U.S. patent application number 13/854198 was filed with the patent office on 2013-08-15 for small peptides specifically bind to colorectal cancers.
This patent application is currently assigned to The Johns Hopkins University. The applicant listed for this patent is The Johns Hopkins University. Invention is credited to John Martin ABRAHAM, Stephen J. MELTZER.
Application Number | 20130209359 13/854198 |
Document ID | / |
Family ID | 40186236 |
Filed Date | 2013-08-15 |
United States Patent
Application |
20130209359 |
Kind Code |
A1 |
ABRAHAM; John Martin ; et
al. |
August 15, 2013 |
Small Peptides Specifically Bind to Colorectal Cancers
Abstract
Cancers are extremely heterogeneous in terms of the frequency
and types of mutations present in different malignant tumors. Thus,
it is likely that uniform clinical treatment is not optimal for all
patients, and that the development of individualized therapeutic
regimens may be beneficial. Multiple, unique small peptides bind to
cell lines derived from different colon adenocarcinomas. Within two
hours of contact, the colorectal cancer cells are able to transfer
a .sup.32P radioisotope from the small peptides to cellular
proteins; the transfer occurs at a substantially higher rate than
in the colorectal cancer cells than in cell lines derived from
other cancers or from normal tissues.
Inventors: |
ABRAHAM; John Martin;
(Woodbine, MD) ; MELTZER; Stephen J.;
(Lutherville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Johns Hopkins University; |
|
|
US |
|
|
Assignee: |
The Johns Hopkins
University
Baltimore
MD
|
Family ID: |
40186236 |
Appl. No.: |
13/854198 |
Filed: |
April 1, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12664302 |
Mar 11, 2010 |
8435490 |
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PCT/US2008/065735 |
Jun 4, 2008 |
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13854198 |
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60936669 |
Jun 21, 2007 |
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61123868 |
Apr 11, 2008 |
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Current U.S.
Class: |
424/1.69 ;
435/15; 435/375; 435/7.23; 530/324; 530/326; 530/327; 530/328 |
Current CPC
Class: |
G01N 33/60 20130101;
A61P 35/00 20180101; A61K 51/08 20130101; C12Q 1/485 20130101 |
Class at
Publication: |
424/1.69 ;
530/328; 530/324; 530/327; 530/326; 435/375; 435/15; 435/7.23 |
International
Class: |
A61K 51/08 20060101
A61K051/08; G01N 33/60 20060101 G01N033/60; C12Q 1/48 20060101
C12Q001/48 |
Goverment Interests
GOVERNMENT FUNDING
[0001] This invention was made using funds from the U.S.
government, particular grants from the National Cancer Institute.
The U.S. government retains certain rights in the invention as
provided in CA077057-09A2 and CA095323-14.
Claims
1. A method of delivering a radioactive isotope to a colon
adenocarcinoma cell, comprising: administering to a colon
adenocarcinoma cell a radioactive isotope-labeled peptide, wherein
the peptide is a substrate for protein kinase A (PKA) comprising
the motif R-X-S/T or R-R/K-X-S/T, whereby the peptide binds to the
cell and the radioactive isotope is internalized and transferred to
cellular proteins.
2. The method of claim 1 wherein the peptide comprises a sequence
R-R-X-S.
3. The method of claim 1 wherein the peptide comprises a sequence
R-R-A/G-S.
4. The method of claim 1 wherein the peptide comprises a sequence
S-R-R-X-S.
5. The method of claim 1 wherein the peptide comprises a sequence
R-R-X-S-G/A.
6. The method of claim 1 wherein the peptide comprises a sequence
G-S-R-R-X-S.
7. The method of claim 1 wherein the peptide comprises a sequence
R-R-X-S-V.
8. The method of claim 1 wherein the peptide comprises a sequence
R-R-X-S-V-G/A.
9. The method of claim 1 wherein the peptide comprises a sequence
R-R-X-S selected from the group consisting of SEQ ID NO: 1-28.
10. The method of claim 1 wherein the peptide comprises from 4-50
amino acid residues.
11. The method of claim 1 wherein the peptide comprises from 4-35
amino acid residues.
12. The method of claim 1 wherein the peptide comprises from 4-30
amino acid residues.
13. The method of claim 1 wherein the peptide comprises from 9-15
amino acid residues.
14. The method of claim 1 wherein the peptide comprises a sequence
R-R-X-S and has at least 90% amino acid residue identity with a
sequence selected from the group consisting of SEQ ID NO: 1-28.
15. The method of claim 1 wherein the radioactive isotope is
.sup.32P or .sup.33P.
16. The method of claim 1 wherein the radioactive isotope is
.sup.125I or .sup.131I.
17. The method of claim 1 wherein the adenocarcinoma cell is in a
patient with a colon tumor or metastasis.
18. The method of claim 17 further comprising the step of:
determining location of the radioactive isotope in the patient by
an imaging technique.
19. The method of claim 17 further comprising the step of:
monitoring size of the tumor.
20. The method of claim 1 wherein the adenocarcinoma cell is in a
xenograft in an animal.
21. The method of claim 1 wherein the adenocarcinoma cell is in
culture.
22. A method to screen a protein kinase A (PKA) substrate peptide
for its ability to deliver radioactive isotope to a colon
adenocarcinoma cell, comprising: (a) contacting a radioactive
isotope-labeled peptide with the colon adenocarcinoma cell of a
patient; and (b) measuring binding of the peptide to the colon
adenocarcinoma cell; and/or (c) measuring transfer of the
radioactive isotope from the peptide to proteins of the
adenocarcinoma cell of the patient.
23. A peptide which has at least 90% homology to a sequence
selected from the group consisting of SEQ ID NO: 1-28, said peptide
able to bind to the surface of a colon adenocarcinoma cell at least
50-fold more than to a normal colon cell.
24. The peptide of claim 23 wherein the peptide comprises from 4-50
amino acid residues.
25. The peptide of claim 23 wherein the peptide comprises from 4-34
amino acid residues.
26. The peptide of claim 23 wherein the peptide comprises from 4-25
amino acid residues.
27. The peptide of claim 23 wherein the peptide comprises from 9-15
amino acid residues.
28. The peptide of claim 23 wherein the peptide comprises a
sequence R-R-X-S.
29. The peptide of claim 23 wherein the peptide comprises a
sequence R-X-S/T.
30. The peptide of claim 23 wherein the peptide comprises a
sequence R-R/K-X/S/T.
31. The peptide of claim 23 which is radiolabeled with a
radioisotope.
32. The peptide of claim 23 which is phosphorylated.
33. The peptide of claim 23 which is isolated and purified.
34. The peptide of claim 31 wherein the radioisotope is .sup.32P or
.sup.33P.
35. The peptide of claim 31 wherein the radioisotope is .sup.125I
or .sup.131I.
36. The peptide of claim 23 which comprises a sequence selected
from the group consisting of SEQ ID NO: 1-28.
37. The peptide of claim 23 wherein the amino acid backbone of the
peptide consists of a sequence selected from the group consisting
of SEQ ID NO: 1-28.
Description
TECHNICAL FIELD OF THE INVENTION
[0002] This invention is related to the area of cancer management.
In particular, it relates to diagnostic and therapeutic approaches
to cancer management.
BACKGROUND OF THE INVENTION
[0003] Recent landmark discoveries have convincingly documented the
extensive genetic heterogeneity among human cancers, particularly
colorectal tumors, by establishing the existence of a small number
of frequently mutated gene "mountains" and a much higher number of
gene "hills" mutated at much lower frequencies [1,2]. This high
degree of diversity among human colorectal cancers suggests that
individualized treatment strategies hold great promise in
successful clinical intervention. Several anticancer
immunotherapies are currently in use, including Herceptin, Rituxin,
and Avastin, a monoclonal antibody directed against VEGF (vascular
endothelial growth factor) that is approved for colorectal cancer
treatment [3-9].
[0004] Radioimmunotherapy (RIT) is an emerging technology with thus
far only two FDA-approved protocols, both directed against
non-Hodgkin's lymphoma (NHL). Each protocol utilizes a monoclonal
antibody directed against the CD20 B-cell marker and can deliver
.sup.90Y (Zevalin) or .sup.131I (Bexxar), each of which generates
electrons (beta particles) that damage DNA, resulting in cell death
[10,11]. Currently, no RIT has yet been approved for the treatment
of colorectal cancer [12].
[0005] There is a continuing need in the art to develop new ways to
detect, image, locate, and ablate cancers.
SUMMARY OF THE INVENTION
[0006] According to one aspect of the invention a method is
provided for delivering a radioactive isotope to a colon
adenocarcinoma cell. A radioactive isotope-labeled peptide is
administered to a colon adenocarcinoma cell. The peptide is a
substrate for protein kinase A (PKA) and comprises the motif
R-X-S/T or R-R/K-X-S/T. The peptide binds to the cell and the
radioactive isotope is internalized and transferred to cellular
proteins.
[0007] Another aspect of the invention is a peptide which has at
least 90% homology to a sequence selected from the group consisting
of SEQ ID NO: 1-28. The peptide is able to bind to the surface of a
colon adenocarcinoma cell at least 50-fold more than to a normal
colon cell.
[0008] According to another aspect of the invention a method is
provided for screening a protein kinase A (PKA) substrate peptide
for its ability to deliver radioactive isotope to a colon
adenocarcinoma cell. A radioactive isotope-labeled peptide is
contacted with the colon adenocarcinoma cell of a patient. Either
binding of the peptide to the colon adenocarcinoma cell or transfer
of the radioactive isotope from the peptide to proteins of the
adenocarcinoma cell of the patient are measured, or both.
[0009] The present invention thus opens up new avenues for
management of cancer, in which small peptide molecules, which can
be readily synthesized or made in recombinant cells, are able to
replace antibodies as targeting agents.
[0010] These and other embodiments which will be apparent to those
of skill in the art upon reading the specification provide the art
with
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1. Schematic diagram of experimental approach. A
bacterial recombinant expression system produced various
gluthathione-S-transferase decapeptide fusion proteins which were
bound to glutathione and labeled with .sup.32P utilizing protein
kinase A (PKA). After washing, the labeled decapeptides were
recovered after thrombin digestion and incubated at various times
with several different cell lines.
[0012] FIG. 2. Levels of binding of decapeptide MA5 to eighteen
different cell lines. The .sup.32P labeled decapeptide MA5 was
incubated for two hours with 10,000 cells, washed three times, and
the radioactive counts of the cells determined by scintillation
counting. Seven cell lines demonstrated avid binding of MA5 and are
shown as bar graphs of the mean and one standard deviation in
triplicate wells. The remaining eleven cell lines, along with one
blank well averaged only 365 cpm. These values are so small as to
not be visible at the scale used in this figure. Further
information on the individual cell lines is provided in the
Supplemental Information.
[0013] FIGS. 3A-3B. Relative levels of binding of nine 32P-labeled
decapeptide variants. (FIG. 3A) Nine .sup.32P-labeled different
decapeptides, varying from one another by only one to three amino
acids, were incubated with Caco-2 cells for two hours, the cells
washed three times, and counts remaining bound to the cells are
shown as a percentage of the total amount of counts for each
decapeptide used. Amino acid substitutions for each variant
(relative to MA1) are underlined and bolded. (FIG. 3B) The
variants, MA1, MA4, and MA5 were incubated with Caco-2 cells for
intervals varying from five minutes to two hours, washed, the
adherent cells dissolved in gel loading buffer and an aliquot run
on a 10%-20% gradient polyacrylamide-SDS gel. The three lanes
marked "24h" (lanes 5, 10, and 15) were incubated with the
respective labeled decapeptides (MA1, MA4, MA5) for two hours,
washed, and the cells incubated with complete medium for 24 hours.
The cells were treated as described for the other lanes of this
figure.
[0014] FIGS. 4A-4D. The majority of the .sup.32P-labeled
decapeptide MA5-bound molecules are released from Caco-2 cells.
(FIG. 4. A) The 32P-labeled decapeptide MA5 was incubated for two
hours with five different cell lines, the cells were washed, and
complete medium was added. Following a 24 hour incubation, the
number of counts per minute released into the medium (R) as well as
the number of counts remaining bound to the cells (B) were
determined. Each bar shows the mean and one standard deviation of
triplicates wells. (FIG. 4. B) Time course for the release and
retention of the .sup.32P-labeled decapeptide MA5. MA5 was
incubated for two hours with Caco-2 cells, the cells washed, and
the cpm released (dashed line) as well as remaining bound (solid
line) to the cells determined for time intervals post-washing. Each
point shows the mean plus/minus one standard deviation of
triplicate determinations. (FIG. 4. C) Radioactive well contents
described as bound (solid line) in FIG. 4B were run on a 16.5%
polyacrylamide-SDS gel and exposed to film. Immediately after
washing (i.e., at 0 hours), the majority of the counts were
visualized as .sup.32P-peptide. Over the next 48 hours, the peptide
counts greatly diminished, with the majority of bound radioactivity
incorporated into cellular proteins. (FIG. 4. D) Aliquots of medium
containing the released (dotted line) .sup.32P-peptide MA5 were
assayed at time intervals after washing, as described in FIG. 4B.
As time progressed, more of the .sup.32P-peptide was released,
reaching a plateau by 24 hours after washing.
[0015] FIG. 5. Schematic diagram of experimental approach. A PCR
product containing 17 random codons was inserted into the BamHI
site of pGEX-2TK producing various glutathione-S-transferase fusion
proteins which were bound to glutathione-sepharose, and labeled
with .sup.32P using protein. kinase A (PKA). After washing and
thrombin digestion, the labeled peptides were incubated with
several different cell lines and assayed.
[0016] FIG. 6. Levels of binding of various .sup.32P-labeled
peptides to Caco2 cells. Different .sup.32P-labeled peptides were
incubated for two hours with 10,000 Caco2 cells, washed three
times, and incubated in complete medium for 24 hours. The amount of
.sup.32P radioisotope that remained permanently incorporated into
cellular proteins is shown as a percentage of uptake of the amount
of peptide added to each cell culture well (mean plus one standard
deviation). The number of amino acids present in each insert is
shown and ranged from 0 to 17 amino acids. The amino acid sequence
(SEQ ID NO: 10-28) of each insert is shown beneath the level of
.sup.32P incorporation attributed to each insert.
[0017] FIG. 7. SDS-polyacrylamide gels of .sup.32P-peptide binding
and radioisotope uptake by Caco2 cells. Four of the MA (Modified
Adjuvant) .sup.32P-peptides shown in FIG. 6 were incubated with
triplicate wells of Caco2 or HeLa cells for two hours. After
washing, 100 .mu.l of gel loading buffer was added and the contents
were run on SDS-polyacrylamide gels (designated as "2 hours").
Identical wells had complete medium added immediately after the
washing step and were incubated for an additional 24 hours, and the
well contents were then run on gels (designated as "24 hours").
Film was developed after an overnight exposure showing the apparent
permanent incorporation of .sup.32P into cellular proteins at 24
hours (marked by * in the MA11 panel). Peptide MA11 bound 215 times
more avidly to Caco2 cells than to HeLa cells at two hours, and 150
times more avidly at 24 hours. Peptide MA20 bound well to both
Caco2 and HeLa cells at two hours, but only Caco2 cells appeared to
possess the cellular machinery needed to incorporate .sup.32P into
cellular proteins. The thin arrow shows the position of the
.sup.32P-labeled peptide, while the bold arrow shows the position
of a relatively low molecular weight labeled intermediate that was
not seen in the HeLa cells.
[0018] FIG. 8. Peptide MA20 binds to multiple cell lines, but
.sup.32P incorporation is only by colon adenocarcinoma derived
lines. The .sup.32P-labeled peptides MA11, MA20 and MA22 were
incubated with seven different cell lines as described in FIG. 7.
MA11 and MA22 bound to and had .sup.32P radioisotope permanently
incorporated by the three colon adenocarcinoma derived cell lines.
MA20 significantly bound to all seven cell lines, including one
derived from a pancreatic adenocarcinoma and one derived from a
lung adenocarcinoma, but had significant levels of .sup.32P
permanently incorporated into cellular proteins only by the three
colon adenocarcinomas. The thin arrow shows the position of the
.sup.32P-labeled peptide, while the bold arrow indicates the
position of a relatively low molecular weight labeled intermediate
that was only seen in colon adenocarcinoma cells.
[0019] FIG. 9. Non-phosphorylated peptide effectively competes with
.sup.32P-labeled MA11peptide in binding to Caco2 cells. Into each
well containing Caco2 cells was added 0.005 .mu.g .sup.32P-labeled
MA11 peptide and the indicated quantity of cold, non-phosphorylated
MA11. After one hour of incubation, adherent cells were washed and
the bound radioactive counts determined.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The inventors have developed a general strategy for
delivering therapeutic or diagnostic moieties to a cancer cell. A
class of peptides have been found which specifically bind to colon
adenocarcinoma cells and deliver a radioisotope to cellular
proteins of the colon adenocarcinoma cells. Surprisingly, peptide
substrates of protein kinase A (PKA) bind to colon adenocarcinoma
cells preferentially relative to other cancer cells lines and
relative to cell lines derived from normal tissues, including
normal colon.
[0021] The peptides can be labeled with any desired radionuclide
according to methods known in the art. Most conveniently, these can
be labeled with isotopes of P, such as .sup.32P or .sup.33P using
PKA. If a non-phosphorus radionuclide is desired, a synthetic
chemistry scheme for attaching is used. Other isotopes which are
used in radiomedicine include technetium-99m, rubidium-82,
thallium-201 chloride, fluoro-deoxy glucose incorporating F-18,
lutetium-177, Yttrium-90, iodine -131, phosphorus-32, boron-10,
actinium-225, bi-213. Any of these or other radionuclides can be
attached to the peptides of the present invention. Different
radionuclides will be selected depending on desired use as a
therapeutic or diagnostic agent. Possible therapeutic radionuclides
include those that decay to provide Auger electrons,
beta-particles, or alpha-particles. Different radionuclides will be
selected depending on the type of imaging device that will be used.
Possible imaging techniques which may be used include Pet, SPECT,
MRI, CAT, etc. Those of skill in the art can readily determine the
appropriate isotope to use.
[0022] The peptides of the present invention appear to be quite
specific for colon adenocarcinoma, particularly relative to normal
colon tissue. The relative difference in the amount and/or rate of
binding between tumor and normal tissue will typically be reflected
in safety and diminished side-effects. In addition, it will be
reflected in the usefulness for imaging, with greater relative
differences providing increased clarity and contrast. The peptides
to be used for radiotherapy or radioimaging will bind at least
20-fold, at least 50-fold, at least 100-fold, at least 150-fold, or
at least 200-fold more to cancer cells than to normal cells of the
same cell tissue type.
[0023] Peptides of the invention are typically between 4 and 50
amino acid residues, or between 4 and 35 amino acid residues, or
between 9 and 30 amino acid residues, or between 9 and 15 amino
acid residues. These are significantly smaller than full antibodies
and even of antibody fragments and derivatives, such as single
chain antibodies. The smaller size provides increased penetration
to tumors, which can be a distinct therapeutic benefit. In some
situations, however, it may be desirable to link additional amino
acid residues to the peptides, for example, to provide additional
functionalities. For example, it may be desirable to attach a
biological toxin to the peptide. Many biological toxins are known
and any can be used if appropriate to provide a desired cell
killing effect. Other non-amino acid moieties may also be added to
the protein for desired benefits. For example, a fluorophore may be
desired and can be used.
[0024] Peptides that will be administered to a patient for imaging
or therapeutic purposes will be safe and suitable for
administration. Such administration may typically by injection into
the blood stream or into particular organs. Alternatively, the
administration can be subcutaneously, intraperitoneally,
intramuscularly, intradermally, sublingually, orally, etc. The
preparations of peptides can be isolated and purified from other
peptides. The preparations can be sterile and/or free of pyrogens.
The peptides can be made by synthetic chemistry, semi-synthetic
chemistry, recombinant organisms, or by isolation from natural
sources and subsequent processing to yield the desired peptide
products. Means for synthesizing peptides, for example in an
automatic peptide synthesizer are well within the skill of the art.
Recombinant techniques for generating peptides are also well known
to the skilled artisan.
[0025] Any peptide can be used, including those that are shown in
SEQ ID NO: 1-40. Preferably the peptide will share at least 90%
identity with the sequence shown in SEQ ID NO: 1-40. The peptide
may have additional portions, as well, as discussed above, for
example to provide additional functionality.
[0026] Peptides can be packaged individually or in libraries to
facilitate screening for a good match between a particular patient
and a peptide. The peptides may be packaged with a radionuclide
attached or the radionuclide may be provided separately. Kits for
attaching a radionuclide may contain both one or more peptides and
an enzyme, such as PKA for attaching a radionuclide the
peptide.
[0027] Decapeptides that can be labeled with a high energy (1.7
Mev) beta emitter (.sup.32P) and can bind avidly to several
different adenocarcinoma cell lines, efficiently delivering this
potential tumor-ablating material to the cells. The decapeptides,
termed MA for Modified Adjuvant, are protein kinase A substrates.
When labeled with a tumor-ablating material such as .sup.32P, these
peptides bind to and transfer the radioisotope to a cell line after
one to two hours of incubation. Moreover, we have shown that
transfer of isotope from these decapeptides is restricted to cell
types derived from primary colon adenocarcinomas. For example,
exposure of certain colon cancer cell lines (e.g., Caco-2) to the
most avidly binding labeled peptide, MA5, for a two-hour period
resulted in the transfer of a radioactive dose of over 29 counts
per minute per cell after a two hour incubation, wash, and
immediate determination of the retained radioactivity.
[0028] The incubation of .sup.32P-labeled decapeptide with certain
cell lines resulted in large amounts of peptide being retained
after a two-hour incubation, but a substantial proportion of this
bound peptide was released after an overnight incubation. For
example, after incubation of the labeled variant MA5 with Caco2
cells for two hours, three wash steps, and overnight incubation in
medium, 88% of the originally retained .sup.32P isotope was
released. However, the 12% that was retained by cells still
represented 5.8 cpm per cell, extrapolating to over 8,300 counts
per cell per day. In addition, radioactivity that was still
retained by cells after overnight medium incubation was permanently
incorporated into a variety of cellular proteins, as demonstrated
by polyacrylamide gel electrophoresis of post-exposure cellular
lysates
[0029] Among 16 cell lines assayed for their ability to bind the
decapeptides, five demonstrated very high retention of isotope
after two-hour incubation. Although all five of these lines
released from 63% to 88% of this radioactivity after an overnight
incubation, the amount of isotope that was retained overnight was
still substantial. All five of these cell lines were derived from
colorectal adenocarcinomas. The eleven cell lines that did not bind
the radioactively labeled decapeptide MA5 were derived from a
variety of tissue origins. These included squamous cell carcinomas
of the cervix, lung, breast, and a fibrosarcoma, as well as normal
kidney, colon, and esophageal tissues.
[0030] The majority of approved immunotherapeutic regimens for
cancer involve an antibody directed against a specific cellular
molecule [27]. These agents can function by binding to the cell
surface and may utilize ADCC, complement activation, or cellular
apoptosis. The antibodies may also be coupled to a tumor-ablating
agent, such as toxins or radioisotopes [14]-[18]. The addition of
isotope to peptides, and their use for both diagnostic and
therapeutic purposes, is an active area of biomedical research
[19]-[22]. Our work utilizes protein kinase A (PKA) substrates
labeled with .sup.32P isotope. A high-energy beta-emitting
radioisotope results in an electron path length range of up to 5
mm, permitting substantial penetration of solid tumors. Due to a
predicted "bystander" effect, one beta particle will penetrate
hundreds or thousands of cells within the tumor, even those not
directly binding the decapeptide. Moreover, since the molecular
weights of these minuscule decapeptides proteins are far lower than
the exclusionary molecular weight limit of the filtering kidneys,
these peptides should be rapidly eliminated in the urine, leading
to reduced systemic toxicity. Thus, it should be feasible for both
a radioactive dose and unbound radioactivity to be eliminated
easily and in a relatively short period of time. We anticipate that
additional known enzyme substrates can be identified as potential
vehicles for the specific delivery of anti-tumor agents to cancer
cells and that potential cancer therapeutic regimens employing this
peptide or other similar substances might be a general strategy for
peptide binding therapy.
[0031] Nineteen different small peptides up to 34 amino acids in
length have been recombinantly produced, each containing an insert
up to 17 residues long, which can be labeled at a conserved nine
amino acid substrate using .sup.32P and protein kinase A (PKA) .
These .sup.32P-labeled peptides bind with unique affinities to cell
lines established from different colon adenocarcinomas and
permanently transfer radioisotope to cellular proteins after two
hours of incubation. The most efficiently binding peptide results
in the permanent uptake of .sup.32P by colon cancer cells over 150
times higher than by cell lines derived from other cancers or
normal tissues. In addition, one .sup.32P-labeled peptide bound to
all cell lines tested, but .sup.32P was processed and permanently
incorporated only by cell lines derived from colon adenocarcinomas,
implying that only this type of cancer cell possesses the machinery
necessary for this processing step. The nineteen different peptides
shown in FIG. 6 were selected from an initial screening panel
containing only 25 peptides. This surprisingly high rate of
obtaining successful peptides enhances the likelihood that this
strategy for individualized therapy development will be feasible.
Finally, a competitive binding assay using cold and
.sup.32P-labeled synthetic MA11 peptide demonstrated that
non-phosphorylated peptide competes very efficiently for binding to
Caco2 cells.
[0032] Most currently approved cancer immunotherapeutic regimens
use an antibody directed against a known cellular molecule; the
antibody may optionally be coupled to a tumor-ablating agent, such
as a radioisotope or a toxin [14-18]. Only two
radioimmunotherapeutic (RUT) treatments are presently FDA-approved;
both are directed against non-Hodgkin's lymphoma utilizing
.sup.131I (Bexxar) or .sup.90Y (Zevalin) via the cell-killing
activity of emitted electrons. .sup.32P radioisotope is a pure beta
emitter, and as shown in Table 1, it has many properties that
compare favorably to .sup.131I and .sup.90Y, in addition to being
readily available and relatively inexpensive. One advantage of
using beta particles to kill tumor cells is that their path range
of up to 5 mm results in a large number of cells being penetrated
by each electron, leading to a cumulative "bystander effect" due to
crossfire from neighboring labeled cells.
TABLE-US-00001 TABLE 1 Comparison of Properties of Radioactive Beta
Emitting Radioisotopes. Maximum Beta Half-life Radioisotope Emits
Energy (MeV) Range (days) .sup.131Iodine Beta 0.6 1.6 mm. (avg.) 8
.sup.90Yttrium Beta 2.3 5 mm. (avg.) 2.7 .sup.32P Beta 1.7 up to 5
mm. 14.3 Bexxar (.sup.131I-anti-CD20) and Zevalin
(.sup.90Y-anti-CD20) are FDA-approved for the treatment of
non-Hodgkin's lymphoma.
[0033] A very active area of biomedical research focuses on the
coupling of radioisotope to peptides, as well as its use in
diagnostic and therapeutic applications [19-22]. Our proposed
application of .sup.32P-labeled small peptides in peptide binding
therapy suggests a number of advantages over traditional RIT based
on monoclonal antibodies. For example, smaller therapeutic
molecules are expected to provide better tumor penetration, and the
average small peptide molecular weight of less than 4,000 Da is
less than 3% of the size of an antibody molecule [23]. Radioactive
halogens such as .sup.131I can be processed and released
prematurely by cells, while the .sup.32P delivered by these small
peptides is permanently incorporated into cancer cell proteins
[24]. A small peptide is less likely to incite the type of host
anti-protein response that can develop when using the much larger
antibodies, and the absence of an Fc immunoglobulin fragment should
result in less nonspecific binding by the liver. The radioisotope
.sup.32P has a long history of clinical use dating to the early
1930's, while today it is still used to treat polycythemia and
essential thrombocythemia [25]. There is a clear need for the
development of effective new treatments for colorectal cancer [26].
Our work suggests that an extremely large library of different
small peptides, each with unique binding and .sup.32P transfer
abilities, can be readily produced either chemically or
biologically, thus increasing the feasibility of developing
individualized treatment regimens for different patients. Cancer
has been shown to be a highly heterogeneous disease, thus the
development of these unique peptide binding therapies could greatly
facilitate individualized patient treatments.
[0034] The above disclosure generally describes the present
invention. All references disclosed herein are expressly
incorporated by reference. A more complete understanding can be
obtained by reference to the following specific examples which are
provided herein for purposes of illustration only, and are not
intended to limit the scope of the invention.
EXAMPLE 1
Materials and Methods
[0035] Production of the recombinant .sup.32P-labeled peptides. As
described in FIG. 5, PCR generated products consisting of 17 random
codons flanked by BamHI sites were cloned into the BamHI site of
pGEX-2TK (GE Healthcare). After transformation into DH5a bacteria,
isolated clones were grown overnight in LB-amp broth, diluted 1/10
in same, grown for two hours, IPTG added to 1 mM, and grown at 37 o
C for five hours. Ten ml of culture were centrifuged and
resuspended in 1 ml of 1.times.TBS containing 100 .mu.g/ml
lysozyme. After two freeze-thaw cycles, the lysate was centrifuged
and mixed with 100 .mu.l Sepharose-Glutathione for one hour, washed
three times with 1.times.TBS, and the bound recombinant fusion
proteins labeled using 32P-.gamma.-ATP and protein kinase A (PKA)
according to the manufacturer's instructions (Sigma, St. Louis,
Mo.). The pellet was washed three times with 1.times. PBS and the
labeled peptide was cleaved and released into the supernatant using
thrombin (GE Healthcare). For each recombinant peptide produced and
assayed, the DNA sequence of the insert in the expression plasmid
was determined.
[0036] Production of the .sup.32P-labeled decapeptides. Different
DNA oligomers were cloned into pGEX-4T-1 (GE Healthcare) which
yield various decapeptides after thrombin cleavage designated MA1
through MA9 (Modified Adjuvant). The protein sequences are: MA1,
GSRRASVGSA (SEQ ID NO: 1); MA2, GSRGASVGGA(SEQ ID NO: 2); MA3,
GSRRGSVGSA(SEQ ID NO: 3); MA4, GSRRGSVASA(SEQ ID NO: 4); MA5,
GSRRASVASA(SEQ ID NO: 5); MA6, GSRRASVGSG(SEQ ID NO: 6); MA7,
GSRGGSVGSA(SEQ ID NO: 7); MA8, GSRGGSVASA(SEQ ID NO: 8); MA9,
GSRGGSVGSG(SEQ ID NO: 9). DH5-.alpha. bacteria containing these
clones were grown overnight in LB (containing 100 .mu.g/ml
ampicillin), diluted 1/10 in LB-Amp and grown at 37.degree. C. for
two hours. IPTG was added to 1 mM and the culture grown at
37.degree. C. for five hours. Ten ml of each culture were
centrifuged and the cell pellet resuspended in 1.times.TBS
containing 100 .mu.g/ml lysozyme. After two cycles of freeze-thaw,
the lysate was centrifuged and the supernatant was mixed with 100
.mu.l of Sepharose-Glutathione for two hours at RT. Each pellet was
washed three times with 1.times.TBS, and the bound recombinant
fusion proteins were labeled with .sup.32P using protein kinase A
(PKA) and .sup.32P-.gamma.-ATP according to the manufacturer's
instructions (Sigma, St. Louis, Mo.). The pellet was washed four
times with 1.times.PBS and the labeled decapeptide was cleaved and
released into the supernatant with thrombin (GE Healthcare).
[0037] Assay of the binding of .sup.32P-labeled decapeptides to
cell lines: Cell lines were grown in complete medium containing 10%
bovine fetal serum (heat inactivated). In each well of a 96-well
plate, 10,000 cells from various cell lines were grown overnight in
complete medium. Ten .mu.l of the labeled-peptide in 1.times.PBS
and 90 .mu.l of complete medium were added to each well and
incubated at 37.degree. C. at various times of up to two hours. The
peptide-medium was removed and one .mu.l added to 100 ul gel
loading buffer and counted by scintillation counting for the probe
control or run on a polyacrylamide-SDS gel (Biorad). The adherent
cells were briefly and gently washed with complete medium three
times and some wells were assayed immediately by adding 100 .mu.l
of gel loading buffer to each well and run on a gel or counted in a
scintillation counter. Other wells had 100 .mu.l complete medium
added and incubated for a further time period. Samples were either
counted in a liquid scintillation counter or run on
polyacrylamide-SDS gels, exposed to x-ray film, and the film
developed.
[0038] Assay of the binding of .sup.32P-labeled peptides to cell
lines. Cell lines were grown in complete medium containing 10% heat
inactivated bovine fetal serum. In each well of a 96-well plate,
10,000 cells from various cell lines were grown overnight. Ten
.mu.l of the .sup.32P-labeled peptide in 1.times.PBS and 90 .mu.l
complete medium were added to each well and incubated at 37.degree.
C. for two hours. The peptide-medium was removed and one .mu.l
added to 100 .mu.l gel loading buffer for scintillation counting
for the probe quantitation or run on a 10%-20% polyacrylamide-SDS
gel (Biorad). The adherent cells were briefly and gently washed
with complete medium three times and some wells were assayed
immediately by adding 100 .mu.l of gel loading buffer to each well
and run on a gel or counted in a scintillation counter. Other
identically treated wells had 200 .mu.l complete medium added and
incubated at 37 o C for an additional 24 hours. The medium was
removed and 100 gel loading buffer added and the samples run on a
gel or counted as described above.
[0039] Production of synthetic .sup.32P-labeled peptide. The 12
amino acid peptide MA11 was chemically synthesized and 0.2 .mu.g
was labeled as described above using 300 .mu.Ci of
.sup.32P-.gamma.-ATP and 30 units of protein kinase A (PKA). After
a five hour labeling reaction, the mixture was microfuged though a
Microcon-10 unit to remove the enzyme from subsequent binding
assays. For the competitive binding assay, 0.005 .mu.g of
.sup.32P-labeled peptide MA11 was added to a well containing 10,000
Caco2 cells and a designated quantity of cold, non-phosphorylated
MA11. After incubation for one hour, the adherent cells were gently
washed and the well contents counted.
EXAMPLE 2
Making Peptides
[0040] We have identified nine decapeptides, differing from one
another by only a few amino acids, that when labeled with .sup.32P
can bind to a number of colorectal carcinoma cell lines. All
decapeptides contain a protein kinase A (PKA) substrate sequence
and are designated as MAs (Modified Adjuvant). FIG. 1 is a
schematic representation of the production of the .sup.32P-labeled
peptides and the experimental design of assays to measure binding
of peptides to cell lines.
[0041] Production of .sup.32P-labeled peptides and binding to colon
adenocarcinoma cells. We produced and identified an additional
series of peptides, up to 34 amino acids in length, whose amino
acid sequences dramatically alter their ability to bind to and
permanently facilitate .sup.32P incorporation into cells. FIG. 5 is
a schematic representation of the experimental design, illustrating
the cloning of a DNA fragment containing 17 randomly generated
codons into the BamHI restriction enzyme site of pGEX-2TK. After
bacterial transformation, individual clones were selected and
expanded to produce a diverse set of .sup.32P-labeled peptides. If
no stop codons were present in the random DNA sequence, then a
34-residue peptide was generated, flanked at its amino end by the
9-residue protein kinase A (PKA) labeling motif and at its carboxy
terminus by an 8-residue sequence. As expected, in several clones,
a stop codon was inserted, resulting in truncated peptides;
however, all of these truncated peptides contained the protein
kinase A (PKA) substrate moiety. These diverse peptides were
incubated with several different cell lines for two hours, adherent
cells were washed three times, and radioactivity remaining bound to
cells was assayed either immediately, or following overnight
incubation in complete medium.
EXAMPLE 3
Cell Binding and Isotope Transfer to Proteins
[0042] FIG. 2 displays the number of .sup.32P counts per minute
(cpm) remaining bound to sixteen different cell lines and blank
wells after a two hour incubation with MA5, the most efficient
binding decapeptide (see below). The Caco-2 colon adenocarcinoma
cell line retained the greatest number of radioactive counts after
a two-hour incubation and subsequent washes with complete medium,
the average value of triplicate wells equaling 298,639 cpm per
10,000 cells. HCT116 colon adenocarcinoma cells retained an average
value of 131,998 cpm per 10,000 cells. Blank wells and nonbinding
cell lines had mean values of less than 550 cpm; bars representing
these values are not visible at the scale used in FIG. 2. For
example, HeLa S3 cervical cancer cells only retained an average of
534 cpm per 10,000, HT1080 fibrosarcoma cells retained 367 cpm, and
the human embryonic kidney cell line 293H retained 429 cpm per
10,000 cells.
[0043] Five of the sixteen cell lines demonstrated very strong
retention of radioactivity when incubated with MA5 (Modified
Adjuvant radioactive peptide) with all five of these being colon
adenocarcinoma cell lines (Caco-2, HCT15, HCT116, LoVo, HT29). In
contrast, the eleven nonbinding cell lines were mostly squamous
cell lines derived from carcinomas of the cervix (HeLa S3), colon
(RKO), lung (1271, A549), esophagus (KYSE-70), a fibrosacroma
(HT1080), or cells cultured from normal kidney (29311), colon
(1459), or esophagus (HEEpiC). Nonbinding cell lines included T84,
derived from a colon adenocarcinoma metastatic to lung, and
SK-BR-3, isolated from a breast adenocarcinoma. The ratio of cpm
retained by Caco-2 (298,639) to the average of the eleven
nonbinding cell lines (365) was 818:1. Caco-2 cells retained
approximately 18% of the total radioactive counts present in the
incubation well after two-hour incubation.
[0044] Nine MA variants were assayed for adherence to Caco-2 cells
after two hours' incubation. The relative binding level and amino
acid composition of each MA variant is displayed in FIG. 3A.
Alteration of only one to three amino acids within the peptide
resulted in retention differences as large as 70-fold, e.g., in
variant MA2 vs. variant MA5.
[0045] To investigate how quickly .sup.32P isotope could be
transferred from the peptide variants and incorporated into
cellular proteins, the three most avidly binding MAs (see FIG. 3A)
were added to replicate wells containing Caco-2 cells, then washed
away at varying time intervals and the cells and supernatant
assayed. As shown in FIG. 3B, substantial percentages of these
.sup.32P-labeled variant decapeptides bound to cells within only a
few minutes, with large amounts of radiolabeled cellular proteins
appearing at two hours after exposing cells to the labeled
peptides. Notably, a parallel experiment in which conditions
described in FIG. 3 were duplicated, but washed cells were
incubated overnight in complete medium (data not shown), still
revealed similar levels of .sup.32P-decapeptide release and
retention for all nine MAs, as described for MA5 in FIG. 2.
[0046] The peptide binding, washing and assay experiment described
for FIG. 2 was then repeated in the seven most avidly binding cell
lines using MA5, except that after three washes of medium, 200
.mu.l of complete medium was added to each well and the cells were
incubated overnight at 37.degree. C. FIG. 4A shows the cpm retained
by cells or released into the medium after this overnight
incubation. Approximately 88% of MA5 radioactive counts initially
retained by the colon cancer cell lines was released into the
medium, while approximately 12% of initially retained radioactive
counts were retained by cells. Caco-2 cells retained the greatest
number of counts, averaging 58,305 cpm in triplicate wells
containing 10,000 cells each. This figure represents approximately
5.8 cpm, or 348 counts per hour, per cell (i.e., when extrapolated
over a potential 2-week exposure period, equivalent to over 87,000
counts per cell).
[0047] FIG. 4B shows the time course of the release of MA5 from the
Caco-2 adenocarcinoma cell line over a 48-hour time period. The
majority of the total counts released over the 48 hour time period
are released by nine hours of incubation. FIGS. 4C and 4D consist
of two autoradiograms showing the locations of the radioactive
molecules described in FIG. 4B on polyacrylamide-SDS gels. The
sizes of the cellular radioactive proteins in the cells are shown
in FIG. 4C; .sup.32P-labeled MA5 released into the medium is shown
in FIG. 4D. There is apparent agreement on the distribution and
overall radioactivity levels in comparing FIG. 4B and FIGS. 4C and
4D. As soon as two hours after the introduction of the radioactive
peptide, a substantial portion of the isotope appears to have been
transferred to higher molecular weight proteins.
[0048] FIG. 6 shows the dramatic variation in levels of permanent
.sup.32P incorporation into the colon adenocarcinoma line Caco2
after washing and overnight medium incubation. As shown above,
cells successfully binding decapeptides after two hours of
incubation released up to 88% of their initially bound .sup.32P
into media after overnight incubation, but still permanently
incorporated high levels of radioisotope into their proteins. The
nineteen different peptides in FIG. 6 are designated MA (Modified
Adjuvant) 10 through 28. Eleven of these 19 contain complete
17-residue inserts, with MA18 permanently transferring .sup.32P to
Caco2 cells over 37 times more efficiently than MA26. The most
efficient permanent radioisotope incorporation into Caco2 cells
occurred after incubation with MA27, which contains only one
randomly inserted amino acid upstream of a stop codon. Peptides
MA16 and MA17 were encoded by the original recombinant expression
vector, leading to low levels of radioisotope incorporation.
EXAMPLE 4
[0049] Visualization of .sup.32P incorporation by gel
autoradiography. Four peptides showing average levels of
radioisotope incorporation were selected for further study;
triplicate-well assays of these peptides are displayed in FIG. 7.
Peptide MA11's insert contained three residues upstream of a stop
codon, resulting in a peptide only 12 amino acids in length.
Despite its relatively short length, this truncated peptide
transferred .sup.32P to Caco2 cells 215 times more efficiently than
to the cervical tumor derived cell line HeLa at two hours. After
washing and overnight incubation in medium, radioactivity retained
by Caco2 cells was more than 150 times greater than that retained
by HeLa cells. As shown in FIG. 7, most .sup.32P bound to Caco2
cells was present in a low-molecular weight (LMW) component (bold
arrow) at 2 hours, but at 24 hours most of this radioactivity had
been incorporated into several different cellular proteins.
[0050] Similar results were observed for peptides MA15 and MA22,
both of which contained 17-residue inserts for a total length of 34
amino acids, and both of which incorporated 23 times more .sup.32P
into Caco2 cells than into HeLa cells after overnight incubation
(FIG. 7). Once again, both MA15 and MA22 showed an intensely
radioactive LMW band (bold arrow) at 2 hours that had almost
completely disappeared at 24 hours, with incorporation of the
remaining .sup.32P into cellular proteins. Originally, we assumed
that this LMW band seen at 2 hours (bold arrow) represented intact
bound .sup.32P-labeled peptide. However, the 34 amino acid peptides
MA15 and MA22 identified these intact 34-aa peptide precursors as
distinct from the intense smaller MW band (bold arrows). Thus, we
concluded that the smaller band was a rapidly processed small
intermediate molecule, which diminished greatly over the ensuring
24 hours during which the .sup.32P was being incorporated into the
cellular proteins.
[0051] Peptide MA20 also contained a 17-residue insert. This
peptide was especially noteworthy, since it was the only one tested
that was able to bind to a cell line not derived from colon
adenocarcinomas and provided key evidence suggesting a possible
cellular processing mechanism. As shown in FIG. 7, MA20 bound at
high levels to both Caco2 and to HeLa cells at two hours. However,
the LMW band (bold arrow) seen with the other three peptides in
FIG. 7 was only visible with Caco2 cells, but not with HeLa cells.
After washing and overnight incubation, Caco2 cells appeared to
have processed the LMW intermediate band (bold arrow) into cellular
proteins, while HeLa cells apparently lacked the ability to
complete this next step (i.e., no radioactive cellular proteins at
these MWs were visualized, and all HeLa bound radioactivity was
still at the same molecular weight as the originally bound
.sup.32P-labeled peptide (thin arrow)). The two bands (thin arrows,
first lane of MA20 and MA22 gels) were the result of incubation of
.sup.32P-labeled peptide in medium containing serum for two hours
at 37.degree. C., and demonstrated apparent partial proteolysis of
the peptide during that time.
EXAMPLE 5
[0052] Only colon adenocarcinoma cells process bound radioactivity
into cellular proteins. FIG. 8 displays the results of incubating
peptides MA11, MA20 and MA22 with seven different carcinoma cell
lines at 2 hours and after overnight incubation. Peptides MA11 and
MA22 exhibited strong binding and transfer of .sup.32P only to the
three colon adenocarcinoma lines (Caco2, HCT116, and HCT15) and not
to cervical (HeLa), fibrosarcoma (1080), pancreatic or lung
adenocarcinoma cells. MA20, in contrast, bound avidly to all seven
cell lines, but its radioactivity was processed into the LMW band
(bold arrow) and later into cellular proteins only by the three
colon adenocarcinoma lines (Caco2, HCT116, and HCT15). HCT116 cells
consistently bound, as well as processed into larger-MW bands,
radioactivity from all three .sup.32P-labeled peptides (MA11, MA20,
and MA22) at a much lower rate than Caco2 and HCT15 cells, but
incorporation into HCT116 cellular proteins was eventually visible
on longer exposures (data not shown).
EXAMPLE 6
[0053] Non-phosphorylated peptide competes with .sup.32P-labeled
peptide for binding to Caco2 cells. The 12-aa peptide MA11 was
chemically synthesized, labeled with .sup.32P, and used in a
competitive binding assay with Caco2 cells against varying amounts
of cold, non-phosphorylated MA11 peptide. FIG. 9 illustrates that
phosphorylation of this peptide was not required to successfully
compete for binding to Caco2 cells, and that increasing amounts of
cold competitor rapidly inhibited the amount of .sup.32P-labeled
peptide that bound to cells.
REFERENCES
[0054] The disclosure of each reference cited is expressly
incorporated herein. [0055] 1. Sjoblom T, Jones S, Wood L D,
Parsons D W, Lin J, et al. (2007) The consensus coding sequences of
human breast and colorectal cancers. Science 314:268-274. [0056] 2.
Wood L D, Parsons D W, Jones S, Lin J, Sjoblom T, et al. (2007) The
genomic landscapes of human breast and colorectal cancers. Science
318:1108-1113. [0057] 3. Slamon D J, Leyland-Jones B, Shak S, Fuchs
H, Paton V, et al. (2001) Use of chemotherapy plus a monoclonal
antibody against HER2 for metastatic breast cancer that
overexpresses HER2. N. Engl. J. Med. 344:783-792. [0058] 4. Romond
E H, Perez E A, Bryant J, Suman V J, Geyer C E, et al. (2005)
Trastuzumab plus adjuvant chemotherapy for operable HER2 positive
breast cancer. N. Engl. J. Med. 353:1673-1684. [0059] 5. Tan-Chiu
E, Yothers G, Romond E, Geyers C E, Ewer M, et al. (2005)
Assessment of cardiac dysfunction in a randomized trial comparing
doxorubicin and cyclophosphamide followed by paclitaxel, with or
without trastuzumab as adjuvant therapy in node positive, human
epidermal growth factor receptor 2-overexpressing breast cancer. J.
Clin. Oncol. 23:7811-7819. [0060] 6. Davis T A, Grillo-Lopez A J,
White C A, McLaughlin P, Czuczman M S, et al. (2000) Rituximab
anti-CD20 monoclonal antibody therapy in non-Hodgkin's lymphoma:
safety and efficacy of retreatment. J. Clin. Oncol. 18:3135-3143.
[0061] 7. Rini B I, Rathmell W K (2007) biological aspects and
binding strategies of vascular endothelial growth factor in renal
cell carcinoma. Clin. Cancer Res. 13:741-746. [0062] 8. Van Cutsem
E, Peeters M, Siena S, Humblet Y, Hendlisz A, et al. (2007)
Open-label phase III trial of panitumumab plus best supportive care
compared with best supportive care alone in patients with
chemotherapy-refractory metastatic colorectal cancer. J. Clin.
Oncol. 25:1658-1664. [0063] 9. Cilley J C, Barfi K, Benson A B,
Mulcahy M F (2007) Bevacizumab in the treatment of colorectal
cancer. Expert. Opin. Biol. Ther. 7:739-749. [0064] 10. Wiseman G
A, White C A, Sparks R B, Erwin W D, Podoloff D A, et al. (2001)
Biodistribution and dosimetry results from a phase III
prospectively randomized controlled trial of Zevalin
radioimmunotherapy for low-grade, follicular, or transformed B-cell
non-Hodgkin's lymphoma. Crit. Rev. Oncol. Hematol. 39:181-194.
[0065] 11. Vose, J M (2004) Bexxar: novel radioimmunotherapy for
the treatment of low-grade and transformed low-grade non-Hodgkin's
lymphoma. Oncologist 9:160-172. [0066] 12. Emir E E, Qureshi U,
Dearling J L J, Boxer G M, Clatworthy I, et al. (2007) Predicting
Response to radioimmunotherapy from the tumor microenvironment of
colorectal cancers. Cancer Res. 67:11896-11905. [0067] 13. Abraham
J M, Sato F, Cheng Y, Paun B, Kan T, et al. (2007) Novel
decapeptides that bind avidly and deliver radioisotope to colon
cancer cells. PLoS ONE 2:e964. [0068] 14. Kreitman R J (2006)
Immunotoxins for targeted cancer therapy AAPS J. 8:532-551. [0069]
15. Kreitman R J, Pastan I (2006) Immunotoxins in the treatment of
hematologic malignancies. Curr. Drug Targets 7:1301-1311. [0070]
16. Kreitman R J (2003) Recombinant toxins for the treatment of
cancer. Curr. Opin. Mol. Ther. 5:44-51. [0071] 17. Boerman O C,
Koppe M J, Postema E J, Corstens F H, Oyen W J (2007) Radionuclide
therapy of cancer with radiolabeled antibodies. Anticancer Agents
Med. Chem. 7:335-343. [0072] 18. Wu A M, Senter P D (2005) Arming
antibodies: prospects and challenges for immunoconjugates. Nat.
Biotech. 23:1137-1146. [0073] 19. Okarvi S M (2004) Peptide-based
radiopharmaceuticals: Future tools for diagnostic imaging of
cancers and other diseases. Med. Res. Rev. 24:357-397. [0074] 20.
Aina O H, Sroka T C, Chen M L, Lam K S (2002) Therapeutic Cancer
Targeting Peptides. Biopolymers 66:184-199. [0075] 21. Wangler C,
Buchmann I, Eisenhut M, Haberkorn U, Mier W (2007) Radiolabeled
peptides and proteins in cancer therapy. Protein Pept. Lett.
14:273-279. [0076] 22. Aina O H, Marik J, Liu, Lau D H, Lam K S
(2005) Identification of novel targeting peptides for human ovarian
cancer cells using "one bead one-compound" combinatorial libraries.
Mol. Cancer Ther. 4:806-813. [0077] 23. Reilly R M (2006)
Radioimmunotherapy of solid tumors: The promise of pretargeting
strategies using bispecific antibodies and radiolabeled haptens. J.
Nucl. Med. 47:196-199. [0078] 24. Jain M, Venkatraman G, Batra S K
(2007) Optimization of radioimmunotherapy of solid tumors:
Biological impediments and their modulation. Clin. Cancer Res.
13:1374-1382. [0079] 25. Brans B, Linden O, Giammarile F, Tennvall
J, Punt C (2006) Clinical applications of newer radionuclide
therapies. Eur. J. Cancer 42:994-1003. [0080] 26. Bertagnolli M
(2005) Radioimmunotherapy for colorectal cancer. Clin. Cancer Res.
11:4337-4338. [0081] 27. Zafir-Lavie I, Michaeli Y, Reiter Y (2007)
Novel antibodies as anticancer agents. Oncogene 26: 3714-3733.
Sequence CWU 1
1
40110PRTArtificial Sequencesynthetic or recombinant sequence 1Gly
Ser Arg Arg Ala Ser Val Gly Ser Ala1 5 10210PRTArtificial
Sequencesynthetic or recombinant sequence 2Gly Ser Arg Gly Ala Ser
Val Gly Gly Ala1 5 10310PRTArtificial Sequencesynthetic or
recombinant sequence 3Gly Ser Arg Arg Gly Ser Val Gly Ser Ala1 5
10410PRTArtificial Sequencesynthetic or recombinant sequence 4Gly
Ser Arg Arg Gly Ser Val Ala Ser Ala1 5 10510PRTArtificial
Sequencesynthetic or recombinant sequence 5Gly Ser Arg Arg Ala Ser
Val Ala Ser Ala1 5 10610PRTArtificial Sequencesynthetic or
recombinant sequence 6Gly Ser Arg Arg Ala Ser Val Gly Ser Gly1 5
10710PRTArtificial Sequencesynthetic or recombinant sequence 7Gly
Ser Arg Gly Gly Ser Val Gly Ser Ala1 5 10810PRTArtificial
Sequencesynthetic or recombinant sequence 8Gly Ser Arg Gly Gly Ser
Val Ala Ser Ala1 5 10910PRTArtificial Sequencesynthetic or
recombinant sequence 9Gly Ser Arg Gly Gly Ser Val Gly Ser Gly1 5
101034PRTArtificial Sequencesynthetic or recombinant sequence 10Gly
Ser Arg Arg Ala Ser Val Gly Ser Ile Ser Thr Arg Pro Ile His1 5 10
15Leu Glu Pro Arg Pro Thr Ala Ala Pro Arg Gly Ser Pro Gly Ile His
20 25 30Arg Asp1112PRTArtificial Sequencesynthetic or recombinant
sequence 11Gly Ser Arg Arg Ala Ser Val Gly Ser Tyr His Val1 5
101219PRTArtificial Sequencesynthetic or recombinant sequence 12Gly
Ser Arg Arg Ala Ser Val Gly Ser Asn Gly Asp Gly Val Phe Ser1 5 10
15Leu Val Arg1314PRTArtificial Sequencesynthetic or recombinant
sequence 13Gly Ser Arg Arg Ala Ser Val Gly Ser Lys Asn Cys Arg Arg1
5 101434PRTArtificial Sequencesynthetic or recombinant sequence
14Gly Ser Arg Arg Ala Ser Val Gly Ser Leu Gly Val Leu Arg Leu Val1
5 10 15Pro Ile Val Arg Arg Val Cys Gln Arg Cys Gly Ser Pro Gly Ile
His 20 25 30Arg Asp1534PRTArtificial Sequencesynthetic or
recombinant sequence 15Gly Ser Arg Arg Ala Ser Val Gly Ser Val Asp
Gly Ala Ser Leu Arg1 5 10 15Cys Ser Ala Gln Asp Pro Pro Pro Lys Asp
Gly Ser Pro Gly Ile His 20 25 30Arg Asp1615PRTArtificial
Sequencesynthetic or recombinant sequence 16Gly Ser Arg Arg Ala Ser
Val Gly Ser Pro Gly Ile His Arg Asp1 5 10 151715PRTArtificial
Sequencesynthetic or recombinant sequence 17Gly Ser Arg Arg Ala Ser
Val Gly Ser Pro Gly Ile His Arg Asp1 5 10 151834PRTArtificial
Sequencesynthetic or recombinant sequence 18Gly Ser Arg Arg Ala Ser
Val Gly Ser Ala Ala Ala Phe Arg Arg Ala1 5 10 15Thr Val Pro Pro Ile
Cys Arg Ala Asp Trp Gly Ser Pro Gly Ile His 20 25 30Arg
Asp199PRTArtificial Sequencesynthetic or recombinant sequence 19Gly
Ser Arg Arg Ala Ser Val Gly Ser1 52034PRTArtificial
Sequencesynthetic or recombinant sequence 20Gly Ser Arg Arg Ala Ser
Val Gly Ser Arg Val Arg Val Trp His Leu1 5 10 15Ser Pro Val Leu Lys
Val Ala Gln Leu Leu Gly Ser Pro Gly Ile His 20 25 30Arg
Asp2134PRTArtificial Sequencesynthetic or recombinant sequence
21Gly Ser Arg Arg Ala Ser Val Gly Ser Val Pro Gly Arg Ser Asn Pro1
5 10 15Pro Ala Leu Pro Gly Val Tyr Glu Leu Ala Gly Ser Pro Gly Ile
His 20 25 30Arg Asp2234PRTArtificial Sequencesynthetic or
recombinant sequence 22Gly Ser Arg Arg Ala Ser Val Gly Ser Val Val
Ala Arg Leu Tyr Val1 5 10 15Arg Gln Pro Gln Asp Ser Asn Ile Thr Phe
Gly Ser Pro Gly Ile His 20 25 30Arg Asp2334PRTArtificial
Sequencesynthetic or recombinant sequence 23Gly Ser Arg Arg Ala Ser
Val Gly Ser Asp Ala His Ser Leu Met Ala1 5 10 15Gly Pro Thr Ala Glu
Ser Leu Glu Phe Leu Gly Ser Pro Gly Ile His 20 25 30Arg
Asp2434PRTArtificial Sequencesynthetic or recombinant sequence
24Gly Ser Arg Arg Ala Ser Val Gly Ser Glu Pro Thr Tyr Asn Thr Thr1
5 10 15Phe Ser Phe Gln Ala Val Asp Tyr Ser Tyr Gly Ser Pro Gly Ile
His 20 25 30Arg Asp2534PRTArtificial Sequencesynthetic or
recombinant sequence 25Gly Ser Arg Arg Ala Ser Val Gly Ser Cys Cys
Gln Arg Met Gly Trp1 5 10 15Gly His Phe Arg Leu Thr Val Gly Asn Phe
Gly Ser Pro Gly Ile His 20 25 30Arg Asp2634PRTArtificial
Sequencesynthetic or recombinant sequence 26Gly Ser Arg Arg Ala Ser
Val Gly Ser Ala Gly Gln Val Thr Ser Ala1 5 10 15Gly Glu Leu Ser Gly
Val Ser His Ile Thr Gly Ser Pro Gly Ile His 20 25 30Arg
Asp2710PRTArtificial Sequencesynthetic or recombinant sequence
27Gly Ser Arg Arg Ala Ser Val Gly Ser Ile1 5 102817PRTArtificial
Sequencesynthetic or recombinant sequence 28Gly Ser Arg Arg Ala Ser
Val Gly Ser Ile Phe Ser Val Asp Trp Val1 5 10
15Ala2918PRTArtificial Sequencesynthetic or recombinant sequence
29Val Pro Arg Gly Ser Arg Arg Ala Ser Val Gly Ser Pro Gly Ile His1
5 10 15Arg Asp3063DNAArtificial Sequencesynthetic or recombinant
sequence 30ggatccnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnngga 60tcc 633137PRTArtificial Sequencesynthetic or
recombinant sequence 31Val Pro Arg Gly Ser Arg Arg Ala Ser Val Gly
Ser Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Gly Ser Pro 20 25 30Gly Ile His Arg Asp
353237PRTArtificial Sequencesynthetic or recombinant sequence 32Val
Pro Arg Gly Ser Arg Arg Ala Ser Val Gly Ser Xaa Xaa Xaa Xaa1 5 10
15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Ser Pro
20 25 30Gly Ile His Arg Asp 353334PRTArtificial Sequencesynthetic
or recombinant sequence 33Gly Ser Arg Arg Ala Ser Val Gly Ser Xaa
Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Gly Ser Pro Gly Ile His 20 25 30Arg Asp344PRTArtificial
Sequencesynthetic or recombinant peptide motif 34Arg Arg Xaa
Ser1354PRTArtificial Sequencesynthetic or recombinant peptide motif
35Arg Arg Xaa Ser1365PRTArtificial Sequencesynthetic or recombinant
peptide motif 36Ser Arg Arg Xaa Ser1 5375PRTArtificial
Sequencesynthetic or recombinant peptide motif 37Arg Arg Xaa Ser
Xaa1 5386PRTArtificial Sequencesynthetic or recombinant peptide
motif 38Gly Ser Arg Arg Xaa Ser1 5395PRTArtificial
Sequencesynthetic or recombinant peptide motif 39Arg Arg Xaa Ser
Val1 5406PRTArtificial Sequencesynthetic or recombinant peptide
motif 40Arg Arg Xaa Ser Val Xaa1 5
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