fibrinogen-Conjugated Liposomes. The
Lipophilic SH reactive reagent with a long spacing arm was
synthesized from maleimide-PEG 2000-NSH ester (Prochem Chemicals,
High Point, N.C., United States of America),
dioleoylphosphatidylethanolanime (DOPE, available from AVANTI.RTM.
Polar Lipids, Inc., Alabaster, Ala., United States of America) and
triethylamine in chloroform (1:1:1.5). Resulting maleimide-PEG
2000-DOPE was purified by flash column. Under stirring, to a
solution of fibrinogen (2 mg/ml) in 0.01M HEPES 0.15 NaCl buffer
pH7.9, containing 10 mM EDTA and 0.08% NaN.sub.3 was added in
5-fold excess of freshly prepared Traut's reagent (2-iminothiolane
hydrochloride) in the same buffer. The reaction was allowed to
proceed for 30 minutes at 0.degree. C.
[0237] SH-fibrinogen was purified using a PD-10 desalting and
buffer exchange column (Amersham Pharmacia Biotech, Piscataway,
N.J., United States of America). PEG 2000-PE, cholesterol,
Dipalmitoyl phosphocholine (AVANTI.RTM. Polar Lipids, Inc. of
Alabaster, Ala., United States of America), DiI (lipid fluorescent
marker available from Molecular Probes, Eugene, Oreg., United
States of America), and maleimide-PEG-2000ADOPE were dissolved in
chloroform and mixed at a molar ratio of 10:43:43:2:2,
respectively, in a round bottom flask. The organic solvent was
removed by evaporation followed by desiccation under vacuum for 2
hours. Liposomes were prepared by hydrating the dried lipid film in
PBS at a lipid concentration of 10 mM. The suspension was then
sonicated 3.times.5 minutes, or until the solution appeared clear,
to form unilamellar liposomes of 100 nM in diameter. To conjugate
thiolated fibrinogen to mateimide containing liposomes, prepared
vesicles and thiolated protein were mixed in 10 mm HEPES, 0.15 M
NaCl and EDTA pH 6.5. The final concentrations for proteins and
liposomes were 0.25 g/L and 2.5 mM, respectively. The
peptide/liposome mixture was incubated for 18 hours at room
temperature. Vesicles were then separated from unconjugated peptide
using a SEPHAROSE.TM. 4B-CL filtration column (Amersham Pharmacia
Biotech of Piscataway, N.J., United States of America).
[0238] Liposomes were fluorescently labeled with DiI fluorescent
marker (Molecular Probes, Inc., Eugene, Oreg., United States of
America) according to the manufacturer's instructions. Labeled
liposomes were administered by tail vein injection to tumor bearing
mice. Tumors were treated with 4 Gy either prior to administration
or after administration of fibrinogen-liposome conjugates. Tumors
were fixed and sectioned at 24 hours following irradiation.
Fluorescence was imaged by ultraviolet microscopy (100.times.).
[0239] Image Analysis. Tumor bearing mice were imaged at one hour
and 24 hours post-administration of radiolabeled proteins. Planar
pinhole gamma camera imaging was performed on a single-head gamma
camera (HELIX.RTM. model from General Electric Medical Systems of
Milwaukee, Wis., United States of America) using a cone-shaped
pinhole collimator with a 4 mm diameter Tungsten aperture. Pinhole
collimation offers the advantage of improved photon detection
efficiency (sensitivity) and spatial resolution when compared with
conventional, parallel multi-hole collimators. Pinhole planar
imaging with a small source-aperture separation can provide
high-resolution images combined with large magnification. Each scan
consisted of a 180 second acquisition (256.times.256 acquisition
matrix) with a 10% energy window centered on 364 keV. The
source-aperture separation was 6.0 cm.
[0240] Prior to imaging analysis in animals, a uniform .sup.131I
disk source was imaged in order to measure the angular dependence
of the pinhole collimator--gamma camera system detection efficiency
with distance from the center of the pinhole. Angular sensitivity,
normalized to 1.0 at the center of the pinhole, was then used to
scale the mouse data in order to correct image counts for this
geometrical effect. A calibration source of known .sup.131I
activity was also scanned at a 6.0 cm source-aperture separation
distance in order to measure system sensitivity along the center of
the pinhole.
[0241] Peptide biodistribution data was assessed using two
measures: (1) tumor-to-background ratio (T/B) of observed activity;
and (2) tumor uptake activity in .mu.Ci. Both types of data were
obtained using region-of-interest (ROI) analysis. For both
measurements an 11.times.11 ROI was used to determine mean counts
within the tumor (.sigma..sub.T) and at five different locations
within the mouse background (.sigma..sub.B). These readings were
scaled to account for geometric sensitivity and the ratio of tumor
uptake to total animal uptake (R) was computed according to the
relation,
R = .sigma. T ( .sigma. T + .sigma. B ) . ##EQU00001##
[0242] Activity uptake in the tumor was then approximated by the
product of the amount of activity administered into the animal
multiplied by the value obtained for R above. Tumor-background
ratios were determined according to the general expression:
( T B ) = .sigma. T .sigma. B . ##EQU00002##
[0243] Fibrinogen Coated Microsphere Localize to Irradiated
Tumors.
[0244] Fibrinogen-coated microspheres were radiolabeled with
.sup.131I and administered by tail vein injection into tumor
bearing mice, and tumors were irradiated with 6 Gy. The specificity
of fibrinogen-coated albumin was determined by measuring the
intensity of gamma detection within regions of interest (ROI) and
well counts of tumor and other tissues. In animals receiving
localized radiation at the tumor site, 90% of the measured
radioactivity was localized to the tumor, and 10% of the
radioactivity was diffusely distributed throughout the entire
animal model. In untreated controls, 10% of radioactive counts were
localized to the tumor (p<0.001).
[0245] During optimization studies, tumors were irradiated
immediately before or immediately after tail vein injection. Both
schedules were effective in achieving .sup.131I-fibrinogen-coated
microsphere binding. However, tumor irradiation subsequent to
microsphere administration achieved increased targeting specificity
when compared to tumors irradiated prior to microsphere
administration. Microspheres lacking the fibrinogen ligand did not
bind irradiated tumors.
[0246] To quantify a level of preferential binding of
fibrinogen-coated microspheres in irradiated tumors, data were
normalized based on background levels of radiation.
Fibrinogen-coated microspheres were 100-fold more abundant in
irradiated tumors compared to non-tumor control tissues. By
contrast, microspheres lacking the fibrinogen ligand were detected
at similar levels in tumor and non-tumor control tissues.
[0247] To determine whether fibrinogen-conjugated microspheres bind
irradiated non-tumor control tissues, the entire hind quarters of
mice bearing hind limb tumors were irradiated, and radiolabeled
fibrinogen-coated microspheres were administered immediately after
irradiation. Well counts of all tissues were performed at 24 hours
after irradiation. 90% of radioactive counts were detected in the
tumor. By contrast, 2% of radioactive counts were detected in
irradiated non-tumor control tissue, demonstrating selective
targeting of fibrinogen-coated microspheres to irradiated
tumors.
[0248] Fibrinogen-Liposome Conjugates Localize to Irradiated
Tumors.
[0249] Fibrinogen-conjugated, fluorescently labeled liposomes were
administered by tail vein into mice bearing tumors on both hind
limbs. The right tumor was treated with radiation and the left
tumor served as the untreated control. Untreated control tumors
showed no fibrinogen-liposome conjugate binding whereas tumors
irradiated immediately before or immediately after tail vein
injection showed fibrinogen adhesion in blood vessels. The
fluorescent marker was observed within the vascular lumen of tumor
microvasculature.
[0250] Studies using radiolabeled fibrinogen-conjugated liposomes
gave similar results. When liposomes were administered after tumor
irradiation, 89% of fibrinogen-coated liposomes localized to
tumors. When liposomes were administered immediately prior to tumor
irradiation, 69% of liposomes showed tumor localization. By
contrast, in untreated controls, a background level of 9% of
fibrinogen-coated liposomes localized to the tumor.
Example 2
Clinical Trials of Radiation-Guided Delivery Using a Peptide Ligand
Ligand Preparation and Administration
[0251] Biapcitide (ACUTECT.RTM. available from Diatide, Inc.,
Londonderry, N.H., United States of America) is a synthetic peptide
that binds to GP-IIb/IIIa receptors on activated platelets (Hawiger
et al., 1989; Hawiger & Timmons, 1992). Biapcitide was labeled
with .sup.99mTc in accordance with a protocol provided by Diatide
Inc.
[0252] Reconstituted .sup.99mTc-labeled biapcitide was administered
to patients at a dose of 100 mcg of biapcitide radiolabeled with 10
mCi of .sup.99mTc. Patients received .sup.99mTc-labeled biapcitide
intravenously immediately prior to irradiation. Patients were then
treated with 10 Gy or more. Patients underwent gamma camera imaging
prior to irradiation and 24 hours following irradiation. Following
planar image acquisition, those patients showing uptake in
irradiated tumors underwent tomographic imaging using SPECT and
repeat imaging at 24 hours. Patients showing no uptake on planer
images during this 24-hour time frame had no further imaging. Each
patient had an internal control, which consisted of a baseline scan
immediately following administration of .sup.99mTc-labeled
biapcitide.
[0253] Patients were treated with X-irradiation ranging from 4 to
18 MV photon using external beam linear accelerator at Vanderbilt
University. Appropriate blocks, wedges, and bolus to deliver
adequate dose to the planned target volume was utilized. The site
of irradiation, treatment intent and normal tissue considerations
determined the radiation dosage and volume. When stereotactic
radiosurgery was used, the dose was prescribed to the tumor
periphery.
[0254] Image Analysis. Image acquisition consisted of both planar
and single photon emission computed tomography (SPECT) studies.
Planar studies were performed on a dual-head gamma camera
(Millenium VG--Variable Geometry model available from General
Electric Medical Systems of Milwaukee, Wis., United States of
America) equipped with low energy high-resolution (LEUR)
collimators. This type of collimator represents a compromise
between sensitivity (photon counting efficiency) and image
resolution. Planar nuclear medicine images were acquired with a
256.times.256 acquisition matrix (pixel size approximately 0.178
cm/pixel) for 10 minutes. In order to maximize collimator-gamma
camera system sensitivity the source-to-detector surface distance
was minimized to the extent that patient geometry allows. The
spatial distribution of fibrinogen within the planar image was
measured using region-of-interest (ROI) analysis. Two different
size ROI's (5.times.5 pixel, and 15.times.15 pixel) was used in
both the tumor and surrounding organs and tissues in the patient.
The rationale for using ROIs with different dimensions is to be
able to quantify image counts while at the same time isolating any
possible influence of ROI size on the results. Tumor-to-background
ratios were computed as the ratio of average counts in the tumor
region divided by average counts in surrounding organs and tissues,
each corrected for background. Background counts was determined
based on ROI analysis of a separate planar acquisition performed in
the absence of a radioactive source.
[0255] Three-dimensional nuclear medicine SPECT examinations were
performed using the same dual-head gamma camera system. Each SPECT
study comprised a 360 scan acquired with a step-and-shoot approach
utilizing the following acquisition parameters: three increments
between views, a 256.times.256.times.64 acquisition matrix, LEUR
collimation and 60 seconds per view. Images were reconstructed
using analytical filtered back-projection and statistical maximum
likelihood techniques with photon attenuation correction and
post-reconstruction deconvolution filtering for approximate
detector response compensation. In this case, correction for
background consisted of subtracting counts acquired in a single
60-second planar view from all views of the SPECT projection data
prior to image reconstruction. SPECT tumor-to-background ratios
were computed using quantitative ROI techniques identical to the
planar studies.
[0256] Results. Administration of a .sup.99mTc-labeled biapcitide,
an RGD peptide mimetic, immediately prior to radiation resulted in
tumor binding in 4 of 4 patients (Hallahan et al., 2001a). Two
patients among this group had second neoplasms that were not
treated with radiation, and binding of .sup.99mTc-labeled
biapcitide was not observed in the untreated tumor. Administration
of the .sup.99mTc-labeled biapcitide within one hour following
radiation also failed to show localization of the targeting
molecule to the tumor (Hallahan et al., 2001a).
Example 3
Response of Tumor Blood Vessels to Ionizing Radiation
[0257] To determine the response of tumor blood vessels to ionizing
radiation, a tumor vascular window and Doppler sonography were used
to measure the change in tumor blood vessels (Donnelly et al.,
2001; Geng et al., 2001). Tumors implanted into the window model
developed blood vessels within 1 week. Tumors were then treated
with radiation and the response of blood vessels was imaged by use
of light microscopy. Radiation doses in the range of 2-3 Gy
increased the vascularity within tumors. In contrast, larger doses
of radiation such as 6 Gy reduced tumor vascularity.
[0258] Established tumors were studied to determine whether there
is a dose-dependent change in blood flow following irradiation.
Tumors in the hind limb were grown to approximately 1 cm in
diameter. Blood flow within tumors was measured by use of power
Doppler (Donnelly et al., 2001). Tumors were treated with 3 Gy or 6
Gy ionizing radiation; and changes in tumor blood flow were
measured using power Doppler sonography. A radiation dose of 3 Gy
achieved an increase in tumor blood flow. In contrast, radiation
doses of 6 Gy or higher markedly reduced tumor blood flow.
Example 4
Preparation of a Recombinant Peptide Library in Phage
[0259] A population of DNA fragments encoding recombinant peptide
sequences was cloned into the T7 SELECT.TM. vector (Novagen,
Madison, Wis., United States of America). Cloning at the EcoR I
restriction enzyme recognition site places the recombinant peptide
in-frame with the 10B protein such that the peptide is displayed on
the capsid protein. The resulting reading frame requires an AAT
initial codon followed by a TCX codon.
[0260] The molar ratio between insert and vector was 1:1.
Size-fractionated cDNA inserts were prepared by gel filtration on
sepharose 4B and ranged from 27 base pairs to 33 base pairs cDNAs
were ligated by use of the DNA ligation kit (Novagen). Recombinant
T7 DNA was packaged according to the manufacturer's instructions
and amplified prior to biopanning in animal tumor models. The
diversity of the library was 10.sup.7.
Example 5
In Vivo Panning for Peptide Ligands to Radiation-Induced
Molecules
[0261] GL261 murine glioma cells and Lewis lung carcinoma cells
were implanted into the hind limb of C57BL6 mice (Hallahan et al.,
1995b; Hallahan et al., 1998; Hallahan & Virudachalam,
1999).
[0262] To determine the optimal time at which peptides bind within
tumors, phage were administered at 1 hour before, at 1 hour after,
and at 4 hours after irradiation of both LLC and GL261 tumors.
Phage were recovered from tumors when administered 4 hours after
irradiation. Phage administered 1 hour before or 1 hour after
irradiation were not recovered from tumors. These data indicate
that the optimal time of administration is beyond 1 hour after
irradiation.
[0263] For in vivo panning, tumors were irradiated with 3 Gy and
approximately 10.sup.10 phage (prepared as described in Example 4)
were administered by tail vein injection into each of the tumor
bearing mice at 4 hours following irradiation. Tumors were
recovered at one hour following injection and amplified in BL21
bacteria. Amplified phage were pooled and re-administered to a
tumor-bearing mouse following tumor irradiation. The phage pool was
sequentially administered to a total of 6 animals. As a control,
wild type phage lacking synthetic peptide inserts were identically
administered to a second experimental group of animals.
[0264] To determine the titer of phage binding in a tumor or in
normal tissue, recovered phage were amplified in BL21 bacteria.
Bacteria were plated and the number of plaques present were
counted. To determine the total phage output per organ, the number
of plaque forming units (PFU) on each plate was divided by the
volume of phage plated and the weight of each organ. Normal
variation was observed as a 2-fold difference in PFU.
[0265] In the present study, background binding within tumor blood
vessels was approximately 10.sup.4 phage. Phage that bound to the
vasculature within irradiated tumors show enrichment in the tumor
relative to other organs and enrichment in the irradiated tumor
relative to the control phage without DNA insert. Phage that home
to irradiated tumors showed a background level of binding in
control organs that was lower than control phage without DNA
insert.
[0266] Following 6 rounds of in vivo panning, fifty recombinant
phage peptides that bound within irradiated tumors were randomly
selected for further analysis. The nucleic acid sequence encoding
recombinant phage was amplified by PCR using primers set forth as
SEQ ID NOs: 14-15 (available from Novagen of Madison, Wis., United
States of America). An individual phage suspension was used as
template. Amplified peptides were sequenced using an ABI PRISM.RTM.
377 sequencer (Applied Biosystems, Foster City, Calif., United
States of America). The sequences of the encoded peptides are
listed in Table 1. Several conserved subsequences were deduced from
the recovered peptides and are presented in Table 2.
[0267] Peptide sequences recovered from both tumor types include
NHVGGSSV (SEQ ID NO: 1), NSLRGDGSSV (SEQ ID NO: 2), and NSVGSRV
(SEQ ID NO: 4).Of the peptide sequences recovered from 6 irradiated
tumors, 56% had the subsequence GSSV (SEQ ID NO: 5), 18% had the
sequence RGDGSSV (SEQ ID NO: 6), and 4% had the sequence GSRV (SEQ
ID NO: 7). Approximately 22-40 of 10.sup.6 injected phage were
recovered from irradiated tumors having a peptide insert comprising
the subsequence GSSV (SEQ ID NO: 5). By contrast, no phage were
from irradiated tumors following administration of 10.sup.6 wild
type phage.
TABLE-US-00001 TABLE 1 Peptides Identified by Invivo Panning of LLC
and GL261 Tumors Number of Phage Number of Phage Recovered from
Recovered from LLC tumors GL261 tumors Peptide Sequence (Frequency)
(Frequency) NHVGGSSV 7 (28%) 12 (48%) (SEQ ID NO: 1) NSLRGDGSSV 7
(28%) 2 (8%) (SEQ ID NO: 2) NSVRGSGSGV 7 (28%) 0 (SEQ ID NO: 3)
NSVGSRV 1 (4%) 3 (12%) (SEQ ID NO: 4) Unique Sequences 3 (12%) 8
(32%)
TABLE-US-00002 TABLE 2 Conserved Motifs within Peptides Identified
by In vivo Panning Conserved Sequence Frequency of Recovery GSSV
(SEQ ID NO: 13) 56% GSXV (SEQ ID NO: 8) 78% NSXRGXGS (SEQ ID NO: 9)
32% NSV (SEQ ID NO: 10) 22% NSXR (SEQ ID NO: 11) 32% NXVG (SEQ ID
NO: 12) 46%
Example 6
Peptide Targeting in Additional Tumors
[0268] The binding properties of phage encoding NHVGGSSV (SEQ ID
NO: 1), NSLRGDGSSV (SEQ ID NO: 2), NSVRGSGSGV (SEQ ID NO: 3), and
NSVGSRV (SEQ ID NO: 4) were additionally characterized in a B16F0
melanoma model. Peptides set forth as SEQ ID NOs: 1 and 2 bound
within the melanoma, lung carcinoma, and glioma tumor models. SEQ
ID NO: 3 bound within glioma and melanoma, and SEQ ID NO: 4 bound
within lung carcinoma and glioma.
Example 7
Characterization of Peptide Binding to Irradiated Tumors
[0269] To determine where recombinant peptides bind in tumor blood
vessels, the biodistribution of biotinylated peptides was assessed.
Tumors were treated with 3 Gy and biotinylated peptides were
administered by tail vein at 4 hours following irradiation. Tumors
were recovered 30 minutes following administration of biotinylated
peptides. Tumors were snap frozen and sectioned on a cryostat.
Frozen sections were then incubated with Avidin-FITC (fluorescein
isothiocyante) and imaged by fluorescent microscopy. Recombinant
peptides (for example, those set forth in Table 1) were observed to
bind the vascular endothelium within tumor blood vessels.
[0270] The anti-.alpha..sub.2b.beta..sub.3 monoclonal antibody was
administered by tail vein to determine whether this receptor is
required for recombinant phage binding in irradiated tumors. Phage
encoding SLRGDGSSV (SEQ ID NO: 5) on the capsid protein were
injected immediately after blocking antibody or control antibody.
Phage were recovered from the tumor and controls organs and
quantified by plaque formation. Radiation induced a 4-fold increase
in phage binding in tumor. Blocking antibody eliminated induction
of phage binding, while control antibody to P-selectin (on
activated platelets) did not reduce phage binding. Thus, the tumor
binding activity of targeting peptide SLRGDGSSV (SEQ ID NO: 5) is
dependent on its interaction with the .alpha..sub.2b.beta..sub.3
receptor.
Example 8
Production of a Phage-Displayed scFv Antibody Library
[0271] A phage-displayed antibody library was constructed based
upon previously published methodologies (see Pope at al., 1996).
Briefly, spleens from outbred newborn and three-to-four week old
mice and rats were used as a source of antibody-encoding genetic
material to produce a library of about 2.times.10.sup.9 members.
The antibody-encoding genetic material was cloned into the pCANTAB
phagemid vector.
[0272] The pCANTAB vector contains an amber stop codon that is
located downstream of the scFv coding sequences and upstream of the
M13 gene III coding sequences. E. coli TG1 cells (a sup E strain of
E. coli) contain a suppressor tRNA that inserts a glutamic acid
residue in response to an UAG (amber) stop codon. The amber stop
codon is about 14% efficient. Therefore, the scFv antibody amino
acid sequences will be fused to M13 phage gene III amino acid
sequences about 14% of the time, and will be produced as a soluble,
non-fusion protein about 86% of the time when the library is grown
in TG1 cells. In contrast, E. coli strain HB2151 does not contain
the amber stop codon, and thus only soluble non-fused scFv will be
produced when the library is grown in HB2151.
Example 9
In Vivo Panning for Antibody Ligands to Radiation-Induced
Molecules
[0273] A phage library comprising diverse single chain antibodies
was prepared in M13 phage. The phage library was exposed to the
radiation-induced neoantigens P-selectin (also called CD62P;
GENBANK.RTM. Accession No. NP.sub.--002996) and/or platelet
membrane glycoprotein IIB (also called CD41; GENBANK.RTM. Accession
Nos. P08514 and NP.sub.--000410) immobilized on glass slides. Phage
were selected based on antigen binding, and selected phage were
pooled as a biased library. For representative in vitro panning
methods, see Fowlkes et al., 1992; Haaparanta & Huse, 1995;
Jung & Pluckthun, 1997; Peter et al., 2000; Holzem at et al.,
2001; Chiu et al., 2000.
[0274] Phage identified by in vitro panning were tested on Western
immunoblots to confirm binding to the P-selectin and platelet
membrane glycoprotein IIB neoantigens. Phage that specifically
bound P-selectin and platelet membrane glycoprotein MB were
subsequently used for in vivo panning to irradiated tumors as
described in Example 5. Wild type phage were used as internal
controls. Antibodies having substantial affinity for irradiated
tumors were identified by observing an increased number of phage in
the irradiated tumor when compared to a number of phage in a
control organ (e.g., liver and lung). Phage antibodies with the
greatest affinity for tumors were identified using the formula:
number of phage in irradiated tumor/number of phage in each
organ.
[0275] Eight antibodies that bound P-selectin and fifteen
antibodies that bound platelet membrane glycoprotein IIB were
recovered following in vivo panning to irradiated tumors.
Representative targeting antibodies identified by this method
include the single chain antibodies set forth as SEQ ID NOs: 18,
20, 22, and 24 (encoded by SEQ ID NOs: 17, 19, 21, and 23,
respectively), which recognize the radiation-induced neoantigens
P-selectin and platelet membrane glycoprotein IIB,
respectively.
Example 10
Conformational Changes Induced in Perlecan
[0276] Mass spectrometry analysis of the samples revealed that
co-culturing HMVEC cells with H460 tumor cells induced several
proteins expression on the HMVEC cells. Among them, CYR61 and
perlecan had been demonstrated to be important for tumor growth and
angiogenesis. Most importantly, several proteins underwent
conformational changes, by exposing some new biotinylation sites as
well as hiding some other sites for biotinylation.
[0277] One such protein was the perlecan precursor (GENBANK.RTM.
Accession Nos. P98160 and NP.sub.--005520; SEQ ID NO: 70). Several
subsequences of the perlecan precursor were found to undergo
conformational changes upon co-culture as evidenced by the blocking
of existing biotinylation sites. These subsequences are presented
in Table 3.
TABLE-US-00003 TABLE 3 Conformational Changes Induced in Perlecan
by Co-Culture Amino acids Biotinylation of SEQ ID Site Subsequence
NO: 70 Blocked? RPEEVCGPTQFR 363-374 Yes LRFDQPDDF 542-550 No
NVRYELAR 617-624 Yes GMLEPVQRPDVVLVGAGY 625-642 Yes AHSVEECRCPIGY
725-737 Yes SGLSCESCDAHF 738-749 No ATATSCRPCPCPY 806-818 Yes
RFSDTCFLDTDGQATCDACAPGYTGR 824-849 Yes RCESCAPGYEGNPIQPGGK 850-868
Yes CRPVNQEIVR 869-878 No RPVNQEIVR 870-878 Yes TCESLGAGGYR
1627-1637 No AVTLECVSAGEPR 3129-3141 No CSATGSPAPTIHWSK 3233-3247
Yes IAHVELADAGQYR 3542-3554 Yes IAHVELADAGQY 3542-3553 Yes
IAHVELADAGQYRCTATN 3542-3559 Yes AHLQVPER 3654-3661 Yes VVPYFTQTPY
3662-3671 Yes NGQKRVPGSPTNL 3704-3716 No VCVCPAGFTGSR 3868-3879 Yes
SAEPLALGR 4004-4012 Yes CLCLPGFSGPR 4164-4174 Yes
[0278] Additional conformational changes were identified when the
co-cultured cells were irradiated with 2 Gy, as new biotinylation
sites were induced. These changes are summarized in Table 4.
TABLE-US-00004 TABLE 4 Conformational Changes Inducedin Perlecan by
Co-Culture and Irradiation Amino acids of New Biotinylation
Subsequence SEQ ID NO: 70 Site Induced? LRFDQPDDF 542-550 No
GHTPTQPGALNQR 648-660 Yes SGLSCESCDAHF 738-749 No CRPVNQEIVR
869-878 No TCESLGAGGYR 1627-1637 No AVTLECVSAGEPR 3129-3141 No
NGQKRVPGSPTNL 3704-3716 No AGLSSGFIGCVR 3810-3821 Yes GCVGEVSVNGK
4075-4085 Yes CQQGSGHGIAESDW 4175-4188 Yes
Example 11
Identification of Additional Targeting Peptides by In Vivo
Panning
[0279] Tumors (Lewis Lung Carcinoma, LLC) were implanted into both
sides of hind limbs of C57 mice, and one side of tumor was treated
with 2 Gy of radiation when the tumors reached a size of 1 cm in
diameter. Six T7 phage-based random peptide libraries were screened
separately by injection through tail veins at 18 hours after the
radiation, and phages were circulated for 1 hour before the mice
were sacrificed to recover phages from the radiated tumor. The
recovered phages were amplified by infecting a bacterial host as
described hereinabove, and used as input for the next round of
biopanning.
[0280] After five rounds of in vivo biopanning, single phage clones
were isolated and the peptide sequence was deduced by sequenced the
relevant fragment in the phage genome. Dozens of peptides were
recovered from the irradiated tumors, with several enriched to be
dominant after the final round of biopanning. The isolated phage
were purified and injected into tumor-bearing mice. Phages in
tissues were visualized using an anti-T7 phage antibody in
conjunction with a FITC-conjugated secondary antibody. DAPI
staining was used to localize the cell nucleus.
[0281] Representative data indicated that the isolated phage
targeted the irradiated tumor cells by the peptide displayed on its
surface. Certain of these peptide sequences are presented in SEQ ID
NOs: 26-60.
Example 12
In Vitro Panning for Nuclear Targeting Peptides
[0282] T7-based linear peptides (.times.12, 16 and 20) were
subjected for biopanning on HUVEC monolayers. After 20 hours
incubation at 37.degree. C., phages recovered from nuclei were
amplified and used for the following round of selection. Titration
result for phages recovered from nuclei and cytoplasm in all the
rounds of screening showed that some nucleus-homing phages had been
enriched in the biopanning process. Phages from the third round
nucleus extraction were sequenced and are presented in SEQ ID NOs:
61-69.
[0283] A BLAST search indicated that the isolated sequence is close
to a well-studied sequence motif which was characterized as
nucleus-exporting signature (NES). However, the cy3-labeled phage
were located in HUVEC nuclei, compared with localization of the
control phage without the peptide insert on cell membrane or in
cytoplasm. Other proteins that contain a Nucleus-Export Sequence
(NES) are as follows:
TABLE-US-00005 HIV-1 Rev LPPLERLTLD HTLV-1 Rex LSAQLYSSLSLD HSV-1
ICP27 IDMLIDLGLDLD EBV Sm LPSPL-ASLTL HSV-VP13/14 NES1 LGRVL-DVLAVM
HSV-VP13/14 NES2 LHTAL-ATVTLK HSV-VP13/14 NES3 LAAGLVLQRLLG MVM NS2
MTKKFGTLTI PKI LALKLAGLDI MAPKK LQKKLEELEL NMD3 LAEMLEDLHI An3
LDQQFAGLDL I.kappa.Ba MVKELQEIRL Cyclin B1 LCQAFSDVIL TFIIIA
LPVLENLTL Consensus .phi.X.sub.2-3.phi.X.sub.2-3.phi.X.phi. .phi. =
L, I, V, F or M, residues with large hydrophobic side chain. X =
any amino acid
[0284] Engeisma et al., 2004 reported two phage-displayed peptides
isolated from an M13 library, S0 and P0, which contained NES-like
sequences and localized to the nuclear membrane.
TABLE-US-00006 S0 LARLFSALSV P0 LSSLFSGLSV Consensus LX2LFX2LSV
Peptide from 17 library FTHALDPGQLAL
Materials and Methods Employed in Examples 13-16
[0285] Linking Compounds. Linking compounds include
1,3,4,6-tetrachloro-3a,6a-diphenylglcouril (a reagent sold under
the registered trademark IODO-GEN0), and MPBA, each available from
Pierce Biotechnology, Inc. (Rockford, Ill., United States of
America). The IODO-GEN.RTM. reagent reacts with tyrosine residues,
while MPBA reacts with cysteine residues, both of which are not on
the peptide HHLGGAKQAGDV (SEQ ID NO: 16). An advantage of the
IODO-GEN.RTM. reagent is that it is supplied in coated tubes and
beads to eliminate contamination of the injectable material,
whereas MPBA is in powder form. Initial experiments use the
IODO-GEN.RTM. reagent to iodinate a poly-tyrosine peptide
derivative of HHLGGAKQAGDV-SGSGS (SEQ ID NO: 26),
HHLGGAKQAGDV-SGSGS-YYYYY (SEQ ID NO: 28), and additional
experiments use MPBA to iodinate poly-Cys.
[0286] Preparation and Radioiodination of Peptides. An
IODO-GEN.RTM.-plated reaction vessel (Pierce Biotechnology, Inc.)
is rinsed with a small amount of sterile saline to remove any loose
microscopic flakes of the iodination reagent. The desired amount of
carrier-free .sup.125I sodium iodide, a specific activity of 100
mCi/mg protein, is added to the reaction vessel, followed by the
reconstituted peptides suspension. The reaction vessel is then
sealed off and the reaction is allowed to proceed for 20 minutes at
room temperature with constant gentle agitation of the reaction
vessel. The iodination process is terminated by removing the
reaction mixture from the reaction vessel into a centrifugation
tube. The reaction mixture is centrifuged at 3,000 rpm for 15
minutes. The supernatant is removed and the residue is
reconstituted in 5 ml sterile normal saline.
[0287] Pinhole Gamma Camera Imaging of Peptide Biodistribution. A
dedicated research single-head gamma camera (20 cm.times.40 cm
active imaging area) fitted with a cone-shaped pinhole collimator
is used for nuclear medicine animal imaging experiments. The
pinhole collimator, equipped with a 4 mm aperture Tungsten insert,
is used to acquire pre-treatment and serial, post-treatment
follow-up images of each animal in order to determine the temporal
distribution of peptide in vivo. Each pinhole acquisition comprises
a planar view acquired for 3 minutes using a 256.times.256 pixel
acquisition matrix. In order to maximize pinhole collimator-gamma
camera system sensitivity, a source-to-aperture distance on the
order of 2 cm to 5 cm is maintained. The spatial distribution of
peptide within each image is measured using quantitative, region of
interest (ROI) analysis. Two different size ROIs are used in both
the tumor region and moue background in order to quantify image
counts and isolate any possible influence of ROI size on
quantification. A 2.times.2 (small) and 11.times.11 (large) pixel
ROI are used to record image counts in the tumor and other organs
in the mouse. The angular dependence of pinhole efficiency is
measured using a flat, uniform sheet source of activity. Image
counts are then corrected for decay and this geometric effect.
[0288] Statistical Considerations. Internal controls are
established in each animal by use of an untreated control tumor
implanted on the left hind limb and irradiation of the right hind
limb tumor, as described in Hallahan, 1995b and Hallahan, 1998.
[0289] Sample Size and Power Analysis. In order to calculate the
statistical significance of differences between groups of mice,
eight mice are studied at each time to determine statistical
significance. In general, a sample size of eight per group gives
about 80% of power to detect a difference of 1.5-fold standard
deviations in the interesting parameters between two groups with a
two-sided statistic equal to 5%.
[0290] Statistical Analysis Plan. Pharmacokinetic parameters are
presented in tabular and graphic form. Pharmacokinetic parameters
such as maximal plasma concentration, time of maximal
concentration, and area under the plasma concentration time curve
are determined using non-compartmental methods. Statistical
analyses are performed using the General Linear Model method of the
Statistical Analysis System (SAS). If significant differences are
indicated by the ANOVA analysis, the Waller-Duncan K-ratio t-test
procedure is used for pairwise comparisons of mean pharmacokinetic
parameter values.
[0291] For the single time point data, tests of hypotheses
concerning correlation between imaging results and results are
completed using the paired t-test or Wilcoxon Signed-Rank test for
the interesting continuous parameters or the McNemar's Chi-square
test for the interesting categorical parameters. For either count
or binary multiple time points data, tests concerning correlation
between imaging results and pharmacokinetic results are made using
the Generalized Estimating Equation (GEE) method statistical
procedure for longitudinal data analysis with multiple observable
vectors for the same subject (Diggle, 1994; Liang, 1986). For
continuous multiple time points data, tests concerning correlation
between groups are completed using the restricted/residual maximum
likelihood (REML)-based repeated measure model (mixed model
analysis; Jennrich, 1986) with various covariance structure.
[0292] The statistical analyses are completed by SAS 6.12
statistical program, or SAS IML macro in this project. Computer
connections, when necessary, are attained via a Novell network
using the Internet Packet eXchange (IPX) protocol.
Example 13
X-Ray-Guided Drug Delivery Via Antibody Delivery Vehicles
[0293] Following platelet activation, several antigens are
expressed on the surface of platelets. Indeed, it has been observed
that irradiation of animal tumors increases the expression of
platelet antigens such as P-selectin and GPIIb/IIIa. As disclosed
herein above, antibodies can be conjugated to radionuclides,
cytotoxic agents, gene therapy vectors, liposomes, and other active
agents. In this Example, the administration of radioimmunoconjugate
delivery vehicles against platelet antigens following irradiation
of tumors is disclosed.
[0294] Anti-GPIIb/IIIa antibodies (R&D Systems, Inc.,
Minneapolis, Minn., United States of America) are labeled with
.sup.131I using 1000-GEN.RTM. reagent (Pierce Biotechnology, Inc.,
Rockford, Ill., United States of America). Labeled antibody is
separated from free iodine by use of column chromatography.
Radioimmunoconjugates are injected into mice by tail vein. Hind
limb tumors are implanted and treated as described herein above.
The optimal time of administration of radioimmunoconjugates is
determined.
[0295] In separate experiments, procoagulants such as DDAVP are
also administered to enhance radioimmunoconjugate binding to
activated platelets in irradiated tumors. Mouse subjects are imaged
by gamma camera as described herein above. PHOSPHORIMAGER.TM.
plates and histologic sections with immunohistochemistry as
described herein above are used to validate image processing. In
the event that certain radioimmunoconjugates do not achieve
specific activity within tumors that is sufficient to image or
treat tumors, multiple radionuclides are incorporated into the
antibody delivery vehicles.
[0296] In additional experiments, Fab' fragments of anti-GPIIIa and
anti-GPIIb antibodies are also employed in binding in a
site-specific manner to irradiated tumors. It is shown herein that
anti-GPIIIa antibody staining in blood vessels following
X-irradiation. There are two approaches in producing antibodies for
site-specific binding. The first is cleavage of the IgG antibody to
form the Fab' fragment. The second approach is the use of phage
antibodies to GPIIIa and GPIIb that are produced in the Vanderbilt
Cancer Center Molecular Discovery Core Laboratory using
phage-display techniques. Each of these approaches yields low
molecular weight antibodies that can be efficiently produced for
clinical studies. Specificities of the GPIIIa (integrin
.beta..sub.3) antibodies and antibody fragments are compared to the
specificities of the GPIIb antibodies and antibody fragments to
establish potentially useful reagents and in that GPIIIa is also
found in .alpha..sub.v.beta..sub.3.
[0297] Experimental Design. The anti-GPIIIa and anti-GPIIb
antibodies (R&D Systems, Inc.) are cleaved to form the Fab'
fragment. This fragment is isolated from the Fc fragment by
columns. In addition, GPIIIa protein is screened with a phage
library within the Vanderbilt Cancer Center Molecular Discovery
Core Laboratory. Antibody from phage is grown up in the bacteria.
Antibodies are then studied for binding in irradiated tumors.
Antibodies are labeled with .sup.131I using IODO-GEN.RTM. reagent
as described above. The molar ratio of .sup.131I to antibody is
optimized to avoid potential reduction in the affinity of antibody
binding due to .sup.131I.
[0298] Tumors are implanted and irradiated as described herein.
Radio-immunoconjugates are administered immediately after
irradiation using tail vein injection. Eight mice are randomly
assigned into experimental and control groups. Imaging and
quantification of .sup.131I are performed as described above.
Statistical analysis is performed as described above.
[0299] Positive control aromas. Radiolabeled fibrinogen is
administered to irradiated tumor bearing mice and compared to
radioimmunoconjugates. These mice are randomly assigned into groups
during the same experiment as radioimmunoconjugates.
[0300] Negative control groups. Non-irradiated control tumors are
implanted in the left hind limb of all mice. Secondly, radiolabeled
anti-.alpha..sub.v and anti-human IgG antibodies are administered
to tumor bearing mice following irradiation to verify that antibody
binding to irradiated tumors is not a generalized phenomenon.
Example 14
X-Ray-Guided Drug Delivery Targeted to Radiation-Induced
Neoantigens in Blood Vessels
[0301] Radiation-induced targets for drug delivery systems will be
most useful if they are not tumor-specific. The vascular
endothelium is an essential component to nearly all neoplasms. As
disclosed herein above, radiation response is similar across a wide
range of tumor types. In particular, P-selectin exocytosis, von
Willebrand Factor release, and platelet aggregation are observed
within all tumor blood vessels following irradiation. In this
Example, antibody delivery vehicles for X-ray-guided drug delivery
to the vascular endothelium of tumors are disclosed. Antibody
delivery vehicles adhere to antigens released into the lumen and
are thus obstructed from circulating beyond the confines of the
tumor. In view of the targeting of vascular endothelium, this
Example is also illustrative of the methods of treating
angiogenesis in accordance with the presently disclosed subject
matter disclosed herein above.
[0302] Additionally, one level of radiation-induced expression of
receptors and adhesion molecules is the activation of inactive
receptors following irradiation of tumor blood vessels. Tumors in
the hind limb of mice were treated with 2 Gy ionizing radiation
followed by sectioning and immunohistochemical staining for the
.beta..sub.3 integrin in the tumor sections. The observed
histologic pattern of staining showed that both platelets and
endothelium stain with anti-.beta..sub.3 antibody after
irradiation, but not prior to irradiation. Thus, therapeutic doses
of irradiation (2 Gy) were and are sufficient to induce the
accumulation of integrin .beta..sub.3 within tumor blood vessels
within 1-4 hours of irradiation.
[0303] Hind limb tumors are implanted into mice and treated with
radiation as described in Hallahan et al., 1998a.
Radioimmunoconjugate delivery vehicles are prepared using
anti-E-selectin and anti-P-selectin antibodies (R&D Systems,
Inc.), IODO-GEN.RTM. reagent (Pierce Biotechnology. Inc.) and
.sup.1311. Radiolabeled antibodies are separated from free
.sup.1311 by use of column chromatography. The delivery vehicles
are injected via tail vein into mice with hind limb tumors
following treatment with irradiation. Mice are imaged with gamma
camera imaging as described herein above. Image processing is
validated by use of PHOSPHORIMAGER.TM. plates, immunofluorescence,
and immunohistochemistry as described herein above.
[0304] One potential limitation of this embodiment of the presently
disclosed subject matter is that anti-E-Selectin antibody binding
occurs in untreated normal tissues such as the lung. The importance
of validation of the tumor specificity for radioimmunoconjugate
delivery vehicles is that the ideal radiation-induced antigens have
substantially no constitutive expression in any tissue, but
prolonged expression in tumor blood vessels. Thus, pharmacokinetics
and biodistribution of the anti-E-selectin and anti-P-selectin
antibody delivery vehicles are also determined.
Example 15
X-Ray-Guided Drug Delivery by Use of a Twelve Amino Acid Segment of
the .gamma. Subunit of Fibrinogen
[0305] This Example pertains to the use of the dodecapeptide
HHLGGAKQAGDV (SEQ ID NO: 16), a segment of the .gamma. subunit of
fibrinogen, to achieve site-specific binding to irradiated tumors.
This peptide segment of the carboxyl terminus of the fibrinogen
.gamma. chain binds to GPIIb/IIIa following platelet activation.
The fibrinogen binding sequence (HHLGGAKQAGDV; SEQ ID NO: 16) is
sufficient for site-specific localization to irradiated tumors.
[0306] Observations. The peptide sequence within fibrinogen that
binds to the activated GPIIb/IIIa receptor is the dodecapeptide
HHLGGAKQAGDV (SEQ ID NO: 16). To determine whether HHLGGAKQAGDV
(SEQ ID NO: 16) binds in irradiated tumors, applicant utilized the
peptide HHLGGAKQAGDV (SEQ ID NO: 16) linked to biotin by a
serine-glycine linker (HHLGGAKQAGDV-SGSGSK-biotin; SEQ ID NO: 30).
This peptide was synthesized in the Vanderbilt University Peptide
Core Lab and biotinylated at the carboxyl terminus. The resulting
HHLGGAKQAGDV-SGSGSK-biotin (SEQ ID NO: 30) was administered by tail
vein injection into tumor bearing mice. B16F0 tumors in the hind
limb were treated with sham irradiation (control), 4 Gy irradiation
followed by HHLGGAKQAGDV-SGSGSK-biotin (SEQ ID NO: 30) injection,
or HHLGGAKQAGDV-SGSGSK-biotin (SEQ ID NO: 30) followed by tumor
irradiation (4 Gy). Tumors were frozen at 4 hours and sectioned for
fluorescence staining. Avidin-FITC was incubated with tumor
sections and imaged by UV microscopy. Avidin-FITC stained blood
vessels were observed in irradiated tumors, but not in untreated
control. Moreover, it was found that HHLGGAKQAGDV (SEQ ID NO: 16)
administration prior to irradiation is a more efficient schedule of
administration as compared to radiation before dodecapeptide
administration.
[0307] Design of Iodination Experiments. Tumors are implanted and
irradiated as described herein above. The synthetic dodecapeptide
encompassing the sequence HHLGGAKQAGDV (SEQ ID NO: 16) on the
carboxyl-terminal segment of fibrinogen .gamma. chain binds to
GPIIb/IIIa is prepared, and a peptide tail for radioiodination
(SGSGS-YYYYY; SEQ ID NO: 32) is added. The peptide tail is
commercially available from PeptidoGenic Research & Co.
(Livermore, Calif., United States of America). A sample from each
batch is sequenced in accordance with standard techniques for
quality control.
[0308] HHLGGAKQAGDV-SGSGS-YYYYY (SEQ ID NO: 28) is labeled with
.sup.131I using IODO-GEN.RTM. reagent as described above. When
tumors are grown to 0.5 cm in diameter, the tail vein of each mouse
subject is cannulated and .sup.131I-labeled
HHLGGAKQAGDV-SGSGS-YYYYY (SEQ ID NO: 28) is injected. The injection
tubing and syringe is counted after the injection to measure
residual .sup.131I. Immediately after administration of
.sup.131I-peptide, tumors are irradiated using techniques described
herein and by Hallahan et al., 1998. Mice are imaged by gamma
camera imaging at 1 and 24 hours after irradiation.
.sup.131I-labeled HHLGGAKQAGDV-SGSGS-YYYYY (SEQ ID NO: 28) binding
to tumors is quantified by gamma camera imaging and direct well
counts from excised tumors as described above. Tissue sections of
all organs are analyzed. Eight tumor-bearing mice are randomly
assigned into each of the experimental and control groups.
Statistical considerations are addressed as described above.
[0309] Positive control groups. Radioiodinated-fibrinogen is
administered to irradiated tumor bearing mice and compared to
radioiodinated-peptide. These mice are randomly assigned into
groups during the same experiment as radiolabeled peptides.
[0310] Negative control groups. Non-irradiated control tumors are
implanted in the left hind limb of all mice. Secondly, radiolabeled
SGSGSGSSGSGSSGSGS-YYYYY (SEQ ID NO: 33) are administered to tumor
bearing mice following irradiation to verify that peptide binding
to irradiated tumors is not a generalized phenomenon.
[0311] It is noted that the three-dimensional conformation of
fibrinogen might facilitate site-specific binding to irradiated
tumors. Alternatively, .sup.131I labeling might interfere with
peptide binding to GPIIb/IIIa. A longer peptide linker and fewer
Tyr residues are options that are employed in each case.
Example 16
Liposome Delivery Vehicle Comprising Twelve Amino Acid Segment of
the .gamma. Subunit of Fibrinogen
[0312] This Example pertains to the preparation of liposomes that
are conjugated to the dodecapeptide HHLGGAKQAGDV (SEQ ID NO: 16), a
segment of the y subunit of fibrinogen, to achieve site-specific
binding to irradiated tumors.
[0313] In initial experiments,
1,1-Dioctadecyl-3,3,3',3'-tetramethylindocarbo-cyanine perchlorate
(DiI), a lipid fluorescent marker, was added to liposome-fibrinogen
conjugates and injected by tail vein. As a control, liposomes
without fibrinogen conjugation were injected. These produced no
increase in fluorescence in irradiated tumors. Fluorescence within
blood vessels of tumors treated with ionizing radiation was
observed for the liposome-fibrinogen conjugates. These findings
support site-directed drug delivery to the fibrinogen receptor in
irradiated tumors.
[0314] Cationic liposomes can be conjugated to antibodies and
peptides (Kirpotin et al., 1997); however, these liposomes bind to
lipophilic proteins in the serum, which reduces the circulation
time. Therefore, polyethylene glycol (PEG) is used to coat the drug
delivery systems. PEG prolongs circulation time (Nam et al., 1999;
Koning et al., 1999).
[0315] In this Example, HHLGGAKQAGDV (SEQ ID NO: 16) is conjugated
to liposomes and encapsulated by PEG. It is then determined whether
both large MW therapeutic proteins and small MW cytotoxic compounds
can be localized to irradiated tumors by liposomes conjugated to
HHLGGAKQAGDV (SEQ ID NO: 16). The linking peptide SGSGS (SEQ ID NO:
31) is placed at the C-terminus, which is linked to liposomes.
Liposomes are conjugated to the SH on Cys at the C-terminus. The
biodistribution of HHLGGAKQAGDV-SGSGSC (SEQ ID NO: 29)-liposome is
studied and the length of the linking peptide is altered as
necessary. In the event that PEG will not achieve membrane fusion
that is comparable to cationic liposomes, the length of the linking
peptide is also altered as necessary.
[0316] Preparation of HHLGGAKQAGDV (SEQ ID NO: 16)--Long
Circulatory Liposomes. Two methods of conjugating liposomes to
peptides are employed. The first method conjugates the liposome to
the N-terminus, and thus the linking peptide is placed at the
N-terminus. This method arranges the conjugate in the following
configuration: liposome-SGSGS-HHLGGAKQAGDVC (SEQ ID NO: 27). The
second method conjugates the liposome to the C-terminus of the
peptide. This method is facilitated by placing a Cys residue at the
C-terminus. This method arranges the conjugate into the
configuration: HHLGGAKQAGDV-SGSGSC (SEQ ID NO: 29)-liposome. These
two methods provide alternatives in the event that one
configuration is useful for site-specific drug delivery over the
other configuration. These methods are also applicable to larger
polypeptides and proteins, including fibrinogen itself.
[0317] Method 1
[0318] Step (1) Synthesis of Maleimide-PGE-PE
[0319] The lipophilic SH reactive reagent with a long spacing arm
is synthesized from maleimide-PEG 2000-NHS ester (Prochem, High
Point, N.C., United States of America),
dioleoylphosphatidylethanolanime (DOPE, Avanti Polar Lipids, inc.,
Alabaster, Ala., United States of America), and triethylamine in
chloroform (1:1:1.5). Resulting maleimide-PEG 2000-DOPE is purified
by flash column.
[0320] Step (2) Preparation of Thiolated HHLGGAKQAGDV (SEQ ID NO:
16)
[0321] Under stirring, to a solution of HHLGGAKQAGDV (SEQ ID NO:
16; 2 mg/mL) in 0.01 M HEPES 0.15 M NaCl buffer pH 7.9, containing
10 mM EDTA and 0.08% sodium azide, is added in five-fold excess of
freshly prepared Traut's Reagent in the same buffer. Reaction is
performed for 30 minutes at 0.degree. C. Thiolated HHLGGAKQAGDV
(SEQ ID NO: 16) is then purified using a desalting PD-10 column
(Amersham Biosciences).
[0322] Preparation of Maleimide-Containing Long Circulating
Liposomes with fluorescent labels. PGE 2000-PE, cholesterol,
Dipalmitoyl phosphocholine (Avanti Polar Lipids), DiI, and
maleimide-PEG-2000-DOPE is dissolved in chloroform and mixed at a
ratio of 10:43:43:2:2 in a round bottom flask as described in
Leserman, 1980. The organic solvent is removed by evaporation
followed by desiccation under vacuum for 2 hours. Liposomes are
prepared by hydrating the dried lipid film in PBS at a lipid
concentration of 10 mM. The suspension is then sonicated 3.times.5
minutes until clear, forming unilamellar liposomes of 100 nM in
diameter.
[0323] Conjugation of thiolated HHLGGAKQAGDV (SEQ ID NO: 16) to
maleimide containing liposomes. Prepared vesicles and thiolated
protein is mixed in 10 mm HEPES, 0.15 M NaCl, and EDTA pH 6.5. The
final concentrations for proteins and liposomes are 0.25 g/L and
2.5 mM, respectively. The mixture is incubated for 18 hours at room
temperature and vesicles are separated from unconjugated protein by
gel filtration on a SEPHAROSE.RTM. 4B-CL column (Amersham
Biosciences).
[0324] Method 2
[0325] To conjugate the peptide to long-circulating liposomes, a
peptide with a Cys residue on the C-terminal is synthesized
(PeptidoGenic Research & Co., Livermore, Calif., United States
of America). A bifunctional PEG (molecular weight 2000) with a
maleic group on one end and NHS group on the other end is used to
conjugate to the aminal group of dioleyol phosphatidyl ethanolamine
(DOPE). The resulting maleic-PEG-DOPE serves as a
sulfhydryl-reactive lipid anchor with a peptide linker between the
lipid portion and the SH-reactive group. Long-circulating liposomes
are prepared by reverse phase evaporation method using a lipid
mixture composed of
DOPC:Cholesterol:PEG-DOPE:maleic-PEG-DOPE:Cy3-DOPE at a ratio of
45:44:5:2:2 (molar ratio). The peptide is then conjugated to the
liposomes at pH 7.0 under inert gas for 24 hours at room
temperature. After the conjugation, the excess of peptide is
removed though a gel filtration step using SEPHACRYL.TM.-100 column
with PBS as eluent. The percentage of conjugation of the peptide to
the liposomes is estimated by the reduction of free peptide
peak.
[0326] Experimental Design. HHLGGAKQAGDV (SEQ ID NO: 16) is
conjugated to liposomes using SH-reactive group as described above.
Liposomes are labeled with gamma emitters and fluorochromes so that
the pharmacokinetics and biodistribution can be measured.
HHLGGAKQAGDV-SGSGSC (SEQ ID NO: 29)-Liposomes are then coated with
PEG as described above. Tumors are implanted and irradiated as
described above. HHLGGAKQAGDV (SEQ ID NO: 16)-conjugated
encapsulated drugs are then injected by tail vein injection.
[0327] Biodistribution is studied by use of gamma emitters and
gamma camera imaging. Both large molecular weight proteins and
small molecular weight compounds (i.e. active agents) are
radiolabeled. A therapeutic protein, tumor necrosis factor is
labeled with .sup.131I by use of IODO-GEN.RTM. reagent as described
above. .sup.131I-TNF is encapsulated in liposomes-HHLGGAKQAGDV (SEQ
ID NO: 16) conjugates and PEG administered by tail vein as
described above.
[0328] Doxorubicin is used to study the biodistribution of a small
MW compound that interacts with radiation. Doxorubicin is
encapsulated in fluorescent liposomes (Avanti Polar Lipids) and
PEG-HHLGGAKQAGDV (SEQ ID NO: 16) conjugates and administered by
tail vein as described above. Methods of preparing fluorescent
liposomes and conjugation of HHLGGAKQAGDV (SEQ ID NO: 16) to
liposomes are described above, Doxorubicin levels in serum and
tumors in the Pharmacokinetic core lab at Vanderbilt University
using standard techniques. Fluorescence microscopy is used to
measure liposomes in tumors using fluorescence quantification
techniques described in Hallahan, 1997a.
[0329] Positive control groups. .sup.131I-labeled
HHLGGAKQAGDV-SGSGS-YYYYY (SEQ ID NO: 28) is administered to one
group of irradiated tumor bearing mice and compared to
biodistribution of encapsulated radiolabeled liposome. These mice
are randomly assigned into groups during the same experiment as
radiolabeled drugs. Radiolabeled drug binding in each group is
quantified and compared to the .sup.131I-labeled
HHLGGAKQAGDV-SGSGS-YYYYY (SEQ ID NO: 28) positive control
group.
[0330] Negative control groups: Firstly, control tumors are
implanted in the left hind limb of all mice and remain
unirradiated. Secondly, SGSGSSGSGSGS-SGSGS (SEQ ID NO: 34) are
conjugated to PEG and liposomes and administered to tumor bearing
mice following irradiation to verify that encapsulated drug binding
to irradiated tumors is not a generalized phenomenon. Eight
tumor-bearing mice are randomly assigned into each of the
experimental and control groups. Statistical considerations are
described above.
Example 17
Anti-P-selectin scFv Binding to Microvasculature of Irradiated
Cancer
[0331] To determine whether anti-P-selectin scFv antibodies bind to
irradiated microvasculature, the binding of four antibodies (4A,
12F, 5H, and 10A) was studied using immunofluoresence microscopy.
Human head and neck squamous cell carcinoma (HNSCC) cell lines were
implanted into the hind limb of nude mice and grown to 10 mm
diameter as in Example 5 (see also Hallahan et al., 1995b; Hallahan
et al., 1998; Hallahan & Virudachalam, 1999). Tumors were
irradiated and dissected 5 hours later. Dissected tumors were snap
frozen and cryosectioned. Immunofluoresence microscopy of each of
the scFv antibodies to human P-selectin demonstrated that the
antigen in these tumor sections was expressed by host (mouse)
cells, indicating that these epitopes are conserved across species.
Each of the scFv antibodies bound to the microvasculature of
irradiated HNSCC, but not to untreated controls.
Example 18
Direct Application of Library to Irradiated Tumors and Endothelial
Cells
[0332] To study the feasibility of selecting antibodies that bind
irradiated endothelial cells, primary culture human umbilical vein
endothelial cells (HUVEC) were used. Negative selection of phage
was first performed by removing all phage antibodies that bind
within an intact umbilical vein and to unirradiated endothelium
from pooled donors. Unbound phage were then incubated with HUVEC at
5 hours after irradiation with 2 Gy. Antibodies were prioritized by
fluorometric microvolume assay technology with an FMAT.TM. 8100
device (PE Biosystems, Foster City, Calif., United States of
America) using irradiated HUVEC in microwells. Selected were scFv
antibodies that bind with high affinity to irradiated HUVEC but do
not bind to untreated HUVEC. Immunofluorescence microscopy of
antibodies developed to irradiated HUVEC showed that several
antibodies did not bind to untreated control cells but did bind to
irradiated HUVEC. These phage-displayed antibodies were not
displaced by anti-P-selectin antibodies indicating that they likely
bound to distinct radiation-induced epitopes on HUVEC. A
determination of which of these antibodies binds to human cancer
microvasculature is presented in Example 21.
[0333] Phage antibodies that bind to irradiated HUVEC and
fibroblasts using a human Fab antibody 17 library are also
selected. Enriched antibodies are prioritized and studied on biopsy
specimens from irradiated HNSCC patients. Antibodies that bind to
human tumor blood vessels are isolated and the radiation-induced
antigen(s) to which they bind are characterized. See Chang et al.,
1991; Garrard et al., 1991; Hoogenboom et al., 1991; Kang et al.,
1991; U.S. Pat. No. 5,837,500.
Example 19
In Vivo Testing of scFv Antibody Binding
[0334] Several scFv antibodies developed to P-selectin and to
.alpha..sub.2b.beta..sub.3 are prioritized by ELISA, BIACORE.RTM.,
and fluorometric microvolume assay technology (the latter using a
FMAT.RTM. 8100 device from PE Biosystems, Foster City, Calif.,
United States of America). These antibodies are tested to determine
which bind to the greatest percentage of tumor specimens from
irradiated patients, while not binding to biopsies of skin and
mucosa. Biopsy specimens are sectioned on the day of antibody
staining, which is performed as described (Schueneman et al.,
2003). Briefly, sections are first incubated with blocking buffer
and washed. Fluorescence labeled scFv and Fab antibodies are then
incubated with the sections under conditions sufficient to allow
binding of the antibodies to targets. Antibody staining of tumor
blood vessels is compared to that of skin and mucosa biopsies from
the same patients. Biopsies from patients are stained for each of
the prioritized antibodies by use of fluorescence microscopy and
image analysis software as has been described (Geng et al., 2001;
Hallahan et al., 2002). Vascular density is also analyzed
simultaneously.
[0335] HNSCC xenografts are implanted subcutaneously in the hind
limb as described in Hallahan et al., 2003. Antibodies and
immunoconjugates with optimal binding are radiolabeled and injected
by tail vein after irradiation of xenografts. The tumor bearing
hind limb is irradiated with 0 Gy (Control), or daily fractionated
radiation (2 Gy.times.7) as described in Schueneman et al., 2003
and Hallahan et al., 2003.
Example 20
Mass Spectrometry Analysis of scFv Antibodies
[0336] To develop a high-throughput screening technique for phage
library antibodies targeted to radiation-induced neoantigens (for
example, P-selectin or .alpha..sub.2b.beta..sub.3 integrin) and
measure tumor specificity of scFv antibodies developed from phage
antibody libraries, a large phage-displayed scFv recombinant
antibody library was developed. The phage library was incubated
with purified P-selectin protein, and high-affinity phage antibody
clones were recovered by washing at pH 1. The antibody clones were
assayed for antigen-binding activity by ELISA. The clones producing
antibodies reactive with P-selectin were grown and induced to
express P-selectin-specific scFv antibodies on a large scale.
[0337] The phage antibody library was also screened for scFv that
bound to expired human platelets obtained from blood banks. Phage
that were nonspecifically bound to inactivated platelets were first
subtracted from the library. Platelets were activated to induce
.alpha..sub.2b.beta..sub.3 integrin in the active conformation.
Bound phage were displaced by the addition of a monoclonal antibody
specific for .alpha..sub.2b. The displaced phage were recovered and
used to produce .alpha..sub.2b antibodies.
[0338] P-selectin and .alpha..sub.2b scFv antibodies were
individually spotted in matrix and evaluated by mass spectrometry
for size to determine sets of 6 that can be effectively
discriminated by mass spectrometry based upon differences in their
molecular weights (approximately 400 mass unit size difference).
Antibodies to P-selectin and to .alpha..sub.2b were administered in
sets of 6 by tail vein injection into mice bearing irradiated
tumors. The tumors were dissected and antibody binding was measured
by MALDI-TOF mass spectrometry.
[0339] Soluble rodent scFv antibodies to P-selectin and to
.alpha..sub.2b were developed, several of which were definitively
measured in matrix by MALDI-TOF mass spectrometry. Of these, 9
soluble rodent scFv antibodies to P-selectin and 9 soluble rodent
scFv antibodies to .alpha..sub.2b were differentially detected in
sets of 3 in mice tumors via MALDI-TOF mass spectrometry. Spectrum
analysis allowed quantification of the amount of the individual
antibodies binding within the tumors.
Example 21
Binding of scFv to Human Cancer Microvasculature
[0340] Using the methods and procedures described hereinabove, scFv
antibodies that are found to bind to HUVEC cells are tested for
binding to human cancer microvasculature either in vivo or in vitro
on biopsy samples.
[0341] Negative selection of the entire phage library
(2.times.10.sup.9) is first performed on untreated vascular
endothelium and platelets. Phage-displayed antibodies that bind to
normal endothelium and platelets are discarded, while phage that do
not bind are used for high throughput screening as follows.
[0342] HUVEC cells are grown to confluence in complete medium and
human serum in 1536-well plates. Cells are irradiated with 3 Gy.
Those scFv phage antibodies that bind to the isolated, irradiated
endothelium are selected by use of an automated colony picker,
followed by high throughput screening using an FMAT.RTM. device (PE
Biosystems, Inc., Foster City, Calif., United States of America),
which is used to quantify fluorescence-labeled phage localized and
concentrated on the irradiated endothelial cell surface.
Example 22
Laser Capture Microdissection
[0343] Microvasculature is identified during laser capture
microdissection (LCM). The use of an LCM system allows selected
single cells or groups of cells to be analyzed. LCM is used to
dissect the vascular endothelium and luminal proteins from a frozen
section of an irradiated tumor. The phage antibody library is added
to these blood vessels and scFv phage antibodies that are recovered
from the irradiated tumor vasculature are selected using an
automated colony picker. Phage undergo several rounds of selection
to reduce nonspecific binding. Identified antibodies are further
selected using FMAT.
Example 23
Antibodies to TIP-1
[0344] A two hybrid system identified the Tax-interacting protein-1
(TIP-1) as a putative receptor for the recombinant HGDPNHVGGSSV
(SEQ ID NO: 71) peptide. Antibodies (monoclonal and antiserum) have
been developed to TIP-1. FIG. 2 presents data with respect to the
binding of an exemplary scFV antibody binding to TIP-1. This
antibody also blocked HGDPNHVGGSSV (SEQ ID NO: 71) peptide binding
to irradiated tumors.
Example 24
TIP-1 Expression in Control and Irradiated Tumors
[0345] TIP-1 expression was studied in irradiated tumors. FIGS. 3A
and 3B shows immunohistochemical staining of TIP-1 in control and
irradiated tumors (3 Gy), respectively. This study showed that
TIP-1 expression increased in the vascular endothelium of
irradiated tumors. FIG. 3A shows little or no TIP-1 was expressed
in untreated tumors. FIG. 3B shows increased immunohistochemical
staining of TIP-1 in tumors at 6 hours after treatment with 3 Gy.
Tumor microvasculature showed little or no TIP-1 expression in
unirradiated tumor vascular endothelium, as compared to robust
staining of TIP-1 protein in the endothelium at 4 hours after
irradiation of tumors.
[0346] Western immunoblots were used to quantify TIP-1 expression
in tumors. Studies were performed using polyclonal antibody. FIG. 4
shows the Western immunoblot of protein extracted from control and
irradiated tumors. In this experiment, cell membrane proteins were
separated from cytoplasmic proteins to determine whether TIP-1 is
expressed at the cell surface. Cell surface expression of TIP-1
could be required for phage peptides to bind within tumors
following injection of the phage into the circulation. FIG. _E
shows increased TIP-1 expression in cell membrane preparation in
response to radiation. These data support the hypothesis that TIP-1
is a putative receptor for HGDPNHVGGSSV (SEQ ID NO: 71) ligand
binding to irradiated tumors.
[0347] To further test this hypothesis, experiments studying TIP-1
blocking antibodies are done. Affinity purification experiments to
test the hypothesis that multiple PDZ containing receptors bind
HGDPNHVGGSSV (SEQ ID NO: 71) in irradiated tumors are also
done.
Example 25
Biodistribution and Pharmacokinetics of Recovered Phage
[0348] To determine the feasibility of imaging the biodistribution
of phage displayed peptides, near infrared (NIR) imaging of Cy7
labeled peptides that were recovered from irradiated tumors was
employed. Tumor specific binding and pharmacokinetics of each of
these phage peptides was tested. Some of the phage that were
recovered from irradiated tumors were compared in order to
determine which peptide has the greatest tumor specific binding.
Phage were first labeled with Cy7 to allow imaging by use of NIR
imaging (Xenogen) within the Vanderbilt Small Animal Imaging Core
Lab. Tumors were implanted into both hind limbs of nude mice and
the right hind limb tumor was irradiated with 3 Gy. The left hind
limb tumor served as an internal negative control. Cy7-labeled
phage were injected into the venous circulation by use of the
jugular catheter. The biodistribution of Cy7 labeled phage was then
imaged by NIR imaging each day for a total of nine days.
[0349] FIGS. 5 and 6 show the NIR imaging of Cy7 labeled phage.
Phage showed tumor binding at 24 hours after irradiation. FIG. 7 is
a bar graph indicating that percentage of total phage localized to
the tumor following clearance from the circulation. Although
several phage peptides show selective binding within the tumor, the
HGDPNHVGGSSV (SEQ ID NO: 71) phage shows very highly selective
binding to irradiated tumors.
[0350] The pharmacokinetics of the HGDPNHVGGSSV (SEQ ID NO: 71)
phage was also compared to that of the RGDGSSV (SEQ ID NO: 75),
HGSSV (SEQ ID NO: 76) and other phage. The HGDPNHVGGSSV (SEQ ID NO:
71) phage was unique in that it demonstrated tumor specific binding
for nine days. FIG. 8 shows the daily NIR imaging of Cy7 labeled
HGDPNHVGGSSV (SEQ ID NO: 71). On day one, the phage peptide
circulates throughout the entire body. By day three, the labeled
phage was cleared from the circulation through the kidney and
collected in the bladder. Tumor binding was the only site other
than the kidney on days 3 through 7. Thereafter, tumor specific
binding was sustained.
[0351] FIG. 9 compares the kinetics of tumor binding by
HGDPNHVGGSSV (SEQ ID NO: 71) phage to that of the RGDGGSSV (SEQ ID
NO: 75) phage. The intensity of NIR images was measured in each
pixel. NIR intensity in tumor and the rest of the body was
tabulated as the percentage of total NIR emission. Immediately
after injection, Cy7 labeled phage was distributed throughout the
entire body (day 0). By day one, binding in tumor was detected and
clearance from the circulation occurred through the kidney. The
percentage of HGDPNHVGGSSV (SEQ ID NO: 71) binding in tumor
continued to increase, while the distribution of this phage
throughout the rest of the body cleared. By day seven, 90% of the
phage was bound within the tumor and tumor specific binding was
sustained beyond nine days. In comparison, the RGOGGSSV (SEQ ID NO:
75) phage achieved 55% binding at day two, but cleared from both
tumor and body by day four. HGDPNHVGGSSV (SEQ ID NO: 71),
therefore, has greater tumor specific binding and prolonged
pharmacokinetics as compared to the next best phage, RGDGGSSV (SEQ
ID NO: 76).
Example 26
Histologic Site of Phage Binding
[0352] To determine the histologic pattern of phage binding, tumors
were fixed at 4 hours and 7 days after irradiation and phage
injection. Sections were stained using the anti-phage antibody and
counter-stained with H & E. FIG. 10 shows staining of the
anti-phage antibody (brown). The HGDPNHVGGSSV (SEQ ID NO: 71) phage
was localized at the vascular endothelium as compared to the
RGDGGSSV (SEQ ID NO: 76) phage, which binds within the lumen of
blood vessels (FIG. 10B). Negative control phage (the phage vector
without insert) showed no binding within irradiated tumors. These
findings indicated that HGDPNHVGGSSV (SEQ ID NO: 71) can be binding
to a radiation-inducible molecule in tumor endothelium. Moreover,
the HGDPNHVGGSSV (SEQ ID NO: 71) phage subsequently traversed the
vascular endothelium and bound to cancer cells (FIG. 10D).
Example 27
Fluorescence-Labeled HGDPNHVGGSSV (SEQ ID NO: 71)
[0353] To determine the feasibility of using this peptide to
recover the putative receptor, HGDPNHVGGSSV (SEQ ID NO: 71) binding
was studied in tumor vasculature and endothelial cells in culture.
HGDPNHVGGSSV (SEQ ID NO: 71) peptide was injected into the
circulation of mice bearing irradiated tumors (3 Gy). HGDPNHVGGSSV
(SEQ ID NO: 71) was first labeled with Texas red. Tumors were
sectioned and counter stained with eosin. FIG. 11 shows that the
Texas red conjugated HGDPNHVGGSSV (SEQ ID NO: 71) peptide
maintained the ability to bind to tumor microvasculature following
irradiation. Fluorescence-labeled HGDPNHVGGSSV (SEQ ID NO: 71)
bound to tumor microvasculature treated with 3 Gy (FIG. 11A), but
not to untreated vessels. NIR imaging of Cy7-labeled HGDPNHVGGSSV
(SEQ ID NO: 71) is shown in the mouse image (FIG. 11A).
Example 28
Tumor Targeting with a TIP-1 Binding Peptide
[0354] To determine whether peptide conjugated to nanoparticle can
be used to target therapy to cancer, the HGDPNHVGGSSV (SEQ 16 NO:
71) peptide was attached to a polyglycine linker and attached to a
protein core by use of biotin. Streptavidin was then labeled with
Cy7 to allow imaging of treated animals. FIG. 12 shows that the
labeled Streptavidin-peptide complex bound within treated tumors
but not untreated control tumors on the left. This system allows
determination of whether peptide conjugates achieve the same level
of binding as the phage displayed peptide shown in FIG. 6.
[0355] Orthopotic tumors have been studied in order to verify that
peptide binding in treated tumors is not limited to subcutaneous
implantation. FIG. 13 shows peptide binding in the glioblastoma
within the brains of treated mouse and in the H460 lung cancer in
the thorax of the treated mouse. FIG. 13C shows peptide binding
within the treated colon carcinoma. FIG. 14 shows peptide binding
in 2 different orthotopic prostate tumors. These studies showed
that all tumor types and all orthotopic tumor models demonstrated
binding of the HGDPNHVGGSSV peptide following treatment with
radiation.
Example 29
HGDPNHVGGSSV (SEQ ID NO: 71) does not Bind to Irradiated Normal
Tissue
[0356] To determine whether HGDPNHVGGSSV (SEQ ID NO: 71) peptide
binds to normal tissues, mice treated with 3 Gy to normal tissue,
followed by tail vein injection of Cy7-labeled
Streptavidin-HGDPNHVGGSSV (SEQ ID NO: 71), were imaged. FIG. 14C
shows peptide binding to the abdominal colon cancer and no binding
in the irradiated liver (red arrow) and no gastrointestinal
binding. FIG. 15 shows binding in the prostate tumor but no binding
within the rest of the abdomen following irradiation of abdominal
organs.
Example 30
Orthotopic Prostate Cancer Models
[0357] Two orthotopic tumor models within the prostates of mice are
studied. The PC3 injection into the prostate of nude mice develops
over 2 weeks (see FIGS. 14A and 14B). A colony of the
prostate-specific conditional PTEN transgenic mice, which develop
tumors within 20-24 weeks (see FIG. 14C), is also available. Tumors
were monitored by ultrasound and PSA levels as described in
Yankeelov et al., 2006. FIG. 14 shows NIR imaging of
labeled-HGDPNHVGGSSV (SEQ ID NO: 71) binding in irradiated (3 Gy)
orthotopic prostate cancers following injection through jugular
catheters. FIG. 14B shows the negative control peptide with no
binding in irradiated PC3 orthotopic tumor (FIG. 14). FIG. 15 shows
the PC3 tumor within the prostate during laparotomy of the
euthanized mouse imaged in FIG. 14A. The arrow indicates the
lobolated tumor in the pelvis. NIR was also used to image each of
the organs (FIG. 15B). Labeled peptide in the prostate tumor was
>20-fold higher than any organ. FIG. 16 shows the transgenic
prostate tumor in the pelvis of the euthanized mouse imaged in FIG.
14C. NIR was also used to image each of the organs (FIG. 16B).
Labeled peptide in the prostate tumor was >20-fold higher than
any organ. These data show that HGDPNHVGGSSV (SEQ ID NO: 71) bound
to the orthotopic prostate tumors.
Example 31
HVGGSSV (SEQ ID NO: 35) Conjugates Bind Endothelial Cells
[0358] To determine the fate of the HGDPNHVGGSSV (SEQ ID NO: 71)
peptide after binding to vascular endothelium, fluorescently
labeled Streptavidin conjugated to HGDPNHVGGSSV (SEQ ID NO: 71) was
employed peptide. Endothelial cells were irradiated and the
fluorescent confocal microscopy was used to image HGDPNHVGGSSV (SEQ
ID NO: 71)-Streptavidin in endothelial cells (see FIG. 16). The
fluorescent-labeled HGDPNHVGGSSV peptide did not bind to untreated
endothelial cells (FIG. 16A). Peptide binds to the surface of
endothelial cells within four hours of irradiation (panel 16B). In
comparison, peptide was then allowed to incubate on endothelial
cells overnight. Panel 16C shows the internalized HGDPNHVGGSSV
peptide adjacent to the nucleus of irradiated vascular endothelium.
These results support that the HGDPNHVGGSSV peptide will facilitate
internalization of gene therapy vectors.
REFERENCES
[0359] All references listed in the instant disclosure, including
but not limited to all patents, patent applications, scientific
journals, and GENBANK.RTM. database entries (including all
annotations available therein) are incorporated herein by reference
in their entireties to the extent that they supplement, explain,
provide a background for or teach methodology, techniques and/or
compositions employed herein. [0360] Aboud-Pirak et al. (1989)
Biochem Pharmacol 38:641-648. [0361] Albini et al., (2000) Am J
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[0527] It will be understood that various details of the presently
disclosed subject matter can be changed without departing from the
scope of the present disclosure. Furthermore, the foregoing
description is for the purpose of illustration only, and not for
the purpose of limitation.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 86 <210> SEQ ID NO 1 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: synthesized <400>
SEQUENCE: 1 Asn His Val Gly Gly Ser Ser Val 1 5 <210> SEQ ID
NO 2 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: synthesized <400> SEQUENCE: 2 Asn Ser Leu
Arg Gly Asp Gly Ser Ser Val 1 5 10 <210> SEQ ID NO 3
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 3 Asn Ser Val Arg
Gly Ser Gly Ser Gly Val 1 5 10 <210> SEQ ID NO 4 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 4 Asn Ser Val Gly Ser Arg Val 1 5
<210> SEQ ID NO 5 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 5
Ser Leu Arg Gly Asp Gly Ser Ser Val 1 5 <210> SEQ ID NO 6
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 6 Arg Gly Asp Gly
Ser Ser Val 1 5 <210> SEQ ID NO 7 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 7 Gly Ser Arg Val 1 <210> SEQ ID NO 8
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 8 Gly Ser Xaa Val 1 <210> SEQ ID NO 9
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (6)..(6) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <400> SEQUENCE: 9 Asn Ser Xaa
Arg Gly Xaa Gly Ser 1 5 <210> SEQ ID NO 10 <211>
LENGTH: 3 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 10 Asn Ser Val 1 <210> SEQ
ID NO 11 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYNTHESIZED <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 11 Asn Ser Xaa Arg 1 <210> SEQ ID NO 12
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (2)..(2) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 12 Asn Xaa Val Gly 1 <210> SEQ ID NO 13
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 13 Gly Ser Ser Val 1
<210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Bacteriophage T7 <400> SEQUENCE: 14
agcggaccag attatcgcta 20 <210> SEQ ID NO 15 <211>
LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:
Bacteriophage T7 <400> SEQUENCE: 15 aaccctcaag acccgttta 19
<210> SEQ ID NO 16 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 16
His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val 1 5 10 <210>
SEQ ID NO 17 <211> LENGTH: 726 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (1)..(726)
<400> SEQUENCE: 17 atg gcc cag gtg aaa ctg cag cag tct ggg
gct gag ctt gtg atg cct 48 Met Ala Gln Val Lys Leu Gln Gln Ser Gly
Ala Glu Leu Val Met Pro 1 5 10 15 ggg gct tca gtg aag atg tcc tgc
aag gct tct ggc tac aca ttc act 96 Gly Ala Ser Val Lys Met Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 gac tac tgg atg cac tgg
gtg aag cag agg cct gga caa ggc ctt gag 144 Asp Tyr Trp Met His Trp
Val Lys Gln Arg Pro Gly Gln Gly Leu Glu 35 40 45 tgg atc gga gcg
att gat act tct gat agt tat act agc tac aat caa 192 Trp Ile Gly Ala
Ile Asp Thr Ser Asp Ser Tyr Thr Ser Tyr Asn Gln 50 55 60 aag ttc
aag ggc aag gcc aca ttg act gta gac gaa tcc tcc agc aca 240 Lys Phe
Lys Gly Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser Thr 65 70 75 80
gcc tac atg cag ctc agc agc ctg aca tct gag gac tct gcg gtc tat 288
Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 85
90 95 tac tgt gca aga aga ggc tac tat agc gca ttt gat tac tgg ggc
caa 336 Tyr Cys Ala Arg Arg Gly Tyr Tyr Ser Ala Phe Asp Tyr Trp Gly
Gln 100 105 110 ggg act acg gtc acc gtc tcc tca ggt gga ggc ggt tca
ggc gga ggt 384 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly 115 120 125 ggc tct ggc ggt ggc gga tcg gac att gag ctc
acc cag tct cca aca 432 Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu
Thr Gln Ser Pro Thr 130 135 140 acc atg gct gca tct cca gga gag aag
gtc acc atc acc tgc cgt gcc 480 Thr Met Ala Ala Ser Pro Gly Glu Lys
Val Thr Ile Thr Cys Arg Ala 145 150 155 160 agc tca agt gta agc tac
atg cac tgg ttc cag cag aag tca ggc acc 528 Ser Ser Ser Val Ser Tyr
Met His Trp Phe Gln Gln Lys Ser Gly Thr 165 170 175 tcc ccc aaa ccc
tgg att tat gac aca tcc aag ctg gct tct gga gtc 576 Ser Pro Lys Pro
Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val 180 185 190 cca gat
cgc ttc agt ggc agt ggg tct ggg acc tct tat tct ctc aca 624 Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr 195 200 205
atc agc tcc atg gag gct gaa gat gct gct act tat tac tgt ctg cag 672
Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln 210
215 220 agg agt agt tac ccg tac acg ttt gga gct ggc acc aag ctg gaa
atc 720 Arg Ser Ser Tyr Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu
Ile 225 230 235 240 aaa cgg 726 Lys Arg <210> SEQ ID NO 18
<211> LENGTH: 242 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 18 Met Ala
Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Met Pro 1 5 10 15
Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20
25 30 Asp Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Glu 35 40 45 Trp Ile Gly Ala Ile Asp Thr Ser Asp Ser Tyr Thr Ser
Tyr Asn Gln 50 55 60 Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp
Glu Ser Ser Ser Thr 65 70 75 80 Ala Tyr Met Gln Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Arg Gly Tyr
Tyr Ser Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly
Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Thr 130 135 140 Thr
Met Ala Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Arg Ala 145 150
155 160 Ser Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Ser Gly
Thr 165 170 175 Ser Pro Lys Pro Trp Ile Tyr Asp Thr Ser Lys Leu Ala
Ser Gly Val 180 185 190 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Ser Tyr Ser Leu Thr 195 200 205 Ile Ser Ser Met Glu Ala Glu Asp Ala
Ala Thr Tyr Tyr Cys Leu Gln 210 215 220 Arg Ser Ser Tyr Pro Tyr Thr
Phe Gly Ala Gly Thr Lys Leu Glu Ile 225 230 235 240 Lys Arg
<210> SEQ ID NO 19 <211> LENGTH: 726 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (1)..(726)
<400> SEQUENCE: 19 atg gcc cag gtc aag ctg cag cag tca gga
cct gag ctg gta aag cct 48 Met Ala Gln Val Lys Leu Gln Gln Ser Gly
Pro Glu Leu Val Lys Pro 1 5 10 15 ggg gct tca gtg aag atg tcc tgc
aag gct tct gga tac aca ttc act 96 Gly Ala Ser Val Lys Met Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 agc tat gtt atg cac tgg
gtg aag cag aag cct ggg cag ggc ctt gag 144 Ser Tyr Val Met His Trp
Val Lys Gln Lys Pro Gly Gln Gly Leu Glu 35 40 45 tgg att gga tat
att aat cct tac aat gat ggt act aag tac aat gag 192 Trp Ile Gly Tyr
Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu 50 55 60 aag ttc
aaa ggc aag gcc gca ctg act tca gac aaa tcc tcc agc aca 240 Lys Phe
Lys Gly Lys Ala Ala Leu Thr Ser Asp Lys Ser Ser Ser Thr 65 70 75 80
gcc tac atg gag ctc agc agc ctg acc tct gag gac tct gcg gtc tat 288
Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 85
90 95 tac tgt gca aga ttt ggt aac tac ggt gct ttg gac tac tgg ggc
caa 336 Tyr Cys Ala Arg Phe Gly Asn Tyr Gly Ala Leu Asp Tyr Trp Gly
Gln 100 105 110 ggg acc acg gtc acc gtc tcc tca ggt gga ggc ggt tca
ggc gga ggt 384 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly 115 120 125 ggc tct ggc ggt ggc gga tcg gac att gag ctc
acc cag tct cca aca 432 Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu
Thr Gln Ser Pro Thr 130 135 140 atc atg tct gca tct cca ggg gag aag
gtc acc ata acc tgc agt gcc 480 Ile Met Ser Ala Ser Pro Gly Glu Lys
Val Thr Ile Thr Cys Ser Ala 145 150 155 160 agc tca agt gta agt tac
atg cac tgg ttc cag cag aag cca ggc act 528 Ser Ser Ser Val Ser Tyr
Met His Trp Phe Gln Gln Lys Pro Gly Thr 165 170 175 tct ccc aaa ccc
tgg att tat ggc aca tcc aac ctg gct tct gga gtc 576 Ser Pro Lys Pro
Trp Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val 180 185 190 cct gtt
cgc ttc agt ggc agt gga tct ggg acc tct tat tct ctc aca 624 Pro Val
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr 195 200 205
atc agc agc atg gag gct gaa gat gct gcc act tat tac tgt caa cag 672
Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 210
215 220 tgg agt agt tac cca ctc acg ttc gga ggg ggg acc aag ctg gaa
ata 720 Trp Ser Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile 225 230 235 240 aaa cgg 726 Lys Arg <210> SEQ ID NO 20
<211> LENGTH: 242 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Construct <400> SEQUENCE: 20 Met Ala
Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 1 5 10 15
Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20
25 30 Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu
Glu 35 40 45 Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys
Tyr Asn Glu 50 55 60 Lys Phe Lys Gly Lys Ala Ala Leu Thr Ser Asp
Lys Ser Ser Ser Thr 65 70 75 80 Ala Tyr Met Glu Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Phe Gly Asn
Tyr Gly Ala Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly
Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Thr 130 135 140 Ile
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala 145 150
155 160 Ser Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly
Thr 165 170 175 Ser Pro Lys Pro Trp Ile Tyr Gly Thr Ser Asn Leu Ala
Ser Gly Val 180 185 190 Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr
Ser Tyr Ser Leu Thr 195 200 205 Ile Ser Ser Met Glu Ala Glu Asp Ala
Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Trp Ser Ser Tyr Pro Leu Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile 225 230 235 240 Lys Arg
<210> SEQ ID NO 21 <211> LENGTH: 795 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (1)..(795)
<400> SEQUENCE: 21 atg gcc cag gtg cag ctg cag gag tca gga
cct ggc ctt gtg aaa ccc 48 Met Ala Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro 1 5 10 15 tca cag tca ctc tcc ctc acc tgt
tct gtc act ggt tac tcc atc act 96 Ser Gln Ser Leu Ser Leu Thr Cys
Ser Val Thr Gly Tyr Ser Ile Thr 20 25 30 agt aat tac tgg ggc tgg
atc cgg aag ttc cca ggg aat aaa atg gag 144 Ser Asn Tyr Trp Gly Trp
Ile Arg Lys Phe Pro Gly Asn Lys Met Glu 35 40 45 tgg atg gga tac
ata agc tac agt ggt agc act agc tac aac cca tct 192 Trp Met Gly Tyr
Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser 50 55 60 ctc aaa
agt cga atc tcc att act aga gac aca tcg aag aat cag ctc 240 Leu Lys
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Leu 65 70 75 80
ttc ctg cag ttg aac tct gta act act gag gac aca gcc aca tat tac 288
Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr 85
90 95 tgt gca aga tat agc ctc ttt aac tac ggt agg agg gac tat gtt
atg 336 Cys Ala Arg Tyr Ser Leu Phe Asn Tyr Gly Arg Arg Asp Tyr Val
Met 100 105 110 gat gcc tgg ggc caa ggg acc acg gtc acc gtc tcc tca
ggt gga ggc 384 Asp Ala Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
Gly Gly Gly 115 120 125 ggt tca ggc gga ggt ggc tct ggc ggt ggc gga
tcg gac att gag ctc 432 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Asp Ile Glu Leu 130 135 140 acc cag tct cca gca acc atg gct gca
tct cca gga gag aaa gtc acc 480 Thr Gln Ser Pro Ala Thr Met Ala Ala
Ser Pro Gly Glu Lys Val Thr 145 150 155 160 atc acc tgc cgt gcc agc
tca act gta agc tac atg cac tgg ttc caa 528 Ile Thr Cys Arg Ala Ser
Ser Thr Val Ser Tyr Met His Trp Phe Gln 165 170 175 cag aag cca ggc
gcc tcc cct aaa ccc tgg att tat gac aca tcc aaa 576 Gln Lys Pro Gly
Ala Ser Pro Lys Pro Trp Ile Tyr Asp Thr Ser Lys 180 185 190 ctg gct
tct gga gtc cca gat cgc ttc agt ggc agt ggg tct ggg aca 624 Leu Ala
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205
gac ttc acc ctc acc att gat cct gtg cag gct gat gat att gca acc 672
Asp Phe Thr Leu Thr Ile Asp Pro Val Gln Ala Asp Asp Ile Ala Thr 210
215 220 tat tac tgt cag cag agt aag gat gat cct cgg acg ttc ggt gga
ggg 720 Tyr Tyr Cys Gln Gln Ser Lys Asp Asp Pro Arg Thr Phe Gly Gly
Gly 225 230 235 240 acc aag ctg gag ctg aaa cgg cgg ccg cag gtg cgc
cgg tgc cgt atc 768 Thr Lys Leu Glu Leu Lys Arg Arg Pro Gln Val Arg
Arg Cys Arg Ile 245 250 255 cgg atc cgc tgg aac cgc gtg ccg cat 795
Arg Ile Arg Trp Asn Arg Val Pro His 260 265 <210> SEQ ID NO
22 <211> LENGTH: 265 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 22 Met
Ala Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 1 5 10
15 Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr
20 25 30 Ser Asn Tyr Trp Gly Trp Ile Arg Lys Phe Pro Gly Asn Lys
Met Glu 35 40 45 Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser
Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp
Thr Ser Lys Asn Gln Leu 65 70 75 80 Phe Leu Gln Leu Asn Ser Val Thr
Thr Glu Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Tyr Ser Leu
Phe Asn Tyr Gly Arg Arg Asp Tyr Val Met 100 105 110 Asp Ala Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu 130 135 140
Thr Gln Ser Pro Ala Thr Met Ala Ala Ser Pro Gly Glu Lys Val Thr 145
150 155 160 Ile Thr Cys Arg Ala Ser Ser Thr Val Ser Tyr Met His Trp
Phe Gln 165 170 175 Gln Lys Pro Gly Ala Ser Pro Lys Pro Trp Ile Tyr
Asp Thr Ser Lys 180 185 190 Leu Ala Ser Gly Val Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Asp Pro
Val Gln Ala Asp Asp Ile Ala Thr 210 215 220 Tyr Tyr Cys Gln Gln Ser
Lys Asp Asp Pro Arg Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu
Glu Leu Lys Arg Arg Pro Gln Val Arg Arg Cys Arg Ile 245 250 255 Arg
Ile Arg Trp Asn Arg Val Pro His 260 265 <210> SEQ ID NO 23
<211> LENGTH: 786 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (1)..(786) <400> SEQUENCE: 23 atg
gcc cag gtg aag ctg cag cag tct gga cct gag ctg gta aag cct 48 Met
Ala Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 1 5 10
15 ggg gct tca gtg aag atg tcc tgc aag gct tct gga tac aca ttc act
96 Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30 agc tat gtt atg cac tgg gtg aag cag agc aat gga aag agc
ctt gag 144 Ser Tyr Val Met His Trp Val Lys Gln Ser Asn Gly Lys Ser
Leu Glu 35 40 45 tgg att gga act att gat cct tac tat ggt ggt act
agc tac aac cag 192 Trp Ile Gly Thr Ile Asp Pro Tyr Tyr Gly Gly Thr
Ser Tyr Asn Gln 50 55 60 aag ttc aag ggc aag gcc aca ttg act gta
gac aaa tcc tcc acc acg 240 Lys Phe Lys Gly Lys Ala Thr Leu Thr Val
Asp Lys Ser Ser Thr Thr 65 70 75 80 gcc tac ata cag ctc aag agc ctg
aca tct gag gac tct gca gtc tat 288 Ala Tyr Ile Gln Leu Lys Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr 85 90 95 tac tgt gca aga tgg gat
ggt tac tac gga ggg ttt tct tac tgg ggc 336 Tyr Cys Ala Arg Trp Asp
Gly Tyr Tyr Gly Gly Phe Ser Tyr Trp Gly 100 105 110 caa ggg acc atg
gtc acc gtc tcc tca ggt gga ggc ggt tca ggc gga 384 Gln Gly Thr Met
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 ggt ggc
tct ggc ggt ggc gga tcg gac att gag ctc acc cag tct cca 432 Gly Gly
Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro 130 135 140
gca atc atg tct gca act cta ggg gag aag gtc acc atg agc tgc agg 480
Ala Ile Met Ser Ala Thr Leu Gly Glu Lys Val Thr Met Ser Cys Arg 145
150 155 160 gcc agc tca aat gta aag tac atg tac tgg tac cag cag aag
tca ggt 528 Ala Ser Ser Asn Val Lys Tyr Met Tyr Trp Tyr Gln Gln Lys
Ser Gly 165 170 175 gcc tcc ccc aaa cta tgg att tat tac aca tcc aac
ctg gct tct gga 576 Ala Ser Pro Lys Leu Trp Ile Tyr Tyr Thr Ser Asn
Leu Ala Ser Gly 180 185 190 gtc cca gct cgc ttc agt ggc agt ggg tct
ggg acc tct tat tct ctc 624 Val Pro Ala Arg Phe Ser Gly Ser Gly Ser
Gly Thr Ser Tyr Ser Leu 195 200 205 aca atc agc agc gtg gag gct gaa
gat gct gcc act tat tac tgc cag 672 Thr Ile Ser Ser Val Glu Ala Glu
Asp Ala Ala Thr Tyr Tyr Cys Gln 210 215 220 cag ttt act agt tcc ccg
tat acg ttc gga tcg ggc acc aag ctg gaa 720 Gln Phe Thr Ser Ser Pro
Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu 225 230 235 240 atc aaa cgg
gcg gcc gca ggt gcg ccg gtg ccg tat ccg gat ccg ctg 768 Ile Lys Arg
Ala Ala Ala Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu 245 250 255 gaa
ccg cgt gcc gca tag 786 Glu Pro Arg Ala Ala 260 <210> SEQ ID
NO 24 <211> LENGTH: 261 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 24 Met
Ala Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 1 5 10
15 Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30 Ser Tyr Val Met His Trp Val Lys Gln Ser Asn Gly Lys Ser
Leu Glu 35 40 45 Trp Ile Gly Thr Ile Asp Pro Tyr Tyr Gly Gly Thr
Ser Tyr Asn Gln 50 55 60 Lys Phe Lys Gly Lys Ala Thr Leu Thr Val
Asp Lys Ser Ser Thr Thr 65 70 75 80 Ala Tyr Ile Gln Leu Lys Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Trp Asp
Gly Tyr Tyr Gly Gly Phe Ser Tyr Trp Gly 100 105 110 Gln Gly Thr Met
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly
Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro 130 135 140
Ala Ile Met Ser Ala Thr Leu Gly Glu Lys Val Thr Met Ser Cys Arg 145
150 155 160 Ala Ser Ser Asn Val Lys Tyr Met Tyr Trp Tyr Gln Gln Lys
Ser Gly 165 170 175 Ala Ser Pro Lys Leu Trp Ile Tyr Tyr Thr Ser Asn
Leu Ala Ser Gly 180 185 190 Val Pro Ala Arg Phe Ser Gly Ser Gly Ser
Gly Thr Ser Tyr Ser Leu 195 200 205 Thr Ile Ser Ser Val Glu Ala Glu
Asp Ala Ala Thr Tyr Tyr Cys Gln 210 215 220 Gln Phe Thr Ser Ser Pro
Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys Arg
Ala Ala Ala Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu 245 250 255 Glu
Pro Arg Ala Ala 260 <210> SEQ ID NO 25 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 25 Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu
Glu Pro Arg 1 5 10 <210> SEQ ID NO 26 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 26 His His Leu Gly Gly Ala Lys Gln Ala Gly
Asp Val Ser Gly Ser Gly 1 5 10 15 Ser <210> SEQ ID NO 27
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 27 Ser Gly Ser Gly
Ser His His Leu Gly Gly Ala Lys Gln Ala Gly Asp 1 5 10 15 Val Cys
<210> SEQ ID NO 28 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 28
His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val Ser Gly Ser Gly 1 5
10 15 Ser Tyr Tyr Tyr Tyr Tyr 20 <210> SEQ ID NO 29
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 29 His His Leu Gly
Gly Ala Lys Gln Ala Gly Asp Val Ser Gly Ser Gly 1 5 10 15 Ser Cys
<210> SEQ ID NO 30 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 30
His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val Ser Gly Ser Gly 1 5
10 15 Ser Lys <210> SEQ ID NO 31 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 31 Ser Gly Ser Gly Ser 1 5 <210> SEQ ID
NO 32 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 32 Ser Ser Gly
Ser Gly Tyr Tyr Tyr Tyr Tyr 1 5 10 <210> SEQ ID NO 33
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 33 Ser Gly Ser Gly
Ser Gly Ser Ser Gly Ser Gly Ser Ser Gly Ser Gly 1 5 10 15 Ser Tyr
Tyr Tyr Tyr Tyr 20 <210> SEQ ID NO 34 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 34 Ser Gly Ser Gly Ser Ser Gly Ser Gly Ser
Gly Ser Ser Gly Ser Gly 1 5 10 15 Ser <210> SEQ ID NO 35
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 35 His Val Gly Gly
Ser Ser Val 1 5 <210> SEQ ID NO 36 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 36 Ser Val Arg Gly Ser Gly Ser Gly Val 1 5
<210> SEQ ID NO 37 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 37
Ser Val Val Arg Asp Gly Ser Glu Val 1 5 <210> SEQ ID NO 38
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 38 Ser Gly Arg Lys
Val Gly Ser Gly Ser Ser Val 1 5 10 <210> SEQ ID NO 39
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 39 Ser Leu Arg Gly
Asp Gly Ser Ser Val 1 5 <210> SEQ ID NO 40 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 40 Ser Val Gly Ser Arg Val 1 5
<210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 41
Thr Arg Arg Ser Tyr Ser Pro Arg His Asn Phe Asn Trp Leu Arg Ile 1 5
10 15 Gly Asp Phe Thr 20 <210> SEQ ID NO 42 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 42 Arg Lys Phe Leu Met Thr Thr
Arg Tyr Ser Arg Val 1 5 10 <210> SEQ ID NO 43 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 43 His Arg Gly Cys Gly Phe Phe
Lys Val Leu 1 5 10 <210> SEQ ID NO 44 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 44 Cys Asp Tyr Gln Ile Tyr Gln Asn Val Phe
Asn Phe 1 5 10 <210> SEQ ID NO 45 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 45 His Leu Ala Arg Asp Ser Gly Leu Cys Ser
Ala Val Pro Asp Pro Asp 1 5 10 15 <210> SEQ ID NO 46
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 46 Leu Thr Pro Pro
Gly Asp Asn Ala Leu Leu Leu Ala 1 5 10 <210> SEQ ID NO 47
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (6)..(6) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 47 Tyr Ser Thr Leu Pro Xaa Thr Asn Phe Cys
Ala Trp Glu Tyr Thr Ala 1 5 10 15 Tyr His His Val 20 <210>
SEQ ID NO 48 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 48
Lys Phe Leu Arg Ser Ala Gly Val Lys Pro Arg Asn Gly Lys Trp Tyr 1 5
10 15 Asp Ser <210> SEQ ID NO 49 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 49 Lys Gly Val Lys Thr Arg Glu Lys Asn Tyr
Thr Pro Arg Met Trp Thr 1 5 10 15 Glu Arg Ala Asp 20 <210>
SEQ ID NO 50 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 50
Lys Thr Ala Lys Lys Asn Val Phe Phe Cys Ser Val 1 5 10 <210>
SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 51
Pro Pro Ser Cys Val Tyr Pro Ser Arg Lys Cys Ser Pro Thr Ile Ile 1 5
10 15 Thr Phe Ser Gln 20 <210> SEQ ID NO 52 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 52 Leu Ser Ile Val Gly Arg Gln
Arg Cys Arg His Val 1 5 10 <210> SEQ ID NO 53 <211>
LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 53 Glu Arg His Val Ser Thr Gln
Pro Leu Leu Lys Glu Ala Asn Ile Lys 1 5 10 15 <210> SEQ ID NO
54 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 54 Arg Gln Pro
Cys Thr Tyr Ile Glu Val Arg Pro 1 5 10 <210> SEQ ID NO 55
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 55 Thr Leu Leu Cys
Thr Ile Lys Glu Cys Ser 1 5 10 <210> SEQ ID NO 56 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 56 Asp Val Ala Cys Val Thr Ile
Asn Leu Pro Asp Val Cys 1 5 10 <210> SEQ ID NO 57 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 57 Ile Tyr Pro Cys Arg Pro Asn
Thr Ala Leu Asn Asp Tyr Cys Ser Leu 1 5 10 15 Tyr <210> SEQ
ID NO 58 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 58 Thr Phe Pro
Cys Lys Pro Leu Arg His Thr Pro Arg Cys Thr Arg 1 5 10 15
<210> SEQ ID NO 59 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 59
Gly Leu Phe Cys Thr Ala Thr Ser Pro His Val Thr Arg Ala Cys Lys 1 5
10 15 Glu Leu <210> SEQ ID NO 60 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 60 Thr Glu Gln Cys Leu Ile His Lys Ser Met
Asn Pro Asn Ser Cys Arg 1 5 10 15 Gly Phe <210> SEQ ID NO 61
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 61 Phe Thr His Ala
Leu Asp Pro Gly Gln Leu Ala Leu 1 5 10 <210> SEQ ID NO 62
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 62 His His Leu Ala
Ser Leu Tyr His His Ser Tyr 1 5 10 <210> SEQ ID NO 63
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 63 Asn Ala Gln Leu
Ser Leu Ser Arg Gly His Leu His Gln Met Ile Gln 1 5 10 15
<210> SEQ ID NO 64 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 64
Lys Ala Arg Leu Pro Pro Glu Pro Ser Phe Thr Val Phe Thr Cys Gly 1 5
10 15 Arg Ala Ser Ala 20 <210> SEQ ID NO 65 <211>
LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 65 Leu Ser Pro Gln Arg Phe Cys
Tyr Gly Tyr Leu Phe Gln Phe Thr Leu 1 5 10 15 Val Leu His Leu 20
<210> SEQ ID NO 66 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 66
Thr Phe Phe Val Ser Thr Arg His Asp Leu Val Ile Cys Leu 1 5 10
<210> SEQ ID NO 67 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 67
Met His Val Glu Arg Val Thr Arg Leu His Thr 1 5 10 <210> SEQ
ID NO 68 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 68 Pro His Phe
Cys Pro Ala Met Lys Leu Ala Ala Ala Leu Glu 1 5 10 <210> SEQ
ID NO 69 <211> LENGTH: 20 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYTHENSIZED <400> SEQUENCE: 69 His Arg Leu
Ser Arg Tyr Arg Pro Arg Leu Gly Pro Tyr Phe Cys Pro 1 5 10 15 Ser
Pro Glu Val 20 <210> SEQ ID NO 70 <211> LENGTH: 4391
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION
NUMBER: P98160 <309> DATABASE ENTRY DATE: 2003-02-28
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(4391) <400>
SEQUENCE: 70 Met Gly Trp Arg Ala Pro Gly Ala Leu Leu Leu Ala Leu
Leu Leu His 1 5 10 15 Gly Arg Leu Leu Ala Val Thr His Gly Leu Arg
Ala Tyr Asp Gly Leu 20 25 30 Ser Leu Pro Glu Asp Ile Glu Thr Val
Thr Ala Ser Gln Met Arg Trp 35 40 45 Thr His Ser Tyr Leu Ser Asp
Asp Glu Asp Met Leu Ala Asp Ser Ile 50 55 60 Ser Gly Asp Asp Leu
Gly Ser Gly Asp Leu Gly Ser Gly Asp Phe Gln 65 70 75 80 Met Val Tyr
Phe Arg Ala Leu Val Asn Phe Thr Arg Ser Ile Glu Tyr 85 90 95 Ser
Pro Gln Leu Glu Asp Ala Gly Ser Arg Glu Phe Arg Glu Val Ser 100 105
110 Glu Ala Val Val Asp Thr Leu Glu Ser Glu Tyr Leu Lys Ile Pro Gly
115 120 125 Asp Gln Val Val Ser Val Val Phe Ile Lys Glu Leu Asp Gly
Trp Val 130 135 140 Phe Val Glu Leu Asp Val Gly Ser Glu Gly Asn Ala
Asp Gly Ala Gln 145 150 155 160 Ile Gln Glu Met Leu Leu Arg Val Ile
Ser Ser Gly Ser Val Ala Ser 165 170 175 Tyr Val Thr Ser Pro Gln Gly
Phe Gln Phe Arg Arg Leu Gly Thr Val 180 185 190 Pro Gln Phe Pro Arg
Ala Cys Thr Glu Ala Glu Phe Ala Cys His Ser 195 200 205 Tyr Asn Glu
Cys Val Ala Leu Glu Tyr Arg Cys Asp Arg Arg Pro Asp 210 215 220 Cys
Arg Asp Met Ser Asp Glu Leu Asn Cys Glu Glu Pro Val Leu Gly 225 230
235 240 Ile Ser Pro Thr Phe Ser Leu Leu Val Glu Thr Thr Ser Leu Pro
Pro 245 250 255 Arg Pro Glu Thr Thr Ile Met Arg Gln Pro Pro Val Thr
His Ala Pro 260 265 270 Gln Pro Leu Leu Pro Gly Ser Val Arg Pro Leu
Pro Cys Gly Pro Gln 275 280 285 Glu Ala Ala Cys Arg Asn Gly His Cys
Ile Pro Arg Asp Tyr Leu Cys 290 295 300 Asp Gly Gln Glu Asp Cys Glu
Asp Gly Ser Asp Glu Leu Asp Cys Gly 305 310 315 320 Pro Pro Pro Pro
Cys Glu Pro Asn Glu Phe Pro Cys Gly Asn Gly His 325 330 335 Cys Ala
Leu Lys Leu Trp Arg Cys Asp Gly Asp Phe Asp Cys Glu Asp 340 345 350
Arg Thr Asp Glu Ala Asn Cys Pro Thr Lys Arg Pro Glu Glu Val Cys 355
360 365 Gly Pro Thr Gln Phe Arg Cys Val Ser Thr Asn Met Cys Ile Pro
Ala 370 375 380 Ser Phe His Cys Asp Glu Glu Ser Asp Cys Pro Asp Arg
Ser Asp Glu 385 390 395 400 Phe Gly Cys Met Pro Pro Gln Val Val Thr
Pro Pro Arg Glu Ser Ile 405 410 415 Gln Ala Ser Arg Gly Gln Thr Val
Thr Phe Thr Cys Val Ala Ile Gly 420 425 430 Val Pro Thr Pro Ile Ile
Asn Trp Arg Leu Asn Trp Gly His Ile Pro 435 440 445 Ser His Pro Arg
Val Thr Val Thr Ser Glu Gly Gly Arg Gly Thr Leu 450 455 460 Ile Ile
Arg Asp Val Lys Glu Ser Asp Gln Gly Ala Tyr Thr Cys Glu 465 470 475
480 Ala Met Asn Ala Arg Gly Met Val Phe Gly Ile Pro Asp Gly Val Leu
485 490 495 Glu Leu Val Pro Gln Arg Gly Pro Cys Pro Asp Gly His Phe
Tyr Leu 500 505 510 Glu His Ser Ala Ala Cys Leu Pro Cys Phe Cys Phe
Gly Ile Thr Ser 515 520 525 Val Cys Gln Ser Thr Arg Arg Phe Arg Asp
Gln Ile Arg Leu Arg Phe 530 535 540 Asp Gln Pro Asp Asp Phe Lys Gly
Val Asn Val Thr Met Pro Ala Gln 545 550 555 560 Pro Gly Thr Pro Pro
Leu Ser Ser Thr Gln Leu Gln Ile Asp Pro Ser 565 570 575 Leu His Glu
Phe Gln Leu Val Asp Leu Ser Arg Arg Phe Leu Val His 580 585 590 Asp
Ser Phe Trp Ala Leu Pro Glu Gln Phe Leu Gly Asn Lys Val Asp 595 600
605 Ser Tyr Gly Gly Ser Leu Arg Tyr Asn Val Arg Tyr Glu Leu Ala Arg
610 615 620 Gly Met Leu Glu Pro Val Gln Arg Pro Asp Val Val Leu Val
Gly Ala 625 630 635 640 Gly Tyr Arg Leu Leu Ser Arg Gly His Thr Pro
Thr Gln Pro Gly Ala 645 650 655 Leu Asn Gln Arg Gln Val Gln Phe Ser
Glu Glu His Trp Val His Glu 660 665 670 Ser Gly Arg Pro Val Gln Arg
Ala Glu Leu Leu Gln Val Leu Gln Ser 675 680 685 Leu Glu Ala Val Leu
Ile Gln Thr Val Tyr Asn Thr Lys Met Ala Ser 690 695 700 Val Gly Leu
Ser Asp Ile Ala Met Asp Thr Thr Val Thr His Ala Thr 705 710 715 720
Ser His Gly Arg Ala His Ser Val Glu Glu Cys Arg Cys Pro Ile Gly 725
730 735 Tyr Ser Gly Leu Ser Cys Glu Ser Cys Asp Ala His Phe Thr Arg
Val 740 745 750 Pro Gly Gly Pro Tyr Leu Gly Thr Cys Ser Gly Cys Ser
Cys Asn Gly 755 760 765 His Ala Ser Ser Cys Asp Pro Val Tyr Gly His
Cys Leu Asn Cys Gln 770 775 780 His Asn Thr Glu Gly Pro Gln Cys Asn
Lys Cys Lys Ala Gly Phe Phe 785 790 795 800 Gly Asp Ala Met Lys Ala
Thr Ala Thr Ser Cys Arg Pro Cys Pro Cys 805 810 815 Pro Tyr Ile Asp
Ala Ser Arg Arg Phe Ser Asp Thr Cys Phe Leu Asp 820 825 830 Thr Asp
Gly Gln Ala Thr Cys Asp Ala Cys Ala Pro Gly Tyr Thr Gly 835 840 845
Arg Arg Cys Glu Ser Cys Ala Pro Gly Tyr Glu Gly Asn Pro Ile Gln 850
855 860 Pro Gly Gly Lys Cys Arg Pro Val Asn Gln Glu Ile Val Arg Cys
Asp 865 870 875 880 Glu Arg Gly Ser Met Gly Thr Ser Gly Glu Ala Cys
Arg Cys Lys Asn 885 890 895 Asn Val Val Gly Arg Leu Cys Asn Glu Cys
Ala Asp Gly Ser Phe His 900 905 910 Leu Ser Thr Arg Asn Pro Asp Gly
Cys Leu Lys Cys Phe Cys Met Gly 915 920 925 Val Ser Arg His Cys Thr
Ser Ser Ser Trp Ser Arg Ala Gln Leu His 930 935 940 Gly Ala Ser Glu
Glu Pro Gly His Phe Ser Leu Thr Asn Ala Ala Ser 945 950 955 960 Thr
His Thr Thr Asn Glu Gly Ile Phe Ser Pro Thr Pro Gly Glu Leu 965 970
975 Gly Phe Ser Ser Phe His Arg Leu Leu Ser Gly Pro Tyr Phe Trp Ser
980 985 990 Leu Pro Ser Arg Phe Leu Gly Asp Lys Val Thr Ser Tyr Gly
Gly Glu 995 1000 1005 Leu Arg Phe Thr Val Thr Gln Arg Ser Gln Pro
Gly Ser Thr Pro 1010 1015 1020 Leu His Gly Gln Pro Leu Val Val Leu
Gln Gly Asn Asn Ile Ile 1025 1030 1035 Leu Glu His His Val Ala Gln
Glu Pro Ser Pro Gly Gln Pro Ser 1040 1045 1050 Thr Phe Ile Val Pro
Phe Arg Glu Gln Ala Trp Gln Arg Pro Asp 1055 1060 1065 Gly Gln Pro
Ala Thr Arg Glu His Leu Leu Met Ala Leu Ala Gly 1070 1075 1080 Ile
Asp Thr Leu Leu Ile Arg Ala Ser Tyr Ala Gln Gln Pro Ala 1085 1090
1095 Glu Ser Arg Val Ser Gly Ile Ser Met Asp Val Ala Val Pro Glu
1100 1105 1110 Glu Thr Gly Gln Asp Pro Ala Leu Glu Val Glu Gln Cys
Ser Cys 1115 1120 1125 Pro Pro Gly Tyr Arg Gly Pro Ser Cys Gln Asp
Cys Asp Thr Gly 1130 1135 1140 Tyr Thr Arg Thr Pro Ser Gly Leu Tyr
Leu Gly Thr Cys Glu Arg 1145 1150 1155 Cys Ser Cys His Gly His Ser
Glu Ala Cys Glu Pro Glu Thr Gly 1160 1165 1170 Ala Cys Gln Gly Cys
Gln His His Thr Glu Gly Pro Arg Cys Glu 1175 1180 1185 Gln Cys Gln
Pro Gly Tyr Tyr Gly Asp Ala Gln Arg Gly Thr Pro 1190 1195 1200 Gln
Asp Cys Gln Leu Cys Pro Cys Tyr Gly Asp Pro Ala Ala Gly 1205 1210
1215 Gln Ala Ala His Thr Cys Phe Leu Asp Thr Asp Gly His Pro Thr
1220 1225 1230 Cys Asp Ala Cys Ser Pro Gly His Ser Gly Arg His Cys
Glu Arg 1235 1240 1245 Cys Ala Pro Gly Tyr Tyr Gly Asn Pro Ser Gln
Gly Gln Pro Cys 1250 1255 1260 Gln Arg Asp Ser Gln Val Pro Gly Pro
Ile Gly Cys Asn Cys Asp 1265 1270 1275 Pro Gln Gly Ser Val Ser Ser
Gln Cys Asp Ala Ala Gly Gln Cys 1280 1285 1290 Gln Cys Lys Ala Gln
Val Glu Gly Leu Thr Cys Ser His Cys Arg 1295 1300 1305 Pro His His
Phe His Leu Ser Ala Ser Asn Pro Asp Gly Cys Leu 1310 1315 1320 Pro
Cys Phe Cys Met Gly Ile Thr Gln Gln Cys Ala Ser Ser Ala 1325 1330
1335 Tyr Thr Arg His Leu Ile Ser Thr His Phe Ala Pro Gly Asp Phe
1340 1345 1350 Gln Gly Phe Ala Leu Val Asn Pro Gln Arg Asn Ser Arg
Leu Thr 1355 1360 1365 Gly Glu Phe Thr Val Glu Pro Val Pro Glu Gly
Ala Gln Leu Ser 1370 1375 1380 Phe Gly Asn Phe Ala Gln Leu Gly His
Glu Ser Phe Tyr Trp Gln 1385 1390 1395 Leu Pro Glu Thr Tyr Gln Gly
Asp Lys Val Ala Ala Tyr Gly Gly 1400 1405 1410 Lys Leu Arg Tyr Thr
Leu Ser Tyr Thr Ala Gly Pro Gln Gly Ser 1415 1420 1425 Pro Leu Ser
Asp Pro Asp Val Gln Ile Thr Gly Asn Asn Ile Met 1430 1435 1440 Leu
Val Ala Ser Gln Pro Ala Leu Gln Gly Pro Glu Arg Arg Ser 1445 1450
1455 Tyr Glu Ile Met Phe Arg Glu Glu Phe Trp Arg Arg Pro Asp Gly
1460 1465 1470 Gln Pro Ala Thr Arg Glu His Leu Leu Met Ala Leu Ala
Asp Leu 1475 1480 1485 Asp Glu Leu Leu Ile Arg Ala Thr Phe Ser Ser
Val Pro Leu Val 1490 1495 1500 Ala Ser Ile Ser Ala Val Ser Leu Glu
Val Ala Gln Pro Gly Pro 1505 1510 1515 Ser Asn Arg Pro Arg Ala Leu
Glu Val Glu Glu Cys Arg Cys Pro 1520 1525 1530 Pro Gly Tyr Ile Gly
Leu Ser Cys Gln Asp Cys Ala Pro Gly Tyr 1535 1540 1545 Thr Arg Thr
Gly Ser Gly Leu Tyr Leu Gly His Cys Glu Leu Cys 1550 1555 1560 Glu
Cys Asn Gly His Ser Asp Leu Cys His Pro Glu Thr Gly Ala 1565 1570
1575 Cys Ser Gln Cys Gln His Asn Ala Ala Gly Glu Phe Cys Glu Leu
1580 1585 1590 Cys Ala Pro Gly Tyr Tyr Gly Asp Ala Thr Ala Gly Thr
Pro Glu 1595 1600 1605 Asp Cys Gln Pro Cys Ala Cys Pro Leu Thr Asn
Pro Glu Asn Met 1610 1615 1620 Phe Ser Arg Thr Cys Glu Ser Leu Gly
Ala Gly Gly Tyr Arg Cys 1625 1630 1635 Thr Ala Cys Glu Pro Gly Tyr
Thr Gly Gln Tyr Cys Glu Gln Cys 1640 1645 1650 Gly Pro Gly Tyr Val
Gly Asn Pro Ser Val Gln Gly Gly Gln Cys 1655 1660 1665 Leu Pro Glu
Thr Asn Gln Ala Pro Leu Val Val Glu Val His Pro 1670 1675 1680 Ala
Arg Ser Ile Val Pro Gln Gly Gly Ser His Ser Leu Arg Cys 1685 1690
1695 Gln Val Ser Gly Ser Pro Pro His Tyr Phe Tyr Trp Ser Arg Glu
1700 1705 1710 Asp Gly Arg Pro Val Pro Ser Gly Thr Gln Gln Arg His
Gln Gly 1715 1720 1725 Ser Glu Leu His Phe Pro Ser Val Gln Pro Ser
Asp Ala Gly Val 1730 1735 1740 Tyr Ile Cys Thr Cys Arg Asn Leu His
Gln Ser Asn Thr Ser Arg 1745 1750 1755 Ala Glu Leu Leu Val Thr Glu
Ala Pro Ser Lys Pro Ile Thr Val 1760 1765 1770 Thr Val Glu Glu Gln
Arg Ser Gln Ser Val Arg Pro Gly Ala Asp 1775 1780 1785 Val Thr Phe
Ile Cys Thr Ala Lys Ser Lys Ser Pro Ala Tyr Thr 1790 1795 1800 Leu
Val Trp Thr Arg Leu His Asn Gly Lys Leu Pro Thr Arg Ala 1805 1810
1815 Met Asp Phe Asn Gly Ile Leu Thr Ile Arg Asn Val Gln Leu Ser
1820 1825 1830 Asp Ala Gly Thr Tyr Val Cys Thr Gly Ser Asn Met Phe
Ala Met 1835 1840 1845 Asp Gln Gly Thr Ala Thr Leu His Val Gln Ala
Ser Gly Thr Leu 1850 1855 1860 Ser Ala Pro Val Val Ser Ile His Pro
Pro Gln Leu Thr Val Gln 1865 1870 1875 Pro Gly Gln Leu Ala Glu Phe
Arg Cys Ser Ala Thr Gly Ser Pro 1880 1885 1890 Thr Pro Thr Leu Glu
Trp Thr Gly Gly Pro Gly Gly Gln Leu Pro 1895 1900 1905 Ala Lys Ala
Gln Ile His Gly Gly Ile Leu Arg Leu Pro Ala Val 1910 1915 1920 Glu
Pro Thr Asp Gln Ala Gln Tyr Leu Cys Arg Ala His Ser Ser 1925 1930
1935 Ala Gly Gln Gln Val Ala Arg Ala Val Leu His Val His Gly Gly
1940 1945 1950 Gly Gly Pro Arg Val Gln Val Ser Pro Glu Arg Thr Gln
Val His 1955 1960 1965 Ala Gly Arg Thr Val Arg Leu Tyr Cys Arg Ala
Ala Gly Val Pro 1970 1975 1980 Ser Ala Thr Ile Thr Trp Arg Lys Glu
Gly Gly Ser Leu Pro Pro 1985 1990 1995 Gln Ala Arg Ser Glu Arg Thr
Asp Ile Ala Thr Leu Leu Ile Pro 2000 2005 2010 Ala Ile Thr Thr Ala
Asp Ala Gly Phe Tyr Leu Cys Val Ala Thr 2015 2020 2025 Ser Pro Ala
Gly Thr Ala Gln Ala Arg Ile Gln Val Val Val Leu 2030 2035 2040 Ser
Ala Ser Asp Ala Ser Pro Pro Pro Val Lys Ile Glu Ser Ser 2045 2050
2055 Ser Pro Ser Val Thr Glu Gly Gln Thr Leu Asp Leu Asn Cys Val
2060 2065 2070 Val Ala Gly Ser Ala His Ala Gln Val Thr Trp Tyr Arg
Arg Gly 2075 2080 2085 Gly Ser Leu Pro Pro His Thr Gln Val His Gly
Ser Arg Leu Arg 2090 2095 2100 Leu Pro Gln Val Ser Pro Ala Asp Ser
Gly Glu Tyr Val Cys Arg 2105 2110 2115 Val Glu Asn Gly Ser Gly Pro
Lys Glu Ala Ser Ile Thr Val Ser 2120 2125 2130 Val Leu His Gly Thr
His Ser Gly Pro Ser Tyr Thr Pro Val Pro 2135 2140 2145 Gly Ser Thr
Arg Pro Ile Arg Ile Glu Pro Ser Ser Ser His Val 2150 2155 2160 Ala
Glu Gly Gln Thr Leu Asp Leu Asn Cys Val Val Pro Gly Gln 2165 2170
2175 Ala His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu Pro
2180 2185 2190 Ala Arg His Gln Thr His Gly Ser Leu Leu Arg Leu His
Gln Val 2195 2200 2205 Thr Pro Ala Asp Ser Gly Glu Tyr Val Cys His
Val Val Gly Thr 2210 2215 2220 Ser Gly Pro Leu Glu Ala Ser Val Leu
Val Thr Ile Glu Ala Ser 2225 2230 2235 Val Ile Pro Gly Pro Ile Pro
Pro Val Arg Ile Glu Ser Ser Ser 2240 2245 2250 Ser Thr Val Ala Glu
Gly Gln Thr Leu Asp Leu Ser Cys Val Val 2255 2260 2265 Ala Gly Gln
Ala His Ala Gln Val Thr Trp Tyr Lys Arg Gly Gly 2270 2275 2280 Ser
Leu Pro Ala Arg His Gln Val Arg Gly Ser Arg Leu Tyr Ile 2285 2290
2295 Phe Gln Ala Ser Pro Ala Asp Ala Gly Gln Tyr Val Cys Arg Ala
2300 2305 2310 Ser Asn Gly Met Glu Ala Ser Ile Thr Val Thr Val Thr
Gly Thr 2315 2320 2325 Gln Gly Ala Asn Leu Ala Tyr Pro Ala Gly Ser
Thr Gln Pro Ile 2330 2335 2340 Arg Ile Glu Pro Ser Ser Ser Gln Val
Ala Glu Gly Gln Thr Leu 2345 2350 2355 Asp Leu Asn Cys Val Val Pro
Gly Gln Ser His Ala Gln Val Thr 2360 2365 2370 Trp His Lys Arg Gly
Gly Ser Leu Pro Val Arg His Gln Thr His 2375 2380 2385 Gly Ser Leu
Leu Arg Leu Tyr Gln Ala Ser Pro Ala Asp Ser Gly 2390 2395 2400 Glu
Tyr Val Cys Arg Val Leu Gly Ser Ser Val Pro Leu Glu Ala 2405 2410
2415 Ser Val Leu Val Thr Ile Glu Pro Ala Gly Ser Val Pro Ala Leu
2420 2425 2430 Gly Val Thr Pro Thr Val Arg Ile Glu Ser Ser Ser Ser
Gln Val 2435 2440 2445 Ala Glu Gly Gln Thr Leu Asp Leu Asn Cys Leu
Val Ala Gly Gln 2450 2455 2460 Ala His Ala Gln Val Thr Trp His Lys
Arg Gly Gly Ser Leu Pro 2465 2470 2475 Ala Arg His Gln Val His Gly
Ser Arg Leu Arg Leu Leu Gln Val 2480 2485 2490 Thr Pro Ala Asp Ser
Gly Glu Tyr Val Cys Arg Val Val Gly Ser 2495 2500 2505 Ser Gly Thr
Gln Glu Ala Ser Val Leu Val Thr Ile Gln Gln Arg 2510 2515 2520 Leu
Ser Gly Ser His Ser Gln Gly Val Ala Tyr Pro Val Arg Ile 2525 2530
2535 Glu Ser Ser Ser Ala Ser Leu Ala Asn Gly His Thr Leu Asp Leu
2540 2545 2550 Asn Cys Leu Val Ala Ser Gln Ala Pro His Thr Ile Thr
Trp Tyr 2555 2560 2565 Lys Arg Gly Gly Ser Leu Pro Ser Arg His Gln
Ile Val Gly Ser 2570 2575 2580 Arg Leu Arg Ile Pro Gln Val Thr Pro
Ala Asp Ser Gly Glu Tyr 2585 2590 2595 Val Cys His Val Ser Asn Gly
Ala Gly Ser Arg Glu Thr Ser Leu 2600 2605 2610 Ile Val Thr Ile Gln
Gly Ser Gly Ser Ser His Val Pro Ser Val 2615 2620 2625 Ser Pro Pro
Ile Arg Ile Glu Ser Ser Ser Pro Thr Val Val Glu 2630 2635 2640 Gly
Gln Thr Leu Asp Leu Asn Cys Val Val Ala Arg Gln Pro Gln 2645 2650
2655 Ala Ile Ile Thr Trp Tyr Lys Arg Gly Gly Ser Leu Pro Ser Arg
2660 2665 2670 His Gln Thr His Gly Ser His Leu Arg Leu His Gln Met
Ser Val 2675 2680 2685 Ala Asp Ser Gly Glu Tyr Val Cys Arg Ala Asn
Asn Asn Ile Asp 2690 2695 2700 Ala Leu Glu Ala Ser Ile Val Ile Ser
Val Ser Pro Ser Ala Gly 2705 2710 2715 Ser Pro Ser Ala Pro Gly Ser
Ser Met Pro Ile Arg Ile Glu Ser 2720 2725 2730 Ser Ser Ser His Val
Ala Glu Gly Glu Thr Leu Asp Leu Asn Cys 2735 2740 2745 Val Val Pro
Gly Gln Ala His Ala Gln Val Thr Trp His Lys Arg 2750 2755 2760 Gly
Gly Ser Leu Pro Ser His His Gln Thr Arg Gly Ser Arg Leu 2765 2770
2775 Arg Leu His His Val Ser Pro Ala Asp Ser Gly Glu Tyr Val Cys
2780 2785 2790 Arg Val Met Gly Ser Ser Gly Pro Leu Glu Ala Ser Val
Leu Val 2795 2800 2805 Thr Ile Glu Ala Ser Gly Ser Ser Ala Val His
Val Pro Ala Pro 2810 2815 2820 Gly Gly Ala Pro Pro Ile Arg Ile Glu
Pro Ser Ser Ser Arg Val 2825 2830 2835 Ala Glu Gly Gln Thr Leu Asp
Leu Lys Cys Val Val Pro Gly Gln 2840 2845 2850 Ala His Ala Gln Val
Thr Trp His Lys Arg Gly Gly Asn Leu Pro 2855 2860 2865 Ala Arg His
Gln Val His Gly Pro Leu Leu Arg Leu Asn Gln Val 2870 2875 2880 Ser
Pro Ala Asp Ser Gly Glu Tyr Ser Cys Gln Val Thr Gly Ser 2885 2890
2895 Ser Gly Thr Leu Glu Ala Ser Val Leu Val Thr Ile Glu Pro Ser
2900 2905 2910 Ser Pro Gly Pro Ile Pro Ala Pro Gly Leu Ala Gln Pro
Ile Tyr 2915 2920 2925 Ile Glu Ala Ser Ser Ser His Val Thr Glu Gly
Gln Thr Leu Asp 2930 2935 2940 Leu Asn Cys Val Val Pro Gly Gln Ala
His Ala Gln Val Thr Trp 2945 2950 2955 Tyr Lys Arg Gly Gly Ser Leu
Pro Ala Arg His Gln Thr His Gly 2960 2965 2970 Ser Gln Leu Arg Leu
His Leu Val Ser Pro Ala Asp Ser Gly Glu 2975 2980 2985 Tyr Val Cys
Arg Ala Ala Ser Gly Pro Gly Pro Glu Gln Glu Ala 2990 2995 3000 Ser
Phe Thr Val Thr Val Pro Pro Ser Glu Gly Ser Ser Tyr Arg 3005 3010
3015 Leu Arg Ser Pro Val Ile Ser Ile Asp Pro Pro Ser Ser Thr Val
3020 3025 3030 Gln Gln Gly Gln Asp Ala Ser Phe Lys Cys Leu Ile His
Asp Gly 3035 3040 3045 Ala Ala Pro Ile Ser Leu Glu Trp Lys Thr Arg
Asn Gln Glu Leu 3050 3055 3060 Glu Asp Asn Val His Ile Ser Pro Asn
Gly Ser Ile Ile Thr Ile 3065 3070 3075 Val Gly Thr Arg Pro Ser Asn
His Gly Thr Tyr Arg Cys Val Ala 3080 3085 3090 Ser Asn Ala Tyr Gly
Val Ala Gln Ser Val Val Asn Leu Ser Val 3095 3100 3105 His Gly Pro
Pro Thr Val Ser Val Leu Pro Glu Gly Pro Val Trp 3110 3115 3120 Val
Lys Val Gly Lys Ala Val Thr Leu Glu Cys Val Ser Ala Gly 3125 3130
3135 Glu Pro Arg Ser Ser Ala Arg Trp Thr Arg Ile Ser Ser Thr Pro
3140 3145 3150 Ala Lys Leu Glu Gln Arg Thr Tyr Gly Leu Met Asp Ser
His Ala 3155 3160 3165 Val Leu Gln Ile Ser Ser Ala Lys Pro Ser Asp
Ala Gly Thr Tyr 3170 3175 3180 Val Cys Leu Ala Gln Asn Ala Leu Gly
Thr Ala Gln Lys Gln Val 3185 3190 3195 Glu Val Ile Val Asp Thr Gly
Ala Met Ala Pro Gly Ala Pro Gln 3200 3205 3210 Val Gln Ala Glu Glu
Ala Glu Leu Thr Val Glu Ala Gly His Thr 3215 3220 3225 Ala Thr Leu
Arg Cys Ser Ala Thr Gly Ser Pro Ala Pro Thr Ile 3230 3235 3240 His
Trp Ser Lys Leu Arg Ser Pro Leu Pro Trp Gln His Arg Leu 3245 3250
3255 Glu Gly Asp Thr Leu Ile Ile Pro Arg Val Ala Gln Gln Asp Ser
3260 3265 3270 Gly Gln Tyr Ile Cys Asn Ala Thr Ser Pro Ala Gly His
Ala Glu 3275 3280 3285 Ala Thr Ile Ile Leu His Val Glu Ser Pro Pro
Tyr Ala Thr Thr 3290 3295 3300 Val Pro Glu His Ala Ser Val Gln Ala
Gly Glu Thr Val Gln Leu 3305 3310 3315 Gln Cys Leu Ala His Gly Thr
Pro Pro Leu Thr Phe Gln Trp Ser 3320 3325 3330 Arg Val Gly Ser Ser
Leu Pro Gly Arg Ala Thr Ala Arg Asn Glu 3335 3340 3345 Leu Leu His
Phe Glu Arg Ala Ala Pro Glu Asp Ser Gly Arg Tyr 3350 3355 3360 Arg
Cys Arg Val Thr Asn Lys Val Gly Ser Ala Glu Ala Phe Ala 3365 3370
3375 Gln Leu Leu Val Gln Gly Pro Pro Gly Ser Leu Pro Ala Thr Ser
3380 3385 3390 Ile Pro Ala Gly Ser Thr Pro Thr Val Gln Val Thr Pro
Gln Leu 3395 3400 3405 Glu Thr Lys Ser Ile Gly Ala Ser Val Glu Phe
His Cys Ala Val 3410 3415 3420 Pro Ser Asp Arg Gly Thr Gln Leu Arg
Trp Phe Lys Glu Gly Gly 3425 3430 3435 Gln Leu Pro Pro Gly His Ser
Val Gln Asp Gly Val Leu Arg Ile 3440 3445 3450 Gln Asn Leu Asp Gln
Ser Cys Gln Gly Thr Tyr Ile Cys Gln Ala 3455 3460 3465 His Gly Pro
Trp Gly Lys Ala Gln Ala Ser Ala Gln Leu Val Ile 3470 3475 3480 Gln
Ala Leu Pro Ser Val Leu Ile Asn Ile Arg Thr Ser Val Gln 3485 3490
3495 Thr Val Val Val Gly His Ala Val Glu Phe Glu Cys Leu Ala Leu
3500 3505 3510 Gly Asp Pro Lys Pro Gln Val Thr Trp Ser Lys Val Gly
Gly His 3515 3520 3525 Leu Arg Pro Gly Ile Val Gln Ser Gly Gly Val
Val Arg Ile Ala 3530 3535 3540 His Val Glu Leu Ala Asp Ala Gly Gln
Tyr Arg Cys Thr Ala Thr 3545 3550 3555 Asn Ala Ala Gly Thr Thr Gln
Ser His Val Leu Leu Leu Val Gln 3560 3565 3570 Ala Leu Pro Gln Ile
Ser Met Pro Gln Glu Val Arg Val Pro Ala 3575 3580 3585 Gly Ser Ala
Ala Val Phe Pro Cys Ile Ala Ser Gly Tyr Pro Thr 3590 3595 3600 Pro
Asp Ile Ser Trp Ser Lys Leu Asp Gly Ser Leu Pro Pro Asp 3605 3610
3615 Ser Arg Leu Glu Asn Asn Met Leu Met Leu Pro Ser Val Arg Pro
3620 3625 3630 Gln Asp Ala Gly Thr Tyr Val Cys Thr Ala Thr Asn Arg
Gln Gly 3635 3640 3645 Lys Val Lys Ala Phe Ala His Leu Gln Val Pro
Glu Arg Val Val 3650 3655 3660 Pro Tyr Phe Thr Gln Thr Pro Tyr Ser
Phe Leu Pro Leu Pro Thr 3665 3670 3675 Ile Lys Asp Ala Tyr Arg Lys
Phe Glu Ile Lys Ile Thr Phe Arg 3680 3685 3690 Pro Asp Ser Ala Asp
Gly Met Leu Leu Tyr Asn Gly Gln Lys Arg 3695 3700 3705 Val Pro Gly
Ser Pro Thr Asn Leu Ala Asn Arg Gln Pro Asp Phe 3710 3715 3720 Ile
Ser Phe Gly Leu Val Gly Gly Arg Pro Glu Phe Arg Phe Asp 3725 3730
3735 Ala Gly Ser Gly Met Ala Thr Ile Arg His Pro Thr Pro Leu Ala
3740 3745 3750 Leu Gly His Phe His Thr Val Thr Leu Leu Arg Ser Leu
Thr Gln 3755 3760 3765 Gly Ser Leu Ile Val Gly Asp Leu Ala Pro Val
Asn Gly Thr Ser 3770 3775 3780 Gln Gly Lys Phe Gln Gly Leu Asp Leu
Asn Glu Glu Leu Tyr Leu 3785 3790 3795 Gly Gly Tyr Pro Asp Tyr Gly
Ala Ile Pro Lys Ala Gly Leu Ser 3800 3805 3810 Ser Gly Phe Ile Gly
Cys Val Arg Glu Leu Arg Ile Gln Gly Glu 3815 3820 3825 Glu Ile Val
Phe His Asp Leu Asn Leu Thr Ala His Gly Ile Ser 3830 3835 3840 His
Cys Pro Thr Cys Arg Asp Arg Pro Cys Gln Asn Gly Gly Gln 3845 3850
3855 Cys His Asp Ser Glu Ser Ser Ser Tyr Val Cys Val Cys Pro Ala
3860 3865 3870 Gly Phe Thr Gly Ser Arg Cys Glu His Ser Gln Ala Leu
His Cys 3875 3880 3885 His Pro Glu Ala Cys Gly Pro Asp Ala Thr Cys
Val Asn Arg Pro 3890 3895 3900 Asp Gly Arg Gly Tyr Thr Cys Arg Cys
His Leu Gly Arg Ser Gly 3905 3910 3915 Leu Arg Cys Glu Glu Gly Val
Thr Val Thr Thr Pro Ser Leu Ser 3920 3925 3930 Gly Ala Gly Ser Tyr
Leu Ala Leu Pro Ala Leu Thr Asn Thr His 3935 3940 3945 His Glu Leu
Arg Leu Asp Val Glu Phe Lys Pro Leu Ala Pro Asp 3950 3955 3960 Gly
Val Leu Leu Phe Ser Gly Gly Lys Ser Gly Pro Val Glu Asp 3965 3970
3975 Phe Val Ser Leu Ala Met Val Gly Gly His Leu Glu Phe Arg Tyr
3980 3985 3990 Glu Leu Gly Ser Gly Leu Ala Val Leu Arg Ser Ala Glu
Pro Leu 3995 4000 4005 Ala Leu Gly Arg Trp His Arg Val Ser Ala Glu
Arg Leu Asn Lys 4010 4015 4020 Asp Gly Ser Leu Arg Val Asn Gly Gly
Arg Pro Val Leu Arg Ser 4025 4030 4035 Ser Pro Gly Lys Ser Gln Gly
Leu Asn Leu His Thr Leu Leu Tyr 4040 4045 4050 Leu Gly Gly Val Glu
Pro Ser Val Pro Leu Ser Pro Ala Thr Asn 4055 4060 4065 Met Ser Ala
His Phe Arg Gly Cys Val Gly Glu Val Ser Val Asn 4070 4075 4080 Gly
Lys Arg Leu Asp Leu Thr Tyr Ser Phe Leu Gly Ser Gln Gly 4085 4090
4095 Ile Gly Gln Cys Tyr Asp Ser Ser Pro Cys Glu Arg Gln Pro Cys
4100 4105 4110 Gln His Gly Ala Thr Cys Met Pro Ala Gly Glu Tyr Glu
Phe Gln 4115 4120 4125 Cys Leu Cys Arg Asp Gly Phe Lys Gly Asp Leu
Cys Glu His Glu 4130 4135 4140 Glu Asn Pro Cys Gln Leu Arg Glu Pro
Cys Leu His Gly Gly Thr 4145 4150 4155 Cys Gln Gly Thr Arg Cys Leu
Cys Leu Pro Gly Phe Ser Gly Pro 4160 4165 4170 Arg Cys Gln Gln Gly
Ser Gly His Gly Ile Ala Glu Ser Asp Trp 4175 4180 4185 His Leu Glu
Gly Ser Gly Gly Asn Asp Ala Pro Gly Gln Tyr Gly 4190 4195 4200 Ala
Tyr Phe His Asp Asp Gly Phe Leu Ala Phe Pro Gly His Val 4205 4210
4215 Phe Ser Arg Ser Leu Pro Glu Val Pro Glu Thr Ile Glu Leu Glu
4220 4225 4230 Val Arg Thr Ser Thr Ala Ser Gly Leu Leu Leu Trp Gln
Gly Val 4235 4240 4245 Glu Val Gly Glu Ala Gly Gln Gly Lys Asp Phe
Ile Ser Leu Gly 4250 4255 4260 Leu Gln Asp Gly His Leu Val Phe Arg
Tyr Gln Leu Gly Ser Gly 4265 4270 4275 Glu Ala Arg Leu Val Ser Glu
Asp Pro Ile Asn Asp Gly Glu Trp 4280 4285 4290 His Arg Val Thr Ala
Leu Arg Glu Gly Arg Arg Gly Ser Ile Gln 4295 4300 4305 Val Asp Gly
Glu Glu Leu Val Ser Gly Arg Ser Pro Gly Pro Asn 4310 4315 4320 Val
Ala Val Asn Ala Lys Gly Ser Val Tyr Ile Gly Gly Ala Pro 4325 4330
4335 Asp Val Ala Thr Leu Thr Gly Gly Arg Phe Ser Ser Gly Ile Thr
4340 4345 4350 Gly Cys Val Lys Asn Leu Val Leu His Ser Ala Arg Pro
Gly Ala 4355 4360 4365 Pro Pro Pro Gln Pro Leu Asp Leu Gln His Arg
Ala Gln Ala Gly 4370 4375 4380 Ala Asn Thr Arg Pro Cys Pro Ser 4385
4390 <210> SEQ ID NO 71 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: SYNTHESIZED <400>
SEQUENCE: 71 His Gly Asp Pro Asn His Val Gly Gly Ser Ser Val 1 5 10
<210> SEQ ID NO 72 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 72
Val Gly Gly Ser Ser Val 1 5 <210> SEQ ID NO 73 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa
can be any naturally occurring amino acid <400> SEQUENCE: 73
Gly Gly Ser Xaa Val 1 5 <210> SEQ ID NO 74 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 74 His Gly Asp Pro Asn His Val
Gly Gly Ser Ser 1 5 10 <210> SEQ ID NO 75 <211> LENGTH:
7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 75 Arg Gly Asp Gly Ser Ser Val 1 5
<210> SEQ ID NO 76 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 76
His Gly Ser Ser Val 1 5 <210> SEQ ID NO 77 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 77 Asn Val Gly His Ser Pro Gly
Ser Asp His Gly 1 5 10 <210> SEQ ID NO 78 <211> LENGTH:
15 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 78 Gly Gly Gly His Gly Asp Pro Asn His Val
Gly Gly Ser Ser Val 1 5 10 15 <210> SEQ ID NO 79 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 79 Ile Tyr Pro Cys Arg Pro Asn
Thr Ala Leu Asn Asp Tyr Cys Ser Leu 1 5 10 15 Tyr <210> SEQ
ID NO 80 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 80 Arg Gln Pro
Cys Thr Tyr Ile Glu Val Arg Pro 1 5 10 <210> SEQ ID NO 81
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (10)..(10) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 81 Thr Leu Leu Cys Thr Ile Lys Glu Cys Xaa 1
5 10 <210> SEQ ID NO 82 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: SYNTHESIZED <400>
SEQUENCE: 82 Thr Arg Arg Ser Tyr Ser Pro Arg His Asn Phe Asn Trp
Leu Arg Ile 1 5 10 15 Gly Asp Phe Thr 20 <210> SEQ ID NO 83
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 83 Arg Lys Phe Leu
Met Thr Thr Arg Tyr Ser Arg Val 1 5 10 <210> SEQ ID NO 84
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (9)..(9) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 84 His Leu Ala Arg Asp Ser Gly Leu Xaa Ser
Ala Val Pro Asp Pro Asp 1 5 10 15 <210> SEQ ID NO 85
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (9)..(9) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (11)..(14) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <400> SEQUENCE: 85 Ser Arg Tyr
Thr Ile Glu Ser Pro Xaa Asp Xaa Xaa Xaa Xaa Glu Ser 1 5 10 15
<210> SEQ ID NO 86 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 86
Gly Gly Ser Ser Val 1 5
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 86 <210>
SEQ ID NO 1 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: synthesized <400> SEQUENCE: 1 Asn His Val
Gly Gly Ser Ser Val 1 5 <210> SEQ ID NO 2 <211> LENGTH:
10 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: synthesized
<400> SEQUENCE: 2 Asn Ser Leu Arg Gly Asp Gly Ser Ser Val 1 5
10 <210> SEQ ID NO 3 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 3
Asn Ser Val Arg Gly Ser Gly Ser Gly Val 1 5 10 <210> SEQ ID
NO 4 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 4 Asn Ser Val
Gly Ser Arg Val 1 5 <210> SEQ ID NO 5 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 5 Ser Leu Arg Gly Asp Gly Ser Ser Val 1 5
<210> SEQ ID NO 6 <211> LENGTH: 7 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 6
Arg Gly Asp Gly Ser Ser Val 1 5 <210> SEQ ID NO 7 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 7 Gly Ser Arg Val 1 <210>
SEQ ID NO 8 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYNTHESIZED <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 8 Gly Ser Xaa Val 1 <210> SEQ ID NO 9
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (6)..(6) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <400> SEQUENCE: 9 Asn Ser Xaa
Arg Gly Xaa Gly Ser 1 5 <210> SEQ ID NO 10 <211>
LENGTH: 3 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 10 Asn Ser Val 1 <210> SEQ
ID NO 11 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYNTHESIZED <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 11 Asn Ser Xaa Arg 1 <210> SEQ ID NO 12
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (2)..(2) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 12 Asn Xaa Val Gly 1 <210> SEQ ID NO 13
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 13 Gly Ser Ser Val 1
<210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Bacteriophage T7 <400> SEQUENCE: 14
agcggaccag attatcgcta 20 <210> SEQ ID NO 15 <211>
LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:
Bacteriophage T7 <400> SEQUENCE: 15 aaccctcaag acccgttta 19
<210> SEQ ID NO 16 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 16
His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val 1 5 10 <210>
SEQ ID NO 17 <211> LENGTH: 726 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (1)..(726)
<400> SEQUENCE: 17 atg gcc cag gtg aaa ctg cag cag tct ggg
gct gag ctt gtg atg cct 48 Met Ala Gln Val Lys Leu Gln Gln Ser Gly
Ala Glu Leu Val Met Pro 1 5 10 15 ggg gct tca gtg aag atg tcc tgc
aag gct tct ggc tac aca ttc act 96
Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20
25 30 gac tac tgg atg cac tgg gtg aag cag agg cct gga caa ggc ctt
gag 144 Asp Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Glu 35 40 45 tgg atc gga gcg att gat act tct gat agt tat act agc
tac aat caa 192 Trp Ile Gly Ala Ile Asp Thr Ser Asp Ser Tyr Thr Ser
Tyr Asn Gln 50 55 60 aag ttc aag ggc aag gcc aca ttg act gta gac
gaa tcc tcc agc aca 240 Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp
Glu Ser Ser Ser Thr 65 70 75 80 gcc tac atg cag ctc agc agc ctg aca
tct gag gac tct gcg gtc tat 288 Ala Tyr Met Gln Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr 85 90 95 tac tgt gca aga aga ggc tac
tat agc gca ttt gat tac tgg ggc caa 336 Tyr Cys Ala Arg Arg Gly Tyr
Tyr Ser Ala Phe Asp Tyr Trp Gly Gln 100 105 110 ggg act acg gtc acc
gtc tcc tca ggt gga ggc ggt tca ggc gga ggt 384 Gly Thr Thr Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 ggc tct ggc
ggt ggc gga tcg gac att gag ctc acc cag tct cca aca 432 Gly Ser Gly
Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Thr 130 135 140 acc
atg gct gca tct cca gga gag aag gtc acc atc acc tgc cgt gcc 480 Thr
Met Ala Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Arg Ala 145 150
155 160 agc tca agt gta agc tac atg cac tgg ttc cag cag aag tca ggc
acc 528 Ser Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Ser Gly
Thr 165 170 175 tcc ccc aaa ccc tgg att tat gac aca tcc aag ctg gct
tct gga gtc 576 Ser Pro Lys Pro Trp Ile Tyr Asp Thr Ser Lys Leu Ala
Ser Gly Val 180 185 190 cca gat cgc ttc agt ggc agt ggg tct ggg acc
tct tat tct ctc aca 624 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Ser Tyr Ser Leu Thr 195 200 205 atc agc tcc atg gag gct gaa gat gct
gct act tat tac tgt ctg cag 672 Ile Ser Ser Met Glu Ala Glu Asp Ala
Ala Thr Tyr Tyr Cys Leu Gln 210 215 220 agg agt agt tac ccg tac acg
ttt gga gct ggc acc aag ctg gaa atc 720 Arg Ser Ser Tyr Pro Tyr Thr
Phe Gly Ala Gly Thr Lys Leu Glu Ile 225 230 235 240 aaa cgg 726 Lys
Arg <210> SEQ ID NO 18 <211> LENGTH: 242 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<400> SEQUENCE: 18 Met Ala Gln Val Lys Leu Gln Gln Ser Gly
Ala Glu Leu Val Met Pro 1 5 10 15 Gly Ala Ser Val Lys Met Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 Asp Tyr Trp Met His Trp
Val Lys Gln Arg Pro Gly Gln Gly Leu Glu 35 40 45 Trp Ile Gly Ala
Ile Asp Thr Ser Asp Ser Tyr Thr Ser Tyr Asn Gln 50 55 60 Lys Phe
Lys Gly Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser Thr 65 70 75 80
Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 85
90 95 Tyr Cys Ala Arg Arg Gly Tyr Tyr Ser Ala Phe Asp Tyr Trp Gly
Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu
Thr Gln Ser Pro Thr 130 135 140 Thr Met Ala Ala Ser Pro Gly Glu Lys
Val Thr Ile Thr Cys Arg Ala 145 150 155 160 Ser Ser Ser Val Ser Tyr
Met His Trp Phe Gln Gln Lys Ser Gly Thr 165 170 175 Ser Pro Lys Pro
Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val 180 185 190 Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr 195 200 205
Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln 210
215 220 Arg Ser Ser Tyr Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu
Ile 225 230 235 240 Lys Arg <210> SEQ ID NO 19 <211>
LENGTH: 726 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <220> FEATURE: <221> NAME/KEY: CDS
<222> LOCATION: (1)..(726) <400> SEQUENCE: 19 atg gcc
cag gtc aag ctg cag cag tca gga cct gag ctg gta aag cct 48 Met Ala
Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 1 5 10 15
ggg gct tca gtg aag atg tcc tgc aag gct tct gga tac aca ttc act 96
Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20
25 30 agc tat gtt atg cac tgg gtg aag cag aag cct ggg cag ggc ctt
gag 144 Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu
Glu 35 40 45 tgg att gga tat att aat cct tac aat gat ggt act aag
tac aat gag 192 Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys
Tyr Asn Glu 50 55 60 aag ttc aaa ggc aag gcc gca ctg act tca gac
aaa tcc tcc agc aca 240 Lys Phe Lys Gly Lys Ala Ala Leu Thr Ser Asp
Lys Ser Ser Ser Thr 65 70 75 80 gcc tac atg gag ctc agc agc ctg acc
tct gag gac tct gcg gtc tat 288 Ala Tyr Met Glu Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr 85 90 95 tac tgt gca aga ttt ggt aac
tac ggt gct ttg gac tac tgg ggc caa 336 Tyr Cys Ala Arg Phe Gly Asn
Tyr Gly Ala Leu Asp Tyr Trp Gly Gln 100 105 110 ggg acc acg gtc acc
gtc tcc tca ggt gga ggc ggt tca ggc gga ggt 384 Gly Thr Thr Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 ggc tct ggc
ggt ggc gga tcg gac att gag ctc acc cag tct cca aca 432 Gly Ser Gly
Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Thr 130 135 140 atc
atg tct gca tct cca ggg gag aag gtc acc ata acc tgc agt gcc 480 Ile
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala 145 150
155 160 agc tca agt gta agt tac atg cac tgg ttc cag cag aag cca ggc
act 528 Ser Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly
Thr 165 170 175 tct ccc aaa ccc tgg att tat ggc aca tcc aac ctg gct
tct gga gtc 576 Ser Pro Lys Pro Trp Ile Tyr Gly Thr Ser Asn Leu Ala
Ser Gly Val 180 185 190 cct gtt cgc ttc agt ggc agt gga tct ggg acc
tct tat tct ctc aca 624 Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr
Ser Tyr Ser Leu Thr 195 200 205 atc agc agc atg gag gct gaa gat gct
gcc act tat tac tgt caa cag 672 Ile Ser Ser Met Glu Ala Glu Asp Ala
Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 tgg agt agt tac cca ctc acg
ttc gga ggg ggg acc aag ctg gaa ata 720 Trp Ser Ser Tyr Pro Leu Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile 225 230 235 240 aaa cgg 726 Lys
Arg <210> SEQ ID NO 20 <211> LENGTH: 242 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Construct
<400> SEQUENCE: 20 Met Ala Gln Val Lys Leu Gln Gln Ser Gly
Pro Glu Leu Val Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Met Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 Ser Tyr Val Met His Trp
Val Lys Gln Lys Pro Gly Gln Gly Leu Glu 35 40 45 Trp Ile Gly Tyr
Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu 50 55 60 Lys Phe
Lys Gly Lys Ala Ala Leu Thr Ser Asp Lys Ser Ser Ser Thr 65 70 75 80
Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 85
90 95 Tyr Cys Ala Arg Phe Gly Asn Tyr Gly Ala Leu Asp Tyr Trp Gly
Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu
Thr Gln Ser Pro Thr 130 135 140 Ile Met Ser Ala Ser Pro Gly Glu Lys
Val Thr Ile Thr Cys Ser Ala 145 150 155 160 Ser Ser Ser Val Ser Tyr
Met His Trp Phe Gln Gln Lys Pro Gly Thr 165 170 175 Ser Pro Lys Pro
Trp Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val 180 185 190 Pro Val
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr 195 200 205
Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 210
215 220 Trp Ser Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile 225 230 235 240 Lys Arg <210> SEQ ID NO 21 <211>
LENGTH: 795 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (1)..(795)
<400> SEQUENCE: 21 atg gcc cag gtg cag ctg cag gag tca gga
cct ggc ctt gtg aaa ccc 48 Met Ala Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro 1 5 10 15 tca cag tca ctc tcc ctc acc tgt
tct gtc act ggt tac tcc atc act 96 Ser Gln Ser Leu Ser Leu Thr Cys
Ser Val Thr Gly Tyr Ser Ile Thr 20 25 30 agt aat tac tgg ggc tgg
atc cgg aag ttc cca ggg aat aaa atg gag 144 Ser Asn Tyr Trp Gly Trp
Ile Arg Lys Phe Pro Gly Asn Lys Met Glu 35 40 45 tgg atg gga tac
ata agc tac agt ggt agc act agc tac aac cca tct 192 Trp Met Gly Tyr
Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser 50 55 60 ctc aaa
agt cga atc tcc att act aga gac aca tcg aag aat cag ctc 240 Leu Lys
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Leu 65 70 75 80
ttc ctg cag ttg aac tct gta act act gag gac aca gcc aca tat tac 288
Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr 85
90 95 tgt gca aga tat agc ctc ttt aac tac ggt agg agg gac tat gtt
atg 336 Cys Ala Arg Tyr Ser Leu Phe Asn Tyr Gly Arg Arg Asp Tyr Val
Met 100 105 110 gat gcc tgg ggc caa ggg acc acg gtc acc gtc tcc tca
ggt gga ggc 384 Asp Ala Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
Gly Gly Gly 115 120 125 ggt tca ggc gga ggt ggc tct ggc ggt ggc gga
tcg gac att gag ctc 432 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Asp Ile Glu Leu 130 135 140 acc cag tct cca gca acc atg gct gca
tct cca gga gag aaa gtc acc 480 Thr Gln Ser Pro Ala Thr Met Ala Ala
Ser Pro Gly Glu Lys Val Thr 145 150 155 160 atc acc tgc cgt gcc agc
tca act gta agc tac atg cac tgg ttc caa 528 Ile Thr Cys Arg Ala Ser
Ser Thr Val Ser Tyr Met His Trp Phe Gln 165 170 175 cag aag cca ggc
gcc tcc cct aaa ccc tgg att tat gac aca tcc aaa 576 Gln Lys Pro Gly
Ala Ser Pro Lys Pro Trp Ile Tyr Asp Thr Ser Lys 180 185 190 ctg gct
tct gga gtc cca gat cgc ttc agt ggc agt ggg tct ggg aca 624 Leu Ala
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205
gac ttc acc ctc acc att gat cct gtg cag gct gat gat att gca acc 672
Asp Phe Thr Leu Thr Ile Asp Pro Val Gln Ala Asp Asp Ile Ala Thr 210
215 220 tat tac tgt cag cag agt aag gat gat cct cgg acg ttc ggt gga
ggg 720 Tyr Tyr Cys Gln Gln Ser Lys Asp Asp Pro Arg Thr Phe Gly Gly
Gly 225 230 235 240 acc aag ctg gag ctg aaa cgg cgg ccg cag gtg cgc
cgg tgc cgt atc 768 Thr Lys Leu Glu Leu Lys Arg Arg Pro Gln Val Arg
Arg Cys Arg Ile 245 250 255 cgg atc cgc tgg aac cgc gtg ccg cat 795
Arg Ile Arg Trp Asn Arg Val Pro His 260 265 <210> SEQ ID NO
22 <211> LENGTH: 265 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 22 Met
Ala Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 1 5 10
15 Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr
20 25 30 Ser Asn Tyr Trp Gly Trp Ile Arg Lys Phe Pro Gly Asn Lys
Met Glu 35 40 45 Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser
Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp
Thr Ser Lys Asn Gln Leu 65 70 75 80 Phe Leu Gln Leu Asn Ser Val Thr
Thr Glu Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Tyr Ser Leu
Phe Asn Tyr Gly Arg Arg Asp Tyr Val Met 100 105 110 Asp Ala Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu 130 135 140
Thr Gln Ser Pro Ala Thr Met Ala Ala Ser Pro Gly Glu Lys Val Thr 145
150 155 160 Ile Thr Cys Arg Ala Ser Ser Thr Val Ser Tyr Met His Trp
Phe Gln 165 170 175 Gln Lys Pro Gly Ala Ser Pro Lys Pro Trp Ile Tyr
Asp Thr Ser Lys 180 185 190 Leu Ala Ser Gly Val Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Asp Pro
Val Gln Ala Asp Asp Ile Ala Thr 210 215 220 Tyr Tyr Cys Gln Gln Ser
Lys Asp Asp Pro Arg Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu
Glu Leu Lys Arg Arg Pro Gln Val Arg Arg Cys Arg Ile 245 250 255 Arg
Ile Arg Trp Asn Arg Val Pro His 260 265 <210> SEQ ID NO 23
<211> LENGTH: 786 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (1)..(786) <400> SEQUENCE: 23 atg
gcc cag gtg aag ctg cag cag tct gga cct gag ctg gta aag cct 48 Met
Ala Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 1 5 10
15 ggg gct tca gtg aag atg tcc tgc aag gct tct gga tac aca ttc act
96 Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30 agc tat gtt atg cac tgg gtg aag cag agc aat gga aag agc
ctt gag 144 Ser Tyr Val Met His Trp Val Lys Gln Ser Asn Gly Lys Ser
Leu Glu 35 40 45 tgg att gga act att gat cct tac tat ggt ggt act
agc tac aac cag 192 Trp Ile Gly Thr Ile Asp Pro Tyr Tyr Gly Gly Thr
Ser Tyr Asn Gln 50 55 60 aag ttc aag ggc aag gcc aca ttg act gta
gac aaa tcc tcc acc acg 240 Lys Phe Lys Gly Lys Ala Thr Leu Thr Val
Asp Lys Ser Ser Thr Thr 65 70 75 80 gcc tac ata cag ctc aag agc ctg
aca tct gag gac tct gca gtc tat 288 Ala Tyr Ile Gln Leu Lys Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr 85 90 95 tac tgt gca aga tgg gat
ggt tac tac gga ggg ttt tct tac tgg ggc 336 Tyr Cys Ala Arg Trp Asp
Gly Tyr Tyr Gly Gly Phe Ser Tyr Trp Gly 100 105 110 caa ggg acc atg
gtc acc gtc tcc tca ggt gga ggc ggt tca ggc gga 384 Gln Gly Thr Met
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 ggt ggc
tct ggc ggt ggc gga tcg gac att gag ctc acc cag tct cca 432 Gly Gly
Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro 130 135 140
gca atc atg tct gca act cta ggg gag aag gtc acc atg agc tgc agg 480
Ala Ile Met Ser Ala Thr Leu Gly Glu Lys Val Thr Met Ser Cys Arg 145
150 155 160 gcc agc tca aat gta aag tac atg tac tgg tac cag cag aag
tca ggt 528 Ala Ser Ser Asn Val Lys Tyr Met Tyr Trp Tyr Gln Gln Lys
Ser Gly 165 170 175 gcc tcc ccc aaa cta tgg att tat tac aca tcc aac
ctg gct tct gga 576 Ala Ser Pro Lys Leu Trp Ile Tyr Tyr Thr Ser Asn
Leu Ala Ser Gly 180 185 190 gtc cca gct cgc ttc agt ggc agt ggg tct
ggg acc tct tat tct ctc 624 Val Pro Ala Arg Phe Ser Gly Ser Gly Ser
Gly Thr Ser Tyr Ser Leu 195 200 205 aca atc agc agc gtg gag gct gaa
gat gct gcc act tat tac tgc cag 672 Thr Ile Ser Ser Val Glu Ala Glu
Asp Ala Ala Thr Tyr Tyr Cys Gln 210 215 220 cag ttt act agt tcc ccg
tat acg ttc gga tcg ggc acc aag ctg gaa 720 Gln Phe Thr Ser Ser Pro
Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu 225 230 235 240 atc aaa cgg
gcg gcc gca ggt gcg ccg gtg ccg tat ccg gat ccg ctg 768 Ile Lys Arg
Ala Ala Ala Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu 245 250 255 gaa
ccg cgt gcc gca tag 786 Glu Pro Arg Ala Ala 260 <210> SEQ ID
NO 24 <211> LENGTH: 261 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Construct <400> SEQUENCE: 24 Met
Ala Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 1 5 10
15 Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30 Ser Tyr Val Met His Trp Val Lys Gln Ser Asn Gly Lys Ser
Leu Glu 35 40 45 Trp Ile Gly Thr Ile Asp Pro Tyr Tyr Gly Gly Thr
Ser Tyr Asn Gln 50 55 60 Lys Phe Lys Gly Lys Ala Thr Leu Thr Val
Asp Lys Ser Ser Thr Thr 65 70 75 80 Ala Tyr Ile Gln Leu Lys Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Trp Asp
Gly Tyr Tyr Gly Gly Phe Ser Tyr Trp Gly 100 105 110 Gln Gly Thr Met
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln
Ser Pro 130 135 140 Ala Ile Met Ser Ala Thr Leu Gly Glu Lys Val Thr
Met Ser Cys Arg 145 150 155 160 Ala Ser Ser Asn Val Lys Tyr Met Tyr
Trp Tyr Gln Gln Lys Ser Gly 165 170 175 Ala Ser Pro Lys Leu Trp Ile
Tyr Tyr Thr Ser Asn Leu Ala Ser Gly 180 185 190 Val Pro Ala Arg Phe
Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu 195 200 205 Thr Ile Ser
Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln 210 215 220 Gln
Phe Thr Ser Ser Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu 225 230
235 240 Ile Lys Arg Ala Ala Ala Gly Ala Pro Val Pro Tyr Pro Asp Pro
Leu 245 250 255 Glu Pro Arg Ala Ala 260 <210> SEQ ID NO 25
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 25 Gly Ala Pro Val
Pro Tyr Pro Asp Pro Leu Glu Pro Arg 1 5 10 <210> SEQ ID NO 26
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 26 His His Leu Gly
Gly Ala Lys Gln Ala Gly Asp Val Ser Gly Ser Gly 1 5 10 15 Ser
<210> SEQ ID NO 27 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 27
Ser Gly Ser Gly Ser His His Leu Gly Gly Ala Lys Gln Ala Gly Asp 1 5
10 15 Val Cys <210> SEQ ID NO 28 <211> LENGTH: 22
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 28 His His Leu Gly Gly Ala Lys Gln Ala Gly
Asp Val Ser Gly Ser Gly 1 5 10 15 Ser Tyr Tyr Tyr Tyr Tyr 20
<210> SEQ ID NO 29 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 29
His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val Ser Gly Ser Gly 1 5
10 15 Ser Cys <210> SEQ ID NO 30 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 30 His His Leu Gly Gly Ala Lys Gln Ala Gly
Asp Val Ser Gly Ser Gly 1 5 10 15 Ser Lys <210> SEQ ID NO 31
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 31 Ser Gly Ser Gly
Ser 1 5 <210> SEQ ID NO 32 <211> LENGTH: 10 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: SYNTHESIZED <400>
SEQUENCE: 32 Ser Ser Gly Ser Gly Tyr Tyr Tyr Tyr Tyr 1 5 10
<210> SEQ ID NO 33 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 33
Ser Gly Ser Gly Ser Gly Ser Ser Gly Ser Gly Ser Ser Gly Ser Gly 1 5
10 15 Ser Tyr Tyr Tyr Tyr Tyr 20 <210> SEQ ID NO 34
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 34 Ser Gly Ser Gly
Ser Ser Gly Ser Gly Ser Gly Ser Ser Gly Ser Gly 1 5 10 15 Ser
<210> SEQ ID NO 35 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 35
His Val Gly Gly Ser Ser Val 1 5 <210> SEQ ID NO 36
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 36 Ser Val Arg Gly
Ser Gly Ser Gly Val 1 5 <210> SEQ ID NO 37 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 37 Ser Val Val Arg Asp Gly Ser
Glu Val 1 5 <210> SEQ ID NO 38 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 38 Ser Gly Arg Lys Val Gly Ser Gly Ser Ser
Val 1 5 10 <210> SEQ ID NO 39 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 39 Ser Leu Arg Gly Asp Gly Ser Ser Val 1 5
<210> SEQ ID NO 40 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 40
Ser Val Gly Ser Arg Val
1 5 <210> SEQ ID NO 41 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: SYNTHESIZED <400>
SEQUENCE: 41 Thr Arg Arg Ser Tyr Ser Pro Arg His Asn Phe Asn Trp
Leu Arg Ile 1 5 10 15 Gly Asp Phe Thr 20 <210> SEQ ID NO 42
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 42 Arg Lys Phe Leu
Met Thr Thr Arg Tyr Ser Arg Val 1 5 10 <210> SEQ ID NO 43
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 43 His Arg Gly Cys
Gly Phe Phe Lys Val Leu 1 5 10 <210> SEQ ID NO 44 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 44 Cys Asp Tyr Gln Ile Tyr Gln
Asn Val Phe Asn Phe 1 5 10 <210> SEQ ID NO 45 <211>
LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 45 His Leu Ala Arg Asp Ser Gly
Leu Cys Ser Ala Val Pro Asp Pro Asp 1 5 10 15 <210> SEQ ID NO
46 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 46 Leu Thr Pro
Pro Gly Asp Asn Ala Leu Leu Leu Ala 1 5 10 <210> SEQ ID NO 47
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (6)..(6) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 47 Tyr Ser Thr Leu Pro Xaa Thr Asn Phe Cys
Ala Trp Glu Tyr Thr Ala 1 5 10 15 Tyr His His Val 20 <210>
SEQ ID NO 48 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 48
Lys Phe Leu Arg Ser Ala Gly Val Lys Pro Arg Asn Gly Lys Trp Tyr 1 5
10 15 Asp Ser <210> SEQ ID NO 49 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 49 Lys Gly Val Lys Thr Arg Glu Lys Asn Tyr
Thr Pro Arg Met Trp Thr 1 5 10 15 Glu Arg Ala Asp 20 <210>
SEQ ID NO 50 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 50
Lys Thr Ala Lys Lys Asn Val Phe Phe Cys Ser Val 1 5 10 <210>
SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 51
Pro Pro Ser Cys Val Tyr Pro Ser Arg Lys Cys Ser Pro Thr Ile Ile 1 5
10 15 Thr Phe Ser Gln 20 <210> SEQ ID NO 52 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 52 Leu Ser Ile Val Gly Arg Gln
Arg Cys Arg His Val 1 5 10 <210> SEQ ID NO 53 <211>
LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 53 Glu Arg His Val Ser Thr Gln
Pro Leu Leu Lys Glu Ala Asn Ile Lys 1 5 10 15 <210> SEQ ID NO
54 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 54 Arg Gln Pro
Cys Thr Tyr Ile Glu Val Arg Pro 1 5 10 <210> SEQ ID NO 55
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 55 Thr Leu Leu Cys
Thr Ile Lys Glu Cys Ser 1 5 10 <210> SEQ ID NO 56 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 56 Asp Val Ala Cys Val Thr Ile
Asn Leu Pro Asp Val Cys 1 5 10 <210> SEQ ID NO 57 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 57 Ile Tyr Pro Cys Arg Pro Asn
Thr Ala Leu Asn Asp Tyr Cys Ser Leu 1 5 10 15 Tyr <210> SEQ
ID NO 58 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 58 Thr Phe Pro Cys Lys Pro Leu Arg His Thr
Pro Arg Cys Thr Arg 1 5 10 15 <210> SEQ ID NO 59 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 59 Gly Leu Phe Cys Thr Ala Thr
Ser Pro His Val Thr Arg Ala Cys Lys 1 5 10 15 Glu Leu <210>
SEQ ID NO 60 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 60
Thr Glu Gln Cys Leu Ile His Lys Ser Met Asn Pro Asn Ser Cys Arg 1 5
10 15 Gly Phe <210> SEQ ID NO 61 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 61 Phe Thr His Ala Leu Asp Pro Gly Gln Leu
Ala Leu 1 5 10 <210> SEQ ID NO 62 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 62 His His Leu Ala Ser Leu Tyr His His Ser
Tyr 1 5 10 <210> SEQ ID NO 63 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 63 Asn Ala Gln Leu Ser Leu Ser Arg Gly His
Leu His Gln Met Ile Gln 1 5 10 15 <210> SEQ ID NO 64
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 64 Lys Ala Arg Leu
Pro Pro Glu Pro Ser Phe Thr Val Phe Thr Cys Gly 1 5 10 15 Arg Ala
Ser Ala 20 <210> SEQ ID NO 65 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 65 Leu Ser Pro Gln Arg Phe Cys Tyr Gly Tyr
Leu Phe Gln Phe Thr Leu 1 5 10 15 Val Leu His Leu 20 <210>
SEQ ID NO 66 <211> LENGTH: 14 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 66
Thr Phe Phe Val Ser Thr Arg His Asp Leu Val Ile Cys Leu 1 5 10
<210> SEQ ID NO 67 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 67
Met His Val Glu Arg Val Thr Arg Leu His Thr 1 5 10 <210> SEQ
ID NO 68 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 68 Pro His Phe
Cys Pro Ala Met Lys Leu Ala Ala Ala Leu Glu 1 5 10 <210> SEQ
ID NO 69 <211> LENGTH: 20 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYTHENSIZED <400> SEQUENCE: 69 His Arg Leu
Ser Arg Tyr Arg Pro Arg Leu Gly Pro Tyr Phe Cys Pro 1 5 10 15 Ser
Pro Glu Val 20 <210> SEQ ID NO 70 <211> LENGTH: 4391
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION
NUMBER: P98160 <309> DATABASE ENTRY DATE: 2003-02-28
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(4391) <400>
SEQUENCE: 70 Met Gly Trp Arg Ala Pro Gly Ala Leu Leu Leu Ala Leu
Leu Leu His 1 5 10 15 Gly Arg Leu Leu Ala Val Thr His Gly Leu Arg
Ala Tyr Asp Gly Leu 20 25 30 Ser Leu Pro Glu Asp Ile Glu Thr Val
Thr Ala Ser Gln Met Arg Trp 35 40 45 Thr His Ser Tyr Leu Ser Asp
Asp Glu Asp Met Leu Ala Asp Ser Ile 50 55 60 Ser Gly Asp Asp Leu
Gly Ser Gly Asp Leu Gly Ser Gly Asp Phe Gln 65 70 75 80 Met Val Tyr
Phe Arg Ala Leu Val Asn Phe Thr Arg Ser Ile Glu Tyr 85 90 95 Ser
Pro Gln Leu Glu Asp Ala Gly Ser Arg Glu Phe Arg Glu Val Ser 100 105
110 Glu Ala Val Val Asp Thr Leu Glu Ser Glu Tyr Leu Lys Ile Pro Gly
115 120 125 Asp Gln Val Val Ser Val Val Phe Ile Lys Glu Leu Asp Gly
Trp Val 130 135 140 Phe Val Glu Leu Asp Val Gly Ser Glu Gly Asn Ala
Asp Gly Ala Gln 145 150 155 160 Ile Gln Glu Met Leu Leu Arg Val Ile
Ser Ser Gly Ser Val Ala Ser 165 170 175 Tyr Val Thr Ser Pro Gln Gly
Phe Gln Phe Arg Arg Leu Gly Thr Val 180 185 190 Pro Gln Phe Pro Arg
Ala Cys Thr Glu Ala Glu Phe Ala Cys His Ser 195 200 205 Tyr Asn Glu
Cys Val Ala Leu Glu Tyr Arg Cys Asp Arg Arg Pro Asp 210 215 220 Cys
Arg Asp Met Ser Asp Glu Leu Asn Cys Glu Glu Pro Val Leu Gly 225 230
235 240 Ile Ser Pro Thr Phe Ser Leu Leu Val Glu Thr Thr Ser Leu Pro
Pro 245 250 255 Arg Pro Glu Thr Thr Ile Met Arg Gln Pro Pro Val Thr
His Ala Pro 260 265 270 Gln Pro Leu Leu Pro Gly Ser Val Arg Pro Leu
Pro Cys Gly Pro Gln 275 280 285 Glu Ala Ala Cys Arg Asn Gly His Cys
Ile Pro Arg Asp Tyr Leu Cys 290 295 300 Asp Gly Gln Glu Asp Cys Glu
Asp Gly Ser Asp Glu Leu Asp Cys Gly 305 310 315 320 Pro Pro Pro Pro
Cys Glu Pro Asn Glu Phe Pro Cys Gly Asn Gly His 325 330 335 Cys Ala
Leu Lys Leu Trp Arg Cys Asp Gly Asp Phe Asp Cys Glu Asp 340 345 350
Arg Thr Asp Glu Ala Asn Cys Pro Thr Lys Arg Pro Glu Glu Val Cys 355
360 365 Gly Pro Thr Gln Phe Arg Cys Val Ser Thr Asn Met Cys Ile Pro
Ala 370 375 380 Ser Phe His Cys Asp Glu Glu Ser Asp Cys Pro Asp Arg
Ser Asp Glu 385 390 395 400 Phe Gly Cys Met Pro Pro Gln Val Val Thr
Pro Pro Arg Glu Ser Ile 405 410 415
Gln Ala Ser Arg Gly Gln Thr Val Thr Phe Thr Cys Val Ala Ile Gly 420
425 430 Val Pro Thr Pro Ile Ile Asn Trp Arg Leu Asn Trp Gly His Ile
Pro 435 440 445 Ser His Pro Arg Val Thr Val Thr Ser Glu Gly Gly Arg
Gly Thr Leu 450 455 460 Ile Ile Arg Asp Val Lys Glu Ser Asp Gln Gly
Ala Tyr Thr Cys Glu 465 470 475 480 Ala Met Asn Ala Arg Gly Met Val
Phe Gly Ile Pro Asp Gly Val Leu 485 490 495 Glu Leu Val Pro Gln Arg
Gly Pro Cys Pro Asp Gly His Phe Tyr Leu 500 505 510 Glu His Ser Ala
Ala Cys Leu Pro Cys Phe Cys Phe Gly Ile Thr Ser 515 520 525 Val Cys
Gln Ser Thr Arg Arg Phe Arg Asp Gln Ile Arg Leu Arg Phe 530 535 540
Asp Gln Pro Asp Asp Phe Lys Gly Val Asn Val Thr Met Pro Ala Gln 545
550 555 560 Pro Gly Thr Pro Pro Leu Ser Ser Thr Gln Leu Gln Ile Asp
Pro Ser 565 570 575 Leu His Glu Phe Gln Leu Val Asp Leu Ser Arg Arg
Phe Leu Val His 580 585 590 Asp Ser Phe Trp Ala Leu Pro Glu Gln Phe
Leu Gly Asn Lys Val Asp 595 600 605 Ser Tyr Gly Gly Ser Leu Arg Tyr
Asn Val Arg Tyr Glu Leu Ala Arg 610 615 620 Gly Met Leu Glu Pro Val
Gln Arg Pro Asp Val Val Leu Val Gly Ala 625 630 635 640 Gly Tyr Arg
Leu Leu Ser Arg Gly His Thr Pro Thr Gln Pro Gly Ala 645 650 655 Leu
Asn Gln Arg Gln Val Gln Phe Ser Glu Glu His Trp Val His Glu 660 665
670 Ser Gly Arg Pro Val Gln Arg Ala Glu Leu Leu Gln Val Leu Gln Ser
675 680 685 Leu Glu Ala Val Leu Ile Gln Thr Val Tyr Asn Thr Lys Met
Ala Ser 690 695 700 Val Gly Leu Ser Asp Ile Ala Met Asp Thr Thr Val
Thr His Ala Thr 705 710 715 720 Ser His Gly Arg Ala His Ser Val Glu
Glu Cys Arg Cys Pro Ile Gly 725 730 735 Tyr Ser Gly Leu Ser Cys Glu
Ser Cys Asp Ala His Phe Thr Arg Val 740 745 750 Pro Gly Gly Pro Tyr
Leu Gly Thr Cys Ser Gly Cys Ser Cys Asn Gly 755 760 765 His Ala Ser
Ser Cys Asp Pro Val Tyr Gly His Cys Leu Asn Cys Gln 770 775 780 His
Asn Thr Glu Gly Pro Gln Cys Asn Lys Cys Lys Ala Gly Phe Phe 785 790
795 800 Gly Asp Ala Met Lys Ala Thr Ala Thr Ser Cys Arg Pro Cys Pro
Cys 805 810 815 Pro Tyr Ile Asp Ala Ser Arg Arg Phe Ser Asp Thr Cys
Phe Leu Asp 820 825 830 Thr Asp Gly Gln Ala Thr Cys Asp Ala Cys Ala
Pro Gly Tyr Thr Gly 835 840 845 Arg Arg Cys Glu Ser Cys Ala Pro Gly
Tyr Glu Gly Asn Pro Ile Gln 850 855 860 Pro Gly Gly Lys Cys Arg Pro
Val Asn Gln Glu Ile Val Arg Cys Asp 865 870 875 880 Glu Arg Gly Ser
Met Gly Thr Ser Gly Glu Ala Cys Arg Cys Lys Asn 885 890 895 Asn Val
Val Gly Arg Leu Cys Asn Glu Cys Ala Asp Gly Ser Phe His 900 905 910
Leu Ser Thr Arg Asn Pro Asp Gly Cys Leu Lys Cys Phe Cys Met Gly 915
920 925 Val Ser Arg His Cys Thr Ser Ser Ser Trp Ser Arg Ala Gln Leu
His 930 935 940 Gly Ala Ser Glu Glu Pro Gly His Phe Ser Leu Thr Asn
Ala Ala Ser 945 950 955 960 Thr His Thr Thr Asn Glu Gly Ile Phe Ser
Pro Thr Pro Gly Glu Leu 965 970 975 Gly Phe Ser Ser Phe His Arg Leu
Leu Ser Gly Pro Tyr Phe Trp Ser 980 985 990 Leu Pro Ser Arg Phe Leu
Gly Asp Lys Val Thr Ser Tyr Gly Gly Glu 995 1000 1005 Leu Arg Phe
Thr Val Thr Gln Arg Ser Gln Pro Gly Ser Thr Pro 1010 1015 1020 Leu
His Gly Gln Pro Leu Val Val Leu Gln Gly Asn Asn Ile Ile 1025 1030
1035 Leu Glu His His Val Ala Gln Glu Pro Ser Pro Gly Gln Pro Ser
1040 1045 1050 Thr Phe Ile Val Pro Phe Arg Glu Gln Ala Trp Gln Arg
Pro Asp 1055 1060 1065 Gly Gln Pro Ala Thr Arg Glu His Leu Leu Met
Ala Leu Ala Gly 1070 1075 1080 Ile Asp Thr Leu Leu Ile Arg Ala Ser
Tyr Ala Gln Gln Pro Ala 1085 1090 1095 Glu Ser Arg Val Ser Gly Ile
Ser Met Asp Val Ala Val Pro Glu 1100 1105 1110 Glu Thr Gly Gln Asp
Pro Ala Leu Glu Val Glu Gln Cys Ser Cys 1115 1120 1125 Pro Pro Gly
Tyr Arg Gly Pro Ser Cys Gln Asp Cys Asp Thr Gly 1130 1135 1140 Tyr
Thr Arg Thr Pro Ser Gly Leu Tyr Leu Gly Thr Cys Glu Arg 1145 1150
1155 Cys Ser Cys His Gly His Ser Glu Ala Cys Glu Pro Glu Thr Gly
1160 1165 1170 Ala Cys Gln Gly Cys Gln His His Thr Glu Gly Pro Arg
Cys Glu 1175 1180 1185 Gln Cys Gln Pro Gly Tyr Tyr Gly Asp Ala Gln
Arg Gly Thr Pro 1190 1195 1200 Gln Asp Cys Gln Leu Cys Pro Cys Tyr
Gly Asp Pro Ala Ala Gly 1205 1210 1215 Gln Ala Ala His Thr Cys Phe
Leu Asp Thr Asp Gly His Pro Thr 1220 1225 1230 Cys Asp Ala Cys Ser
Pro Gly His Ser Gly Arg His Cys Glu Arg 1235 1240 1245 Cys Ala Pro
Gly Tyr Tyr Gly Asn Pro Ser Gln Gly Gln Pro Cys 1250 1255 1260 Gln
Arg Asp Ser Gln Val Pro Gly Pro Ile Gly Cys Asn Cys Asp 1265 1270
1275 Pro Gln Gly Ser Val Ser Ser Gln Cys Asp Ala Ala Gly Gln Cys
1280 1285 1290 Gln Cys Lys Ala Gln Val Glu Gly Leu Thr Cys Ser His
Cys Arg 1295 1300 1305 Pro His His Phe His Leu Ser Ala Ser Asn Pro
Asp Gly Cys Leu 1310 1315 1320 Pro Cys Phe Cys Met Gly Ile Thr Gln
Gln Cys Ala Ser Ser Ala 1325 1330 1335 Tyr Thr Arg His Leu Ile Ser
Thr His Phe Ala Pro Gly Asp Phe 1340 1345 1350 Gln Gly Phe Ala Leu
Val Asn Pro Gln Arg Asn Ser Arg Leu Thr 1355 1360 1365 Gly Glu Phe
Thr Val Glu Pro Val Pro Glu Gly Ala Gln Leu Ser 1370 1375 1380 Phe
Gly Asn Phe Ala Gln Leu Gly His Glu Ser Phe Tyr Trp Gln 1385 1390
1395 Leu Pro Glu Thr Tyr Gln Gly Asp Lys Val Ala Ala Tyr Gly Gly
1400 1405 1410 Lys Leu Arg Tyr Thr Leu Ser Tyr Thr Ala Gly Pro Gln
Gly Ser 1415 1420 1425 Pro Leu Ser Asp Pro Asp Val Gln Ile Thr Gly
Asn Asn Ile Met 1430 1435 1440 Leu Val Ala Ser Gln Pro Ala Leu Gln
Gly Pro Glu Arg Arg Ser 1445 1450 1455 Tyr Glu Ile Met Phe Arg Glu
Glu Phe Trp Arg Arg Pro Asp Gly 1460 1465 1470 Gln Pro Ala Thr Arg
Glu His Leu Leu Met Ala Leu Ala Asp Leu 1475 1480 1485 Asp Glu Leu
Leu Ile Arg Ala Thr Phe Ser Ser Val Pro Leu Val 1490 1495 1500 Ala
Ser Ile Ser Ala Val Ser Leu Glu Val Ala Gln Pro Gly Pro 1505 1510
1515 Ser Asn Arg Pro Arg Ala Leu Glu Val Glu Glu Cys Arg Cys Pro
1520 1525 1530 Pro Gly Tyr Ile Gly Leu Ser Cys Gln Asp Cys Ala Pro
Gly Tyr 1535 1540 1545 Thr Arg Thr Gly Ser Gly Leu Tyr Leu Gly His
Cys Glu Leu Cys 1550 1555 1560 Glu Cys Asn Gly His Ser Asp Leu Cys
His Pro Glu Thr Gly Ala 1565 1570 1575 Cys Ser Gln Cys Gln His Asn
Ala Ala Gly Glu Phe Cys Glu Leu 1580 1585 1590 Cys Ala Pro Gly Tyr
Tyr Gly Asp Ala Thr Ala Gly Thr Pro Glu 1595 1600 1605 Asp Cys Gln
Pro Cys Ala Cys Pro Leu Thr Asn Pro Glu Asn Met 1610 1615 1620 Phe
Ser Arg Thr Cys Glu Ser Leu Gly Ala Gly Gly Tyr Arg Cys 1625 1630
1635 Thr Ala Cys Glu Pro Gly Tyr Thr Gly Gln Tyr Cys Glu Gln Cys
1640 1645 1650 Gly Pro Gly Tyr Val Gly Asn Pro Ser Val Gln Gly Gly
Gln Cys 1655 1660 1665 Leu Pro Glu Thr Asn Gln Ala Pro Leu Val Val
Glu Val His Pro 1670 1675 1680 Ala Arg Ser Ile Val Pro Gln Gly Gly
Ser His Ser Leu Arg Cys 1685 1690 1695 Gln Val Ser Gly Ser Pro Pro
His Tyr Phe Tyr Trp Ser Arg Glu
1700 1705 1710 Asp Gly Arg Pro Val Pro Ser Gly Thr Gln Gln Arg His
Gln Gly 1715 1720 1725 Ser Glu Leu His Phe Pro Ser Val Gln Pro Ser
Asp Ala Gly Val 1730 1735 1740 Tyr Ile Cys Thr Cys Arg Asn Leu His
Gln Ser Asn Thr Ser Arg 1745 1750 1755 Ala Glu Leu Leu Val Thr Glu
Ala Pro Ser Lys Pro Ile Thr Val 1760 1765 1770 Thr Val Glu Glu Gln
Arg Ser Gln Ser Val Arg Pro Gly Ala Asp 1775 1780 1785 Val Thr Phe
Ile Cys Thr Ala Lys Ser Lys Ser Pro Ala Tyr Thr 1790 1795 1800 Leu
Val Trp Thr Arg Leu His Asn Gly Lys Leu Pro Thr Arg Ala 1805 1810
1815 Met Asp Phe Asn Gly Ile Leu Thr Ile Arg Asn Val Gln Leu Ser
1820 1825 1830 Asp Ala Gly Thr Tyr Val Cys Thr Gly Ser Asn Met Phe
Ala Met 1835 1840 1845 Asp Gln Gly Thr Ala Thr Leu His Val Gln Ala
Ser Gly Thr Leu 1850 1855 1860 Ser Ala Pro Val Val Ser Ile His Pro
Pro Gln Leu Thr Val Gln 1865 1870 1875 Pro Gly Gln Leu Ala Glu Phe
Arg Cys Ser Ala Thr Gly Ser Pro 1880 1885 1890 Thr Pro Thr Leu Glu
Trp Thr Gly Gly Pro Gly Gly Gln Leu Pro 1895 1900 1905 Ala Lys Ala
Gln Ile His Gly Gly Ile Leu Arg Leu Pro Ala Val 1910 1915 1920 Glu
Pro Thr Asp Gln Ala Gln Tyr Leu Cys Arg Ala His Ser Ser 1925 1930
1935 Ala Gly Gln Gln Val Ala Arg Ala Val Leu His Val His Gly Gly
1940 1945 1950 Gly Gly Pro Arg Val Gln Val Ser Pro Glu Arg Thr Gln
Val His 1955 1960 1965 Ala Gly Arg Thr Val Arg Leu Tyr Cys Arg Ala
Ala Gly Val Pro 1970 1975 1980 Ser Ala Thr Ile Thr Trp Arg Lys Glu
Gly Gly Ser Leu Pro Pro 1985 1990 1995 Gln Ala Arg Ser Glu Arg Thr
Asp Ile Ala Thr Leu Leu Ile Pro 2000 2005 2010 Ala Ile Thr Thr Ala
Asp Ala Gly Phe Tyr Leu Cys Val Ala Thr 2015 2020 2025 Ser Pro Ala
Gly Thr Ala Gln Ala Arg Ile Gln Val Val Val Leu 2030 2035 2040 Ser
Ala Ser Asp Ala Ser Pro Pro Pro Val Lys Ile Glu Ser Ser 2045 2050
2055 Ser Pro Ser Val Thr Glu Gly Gln Thr Leu Asp Leu Asn Cys Val
2060 2065 2070 Val Ala Gly Ser Ala His Ala Gln Val Thr Trp Tyr Arg
Arg Gly 2075 2080 2085 Gly Ser Leu Pro Pro His Thr Gln Val His Gly
Ser Arg Leu Arg 2090 2095 2100 Leu Pro Gln Val Ser Pro Ala Asp Ser
Gly Glu Tyr Val Cys Arg 2105 2110 2115 Val Glu Asn Gly Ser Gly Pro
Lys Glu Ala Ser Ile Thr Val Ser 2120 2125 2130 Val Leu His Gly Thr
His Ser Gly Pro Ser Tyr Thr Pro Val Pro 2135 2140 2145 Gly Ser Thr
Arg Pro Ile Arg Ile Glu Pro Ser Ser Ser His Val 2150 2155 2160 Ala
Glu Gly Gln Thr Leu Asp Leu Asn Cys Val Val Pro Gly Gln 2165 2170
2175 Ala His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu Pro
2180 2185 2190 Ala Arg His Gln Thr His Gly Ser Leu Leu Arg Leu His
Gln Val 2195 2200 2205 Thr Pro Ala Asp Ser Gly Glu Tyr Val Cys His
Val Val Gly Thr 2210 2215 2220 Ser Gly Pro Leu Glu Ala Ser Val Leu
Val Thr Ile Glu Ala Ser 2225 2230 2235 Val Ile Pro Gly Pro Ile Pro
Pro Val Arg Ile Glu Ser Ser Ser 2240 2245 2250 Ser Thr Val Ala Glu
Gly Gln Thr Leu Asp Leu Ser Cys Val Val 2255 2260 2265 Ala Gly Gln
Ala His Ala Gln Val Thr Trp Tyr Lys Arg Gly Gly 2270 2275 2280 Ser
Leu Pro Ala Arg His Gln Val Arg Gly Ser Arg Leu Tyr Ile 2285 2290
2295 Phe Gln Ala Ser Pro Ala Asp Ala Gly Gln Tyr Val Cys Arg Ala
2300 2305 2310 Ser Asn Gly Met Glu Ala Ser Ile Thr Val Thr Val Thr
Gly Thr 2315 2320 2325 Gln Gly Ala Asn Leu Ala Tyr Pro Ala Gly Ser
Thr Gln Pro Ile 2330 2335 2340 Arg Ile Glu Pro Ser Ser Ser Gln Val
Ala Glu Gly Gln Thr Leu 2345 2350 2355 Asp Leu Asn Cys Val Val Pro
Gly Gln Ser His Ala Gln Val Thr 2360 2365 2370 Trp His Lys Arg Gly
Gly Ser Leu Pro Val Arg His Gln Thr His 2375 2380 2385 Gly Ser Leu
Leu Arg Leu Tyr Gln Ala Ser Pro Ala Asp Ser Gly 2390 2395 2400 Glu
Tyr Val Cys Arg Val Leu Gly Ser Ser Val Pro Leu Glu Ala 2405 2410
2415 Ser Val Leu Val Thr Ile Glu Pro Ala Gly Ser Val Pro Ala Leu
2420 2425 2430 Gly Val Thr Pro Thr Val Arg Ile Glu Ser Ser Ser Ser
Gln Val 2435 2440 2445 Ala Glu Gly Gln Thr Leu Asp Leu Asn Cys Leu
Val Ala Gly Gln 2450 2455 2460 Ala His Ala Gln Val Thr Trp His Lys
Arg Gly Gly Ser Leu Pro 2465 2470 2475 Ala Arg His Gln Val His Gly
Ser Arg Leu Arg Leu Leu Gln Val 2480 2485 2490 Thr Pro Ala Asp Ser
Gly Glu Tyr Val Cys Arg Val Val Gly Ser 2495 2500 2505 Ser Gly Thr
Gln Glu Ala Ser Val Leu Val Thr Ile Gln Gln Arg 2510 2515 2520 Leu
Ser Gly Ser His Ser Gln Gly Val Ala Tyr Pro Val Arg Ile 2525 2530
2535 Glu Ser Ser Ser Ala Ser Leu Ala Asn Gly His Thr Leu Asp Leu
2540 2545 2550 Asn Cys Leu Val Ala Ser Gln Ala Pro His Thr Ile Thr
Trp Tyr 2555 2560 2565 Lys Arg Gly Gly Ser Leu Pro Ser Arg His Gln
Ile Val Gly Ser 2570 2575 2580 Arg Leu Arg Ile Pro Gln Val Thr Pro
Ala Asp Ser Gly Glu Tyr 2585 2590 2595 Val Cys His Val Ser Asn Gly
Ala Gly Ser Arg Glu Thr Ser Leu 2600 2605 2610 Ile Val Thr Ile Gln
Gly Ser Gly Ser Ser His Val Pro Ser Val 2615 2620 2625 Ser Pro Pro
Ile Arg Ile Glu Ser Ser Ser Pro Thr Val Val Glu 2630 2635 2640 Gly
Gln Thr Leu Asp Leu Asn Cys Val Val Ala Arg Gln Pro Gln 2645 2650
2655 Ala Ile Ile Thr Trp Tyr Lys Arg Gly Gly Ser Leu Pro Ser Arg
2660 2665 2670 His Gln Thr His Gly Ser His Leu Arg Leu His Gln Met
Ser Val 2675 2680 2685 Ala Asp Ser Gly Glu Tyr Val Cys Arg Ala Asn
Asn Asn Ile Asp 2690 2695 2700 Ala Leu Glu Ala Ser Ile Val Ile Ser
Val Ser Pro Ser Ala Gly 2705 2710 2715 Ser Pro Ser Ala Pro Gly Ser
Ser Met Pro Ile Arg Ile Glu Ser 2720 2725 2730 Ser Ser Ser His Val
Ala Glu Gly Glu Thr Leu Asp Leu Asn Cys 2735 2740 2745 Val Val Pro
Gly Gln Ala His Ala Gln Val Thr Trp His Lys Arg 2750 2755 2760 Gly
Gly Ser Leu Pro Ser His His Gln Thr Arg Gly Ser Arg Leu 2765 2770
2775 Arg Leu His His Val Ser Pro Ala Asp Ser Gly Glu Tyr Val Cys
2780 2785 2790 Arg Val Met Gly Ser Ser Gly Pro Leu Glu Ala Ser Val
Leu Val 2795 2800 2805 Thr Ile Glu Ala Ser Gly Ser Ser Ala Val His
Val Pro Ala Pro 2810 2815 2820 Gly Gly Ala Pro Pro Ile Arg Ile Glu
Pro Ser Ser Ser Arg Val 2825 2830 2835 Ala Glu Gly Gln Thr Leu Asp
Leu Lys Cys Val Val Pro Gly Gln 2840 2845 2850 Ala His Ala Gln Val
Thr Trp His Lys Arg Gly Gly Asn Leu Pro 2855 2860 2865 Ala Arg His
Gln Val His Gly Pro Leu Leu Arg Leu Asn Gln Val 2870 2875 2880 Ser
Pro Ala Asp Ser Gly Glu Tyr Ser Cys Gln Val Thr Gly Ser 2885 2890
2895 Ser Gly Thr Leu Glu Ala Ser Val Leu Val Thr Ile Glu Pro Ser
2900 2905 2910 Ser Pro Gly Pro Ile Pro Ala Pro Gly Leu Ala Gln Pro
Ile Tyr 2915 2920 2925 Ile Glu Ala Ser Ser Ser His Val Thr Glu Gly
Gln Thr Leu Asp 2930 2935 2940 Leu Asn Cys Val Val Pro Gly Gln Ala
His Ala Gln Val Thr Trp 2945 2950 2955
Tyr Lys Arg Gly Gly Ser Leu Pro Ala Arg His Gln Thr His Gly 2960
2965 2970 Ser Gln Leu Arg Leu His Leu Val Ser Pro Ala Asp Ser Gly
Glu 2975 2980 2985 Tyr Val Cys Arg Ala Ala Ser Gly Pro Gly Pro Glu
Gln Glu Ala 2990 2995 3000 Ser Phe Thr Val Thr Val Pro Pro Ser Glu
Gly Ser Ser Tyr Arg 3005 3010 3015 Leu Arg Ser Pro Val Ile Ser Ile
Asp Pro Pro Ser Ser Thr Val 3020 3025 3030 Gln Gln Gly Gln Asp Ala
Ser Phe Lys Cys Leu Ile His Asp Gly 3035 3040 3045 Ala Ala Pro Ile
Ser Leu Glu Trp Lys Thr Arg Asn Gln Glu Leu 3050 3055 3060 Glu Asp
Asn Val His Ile Ser Pro Asn Gly Ser Ile Ile Thr Ile 3065 3070 3075
Val Gly Thr Arg Pro Ser Asn His Gly Thr Tyr Arg Cys Val Ala 3080
3085 3090 Ser Asn Ala Tyr Gly Val Ala Gln Ser Val Val Asn Leu Ser
Val 3095 3100 3105 His Gly Pro Pro Thr Val Ser Val Leu Pro Glu Gly
Pro Val Trp 3110 3115 3120 Val Lys Val Gly Lys Ala Val Thr Leu Glu
Cys Val Ser Ala Gly 3125 3130 3135 Glu Pro Arg Ser Ser Ala Arg Trp
Thr Arg Ile Ser Ser Thr Pro 3140 3145 3150 Ala Lys Leu Glu Gln Arg
Thr Tyr Gly Leu Met Asp Ser His Ala 3155 3160 3165 Val Leu Gln Ile
Ser Ser Ala Lys Pro Ser Asp Ala Gly Thr Tyr 3170 3175 3180 Val Cys
Leu Ala Gln Asn Ala Leu Gly Thr Ala Gln Lys Gln Val 3185 3190 3195
Glu Val Ile Val Asp Thr Gly Ala Met Ala Pro Gly Ala Pro Gln 3200
3205 3210 Val Gln Ala Glu Glu Ala Glu Leu Thr Val Glu Ala Gly His
Thr 3215 3220 3225 Ala Thr Leu Arg Cys Ser Ala Thr Gly Ser Pro Ala
Pro Thr Ile 3230 3235 3240 His Trp Ser Lys Leu Arg Ser Pro Leu Pro
Trp Gln His Arg Leu 3245 3250 3255 Glu Gly Asp Thr Leu Ile Ile Pro
Arg Val Ala Gln Gln Asp Ser 3260 3265 3270 Gly Gln Tyr Ile Cys Asn
Ala Thr Ser Pro Ala Gly His Ala Glu 3275 3280 3285 Ala Thr Ile Ile
Leu His Val Glu Ser Pro Pro Tyr Ala Thr Thr 3290 3295 3300 Val Pro
Glu His Ala Ser Val Gln Ala Gly Glu Thr Val Gln Leu 3305 3310 3315
Gln Cys Leu Ala His Gly Thr Pro Pro Leu Thr Phe Gln Trp Ser 3320
3325 3330 Arg Val Gly Ser Ser Leu Pro Gly Arg Ala Thr Ala Arg Asn
Glu 3335 3340 3345 Leu Leu His Phe Glu Arg Ala Ala Pro Glu Asp Ser
Gly Arg Tyr 3350 3355 3360 Arg Cys Arg Val Thr Asn Lys Val Gly Ser
Ala Glu Ala Phe Ala 3365 3370 3375 Gln Leu Leu Val Gln Gly Pro Pro
Gly Ser Leu Pro Ala Thr Ser 3380 3385 3390 Ile Pro Ala Gly Ser Thr
Pro Thr Val Gln Val Thr Pro Gln Leu 3395 3400 3405 Glu Thr Lys Ser
Ile Gly Ala Ser Val Glu Phe His Cys Ala Val 3410 3415 3420 Pro Ser
Asp Arg Gly Thr Gln Leu Arg Trp Phe Lys Glu Gly Gly 3425 3430 3435
Gln Leu Pro Pro Gly His Ser Val Gln Asp Gly Val Leu Arg Ile 3440
3445 3450 Gln Asn Leu Asp Gln Ser Cys Gln Gly Thr Tyr Ile Cys Gln
Ala 3455 3460 3465 His Gly Pro Trp Gly Lys Ala Gln Ala Ser Ala Gln
Leu Val Ile 3470 3475 3480 Gln Ala Leu Pro Ser Val Leu Ile Asn Ile
Arg Thr Ser Val Gln 3485 3490 3495 Thr Val Val Val Gly His Ala Val
Glu Phe Glu Cys Leu Ala Leu 3500 3505 3510 Gly Asp Pro Lys Pro Gln
Val Thr Trp Ser Lys Val Gly Gly His 3515 3520 3525 Leu Arg Pro Gly
Ile Val Gln Ser Gly Gly Val Val Arg Ile Ala 3530 3535 3540 His Val
Glu Leu Ala Asp Ala Gly Gln Tyr Arg Cys Thr Ala Thr 3545 3550 3555
Asn Ala Ala Gly Thr Thr Gln Ser His Val Leu Leu Leu Val Gln 3560
3565 3570 Ala Leu Pro Gln Ile Ser Met Pro Gln Glu Val Arg Val Pro
Ala 3575 3580 3585 Gly Ser Ala Ala Val Phe Pro Cys Ile Ala Ser Gly
Tyr Pro Thr 3590 3595 3600 Pro Asp Ile Ser Trp Ser Lys Leu Asp Gly
Ser Leu Pro Pro Asp 3605 3610 3615 Ser Arg Leu Glu Asn Asn Met Leu
Met Leu Pro Ser Val Arg Pro 3620 3625 3630 Gln Asp Ala Gly Thr Tyr
Val Cys Thr Ala Thr Asn Arg Gln Gly 3635 3640 3645 Lys Val Lys Ala
Phe Ala His Leu Gln Val Pro Glu Arg Val Val 3650 3655 3660 Pro Tyr
Phe Thr Gln Thr Pro Tyr Ser Phe Leu Pro Leu Pro Thr 3665 3670 3675
Ile Lys Asp Ala Tyr Arg Lys Phe Glu Ile Lys Ile Thr Phe Arg 3680
3685 3690 Pro Asp Ser Ala Asp Gly Met Leu Leu Tyr Asn Gly Gln Lys
Arg 3695 3700 3705 Val Pro Gly Ser Pro Thr Asn Leu Ala Asn Arg Gln
Pro Asp Phe 3710 3715 3720 Ile Ser Phe Gly Leu Val Gly Gly Arg Pro
Glu Phe Arg Phe Asp 3725 3730 3735 Ala Gly Ser Gly Met Ala Thr Ile
Arg His Pro Thr Pro Leu Ala 3740 3745 3750 Leu Gly His Phe His Thr
Val Thr Leu Leu Arg Ser Leu Thr Gln 3755 3760 3765 Gly Ser Leu Ile
Val Gly Asp Leu Ala Pro Val Asn Gly Thr Ser 3770 3775 3780 Gln Gly
Lys Phe Gln Gly Leu Asp Leu Asn Glu Glu Leu Tyr Leu 3785 3790 3795
Gly Gly Tyr Pro Asp Tyr Gly Ala Ile Pro Lys Ala Gly Leu Ser 3800
3805 3810 Ser Gly Phe Ile Gly Cys Val Arg Glu Leu Arg Ile Gln Gly
Glu 3815 3820 3825 Glu Ile Val Phe His Asp Leu Asn Leu Thr Ala His
Gly Ile Ser 3830 3835 3840 His Cys Pro Thr Cys Arg Asp Arg Pro Cys
Gln Asn Gly Gly Gln 3845 3850 3855 Cys His Asp Ser Glu Ser Ser Ser
Tyr Val Cys Val Cys Pro Ala 3860 3865 3870 Gly Phe Thr Gly Ser Arg
Cys Glu His Ser Gln Ala Leu His Cys 3875 3880 3885 His Pro Glu Ala
Cys Gly Pro Asp Ala Thr Cys Val Asn Arg Pro 3890 3895 3900 Asp Gly
Arg Gly Tyr Thr Cys Arg Cys His Leu Gly Arg Ser Gly 3905 3910 3915
Leu Arg Cys Glu Glu Gly Val Thr Val Thr Thr Pro Ser Leu Ser 3920
3925 3930 Gly Ala Gly Ser Tyr Leu Ala Leu Pro Ala Leu Thr Asn Thr
His 3935 3940 3945 His Glu Leu Arg Leu Asp Val Glu Phe Lys Pro Leu
Ala Pro Asp 3950 3955 3960 Gly Val Leu Leu Phe Ser Gly Gly Lys Ser
Gly Pro Val Glu Asp 3965 3970 3975 Phe Val Ser Leu Ala Met Val Gly
Gly His Leu Glu Phe Arg Tyr 3980 3985 3990 Glu Leu Gly Ser Gly Leu
Ala Val Leu Arg Ser Ala Glu Pro Leu 3995 4000 4005 Ala Leu Gly Arg
Trp His Arg Val Ser Ala Glu Arg Leu Asn Lys 4010 4015 4020 Asp Gly
Ser Leu Arg Val Asn Gly Gly Arg Pro Val Leu Arg Ser 4025 4030 4035
Ser Pro Gly Lys Ser Gln Gly Leu Asn Leu His Thr Leu Leu Tyr 4040
4045 4050 Leu Gly Gly Val Glu Pro Ser Val Pro Leu Ser Pro Ala Thr
Asn 4055 4060 4065 Met Ser Ala His Phe Arg Gly Cys Val Gly Glu Val
Ser Val Asn 4070 4075 4080 Gly Lys Arg Leu Asp Leu Thr Tyr Ser Phe
Leu Gly Ser Gln Gly 4085 4090 4095 Ile Gly Gln Cys Tyr Asp Ser Ser
Pro Cys Glu Arg Gln Pro Cys 4100 4105 4110 Gln His Gly Ala Thr Cys
Met Pro Ala Gly Glu Tyr Glu Phe Gln 4115 4120 4125 Cys Leu Cys Arg
Asp Gly Phe Lys Gly Asp Leu Cys Glu His Glu 4130 4135 4140 Glu Asn
Pro Cys Gln Leu Arg Glu Pro Cys Leu His Gly Gly Thr 4145 4150 4155
Cys Gln Gly Thr Arg Cys Leu Cys Leu Pro Gly Phe Ser Gly Pro 4160
4165 4170 Arg Cys Gln Gln Gly Ser Gly His Gly Ile Ala Glu Ser Asp
Trp 4175 4180 4185 His Leu Glu Gly Ser Gly Gly Asn Asp Ala Pro Gly
Gln Tyr Gly 4190 4195 4200 Ala Tyr Phe His Asp Asp Gly Phe Leu Ala
Phe Pro Gly His Val 4205 4210 4215
Phe Ser Arg Ser Leu Pro Glu Val Pro Glu Thr Ile Glu Leu Glu 4220
4225 4230 Val Arg Thr Ser Thr Ala Ser Gly Leu Leu Leu Trp Gln Gly
Val 4235 4240 4245 Glu Val Gly Glu Ala Gly Gln Gly Lys Asp Phe Ile
Ser Leu Gly 4250 4255 4260 Leu Gln Asp Gly His Leu Val Phe Arg Tyr
Gln Leu Gly Ser Gly 4265 4270 4275 Glu Ala Arg Leu Val Ser Glu Asp
Pro Ile Asn Asp Gly Glu Trp 4280 4285 4290 His Arg Val Thr Ala Leu
Arg Glu Gly Arg Arg Gly Ser Ile Gln 4295 4300 4305 Val Asp Gly Glu
Glu Leu Val Ser Gly Arg Ser Pro Gly Pro Asn 4310 4315 4320 Val Ala
Val Asn Ala Lys Gly Ser Val Tyr Ile Gly Gly Ala Pro 4325 4330 4335
Asp Val Ala Thr Leu Thr Gly Gly Arg Phe Ser Ser Gly Ile Thr 4340
4345 4350 Gly Cys Val Lys Asn Leu Val Leu His Ser Ala Arg Pro Gly
Ala 4355 4360 4365 Pro Pro Pro Gln Pro Leu Asp Leu Gln His Arg Ala
Gln Ala Gly 4370 4375 4380 Ala Asn Thr Arg Pro Cys Pro Ser 4385
4390 <210> SEQ ID NO 71 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: SYNTHESIZED <400>
SEQUENCE: 71 His Gly Asp Pro Asn His Val Gly Gly Ser Ser Val 1 5 10
<210> SEQ ID NO 72 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 72
Val Gly Gly Ser Ser Val 1 5 <210> SEQ ID NO 73 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa
can be any naturally occurring amino acid <400> SEQUENCE: 73
Gly Gly Ser Xaa Val 1 5 <210> SEQ ID NO 74 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 74 His Gly Asp Pro Asn His Val
Gly Gly Ser Ser 1 5 10 <210> SEQ ID NO 75 <211> LENGTH:
7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 75 Arg Gly Asp Gly Ser Ser Val 1 5
<210> SEQ ID NO 76 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 76
His Gly Ser Ser Val 1 5 <210> SEQ ID NO 77 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 77 Asn Val Gly His Ser Pro Gly
Ser Asp His Gly 1 5 10 <210> SEQ ID NO 78 <211> LENGTH:
15 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SYNTHESIZED
<400> SEQUENCE: 78 Gly Gly Gly His Gly Asp Pro Asn His Val
Gly Gly Ser Ser Val 1 5 10 15 <210> SEQ ID NO 79 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
SYNTHESIZED <400> SEQUENCE: 79 Ile Tyr Pro Cys Arg Pro Asn
Thr Ala Leu Asn Asp Tyr Cys Ser Leu 1 5 10 15 Tyr <210> SEQ
ID NO 80 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 80 Arg Gln Pro
Cys Thr Tyr Ile Glu Val Arg Pro 1 5 10 <210> SEQ ID NO 81
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (10)..(10) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 81 Thr Leu Leu Cys Thr Ile Lys Glu Cys Xaa 1
5 10 <210> SEQ ID NO 82 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: SYNTHESIZED <400>
SEQUENCE: 82 Thr Arg Arg Ser Tyr Ser Pro Arg His Asn Phe Asn Trp
Leu Arg Ile 1 5 10 15 Gly Asp Phe Thr 20 <210> SEQ ID NO 83
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <400> SEQUENCE: 83 Arg Lys Phe Leu
Met Thr Thr Arg Tyr Ser Arg Val 1 5 10 <210> SEQ ID NO 84
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (9)..(9) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 84 His Leu Ala Arg Asp Ser Gly Leu Xaa Ser
Ala Val Pro Asp Pro Asp 1 5 10 15 <210> SEQ ID NO 85
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: SYNTHESIZED <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (9)..(9) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (11)..(14) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <400> SEQUENCE: 85 Ser Arg Tyr
Thr Ile Glu Ser Pro Xaa Asp Xaa Xaa Xaa Xaa Glu Ser 1 5 10 15
<210> SEQ ID NO 86 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: SYNTHESIZED <400> SEQUENCE: 86
Gly Gly Ser Ser Val 1 5
* * * * *