U.S. patent application number 12/559305 was filed with the patent office on 2010-03-25 for molecules that selectively home to vasculature of premalignant or malignant lesions of the pancreas and other organs.
This patent application is currently assigned to BURNHAM INSTITUTE FOR MEDICAL RESEARCH. Invention is credited to Douglas Hanahan, Johanna A. Joyce, Pirjo Laakkonen, Erkki Ruoslahti.
Application Number | 20100076175 12/559305 |
Document ID | / |
Family ID | 34549491 |
Filed Date | 2010-03-25 |
United States Patent
Application |
20100076175 |
Kind Code |
A1 |
Hanahan; Douglas ; et
al. |
March 25, 2010 |
MOLECULES THAT SELECTIVELY HOME TO VASCULATURE OF PREMALIGNANT OR
MALIGNANT LESIONS OF THE PANCREAS AND OTHER ORGANS
Abstract
The invention provides a conjugate that includes a therapeutic
moiety linked to a peptide or peptidomimetic that selectively homes
to vasculature of premalignant pancreas. The peptide or
peptidomimetic contains at least 5 contiguous amino acids of an
amino acid sequence selected from CRSRKG (SEQ ID NO:9) and CEYQLDVE
(SEQ ID NO:34), or a conservative variant or peptidomimetic
thereof. The invention additionally provides a conjugate containing
a therapeutic moiety linked to a peptide or peptidomimetic that
selectively homes to pancreatic tumor cells and pancreatic tumor
vasculature, the peptide or peptidomimetic comprising at least 5
contiguous amino acids of an amino acid sequence selected from
CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID NO:19), and VGVGEWSV (SEQ
ID NO:35), or a conservative variant or peptidomimetic thereof.
Inventors: |
Hanahan; Douglas; (San
Francisco, CA) ; Ruoslahti; Erkki; (Buellton, CA)
; Joyce; Johanna A.; (New York, NY) ; Laakkonen;
Pirjo; (Helsinki, FI) |
Correspondence
Address: |
MCDERMOTT, WILL & EMERY
11682 EL CAMINO REAL, SUITE 400
SAN DIEGO
CA
92130-2047
US
|
Assignee: |
BURNHAM INSTITUTE FOR MEDICAL
RESEARCH
La Jolla
CA
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Oakland
CA
|
Family ID: |
34549491 |
Appl. No.: |
12/559305 |
Filed: |
September 14, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10977367 |
Oct 29, 2004 |
7598341 |
|
|
12559305 |
|
|
|
|
60516118 |
Oct 31, 2003 |
|
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Current U.S.
Class: |
530/328 ;
530/329 |
Current CPC
Class: |
C07K 7/06 20130101; C07K
14/4748 20130101; A61K 49/0043 20130101; A61K 49/0056 20130101;
A61K 38/00 20130101; A61K 47/62 20170801 |
Class at
Publication: |
530/328 ;
530/329 |
International
Class: |
C07K 7/06 20060101
C07K007/06 |
Goverment Interests
[0002] This invention was made with government support under
CA82713, awarded by the National Cancer Institute, and DAMD
17-02-1-0315, awarded by the Department of Defense. The government
has certain rights in this invention.
Claims
1. A peptide or peptidomimetic, having a length of less than 60
amino acid residues and comprising at least 5 contiguous amino
acids of an amino acid sequence selected from CKGAKAR (SEQ ID
NO:19), FRVGVADV (SEQ ID NO:27), CEYQLDVE (SEQ ID NO:34) and
VGVGEWSV (SEQ ID NO:35), or a conservative variant or
peptidomimetic thereof.
2. The peptide or peptidomimetic of claim 1, wherein said peptide
or peptidomimetic is a peptide.
3. The peptide or peptidomimetic of claim 1, having a length of
less than 40 amino acid residues.
4. The peptide or peptidomimetic of claim 1, having a length of
less than 20 amino acid residues.
5. The peptide or peptidomimetic of claim 1, having a length of
less than 10 amino acid residues.
6. A conjugate, comprising a therapeutic moiety linked to a peptide
or peptidomimetic that selectively homes to vasculature of
premalignant pancreas, said peptide or peptidomimetic comprising at
least 5 contiguous amino acids of the amino acid sequence CEYQLDVE
(SEQ ID NO:34), or a conservative variant or peptidomimetic
thereof, or wherein said peptide or peptidomimetic binds
specifically to a cognate receptor for SEQ ID NO:34.
7. The conjugate of claim 6, wherein said peptide or peptidomimetic
has a length of less than 100 residues.
8. The conjugate of claim 6, wherein said peptide or peptidomimetic
has a length of less than 50 residues.
9. The conjugate of claim 6, wherein said peptide or peptidomimetic
has a length of less than 25 residues.
10. The conjugate of claim 6, wherein said peptide or
peptidomimetic is a peptide.
11-22. (canceled)
23. A conjugate, comprising a therapeutic moiety linked to a
peptide or peptidomimetic that selectively homes to pancreatic
tumor cells and pancreatic tumor vasculature, said peptide or
peptidomimetic comprising at least 5 contiguous amino acids of an
amino acid sequence selected from CKGAKAR (SEQ ID NO:19) and
VGVGEWSV (SEQ ID NO:35), or a conservative variant or
peptidomimetic thereof, or wherein said peptide or peptidomimetic
binds specifically to a cognate receptor for SEQ ID NO:19 or SEQ ID
NO:35.
24. The conjugate of claim 23, wherein said peptide or
peptidomimetic has a length of less than 100 residues.
25. The conjugate of claim 23, wherein said peptide or
peptidomimetic has a length of less than 50 residues.
26. The conjugate of claim 23, wherein said peptide or
peptidomimetic has a length of less than 25 residues.
27. The conjugate of claim 23, wherein said peptide or
peptidomimetic is a peptide.
28. (canceled)
29. The conjugate of claim 23, wherein said peptide or
peptidomimetic comprises at least 5 contiguous amino acids of
CKGAKAR (SEQ ID NO:19) or a conservative variant or peptidomimetic
thereof.
30. The conjugate of claim 23, wherein said peptide or
peptidomimetic comprises at least 5 contiguous amino acids of
VGVGEWSV (SEQ ID NO:35) or a conservative variant or peptidomimetic
thereof.
31-43. (canceled)
44. The conjugate of claim 23, wherein said therapeutic moiety is
an angiogenic inhibitor.
45. The conjugate of claim 44, wherein said angiogenic inhibitor is
selective for mature tumor vasculature.
46. The conjugate of claim 23, wherein said therapeutic moiety is a
cytotoxic agent.
47-54. (canceled)
55. A conjugate, comprising a therapeutic moiety linked to a
peptide or peptidomimetic that selectively homes to premalignant
and malignant pancreatic vasculature, said peptide or
peptidomimetic comprising at least 5 contiguous amino acids of an
amino acid sequence selected from FRVGVADV (SEQ ID NO:27), or a
conservative variant or peptidomimetic thereof, or wherein said
peptide or peptidomimetic binds specifically to a cognate receptor
for SEQ ID NO:27.
56-67. (canceled)
68. The conjugate of claim 55, wherein said peptide or
peptidomimetic has a length of less than 100 residues.
69. The conjugate of claim 55, wherein said peptide or
peptidomimetic has a length of less than 50 residues.
70. The conjugate of claim 55, wherein said peptide or
peptidomimetic has a length of less than 25 residues.
71. The conjugate of claim 55, wherein said peptide or
peptidomimetic is a peptide.
72. The conjugate of claim 55, wherein said therapeutic moiety is
an angiogenic inhibitor.
73. The conjugate of claim 72, wherein said angiogenic inhibitor is
selective for mature tumor vasculature.
74. The conjugate of claim 55, wherein said therapeutic moiety is a
cytotoxic agent.
75. A multivalent conjugate, comprising a therapeutic moiety linked
to at least two peptides or peptidomimetics that selectively home
to vasculature of premalignant pancreas, each of said peptides or
peptidomimetics comprising at least 5 contiguous amino acids of the
amino acid sequence CEYQLDVE (SEQ ID NO:34), or wherein each of
said peptides or peptidomimetics binds specifically to a cognate
receptor for SEQ ID NO:34; wherein said at least two peptides or
peptidomimetics selectively home to pancreatic tumor cells and
pancreatic tumor vasculature, each of said peptides or
peptidomimetics comprising at least 5 contiguous amino acids of an
amino acid sequence selected from CKGAKAR (SEQ ID NO:19), and
VGVGEWSV (SEQ ID NO:35), or a conservative variant or
peptidomimetic thereof, or wherein each of said peptides or
peptidomimetics binds specifically to a cognate receptor for an
amino acid sequence selected from CKGAKAR (SEQ ID NO:19) and
VGVGEWSV (SEQ ID NO:35); or wherein said at least two peptides or
peptidomimetics selectively home to premalignant and malignant
vasculature, each of said peptides or peptidomimetics comprising at
least 5 contiguous amino acids of the amino acid sequence FRVGVADV
(SEQ ID NO:27), or a conservative variant or peptidomimetic
thereof, or wherein each of said peptides or peptidomimetics binds
specifically to a cognate receptor for the amino acid sequence
FRVGVADV (SEQ ID NO:27).
76-93. (canceled)
Description
[0001] This application is a continuation of U.S. application Ser.
No. 10/977,367, filed Oct. 29, 2004, which claims the benefit of
U.S. provisional Ser. No. 60/516,118, filed Oct. 31, 2003, each of
which the entire contents are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] A hurdle to advances in preventing and treating cancer is
the lack of agents that effectively target a cancer or
pre-cancerous tissue while sparing normal tissues. Radiation
therapy and surgery, which are typically localized treatments, can
cause substantial damage to normal tissue in the treatment field,
resulting in scarring and loss of normal tissue. Furthermore,
chemotherapy, which is typically a systemic treatment, can cause
substantial damage to normal organs such as non-cancerous skin,
bone marrow, mucosa, and small intestine, in particular because
these tissues undergo rapid cell turnover and continuous cell
division. As a result, undesirable side effects such as nausea,
loss of hair and drop in blood cell count can result from systemic
treatment with a chemotherapeutic agent. Such undesirable side
effects often limit the amount of drug that can be safely
administered, thereby reducing patient survival rates and quality
of life.
[0005] Selective delivery of therapeutics such as anti-angiogenic
agents to vasculature that supports tumors would result in less
toxic therapy since rapidly proliferating normal cells would be
spared. Similarly, selective delivery of anti-angiogenic agents to
vasculature of premalignant tissues would provide a prophylactic
strategy for reducing the risk of cancer. However, to date, it has
been difficult to produce drugs that are delivered specifically to
tumor vasculature or to vasculature of premalignant tissues. Thus,
there is a need for molecules that selectively target tumor tissues
and vasculature, such as pancreatic tumors and vasculature, as well
as for molecules that selectively target premalignant tissues and
vasculature, such as premalignant pancreas and vasculature. The
present invention satisfies these needs and provides related
advantages as well.
[0006] 2. Background Information
[0007] A hurdle to advances in preventing and treating cancer is
the lack of agents that effectively target a cancer or
pre-cancerous tissue while sparing normal tissues. Radiation
therapy and surgery, which are typically localized treatments, can
cause substantial damage to normal tissue in the treatment field,
resulting in scarring and loss of normal tissue. Furthermore,
chemotherapy, which is typically a systemic treatment, can cause
substantial damage to normal organs such as non-cancerous skin,
bone marrow, mucosa, and small intestine, in particular because
these tissues undergo rapid cell turnover and continuous cell
division. As a result, undesirable side effects such as nausea,
loss of hair and drop in blood cell count can result from systemic
treatment with a chemotherapeutic agent. Such undesirable side
effects often limit the amount of drug that can be safely
administered, thereby reducing patient survival rates and quality
of life.
[0008] Selective delivery of therapeutics such as anti-angiogenic
agents to vasculature that supports tumors would result in less
toxic therapy since rapidly proliferating normal cells would be
spared. Similarly, selective delivery of anti-angiogenic agents to
vasculature of premalignant tissues would provide a prophylactic
strategy for reducing the risk of cancer. However, to date, it has
been difficult to produce drugs that are delivered specifically to
tumor vasculature or to vasculature of premalignant tissues. Thus,
there is a need for molecules that selectively target tumor tissues
and vasculature, such as pancreatic tumors and vasculature, as well
as for molecules that selectively target premalignant tissues and
vasculature, such as premalignant pancreas and vasculature. The
present invention satisfies these needs and provides related
advantages as well.
SUMMARY OF THE INVENTION
[0009] The invention provides a peptide or peptidomimetic having a
length of less than 60 amino acid residues and containing at least
5 contiguous amino acids of CRGRRST (SEQ ID NO:5), CRSRKG (SEQ ID
NO:9), CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID NO:19), FRVGVADV
(SEQ ID NO:27), CEYQLDVE (SEQ ID NO:34) and VGVGEWSV (SEQ ID
NO:35), or a conservative variant or peptidomimetic thereof. In
embodiments of the invention, the peptide or peptidomimetic
contains 40 amino acids, 20 amino acids or 10 amino acids.
[0010] The invention provides a conjugate that includes a
therapeutic moiety linked to a peptide or peptidomimetic that
selectively homes to vasculature of premalignant pancreas. The
peptide or peptidomimetic contains at least 5 contiguous amino
acids of an amino acid sequence selected from CRSRKG (SEQ ID NO:9)
and CEYQLDVE (SEQ ID NO:34), or a conservative variant or
peptidomimetic thereof. In embodiments of the invention, the
peptide or peptidomimetic has a length of less than 100 residues,
less than 50 residues and less than 25 residues. Also provided by
the invention is a conjugate containing a therapeutic moiety linked
to a peptide or peptidomimetic that selectively homes to
vasculature of premalignant pancreas, in which the peptide or
peptidomimetic binds specifically to a cognate receptor for SEQ ID
NO:9 or SEQ ID NO:34.
[0011] The invention additionally provides a conjugate containing a
therapeutic moiety linked to a peptide or peptidomimetic that
selectively homes to pancreatic tumor cells and pancreatic tumor
vasculature, the peptide or peptidomimetic comprising at least 5
contiguous amino acids of an amino acid sequence selected from
CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID NO:19), and VGVGEWSV (SEQ
ID NO:35), or a conservative variant or peptidomimetic thereof. In
embodiments of the invention, the peptide or peptidomimetic has a
length of less than 100 residues, less than 50 residues and less
than 25 residues. Also provided by the invention is a conjugate
that contains therapeutic moiety linked to a peptide or
peptidomimetic that selectively homes to pancreatic tumor cells and
pancreatic tumor vasculature, wherein the peptide or peptidomimetic
binds specifically to a cognate receptor for SEQ ID NO:15, SEQ ID
NO:19 or SEQ ID NO:35.
[0012] The invention provides a conjugate that contains a
therapeutic moiety linked to a peptide or peptidomimetic that
selectively homes to premalignant and malignant pancreatic
vasculature, the peptide or peptidomimetic containing at least 5
contiguous amino acids of an amino acid sequence selected from
CRGRRST (SEQ ID NO:5) and FRVGVADV (SEQ ID NO:27), or a
conservative variant or peptidomimetic thereof. In embodiments of
the invention, the peptide or peptidomimetic has a length of less
than 100 residues, 50 residues and less than 25 residues. Also
provided by the invention is a conjugate that contains a
therapeutic moiety linked to a peptide or peptidomimetic that
selectively homes to pancreatic tumor cells and pancreatic tumor
vasculature, wherein the peptide or peptidomimetic binds
specifically to a cognate receptor for SEQ ID NO:5 or SEQ ID
NO:27
[0013] The conjugates of the invention can be linked to a variety
of moieties. In one embodiment, the moiety is a therapeutic moiety.
In another embodiment, the moiety is a detectable moiety.
[0014] The invention provides a multivalent conjugate, containing a
therapeutic moiety linked to at least two peptides or
peptidomimetics that selectively home to vasculature of
premalignant pancreas, each of the peptides or peptidomimetics
containing at least 5 contiguous amino acids of an amino acid
sequence selected from CRSRKG (SEQ ID NO:9) and CEYQLDVE (SEQ ID
NO:34).
[0015] The invention also provides a multivalent conjugate that
contains a therapeutic moiety linked to at least two peptides or
peptidomimetics that selectively home to vasculature of
premalignant pancreas, wherein each of the peptides or
peptidomimetics binds specifically to a cognate receptor for SEQ ID
NO:9 or SEQ ID NO:34.
[0016] The invention further provides a multivalent conjugate that
contains a therapeutic moiety linked to at least two peptides or
peptidomimetics that selectively home to pancreatic tumor cells and
pancreatic tumor vasculature, each of the peptides or
peptidomimetics containing at least 5 contiguous amino acids of an
amino acid sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR
(SEQ ID NO:19), and VGVGEWSV (SEQ ID NO:35), or a conservative
variant or peptidomimetic thereof.
[0017] Also provided by the invention is a multivalent conjugate
that contains a therapeutic moiety linked to at least two peptides
or peptidomimetics that selectively home to premalignant and
malignant pancreatic vasculature, wherein each of the peptides or
peptidomimetics binds specifically to a cognate receptor for an
amino acid sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR
(SEQ ID NO:19), and VGVGEWSV (SEQ ID NO:35).
[0018] The invention provides a multivalent conjugate that contains
a therapeutic moiety linked to at least two peptides or
peptidomimetics that selectively home to premalignant and malignant
vasculature, each of the peptides or peptidomimetics containing at
least 5 contiguous amino acids of an amino acid sequence selected
from CRGRRST (SEQ ID NO:5) and FRVGVADV (SEQ ID NO:27), or a
conservative variant or peptidomimetic thereof.
[0019] Also provided is a multivalent conjugate that contains a
therapeutic moiety linked to at least two peptides or
peptidomimetics that selectively home to pancreatic tumor cells and
pancreatic tumor vasculature, wherein each of the peptides or
peptidomimetics binds specifically to a cognate receptor for an
amino acid sequence selected from CRGRRST (SEQ ID NO:5) and
FRVGVADV (SEQ ID NO:27).
[0020] The invention provides a method of directing a moiety to a
pancreatic premalignant lesion in an individual. The method
involves administering to the individual a conjugate containing a
moiety linked to (a) a peptide or peptidomimetic that selectively
homes to vasculature of premalignant pancreas, the peptide or
peptidomimetic containing at least 5 contiguous amino acids of an
amino acid sequence selected from CRSRKG (SEQ ID NO:9) and CEYQLDVE
(SEQ ID NO:34), or a conservative variant or peptidomimetic
thereof, or (b) a peptide or peptidomimetic that selectively homes
to premalignant and malignant pancreatic vasculature, the peptide
or peptidomimetic containing at least 5 contiguous amino acids of
an amino acid sequence selected from CRGRRST (SEQ ID NO:5) and
FRVGVADV (SEQ ID NO:27), or a conservative variant or
peptidomimetic thereof, thereby directing the moiety to the
vasculature of the pancreatic premalignant lesion. In one
embodiment, the moiety is a therapeutic moiety, such as an
angiogenic inhibitor. In another embodiment, the moiety is a
diagnostic moiety.
[0021] The invention provides a method of imaging pancreatic
premalignant lesions in an individual. The method involves: (a)
administering to the individual a conjugate containing a detectable
moiety linked to a peptide or peptidomimetic that selectively homes
to vasculature of premalignant pancreas, the peptide or
peptidomimetic containing at least 5 contiguous amino acids of an
amino acid sequence selected from CRSRKG (SEQ ID NO:9) and CEYQLDVE
(SEQ ID NO:34), or a conservative variant or peptidomimetic
thereof, and (b) detecting the conjugate, thereby imaging
pancreatic premalignant lesions.
[0022] Further provided by the invention is a method of treating a
pancreatic premalignant lesion in an individual. The method
involves administering to the individual a conjugate containing a
therapeutic moiety linked to: (a) a peptide or peptidomimetic that
selectively homes to premalignant and malignant pancreatic
vasculature, the peptide or peptidomimetic containing at least 5
contiguous amino acids of an amino acid sequence selected from
CRGRRST (SEQ ID NO:5), and FRVGVADV (SEQ ID NO:27), or a
conservative variant or peptidomimetic thereof, or (b) a peptide or
peptidomimetic that selectively homes to vasculature of
premalignant pancreas containing at least 5 contiguous amino acids
of an amino acid sequence selected from CRSRKG (SEQ ID NO:9) and
CEYQLDVE (SEQ ID NO:34), thereby directing the therapeutic moiety
to the pancreatic premalignant lesion in the individual to treat
the pancreatic premalignant lesion. In one embodiment, the moiety
is a therapeutic moiety, such as an angiogenic inhibitor.
[0023] The invention provides of directing a moiety to pancreatic
tumor cells and pancreatic tumor vasculature in an individual. The
method involves administering to the individual a conjugate
containing a moiety linked to: (a) a peptide or peptidomimetic that
selectively homes to pancreatic tumor cells and pancreatic tumor
vasculature, the peptide or peptidomimetic containing at least 5
contiguous amino acids of an amino acid sequence selected from
CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID NO:19), FRVGVADV and
VGVGEWSV (SEQ ID NO:35), or a conservative variant or
peptidomimetic thereof, or (b) a peptide or peptidomimetic that
selectively homes to premalignant and malignant pancreatic
vasculature, the peptide or peptidomimetic containing at least 5
contiguous amino acids of an amino acid sequence selected from
CRGRRST (SEQ ID NO:5) and FRVGVADV (SEQ ID NO:27), or a
conservative variant or peptidomimetic thereof, thereby directing
the moiety to pancreatic tumor cells and pancreatic tumor
vasculature.
[0024] The invention provides a method of imaging pancreatic tumors
and pancreatic tumor vasculature in an individual. The method
involves (a) administering to the individual a conjugate containing
a detectable moiety linked to a peptide or peptidomimetic that
selectively homes to pancreatic tumor cells and pancreatic tumor
vasculature, the peptide or peptidomimetic containing at least 5
contiguous amino acids of an amino acid sequence selected from
CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID NO:19) and VGVGEWSV (SEQ ID
NO:35), or a conservative variant or peptidomimetic thereof, and
(b) detecting the conjugate, thereby imaging the pancreatic tumors
and pancreatic tumor vasculature.
[0025] The invention provides a method of reducing the severity of
pancreatic cancer in an individual. The method involves
administering to the individual a conjugate containing a
therapeutic moiety linked to:
[0026] (a) a peptide or peptidomimetic that selectively homes to
pancreatic tumor cells and pancreatic tumor vasculature, the
peptide or peptidomimetic containing at least 5 contiguous amino
acids of an amino acid sequence selected from CKAAKNK (SEQ ID
NO:15), CKGAKAR (SEQ ID NO:19), FRVGVADV and VGVGEWSV (SEQ ID
NO:35), or a conservative variant or peptidomimetic thereof, or (b)
a peptide or peptidomimetic that selectively homes to premalignant
and malignant pancreatic vasculature, the peptide or peptidomimetic
containing at least 5 contiguous amino acids of an amino acid
sequence selected from CRGRRST (SEQ ID NO:5) and FRVGVADV (SEQ ID
NO:27), or a conservative variant or peptidomimetic thereof,
thereby directing the therapeutic moiety to pancreatic tumor cells
or pancreatic tumor vasculature in the individual to reduce the
severity of the pancreatic cancer.
[0027] The invention provides a method of staging tumor progression
in an individual having or suspected of having a pancreatic
premalignant lesion or pancreatic tumor. The method involves: (a)
administering to the individual at least one conjugate containing a
detectable moiety linked to (i) a peptide or peptidomimetic that
selectively homes to vasculature of premalignant pancreas, the
peptide or peptidomimetic specifically binding a cognate receptor
for CRSRKG (SEQ ID NO:9) or CEYQLDVE (SEQ ID NO:34), or (ii) a
peptide or peptidomimetic that selectively homes to pancreatic
tumor cells and pancreatic tumor vasculature, the peptide or
peptidomimetic specifically binding a cognate receptor for an amino
acid sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID
NO:19) and VGVGEWSV (SEQ ID NO:35); and (b) detecting the
conjugate, wherein detection of the conjugate containing a peptide
or peptidomimetic that selectively homes to vasculature of
premalignant pancreas indicates a premalignant stage of tumor
progression in the individual and wherein detection of the
conjugate containing a peptide or peptidomimetic that selectively
homes to pancreatic tumor cells and pancreatic tumor vasculature
indicates a malignant stage of tumor progression in the
individual.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 shows that phage-displayed peptides selectively home
to premalignant pancreas or pancreatic tumor cells. (A) Ex vivo
screening on angiogenic islets or tumors derived from RIP1-Tag2
mice using the CX.sub.7C peptide library displayed on T7 phage. The
enriched phage pools were used for subsequent in vivo homing in
RIP1-Tag2 mice; (B) angiogenic islets (3 rounds of selection); (C)
tumors (2 rounds of selection); (D) In vivo homing of individual
phage to RIP1-Tag2 angiogenic islets and tumors.
[0029] FIG. 2 shows tumor progression stage-specific homing of
fluorescein-conjugated peptides in RIP1-Tag2 model. Visualization
of an angiogenic islet-selective peptide (RSR) homing is shown in
normal islet (A), angiogenic islet (B), and tumor (C). Homing
profiles are also shown for a tumor-selective peptide (KAA) to
normal islet (D), angiogenic islet (E), and tumor (F), as well as
of a peptide (RGR) that homes to both angiogenic islets and tumors
(G) normal islet (H) angiogenic islet, and (I) tumor. Control
tissues were (J) kidney, (K) brain, and (L) liver. Magnification
shown is 200.times..
[0030] FIG. 3 shows co-localization of fluorescein-conjugated
peptides with vascular markers in RIP1-Tag2 islet lesions. RSR
peptide localization in an angiogenic islet is shown in panel A and
D, while co-staining for MECA-32 and the merge are shown in panels
B and C. Co-staining for NG2 is shown in panel E, with the merge in
panel F. KAA peptide localization in a tumor is shown in panels G
and J, while co-staining for MECA-32 and the merge are shown in
panels H and I. Co-staining for NG2 is shown in panel K, with the
merge in panel L. RGR peptide localization in an angiogenic islet
is shown in panel M and P, while co-staining for MECA-32 and the
merge are shown in panels N and O. Co-staining for NG2 is shown in
panel Q, with the merge in panel R. Scale bar shown is 10 mm.
[0031] FIG. 4 shows evaluation of the specificity of selected
homing phage and peptides. (A) Bar graph showing homing efficiency
of individual phage to a pancreatic islet tumor in a RIP1-Tag2
mouse, a bTC3-derived subcutaneous transplant tumor in a nude
mouse, and a squamous cell carcinoma in a K14-HPV16 mouse. (B)
Table summarizing the relative homing of fluorescein-conjugated
peptides to different tumor models; +++ indicates strong homing, as
revealed by the fluorescent intensity of i.v. injected peptide, ++
indicates moderate homing, + indicates weak homing, - indicates
absence of homing. Representative images of fluorescein-conjugated
KAR peptide homing to a RIP1-Tag2 pancreatic islet tumor (C), a
bTC3 subcutaneous tumor (D), and an MDA subcutaneous tumor (E) are
also shown. Magnification shown is 200.times..
[0032] FIG. 5 shows binding of RGR phage to PDGFR.beta.. (A) Bar
graph showing binding of RGR or RSR phage to 293 cells transfected
with either the PDGFR.beta., VEGFR2, or non-transfected cells. (B)
Co-localization of fluorescein-conjugated RGR-peptide (a) with the
PDGFR.beta. antibody (b) and merged images (c, d) in RIP1-Tag2.
Magnification shown is 400.times..
DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention is directed to the discovery of homing
molecules that selectively home to vasculature of premalignant
tissues and further directed to molecules that selectively home to
tumor cells and tumor vasculature, such as premalignant pancreatic
tissue and pancreatic tumor cells and tumor vasculature. As
disclosed herein in Example I, peptides specific for premalignant
pancreatic lesions or for pancreatic tumor cells and pancreatic
tumor vasculature were isolated using a combination of in vivo and
ex vivo selections using 12 week old RIP1-Tag2 mice.
[0034] The RIP1-Tag2 transgenic mouse is a prototypical mouse model
of multistage tumorigenesis of islet cell carcinoma (Hanahan,
Nature 315:115-122 (1985)). RIP1-Tag2 transgenic mice express the
SV40 T antigens (Tag) under the control of the insulin gene
promoter, which elicits the sequential development of tumors in the
islets of Langerhans over a period of 12-14 weeks. Hyperplastic
islets begin to appear at around 4 weeks of age, and angiogenesis
is activated a few weeks later in a subset of the hyperplastic
islets, producing angiogenic (dysplastic) islets (Bergers et al.,
Int. J. Dev. Biol. 42:995-1002 (1998); Folkman et al., Nature
339:58-61 (1989)). Solid tumors form beginning at 9-10 weeks,
initially presenting as small nodules that grow and progress to
large islet tumors with well defined margins as well as two classes
of invasive carcinoma (Lopez and Hanahan, Cell 1:339-353 (2002)).
As is described in Examples I and II, stage-specific molecular
markers accessible via the circulation were identified, either on
the surface of endothelial cells, their peri-endothelial support
cells (pericytes and smooth muscle cells) or even tumor cells
themselves (as a result of the hemorrhagic, leaky angiogenic
vasculature). Phage pools that homed preferentially to different
stages during RIP1-Tag2 tumorigenesis were identified using ex vivo
and in vitro selections, as described in Examples I and II. Also
identified were `pan-angiogenic` markers shared by many types of
tumors (Example IV).
[0035] Ex vivo selections were performed using suspensions of
either angiogenic islets or solid tumors, and resulted in a 7 or
8-fold enrichment of phage relative to enrichment of nonrecombinant
phage lacking displayed peptides, respectively (FIG. 1A).
Sequential in vivo selection using angiogenic islets resulted in a
7-fold enrichment (FIG. 1B); and sequential in vivo using solid
tumors resulted in an 8-fold enrichment (FIG. 1C). Peptides
displayed on several of the selected phage clones were found to be
highly selective for either angiogenic islets in comparison to
solid tumors, or for solid tumors in comparison to angiogenic
islets (FIG. 1D).
[0036] Sequencing of phage from the selected pools identified a
number of peptide sequences that were represented more than once,
and these were tested for their ability to bind cell suspensions
prepared from angiogenic islets and tumors. Six of the phage
selected for further analysis were from the tumor screen (referred
to as KAA, RGR, RSR, VGVA, VGVG and KAR), and one (EYQ) was picked
from the angiogenic screen. Peptide sequences corresponding to each
of these peptide motifs are set forth as SEQ ID NO:15 (CKAAKNK),
SEQ ID NO:5 (CRGRRST), SEQ ID NO:9 (CRSRKG), SEQ ID NO:27
(FRVGVADV), SEQ ID NO:35 (VGVGEWSV), SEQ ID NO:19 (CKGAKAR) and SEQ
ID NO:34 (CEYQLDVE), respectively.
[0037] Each of the identified peptides was linear, although the
phage library used (CX.sub.7C) was designed to express primarily
cyclized peptides, with a minority of linear peptides. However,
linear peptides form in this library, for example, by the
occurrence of a stop codon in a random insert, causing truncation
of the peptide, and by occurrence of a frameshift mutation that
mutates the second cysteine (which is required for cyclization)
into valine.
[0038] To confirm homing specificity of the selected peptides,
purified peptides were intravenously injected into 8-week or
12-week old RIP-Tag2 mice to examine peptide localization at early
(angiogenic) or late (malignant) stages of tumor progression,
respectively. The observed peptide localization profiles in each
case was similar to those of cognate phage. As is described in
Example IV, the peptides were found to be highly selective for
premalignant angiogenic islets (RSR and EYQ peptides), malignant
solid tumors (KAA and KAR peptides), or both angiogenic islets and
tumors (RGR and VGVA peptides), and did not appreciably home to
normal islets, kidney, brain, liver, lung or spleen.
[0039] To further characterize homing selectivity, tissues were
collected following i.v. infusion with fluorescein-conjugated RSR,
KAA, RGR peptides, sectioned, and evaluated with endothelial cell
and endothelial lumen markers and a neovascular pericyte marker.
All three peptides (RSR, KAA, and RGR) co-localized both with the
endothelial cell and pericyte markers, indicating that each homes
to and binds a moiety associated with both cell types (see, for
example, FIG. 3). Co-localization of these peptides with the same
markers in adjacent exocrine pancreas or in normal pancreatic
islets was not observed.
[0040] As further disclosed in Example IV, homing selectivity of
premalignant and/or malignant tissue peptides RGR, KAA, RSR, VGVA,
VGVG, and KAR was analyzed for the ability of these peptides to
home to endothelium in tumors of different tissue origins and
localized to different anatomical locations. In particular, two
subcutaneously implanted tumors and a tumor produced in a
transgenic animal model were examined for accumulation of
fluorescein-labeled peptides following intravenous injection. As
shown in FIG. 4A, different homing specificities were observed for
each peptide in the various tumor microenvironments. In particular,
as shown in FIG. 4A, peptides KAA, RGR, RSR, VGVA, and VGVG did not
home appreciably to HPV tumor, whereas peptide KAR homed to this
tumor type. In addition, peptides KAA, RGR, VGVA, and KAR did not
home appreciably to TC3 tumor, whereas peptides RSR and VGVG homed
to this tumor type. As shown in FIG. 4B, peptides RGR and RSR did
not home appreciably to MDA tumor, whereas peptide KAA homed to
this tumor type.
[0041] As further disclosed in Example V, a receptor for the
peptide RGR was identified using a combination of sequence
searching and cell biological methods. Specifically, using sequence
searching methods, a ligand for PDGFR-.beta. was identified to
contain the sequence RGRRS. Using cell biological methods, phage
containing the RGR sequence (CRGRRST; SEQ ID NO:5) was shown to
bind to PDGFR-.beta. expressing cells, but not to cells expressing
a similar receptor (VSGFR2) or to control cells. The association of
the CRGRRST peptide with PDGFR.beta. was confirmed when
intravenously injected fluorescein-conjugated CRGRRST peptide was
shown to co-localize with PDGFR.beta., as visualized by subsequent
immunostaining of tissue sections from RIP1-Tag2 tumors.
Peptides and Peptidomimetics
[0042] Based on the above findings, the present invention provides
several isolated peptides and peptidomimetics that home to
vasculature of premalignant pancreas, to pancreatic tumor cells and
pancreatic tumor vasculature or to both premalignant and/or
malignant pancreatic vasculature. In an embodiment, the invention
provides an isolated peptide or peptidomimetic that selectively
homes to vasculature of premalignant pancreas. The peptide contains
at least 4 contiguous amino acids of the amino acid sequence CRSRKG
(SEQ ID NO:9) or CEYQLDVE (SEQ ID NO:34), or a conservative variant
or peptidomimetic thereof and has a length of less than 50
residues, such as less than 40 residues, less than 30 residues,
less than 20 residues, less than 10 residues, or less than 8
residues.
[0043] In one embodiment, a peptide of the invention, as well as a
peptide contained in a conjugate of the invention, contains at
least 4 contiguous amino acids of the amino acid sequence CRSRKG
(SEQ ID NO:9), or a conservative variant of peptidomimetic thereof.
The peptide also can contain at least 5 contiguous amino acids of
the amino acid sequence CRSRKG (SEQ ID NO:9), or at least 6
contiguous amino acids of the amino acid sequence CRSRKG (SEQ ID
NO:9), or a conservative variant of peptidomimetic thereof.
Exemplary peptide sequences that contain at least 4 contiguous
amino acids of the amino acid sequence CRSRKG (SEQ ID NO:9)
include, but are not limited to, RSRX.sub.1G wherein X.sub.1 is a
basic amino acid (SEQ ID NO:6), CRSRX.sub.1G wherein X.sub.1 is a
basic amino acid (SEQ ID NO:7), and RSRKG (SEQ ID NO:8). X.sub.1
can be, for example, arginine, histidine and lysine.
[0044] In another embodiment, a peptide of the invention, as well
as a peptide contained in a conjugate of the invention, contains at
least 4 contiguous amino acids of the amino acid sequence CEYQLDVE
(SEQ ID NO:34), or a conservative variant of peptidomimetic
thereof. The peptide also can contain at least 5 contiguous amino
acids of the amino acid sequence CEYQLDVE (SEQ ID NO:34), at least
6 contiguous amino acids of the amino acid sequence CEYQLDVE (SEQ
ID NO:34), at least 7 contiguous amino acids of the amino acid
sequence CEYQLDVE (SEQ ID NO:34), or at least 8 contiguous amino
acids of the amino acid sequence CEYQLDVE (SEQ ID NO:34) or a
conservative variant of peptidomimetic thereof. Exemplary peptide
sequences that contain at least 4 contiguous amino acids of the
amino acid sequence CEYQLDVE (SEQ ID NO:34) include, but are not
limited to CEYQL (SEQ ID NO:28), EYQLD (SEQ ID NO:29), EYQLDV (SEQ
ID NO:30), EYQLDVE (SEQ ID NO:31), YQLDV (SEQ ID NO:32), and YQLDVE
(SEQ ID NO:33).
[0045] The invention also provides an isolated peptide or
peptidomimetic that selectively homes to pancreatic tumor cells and
pancreatic tumor vasculature. The peptide contains at least 4
contiguous amino acids of the amino acid sequence CKAAKNK (SEQ ID
NO:15), CKGAKAR (SEQ ID NO:19) or VGVGEWSV (SEQ ID NO:35), or a
conservative variant or peptidomimetic thereof and has a length of
less than 50 residues, such as less than 40 residues, less than 30
residues, less than 20 residues, less than 10 residues, and less
than 8 residues.
[0046] In one embodiment, a peptide of the invention, as well as a
peptide contained in a conjugate of the invention, contains at
least 4 contiguous amino acids of the amino acid sequence CKAAKNK
(SEQ ID NO:15), or a conservative variant of peptidomimetic
thereof. The peptide also can contain at least 5 contiguous amino
acids of the amino acid sequence CKAAKNK (SEQ ID NO:15), at least 6
contiguous amino acids of the amino acid sequence CKAAKNK (SEQ ID
NO:15), at least 7 contiguous amino acids of the amino acid
sequence CKAAKNK (SEQ ID NO:15), or a conservative variant of
peptidomimetic thereof. Exemplary peptide sequences that contain at
least 4 contiguous amino acids of the amino acid sequence CKAAKNK
(SEQ ID NO:15) include, but are not limited to CKAX.sub.1K wherein
X.sub.1 is a basic amino acid (SEQ ID NO:10), CKAX.sub.1KN wherein
X.sub.1 is a basic amino acid (SEQ ID NO:11), CKAAK (SEQ ID NO:12),
CKAAKN (SEQ ID NO:13) and KAAKN (SEQ ID NO:14). X.sub.1 can be, for
example, arginine, histidine and lysine.
[0047] In another embodiment, a peptide of the invention, as well
as a peptide contained in a conjugate of the invention, contains at
least 4 contiguous amino acids of the amino acid CKGAKAR (SEQ ID
NO:19), or a conservative variant of peptidomimetic thereof. The
peptide also can contain at least 5 contiguous amino acids of the
amino acid sequence CKGAKAR (SEQ ID NO:19), at least 6 contiguous
amino acids of the amino acid sequence CKGAKAR (SEQ ID NO:19), at
least 7 contiguous amino acids of the amino acid sequence CKGAKAR
(SEQ ID NO:19) or a conservative variant of peptidomimetic thereof.
Exemplary peptide sequences that contain at least 4 contiguous
amino acids of the amino acid sequence CKGAKAR (SEQ ID NO:19)
include, but are not limited to AKAR (SEQ ID NO:16), GAKAR (SEQ ID
NO:17), KGAKAR (SEQ ID NO:18), and CKGAKA (SEQ ID NO:20).
[0048] In a further embodiment, a peptide of the invention, as well
as a peptide contained in a conjugate of the invention, contains at
least 4 contiguous amino acids of the amino acid sequence VGVGEWSV
(SEQ ID NO:35), or a conservative variant of peptidomimetic
thereof. The peptide also can contain at least 5 contiguous amino
acids of the amino acid sequence VGVGEWSV (SEQ ID NO:35), at least
6 contiguous amino acids of the amino acid sequence VGVGEWSV (SEQ
ID NO:35), at least 7 contiguous amino acids of the amino acid
sequence VGVGEWSV (SEQ ID NO:35), or at least 8 contiguous amino
acids of the amino acid sequence VGVGEWSV (SEQ ID NO:35), or a
conservative variant of peptidomimetic thereof. Exemplary peptide
sequences that contain at least 4 contiguous amino acids of the
amino acid sequence VGVGEWSV (SEQ ID NO:35) include, but are not
limited to VGVG (SEQ ID NO:36), VGVGE (SEQ ID NO:37).
[0049] The invention also provides an isolated peptide or
peptidomimetic that selectively homes to premalignant and malignant
pancreatic vasculature. The peptide contains at least 4 contiguous
amino acids of the amino acid sequence CRGRRST (SEQ ID NO:5) or
FRVGVADV (SEQ ID NO:27), or a conservative variant or
peptidomimetic thereof and has a length of less than 50 residues,
such as less than 40 residues, less than 30 residues, less than 20
residues, less than 10 residues, and less than 8 residues.
[0050] In one embodiment, a peptide of the invention, as well as a
peptide contained in a conjugate of the invention, contains at
least 4 contiguous amino acids of the amino acid sequence CRGRRST
(SEQ ID NO:5), or a conservative variant of peptidomimetic thereof.
The peptide also can contain at least 5 contiguous amino acids of
the amino acid sequence CRGRRST (SEQ ID NO:5), at least 6
contiguous amino acids of the amino acid sequence CRGRRST (SEQ ID
NO:5), or at least 7 contiguous amino acids of the amino acid
sequence CRGRRST (SEQ ID NO:5), or a conservative variant of
peptidomimetic thereof. Exemplary peptide sequences that contain at
least 4 contiguous amino acids of the amino acid sequence CRGRRST
(SEQ ID NO:5) include, but are not limited to, RGRR (SEQ ID NO:1);
RGRRS (SEQ ID NO:2); RGRRST (SEQ ID NO:3); and CRGRRS (SEQ ID
NO:4).
[0051] In another embodiment, a peptide of the invention, as well
as a peptide contained in a conjugate of the invention, contains at
least 4 contiguous amino acids of the amino acid sequence FRVGVADV
(SEQ ID NO:27), or a conservative variant of peptidomimetic
thereof. The peptide also can contain at least 5 contiguous amino
acids of the amino acid sequence FRVGVADV (SEQ ID NO:27), at least
6 contiguous amino acids of the amino acid sequence FRVGVADV (SEQ
ID NO:27), at least 7 contiguous amino acids of the amino acid
sequence FRVGVADV (SEQ ID NO:27), or at least 8 contiguous amino
acids of the amino acid sequence FRVGVADV (SEQ ID NO:27). Exemplary
peptide sequences that contain at least 4 contiguous amino acids of
the amino acid sequence FRVGVADV (SEQ ID NO:27) include, but are
not limited to, RVGV (SEQ ID NO:21), RVGVA (SEQ ID NO:22), RVGVAD
(SEQ ID NO:23), VGVAD (SEQ ID NO:24), VGVADV (SEQ ID NO:25), and
RVGVADV (SEQ ID NO:26).
[0052] As disclosed herein in Example V, a receptor for peptide
sequence CRGRRST (SEQ ID NO:5) was identified as PDGFR.beta..
Therefore, the invention provides an isolated peptide or
peptidomimetic that selectively homes to premalignant and malignant
pancreatic vasculature, wherein the peptide or peptidomimetic has
an ability to bind to a PDGFR.beta., and has a length of less than
50 residues. The isolated peptide or peptidomimetic can have, for
example, a length of less than 40 residues, less than 30 residues,
less than 20 residues, less than 10 residues, and less than 8
residues.
[0053] It is understood that a homing molecule useful in the
invention can be, without limitation, a peptide or peptidomimetic.
As used herein, the term "peptide" is used broadly to mean
peptides, proteins, fragments of proteins and the like. The term
"peptidomimetic," as used herein, means a peptide-like molecule
that has the activity of the peptide upon which it is structurally
based. Such peptidomimetics include chemically modified peptides,
peptide-like molecules containing non-naturally occurring amino
acids, and peptoids, and have an activity such as the selective
homing activity of the peptide upon which the peptidomimetic is
derived (see, for example, Goodman and Ro, Peptidomimetics for Drug
Design, in "Burger's Medicinal Chemistry and Drug Discovery" Vol. 1
(ed. M. E. Wolff; John Wiley & Sons (1995), pages 803-861).
[0054] A variety of peptidomimetics are known in the art including,
but not limited to, peptide-like molecules that contain a
constrained amino acid, a non-peptide component that mimics peptide
secondary structure, or an amide bond isostere. A peptidomimetic
that contains a constrained, non-naturally occurring amino acid can
include, without limitation, an .alpha.-methylated amino acid;
.alpha., .alpha.-dialkylglycine or .alpha.-aminocycloalkane
carboxylic acid; an N.sup..alpha.--C.sup..alpha. cyclized amino
acid; an N.sup..alpha.-methylated amino acid; a .beta.- or
.gamma.-amino cycloalkane carboxylic acid; an
.alpha.,.beta.-unsaturated amino acid; a .beta.,.beta.-dimethyl or
.beta.-methyl amino acid; a .beta.-substituted-2,3-methano amino
acid; an N--C.sup..delta. or C.sup..alpha.--C.sup..delta. cyclized
amino acid; a substituted proline or another amino acid mimetic. A
peptidomimetic that mimics peptide secondary structure can contain,
without limitation, a nonpeptidic .beta.-turn mimic; .gamma.-turn
mimic; mimic of .beta.-sheet structure; or mimic of helical
structure, each of which is well known in the art. As non-limiting
examples, a peptidomimetic also can be a peptide-like molecule that
contains an amide bond isostere such as a retro-inverso
modification; reduced amide bond; methylenethioether or
methylene-sulfoxide bond; methylene ether bond; ethylene bond;
thioamide bond; trans-olefin or fluoroolefin bond;
1,5-disubstituted tetrazole ring; ketomethylene or
fluoroketomethylene bond or another amide isostere. One skilled in
the art understands that these and other peptidomimetics are
encompassed within the meaning of the term "peptidomimetic" as used
herein.
[0055] Methods for identifying a peptidomimetic are well known in
the art and include, for example, the screening of databases that
contain libraries of potential peptidomimetics. For example, the
Cambridge Structural Database contains a collection of greater than
300,000 compounds that have known crystal structures (Allen et al.,
Acta Crystallogr. Section B, 35:2331 (1979)). This structural
depository is continually updated as new crystal structures are
determined and can be screened for compounds having suitable
shapes, for example, the same shape as a peptide of the invention,
as well as potential geometrical and chemical complementarity to a
target molecule. Where no crystal structure of a peptide of the
invention is available, a structure can be generated using, for
example, the program CONCORD (Rusinko et al., J. Chem. Inf. Comput.
Sci. 29:251 (1989)). Another database, the Available Chemicals
Directory (Molecular Design Limited, Informations Systems; San
Leandro, Calif.), contains about 100,000 compounds that are
commercially available and also can be searched to identify
potential peptidomimetics of a peptide of the invention, for
example, with activity in selectively homing to vasculature of
premalignant pancreas or pancreatic tumor cells and pancreatic
tumor vasculature.
[0056] The peptides and peptidomimetics of the invention, including
the bifunctional and multivalent peptides and peptidomimetics
described herein below, can have a variety of lengths. A peptide or
peptidomimetic of the invention, or the peptide or peptidomimetic
portion of a conjugate of the invention, can have, for example, a
relatively short length of less than 6, less than 7, less than 8,
less than 9, less than 10, less than 12, less than 15, less than
20, less than 25, less than 30, less than 35, less than 40, less
than 45, less than 50, less than 60, less than 70 or less than 80
residues. A peptide or peptidomimetic of the invention, or
conjugate containing the peptide or peptidomimetic, also can be
useful in the context of a significantly longer sequence as
described further below. As used herein, the term "residue" refers
to an amino acid or analog thereof.
[0057] In various embodiments, the peptide or peptidomimetic
portion of the conjugate has a defined length. The peptide or
peptidomimetic, or the peptide or peptidomimetic portion of the
conjugate, can have, for example, a length of at most 10, at most
20, most 10, at most 30, most 10, at most 40, most 10, at most 50,
most 10, at most 100, most 10, at most 150, most 10, at most 200,
most 10, at most 250, most 10, at most 300, most 10, at most 400,
most 10, at most 500, most 10, at most 600, most 10, at most 700,
most 10, at most 800, most 10, at most 900, most 10, at most 1000
or most 10, at most 2000 residues. The peptide or peptidomimetic,
or the peptide or peptidomimetic portion of the conjugate, also can
have, for example, a length of less than 60, less than 50, less
than 40, less than 30, less than 25, less than 20, less than 15, or
less than 10 residues. It is understood that the term "peptide or
peptidomimetic portion of the conjugate" means the total number of
residues in the peptide or peptidomimetic and any contiguous
protein, peptide or peptidomimetic, such as a therapeutic protein
or pro-apoptotic peptide.
Chimeric Proteins
[0058] As disclosed herein, a peptide or peptidomimetic of the
invention can maintain homing activity in the context of a
significantly longer sequence. As a non-limiting example, the
peptides referenced as SEQ ID NOS:5, 9, 15, 19, 27, 34 and 35
demonstrated homing to respective targets when fused to a phage
coat protein, confirming that a peptide of the invention can have
selective homing activity when embedded in a larger protein
sequence (see Example I). Thus, the invention provides a chimeric
protein containing a peptide or peptidomimetic of the invention,
fused to a heterologous protein. In one embodiment, the invention
provides a chimeric protein containing a peptide or peptidomimetic
that selectively homes to vasculature of premalignant pancreas and
contains at least 5 contiguous amino acids of an amino acid
sequence selected from SEQ ID NO:9 or SEQ ID NO:34, or a
conservative variant or peptidomimetic of one or these sequences,
fused to a heterologous protein.
[0059] In another embodiment, the invention provides a chimeric
protein containing a peptide or peptidomimetic that selectively
homes to pancreatic tumor cells and pancreatic tumor vasculature
and contains at least 5 contiguous amino acids of an amino acid
sequence selected from SEQ ID NO:15, SEQ ID NO:19, or SEQ ID NO:35,
or a conservative variant or peptidomimetic of one or these
sequences, fused to a heterologous protein.
[0060] In a further embodiment, the invention provides a chimeric
protein containing a peptide or peptidomimetic that selectively
homes to premalignant and malignant pancreatic vasculature and
contains at least 5 contiguous amino acids of an amino acid
sequence selected from SEQ ID NO:5 or SEQ ID NO:27, or a
conservative variant or peptidomimetic of one or these sequences,
fused to a heterologous protein.
[0061] A variety of heterologous proteins can be fused to one of
these peptides or peptidomimetics. The term "heterologous," as used
herein in reference to a protein fused to a peptide or
peptidomimetic of the invention, means a protein derived from a
source other than the gene encoding the fused peptide or upon which
the fused homing peptidomimetic is derived. A heterologous protein
can be, without limitation, a heterologous protein having a
therapeutic activity, an antibody or an antigen-binding fragment. A
chimeric protein of the invention can have a variety of lengths
including, but not limited to, up to 100, up to 200, up to 300, up
to 400, up to 500, up to 800, up to 1000 or up to 2000 residues or
more.
Bifunctional Peptides
[0062] The invention also provides bifunctional peptides that
contain a peptide that selectively homes to vasculature of
premalignant pancreas fused to a second peptide having a separate
function. Also provided by the invention are bifunctional peptides
that contain a peptide that selectively homes pancreatic tumor
cells and pancreatic tumor vasculature fused to a second peptide
having a separate function. Further provided by the invention are
bifunctional peptides that contain a peptide that selectively homes
to premalignant or malignant pancreatic vasculature fused to a
second peptide having a separate function. Such bifunctional
peptides have at least two functions conferred by different
portions of the peptide and can, for example, display
anti-angiogenic activity or pro-apoptotic activity in addition to
selective homing activity. As non-limiting examples, the invention
provides bifunctional peptides such as
CRSRKG-GG-.sub.D(KLAKLAK).sub.2, CEYQLDVE-GG-.sub.D(KLAKLAK).sub.2,
CRGRRST-GG-.sub.D(KLAKLAK).sub.2, CKAAKNK-GG-.sub.D(KLAKLAK).sub.2,
FRVGVADV-GG-.sub.D(KLAKLAK).sub.2,
CSRPPRSEC-GG-.sub.D(KLAKLAK).sub.2,
VGVGEWSV-GG-.sub.D(KLAKLAK).sub.2 or
CKGAKAR-GG-.sub.D(KLAKLAK).sub.2. In such peptides, the CRSRKG (SEQ
ID NO:9), CEYQLDVE (SEQ ID NO:34), CRGRRST (SEQ ID NO:5), CKAAKNK
(SEQ ID NO:15), FRVGVADV (SEQ ID NO:27), VGVGEWSV (SEQ ID NO:35),
or CKGAKAR (SEQ ID NO:19) portion exhibits selective homing
activity, while the .sub.D(KLAKLAK).sub.2 portion exhibits
pro-apoptotic activity.
Homing Molecules that are Antibodies
[0063] The conjugates and methods of the invention can be practiced
with a homing antibody or antigen-binding fragment thereof that
selectively homes to vasculature of premalignant pancreas; that
selectively homes to pancreatic tumor cells and pancreatic tumor
vasculature or that selectively homes to premalignant and malignant
pancreatic vasculature. As used herein, the term "antibody" is used
in its broadest sense to include polyclonal and monoclonal
antibodies, as well as polypeptide fragments of antibodies that
retain binding activity for the respective cognate receptor of at
least about 1.times.10.sup.5 M.sup.-1. One skilled in the art
understands that antibody fragments including, without limitation,
Fab, F(ab').sub.2 and Fv fragments, can retain binding activity for
a cognate receptor and, thus, are included within the definition of
antibody. In addition, the term "antibody," as used herein,
encompasses non-naturally occurring antibodies and fragments
usually containing, at a minimum, one V.sub.H and one V.sub.L
domain, such as chimeric antibodies, humanized antibodies and
single chain Fv fragments (scFv) that specifically or selectively
bind the appropriate cognate receptor. Such non-naturally occurring
antibodies can be constructed using solid phase peptide synthesis,
produced recombinantly or obtained by screening phage-displayed or
other combinatorial libraries such as those consisting of variable
heavy and light chains as described in Borrebaeck (Ed.), Antibody
Engineering (Second edition) New York: Oxford University Press
(1995)) using, for example, an assay described herein below.
[0064] Homing molecules that are antibodies also can be prepared
using a cognate receptor fusion protein or a synthetic peptide
encoding a portion of a cognate receptor. One skilled in the art
understands that purified human or other cognate receptors, which
can be produced recombinantly, including peptide portions of a
cognate receptor such as synthetic peptide fragments can be used as
immunogens. It is understood that fragments of the cognate receptor
for an amino acid sequence selected from SEQ ID NOS:5, 9, 15, 19,
27, 34 or 35 useful as immunogens include fragments of the cognate
receptor that serve to produce anti-cognate receptor antibodies
that are readily internalized into cells expressing cell-surface
cognate receptor for an amino acid sequence selected from SEQ ID
NOS:5, 9, 15, 19, 27, 34 or 35. One skilled in the art further
understands that non-immunogenic fragments or synthetic peptides of
a cognate receptor for an amino acid sequence selected from SEQ ID
NOS:5, 9, 15, 19, 27, 34 or 35 can be made immunogenic by coupling
the hapten to a carrier molecule such as bovine serum albumin (BSA)
or keyhole limpet hemocyanin (KLH). In addition, various other
carrier molecules and methods for coupling a hapten to a carrier
molecule are well known in the art as described, for example, by
Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring
Harbor Laboratory Press, 1988)).
Conjugates
[0065] The invention provides a conjugate that includes a
therapeutic moiety linked to a peptide or peptidomimetic that
selectively homes to vasculature of premalignant pancreas. The
peptide or peptidomimetic contains at least 5 contiguous amino
acids of an amino acid sequence selected from CRSRKG (SEQ ID NO:9)
and CEYQLDVE (SEQ ID NO:34), or a conservative variant or
peptidomimetic thereof. In embodiments of the invention, the
peptide or peptidomimetic has a length of less than 100 residues,
less than 50 residues and less than 25 residues. Also provided by
the invention is a conjugate containing a therapeutic moiety linked
to a peptide or peptidomimetic that selectively homes to
vasculature of premalignant pancreas, in which the peptide or
peptidomimetic binds specifically to a cognate receptor for SEQ ID
NO:9 or SEQ ID NO:34.
[0066] The invention additionally provides a conjugate containing a
therapeutic moiety linked to a peptide or peptidomimetic that
selectively homes to pancreatic tumor cells and pancreatic tumor
vasculature, the peptide or peptidomimetic comprising at least 5
contiguous amino acids of an amino acid sequence selected from
CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID NO:19), and VGVGEWSV (SEQ
ID NO:35), or a conservative variant or peptidomimetic thereof. In
embodiments of the invention, the peptide or peptidomimetic has a
length of less than 100 residues, less than 50 residues and less
than 25 residues. Also provided by the invention is a conjugate
that contains therapeutic moiety linked to a peptide or
peptidomimetic that selectively homes to pancreatic tumor cells and
pancreatic tumor vasculature, wherein the peptide or peptidomimetic
binds specifically to a cognate receptor for SEQ ID NO:15, SEQ ID
NO:19 or SEQ ID NO:35.
[0067] The invention provides a conjugate that contains a
therapeutic moiety linked to a peptide or peptidomimetic that
selectively homes to premalignant and malignant pancreatic
vasculature, the peptide or peptidomimetic containing at least 5
contiguous amino acids of an amino acid sequence selected from
CRGRRST (SEQ ID NO:5) and FRVGVADV (SEQ ID NO:27), or a
conservative variant or peptidomimetic thereof. In embodiments of
the invention, the peptide or peptidomimetic has a length of less
than 100 residues, less than 50 residues and less than 25 residues.
Also provided by the invention is a conjugate that contains a
therapeutic moiety linked to a peptide or peptidomimetic that
selectively homes to pancreatic tumor cells and pancreatic tumor
vasculature, wherein the peptide or peptidomimetic binds
specifically to a cognate receptor for SEQ ID NO:5 or SEQ ID
NO:27.
[0068] The conjugates and methods of the invention disclosed herein
involve homing molecules. As used herein, the term "molecule" is
used broadly to mean a polymeric or non-polymeric organic chemical
such as a small molecule drug; a nucleic acid molecule such as an
RNA, a cDNA or other DNA, or an oligonucleotide; a peptide or
peptidomimetic; or a protein such as an antibody or a growth factor
receptor or a fragment thereof such as an Fv, Fd, or Fab fragment
of an antibody containing the antigen-binding domain.
[0069] The phrase "homing molecule that selectively homes to
vasculature of premalignant pancreas," as used herein, means any
molecule that preferentially localizes in vivo to vasculature of
premalignant pancreas as compared to vasculature of malignant
pancreas and vasculature of normal pancreas. Similarly, the phrase
"peptide or peptidomimetic that selectively homes to vasculature of
premalignant pancreas" means a peptide or peptidomimetic that
preferentially localizes in vivo to vasculature of premalignant
pancreas as compared to vasculature of malignant pancreas and
vasculature of normal pancreas. As disclosed herein, such a homing
molecule can be a peptide or peptidomimetic. It is understood that
a peptide or peptidomimetic that selectively homes to vasculature
of premalignant pancreas can home to the supporting vasculature of
a variety of premalignant lesions in addition to premalignant
pancreas, or can exhibit preferential homing to vasculature of
premalignant lesions in a subset of tissue types including
premalignant pancreas, or can exhibit significant homing
exclusively to vasculature of premalignant pancreas.
[0070] Selective homing of a peptide or peptidomimetic that
selectively homes to vasculature of premalignant pancreas generally
is characterized by at least a two-fold greater localization within
vasculature of premalignant pancreas as compared to vasculature of
malignant pancreas and normal pancreatic vasculature. Such a
peptide or peptidomimetic can be characterized, for example, by
5-fold, 10-fold, 20-fold or more greater localization within
vasculature of premalignant pancreas as compared to vasculature of
malignant pancreas and normal pancreatic vasculature. As discussed
above, it is understood that a peptide or peptidomimetic that
selectively homes to vasculature of premalignant pancreas can home,
in part, to vasculature of one or more other premalignant
tissues.
[0071] As used herein, the term "premalignant" means a precancerous
state of a tissue having a an abnormality in which cancer is more
likely to occur than in a normal tissue of the same type. Such an
abnormality can be characterized based on histological
abnormalities of cytology and/or architecture or biochemical
differences between the precancerous versus normal states of the
tissue. Particular differences depend on the particular type of
tissue undergoing a premalignant process and are described in the
art, for example, as metaplasia, dysplasia, hyperplasia, carcinoma
in situ, angiogenic and the like, depending on the degree of
structural and/or functional change compared to normal.
[0072] The phrase "homing molecule that selectively homes to
pancreatic tumor cells and pancreatic tumor vasculature," as used
herein, means any molecule that preferentially localizes in vivo to
pancreatic tumor cells and pancreatic tumor vasculature as compared
to premalignant pancreatic tumor cells and vasculature, and normal
pancreatic tumor cells and vasculature. Similarly, the phrase
"peptide or peptidomimetic that selectively homes to pancreatic
tumor cells and pancreatic tumor vasculature" means a peptide or
peptidomimetic that preferentially localizes in vivo to pancreatic
tumor cells and pancreatic tumor vasculature as compared to
premalignant pancreatic cells and vasculature, and normal
pancreatic cells and vasculature. It is understood that a peptide
or peptidomimetic that selectively homes to pancreatic tumor cells
and pancreatic tumor vasculature can home to the supporting
vasculature of a variety of malignant lesions in addition to
pancreatic tumor cells and vasculature, or can exhibit preferential
homing to vasculature of premalignant lesions in a subset of tissue
types including premalignant pancreas, or can exhibit significant
homing exclusively to pancreatic tumor cells and pancreatic tumor
vasculature.
[0073] Selective homing of a peptide or peptidomimetic that
selectively homes to pancreatic tumor cells and pancreatic tumor
vasculature generally is characterized by at least a two-fold
greater localization within pancreatic tumor cells and pancreatic
tumor vasculature as compared to pancreatic premalignant cells and
premalignant pancreatic vasculature and normal pancreatic cells and
vasculature. Such a peptide or peptidomimetic can be characterized,
for example, by 5-fold, 10-fold, 20-fold or more greater
localization within pancreatic tumor cells and pancreatic tumor
vasculature as compared to premalignant cells and premalignant
pancreatic vasculature and normal pancreatic cells and vasculature.
As discussed above, it is understood that a peptide or
peptidomimetic that selectively homes to pancreatic tumor cells and
pancreatic tumor vasculature can additionally localize to tumor
cells and vasculature of one or more other malignant tissues in
addition to selectively homing to pancreatic tumor cells and
pancreatic tumor vasculature.
[0074] A peptide or peptidomimetic of the invention can be
characterized by having the ability to preferentially localize to
both premalignant and malignant pancreatic vasculature. Such a
peptide or peptidomimetic can have greater selectivity for
premalignant vasculature, greater selectivity for malignant
vasculature, or can have similar selectivities for both
premalignant and malignant vasculature. The phrase "homing molecule
that selectively homes to premalignant and malignant pancreatic
vasculature," as used herein, means any molecule that
preferentially localizes in vivo to premalignant and malignant
pancreatic vasculature as compared to normal pancreatic
vasculature. Similarly, the phrase "peptide or peptidomimetic that
selectively homes to premalignant and malignant pancreatic
vasculature" means a peptide or peptidomimetic that preferentially
localizes in vivo to premalignant and malignant pancreatic
vasculature as compared to normal pancreatic vasculature. It is
understood that a peptide or peptidomimetic that selectively homes
to premalignant and malignant pancreatic vasculature can home to
the supporting vasculature of a variety of premalignant and
malignant lesions in addition to premalignant and malignant
pancreatic vasculature, or can exhibit preferential homing to
vasculature of premalignant and malignant lesions in a subset of
tissue types including premalignant and malignant pancreas, or can
exhibit significant homing exclusively to premalignant and
malignant pancreatic vasculature. It is also understood that a
peptide or peptidomimetic that selectively homes to premalignant
and malignant pancreatic vasculature can home to tumor cells, such
as pancreatic tumor cells, for example, via compromised, damaged or
otherwise leaky malignant pancreatic vasculature.
[0075] Selective homing of a peptide or peptidomimetic that
selectively homes to premalignant and malignant pancreatic
vasculature generally is characterized by at least a two-fold
greater localization within premalignant or malignant pancreatic
vasculature as compared to vasculature of normal pancreas. Such a
peptide or peptidomimetic can be characterized, for example, by
5-fold, 10-fold, 20-fold or more greater localization within
premalignant and malignant pancreatic vasculature as compared to
vasculature of normal pancreas. As discussed above, it is
understood that a peptide or peptidomimetic that selectively homes
to premalignant and malignant pancreatic vasculature can
additionally localize to premalignant and malignant vasculature of
one or more other tissues in addition to selectively homing to
premalignant and malignant pancreatic vasculature. In addition, a
peptide or peptidomimetic that selectively homes to vasculature of
premalignant pancreas that also selectively homes, to a lesser
extent, to pancreatic tumor cells and pancreatic tumor vasculature
can be characterized as a peptide or peptidomimetic that
selectively homes to premalignant and malignant pancreatic
vasculature. Similarly, a peptide or peptidomimetic that
selectively homes to pancreatic tumor cells and pancreatic tumor
vasculature that also selectively homes, to a lesser extent, to
premalignant pancreas can be characterized as a peptide or
peptidomimetic that selectively homes to premalignant and malignant
pancreatic vasculature.
Conservative Variants
[0076] The present invention also provides a peptide or
peptidomimetic or conjugate containing a peptide or peptidomimetic
that includes an amino acid sequence that is a conservative
variant, for example, of at least five contiguous amino acids of an
amino acid sequence selected from CRGRRST (SEQ ID NO:5), CRSRKG
(SEQ ID NO:9); CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID NO:19),
FRVGVADV (SEQ ID NO:27), CEYQLDVE (SEQ ID NO:34), and VGVGEWSV (SEQ
ID NO:35). As used herein, a "conservative variant" is an amino
acid sequence in which a first amino acid is replaced by a second
amino acid or amino acid analog having at least one similar
biochemical property, which can be, for example, similar size,
charge, hydrophobicity or hydrogen-bonding capacity. For example, a
first hydrophobic amino acid can be conservatively substituted with
a second (non-identical) hydrophobic amino acid such as alanine,
valine, leucine, or isoleucine, or an analog thereof. Similarly, a
first basic amino acid can be conservatively substituted with a
second basic amino acid such as arginine or lysine, or an analog
thereof. In the same way, a first acidic amino acid can be
conservatively substituted with a second acidic amino acid such as
aspartic acid or glutamic acid, or an analog thereof, or an
aromatic amino acid such as phenylalanine can be conservatively
substituted with a second aromatic amino acid or amino acid analog,
for example, tyrosine.
Multivalent Conjugates
[0077] The invention provides a multivalent conjugate, containing a
therapeutic moiety linked to at least two peptides or
peptidomimetics that selectively home to vasculature of
premalignant pancreas, each of the peptides or peptidomimetics
containing at least 5 contiguous amino acids of an amino acid
sequence selected from CRSRKG (SEQ ID NO:9) and CEYQLDVE (SEQ ID
NO:34).
[0078] The invention further provides a multivalent conjugate that
contains a therapeutic moiety linked to at least two peptides or
peptidomimetics that selectively home to pancreatic tumor cells and
pancreatic tumor vasculature, each of the peptides or
peptidomimetics containing at least 5 contiguous amino acids of an
amino acid sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR
(SEQ ID NO:19), and VGVGEWSV (SEQ ID NO:35), or a conservative
variant or peptidomimetic thereof.
[0079] The invention provides a multivalent conjugate that contains
a therapeutic moiety linked to at least two peptides or
peptidomimetics that selectively home to premalignant and malignant
vasculature, each of the peptides or peptidomimetics containing at
least 5 contiguous amino acids of an amino acid sequence selected
from CRGRRST (SEQ ID NO:5) and FRVGVADV (SEQ ID NO:27), or a
conservative variant or peptidomimetic thereof.
[0080] A multivalent conjugate of the invention containing multiple
peptide or peptidomimetics can include, for example, two or more,
three or more, five or more, ten or more, twenty or more, thirty or
more, forty or more, fifty or more, 100 or more, 200 or more, 300
or more, 400 or more, 500 or more or 1000 or more peptides or
peptidomimetics. In one embodiment, the peptides or peptidomimetics
have an identical amino acid sequence. In another embodiment, the
multivalent conjugate includes peptides or peptidomimetics having
non-identical amino acid sequences.
[0081] A multivalent conjugate of the invention can be linked to a
variety of moieties. Moieties useful in a multivalent conjugate of
the invention include, without limitation, phage, retroviruses,
adenoviruses, adeno-associated viruses and other viruses, cells,
liposomes, polymeric matrices, non-polymeric matrices or particles
such as gold particles, microdevices and nanodevices, and
nano-scale semiconductor materials.
[0082] Liposomes consisting, without limitation, of phospholipids
or other lipids, are nontoxic, physiologically acceptable and
metabolizable carriers that are readily made to be incorporated
into a conjugate of the invention (Gregoriadis, Liposome
Technology, Vol. 1 (CRC Press, Boca Raton, Fla. (1984)). It is
understood that the liposome or other polymeric matrix additionally
can include one or more other components if desired, such as,
without limitation, one or any combination of therapeutic agents,
anti-angiogenic agents or cytotoxic agents.
[0083] As disclosed herein, peptides CEYQLDVE (SEQ ID NO:34) and
CRSRKG (SEQ ID NO:9) recognize a target "receptor" that is
expressed in vasculature of premalignant pancreas but is
essentially absent or inaccessible for binding via the circulation
in vasculature of malignant pancreas or normal pancreas. Also as
disclosed herein, peptides CKAAKNK (SEQ ID NO:15); CKGAKAR (SEQ ID
NO:19); and VGVG (SEQ ID NO:x) recognize a target "receptor" that
is expressed in pancreatic tumor cells and pancreatic tumor
vasculature but is essentially absent or inaccessible for binding
via the circulation in the vasculature or premalignant pancreas or
normal pancreas. Further, as disclosed herein, peptides CRGRRST
(SEQ ID NO:5) or FRVGVADV (SEQ ID NO:27) recognize a target
"receptor" that is expressed in premalignant and malignant
pancreatic vasculature but is essentially absent or inaccessible
for binding via the circulation in the vasculature of normal
pancreas. The cell surface and cell-type selective expression of
the target receptor form the basis for the selective homing
activity of the specific peptides described above and related
peptides, peptidomimetics and other molecules.
[0084] Based on these discoveries, it is clear that molecules
structurally unrelated to SEQ ID NOS: 9 or 34 but that bind the
same cognate receptors also have the same characteristic of
selectively homing to vasculature of premalignant pancreas and
other premalignant tissues. Such molecules can be identified by the
ability to specifically bind to, or to compete with SEQ ID NO:9 or
34 for specific binding to cells expressing their respective
cognate receptors. It is also clear that molecules structurally
unrelated to SEQ ID NOS:15 and 19 but that bind the same cognate
receptors also have the same characteristic of selectively homing
to pancreatic tumor cells and pancreatic tumor vasculature and
cells and vasculature of other malignant tissues. Such molecules
can be identified by the ability to specifically bind to, or to
compete with SEQ ID NOS:15 or 19 for specific binding to cells
expressing their respective cognate receptors. Further, it is clear
that molecules structurally unrelated to SEQ ID NOS:5, 27 or 35 but
that bind the same cognate receptors also have the same
characteristic of selectively homing to premalignant and malignant
pancreatic vasculature and premalignant and malignant vasculature
of other tissues. Such molecules can be identified by the ability
to specifically bind to, or to compete with SEQ ID NOS:5, 27 or 35
for specific binding to cells expressing their respective cognate
receptors. Selective homing to vasculature of premalignant
pancreas; pancreatic tumor cells and pancreatic tumor vasculature;
or premalignant and malignant pancreatic vasculature, readily can
be confirmed using in vivo panning as disclosed herein in Example I
(see, also, U.S. Pat. No. 5,622,699).
[0085] A homing molecule of the invention, such as a peptide or
peptidomimetic, specifically binds the indicated cognate receptor.
As used herein, the term "specifically binds" or "specifically
binding" means binding that is measurably different from a
non-specific interaction. Specific binding can be measured, for
example, by determining binding of a molecule compared to binding
of a control molecule, which generally is a molecule of similar
structure that does not have binding activity. In this case,
specific binding is indicated if the molecule has measurably higher
affinity for cells expressing the cognate receptor, for example,
than for cells that do not express the cognate receptor.
Specificity of binding can be determined, for example, by
competitive inhibition of the binding of a known binding molecule
such SEQ ID NO:9 or 34 to identify molecules that selectively home
to vasculature of premalignant pancreas; by competitive inhibition
of the binding of a known binding molecule such as SEQ ID NO:15, 19
or 35 to identify molecules that selectively home to pancreatic
tumor cells and pancreatic tumor vasculature; or by competitive
inhibition of the binding of a known binding molecule such as SEQ
ID NO:5 or 27 to identify molecules that selectively home to
premalignant and malignant pancreatic vasculature.
[0086] The term "binds specifically," as used herein, includes both
low and high affinity specific binding. Specific binding can be
exhibited, for example, by a low affinity homing molecule having a
Kd of at least about 10.sup.-4 M. For example, if the cognate
receptor has more than one binding site, a homing molecule having
low affinity can be useful for targeting, for example, vasculature
of premalignant pancreas; pancreatic tumor cells and pancreatic
tumor vasculature; or premalignant and malignant pancreatic
vasculature. Specific binding also can be exhibited by a high
affinity homing molecule, for example, a homing molecule having a
Kd of at least about 10.sup.-5 M. Such a molecule can have, for
example, a Kd of at least about 10.sup.-6 M, at least about
10.sup.-7 M, at least about 10.sup.-8 M, at least about 10.sup.-9
M, at least about 10.sup.-10 M, or can have a Kd of at least about
10.sup.-11 M or 10.sup.-12 M or greater. Both low and high affinity
homing molecules are useful and are encompassed by the invention.
Low affinity homing molecules are useful in targeting, for example,
multivalent conjugates such as viruses and other particles. High
affinity homing molecules are useful in targeting, for example,
multivalent and univalent conjugates.
[0087] Thus, the invention further provides a conjugate that
contains a therapeutic moiety linked to a peptide or peptidomimetic
that selectively homes to vasculature of premalignant pancreas and
that specifically binds a cognate receptor for SEQ ID NO:9 or 34.
Also provided is a conjugate that contains a therapeutic moiety
linked to a peptide or peptidomimetic that selectively homes to
pancreatic tumor cells and pancreatic tumor vasculature and which
specifically binds a cognate receptor for an amino acid sequence
selected from SEQ ID NO:15, 19 or 35. Further provided is a
conjugate that contains a therapeutic moiety linked to a peptide or
peptidomimetic that selectively homes to premalignant and malignant
pancreatic vasculature and that specifically binds a cognate
receptor for SEQ ID NO:5 or 27. As is shown herein in Example V,
the PDGFR.beta. is a cognate receptor for SEQ ID NO:5. Thus, in one
embodiment, the conjugate contains a peptide or peptidomimetic that
specifically binds PDGFR.beta..
[0088] In one embodiment, any of such conjugates can contain a
peptide or peptidomimetic that is not an antibody or
antigen-binding fragment thereof. In another embodiment, the
peptide or peptidomimetic portion of the conjugate can have a
length of at most 200 residues, or a length of at most 50
residues.
[0089] The invention provides method of directing a moiety to a
pancreatic premalignant lesion in an individual. The method
involves administering to the individual a conjugate containing a
moiety linked to (a) a peptide or peptidomimetic that selectively
homes to vasculature of premalignant pancreas, the peptide or
peptidomimetic containing at least 5 contiguous amino acids of an
amino acid sequence selected from CRSRKG (SEQ ID NO:9) and CEYQLDVE
(SEQ ID NO:34), or a conservative variant or peptidomimetic
thereof, or (b) a peptide or peptidomimetic that selectively homes
to premalignant and malignant pancreatic vasculature, the peptide
or peptidomimetic containing at least 5 contiguous amino acids of
an amino acid sequence selected from CRGRRST (SEQ ID NO:5) and
FRVGVADV (SEQ ID NO:27), or a conservative variant or
peptidomimetic thereof, thereby directing the moiety to the
vasculature of the pancreatic premalignant lesion. In one
embodiment, the moiety is a therapeutic moiety, such as an
angiogenic inhibitor. In another embodiment, the moiety is a
diagnostic moiety. Exemplary moieties useful in this method are
described herein below.
[0090] The invention also provides a method of directing a moiety
to pancreatic tumor cells and pancreatic tumor vasculature in an
individual. The method involves administering to the individual a
conjugate containing a moiety linked to: (a) a peptide or
peptidomimetic that selectively homes to pancreatic tumor cells and
pancreatic tumor vasculature, the peptide or peptidomimetic
containing at least 5 contiguous amino acids of an amino acid
sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID
NO:19), FRVGVADV and VGVGEWSV (SEQ ID NO:35), or a conservative
variant or peptidomimetic thereof, or (b) a peptide or
peptidomimetic that selectively homes to premalignant and malignant
pancreatic vasculature, the peptide or peptidomimetic containing at
least 5 contiguous amino acids of an amino acid sequence selected
from CRGRRST (SEQ ID NO:5) and FRVGVADV (SEQ ID NO:27), or a
conservative variant or peptidomimetic thereof, thereby directing
the moiety to pancreatic tumor cells and pancreatic tumor
vasculature.
Imaging
[0091] Selective delivery of diagnostic agents to vasculature that
supports tumors provides a tool for diagnosis of early or late
stage cancers, such pancreatic cancer. The pancreas is an organ of
the digestive system that contains exocrine and endocrine
components. The exocrine component is a compound gland with
branched ducts and serous secretory units. The endocrine component
synthesizes and secretes into the blood, hormones that regulate
glucose, lipid and protein metabolism of the body. This component
is dispersed within the exocrine component as distinct cell masses
called islets of Langerhans. Islets of Langerhans are polygonal
endocrine cells arranged in short, irregular cords that are
profusely invested with a network of fenestrated capillaries. As is
shown herein, a peptide or peptidomimetic of the invention can be
used to distinguish between normal pancreatic vasculature and
premalignant or malignant pancreatic vasculature. Therefore, when
linked to a detectable moiety, a peptide or peptidomimetic of the
invention can be used to visualize, or otherwise render detectable,
early changes in pancreatic vascular associated with a precancerous
state, as well as later changes in pancreatic vascular associated
with a more advanced or malignant state.
[0092] The invention provides a method for imaging pancreatic
premalignant lesions in an individual. The method involves: (a)
administering to the individual a conjugate containing a detectable
moiety linked to a peptide or peptidomimetic that selectively homes
to vasculature of premalignant pancreas, the peptide or
peptidomimetic containing at least 5 contiguous amino acids of an
amino acid sequence selected from CRSRKG (SEQ ID NO:9) and CEYQLDVE
(SEQ ID NO:34), or a conservative variant or peptidomimetic
thereof, and (b) detecting the conjugate, thereby imaging
pancreatic premalignant lesions.
[0093] The invention also provides a method of imaging pancreatic
tumors and pancreatic tumor vasculature in an individual. The
method involves (a) administering to the individual a conjugate
containing a detectable moiety linked to a peptide or
peptidomimetic that selectively homes to pancreatic tumor cells and
pancreatic tumor vasculature, the peptide or peptidomimetic
containing at least 5 contiguous amino acids of an amino acid
sequence selected from CKAAKNK (SEQ ID NO:15) and CKGAKAR (SEQ ID
NO:19), or a conservative variant or peptidomimetic thereof, and
(b) detecting the conjugate, thereby imaging the pancreatic tumors
and pancreatic tumor vasculature.
[0094] As used herein, the term "detectable moiety" means any
molecule that can be administered in vivo and subsequently
detected. Exemplary detectable moieties useful in the conjugates
and methods of the invention include, without limitation,
radiolabels and fluorescent molecules. Exemplary radionuclides
include indium-111, technetium-99, carbon-11, and carbon-13.
Fluorescent molecules include, without limitation, fluorescein,
allophycocyanin, phycoerythrin, rhodamine, and Texas red.
[0095] The methods of the invention for imaging the vasculature of
a premalignant tissue such as premalignant pancreas, or for imaging
of tumor cells and tumor vasculature, such as pancreatic tumor
cells and pancreatic rumor vasculature can be useful for early
detection of premalignant lesions or malignancies including but not
limited to, pancreatic premalignant lesions and tumors. Following
administration of a conjugate of the invention containing a
detectable moiety, the vasculature of premalignant tissue or tumor
tissue is visualized. If the image is positive for the presence of
such vasculature, further evaluation can be performed for the size
of the tumor, if any, and the quantity of vascular infiltration.
These results provide valuable information to the clinician with
regard to the stage of development of the cancer and the presence
or probability of metastasis. It is understood that the methods of
the invention are application to a variety of types of premalignant
lesions and cancers of organs including, yet not limited to,
cancers of digestive tract, such as head and neck cancers,
esophageal cancer, stomach cancer, pancreatic cancer, liver
cancer,
[0096] colon and rectal cancer, anal cancer; cancers of genital and
urinary systems, such as kidney cancer, bladder cancer, testicular
cancer, prostate cancer; cancers of the nervous system, such as
brain cancer; bone cancer; nasopharyngeal cancer; retroperitoneal
sarcomas; soft tissue cancers; thyroid cancer; breast cancer;
ovarian cancer; gynecological cancers; choriocarcinoma and other
types of cancers.
[0097] In a method of the invention for imaging vasculature of a
premalignant tissue, the conjugate administered contains a
detectable moiety that allows detection or visualization of the
vasculature of the premalignant tissue such as a pancreatic
premalignant lesion. In a method of the invention for imaging calls
and vasculature of a malignant tissue, the conjugate administered
contains a detectable moiety that allows detection or visualization
of pancreatic tumors and pancreatic tumor vasculature. For such in
vivo diagnostic imaging, a peptide or peptidomimetic is linked to a
detectable moiety that, upon administration to the subject, is
detectable external to the subject. Such a detectable moiety can
be, for example, a gamma ray emitting radionuclide such as
indium-113, indium-115 or technetium-99; following administration
to a subject, the conjugate can be visualized using a solid
scintillation detector.
[0098] A detectable moiety useful in the invention can be, for
example, a paramagnetic ion such as a magnesium, manganese, iron
oxide, dysprosium or gadolinium ion. Exemplary iron oxides include
dextran-coated superparamagnetic iron oxide, carboxydextran-coated
superparamagnetic iron oxide, ultrasmall superparamagnetic iron
oxide, monocrystalline iron oxide nanocompound and ferromagnetic
iron lignosulfonate. Exemplary gadolinium-based paramagnetic
moieties include Gd-DTPA, dextran-Gd-DTPA,
Gd-DTPA-24-cascade-polymer, Gd-DTPA-polylysine, Gd-melanin polymer
and 6-dendrimer-Gd-DTPA. When linked to a peptide or peptidomimetic
of the invention, such a paramagnetic ion can be used for enhanced
magnetic resonance imaging (MRI). A paramagnetic ion can be linked
directly to a peptide or peptidomimetic or linked indirectly, for
example, by being contained in a liposome that is linked to the
peptide or peptidomimetic.
[0099] A detectable moiety also can be an agent that facilitates
detection of premalignant and malignant tissue in vitro. For
example, a conjugate can contain a peptide or peptidomimetic of the
invention linked to an enzyme, which produces a visible signal when
an appropriate substrate is present. A detectable moiety useful in
such a conjugate can be, for example, alkaline phosphatase or
luciferase or the like, and can be detected by immunohistochemistry
using routine techniques.
Therapy
[0100] The invention provides a method of treating a pancreatic
premalignant lesion in an individual. The method involves
administering to the individual a conjugate containing a
therapeutic moiety linked to: (a) a peptide or peptidomimetic that
selectively homes to premalignant and malignant pancreatic
vasculature, the peptide or peptidomimetic containing at least 5
contiguous amino acids of an amino acid sequence selected from
CRGRRST (SEQ ID NO:5), and FRVGVADV (SEQ ID NO:27), or a
conservative variant or peptidomimetic thereof, or (b) a peptide or
peptidomimetic that selectively homes to vasculature of
premalignant pancreas containing at least 5 contiguous amino acids
of an amino acid sequence selected from CRSRKG (SEQ ID NO:9) and
CEYQLDVE (SEQ ID NO:34), thereby directing the therapeutic moiety
to the pancreatic premalignant lesion in the individual to treat
the pancreatic premalignant lesion. In one embodiment, the moiety
is a therapeutic moiety, such as an angiogenic inhibitor. In one
embodiment, the peptide or peptidomimetic selectively homes to
vasculature of premalignant pancreas.
[0101] The invention also provides a method of reducing the
severity of pancreatic cancer in an individual. The method involves
administering to the individual a conjugate containing a
therapeutic moiety linked to: (a) a peptide or peptidomimetic that
selectively homes to pancreatic tumor cells and pancreatic tumor
vasculature, the peptide or peptidomimetic containing at least 5
contiguous amino acids of an amino acid sequence selected from
CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID NO:19), FRVGVADV and
VGVGEWSV (SEQ ID NO:35), or a conservative variant or
peptidomimetic thereof, or (b) a peptide or peptidomimetic that
selectively homes to premalignant and malignant pancreatic
vasculature, the peptide or peptidomimetic containing at least 5
contiguous amino acids of an amino acid sequence selected from
CRGRRST (SEQ ID NO:5) and FRVGVADV (SEQ ID NO:27), or a
conservative variant or peptidomimetic thereof, thereby directing
the therapeutic moiety to pancreatic tumor cells or pancreatic
tumor vasculature in the individual to reduce the severity of the
pancreatic cancer.
Therapeutic Moieties
[0102] A variety of therapeutic moieties are useful in the
conjugates and methods of the invention, including, without
limitation, anti-angiogenic agents and cytotoxic agents, such as
those that target a DNA-associated process. As used herein, the
term "therapeutic moiety" is used broadly to mean a physical,
chemical, or biological material that can be linked to a homing
molecule and that alters biological activity in a normal or
pathologic tissue upon administration. A therapeutic moiety,
therefore, is potentially useful for the treatment of disease
conditions. A therapeutic moiety can be any natural or nonnatural
material including a biological material, such as a cell or phage;
an organic chemical, such as a small molecule; a radionuclide; a
nucleic acid molecule or oligonucleotide; a polypeptide; or a
peptide or peptidomimetic. Therapeutic moieties useful in the
invention include, without limitation, anti-angiogenic agents;
cancer chemotherapeutic agents; cytotoxic agents; pro-apoptotic
agents. A therapeutic moiety useful in the invention can be
expressed on, contained in, or linked to any of the following:
phage or other virus, cell, liposome, polymeric or non-polymeric
matrix, gold or other particle, or a microdevice, nanodevice, or
nano-scale semiconductor material. These and other materials known
in the art can be components of the conjugates of the
invention.
[0103] A therapeutic moiety useful in a conjugate of the invention
can be, for example, an anti-angiogenic agent. As used herein, the
term "anti-angiogenic agent" means a molecule that reduces or
inhibits angiogenesis. An anti-angiogenic agent useful in the
conjugates and methods of the invention can be, for example, an
inhibitor or neutralizing antibody that reduces the expression or
signaling of an angiogenic factor such as vascular endothelial
growth factor (VEGF), which is a major inducer of angiogenesis in
normal and pathological conditions, and is essential in embryonic
vasculogenesis. The biological effects of VEGF include stimulation
of endothelial cell proliferation, survival, migration and tube
formation, and regulation of vascular permeability. An
anti-angiogenic agent also can inhibit another angiogenic factor
such as a member of the fibroblast growth factor (FGF) family such
as FGF-1 (acidic), FGF-2 (basic), FGF-4 or FGF-5 (Slavin et al.,
Cell Biol. Int. 19:431-444 (1995); Folkman and Shing, J. Biol.
Chem. 267:10931-10934 (1992)) or angiopoietin-1, a factor that
signals through the endothelial cell-specific Tie2 receptor
tyrosine kinase (Davis et al., Cell 87:1161-1169 (1996); and Suri
et al., Cell 87:1171-1180 (1996)), or the receptor of one of these
angiogenic factors. It is understood that a variety of mechanisms
can act to inhibit activity of an angiogenic factor including,
without limitation, direct inhibition of receptor binding or of
secretion of the angiogenic factor into the extracellular space,
and inhibition of signaling, expression or function of the
angiogenic factor.
[0104] A variety of anti-angiogenic agents useful in the invention
are known in the art and can be prepared by routine methods. See,
for example, Hagedorn and Bikfalvi, Crit. Rev. Oncol. Hematol.
34:89-110 (2000) and Kirsch et al., J. Neurooncol. 50:149-163
(2000). Anti-angiogenic agents include, without limitation, small
molecules; proteins such as angiogenic factors and receptors,
transcription factors, and antibodies and antigen-binding fragments
thereof; peptides and peptidomimetics; and nucleic acid molecules
including ribozymes, antisense oligonucleotides, and nucleic acid
molecules encoding, for example, dominant negative angiogenic
factors and receptors, transcription factors, and antibodies and
antigen-binding fragments thereof. Exemplary anti-angiogenic agents
useful in the conjugates and methods of the invention include, yet
are not limited to, angiostatin, endostatin, metastatin and 2ME2
(EntreMed; Rockville, Md.); anti-VEGF antibodies such as Avastin
(Genentech; South San Francisco, Calif.); VEGFR-2 inhibitors such
as the small molecules SU5416 and SU6668, (SUGEN; South San
Francisco, Calif.); heparin-binding fragments of fibronectin;
modified forms of antithrombin; collagenase inhibitors; basement
membrane turnover inhibitors; angiostatic steroids; platelet factor
4, and fragments and peptides thereof; thrombospondin, and
fragments and peptides thereof; and doxorubicin (O'Reilly et al.,
Cell 79:315-328 (1994)); O'Reilly et al., Cell 88: 277-285 (1997);
Homandberg et al., Am. J. Path. 120:327-332 (1985); Biochim.
Biophys. Acta 874:61-71 (1986); and O'Reilly et al., Science
285:1926-1928 (1999)). It is understood that these as well as other
anti-angiogenic agents known in the art or that can be prepared by
routine methods are encompassed by the term "anti-angiogenic agent"
and can be used in the various conjugates and methods of the
invention.
[0105] It is understood by those skilled in the art that an
anti-angiogenic agent can be particularly efficacious when targeted
to a specific stage of tumor progression (see, for example, Bergers
et al., Science 284:808-812 (1999); and Bergers et al., J. Clin.
Invest. 111:1287-1295 (2003)). Thus, in one embodiment, an
anti-angiogenic agent useful in the invention is "effective against
premalignant vasculature." As used herein, the term
"anti-angiogenic agent effective against premalignant vasculature"
means an angiogenic agent that can significantly reduce the number
of angiogenic lesions during the premalignant phase of
carcinogenesis, before solid tumors have formed. Such an
anti-angiogenic agent is an anti-angiogenic agent effective against
premalignant vasculature whether or not the agent also
significantly reduces tumor burden or extends life-span in animals
with tumors, including animals with small solid tumors or animals
having large tumors and end-stage disease. As non-limiting
examples, an anti-angiogenic agent effective against pre-malignant
vasculature can be BB-94 (batimastat), a broad-spectrum inhibitor
of matrix metalloproteinases (Talbot and Brown, Eur. J. Cancer 32A:
2528 (1996)); SU5416 (SUGEN), a small molecule inhibitor of
VEGFR-2; or endostatin, a carboxy-terminal fragment of collagen
XVIII (O'Reilly et al., Cell 88: 277 (1997); and Boehm et al.,
Nature 390: 404 (1997)), alone or combined with angiostatin, an
internal fragment of plasminogen (O'Reilly et al., Cell 79: 314
(1994); and O'Reilly et al., Nature Med. 2: 689 (1996)). See, also,
Bergers et al., supra, 1999; Bergers et al., supra, 2003.
[0106] An anti-angiogenic agent useful in the invention also can be
an anti-angiogenic agent effective against tumor vasculature. As
used herein, the term "anti-angiogenic agent effective against
tumor vasculature" means an angiogenic agent that can significantly
reduce tumor burden or extend life-span of animals having solid,
vascularized tumors. Such an anti-angiogenic agent is an
anti-angiogenic agent effective against tumor vasculature if there
is efficacy against one or more of the following: small solid
tumors, tumors with well-defined margins, invasive tumors or
end-stage cancer, whether or not the agent significantly reduces
the number of angiogenic lesions during the pre-malignant phase of
carcinogenesis. As non-limiting examples, an anti-angiogenic agent
effective against tumor vasculature can be efficacious only against
small vascularized tumors, or against large tumors as well as small
vascularized tumors. An anti-angiogenic agent effective against
tumor vasculature can be, without limitation, an anti-angiogenic
agent such as BB-94 (batimastat), endostatin, or angiostatin, which
is effective against small tumors without significant efficacy on
the large tumors characteristic of end-stage cancer (Bergers et
al., supra, 1999). An anti-angiogenic agent effective against tumor
vasculature further can be an anti-angiogenic agent effective
against small tumors as well as large tumors in animals with short
life expectancy such as, without limitation, AGM-1470 (TNP470), a
small molecule inhibitor of endothelial cell proliferation (Ingber
et al., Nature 348:555 (1990); Griffith et al., Chem. Biol. 4:461
(1997); Sin et al., Proc. Natl. Acad. Sci. U.S.A. 94:6099 (1997);
and Castronovo and Belotti, Eur. J. Cancer 32A:2520 (1996)).
[0107] A therapeutic moiety useful in a conjugate of the invention
can be, for example, a cytotoxic agent. As used herein, the term
"cytotoxic agent" means any molecule that results in cell death by
any mechanism. Exemplary cytotoxic agents useful in a conjugate of
the invention encompass, without limitation, taxanes such as
docetaxel; anthracyclins such as doxorubicin; alkylating agents;
vinca alkaloids; anti-metabolites; platinum agents such as
cisplatin or carboplatin; steroids such as methotrexate;
antibiotics such as adriamycin; antimicrobial peptides, described
herein below; and other cancer chemotherapeutic agents, which are
chemical agents that inhibit the proliferation, growth, life-span
or metastatic activity of cancer cells.
[0108] Effective cytotoxic agents useful in the invention include
those that target DNA, for example, alkylating agents, agents that
intercalate into DNA, and agents that result in double-stranded DNA
breaks. Exemplary DNA-targeted drugs include, without limitation,
cyclophosphamide, melphalan, mitomycin C, bizelesin, cisplatin,
doxorubicin, etoposide, mitoxantrone, SN-38, Et-743, actinomycin D,
bleomycin, TLK286 and SGN-15 (Hurley, supra, 2002). It is
understood that DNA-targeting cytotoxic agents can be particular
useful when combined in conjugates with a homing molecule that
localizes, at least in part, to the nuclei of cells. Thus, in one
embodiment the invention provides a conjugate containing a
therapeutic moiety linked to a peptide or peptidomimetic that
selectively homes to vasculature of premalignant pancreas and
contains at least 5 contiguous amino acids of an amino acid
sequence selected from CRSRKG (SEQ ID NO:9) and CEYQLDVE (SEQ ID
NO:34), or a conservative variant or peptidomimetic thereof, where
the therapeutic moiety is a cytotoxic agent that targets a
DNA-associated process. In a further embodiment, the invention
provides a conjugate containing a therapeutic moiety linked to a
peptide or peptidomimetic that selectively homes to pancreatic
tumor cells and pancreatic tumor vasculature and contains at least
5 contiguous amino acids of an amino acid sequence selected from
CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID NO:19), and VGVGEWSV (SEQ
ID NO:35), or a conservative variant or peptidomimetic thereof.
Useful cytotoxic agents that target a DNA-associated process
include, without limitation, alkylating agents, anti-tumor
antibiotics and sequence-selective agents and further encompass
agents such as cyclophosphamide, melphalan, mitomycin C, bizelesin,
cisplatin, doxorubicin, etoposide, mitoxantrone, SN-38, Et-743,
actinomycin D, bleomycin and TLK286.
[0109] Taxanes are cytotoxic agents useful in a conjugate of the
invention. Useful taxanes include, without limitation, docetaxel
(Taxotere; Aventis Pharmaceuticals, Inc.; Parsippany, N.J.) and
paclitaxel (Taxol; Bristol-Myers Squibb; Princeton, N.J.). See, for
example, Chan et al., J. Clin. Oncol. 17:2341-2354 (1999), and
Paridaens et al., J. Clin. Oncol. 18:724 (2000).
[0110] A cytotoxic agent useful in a conjugate of the invention
also can be an anthracyclin such as doxorubicin, idarubicin or
daunorubicin. Doxorubicin is a commonly used cancer
chemotherapeutic agent (Stewart and Ratain, In: "Cancer: Principles
and practice of oncology" 5th ed., chap. 19 (eds. DeVita, Jr., et
al.; J. P. Lippincott 1997); Harris et al., In "Cancer: Principles
and practice of oncology," supra, 1997). In addition, doxorubicin
has anti-angiogenic activity that can contribute to its
effectiveness in treating cancer (Folkman, supra, 1997; Steiner, In
"Angiogenesis: Key principles-Science, technology and medicine,"
pp. 449-454 (eds. Steiner et al.; Birkhauser Verlag, 1992)).
[0111] An alkylating agent such as melphalan or chlorambucil also
can be a cytotoxic agent useful in a conjugate of the invention.
Similarly, a vinca alkaloid such as vindesine, vinblastine or
vinorelbine; or an antimetabolite such as 5-fluorouracil,
5-fluorouridine or a derivative thereof can be a cytotoxic agent
that can be linked to a homing molecule in a conjugate of the
invention.
[0112] Cytotoxic agents useful in the conjugates of the invention
also include, without limitation, platinum agents. Such a platinum
agent can be, for example, cisplatin or carboplatin as described,
for example, in Crown, Seminars in Oncol. 28:28-37 (2001). Other
cytotoxic agents useful in a conjugate of the invention include,
but are not limited to, methotrexate, mitomycin-C, adriamycin,
ifosfamide and ansamycins.
[0113] A cytotoxic agent also can be, for example, an antimicrobial
peptide. In one embodiment, the invention provides a conjugate in
which a peptide or peptidomimetic that selectively homes to
vasculature of premalignant pancreas that is linked to an
antimicrobial peptide, where the conjugate is selectively
internalized by vasculature of a As used herein, the term
"antimicrobial peptide" means a naturally occurring or synthetic
peptide having antimicrobial activity, which is the ability to kill
or slow the growth of one or more microbes and that has low
mammalian cell toxicity when not linked to a homing molecule. An
antimicrobial peptide can kill or slow the growth of, for example,
one or more strains of bacteria including a Gram-positive or
Gram-negative bacteria, or a fungi or protozoa. Thus, an
antimicrobial peptide can have, for example, bacteriostatic or
bacteriocidal activity against, for example, one or more strains of
Escherichia coli, Pseudomonas aeruginosa or Staphylococcus aureus.
While not wishing to be bound by the following, an antimicrobial
peptide can have biological activity due to the ability to form ion
channels through membrane bilayers as a consequence of
self-aggregation.
[0114] An antimicrobial peptide is typically highly basic and can
have a linear or cyclic structure. As discussed further below, an
antimicrobial peptide can have an amphipathic .alpha.-helical
structure (see U.S. Pat. No. 5,789,542; Javadpour et al., J. Med.
Chem. 39:3107-3113 (1996); and Blondelle and Houghten, Biochem. 31:
12688-12694 (1992). An antimicrobial peptide also can be, for
example, a .beta.-strand/sheet-forming peptide as described in
Mancheno et al., J. Peptide Res. 51:142-148 (1998).
[0115] An antimicrobial peptide can be a naturally occurring or
synthetic peptide. Naturally occurring antimicrobial peptides have
been isolated from biological sources such as bacteria, insects,
amphibians, and mammals and are thought to represent inducible
defense proteins that can protect the host organism from bacterial
infection. Naturally occurring antimicrobial peptides include the
gramicidins, magainins, mellitins, defensins and cecropins (see,
for example, Maloy and Kari, Biopolymers 37:105-122 (1995);
Alvarez-Bravo et al., Biochem. J. 302:535-538 (1994); Bessalle et
al., FEBS 274:151-155 (1990); and Blondelle and Houghten in Bristol
(Ed.), Annual Reports in Medicinal Chemistry pages 159-168 Academic
Press, San Diego)). As discussed further below, an antimicrobial
peptide also can be an analog of a natural peptide, especially one
that retains or enhances amphipathicity.
[0116] An antimicrobial peptide incorporated within a conjugate of
the invention has low mammalian cell toxicity when not linked to a
homing molecule of the invention. Mammalian cell toxicity readily
can be assessed using routine assays. For example, mammalian cell
toxicity can be assayed by lysis of human erythrocytes in vitro as
described in Javadpour et al., supra, 1996. An antimicrobial
peptide having low mammalian cell toxicity is not lytic to human
erythrocytes or requires concentrations of greater than 100 .mu.M
for lytic activity, preferably concentrations greater than 200,
300, 500 or 1000 .mu.M for lytic activity.
[0117] It is understood by one skilled in the art of medicinal
oncology that these and other agents are useful therapeutic
moieties, which can be used separately or together in the
conjugates and methods of the invention. It further is understood
that a conjugate of the invention can contain one or more of such
therapeutic moieties and that additional components can be included
as part of the conjugate, if desired. As an example, in some cases
it can be desirable to utilize an oligopeptide spacer between the
homing molecule and the therapeutic agent (Fitzpatrick and Garnett,
Anticancer Drug Des. 10:1-9 (1995)).
Staging Tumor Progression
[0118] The invention provides a method of staging tumor progression
in an individual having or suspected of having a pancreatic
premalignant lesion or pancreatic tumor. The method involves: (a)
administering to the individual at least one conjugate containing a
detectable moiety linked to (i) a peptide or peptidomimetic that
selectively homes to vasculature of premalignant pancreas, the
peptide or peptidomimetic specifically binding a cognate receptor
for CRSRKG (SEQ ID NO:9) or CEYQLDVE (SEQ ID NO:34), or (ii) a
peptide or peptidomimetic that selectively homes to pancreatic
tumor cells and pancreatic tumor vasculature, the peptide or
peptidomimetic specifically binding a cognate receptor for an amino
acid sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID
NO:19) and VGVGEWSV (SEQ ID NO:35); and (b) detecting the
conjugate, wherein detection of the conjugate containing a peptide
or peptidomimetic that selectively homes to vasculature of
premalignant pancreas indicates a premalignant stage of tumor
progression in the individual and wherein detection of the
conjugate containing a peptide or peptidomimetic that selectively
homes to pancreatic tumor cells and pancreatic tumor vasculature
indicates a malignant stage of tumor progression in the individual.
Exemplary moieties useful in this method are described herein
above.
[0119] A stage of a tumor refers to the degree of progression of a
tumor. A premalignant stage of tumor progression in a tissue means
precancerous state of the tissue, which can be characterized, for
example, by abnormal tissue structural and functional changes in
comparison to a normal state of the tissue. Such changes are
described in the art, for example, as metaplasia, dysplasia,
hyperplasia, carcinoma in situ, angiogenic and the like, depending
on the degree of structural and/or functional changes. Histological
and biochemical changes characteristic of precancerous tissues
depend upon the type of tumor and can be determined by those
skilled in the art. A malignant stage of tumor progression means
that the tumor is established, as indicated by, for example,
increased proliferation rate compared to normal. Various stages of
tumor development are well known to those of skill in the art, as
exemplified in Markman, "Basic Cancer Medicine," Saunders, (ed.
Zorab, R.) (1997). For example, malignant cancers can be staged
into three general stages--localized, regional spread, and distant
spread. Cancers also can be staged using the TNM system, which
considers the extent of direct spread within affected and nearby
tissues, the extent of spread to nearby lymph nodes, and the extent
of spread to distant organs. Based on these features, spread of
cancers can be summarized by assigning Roman numerals from 0
through IV. Those skilled in the art can select an appropriate
staging system for a particular type of cancer.
Routes of Administration
[0120] It is understood that a variety of routes of administration
are useful in the methods of the invention. Such routes encompass
systemic and local administration and include, without limitation,
oral administration, topical application, intravenous injection,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, transdermal diffusion or electrophoresis, local
injection, and extended release delivery devices including locally
implanted extended release devices such as bioerodible or
reservoir-based implants.
[0121] The following examples are intended to illustrate but not
limit the present invention.
Example I
Isolation of Stage-Specific Phage
[0122] This example describes the isolation of phage that
selectively home to premalignant lesions and malignant tumors.
[0123] To isolate peptides that selectively home to premalignant
lesions and malignant tumors, the RIP1-Tag2 mouse model was used.
RIP1-Tag2 mice develop multifocal angiogenic islet progenitors and
then solid tumors in a stepwise manner, such that at 12 weeks of
age, each mouse typically has approximately 50 angiogenic islets
and 2-6 small tumors. Using 12 week-old mice, phage that bind to
angiogenic islet progenitors and/or tumors in the same mouse were
identified.
[0124] The generation of RIP1-Tag2 mice is described, for example,
in Hanahan, Nature 315:115-122 (1985). Angiogenic islets were
isolated from 8 and 12 week old RIP1-Tag2 mice by collagenase
digestion of the excised pancreas, and selected based on their red,
hemorrhagic appearance (Parangi et al. Cancer Res. 55: 6071-6076
(1995)). Tumors were microdissected from the excised pancreas of
12-week old RIP1-Tag2 mice and the surrounding exocrine tissue
carefully removed. The synchronicity of tumorigenesis in the
RIP1-Tag2 model allowed for simultaneous isolation of angiogenic
islets and tumors from the same mouse at 12 weeks of age, such that
homing of individual phage to different stages in tumor progression
in an individual mouse/pancreas could be compared.
[0125] In order to enrich for phage that bind to RIP1-Tag2 target
cells (endothelial, perivascular and tumor cells), a pre-selection
step was performed on cell suspensions prepared from premalignant
and malignant pancreatic lesions. Pre-selection methods are
described, for example, in Laakkonen et al. Nat. Med. 8:751-755
(2002) and Porkka et al. Proc. Natl. Acad. Sci. USA 99, 7444-7449
(2002). The pre-selection step involved two rounds of ex vivo
selection from a CX.sub.7C peptide library on cell suspensions from
angiogenic islets or solid tumors. For the ex vivo selections, cell
suspensions were prepared from the different RIP1-Tag2 lesions in
12 week old RIP1-Tag2 mice and incubated overnight at 4.degree. C.
with 10.sup.9 plaque forming units (p.f.u.) of a T7 phage (Novagen)
displaying the CX.sub.7C peptide library. The cells were washed to
remove unbound phage, and bound phage were rescued and amplified in
E. coli. This procedure resulted in enrichment in phage that bound
to tumor, endothelial and other stromal cells present in the
suspension.
[0126] Ex vivo screening yielded phage pools that bound 7- to
8-fold over a control, non-recombinant phage to their respective
target cells (see FIG. 1A). These enriched phage pools were used in
subsequent in vivo rounds to select for phage that homed
specifically to either angiogenic islets or tumors in RIP1-Tag2
mice.
[0127] For in vivo selection of phage that home specifically to
premalignant lesions and malignant tumors, the ex vivo pre-selected
phage pool was injected intravenously into 12 week old RIP1-Tag2
mice through the tail vein, allowed to circulate for 7 minutes and
heart-perfused with PBS to remove unbound intravascular phage. As
the vasculature is preferentially available for the phage to bind
in this selection, there is an enrichment of phage that bind to the
endothelium of the target tissue.
[0128] The RIP1-Tag2 lesions and control tissues (brain, kidney,
spleen, lung, `white` pancreas lacking hemorrhagic lesions and
liver were excised to allow for comparison of homing efficiencies.
Cell suspensions were prepared by mechanical disruption of the
tissues. Tissues were washed to remove unbound phage, and the bound
phage rescued and amplified by adding E. coli. The phage pool was
re-injected into mice at a similar disease stage, and the cycle
repeated. In each experiment, non-recombinant control phage were
used as a control for relative selectivity.
[0129] Three rounds of in vivo selection on angiogenic islets
resulted in a phage pool that selectively homed to angiogenic
islets. The homing to angiogenic islets was 7-fold higher than to
tumors in the same mouse (FIG. 1B). There was no homing to control
organs. The tumor selection yielded a pool that showed an 8-fold
preference for tumors versus angiogenic islets in the same mouse
following two rounds of in vivo selection (FIG. 1C).
[0130] Sets of 96 phage clones were randomly collected from each
homing phage population that selectively homed to premalignant
angiogenic islets or pancreatic tumor cells. Peptide-encoding DNA
inserts from collected phage clones were amplified by PCR, and the
PCR products sequenced. Phage representing the most frequently
appearing peptide motifs were individually tested for their ability
to selectively home to the lesions on which they were selected,
relative to other stages in the tumorigenesis pathway and to
control organs. Six of the phage selected for further analysis were
from the tumor screen (referred to as KAA, RGR, RSR, VGVA, VGVG and
KAR), and one (EYQ) was picked from the angiogenic screen. Peptide
sequences corresponding to each of these peptide motifs are shown
in Table 1.
[0131] The identified RIP1-Tag2 homing phage fell into three
classes based on their ability to home either to angiogenic islets
or to tumors in vivo (FIG. 1D) and their ex vivo binding patterns.
The identified classes were tumor-selective phage (KAA, KAR and
VGVG); angiogenic islet-selective phage (RSR and EYQ); and phage
that home to both types of lesion (VGVA and RGR) (See Table 1 for
peptide sequences). Some of the selected peptides that share
similar peptide motifs also display similar homing patterns. For
example, KAA and KAR (CKAAKNK and CKGAKAR=XBXXBXB, where B
represents basic residues and X denotes uncharged residues) both
preferentially home to tumors over angiogenic islets. However,
other related peptides such as RGR and RSR(CRGRRST and
CRSRKG=XBXBBX) have quite different homing capabilities.
TABLE-US-00001 TABLE I Peptide sequence Extended motif Mouse
protein Accession Peptide (SEQ ID NO: ) (SEQ ID NO: ) with the
motif number RGR CRGRRST (5) RGRRS (2) PDGF-B P31240 RGRR (1)
Stromal interaction Q9P246 molecule 2 RSR CRSRKG (9) CRSR-G (38)
Cadherin EGF 035161 LAG receptor 1 KAA CKAAKNK (15) CKA-K (39)
WNT-2 NP076142 KAR CKGAKAR (19) CKGAKA (20) Collagen XII Q60847
AKAR (16) Collagen XII Q60847 GAKAR (17) Claudin 9 Q9Z0S7 VGVA
FRVGVADV (27)) F-VGVADV (40) Collagen XII Q60847 RVGV (21) Collagen
XII Q60847 EYQ CEYQLDVE (34) CEYQL (28) Semaphorin 4C Q64151 YQLDV
(32) FGFR-1 P16092 YQLDV (32) Tie-1 Q06806 Table 1. Candidate mouse
proteins sharing motifs with peptides. Peptides were analyzed using
a BLAST (NCBI) search against the SWISSPROT database, using the
option for short nearly exact matches, to identify mouse proteins
with homologous sequences.
[0132] Thus, a combination of ex vivo and in vivo phage screening
was used to obtain peptides that selectively home to either
premalignant pancreatic tissue, malignant pancreatic tissue or both
premalignant and malignant pancreatic tissue.
Example II
Tumor Stage-Specific Homing of Fluorescein-Conjugated Peptides In
Vivo
[0133] This example describes that selective phage homing was due
to the displayed peptide.
[0134] To confirm that selective phage homing was due to the
displayed peptide sequences, localization of fluorescein-conjugated
peptides after intravenous injection was observed. For this
analysis, one peptide from each homing class was selected as
follows: CRSRKG (SEQ ID NO:9), referred to as RSR
(angiogenic-selective), CKAAKNK (SEQ ID NO:15), referred to as KAA
(tumor-selective) and CRGRRST (SEQ ID NO:5), referred to as RGR
(angiogenic- and tumor-homing). Eight week old RIP1-Tag2 mice were
used to examine peptide localization during the angiogenic switch,
and 12-week old RIP1-Tag2 mice were used to visualize both
angiogenic islets and tumors.
[0135] Fluorescein-conjugated peptides corresponding to phage
insert sequences were synthesized using an automated peptide
synthesizer with standard solid-phase fluorenylmethoxycarbonyl
(Fmoc) chemistry. 100 mg of each individual fluorescein-conjugated
peptide was injected intravenously into the tail vein of RIP1-Tag2
mice at 8 or 12 weeks of age, and into normal BL/6 mice. The
peptide was allowed to circulate for 7 minutes, followed by heart
perfusion first with PBS and then with Zn-buffered formalin. The
RIP1-Tag2 pancreas and control organs (brain, kidney, liver, lung
and spleen) were removed, fixed for one hour in formalin, washed
with 1.times.PBS, placed in 30% sucrose for several hours, washed
with 1.times.PBS, and embedded in OCT (Tissue-Tek). Each peptide
was injected into at least three individual RIP1-Tag2 or normal
mice at each of the different stages. To examine the localization
of injected fluorescein-conjugated peptides, frozen sections (10 mm
thick) were cut on a cryostat, mounted in Vectashield Mounting
Medium with DAPI (Vector Laboratories) and visualized under an
inverted fluorescent microscope or a confocal microscope (Zeiss LSM
510 META).
[0136] FIG. 2 shows specific homing of RSR, KAA and RGR peptides to
normal islets, angiogenic islets and tumors. Visualization of
angiogenic islet-selective peptide RSR homing is shown in normal
islet (A), angiogenic islet (B), and tumor (C). Visualization of
tumor-selective peptide KAA homing is shown in normal islet (D),
angiogenic islet (E), and tumor (F). Visualization of angiogenic
islet- and tumor-selective peptide RGR homing is shown in (G)
normal islet, (H) angiogenic islet, and (I) tumor. Control tissues
from a RIP1-Tag2 mouse injected with fluorescein-conjugated
RGR-peptide are shown in (J) kidney; (K) brain, and (L) liver.
Similar absence of fluorescence in control tissues was observed for
the other injected peptides, indicative of a lack of homing to
control tissues.
[0137] As can be seen in FIG. 2, RSR shows abundant accumulation in
RIP1-Tag2 angiogenic islets, but little or no localization in
tumors or normal islets. KAA shows abundant localization in
RIP1-Tag2 tumors but little or no localization in angiogenic
islets, or normal islets. Finally, RGR localizes in both RIP1-Tag2
angiogenic islets and tumors but little or no localization in
normal islets. Fluorescence detected in kidney was assessed as
non-specific, likely resulting from uptake from glomerular filtrate
(FIG. 2J). Unexpectedly RSR, which was selected from the tumor
phage screening, preferentially bound to angiogenic islets. This
result indicates that the epitope bound by RSR is present both in
tumors and angiogenic islets, but is more abundant in angiogenic
islets. The observed peptide localization profiles in each case
were similar to localization profiles of the cognate phage (compare
FIG. 1D and FIG. 2), with each peptide falling into the same of the
three homing classes. In addition, control peptides did not show
specific homing to any of the RIP1-Tag2 lesional stages or to a
number of normal tissues
[0138] Thus, selective homing was confirmed to be due to the
displayed peptide sequences, rather than the phage.
Example III
Co-localization of Fluorescein-Conjugated Peptides with Vascular
Markers in RIP1-Tag2 Premalignant and Malignant Lesions
[0139] This example describes co-localization of
fluorescein-conjugated peptides with vascular markers.
[0140] To confirm that intravenous administration of phage
libraries selects for phage carrying peptides that bind to
endothelial molecules specific for the target vasculature, tissues
were collected following i.v. infusion with the various
fluorescein-conjugated peptides, sectioned, and evaluated with
endothelial cell markers.
[0141] For immunohistochemistry, frozen slides were pre-incubated
with blocking buffer (1.times.PNB from NEN Biosciences) for one
hour, washed several times in 1.times.PBS and incubated with the
primary antibody of interest overnight at 4.degree. C. The
cell-specific antibodies used were rat monoclonal anti-mouse CD31
(1:200; BD Pharmingen), rat monoclonal anti-mouse MECA-32 (1:200;
BD Pharmingen), rabbit polyclonal anti-mouse NG2 (1:200; Chemicon),
and rat monoclonal anti-mouse PDGFRb (CD140b) (1:200; eBioscience).
The corresponding secondary antibodies; Cy-3 donkey anti-rabbit IgG
and Rhodamine Red donkey anti-rat IgG (Jackson ImmunoResearch),
were used at a 1:200 dilution and incubated for one hour at room
temperature. The following species-matched immunoglobulins were
used as negative controls; rabbit IgG (Vector Laboratories) and rat
IgG (Jackson ImmunoResearch) at a 1:200 dilution. The slides were
washed several times in 1.times.PBS and mounted in Vectashield
Mounting Medium with DAPI (Vector Laboratories). Hematoxylin and
eosin (H&E) staining was performed for histological grading of
adjacent sections by standard methods, and lesions were graded as
previously described (Lopez and Hanahan, 2002).
[0142] The primary analysis involved immunostaining with a mouse
pan-endothelial cell antigen (MECA-32) antibody that recognizes a
dimer of 50-55 kDa protein subunits present on all endothelial
cells (Hallman et al., 1995; Leppink et al., 1989) (FIG. 3
B,C,H,I,N,O). Additional analyses involved immunostaining to reveal
CD31/PE-CAM, or systemic infusion of a fluorescent-labeled lectin
that binds to the endothelial lumen. In addition, tissue sections
from peptide-infused mice were stained with an antibody recognizing
NG2, a marker of the neovascular pericytes (Schlingemann et al.,
1990, 1991) (FIG. 3 E,F,K,L,Q,R).
[0143] RSR peptide localization in an angiogenic islet is shown in
panel A and D (green), while co-staining for MECA-32 (red) and the
merge are shown in panels B and C. Co-staining for NG2 (red) is
shown in panel E, with the merge in panel F. KAA peptide
localization in a tumor is shown in panels G and J (green), while
co-staining for MECA-32 (red) and the merge are shown in panels H
and I. Co-staining for NG2 (red) is shown in panel K, with the
merge in panel L. RGR peptide localization in an angiogenic islet
is shown in panel M and P (green), while co-staining for MECA-32
(red) and the merge are shown in panels N and O. Co-staining for
NG2 (red) is shown in panel Q, with the merge in panel R.
[0144] As is shown in FIG. 3, all three peptides (RSR, KAA, and
RGR) show some co-localization both with endothelial cell and
pericyte markers, indicating that each homes to and binds moieties
associated with both cell types (FIG. 3). There was no
co-localization of these peptides with MECA-32 or NG2 in the
adjacent exocrine pancreas or in normal pancreatic islets.
[0145] Homing of peptides representing all three classes of binding
specificity to both pericytes and endothelial cells was unexpected.
This result supports studies indicating that RIP1-Tag2 tumor
vasculature is leaky, as evidenced by extensive micro-hemorrhaging
(Parangi et al., 1995) and morphometric analysis (Hashizume et al.,
2000; Morikawa et al., 2002; Thurston et al., 1998), by indicating
that a circulating phage pool can have access to the extra-luminal
vascular microenvironment, where receptors on pericytes and in the
extracellular matrix can be accessible. The ex vivo pre-selection
step used to enrich for RIP1-Tag2 specific targets similarly can
select for non-luminal endothelial binding partners.
[0146] Thus, this example shows that peptides that selectively home
to premalignant, malignant or both premalignant and malignant
pancreatic tissue, co-localize with endothelial cell and pericyte
markers.
Example IV
Tumor Specificity of Homing Phage and Peptides
[0147] This example describes the specificity of in vivo phage
homing to angiogenic islets and/or tumors in the pancreas, and to
the angiogenic vasculature in other tumor types.
[0148] To determine whether phage identified to home to pancreatic
tumors also home to other types of tumors, these phage were tested
using animals having various types of tumors, including bTC3
subcutaneous tumor and MDA MB-435 subcutaneous tumor. bTC3
transplant tumors arise following subcutaneous inoculation of nude
mice with cultured islet tumor-derived (bTC3) cells (Efrat et al.,
1988). Since the vasculature of a subcutaneously grown bTC3 tumor
derives from skin, we also tested another subcutaneous transplant
tumor, arising from inoculation of the MDA-MB-435 human breast
carcinoma cell line. Finally, K14-HPV16 mice, another well-studied
transgenic mouse model of cancer that develop tumors of the
squamous epithelial cells of the skin (Arbeit et al., 1994;
Coussens et al., 1996), was used to compare RIP1-Tag2 islet tumors
to a tumor with similar multistage pathogenesis arising in a
different tissue.
[0149] Tumors were dissected from the ear or chest of K14-HPV16
mice. For the bTC3 allograft models, 10.sup.6 bTC3 tumor cells
(Efrat et al., 1988) were inoculated under the skin of the rear
flank of nu/nu mice in a BALB/c background and allowed to grow
until approximately 5 mm in size, and then used for experimental
analysis. MDA-MB-435 xenograft models were generated by inoculating
10.sup.6 tumor cells subcutaneously in the chest of nu/nu Balb/c
mice. Tumors were used for the homing/binding experiments at 8-12
weeks after injection of the tumor cells.
[0150] FIG. 4A is bar graph showing homing efficiency of individual
phage to a pancreatic tumor in a RIP1-Tag2 mouse, a bTC3-derived
subcutaneous transplant tumor in a nude mouse, and a squamous cell
carcinoma in a K14-HPV16 mouse.
[0151] FIG. 4B is a table summarizing the relative homing of
fluorescein-conjugated peptides to different tumor models. +++
indicates strong homing, as revealed by the fluorescent intensity
of i.v. injected peptide, ++ indicates moderate homing, + indicates
weak homing, - indicates absence of homing.
[0152] FIGS. 4C, D and E show representative images of
fluorescein-conjugated KAR peptide homing to a RIP1-Tag2 pancreatic
islet tumor (C); a bTC3 subcutaneous tumor (D); and an MDA
subcutaneous tumor (E).
[0153] As indicated by FIG. 4A, the relative homing efficiencies in
the various tumor models of the phage from the RIP1-Tag2 tumor
screen fall broadly into two categories: those that selectively
home to RIP1-Tag2 tumors (KAA, RGR, VGVA), and those that show a
more general homing to other tumors in addition to RIP1-Tag2 (VGVG,
KAR). The phage homing data were supported by i.v. injection of
fluorescein-conjugated peptides corresponding to the phage, as
shown in FIGS. 4C, D and E.
[0154] Thus, this example shows that certain peptides selectively
home to pancreatic tumors while others selectively home to other
types of tumors in addition to pancreatic tumors.
Example V
Identification of Receptors for RGR Peptides
[0155] This example describes the identification of candidate
vascular receptors for peptides by sequence homology
comparisons.
[0156] The set of peptides that selectively home to angiogenic
premalignant lesions in the pancreas were used in database searches
to identify mouse proteins containing sequences homologous to
peptide sequences. Table 1 lists candidate proteins of interest
that contain such homologies. Many of the candidate proteins have
been previously associated with the vasculature, and could
correspond to putative ligands mimicked by the phage-displayed
peptides. One protein, collagen XII, was found to share homology
with two peptides; KAR (CKGAKAR) and VGVA (FRVGVADV). It is
interesting to note that collagen XII was also identified by gene
expression profiling as a gene that is over-expressed in tumor
endothelial cells (St. Croix et al., 2000; and
http://mendel.imp.univie.ac.at/SEQUENCES/TEMS/mainpgs/temtable.html).
[0157] Another homology was observed for the RGR peptide (CRGRRST).
Specifically, as is shown in Table 1, the sequence RGRRS is
contained in the B chain of the pro-form of platelet-derived growth
factor (PDGF-B), a known ligand for the transmembrane receptor
tyrosine kinase PDGFR.beta.. The RGR sequence homology spans the
pro-peptide cleavage site of pro-PDGF-B (Johnsson et al., 1984).
Therefore, PDGFR.beta. was considered a candidate receptor for the
RGR peptide.
[0158] To confirm that the RGR peptide binds specifically to
PDGFR.beta., phage displaying this peptide were incubated with 293T
cells overexpressing PDGFR.beta.. Recombinant 293T cells were
prepared by transfecting with plasmids encoding PDGFR.beta. or
VEGFR-2 (Borges et al., 2000) using Fugene transfection reagent
(Roche Diagnostics). Briefly, 10 .mu.g of plasmid was mixed with
700 .mu.l of DMEM without serum and 30 .mu.l of Fugene reagent, and
incubated for 15 minutes at room temperature before adding the
mixture to the cells. Forty-eight hours post-transfection the cells
were detached from the culture plates using EDTA and washed once
with PBS. Recombinant phage displaying the RSR, RGR peptides and
control nonrecombinant phage (about 1.times.10.sup.9 pfu) were
incubated with the transfected cells for 2 hours at 4.degree. C.,
followed by 5 washes with 1% BSA in PBS to remove the unbound
phage. The bound phage were rescued by adding bacteria, and the
binding efficiencies were determined by plaque assay.
[0159] The results of these studies are shown in FIG. 5. FIG. 5A is
a bar graph showing binding of RGR or RSR phage to 293 cells
transfected with either PDGFR.beta., VEGFR2, or non-transfected
cells. FIG. 5B shows co-localization of fluorescein-conjugated
RGR-peptide (panel a, green) with the PDGFR.beta. antibody (panel
b, red) and merged images (panels c and d) in RIP1-Tag2.
[0160] As shown in FIG. 5, binding of RGR phage was 20-fold more
efficient to PDGFR.beta.-transfected cells than non-transfected
cells. In contrast, no binding above background was detected toward
cells transfected with vascular endothelial growth factor receptor
2 (VEGFR2) (FIG. 5A). Moreover, RSR phage, which has a peptide
sequence similar to RGR, displayed no specific binding was observed
either to PDGFR.beta. or VEGFR2 transfected cells (FIG. 5A). The
association of RGR with PDGFR.beta. was confirmed when
intravenously injected fluorescein-conjugated RGR peptide was shown
to co-localize with PDGFR.beta. visualized by subsequent immuno
staining of tissue sections from RIP1-Tag2 tumors. Merging of the
RGR-FITC image (FIG. 5B, panel a) with the antibody staining for
PDGFR.beta. (FIG. 5B, panel b) revealed almost complete
co-localization (FIG. 5B, panels c and d). These results indicate
that PDGFR.beta. is a receptor for the RGR peptide.
[0161] Thus, this example shows that PDGFR.beta. is a receptor for
the RGR peptide (CRGRRST).
[0162] All journal articles, references and patent citations
provided above, in parentheses or otherwise, whether previously
stated or not, are incorporated herein by reference in their
entirety.
[0163] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
4014PRTArtificial Sequencesynthetic peptide 1Arg Gly Arg
Arg125PRTArtificial Sequencesynthetic peptide 2Arg Gly Arg Arg Ser1
536PRTArtificial Sequencesynthetic peptide 3Arg Gly Arg Arg Ser
Thr1 546PRTArtificial Sequencesynthetic peptide 4Cys Arg Gly Arg
Arg Ser1 557PRTArtificial Sequencesynthetic peptide 5Cys Arg Gly
Arg Arg Ser Thr1 565PRTArtificial Sequencesynthetic peptide 6Arg
Ser Arg Xaa Gly1 576PRTArtificial Sequencesynthetic peptide 7Cys
Arg Ser Arg Xaa Gly1 585PRTArtificial Sequencesynthetic peptide
8Arg Ser Arg Lys Gly1 596PRTArtificial Sequencesynthetic peptide
9Cys Arg Ser Arg Lys Gly1 5105PRTArtificial Sequencesynthetic
peptide 10Cys Lys Ala Xaa Lys1 5116PRTArtificial Sequencesynthetic
peptide 11Cys Lys Ala Xaa Lys Asn1 5125PRTArtificial
Sequencesynthetic peptide 12Cys Lys Ala Ala Lys1 5136PRTArtificial
Sequencesynthetic peptide 13Cys Lys Ala Ala Lys Asn1
5145PRTArtificial Sequencesynthetic peptide 14Lys Ala Ala Lys Asn1
5157PRTArtificial Sequencesynthetic peptide 15Cys Lys Ala Ala Lys
Asn Lys1 5164PRTArtificial Sequencesynthetic peptide 16Ala Lys Ala
Arg1175PRTArtificial Sequencesynthetic peptide 17Gly Ala Lys Ala
Arg1 5186PRTArtificial Sequencesynthetic peptide 18Lys Gly Ala Lys
Ala Arg1 5197PRTArtificial Sequencesynthetic peptide 19Cys Lys Gly
Ala Lys Ala Arg1 5206PRTArtificial Sequencesynthetic peptide 20Cys
Lys Gly Ala Lys Ala1 5214PRTArtificial Sequencesynthetic peptide
21Arg Val Gly Val1225PRTArtificial Sequencesynthetic peptide 22Arg
Val Gly Val Ala1 5236PRTArtificial Sequencesynthetic peptide 23Arg
Val Gly Val Ala Asp1 5245PRTArtificial Sequencesynthetic peptide
24Val Gly Val Ala Asp1 5256PRTArtificial Sequencesynthetic peptide
25Val Gly Val Ala Asp Val1 5267PRTArtificial Sequencesynthetic
peptide 26Arg Val Gly Val Ala Asp Val1 5278PRTArtificial
Sequencesynthetic peptide 27Phe Arg Val Gly Val Ala Asp Val1
5285PRTArtificial Sequencesynthetic peptide 28Cys Glu Tyr Gln Leu1
5295PRTArtificial Sequencesynthetic peptide 29Glu Tyr Gln Leu Asp1
5306PRTArtificial Sequencesynthetic peptide 30Glu Tyr Gln Leu Asp
Val1 5317PRTArtificial Sequencesynthetic peptide 31Glu Tyr Gln Leu
Asp Val Glu1 5325PRTArtificial Sequencesynthetic peptide 32Tyr Gln
Leu Asp Val1 5336PRTArtificial Sequencesynthetic peptide 33Tyr Gln
Leu Asp Val Glu1 5348PRTArtificial Sequencesynthetic peptide 34Cys
Glu Tyr Gln Leu Asp Val Glu1 5358PRTArtificial Sequencesynthetic
peptide 35Val Gly Val Gly Glu Trp Ser Val1 5364PRTArtificial
Sequencesynthetic peptide 36Val Gly Val Gly1375PRTArtificial
Sequencesynthetic peptide 37Val Gly Val Gly Glu1 5386PRTArtificial
Sequencesynthetic peptide 38Cys Arg Ser Arg Xaa Gly1
5395PRTArtificial Sequencesynthetic peptide 39Cys Lys Ala Xaa Lys1
5408PRTArtificial Sequencesynthetic peptide 40Phe Xaa Val Gly Val
Ala Asp Val1 5
* * * * *
References